Curcumin suppresses tumorigenesis by ferroptosis in breast cancer

Xuelei Cao; Yao Li; Yongbin Wang; Tao Yu; Chao Zhu; Xuezhi Zhang; Jialiang Guan

doi:10.1371/journal.pone.0261370

. 2022 Jan 18;17(1):e0261370. doi: 10.1371/journal.pone.0261370

Curcumin suppresses tumorigenesis by ferroptosis in breast cancer

Xuelei Cao ^1,^#, Yao Li ^2,^#, Yongbin Wang ¹, Tao Yu ¹, Chao Zhu ^3,^*, Xuezhi Zhang ^4,^*, Jialiang Guan ^1,^*

Editor: Irina V Balalaeva⁵

PMCID: PMC8765616 PMID: 35041678

Abstract

Breast cancer (BC) is one of the most common malignant tumors found in females. Previous studies have demonstrated that curcumin, which is a type of polyphenol compound extracted from Curcuma longa underground rhizome, is able to inhibit the survival of cancer cells. However, the functional role and mechanism of curcumin in BC are still unclear. The Cell Counting Kit-8 assay was performed to examine the effects of curcumin on cell viability in the BC cell lines MDA-MB-453 and MCF-7. The levels of lipid reactive oxygen species (ROS), malondialdehyde (MDA) production, and intracellular Fe²⁺ were determined to assess the effects of curcumin on cell ferroptosis. Western blot analysis was also carried out to detect the protein levels. Finally, the antitumorigenic effect of curcumin on BC was identified in a xenograft tumor model. In the present study, the results indicated that curcumin could dose-dependently suppress the viability of both MDA-MB-453 and MCF-7 cells. Further studies revealed that curcumin facilitated solute carrier family 1 member 5 (SLC1A5)-mediated ferroptosis in both MDA-MB-453 and MCF-7 cells by enhancing lipid ROS levels, lipid peroxidation end-product MDA accumulation, and intracellular Fe²⁺ levels. In vivo experiments demonstrated that curcumin could significantly hamper tumor growth. Collectively, the results demonstrated that curcumin exhibited antitumorigenic activity in BC by promoting SLC1A5-mediated ferroptosis, which suggests its use as a potential therapeutic agent for the treatment of BC.

Introduction

Breast cancer (BC) is one of the most frequently diagnosed cancers and the leading cause of cancer-related death among women [1]. BC accounts for 30% of all cancer cases and 14% of all cancer-related deaths among women [2]. In recent years, the incidence of BC has continued to rise, which affects human health and the quality of life, and causes a massive burden to the medical industry and economy. Due to the lack of notably early symptoms and standardized physical examinations, the majority of patients with BC are diagnosed with metastasis, which results in a poor prognosis [3]. Surgery, chemotherapy, and radiotherapy are most commonly used for the treatment of BC. However, chemotherapeutic drugs generally have the disadvantages of being costly and causing side effects, including emesis, nausea, alopecia, myelosuppression, and thromboembolism [4, 5]. Therefore, it is of considerable significance to identify safe, effective, and widely sourced anticancer drugs with limited side effects for the treatment of BC.

Accumulating evidence has demonstrated that ingredients extracted from Chinese herbal medicines and natural plants can be considered novel approaches to prevent and cure tumors [6]. Curcumin is the main active material that is separated from the Curcuma longa underground rhizome [7]. Curcumin has a widespread function in tumor prevention and treatment [8]. Curcumin exhibits antitumor effects on various cancers via the regulation of tumor-related genes and signaling pathways [9]. Recent studies have shown that curcumin exhibits antitumor effects on BC [10]. However, the functional roles and mechanisms of curcumin in BC have not been clearly elucidated.

Ferroptosis is a type of iron-dependent programmed cell death, which is different from apoptosis, necrosis and autophagy [11]. The primary mechanism underlying ferroptosis involves the action of divalent iron or lipoxygenase, which catalyzes the metabolism of unsaturated fatty acids on the cell membrane, resulting in lipid peroxidation that eventually induces cell death [12]. Ferroptosis plays an essential role in the occurrence and development of cancer [13, 14]. Moreover, recent studies have shown that the induction of cell ferroptosis may become an effective cancer treatment strategy [15]. It has been reported that danshen, a traditional Chinese medicine, improves survival of patients with BC and induces ferroptosis and apoptosis of BC cells [16]. Similarly, an additional study published in 2018 reported that certain natural compounds exerted antitumor activities via the induction of non-apoptotic programmed cell death, including ferroptosis, which provided an effective therapeutic strategy for patients with cancer [17]. The aforementioned studies indicate that ferroptosis plays an important role in the occurrence and progression of this disease. However, whether curcumin exhibits antitumor effects by regulating cell ferroptosis in BC remains unknown.

In the present study, curcumin treatment significantly inhibited BC cell viability in a dose-dependent manner. Moreover, administration of curcumin to BC cells induced ferroptosis by enhancing the levels of lipid reactive oxygen species (ROS), malondialdehyde (MDA), which is one of the most vital end-products of lipid peroxidation, and intracellular Fe²⁺. Treatment of BC cells with curcumin significantly suppressed tumorigenesis by upregulating solute carrier family 1 member 5 (SLC1A5) expression, which is an essential transporter of glutamine. Based on these results, it may be concluded that therapeutic interventions mediated through the use of curcumin-induced ferroptosis can potentially provide a promising strategy for the treatment of BC.

Materials and methods

Cell culture

The human BC cell lines (MDA-MB-453 and MCF-7) were purchased from Shanghai Institutes for Biological Sciences Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium (Gibco; Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, USA) and 2 mM L-glutamine at 37°C in an incubator with a humidified atmosphere containing 5% CO₂.

Cell treatment

Curcumin (purity>98%; Sigma-Aldrich; Merck KGaA, USA) was dissolved in DMSO at a concentration of 0, 1, 2, 5, 10, 20, and 50 μΜ. The stock solutions were stored and diluted to specific concentrations in cell culture medium for cell treatment. Both MDA-MB-453 and MCF-7 cells were preincubated with various inhibitors, such as ZVAD-FMK (Sigma-Aldrich; Merck KGaA, USA), ferrostatin-1 (Fer-1; Sigma-Aldrich; Merck KGaA, USA), deferoxamine (DFO; Sigma-Aldrich; Merck KGaA, USA), and necro-sulfonamide (NS; Sigma-Aldrich; Merck KGaA, USA), for 2 h. 10 nM erastin (MedChem Express, USA) was used as a control for 24 h, following treatment with different concentrations of curcumin for 48 h.

Cell transfection

A total of 2x10⁵ cells were seeded per well and grown to 40–60% confluence. The SLC1A5 small interfering RNA (si-SLC1A5), and blank plasmid were purchased from GenePharma (Shanghai, China). The vectors were transfected into MDA-MB-453 and MCF-7 cells using Lipofectamine^® 3000 kits (Invitrogen; Thermo Fisher Scientific, USA) according to the manufacturer’s protocols. Following transfection, the cells were incubated for 48 h, and the transfection efficiency was determined by western blot analysis.

Cell viability analysis

A total of 100 μl cell suspension (5×10³ cells) was plated in 96-well plates and incubated at 37˚C with 5% CO₂. The cells were grown to ~70% confluence and subsequently treated with curcumin or transfected with si-SLC1A5. Following transfection, the cells were incubated for 24 h and 10 μl Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology, China) solution was added to each well. The samples were incubated for 60 min and the absorbance of each well was assessed at 450 nm using a microplate reader (Thermo Fisher Scientific, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted with TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, USA) according to the supplier’s instructions. A total of 1 μg total RNA was reverse transcribed into single-strand complementary DNA (cDNA) using the One Step PrimeScript miRNA cDNA Synthesis Kit (TaKaRa Bio, Japan). RT-qPCR was performed in triplicate using SYBR Green PCR Master Mix (Life Technologies, USA) following the manufacturer’s protocols. The relative expression levels of acyl-CoA synthetase long-chain family member 4 (ACSL4), nicotinamide adenine dinucleotide phosphate-oxidase 1 (NOX1), glutathione peroxidase 4 (GPX4), and ferritin light chain (FTL) were normalized using β-actin as reference. The values were calculated using the 2^-ΔΔCt method. The sequences of the primers used for RT-qPCR were the following: ACSL4 (SLC1A5) forward, 5’-TTTTGCGAGCTTTCCGAGTG-3’ and reverse, 5’-AGCCGACAATAAAGTACGCAA-3’; NOX1 forward, 5’-TTGGGTCAACATTGGCCTGT-3’ and reverse. 5’-AAGGACAGCAGATTGCGACA-3’; GPX4 forward, 5’-ATTGGTCGGCTGGACGAG-3’ and reverse, 5’-TCGATGTCCTTGGCGGAAAA-3’; FTL forward, 5’-GCCACTTCTTCCGCGAATTG-3’ and reverse, 5’-TTCATGGCGTCTGGGGTTTT-3’; Sodium-coupled neutral amino acid transporter 1 (SLC38A1) forward, 5’-AACCTCCTTAGGCATGTCTGT-3’ and reverse, 5’-GCAAAGGCGAGTCCCAAAAT-3’ and β-actin forward, 5’-TCCCTGGAGAAGAGCTACGA-3’ and reverse, 5’-AGCACTGTGTTGGCGTACAG-3’.

Western blot analysis

Total protein was extracted by cell lysis using RIPA Lysis Buffer (Beyotime Institute of Biotechnology, China). The lysate was centrifuged at 12,000 rpm at 4˚C for 10 min, and the supernatant was transferred to a new tube to quantify total protein using the BCA assay. Subsequently, electrophoresis was conducted with 12% SDS gels followed by transfer onto PVDF membranes (EMD Millipore). Following blocking with 5% (w/v) non-fat dry milk, the membranes were incubated with primary antibodies against β-actin (ab8227, 1:1,000), SLC1A5 (ab237704, 1:1,000), aspartate aminotransferase (GOT1; ab239487, 1:1,000 dilution), and glutaminase 2 (GLS2; ab113509, 1:1,000) and all antibodies were purchased from Abcam. Subsequently, the appropriate HRP-conjugated secondary antibodies (1:5,000; ProteinTech Group) were applied. The protein bands were detected using a chemiluminescence-based method (Pierce; Thermo Fisher Scientific, USA) on a Tanon 5200 Imaging system (Tanon Science & Technology Co., China). The expression levels of the proteins in each sample were normalized to those of β-actin.

