Nrf2 depletion enhanced curcumin therapy effect in gastric cancer by inducing the excessive accumulation of ROS

Abstract

Gastric cancer (GC) is the most common malignant tumor of the gastrointestinal tract and currently has a poor clinical outcome. Turmeric’s rhizome contains a polyphenolic component called curcumin (Cur), which has been demonstrated to inhibit a variety of tumor cells, such as pancreatic, colon, lung and gastric cancers. However, it remains to be elucidated how Cur functions in GC and what molecular processes underlie it. Here, Cur showed a stronger inhibitory effect on GC cells AGS and HGC27. In addition, Cur’s inhibition of GC cells growth was accompanied by increased ROS production, triggering of the Keap1-Nrf2 signaling pathway, and increased transcription of its downstream antioxidant genes HO-1, GCLM, and NQO1. However, when a ROS scavenger NAC was used, the inhibitory effect of Cur on GC cells was reversed. Nuclear factor erythroid 2-related factor 2 (Nrf2) is overexpressed or activated in cancers to shield cancer cells from oxidative damage by responding to oxidative stress (OS). Cur has been found to act as an activator of Nrf2. Notably, compared with Nrf2 knockdown and Cur alone, the combination of the two dramatically increased Cur-induced ROS overaccumulation and inhibition of GC cells proliferation, migration, and invasive abilities. Consistent with in vitro experiments, Cur combined with Nrf2 knockdown significantly inhibited tumor growth in nude mice transplanted with AGS cells. Therefore, we concluded that Nrf2 depletion enhanced Cur therapy effect in GC by inducing the excessive accumulation of ROS, indicating that this is a promising treatment strategy.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-81375-1.

Introduction

Gastric cancer (GC) is a malignant tumor that originates from the gastric mucosal epithelium. Among cancers, GC ranks fifth in incidence and fourth in mortality¹. Early GC typically presents asymptomatic and is therefore diagnosed at an advanced stage². Patients with advanced GC have a very bad prognosis and less than 10% of them survive for five years³. For advanced gastric cancer, the traditional palliative treatment is multidrug cytotoxic chemotherapy. Although it has been proven that the clinical efficacy of the combination of two or even three drugs is increased, there is a corresponding increase in the side effect profile and treatment-limiting toxicity^4,5. To improve the survival rate and overall quality of life for GC patients, further insights into GC and the development of efficient chemotherapeutic drugs are needed.

A naturally occurring polyphenolic substance called curcumin (Cur) is extracted from the dried rhizome of Curcuma longa L⁶. Its pharmacological properties span a wide spectrum, such as anti-inflammatory⁷, antibacterial⁸, antiviral⁹, antioxidant¹⁰ and antitumor¹¹ activities. In recent years, several studies have demonstrated that Cur has toxic effects on several malignant tumors. For example, Cur regulated p38/MAPK phosphorylation by inducing ROS production, which triggered apoptosis in chemotherapy-resistant lung cancer cells¹². In bladder cancer, Cur inhibited microRNA-7641 to mediate p16 upregulation to promote apoptosis¹³. Cur has been shown to have anticancer effects on GC^14,15. In addition, Cur can be used in the prevention and treatment of colorectal cancer and has shown good efficacy and oral safety in clinical studies¹⁶.

Despite its efficacy in cancer, Cur has some drawbacks that should not be overlooked. Some studies have found that Cur may affect iron metabolism, and therefore should be taken with caution in patients with borderline or depleted iron stores who have anemia from cancer and chronic disease¹⁷. Low bioavailability of Cur is another indisputable fact. After oral administration, Cur is mainly metabolized by the liver, kidneys and intestines, and undergoes phase I and II biotransformation, most of it is excreted through the feces, and only a small amount of the drug remaining in the body is difficult to exert its pharmacological activity¹⁸. Increasing the concentration of Cur may be a good strategy, however, one study showed that high concentrations of Cur increased hepatotoxicity¹⁹. Current research focuses on the development of Cur derivatives and innovation of nanodelivery systems to overcome low bioavailability of Cur²⁰. This indicates Cur is worthy of further development and utilization.

Oxidative stress (OS) is the result of an imbalance between the production of ROS in the body and the inhibitory effect of free radicals by the antioxidant system²¹. ROS and cancer have been linked in numerous research. Cancer cells usually have higher levels of ROS than healthy cells, but ROS have a double-edged role in tumor development²². Increased ROS levels are considered to promote the occurrence, development and metastasis of tumor by inducing DNA damage and chromosomal abnormalities to promote activation of proto-oncogenes and inactivation of oncogenes^23,24. However, by causing chromosomal and protein disruption, high intracellular ROS generation might result in cell death. To eliminate excessive intracellular ROS, cancer cells must activate antioxidant defense system in response to OS²⁵. Cur was found to induce OS in cancer cells, promote ROS production and trigger apoptosis²⁶. Therefore, we recognize that ROS are involved in antitumor function.

In order to shield cells from oxidative damage, nuclear factor E2-related factor 2 (Nrf2) regulates the expression of antioxidant genes, which functions as a fundamental regulator of the cellular antioxidant stress response²⁷. Under physiological conditions, Nrf2 undergoes ubiquitination degradation in the cytoplasm in conjunction with Kelch-like ECH-associated protein 1 (Keap1) and is unable to enter the cell and exert transcriptional activity. After oxidizing Keap1’s cysteine residues, Nrf2 separates from Keap1 and moves into the nucleus, where it attaches to the antioxidant response elements (AREs) to trigger the transcription of antioxidant genes further down the line. This process occurs when the cells experience OS²⁸. It was found that Nrf2 was upregulated in GC tissues and was closely associated with poor patient prognosis²⁹. Therefore, inhibition of Nrf2 can be used to treat GC. In hepatocellular carcinoma, disulfiram/copper increased the expression level of Nrf2 and induced ferroptosis, whereas Nrf2 silencing increased the susceptibility to disulfiram/copper-induced ferroptosis³⁰. Additionally, through stimulation of the Keap1-Nrf2 signaling pathway, Cur has been shown to promote the production of antioxidant enzymes and have preventive effects on a variety of spontaneous tumors in animal models^31,32. However, it remains unknown whether Cur activates the Keap1-Nrf2 signaling pathway and whether Nrf2 depletion increases Cur therapy effect in GC.

In the current investigation, we have verified that Cur may cause ROS buildup to impede GC cells growth while triggering the Keap1-Nrf2 signaling pathway to provide cytoprotective benefits. It’s interesting to note that Nrf2 knockdown dramatically enhanced Cur-induced inhibition and ROS overaccumulation in GC. The findings of this investigation could broaden Cur’s therapeutic use.

Results

Cur inhibited the growth and induced the death of GC cells

Figure 1A shows the chemical structure of curcumin (Cur). Using the CCK-8 asay, we assessed the viability of human gastric mucosal epithelial cell line GES-1 (Fig. 1B), GC cell lines AGS (Fig. 1C), and HGC27 (Fig. 1D) after treating them with varying concentrations of Cur for 24 h. This allowed us to ascertain the growth inhibitory effect of Cur on GC cells. The outcomes shown that Cur may, in a concentration-dependent manner, dramatically reduce the viability of GES-1, AGS, and HGC27. The three types of cells had different sensitivities to Cur. The growth inhibitory impact of Cur on AGS and HGC27 cells was stronger than that on GES-1 cells. The IC50 values of GES-1, AGS and HGC27 cells were 57.59 µM, 22.21 µM and 17.87 µM, respectively (Fig. 1E). Based on these preliminary results, we selected 0, 10, 20 and 30 µM as the experimental concentration ranges for AGS and HGC27 cells in subsequent experiments. Additionally, Cur’s cytotoxic effects on AGS and HGC27 cells were seen using light microscopy. The results (Fig. 1F) showed that compared with untreated cells, GC cells were considerably fewer in number, and the cells became rounded or even disintegrated after Cur treatment for 24 h.

Fig. 1.