Nude mice model

Female BALB/c nude mice (age, 6–8 weeks) were purchased from Guangdong Medical Laboratory Animal Center (Foshan, China). The animal model experiments were approved by the Ethical Committee of The Affiliated Hospital of Qingdao University. MCF-7 cells (5x10⁶) were suspended in serum-free DMEM and subsequently injected into the right posterior flanks of the mice. Following two weeks of tumor growth, 40 mice were randomly divided into the four following groups (n = 10 per group): Control group, curcumin group, curcumin + DFO group, and curcumin + NS group. The mice in the curcumin group were treated with 30 mg/kg/d curcumin (Intragastric administration). DFO and NS were administered by intraperitoneal injection at concentrations of 30 mg/kg and 30 mg/kg, respectively, three times a week following administration of curcumin. The mice in the control group were fed with 0.9% sodium chloride and 1% DMSO. The tumor growth in the mice was examined every 3 days. The mice were sacrificed by intraperitoneal injection of pentobarbital sodium (200 mg/kg) following administration of curcumin for 4 weeks, and the size of each tumor was measured. The tumor tissues were collected for subsequent experiments and the expression levels of SLC1A5 (1:500; Cell Signaling Technology) and Ki-67 (1:500; Cell Signaling Technology) were evaluated by immunohistochemical staining according to the manufacturer’s instructions and previous methodologies [18].

Iron determination assay

The intracellular ferrous iron (Fe²⁺) levels were determined using the iron assay kit purchased from Abcam (cat. no. ab83366) according to the manufacturer’s instructions. The experiment was repeated three times for each group.

MDA assay

The intracellular MDA concentration in cell lysates or tissues was assessed using a lipid peroxidation assay kit (cat. no. ab118970, Abcam) according to the manufacturer’s instructions. The reaction of MDA in the samples with thiobarbituric acid (TBA) resulted in the generation of a MDA-TBA adduct. The MDA-TBA adduct was quantified colorimetrically [optical density (OD) = 532 nm]. The experiments were repeated three times for each group.

Glutamine uptake assay

BC cells were cultured in six-well plates in the glutamine-free DMEM/F-12 medium (Invitrogen; Thermo Fisher Scientific, USA). Following collection and counting, the cells were incubated with 200 nM [³H]-L-glutamine (PerkinElmer; Thermo Fisher Scientific, USA) in glutamine-free DMEM/F-12 medium (Gibco; Thermo Fisher Scientific, USA) for 15 min at 37˚C in the presence of curcumin or with curcumin + SLC1A5 small interfering RNA (siRNA). The cells were collected, transferred to filter paper using a 96-well plate harvester, dried, and exposed to scintillation fluid. The counts were measured using a liquid scintillation counter (PerkinElmer; Thermo Fisher Scientific).

Determination of lipid ROS levels

The cells (2×10⁵) were seeded in a 6-well plate and treated with curcumin or erastin for 24 h. All cells were cultured in DMEM medium with 5 μM BODIPY-C11 (Thermo Fisher Scientific, USA) for 45 min at room temperature. Following incubation, the cells were collected and washed twice with PBS buffer. Subsequently, the cells were resuspended in 500 μl PBS, and subsequently filtered on a 0.4 μm nylon cell strainer. Finally, they were analyzed by flow cytometry to detect the levels of ROS within the cells. The fluorescence intensities of the cells were determined using a CytoFLEX flow cytometer (Beckman Coulter, USA). Each experiment was repeated three times.

Statistical analysis

All experiments were repeated at least three times. The data are presented as mean ± SD. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, USA). The unpaired Student’s t-test and one-way ANOVA were used to compare the means of two and three or more groups, respectively. Pairwise group comparisons were conducted using the Tukey’s post hoc test following ANOVA. P<0.05 was considered to indicate a statistically significant difference.

Results

Curcumin contributes to erastin-induced ferroptosis in BC cells

Previous studies have confirmed that curcumin functions as an antineoplastic agent in various solid tumors [8]. Results of the CCK-8 assay indicated that curcumin exhibited dose-dependent inhibitory effects on both MDA-MB-453 and MCF-7 cells (IC₅₀-MDA-MB-453 = 19.73 μM, Fig 1A; IC₅₀-MCF-7 = 20.46 μM, Fig 1B). In addition, the data indicated that the corresponding volume of the DMSO solvent exhibited no cytotoxicity on MDA-MB-453 and MCF-7 cells compared with that of 20 μM curcumin (S1 Fig). Based on these findings, 20 μM curcumin was selected as the follow-up experimental dose. Moreover, the effects of this compound were examined on iron-dependent cell death of BC cells. As shown in Fig 1C and 1D, treatment with erastin, a ferroptosis activator, significantly inhibited cell viability compared with that of the control group, whereas co-treatment of curcumin and erastin strongly reduced BC cell viability. Treatment of the cells with ferrostatin-1 (Fer-1), a ferroptosis inhibitor, notably restored the inhibitory effect of curcumin or erastin on BC cell viability, whereas pretreatment of the cells with an apoptotic (ZVAD-FMK) or necroptotic inhibitor (NS) did not improve cell survival. Taken together, these results indicated that curcumin exhibited an antitumor effect on BC cells by inducing cell ferroptosis.

Fig 1 — A and B: BC cells were treated with CUN at 0, 1, 2, 5, 10, 20 and 50 μM for 48 h. The CCK-8 assay was used to assess cell viability. C and D: BC cells were preincubated with various inhibitors, including ZVAD-FMK (50 μM), NS (50 μM), ferrostatin-1 (50 nM) or erastin (10 nM) for 2 h followed by CUN (20 μM) treatment for 48 h. The CCK-8 assay was performed to detect cell viability in both MDA-MB-453 and MCF-7 cells. Each data point is expressed as mean ± SEM of 3–5 independent tests. **P<0.01, ***P<0.001. CUN, curcumin; BC, breast cancer; CCK-8, cell counting kit-8; NS, necro-sulfonamide; SEM, standard error of the mean.

Curcumin promotes lipid peroxidation and iron accumulation during activation of ferroptosis

Accumulating evidence has shown that cell ferroptosis is mainly caused by intracellular lipid peroxidation and by accumulation of lethal ROS during iron metabolism [19]. As shown in Fig 2A and 2B, treatment of the cells with erastin significantly enhanced accumulation of lipid ROS compared with the control group. Similarly, treatment of the two BC cell lines with 20 μM curcumin increased lipid ROS levels, as demonstrated by flow cytometry using the fluorescent probe C11-BODIPY. Moreover, both MDA-MB-453 and MCF-7 cells treated with erastin or curcumin indicated a rise in MDA levels, which is one of the most vital end-products of lipid peroxidation (Fig 2C). Furthermore, higher levels of intracellular Fe²⁺ were detected in the erastin or curcumin groups than those noted in the control group (Fig 2D). However, treatment of the cells with the ferroptosis inhibitor DFO caused downregulation of intracellular Fe²⁺ levels (S2A Fig). It is interesting to note that treatment of the cells with curcumin or erastin significantly decreased the mRNA levels of GPX4 and ferritin light chain (FTL; Fig 2E and 2F), whereas it increased the mRNA levels of ACSL4 and NOX1 (Fig 2E and 2F). Collectively, these results indicated that curcumin significantly increased ferroptosis in BC cells.

Fig 2 — A and B: The levels of lipid ROS were evaluated in MDA-MB-453 and MCF-7 cells treated with CUN for 48 h or erastin for 2 h by flow cytometry using C11-BODIPY; C: The MDA accumulation was examined by a lipid peroxidation assay kit; D: The intracellular Fe²⁺ levels in the BC cell lines were measured by an iron assay kit; E and F: qRT-PCR was performed to examine the mRNA levels of genes associated with ferroptosis. Each data point is expressed as mean ± SEM of 3–5 independent tests. **P<0.01, ***P<0.001. CUN, curcumin; ROS, reactive oxygen species; MDA, malondialdehyde; BC, breast cancer; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean.

Increased glutamine uptake is essential for curcumin-induced ferroptosis in BC cells

Previous studies have confirmed that abnormal glutamine metabolism may contribute to ferroptosis by the accumulation of lipid peroxidation products in cancer cells (Fig 3A) [20]. Glutamine uptake is mainly dependent on specific transporters, such as cysteine-preferring transporter 2 (ASCT2; SLC1A5) and SLC38A1 [21]. As expected, curcumin significantly enhanced the mRNA levels of SLC1A5, but not those of SLC38A1 in MDA-MB-453 and MCF-7 cells (S3 Fig). Moreover, SLC1A5 has been shown to mediate uptake of glutamine, which is a conditionally essential amino acid used in rapidly proliferating tumor cells [22]. The data indicated that curcumin treatment significantly enhanced glutamine uptake (Fig 3B) in both BC cell types compared with that of the control group. Subsequently, the effects of curcumin were examined on the expression of crucial glutamine metabolism genes, such as SLC1A5, GLS2, and GOT1 by western blot analysis. Both BC cell lines were treated with curcumin, which resulted in a significant increase in the protein levels of SLC1A5 compared with those of the control group (Fig 3C and 3D). However, the protein levels of GLS2 and GOT1 remained unaltered. Moreover, both cell lines were treated with an inhibitor of GLS2 (compound 968; C-968) and GOT1 (AOA), which significantly restored the inhibitory effect of curcumin on cell viability (Fig 3E). Furthermore, curcumin induced high levels of lipid ROS (Fig 3F), MDA (Fig 3G), and intracellular Fe²⁺ (Fig 3H, S2B Fig) in BC cells. These effects were alleviated by C-968 or AOA in both MDA-MB-453 and MCF-7 cells. Overall, these data suggested that curcumin induced ferroptosis by the upregulation of glutamine uptake in BC cells.

Fig 3 — A: Schematic overview of the glutaminolysis pathway in ferroptosis; B: The [³H]-_L-glutamine uptake was determined using a liquid scintillation counter; C and D: Western blot analysis was performed to detect the protein levels of SLC1A5, GOT1, and GLS2 in both BC cell lines; E: The CCK-8 assay was used to examine cell viability; F: Flow cytometry was employed using C11-BODIPY to detect the levels of lipid ROS in cells treated with curcumin or erastin; G: The MDA accumulation was examined by a lipid peroxidation assay kit; H: The intracellular Fe²⁺ levels were measured in both BC cell lines by an iron assay kit. Each data point is expressed as the mean ± SEM of 3–5 independent tests. **P<0.01, ***P<0.001. ^##P<0.01, compared with the curcumin treatment alone group. CUN, curcumin; BC, breast cancer; SLC1A5, solute carrier family 1 member 5; GOT1, aspartate aminotransferase; GLS2, glutaminase 2; CCK-8, cell counting kit-8; ROS, reactive oxygen species; MDA, malondialdehyde; SEM, standard error of the mean.