Cur inhibited the growth and induced the death of GC cells. (A) The chemical structure of curcumin (Cur). (B–E) GES-1, AGS and HGC27 cells were treated with different concentrations of Cur for 24 h. Cytotoxicity was detected by the CCK8 assay. IC50 was obtained by GraphPad Prism software. (F) Cell morphology of AGS and HGC27 after treatment with concentration of Cur (0, 10, 20, 30 µM) at 24 h (Scale: 500μm). n = 3, **p < 0.01, ****p < 0.0001 and ns, no statistical significance, compared with 0 µM Cur.

Cur inhibited the colony formation, migration and invasion abilities of GC cells

Subsequently, we used a colony formation experiment to examine Cur’s impact on GC cells colony formation. As illustrated in Fig. 2A,B, following a 10-day Cur (0, 2.5, 5 and 10 µM) treatment, a notable concentration-dependent reduction in the quantity of GC cell colonies was noted. Furthermore, the impact of Cur on GC cells migratory ability was examined using the wound-healing (Fig. 2C–F) and Transwell assays (Fig. 2G,H). Following treatment with Cur (0, 10, 20, and 30 µM), GC cells’ capacity to migrate was greatly inhibited. Similarly, Cur decreased the invasiveness of GC cells, as shown by a Matrigel-based Transwell assay (Fig. 2I,J).

Fig. 2.

Cur inhibited the colony formation, migration and invasion abilities of GC cells. (A,B) AGS and HGC27 cells were treated with Cur for 10 days, and colony formation was observed and quantified. (C–F) AGS and HGC27 cells were treated with the indicated concentrations of Cur for 48 h to compare the wound healing area and quantitative analysis was done. (G–J) AGS and HGC27 cells were treated with Cur for 24 h. Transwell assay was used to compare the migratory and invasive capacities of GC cells. Scale: 500 μm. Data were presented as Mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ns, no statistical significance, compared with the 0 µM Cur.

Analysis of differentially expressed proteins in Cur-induced GC cells

Label-free quantitative proteomics was utilized to screen the differentially expressed proteins in order to comprehend Cur’s function in GC. Quantitative proteomics discovered 4183 proteins in total. FC > 1.5 and p < 0.05 were used to screen 154 differentially expressed proteins, comprising 86 up-regulated and 68 down-regulated proteins. In addition, cell cycle-related proteins CDCA2, CDCA5, CDCA8 and CDC123 were significantly up-regulated, suggesting that Cur may regulate the cell cycle (Fig. 3A). GO enrichment analysis of all differentially expressed proteins was performed using Blast2Go software³³. According to the findings (Fig. 3B), proteins that were differentially expressed were primarily enriched in biological regulation and cellular processes under biological process (BP); under conditions related to cellular components (CC), proteins were primarily enriched in cells and cell parts; and molecular function (MF) was primarily binding and catalytic activity. All differentially expressed proteins were subjected to pathway enrichment analysis using the KEGG pathway database³⁴. These proteins were most prevalent in ferroptosis, followed by the cell cycle, as seen in Fig. 3C. Based on the protein interactions in the STRING database, protein interaction network maps were constructed for the differentially expressed proteins using CytoScape software. We found that proteins closely associated with ferroptosis included GCLM, GCLC, HO-1, NQO1, FTL and FTH1. In addition, there were proteins closely associated with the cell cycle, including the cell division cycle proteins CDC45, CDCA2, CDCA5, and CDCA8 (Fig. 3D). This aligned with the findings of the KEGG analysis. We detected apoptosis of GC cells by flow cytometry after 24 h of Cur treatment (Fig. 3E). The outcomes demonstrated that Cur caused GC cells to undergo apoptosis. Interestingly, a portion of GC cells were concentrated within the FITC-/PI-region, suggesting that Cur may induce other types of cell death besides apoptosis, such as ferroptosis analyzed by proteomics sequencing. However, it remains to be demonstrated whether Cur induces ferroptosis in GC cells or not, which needs to be demonstrated by further studies. We also examined the temporal distribution of the cell cycle (Fig. 3F) and found that Cur treatment shortened the G1 and S phases and arrested the G2 phase in AGS cells, whereas it caused cellular arrest in the G1 and G2 phases and shortened the S phase in HGC27 cells.

Fig. 3.

Analysis of differentially expressed proteins in Cur-induced GC cells. AGS cells were treated with 20 µM Cur or an equivalent amount of solvent as a comparison. Label-free quantitative proteomics technology was used to screen for differentially expressed proteins. n = 3. (A) Volcano plot of differentially expressed proteins. Blue markers are significantly down-regulated proteins (FC < 0.667 and p < 0.5), red markers are significantly up-regulated proteins (FC > 1.5 and p < 0.05), and gray markers are proteins not differentially expressed. (B) GO enrichment analysis of all differentially expressed proteins. (C) KEGG pathway enrichment analysis of all differentially expressed proteins. The top 20 pathways with the highest number of enriched genes were selected. (D) All differentially expressed proteins construct a protein interaction network diagram. (E,F) After treatment of GC cells with 0 or 20 µM Cur for 24 h, apoptosis and cell cycle were detected by flow cytometry.

Cur induced ROS production and activation of the Keap1-Nrf2 signaling pathway

As important signaling molecules, ROS are involved in cellular signal transduction and maintenance of redox homeostasis in aerobic organisms³⁵. ROS is not only a protector of tumor cells but also sharp sword to kill them³⁶. In this study, The DCFH-DA probe was utilized to identify ROS generation in GC cells. After 24 h of Cur treatment, intracellular DCF fluorescence values were detected using a fluorescent enzyme marker. The findings demonstrated that Cur induced concentration-dependent production of ROS in GC cells (Fig. 4A,B). To clarify the functional significance of ROS in more detail, intracellular ROS accumulation was effectively inhibited by employing N-acetylcysteine (NAC), a potent scavenger of ROS (Fig. 4C,D). Moreover, the CCK8 assay was used to assess cell viability. The growth inhibiting impact of Cur on GC cells was greatly decreased by NAC pretreatment, as demonstrated in Fig. 4E,F. These data indicated that Cur suppressed cell viability in GC cells by triggering ROS production. As an antioxidant, GSH is essential for preserving cellular redox homeostasis³⁷. Thus, 24 h following Cur administration, the intracellular GSH level in GC cells were assessed. The findings demonstrated that within 24 h, GSH level dropped in a concentration-dependent manner (Fig. 4G,H).

Fig. 4.

Cur induced ROS production and activation of the Keap1-Nrf2 signaling pathway. (A,B) The fluorescence values of DCF in Cur-treated AGS and HGC27 cells were examined using a fluorescent enzyme labeler to detect ROS accumulation. (C,D) The effect of NAC on regulating Cur-mediated accumulation of ROS in AGS and HGC27 cells. (E,F) The effect of NAC on regulating Cur-mediated growth inhibition in AGS and HGC27 cells. (G,H) The GSH level was measured in Cur-treated AGS and HGC27 cells. (I,J) Keap1, Nrf2, HO-1, GCLM and NQO1 mRNA levels were measured after Cur treatment. (K,L) Western blotting analysis of Keap1, Nrf2, HO-1, GCLM and NQO1 protein expression. GAPDH was used as an internal control. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ns, no statistical significance.

It is well known that in reaction to ROS, the cellular transcription factor Nrf2 is increased. When OS occurs, Nrf2 was translocated to the nucleus to activate its downstream antioxidant genes, such as GCLM, GCLC, HO-1, NQO1, etc., to protect cells from oxidative damage³⁸. Our results showed that 12 h after Cur addition (Fig. 4I,J), Cur decreased the mRNA level of Keap1 and increased the mRNA levels of Nrf2, HO-1, GCLM and NQO1 in GC cells. Cur considerably raised the expression of Nrf2, HO-1, GCLM and NQO1 in GC cells at the protein level and dramatically decreased the expression of Keap1 at 24 h after Cur addition (Fig. 4K,L). Proteomic sequencing analysis showed increased expression of Nrf2 target genes HO-1, GCLM, and NQO1 (Supplement Fig. 1). These results suggested that Cur triggered ROS production in GC cells causing OS and activated the Keap1-Nrf2 signaling pathway to counteract OS.