Curcumin promotes ferroptosis by upregulating SLC1A5 expression in BC cells

To further assess the regulation of curcumin-induced ferroptosis by SLC1A5 in BC cells, SLC1A5 siRNA (si-SLC1A5) was transfected into MDA-MB-453 and MCF-7 cells in order to decrease its expression levels (Fig 4A). The CCK-8 assay indicated that curcumin treatment significantly reduced cell viability, which was ameliorated by the inhibition of SLC1A5 expression in both cell lines (Fig 4B). Moreover, knockdown of SLC1A5 reduced the effects of curcumin on glutamine uptake (Fig 4C). Similarly, the induction of lipid ROS (Fig 4D and 4E), MDA production (Fig 4F), and intracellular Fe²⁺ levels (Fig 4G), which was caused by curcumin treatment in both BC cell lines was reversed by suppression of SLC1A5. Taken together, these data indicated that curcumin exhibited its antitumor effect on BC by promoting SLC1A5-mediated ferroptosis in vitro.

Fig 4 — A: Western blot analysis was performed to examine the protein levels of SLC1A5 in both BC cell lines following transfection with si-SLC1A5 or si-NC; B: The CCK-8 assay was used to examine the effects of SLC1A5 knockdown on cell viability in both BC cell lines; C: The [³H]-_L-glutamine uptake was determined using a liquid scintillation counter; D and E: Flow cytometry was employed using C11-BODIPY to detect the levels of lipid ROS in BC cell lines treated with curcumin or erastin; F: The MDA accumulation in both BC cells was examined by a lipid peroxidation assay kit; G: The intracellular Fe²⁺ levels in both BC cell lines were measured by an iron assay kit. Each data point is expressed as mean ± SEM of 3–5 independent tests. **P<0.01. CUN, curcumin; SLC1A5, solute carrier family 1 member 5; BC, breast cancer; si-SLC1A5, SLC1A5 small interfering RNA; si-NC, NC-small interfering RNA; CCK-8, cell counting kit-8; ROS, reactive oxygen species; MDA, malondialdehyde; SEM, standard error of the mean.

Curcumin inhibits tumor growth of BC in vivo

Based on the previous in vitro results of curcumin on BC cells, its effects were explored on tumor growth of BC in vivo. As shown in Fig 5A, curcumin treatment did not affect body weight, indicating that it was safe in mice. Subsequently, the effects of curcumin were examined on tumor growth of BC in vivo. As shown in Fig 5B–5D, the tumor volume and tumor weight in the treatment group that received curcumin at a dose of 30 mg/kg/d were decreased compared with those of the control group. In contrast to these findings, the administration of curcumin and DFO reversed the inhibitory effect of curcumin on tumor growth, whereas the addition of NS, a necroptotic inhibitor, did not affect the efficiency of curcumin, which was consistent with the results of the in vitro experiments indicating that curcumin exhibited an antitumor effect on BC cells by inducing cell ferroptosis. In addition, immunohistochemical staining demonstrated that curcumin administration reduced the expression levels of Ki-67 in xenograft tissues compared with those of the control group, whereas it increased the expression levels of SLC1A5 (Fig 5E and 5F). Furthermore, curcumin administration resulted in elevated MDA production (Fig 5G) and increased iron content in tumor tissues (Fig 5H) compared with that of the control group, whereas it decreased the content of glutathione (GSH; Fig 5I). Moreover, DFO (ferroptosis inhibitor) effectively suppressed curcumin-induced ferroptosis (Fig 5B–5I), whereas NS could not change the effects of curcumin-induced cell death. In conclusion, the findings suggested that curcumin exerted its antitumor effect on BC by promoting SLC1A5-mediated ferroptosis.

Discussion

Currently, BC exhibits the highest morbidity and mortality rates in women. Although surgery, chemotherapy, and radiotherapy are effective treatment strategies for BC, patients are still suffering from the side effects of chemoradiotherapy. Therefore, it is important to assess the mechanism of BC and seek novel strategies to reduce cancer mortality. In the present study, the results indicated that curcumin significantly suppressed cell viability and tumor growth in BC. Moreover, BC cells treated with curcumin triggered cancer cell ferroptosis. Curcumin exhibited antitumorigenic effects on BC via the upregulation of SLC1A5-mediated ferroptosis.

Increasing evidence has confirmed that therapeutic drugs targeting cell ferroptosis can effectively interfere with cell proliferation and inhibit tumor progression [23]. Ferroptosis plays a vital role in the interaction of cancer-acquired drug resistance and immune evasion [24]. For example, inhibition of nuclear factor erythroid 2-related factor reversed cisplatin resistance by upregulating the expression levels of glutathione peroxidase 4, which is a regulator of ferroptosis, in order to induce ferroptosis in head and neck cancer [25]. Sun et al. reported that metallothionein-1G contributed to sorafenib resistance by suppressing ferroptosis in hepatocellular carcinoma [26]. Moreover, other studies have shown that glutamine metabolism is conducive to cell ferroptosis by enhancing the accumulation of oxidizable lipids [15, 27]. SLC1A5, which acts as an essential transporter for glutamine uptake, is associated with the progression of several tumors [28]. For example, inhibition of SLC1A5 restricted the progression of non-small cell lung cancer by decreasing glutamine consumption, cell growth, and inducing cell autophagy and apoptosis [29]. In the present study, the data indicated that inhibition of SLC1A5 promoted BC cell viability. Moreover, several studies have shown that the expression levels of SLC1A5 are higher in various solid cancers [30], while suppression of SLC1A5 activity by treatment with the glutamine transporter inhibitor L-γ-Glutamyl-p-nitroanilide or by transfecting siRNA significantly inhibits tumorigenesis [31]. In the present study, suppression of SLC1A5 inhibited ferroptosis of BC cells by reducing the levels of glutamine uptake and lipid ROS, while curcumin treatment significantly reversed this effect. Taken together, the data suggest that targeting SLC1A5 may be an effective therapeutic target for cancer treatment, particularly to target glutamine metabolism and cell biological behavior.

Accumulating evidence has confirmed that curcumin can suppress cell proliferation and induce cell apoptosis in various cancer types [32]. Moreover, curcumin exhibits antimetastatic activity in BC cells and inhibits the proliferation of cancer stem-like cells [33], whereas it overcomes chemoresistance of cancer by suppressing the expression levels of multiple antiapoptotic proteins [34]. It is interesting to note that curcumin may act as a potential therapeutic agent and as an adjunct therapy in BC [35]. In the present study, the data indicated that treatment with curcumin notably inhibited BC cell viability and tumor growth. It is important to note that SLC1A5-mediated glutamine metabolism played an important role in regulating ferroptosis in cancer cells. As expected, the data indicated that suppression of curcumin in BC cell growth was induced by upregulation of SLC1A5-mediated ferroptosis. Similarly, several studies have demonstrated that treatment with curcumin can promote ferroptosis in non-small-cell lung cancer [36] and glioblastoma [37]. In contrast to these findings, Guerrero-Hue et al. demonstrated that curcumin reduced ferroptosis by inhibiting myoglobin-mediated induction of heme oxygenase-1 (HO-1) and ferritin, which contributed to cell ferroptosis [38]. A previous study by Li et al. confirmed that curcumin promoted BC cell ferroptosis by upregulating HO-1 levels [39]. Finally, it has been shown that HO-1 catabolizes myoglobin-derived heme to biliverdin, carbon monoxide, and iron, which is subsequently stored in ferritin [40].

In conclusion, the results of the present study indicated that curcumin inhibited cell proliferation and induced cell ferroptosis in BC. Moreover, curcumin exhibits its antitumor effect on BC by enhancing SLC1A5 expression to induce ferroptosis both in vitro and in vivo. Overall, the present study provided novel insights into the action of curcumin as an active anticancer agent, and supported the notion for its use as a potential antitumor drug for BC treatment. Moreover, the mechanism by which curcumin regulates SLC1A5-mediated ferroptosis requires further investigation.

Supporting information

S1 Fig. Effects of CUN on cell viability.

The CCK-8 assay was performed to assess cell viability in both BC cell lines treated with curcumin or DMSO. ***P<0.001, compared with the DMSO group. CUN, curcumin; CCK-8, cell counting kit-8; BC, breast cancer.

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S2 Fig. Effects of CUN on the intracellular Fe²⁺ expression.

Both BC cells were preincubated with DFO (50 μM), for 2 h followed by CUN treatment for 48 h. A, B: The intracellular Fe²⁺ expression in both BC cell lines was measured by an iron assay kit. **P<0.001, compared with the CUN group. CUN, curcumin; BC, breast cancer; DFO, deferoxamine.

(TIF)

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S3 Fig. Effects of curcumin on gene expression.

A and B: qRT-PCR was used to examine the mRNA expression levels of SLCA5 and SLC38A1. ^###P<0.001. compared with the control group.

(TIF)

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S1 Raw images. Raw data images of western blot analysis corresponding to Figs 3 and 4.

(PDF)

Click here for additional data file.^{(1.9MB, pdf)}

Abbreviations

ACSL4: Acyl-CoA synthetase long-chain family member 4
CCK-8: Cell counting kit-8
cDNA: Complementary DNA
C-968: Compound 968
DMEM: Dulbecco’s modified Eagle’s medium
FBS: Fetal bovine serum
Fer-1: Ferrostatin-1
FTL: Ferritin light chain
GPX4: Glutathione peroxidase 4
MDA: Malondialdehyde
NOX1: Nicotinamide adenine dinucleotide phosphate-oxidase 1
NS: Necro-sulfonamide
PVDF: Polyvinylidene difluoride
qRT-PCR: Quantitative real-time polymerase chain reaction
ROS: Reactive oxygen species