Cur combined with Nrf2 knockdown effectively inhibited the proliferation, migration and invasion of GC cells

Nrf2 is activated or overexpressed in cancers to protect cancer cells from oxidative damage in response to OS³⁹. Therefore, we hypothesized that knocking down Nrf2 might increase the ability of Cur to impede the growth, migration and infiltration of GC cells. Thus, as shown in Fig. 5A,B, we transfected GC cells with siNrf2. The knockdown efficiency of the three siNrf2 sequences in GC cells was verified by rt-qPCR, and we finally chose sequence 3 with the highest knockdown efficiency for upcoming investigations. Next, the findings of the CCK-8 assay verified both Nrf2 knockdown alone and Cur (20 µΜ) alone inhibited the proliferative ability of GC cells relative to the siNC group, and the inhibition of GC cells proliferation was more pronounced when co-treated with siNrf2 and Cur (20 µΜ) (Fig. 5C,D). Utilizing both the wound-healing (Fig. 5E–H) and Transwell (Fig. 5I,J) assays, the effect of Nrf2 knockdown in combination with Cur on GC cells migration was investigated. We discovered that Nrf2 knockdown amplified Cur’s inhibitory impact on GC cells migration. Similarly, Transwell assay using Matrigel demonstrated that Nrf2 knockdown combined with Cur treatment inhibited GC cells invasive capacity more significantly than Nrf2 knockdown alone and Cur treatment alone (Fig. 5K,L).

Fig. 5.

Cur combined with Nrf2 knockdown effectively inhibited the proliferation, migration and invasion of GC cells. (A,B) rt-qPCR validated the efficiency of three siRNA sequences to knock down Nrf2 in AGS and HGC27 cells. (C,D) After GC cells were transfected with siNC and siNrf2 for 24 h, they were treated with Cur (0 and 20 µM) for 24 h. Cell viability was detected by the CCK8 assay. (E–H) The migratory capacity of GC cells was compared using a wound healing assay and the wound healing area was quantified. (I–L) GC cells were transfected with siNC and siNrf2 or treated with Cur (0 and 20 µM) for 24 h. Transwell assay was used to compare the migratory and invasive capacities of GC cells. Scale: 500 μm. Data are presented as the mean ± SD. n = 3. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Nrf2 depletion increased Cur-induced excessive accumulation of ROS and attenuated Cur-induced activation of the Keap1-Nrf2 signaling pathway

As previously reported, Cur could hinder the growth of GC cells by promoting ROS production while blocking oxidative damage to GC cells by turning up the Keap1-Nrf2 signaling pathway, but Nrf2 knockdown increased the growth inhibitory effect of Cur on GC cells. Therefore, we verified whether Nrf2 knockdown increased the toxicity of Cur in GC cells by increasing the excessive ROS amassment produced by Cur and inhibiting the antioxidative stress impact of the Keap1-Nrf2 signaling pathway. As shown in Fig. 6A,B, Nrf2 knockdown increased Cur-induced excessive ROS accumulation in GC cells. In addition, Nrf2 knockdown combined with Cur treatment further reduced GSH level in GC cells (Fig. 6C,D). At the mRNA level, Nrf2 knockdown inhibited the Keap1-Nrf2 signaling pathway and downstream antioxidant factors HO-1, GCLM, and NQO1; this inhibitory effect could be partially reversed by Cur (Fig. 6E,F). At the protein level, Cur partly eradicated the inhibiting impact of Nrf2 knockdown on the Keap1-Nrf2 signaling pathway and downstream antioxidant genes (Fig. 6G,H). We also extracted the nuclear proteins of GC cells transfected with siNrf2 and treated with Cur, and verified the protein expression of Nrf2 in the nucleus of GC cells in each treatment group, and found that Cur not only increased the protein expression of Nrf2 in the nucleus of GC cells, but also partially reversed the protein expression of Nrf2 in the nucleus caused by siNrf2 (Fig. 6I,J). These findings implied that Nrf2 knockdown reduced Nrf2 translocation to the nucleus, weakening the activation of Nrf2 by Cur. This increased ROS buildup and amplified toxic effects of Cur on GC cells.