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

References

1.Spronk I, Schellevis FG, Burgers JS, de Bock GH, Korevaar JC. Incidence of isolated local breast cancer recurrence and contralateral breast cancer: A systematic review. Breast. 2018;39:70–9. Epub 2018/04/06. doi: 10.1016/j.breast.2018.03.011 . [DOI] [PubMed] [Google Scholar]
2.Hamood R, Hamood H, Merhasin I, Keinan-Boker L. Hormone therapy and osteoporosis in breast cancer survivors: assessment of risk and adherence to screening recommendations. Osteoporos Int. 2019;30(1):187–200. Epub 2018/11/11. doi: 10.1007/s00198-018-4758-4 . [DOI] [PubMed] [Google Scholar]
3.Ahern TP, Sprague BL, Bissell MCS, Miglioretti DL, Buist DSM, Braithwaite D, et al. Family History of Breast Cancer, Breast Density, and Breast Cancer Risk in a U.S. Breast Cancer Screening Population. Cancer Epidemiol Biomarkers Prev. 2017;26(6):938–44. Epub 2017/01/18. doi: 10.1158/1055-9965.EPI-16-0801 ; PubMed Central PMCID: PMC5457358. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Nawa-Nishigaki M, Kobayashi R, Suzuki A, Hirose C, Matsuoka R, Mori R, et al. Control of Nausea and Vomiting in Patients Receiving Anthracycline/Cyclophosphamide Chemotherapy for Breast Cancer. Anticancer Res. 2018;38(2):877–84. Epub 2018/01/29. doi: 10.21873/anticanres.12297 . [DOI] [PubMed] [Google Scholar]
5.Kawazoe H, Murakami A, Yamashita M, Nishiyama K, Kobayashi-Taguchi K, Komatsu S, et al. Patient-related Risk Factors for Nausea and Vomiting with Standard Antiemetics in Patients with Breast Cancer Receiving Anthracycline-based Chemotherapy: A Retrospective Observational Study. Clin Ther. 2018;40(12):2170–9. Epub 2018/11/06. doi: 10.1016/j.clinthera.2018.10.004 . [DOI] [PubMed] [Google Scholar]
6.Luo H, Vong CT, Chen H, Gao Y, Lyu P, Qiu L, et al. Naturally occurring anti-cancer compounds: shining from Chinese herbal medicine. Chin Med. 2019;14:48. Epub 2019/11/14. doi: 10.1186/s13020-019-0270-9 ; PubMed Central PMCID: PMC6836491. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Bandyopadhyay D. Farmer to pharmacist: curcumin as an anti-invasive and antimetastatic agent for the treatment of cancer. Front Chem. 2014;2:113. Epub 2015/01/08. doi: 10.3389/fchem.2014.00113 ; PubMed Central PMCID: PMC4275038. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Giordano A, Tommonaro G. Curcumin and Cancer. Nutrients. 2019;11(10). Epub 2019/10/09. doi: 10.3390/nu11102376 ; PubMed Central PMCID: PMC6835707. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Kunnumakkara AB, Bordoloi D, Harsha C, Banik K, Gupta SC, Aggarwal BB. Curcumin mediates anticancer effects by modulating multiple cell signaling pathways. Clin Sci (Lond). 2017;131(15):1781–99. Epub 2017/07/07. doi: 10.1042/CS20160935 . [DOI] [PubMed] [Google Scholar]
10.Mbese Z, Khwaza V, Aderibigbe BA. Curcumin and Its Derivatives as Potential Therapeutic Agents in Prostate, Colon and Breast Cancers. Molecules. 2019;24(23). ARTN 4386 doi: 10.3390/molecules24234386 WOS:000507294400185. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Mou Y, Wang J, Wu J, He D, Zhang C, Duan C, et al. Ferroptosis, a new form of cell death: opportunities and challenges in cancer. J Hematol Oncol. 2019;12(1):34. Epub 2019/03/31. doi: 10.1186/s13045-019-0720-y ; PubMed Central PMCID: PMC6441206. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Imai H, Matsuoka M, Kumagai T, Sakamoto T, Koumura T. Lipid Peroxidation-Dependent Cell Death Regulated by GPx4 and Ferroptosis. Curr Top Microbiol Immunol. 2017;403:143–70. Epub 2017/02/17. doi: 10.1007/82_2016_508 . [DOI] [PubMed] [Google Scholar]
13.Shen Z, Song J, Yung BC, Zhou Z, Wu A, Chen X. Emerging Strategies of Cancer Therapy Based on Ferroptosis. Adv Mater. 2018;30(12):e1704007. Epub 2018/01/23. doi: 10.1002/adma.201704007 ; PubMed Central PMCID: PMC6377162. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Lu B, Chen XB, Ying MD, He QJ, Cao J, Yang B. The Role of Ferroptosis in Cancer Development and Treatment Response. Front Pharmacol. 2017;8:992. Epub 2018/01/30. doi: 10.3389/fphar.2017.00992 ; PubMed Central PMCID: PMC5770584. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Xu T, Ding W, Ji X, Ao X, Liu Y, Yu W, et al. Molecular mechanisms of ferroptosis and its role in cancer therapy. J Cell Mol Med. 2019;23(8):4900–12. Epub 2019/06/25. doi: 10.1111/jcmm.14511 ; PubMed Central PMCID: PMC6653007. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Lin YS, Shen YC, Wu CY, Tsai YY, Yang YH, Lin YY, et al. Danshen Improves Survival of Patients With Breast Cancer and Dihydroisotanshinone I Induces Ferroptosis and Apoptosis of Breast Cancer Cells. Front Pharmacol. 2019;10:1226. Epub 2019/11/19. doi: 10.3389/fphar.2019.01226 ; PubMed Central PMCID: PMC6836808. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Ye J, Zhang R, Wu F, Zhai L, Wang K, Xiao M, et al. Non-apoptotic cell death in malignant tumor cells and natural compounds. Cancer Lett. 2018;420:210–27. Epub 2018/02/08. doi: 10.1016/j.canlet.2018.01.061 . [DOI] [PubMed] [Google Scholar]
18.Zan L, Chen Q, Zhang L, Li X. Epigallocatechin gallate (EGCG) suppresses growth and tumorigenicity in breast cancer cells by downregulation of miR-25. Bioengineered. 2019;10(1):374–82. Epub 2019/08/23. doi: 10.1080/21655979.2019.1657327 ; PubMed Central PMCID: PMC6738446. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Niu Y, Zhang J, Tong Y, Li J, Liu B. Physcion 8-O-beta-glucopyranoside induced ferroptosis via regulating miR-103a-3p/GLS2 axis in gastric cancer. Life Sci. 2019;237:116893. Epub 2019/10/14. doi: 10.1016/j.lfs.2019.116893 . [DOI] [PubMed] [Google Scholar]
20.Luo M, Wu L, Zhang K, Wang H, Zhang T, Gutierrez L, et al. miR-137 regulates ferroptosis by targeting glutamine transporter SLC1A5 in melanoma. Cell Death Differ. 2018;25(8):1457–72. Epub 2018/01/20. doi: 10.1038/s41418-017-0053-8 ; PubMed Central PMCID: PMC6113319. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.DeBerardinis RJ, Cheng T. Q’s next: the diverse functions of glutamine in metabolism, cell biology and cancer. Oncogene. 2010;29(3):313–24. Epub 2009/11/03. doi: 10.1038/onc.2009.358 ; PubMed Central PMCID: PMC2809806. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Scalise M, Pochini L, Console L, Losso MA, Indiveri C. The Human SLC1A5 (ASCT2) Amino Acid Transporter: From Function to Structure and Role in Cell Biology. Front Cell Dev Biol. 2018;6:96. Epub 2018/09/21. doi: 10.3389/fcell.2018.00096 ; PubMed Central PMCID: PMC6131531. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Hassannia B, Vandenabeele P, Vanden Berghe T. Targeting Ferroptosis to Iron Out Cancer. Cancer Cell. 2019;35(6):830–49. Epub 2019/05/21. doi: 10.1016/j.ccell.2019.04.002 . [DOI] [PubMed] [Google Scholar]
24.Angeli JPF, Krysko DV, Conrad M. Ferroptosis at the crossroads of cancer-acquired drug resistance and immune evasion. Nat Rev Cancer. 2019;19(7):405–14. doi: 10.1038/s41568-019-0149-1 WOS:000472883000014. [DOI] [PubMed] [Google Scholar]
25.Shin D, Kim EH, Lee J, Roh JL. Nrf2 inhibition reverses resistance to GPX4 inhibitor-induced ferroptosis in head and neck cancer. Free Radical Bio Med. 2018;129:454–62. doi: 10.1016/j.freeradbiomed.2018.10.426 WOS:000450298400042. [DOI] [PubMed] [Google Scholar]
26.Sun XF, Niu XH, Chen RC, He WY, Chen D, Kang R, et al. Metallothionein-1G Facilitates Sorafenib Resistance Through Inhibition of Ferroptosis. Hepatology. 2016;64(2):488–500. doi: 10.1002/hep.28574 WOS:000380034500019. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Gao MH, Monian P, Quadri N, Ramasamy R, Jiang XJ. Glutaminolysis and Transferrin Regulate Ferroptosis. Mol Cell. 2015;59(2):298–308. doi: 10.1016/j.molcel.2015.06.011 WOS:000362457000016. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Jiang HL, Zhang N, Tang TZ, Feng F, Sun HP, Qu W. Target the human Alanine/Serine/Cysteine Transporter 2(ASCT2): Achievement and Future for Novel Cancer Therapy. Pharmacol Res. 2020;158. ARTN 104844 doi: 10.1016/j.phrs.2020.104844 WOS:000542124500056. [DOI] [PubMed] [Google Scholar]
29.Hassanein M, Qian J, Hoeksema MD, Wang J, Jacobovitz M, Ji XM, et al. Targeting SLC1a5-mediated glutamine dependence in non-small cell lung cancer. Int J Cancer. 2015;137(7):1587–97. doi: 10.1002/ijc.29535 WOS:000358012600007. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Zhuang X, Tong HC, Ding Y, Wu LY, Cai JS, Si Y, et al. Long noncoding RNA ABHD11-AS1 functions as a competing endogenous RNA to regulate papillary thyroid cancer progression by miR-199a-5p/SLC1A5 axis. Cell Death Dis. 2019;10. ARTN 620 doi: 10.1038/s41419-019-1850-4 WOS:000481964400002. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Wang L, Liu Y, Zhao TL, Li ZZ, He JY, Zhang BJ, et al. Topotecan induces apoptosis via ASCT2 mediated oxidative stress in gastric cancer. Phytomedicine. 2019;57:117–28. doi: 10.1016/j.phymed.2018.12.011 WOS:000465081700013. [DOI] [PubMed] [Google Scholar]
32.Calibasi-Kocal G, Pakdemirli A, Bayrak S, Ozupek NM, Sever T, Basbinar Y, et al. Curcumin effects on cell proliferation, angiogenesis and metastasis in colorectal cancer. J Buon. 2019;24(4):1482–7. WOS:000481595900022. [PubMed] [Google Scholar]
33.Hu CX, Li MJ, Guo TT, Wang SX, Huang WP, Yang K, et al. Anti-metastasis activity of curcumin against breast cancer via the inhibition of stem cell-like properties and EMT. Phytomedicine. 2019;58. ARTN 152740 doi: 10.1016/j.phymed.2018.11.001 WOS:000473047100001. [DOI] [PubMed] [Google Scholar]
34.Mortezaee K, Salehi E, Mirtavoos-mahyari H, Motevaseli E, Najafi M, Farhood B, et al. Mechanisms of apoptosis modulation by curcumin: Implications for cancer therapy. Journal of Cellular Physiology. 2019;234(8):12537–50. doi: 10.1002/jcp.28122 WOS:000467240800036. [DOI] [PubMed] [Google Scholar]
35.Kumar P, Kadakol A, Shasthrula PK, Mundhe NA, Jamdade VS, Barua CC, et al. Curcumin as an Adjuvant to Breast Cancer Treatment. Anti-Cancer Agent Me. 2015;15(5):647–56. doi: 10.2174/1871520615666150101125918 WOS:000354610900011. [DOI] [PubMed] [Google Scholar]
36.Tang X, Ding H, Liang ML, Chen X, Yan YX, Wan NS, et al. Curcumin induces ferroptosis in non-small-cell lung cancer via activating autophagy. Thoracic Cancer. 2021;12(8):1219–30. doi: 10.1111/1759-7714.13904 WOS:000624623600001. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Chen TC, Chuang JY, Ko CY, Kao TJ, Yang PY, Yu CH, et al. AR ubiquitination induced by the curcumin analog suppresses growth of temozolomide-resistant glioblastoma through disrupting GPX4-Mediated redox homeostasis. Redox Biol. 2020;30. ARTN 101413 doi: 10.1016/j.redox.2019.101413 WOS:000513891900003. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Guerrero-Hue M, Garcia-Caballero C, Palomino-Antolin A, Rubio-Navarro A, Vazquez-Carballo C, Herencia C, et al. Curcumin reduces renal damage associated with rhabdomyolysis by decreasing ferroptosis-mediated cell death. Faseb Journal. 2019;33(8):8961–75. doi: 10.1096/fj.201900077R WOS:000478832900022. [DOI] [PubMed] [Google Scholar]
39.Li RH, Zhang J, Zhou YF, Gao Q, Wang R, Fu YR, et al. Transcriptome Investigation and In Vitro Verification of Curcumin-Induced HO-1 as a Feature of Ferroptosis in Breast Cancer Cells. Oxid Med Cell Longev. 2020;2020. Artn 3469840 doi: 10.1155/2020/3469840 WOS:000596448900002. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Stocker R. Induction of haem oxygenase as a defence against oxidative stress. Free Radic Res Commun. 1990;9(2):101–12. Epub 1990/01/01. doi: 10.3109/10715769009148577 . [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0261370.r001