Fig. 6.

Nrf2 depletion increased Cur-induced excessive accumulation of ROS and attenuated Cur-induced activation of the Keap1-Nrf2 signaling pathway. Determination of ROS (A,B) and GSH (C,D) levels via Fluorence microplate reader in GC cells transfected with siNC and siNrf2 under 20 µM Cur treatment. (E,F) The relative Keap1, Nrf2, HO-1, GCLM and NQO1 mRNA levels via rt-qPCR in GC cells transfected with siNC and siNrf2 under 20 µM Cur treatment. (G,H) The relative Keap1, Nrf2, HO-1, GCLM and NQO1 protein expression via Western blot in GC cells transfected with siNC and siNrf2 under 20 µM Cur treatment. (I,J) The relative nucl-Nrf2 protein expression in GC cells transfected with siNC and siNrf2 under 20 µM Cur treatment. GAPDH and Histone H3 were used as internal controls. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ns, no statistical significance.

Nrf2 depletion increased the growth inhibitory effect of Cur on AGS cells in vivo

To further confirm the antitumor effect of Nrf2 knockdown combined with Cur in vivo, we transfected AGS cells with Nrf2-interfering lentiviral vectors or empty vectors to establish an AGS cell xenograft model. We observed that Cur treatment or combined with Nrf2 knockdown didn’t have a significant impact on mice’s body weight (Fig. 7A), and Cur alone or Nrf2 knockdown alone resulted in significant reductions in tumor volume and tumor weight compared to controls, whereas the combination of the two showed a more pronounced antitumor effect (Fig. 7B-D).  In addition, we performed HE staining on the heart, liver, spleen, and kidney tissues of nude mice in each group to observe whether our treatment would damage these organs. The results showed that our treatment was safe and did not burden the organs of nude mice (Fig. 7E). Western blot results of tumor tissues from all groups of mice showed that Cur activated the Keap1-Nrf2 signaling pathway and partially reversed the inhibitory effect of Nrf2 knockdown on the Keap1-Nrf2 signaling pathway (Fig. 7F,G). These results confirmed that Nrf2 depletion could indeed increase the growth inhibitory effect of Cur in vivo tumors.

Fig. 7.

Nrf2 depletion increased the growth inhibitory effect of Cur on AGS cells in vivo. AGS cells infected with Nrf2-interfering lentivirus and control lentivirus were injected into the axilla of the left anterior upper limb of nude mice, followed by intraperitoneal injection of the drug. The growth curve of mice weight (A) and tumor volume (B) in each group of mice. (C) Representative images of tumors in each group of mice. (D) The tumor weight of each group. (E) Representative images of the heart, liver, spleen and kidney of different treatment groups. (F,G) Relative expression of Keap1, Nrf2, HO-1, GCLM and NQO1 proteins in tumor tissues of mice in each group. Scale: 100 μm. n = 3; *p < 0.05, ***p < 0.001, ****p < 0.0001.

Discussion

Gastric cancer is a common primary malignant tumor. Although there have been major breakthroughs in the treatment of GC, the therapeutic situation is still very serious due to its late diagnosis, side effects of chemotherapy, the occurrence of drug resistance and easy recurrence⁴⁰. Natural plant extracts, because of their varied chemical structures and biological activities, are currently acknowledged as a major source of anti-tumor medicines. Moreover, they exhibit comparatively reduced toxicity and side effects in comparison to chemically synthesized drugs⁴¹. Revealing the mechanism of action underlying these natural plant extracts has emerged as a prominent research focus⁴². Previous studies confirmed that Cur is a plant extract with good safety, and it primarily inhibits the proliferation and movement of tumor cells to provide its anticancer action. For example, Cur treatment inhibited the proliferation, migration and invasion of colorectal cancer cells⁴³. Cur inhibited the proliferation, migration and invasion of the GC cell line SGC7901¹⁵. These investigations allowed us to demonstrate that Cur therapy decreased the proliferation, colony formation, migration, and invasion capacities of the GC cell lines AGS and HGC27 in vitro and hampered GC growth in vivo. As a result, Cur seems like a good medication option for the management of GC.

ROS is a collective term for unstable molecules containing hydrogen peroxide (H₂O₂), hydroxyl radicals (OH⁻), etc⁴⁴. ROS may paradoxically contribute to the growth and demise of cancer cells, according to mounting data⁴⁵. At low to moderate levels, ROS function as signaling transducers that induce cancer cells proliferation, migration, invasion, and angiogenesis^46,47. However, elevated ROS levels exert cytotoxic effects on cancer cells, ultimately leading to cellular demise²¹. Therefore, both scavenging ROS and promoting ROS production as targeted approaches for cancer cells eradication hold great promise as anticancer therapeutic strategies, despite their inherent contradictions and complexities. In this work, Cur therapy reduced proliferation of GC cells and elevated ROS generation, but the ROS scavenger NAC was able to counteract the inhibitory effect of Cur therapy. Put another way, by raising ROS levels, Cur prevented GC cells from proliferating.

To prevent the overproduction of ROS within cells, cancer cells protect themselves from OS by activating the antioxidant defense system²⁵. Nrf2 is an important regulator of cancer cells’ antioxidant response⁴⁸. In cancers, Nrf2 is activated and overexpressed to prevent OS and to ensure cancer cells survival³⁹. Under normal conditions, Nrf2 level is regulated by negative feedback from Keap1. Keap1 acts on Nrf2 to mediate its proteasomal degradation. However, upon cellular exposure to OS, Keap1 undergoes a conformational change, releasing Nrf2 into the nucleus where it binds and activates AREs⁴⁹. Numerous antioxidant enzymes’ expression is regulated by the AREs, such as heme oxygenase 1, NAD(P)H quinone dehydrogenase 1, catalase, superoxide dismutase, thioredoxin reductase 1, and glutathione peroxidase⁵⁰. Therefore, cancer cells protect themselves from excessive ROS. Previous studies have confirmed that Cur can counteract OS by interacting with Cys151 in Keap1 to increase nuclear accumulation of Nrf2⁵¹. Nrf2 was found to be overexpressed in GC tissues and cells, suggesting that it is a pro-oncogene in GC⁵². Therefore, we conjecture that Nrf2 inhibition may increase Cur-induced OS and death injury in GC cells. In our investigation, the antioxidant genes HO-1, GCLM, and NQO1 were expressed both on the mRNA and protein levels as a result of Cur therapy activating the Keap1-Nrf2 signaling pathway. Furthermore, in comparison to the NC group, GC cells produced more ROS after Nrf2 was knocked down, and their ability to proliferate, migrate, and invade was also markedly reduced. Interestingly, Nrf2 knockdown combined with Cur treatment significantly enhanced ROS production and inhibition in GC cells compared with Nrf2 knockdown alone and Cur alone. These results suggested that Nrf2 depletion combined with Cur treatment is a potential therapeutic strategy for GC.

In addition, Label-free quantitative proteomics was used to screen for differential proteins in GC cells treated with or without Cur. The results of KEGG enrichment analysis and PPI network showed that ferroptosis and cell cycle were highly enriched in Cur-treated GC cells. The effect of Cur on the cell cycle of GC have been studied in great detail^53,54. According to our findings, Cur treatment caused AGS cells to shorten phases G1 and S, G2 phases to extend, while HGC27 cells were extended phases G1 and G2 and S phases were shortened. These results are consistent with previous studies. In previous studies, Cur induced ferroptosis in lung, breast, thyroid and colorectal cancers^55–58. However, the ferroptosis of Cur in GC is still rarely reported. The results of our apoptosis experiments suggested that Cur may induce cell death other than apoptosis in GC cells, possibly ferroptosis analyzed by proteomics sequencing, which needs to be confirmed by further basic experimental studies.