Decision Letter 0

Irina V Balalaeva

11 Aug 2021

PONE-D-21-20659

Curcumin suppresses tumorigenesis by ferroptosis in breast cancer

PLOS ONE

Dear Dr. Guan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: I Don't Know

**********

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Although the authors showed that curcumin suppressed viability of breast cancer cells and tumor growth at relatively high concentrations, it is also toxic to non-tumor cells such as mouse hippocampal HT22, PC12 and NIH-3T3 cells (Hirata et al., ACS Chem Neurosci 11, 76-85, 2020; Meng et al., PLOS ONE 9, e85570, 2014; Zhang et al., Mol Immunol 116, 191-198, 2019). Therefore, the cytotoxic effect of curcumin is not specific for breast cancer cells.

Line 61-62. This sentence is inaccurate, or misleading information.

Lin et al. analyzed a cohort of 79,335 patients with breast cancer between 2000 and 2010 and showed that danshen improves survival of breast cancer patients. They also showed that dihydroisotanshinone I (DT), a pure compound present in danshen, inhibits the growth of breast carcinoma cells, including MCF-7 and MDA-MB-231 and induces both apoptosis and ferroptosis in those cultured cells. DT-induced ferroptosis is characterized by reducing GSH content and GPX4 protein, and increasing lipid peroxidation. Therefore, there is no direct evidence that danshen prolonged the survival of patients with breast cancer by inducing ferroptosis.

Figure 2D, Figure 3H:

1. According to these results, curcumin increased ferrous ion levels in MCF-7 and MDA-MB-453 cells. However, curcumin has effective ferrous ions chelating capacity (Ak and Gulcin, Chemico-Biol Int 174, 27-37, 2008). In fact, curcumin reduces ferrous ions in cultured cells (Hirata et al., ACS Chem Neurosci 11, 76-85, 2020) and in in vitro assay (Shome et al., Biotechnology and Applied Biochemistry 68, 603-615 Biotechnology and Applied Biochemistry, 2021) at concentrations similar to those reported in this study. The authors need to examine the effect defferoxamine on ferrous ion levels as a positive control.

2. Please check whether the unit of ferrous ion is correct.

3. The relative of intracellular Fe2+ expression …: Intracellular Fe2+ level in both …

Minor points:

Figure 1: Is the concentration of erastin 10 nM?

Figure legends: The number of samples used in the experiments and statistics needs to be clarified.

Reviewer #2: In this article, the authors performed a series of in vitro and in vivo experiments to find out novel anti-tumorigenic mechanistic insight of curcumin. And they revealed that curcumin administration induced ferroptosis by enhancing the levels of lipid reactive oxygen species (ROS), malondialdehyde (MDA) of lipid peroxidation, and intracellular Fe2+ level in BC cells. Furthermore, treatment with curcumin significantly suppresses tumorigenesis in BC via upregulating SLC1A5 expression, which is an essential transporter for glutamine uptake.

As a whole, the authors performed thorough experimental plan, and their finding are quite interesting. However, the authors need further improvement of this article to grown-up to publishing-quality. The followings are the issues that the authors should be addressed.

#Because there are some other published article in which Li R, et al, revealed the anti-tumorigenic effect of curcumin by altering other ferroptosis associated gene (HO-1) in breast cancer area (Oxid Med Cell Longev. 2020 Nov 18;2020:3469840), the authors should add this article as reference and should add some discussion with regard to this article.

#As for Figure2A,2B, and 3F, please add the title of X-axis. Lipid ROS level?

#How about changing the position of Figure 5F to 5A in order to explain the less-toxic effect of curcumin to mice?

Reviewer #3: In this article, the authors show that curcumin exhibits anti-tumorigenesis activity in breast cancer cell lines by promoting SLC1A5-mediated ferroptosis (lipid ROS, lipid peroxidation end-product MDA accumulation, and intracellular Fe2+ levels). Objectives are clear, experiments are well designed, and methodology is adequate to obtain the results. The article is well written

MAJOR ISSUES

1) Which diluent is used to dissolve curcumin? It must be checked that the vehicle does not reduce cells viability.

2) Authors explain the role of ASCT2, SLC1A5 and SCL38A1 in glutamine uptake, but they only measured SLC1A5. Then they argue about the role of SLC1A5 in ferroptosis induced by curcumin but not about other receptors. Other receptors should be measured to confirm SLC1A5 implication.

3) Inmunohistochemistry staining should be quantified; a representative image is not enough to conclude Ki-67 and SLC1A5 increase.

4) To confirm the role of ferroptosis in vivo, authors should perform the same experiment but treating mice with ferrostatin and other cell death inhibitors, such as ZVAD and NS. In addition, authors should measure other ferroptosis markers than just MDA in the tumor to conclude the role of this pathway.

5) In the discussion sections, authors report that curcumin promotes ferroptosis. However, there are contradictory data in the bibliography about this issue, see reference 38 (Guerrero-Hue et al.). Authors should discuss about the possible dual role of curcumin on ferroptosis.

6) Authors should add in the figure legend the time in which each experiment was performed.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Juan Antonio Moreno

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PLoS One. 2022 Jan 18;17(1):e0261370. doi: 10.1371/journal.pone.0261370.r002

Author response to Decision Letter 0

29 Sep 2021

RE: Manuscript ID: PONE-D-21-20659

Dear Dr. Balalaeva,

We are indeed very grateful to the careful and thoughtful comments of three reviewers and your suggestions on our manuscript entitled “Curcumin suppresses tumorigenesis by ferroptosis in breast cancer” by Cao X et al. Based on the suggestions from you and the reviewers, we have carried out some additional experiments and revised the manuscript. In this letter, we have listed our responses to the specific comments/questions raised by each reviewer, and incorporated all necessary changes in the revision.

Editor:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Response: We revised our manuscript to meet PLOS ONE's style requirements.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Response: We updated the Data Availability statement in this revision by stating that “All relevant data are within the paper and its Supporting information files”.

Response: Raw images of Western blot from Figs 3 and 4 were provided as S1 Raw images in this revision.

Response: An ORCID iD for the corresponding author was updated in this submission.

Again, we really appreciate the reviewer’s careful reading and suggestions on our manuscript, and thank you very much for your consideration of our paper for publication in Plos one.

Sincerely yours,

Jialiang Guan,

The Department of Emergency Internal Medicine,

The Affiliated Hospital of Qingdao University,

E-mail: gjlqdfy@126.com

Reviewer #1:

Question #1: Although the authors showed that curcumin suppressed viability of breast cancer cells and tumor growth at relatively high concentrations, it is also toxic to non-tumor cells such as mouse hippocampal HT22, PC12 and NIH-3T3 cells (Hirata et al., ACS Chem Neurosci 11, 76-85, 2020; Meng et al., PLOS ONE 9, e85570, 2014; Zhang et al., Mol Immunol 116, 191-198, 2019). Therefore, the cytotoxic effect of curcumin is not specific for breast cancer cells.

Response #1: Thanks for your nice comments on our article. Recently, several studies have demonstrated that curcumin had anticancer and chemoprevention effects on breast cancer through inhibiting cancer proliferation [1] and tumor metastasis [2], as well as enhancing the sensitivity of breast cancer cells to chemotherapeutic drugs [3-5]. Moreover, our data showed that 2-50 μM curcumin significantly reduced the viabilities of breast cancer cell lines (MDA-MB-453 and MCF-7). Therefore, the above results indicated the curcumin serves as an adjunct therapy in breast cancer.

Question #2: Line 61-62. This sentence is inaccurate, or misleading information.

Response #2: We rewrote this sentence as “It was reported that danshen, a traditional Chinese medicine, improved survival of patients with breast cancer and induced ferroptosis and apoptosis of breast cancer cells”. (Line 62-63, page 3 in this revision.)

Question #3: Lin et al. analyzed a cohort of 79,335 patients with breast cancer between 2000 and 2010 and showed that danshen improves survival of breast cancer patients. They also showed that dihydroisotanshinone I (DT), a pure compound present in danshen, inhibits the growth of breast carcinoma cells, including MCF-7 and MDA-MB-231 and induces both apoptosis and ferroptosis in those cultured cells. DT-induced ferroptosis is characterized by reducing GSH content and GPX4 protein, and increasing lipid peroxidation. Therefore, there is no direct evidence that danshen prolonged the survival of patients with breast cancer by inducing ferroptosis.

Response #3: In this study, the results showed that the use of Danshen ≥84 g was highly associated with decreased mortality (the adjusted HR of Danshen ≥84 g users was 0.54 [95% CI, 0.46–0.63] (p <0.001). Moreover, the use of Danshen for >28 days remained highly associated with decreased mortality (the adjusted HR of Danshen users for >28 days was 0.55 [95% CI, 0.49–0.62] (p <0.001). Thus, these data demonstrate the protective effects of a higher dose or longer use of Danshen for patients with breast cancer.

Question #4: Figure 2D, Figure 3H:

(1). According to these results, curcumin increased ferrous ion levels in MCF-7 and MDA-MB-453 cells. However, curcumin has effective ferrous ions chelating capacity (Ak and Gulcin, Chemico-Biol Int 174, 27-37, 2008). In fact, curcumin reduces ferrous ions in cultured cells (Hirata et al., ACS Chem Neurosci 11, 76-85, 2020) and in in vitro assay (Shome et al., Biotechnology and Applied Biochemistry 68, 603-615 Biotechnology and Applied Biochemistry, 2021) at concentrations similar to those reported in this study. The authors need to examine the effect defferoxamine on ferrous ion levels as a positive control.

Response #4(1): As suggested, we examined the effect deferoxamine (DFO) on the curcumin-induced increase in intracellular iron, and the results as showed in supplementary Fig. 2. We added this information in line 210-212, page 9 in this revision.

(2). Please check whether the unit of ferrous ion is correct.

Response #4(2): As suggested, we checked the unit of ferrous and revised this error in Figs 2 and 3 in this revision.

(3). The relative of intracellular Fe2+ expression …: Intracellular Fe2+ level in both …

Response #4(3): We corrected the “The relative of intracellular Fe2+ expression” into “The intracellular Fe2+ level”. (Line 483 and 493, page 17 in this revision)

Minor points:

Question #5: Figure 1: Is the concentration of erastin 10 nM?

Response #5: Thanks for your carefully checking. In this study, the concentration of curcumin in both breast cancer cells was 10 nM.

Question #6: Figure legends: The number of samples used in the experiments and statistics needs to be clarified.

Response #6: We have added the detailed information for the samples used in the figure legend in this revision. (Line 477, 485, 494, 504 and 513, page 17-18 in this revision)

Reviewer #2

In this article, the authors performed a series of in vitro and in vivo experiments to find out novel anti-tumorigenic mechanistic insight of curcumin. And they revealed that curcumin administration induced ferroptosis by enhancing the levels of lipid reactive oxygen species (ROS), malondialdehyde (MDA) of lipid peroxidation, and intracellular Fe2+ level in BC cells. Furthermore, treatment with curcumin significantly suppresses tumorigenesis in BC via upregulating SLC1A5 expression, which is an essential transporter for glutamine uptake.

Question #1: Because there are some other published articles in which Li R, et al, revealed the anti-tumorigenic effect of curcumin by altering other ferroptosis associated gene (HO-1) in breast cancer area (Oxid Med Cell Longev. 2020 Nov 18; 2020:3469840), the authors should add this article as reference and should add some discussion with regard to this article.

Response #1: As suggested, we cited this reference as Ref.39 and added some discussion in line 304-308, page 12 in this revision.

Question #2: As for Figure2A,2B, and 3F, please add the title of X-axis. Lipid ROS level?

Response #2: As suggested, we added the title of X-axis as “Lipid ROS content” in Figure 2A, 2B and 3F.

Question #3: How about changing the position of Figure 5F to 5A in order to explain the less-toxic effect of curcumin to mice?

Response #3: As suggested, we exchanged the position of Figure 5F and 5A.

Reviewer #3

In this article, the authors show that curcumin exhibits anti-tumorigenesis activity in breast cancer cell lines by promoting SLC1A5-mediated ferroptosis (lipid ROS, lipid peroxidation end-product MDA accumulation, and intracellular Fe2+ levels). Objectives are clear, experiments are well designed, and methodology is adequate to obtain the results. The article is well written.

Major issues

Question #1: Which diluent is used to dissolve curcumin? It must be checked that the vehicle does not reduce cells viability.

Response #1: As suggested, we provided the dilution method of curcumin and test the cell viability of DMSO solvent. As shown in supplementary Fig. 1, the corresponding volume of DMSO had no cytotoxicity on MDA-MB-453 and MCF-7 cells (supplementary Fig. 1). (Line 86-88, page 4 and line 189-191, page 8 in this revision.)

Question #2: Authors explain the role of ASCT2, SLC1A5 and SLC38A1 in glutamine uptake, but they only measured SLC1A5. Then they argue about the role of SLC1A5 in ferroptosis induced by curcumin but not about other receptors. Other receptors should be measured to confirm SLC1A5 implication.

Response #2: ASCT2 is encoded by SLC1A5 that was evaluated in this original manuscript. As suggested, we further confirmed the mRNA expression of SLC38A1 in MDA-MB-453 and MCF-7 cells after treated with or without curcumin. As shown in supplementary Fig. 3, curcumin significantly enhanced the expression SLC1A5, but had no obvious effect on the expression of SLC38A1. We added this information in line 221-222, page 9 in this revision.

Question #3: Immunohistochemistry staining should be quantified; a representative image is not enough to conclude Ki-67 and SLC1A5 increase.

Response #3: As suggested, we provided the quantified results of Immunohistochemistry staining as Figure 5F in this revision.

Question #4: To confirm the role of ferroptosis in vivo, authors should perform the same experiment but treating mice with ferrostatin and other cell death inhibitors, such as ZVAD and NS. In addition, authors should measure other ferroptosis markers than just MDA in the tumor to conclude the role of this pathway.

Response #4: As suggested, to confirm that curcumin inhibited tumor growth in vivo by ferroptosis, we treated mice with curcumin, curcumin + DFO and curcumin + NS. As shown in Figure 5B-D in this revision, the administration of curcumin + DFO obviously reversed the inhibitory effect of curcumin on tumor growth, whereas the addition of NS, did not impact the efficiency of curcumin, which was consistent with the results of in vitro experiments that curcumin showed an antitumor effect on BC cells by inducing cell ferroptosis. (Line 248-255, page 10 in this revision.)

In addition, we supplemented the results of other ferroptosis markers such as GSH and iron in tumor tissues of mice and the results were shown in Figure 5G-I. (Line 259-262, page 10 in this revision.)

Question #5: In the discussion sections, authors report that curcumin promotes ferroptosis. However, there are contradictory data in the bibliography about this issue, see reference 38 (Guerrero-Hue et al.). Authors should discuss about the possible dual role of curcumin on ferroptosis.

Response #5: Thanks for your comments, we have supplemented the possible dual role of curcumin on ferroptosis in the Discussion section. (Line 304-308, page 12 in this revision.)

Question #6: Authors should add in the figure legend the time in which each experiment was performed.

Response #6: As suggested, we added the treatment time in which each experiment in the figure legend. (Line 474, 476, 482 and 508, page 17-18 in this revision.)

References in this letter:

1. Mohammed F, Rashid-Doubell F, Taha S, Cassidy S, Fredericks S. Effects of curcumin complexes on MDAMB231 breast cancer cell proliferation. Int J Oncol. 2020;57(2):445-55. Epub 2020/07/07. doi: 10.3892/ijo.2020.5065. PubMed PMID: 32626932; PubMed Central PMCID: PMCPMC7307592.

2. Hu CX, Li MJ, Guo TT, Wang SX, Huang WP, Yang K, et al. Anti-metastasis activity of curcumin against breast cancer via the inhibition of stem cell-like properties and EMT. Phytomedicine. 2019;58. doi: ARTN 152740

10.1016/j.phymed.2018.11.001. PubMed PMID: WOS:000473047100001.

3. Xiong K, Zhang Y, Wen Q, Luo J, Lu Y, Wu Z, et al. Co-delivery of paclitaxel and curcumin by biodegradable polymeric nanoparticles for breast cancer chemotherapy. Int J Pharm. 2020;589:119875. Epub 2020/09/13. doi: 10.1016/j.ijpharm.2020.119875. PubMed PMID: 32919003.

4. Kalluru H, Kondaveeti SS, Telapolu S, Kalachaveedu M. Turmeric supplementation improves the quality of life and hematological parameters in breast cancer patients on paclitaxel chemotherapy: A case series. Complement Ther Clin Pract. 2020;41:101247. Epub 2020/10/26. doi: 10.1016/j.ctcp.2020.101247. PubMed PMID: 33099272.

5. Wen C, Fu L, Huang J, Dai Y, Wang B, Xu G, et al. Curcumin reverses doxorubicin resistance via inhibition the efflux function of ABCB4 in doxorubicinresistant breast cancer cells. Mol Med Rep. 2019;19(6):5162-8. Epub 2019/05/07. doi: 10.3892/mmr.2019.10180. PubMed PMID: 31059026; PubMed Central PMCID: PMCPMC6522915.

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PLoS One. doi: 10.1371/journal.pone.0261370.r003

Decision Letter 1

Irina V Balalaeva

19 Oct 2021

PONE-D-21-20659R1Curcumin suppresses tumorigenesis by ferroptosis in breast cancerPLOS ONE

Dear Dr. Guan,

ACADEMIC EDITOR: Please, check the concentrations of the compounds in the experiments, especially those pointed at by the Reviewers.

Please submit your revised manuscript by Dec 03 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Irina V. Balalaeva, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have not adequately addressed my concerns.

Question #4/Response #4(1)

(1) The authors did not discuss the chelating ability of curcumin and ignored the related references (Ak and Guicin, 2008; Hirata et al., 2020, Shome et al., 2021). The chelating ability of curcumin is opposite action the authors reported in this study and therefore the authors should discuss the discrepancy.

The authors examined the effect of deferoxamine (DFO) on the curcumin-induced increase in intracellular Fe2+ (Figure S2). DFO only affected curcumin-induced increase in intracellular ferrous ions. The results is strange. Why DFO did not decrease the control level of ferrous ions?

Question #4/Response #4(2)

(2) The authors correct the unit of ferrous ion μg/μg protein to nmol/μg protein. According to the abcam’s protocol (ab83366, abcam) the authors used, rat liver lysate contains approximately 0.1 nmoles Fe2+/mg tissue, which is approximately 10,000 times less compared the amount of Fe2+ the authors reported (10 µmoles/µg protein).

Question #5/Response #5

Erastin at 10 nM cannot cause ferroptotic cell death in cultured cells. Most cancer cells are required 5-10 µM erastin to cause ferroptosis.

Reviewer #2: The authors have satisfactorily addressed the issues raised by reviewers and have improved the manuscript. Therefore the revised article has reached to sufficient quality.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

PLoS One. 2022 Jan 18;17(1):e0261370. doi: 10.1371/journal.pone.0261370.r004

Author response to Decision Letter 1

29 Oct 2021

Response to reviewer

Reviewer #1:

Question #4/Response #4(1): The authors did not discuss the chelating ability of curcumin and ignored the related references (Ak and Guicin, 2008; Hirata et al., 2020, Shome et al., 2021). The chelating ability of curcumin is opposite action the authors reported in this study and therefore the authors should discuss the discrepancy.

The authors examined the effect of deferoxamine (DFO) on the curcumin-induced increase in intracellular Fe2+ (Figure S2). DFO only affected curcumin-induced increase in intracellular ferrous ions. The results are strange. Why DFO did not decrease the control level of ferrous ions?

Response: We felt great thanks for your professional review work on our article. As suggested, we added the related references (Ak and Guicin, 2008; Hirata et al., 2020, Shome et al., 2021) as Ref. 41-43 and discussed the discrepancy between these studies and our work (line 308-318, page 12 in this revision).

In addition, as shown in supplementary Fig. 2, our data showed that DFO did not only affect curcumin-induced increase in intracellular ferrous ions, but also decrease the control level of ferrous ions (the second column).

Question #4/Response #4(2): The authors correct the unit of ferrous ion μg/μg protein to nmol/μg protein. According to the abcam’s protocol (ab83366, abcam) the authors used, rat liver lysate contains approximately 0.1 nmoles Fe2+/mg tissue, which is approximately 10,000 times less compared the amount of Fe2+ the authors reported (10 µmoles/µg protein).

Response: In this study, the unit of ferrous was (nmol/μg protein) × 103 in Fig. 2 and 3, which is equivalent to nmol/mg protein.

Question #5/Response #5: Erastin at 10 nM cannot cause ferroptotic cell death in cultured cells. Most cancer cells are required 5-10 µM erastin to cause ferroptosis.

Response: We are very sorry for our carelessness in writing. After careful checking, we verified that the dose of erastin used in this experiment was 10 µM. Special thank you very much for your careful review and we express our sincere apologies for this error. We corrected this mistake in this revision (line 90, page 4 and line 501, page 18 in this revision).

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PLoS One. doi: 10.1371/journal.pone.0261370.r005

Decision Letter 2

Irina V Balalaeva

12 Nov 2021

PONE-D-21-20659R2Curcumin suppresses tumorigenesis by ferroptosis in breast cancerPLOS ONE

Dear Dr. Guan,

Academic editor: Please, check the results mentioned by the Reviewer.

Please submit your revised manuscript by Dec 27 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Irina V. Balalaeva, PhD

Academic Editor

PLOS ONE

Journal Requirements:

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

**********

6. Review Comments to the Author

Reviewer #1: The author’s response: In addition, as shown in supplementary Fig. 2, our data showed that DFO did not only affect curcumin-induced increase in intracellular ferrous ions, but also decrease the control level of ferrous ions (the second column).

The authors did not respond my concern as follows:

DFO only affected curcumin-induced increase in intracellular ferrous ions (Figure S2A, the 3rd column vs 4th column) but did not affect the control level of ferrous ions (Figure S2A, the first column vs second column). The results is strange.

The English writing standard needs to be improved further.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

PLoS One. 2022 Jan 18;17(1):e0261370. doi: 10.1371/journal.pone.0261370.r006

Author response to Decision Letter 2

24 Nov 2021

Reviewer #1:

Question #1: The authors did not respond my concern as follows:

Response #1: We retest the intracellular ferrous ions in MDA-MB-453 and MCF-7 cells with or without the presence of DFO. As shown in Figure 1 in this response, no obvious difference was observed between the intracellular ion level in of MDA-MB-453 and MCF-7 cells with or without the presence of 50 μM deferoxamine, which was also found in recent published researches [1-3]. Although deferoxamine mesylate was an iron chelator that bound free iron in a stable complex, we speculated that it might not affect the relatively low normal ferrous ions level in some cells but effectively reverse the iron accumulation induced by ferroptosis inducer.

Question #2: The English writing standard needs to be improved further.

Response #2: As suggested, we improved the quality of our language by an English language editing service of Spandidos Publications. Based on the editing report, we revised our manuscript and also uploaded the certificate with this revision.

References in this response:

1. Tu H, Zhou YJ, Tang LJ, Xiong XM, Zhang XJ, Ali Sheikh MS, et al. Combination of ponatinib with deferoxamine synergistically mitigates ischemic heart injury via simultaneous prevention of necroptosis and ferroptosis. Eur J Pharmacol. 2021;898:173999. Epub 2021/03/07. doi: 10.1016/j.ejphar.2021.173999. PubMed PMID: 33675785.

2. Yang J, Zhou Y, Xie S, Wang J, Li Z, Chen L, et al. Metformin induces Ferroptosis by inhibiting UFMylation of SLC7A11 in breast cancer. J Exp Clin Cancer Res. 2021;40(1):206. Epub 2021/06/25. doi: 10.1186/s13046-021-02012-7. PubMed PMID: 34162423; PubMed Central PMCID: PMCPMC8223374.

3. Song Z, Xiang X, Li J, Deng J, Fang Z, Zhang L, et al. Ruscogenin induces ferroptosis in pancreatic cancer cells. Oncol Rep. 2020;43(2):516-24. Epub 2020/01/03. doi: 10.3892/or.2019.7425. PubMed PMID: 31894321; PubMed Central PMCID: PMCPMC6967081.

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PLoS One. doi: 10.1371/journal.pone.0261370.r007

Decision Letter 3

Irina V Balalaeva

1 Dec 2021

Curcumin suppresses tumorigenesis by ferroptosis in breast cancer

PONE-D-21-20659R3

Dear Dr. Guan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Irina V. Balalaeva, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0261370.r008

Acceptance letter

Irina V Balalaeva

7 Jan 2022

PONE-D-21-20659R3

Curcumin suppresses tumorigenesis by ferroptosis in breast cancer

Dear Dr. Guan:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Irina V. Balalaeva

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Effects of CUN on cell viability.

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S2 Fig. Effects of CUN on the intracellular Fe²⁺ expression.

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S3 Fig. Effects of curcumin on gene expression.

A and B: qRT-PCR was used to examine the mRNA expression levels of SLCA5 and SLC38A1. ^###P<0.001. compared with the control group.

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S1 Raw images. Raw data images of western blot analysis corresponding to Figs 3 and 4.

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0261370.ref001] 1.Spronk I, Schellevis FG, Burgers JS, de Bock GH, Korevaar JC. Incidence of isolated local breast cancer recurrence and contralateral breast cancer: A systematic review. Breast. 2018;39:70–9. Epub 2018/04/06. doi: 10.1016/j.breast.2018.03.011 . [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref002] 2.Hamood R, Hamood H, Merhasin I, Keinan-Boker L. Hormone therapy and osteoporosis in breast cancer survivors: assessment of risk and adherence to screening recommendations. Osteoporos Int. 2019;30(1):187–200. Epub 2018/11/11. doi: 10.1007/s00198-018-4758-4 . [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref003] 3.Ahern TP, Sprague BL, Bissell MCS, Miglioretti DL, Buist DSM, Braithwaite D, et al. Family History of Breast Cancer, Breast Density, and Breast Cancer Risk in a U.S. Breast Cancer Screening Population. Cancer Epidemiol Biomarkers Prev. 2017;26(6):938–44. Epub 2017/01/18. doi: 10.1158/1055-9965.EPI-16-0801 ; PubMed Central PMCID: PMC5457358. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref004] 4.Nawa-Nishigaki M, Kobayashi R, Suzuki A, Hirose C, Matsuoka R, Mori R, et al. Control of Nausea and Vomiting in Patients Receiving Anthracycline/Cyclophosphamide Chemotherapy for Breast Cancer. Anticancer Res. 2018;38(2):877–84. Epub 2018/01/29. doi: 10.21873/anticanres.12297 . [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref005] 5.Kawazoe H, Murakami A, Yamashita M, Nishiyama K, Kobayashi-Taguchi K, Komatsu S, et al. Patient-related Risk Factors for Nausea and Vomiting with Standard Antiemetics in Patients with Breast Cancer Receiving Anthracycline-based Chemotherapy: A Retrospective Observational Study. Clin Ther. 2018;40(12):2170–9. Epub 2018/11/06. doi: 10.1016/j.clinthera.2018.10.004 . [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref006] 6.Luo H, Vong CT, Chen H, Gao Y, Lyu P, Qiu L, et al. Naturally occurring anti-cancer compounds: shining from Chinese herbal medicine. Chin Med. 2019;14:48. Epub 2019/11/14. doi: 10.1186/s13020-019-0270-9 ; PubMed Central PMCID: PMC6836491. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref007] 7.Bandyopadhyay D. Farmer to pharmacist: curcumin as an anti-invasive and antimetastatic agent for the treatment of cancer. Front Chem. 2014;2:113. Epub 2015/01/08. doi: 10.3389/fchem.2014.00113 ; PubMed Central PMCID: PMC4275038. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref008] 8.Giordano A, Tommonaro G. Curcumin and Cancer. Nutrients. 2019;11(10). Epub 2019/10/09. doi: 10.3390/nu11102376 ; PubMed Central PMCID: PMC6835707. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref009] 9.Kunnumakkara AB, Bordoloi D, Harsha C, Banik K, Gupta SC, Aggarwal BB. Curcumin mediates anticancer effects by modulating multiple cell signaling pathways. Clin Sci (Lond). 2017;131(15):1781–99. Epub 2017/07/07. doi: 10.1042/CS20160935 . [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref010] 10.Mbese Z, Khwaza V, Aderibigbe BA. Curcumin and Its Derivatives as Potential Therapeutic Agents in Prostate, Colon and Breast Cancers. Molecules. 2019;24(23). ARTN 4386 doi: 10.3390/molecules24234386 WOS:000507294400185. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref011] 11.Mou Y, Wang J, Wu J, He D, Zhang C, Duan C, et al. Ferroptosis, a new form of cell death: opportunities and challenges in cancer. J Hematol Oncol. 2019;12(1):34. Epub 2019/03/31. doi: 10.1186/s13045-019-0720-y ; PubMed Central PMCID: PMC6441206. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref012] 12.Imai H, Matsuoka M, Kumagai T, Sakamoto T, Koumura T. Lipid Peroxidation-Dependent Cell Death Regulated by GPx4 and Ferroptosis. Curr Top Microbiol Immunol. 2017;403:143–70. Epub 2017/02/17. doi: 10.1007/82_2016_508 . [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref013] 13.Shen Z, Song J, Yung BC, Zhou Z, Wu A, Chen X. Emerging Strategies of Cancer Therapy Based on Ferroptosis. Adv Mater. 2018;30(12):e1704007. Epub 2018/01/23. doi: 10.1002/adma.201704007 ; PubMed Central PMCID: PMC6377162. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref014] 14.Lu B, Chen XB, Ying MD, He QJ, Cao J, Yang B. The Role of Ferroptosis in Cancer Development and Treatment Response. Front Pharmacol. 2017;8:992. Epub 2018/01/30. doi: 10.3389/fphar.2017.00992 ; PubMed Central PMCID: PMC5770584. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref015] 15.Xu T, Ding W, Ji X, Ao X, Liu Y, Yu W, et al. Molecular mechanisms of ferroptosis and its role in cancer therapy. J Cell Mol Med. 2019;23(8):4900–12. Epub 2019/06/25. doi: 10.1111/jcmm.14511 ; PubMed Central PMCID: PMC6653007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref016] 16.Lin YS, Shen YC, Wu CY, Tsai YY, Yang YH, Lin YY, et al. Danshen Improves Survival of Patients With Breast Cancer and Dihydroisotanshinone I Induces Ferroptosis and Apoptosis of Breast Cancer Cells. Front Pharmacol. 2019;10:1226. Epub 2019/11/19. doi: 10.3389/fphar.2019.01226 ; PubMed Central PMCID: PMC6836808. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref017] 17.Ye J, Zhang R, Wu F, Zhai L, Wang K, Xiao M, et al. Non-apoptotic cell death in malignant tumor cells and natural compounds. Cancer Lett. 2018;420:210–27. Epub 2018/02/08. doi: 10.1016/j.canlet.2018.01.061 . [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref018] 18.Zan L, Chen Q, Zhang L, Li X. Epigallocatechin gallate (EGCG) suppresses growth and tumorigenicity in breast cancer cells by downregulation of miR-25. Bioengineered. 2019;10(1):374–82. Epub 2019/08/23. doi: 10.1080/21655979.2019.1657327 ; PubMed Central PMCID: PMC6738446. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref019] 19.Niu Y, Zhang J, Tong Y, Li J, Liu B. Physcion 8-O-beta-glucopyranoside induced ferroptosis via regulating miR-103a-3p/GLS2 axis in gastric cancer. Life Sci. 2019;237:116893. Epub 2019/10/14. doi: 10.1016/j.lfs.2019.116893 . [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref020] 20.Luo M, Wu L, Zhang K, Wang H, Zhang T, Gutierrez L, et al. miR-137 regulates ferroptosis by targeting glutamine transporter SLC1A5 in melanoma. Cell Death Differ. 2018;25(8):1457–72. Epub 2018/01/20. doi: 10.1038/s41418-017-0053-8 ; PubMed Central PMCID: PMC6113319. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref021] 21.DeBerardinis RJ, Cheng T. Q’s next: the diverse functions of glutamine in metabolism, cell biology and cancer. Oncogene. 2010;29(3):313–24. Epub 2009/11/03. doi: 10.1038/onc.2009.358 ; PubMed Central PMCID: PMC2809806. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref022] 22.Scalise M, Pochini L, Console L, Losso MA, Indiveri C. The Human SLC1A5 (ASCT2) Amino Acid Transporter: From Function to Structure and Role in Cell Biology. Front Cell Dev Biol. 2018;6:96. Epub 2018/09/21. doi: 10.3389/fcell.2018.00096 ; PubMed Central PMCID: PMC6131531. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref023] 23.Hassannia B, Vandenabeele P, Vanden Berghe T. Targeting Ferroptosis to Iron Out Cancer. Cancer Cell. 2019;35(6):830–49. Epub 2019/05/21. doi: 10.1016/j.ccell.2019.04.002 . [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref024] 24.Angeli JPF, Krysko DV, Conrad M. Ferroptosis at the crossroads of cancer-acquired drug resistance and immune evasion. Nat Rev Cancer. 2019;19(7):405–14. doi: 10.1038/s41568-019-0149-1 WOS:000472883000014. [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref025] 25.Shin D, Kim EH, Lee J, Roh JL. Nrf2 inhibition reverses resistance to GPX4 inhibitor-induced ferroptosis in head and neck cancer. Free Radical Bio Med. 2018;129:454–62. doi: 10.1016/j.freeradbiomed.2018.10.426 WOS:000450298400042. [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref026] 26.Sun XF, Niu XH, Chen RC, He WY, Chen D, Kang R, et al. Metallothionein-1G Facilitates Sorafenib Resistance Through Inhibition of Ferroptosis. Hepatology. 2016;64(2):488–500. doi: 10.1002/hep.28574 WOS:000380034500019. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref027] 27.Gao MH, Monian P, Quadri N, Ramasamy R, Jiang XJ. Glutaminolysis and Transferrin Regulate Ferroptosis. Mol Cell. 2015;59(2):298–308. doi: 10.1016/j.molcel.2015.06.011 WOS:000362457000016. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref028] 28.Jiang HL, Zhang N, Tang TZ, Feng F, Sun HP, Qu W. Target the human Alanine/Serine/Cysteine Transporter 2(ASCT2): Achievement and Future for Novel Cancer Therapy. Pharmacol Res. 2020;158. ARTN 104844 doi: 10.1016/j.phrs.2020.104844 WOS:000542124500056. [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref029] 29.Hassanein M, Qian J, Hoeksema MD, Wang J, Jacobovitz M, Ji XM, et al. Targeting SLC1a5-mediated glutamine dependence in non-small cell lung cancer. Int J Cancer. 2015;137(7):1587–97. doi: 10.1002/ijc.29535 WOS:000358012600007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref030] 30.Zhuang X, Tong HC, Ding Y, Wu LY, Cai JS, Si Y, et al. Long noncoding RNA ABHD11-AS1 functions as a competing endogenous RNA to regulate papillary thyroid cancer progression by miR-199a-5p/SLC1A5 axis. Cell Death Dis. 2019;10. ARTN 620 doi: 10.1038/s41419-019-1850-4 WOS:000481964400002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref031] 31.Wang L, Liu Y, Zhao TL, Li ZZ, He JY, Zhang BJ, et al. Topotecan induces apoptosis via ASCT2 mediated oxidative stress in gastric cancer. Phytomedicine. 2019;57:117–28. doi: 10.1016/j.phymed.2018.12.011 WOS:000465081700013. [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref032] 32.Calibasi-Kocal G, Pakdemirli A, Bayrak S, Ozupek NM, Sever T, Basbinar Y, et al. Curcumin effects on cell proliferation, angiogenesis and metastasis in colorectal cancer. J Buon. 2019;24(4):1482–7. WOS:000481595900022. [PubMed] [Google Scholar]

[pone.0261370.ref033] 33.Hu CX, Li MJ, Guo TT, Wang SX, Huang WP, Yang K, et al. Anti-metastasis activity of curcumin against breast cancer via the inhibition of stem cell-like properties and EMT. Phytomedicine. 2019;58. ARTN 152740 doi: 10.1016/j.phymed.2018.11.001 WOS:000473047100001. [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref034] 34.Mortezaee K, Salehi E, Mirtavoos-mahyari H, Motevaseli E, Najafi M, Farhood B, et al. Mechanisms of apoptosis modulation by curcumin: Implications for cancer therapy. Journal of Cellular Physiology. 2019;234(8):12537–50. doi: 10.1002/jcp.28122 WOS:000467240800036. [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref035] 35.Kumar P, Kadakol A, Shasthrula PK, Mundhe NA, Jamdade VS, Barua CC, et al. Curcumin as an Adjuvant to Breast Cancer Treatment. Anti-Cancer Agent Me. 2015;15(5):647–56. doi: 10.2174/1871520615666150101125918 WOS:000354610900011. [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref036] 36.Tang X, Ding H, Liang ML, Chen X, Yan YX, Wan NS, et al. Curcumin induces ferroptosis in non-small-cell lung cancer via activating autophagy. Thoracic Cancer. 2021;12(8):1219–30. doi: 10.1111/1759-7714.13904 WOS:000624623600001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref037] 37.Chen TC, Chuang JY, Ko CY, Kao TJ, Yang PY, Yu CH, et al. AR ubiquitination induced by the curcumin analog suppresses growth of temozolomide-resistant glioblastoma through disrupting GPX4-Mediated redox homeostasis. Redox Biol. 2020;30. ARTN 101413 doi: 10.1016/j.redox.2019.101413 WOS:000513891900003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref038] 38.Guerrero-Hue M, Garcia-Caballero C, Palomino-Antolin A, Rubio-Navarro A, Vazquez-Carballo C, Herencia C, et al. Curcumin reduces renal damage associated with rhabdomyolysis by decreasing ferroptosis-mediated cell death. Faseb Journal. 2019;33(8):8961–75. doi: 10.1096/fj.201900077R WOS:000478832900022. [DOI] [PubMed] [Google Scholar]

[pone.0261370.ref039] 39.Li RH, Zhang J, Zhou YF, Gao Q, Wang R, Fu YR, et al. Transcriptome Investigation and In Vitro Verification of Curcumin-Induced HO-1 as a Feature of Ferroptosis in Breast Cancer Cells. Oxid Med Cell Longev. 2020;2020. Artn 3469840 doi: 10.1155/2020/3469840 WOS:000596448900002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261370.ref040] 40.Stocker R. Induction of haem oxygenase as a defence against oxidative stress. Free Radic Res Commun. 1990;9(2):101–12. Epub 1990/01/01. doi: 10.3109/10715769009148577 . [DOI] [PubMed] [Google Scholar]

PERMALINK

Curcumin suppresses tumorigenesis by ferroptosis in breast cancer

Xuelei Cao

Yao Li

Yongbin Wang

Tao Yu

Chao Zhu

Xuezhi Zhang

Jialiang Guan

Roles

Abstract

Introduction

Materials and methods

Cell culture

Cell treatment

Cell transfection

Cell viability analysis

Quantitative real-time polymerase chain reaction (qRT-PCR)

Western blot analysis

Nude mice model

Iron determination assay

MDA assay

Glutamine uptake assay

Determination of lipid ROS levels

Statistical analysis

Results

Curcumin contributes to erastin-induced ferroptosis in BC cells

Fig 1. Treatment with CUN upregulates erastin-induced ferroptosis in BC cells.

Curcumin promotes lipid peroxidation and iron accumulation during activation of ferroptosis

Fig 2. CUN induces cell ferroptosis by upregulation of lipid peroxidation and iron accumulation.

Increased glutamine uptake is essential for curcumin-induced ferroptosis in BC cells

Fig 3. Suppression of glutamine uptake is essential for CUN-induced ferroptosis in BC.

Curcumin promotes ferroptosis by upregulating SLC1A5 expression in BC cells

Fig 4. Treatment with CUN facilitates ferroptosis via upregulation of SLC1A5 expression in BC.

Curcumin inhibits tumor growth of BC in vivo

Fig 5. CUN suppresses tumor growth in vivo.

Discussion

Supporting information

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Irina V Balalaeva

Roles

Author response to Decision Letter 0

Decision Letter 1

Irina V Balalaeva

Roles

Author response to Decision Letter 1

Decision Letter 2

Irina V Balalaeva

Roles

Author response to Decision Letter 2

Decision Letter 3

Irina V Balalaeva

Roles

Acceptance letter

Irina V Balalaeva

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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