Conclusion

In summary, our study proved that Cur reduced GC cells growth by triggering ROS and shielded GC cells from ROS damage by activating the Keap1-Nrf2 signaling pathway. Knocking down Nrf2 combined with Cur treatment could significantly increase ROS production, exhibiting a synergic cytotoxic effect. In addition, Nrf2 depletion could also increase the growth inhibitory effect of Cur on in vivo tumors. The findings offer the theoretical justification for the combined synergistic antitumor effect of Cur and Nrf2 knockdown in GC, as well as theoretical direction for the selection of therapies for GC.

Materials and methods

Cell culture

The Chinese Academy of Sciences’ Cell Bank provided the GES-1, AGS, and HGC27 cell lines. Every cell line was grown in full media supplemented with 10% fetal calf serum (Gibco, Thermo Fisher Scientific, USA), 1% penicillin/streptomycin (Procell, Wuhan, China), and RPMI-1640 (Procell, Wuhan, China). The cells were grown in an incubator with humidity, 5% CO₂, at a temperature of 37 °C.

Reagents

After buying curcumin (Cur) from Sigma-Aldrich, the substance was dissolved in dimethyl sulfoxide (DMSO) to produce a 100 mM stock solution, which was then kept for backup at -80 °C. N-acetylcysteine (NAC) was obtained from Aladdin and prepared ready to use.

Cell viability assay

96-well culture plates (Labgic, Beijing, China) were injected with Cells (8 × 10³/well) for 24 h. After that, the cells were cultured for 24 h with a graded dilution of Cur. Each well received 10 µL of CCK-8 detection reagent, which was then incubated for 1 h at 37 °C. An enzyme meter was used to detect the absorbance at 450 nm. The IC50 value was calculated with the use of GraphPad Prism 8.

Clonogenic assay

After being injected into 6-well plates, cells (8 × 10²/well) were grown for 24 h. Then, various concentrations of Cur were applied to the cells. Every three days, the medium was switched out. Following a 10-day period, the cells were stained using 1% crystal violet solution (Solarbio, Beijing, China) for 20 min. Colony photos were obtained and counted.

Wound-healing assay

GC cell monolayers planted in 6-well plates were scratch wounds made with sterile 100 µL pipette tips. The wells were then filled with a medium containing 2% serum, and Cur was allowed to incubate for 48 h. An inverted microscope was used to observe the scratch wound at 0 and 48 h. Data were analyzed using ImageJ software.

Transwell migration and invasion assays

For the cell invasion test, Transwell chambers containing Matrigel (ABW, Shanghai, China) were utilized, and for the cell migration test, Transwell chambers devoid of Matrigel. To sum up, the lower chamber was filled with 600 µL of complete medium containing 20% FBS. 200 µL of serum-free RPMI-1640 and cells (5 × 10⁴ AGS cells/well, 8 × 10⁴ HGC27 cells/well, Cur treatment for 24 h) were added to the upper. Following a 24-h incubation period, the cells were fixed for 30 min using 4% paraformaldehyde and stained for 20 min using 1% crystal violet. Pictures were taken using an inverted microscope (Nikon, Tokyo, Japan), and were analyzed by ImageJ software.

Proteomics sequencing

After using 0 and 20 µM Cur to act on AGS cells for 24 h, the cells were collected and added with RIPA lysis solution to fully lysed the cells. The processed samples were subjected to proteomic sequencing and analyzed by Applied protein technolocy (Shanghai, China). Data are available via the PRIDE repository with identifier PXD050978.

Cell cycle

Cell cycle assay kit (Beyotime, Shanghai, China) was used to detect the cell cycle. The cells to be tested were collected in 1.5 ml EP tubes, the cells were washed by adding pre-cooled PBS, the supernatant was carefully aspirated after centrifugation, mixed with pre-cooled 70% ethanol by gently blowing, and then fixed at 4℃ for 24 h. The cells were configured with propidium iodide staining solution, and the cells were resuspended with it, and then incubated at 37℃ in an incubator for 30 min under the protection of light, and then detected by using a flow cytometer.

Apoptosis

Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) was used to detect apoptosis. Cells to be tested were collected and washed twice using pre-cooled PBS, about 1 × 10⁵ cells were taken, and the cells were gently resuspended by adding Annexin V-FITC conjugate, followed by mixing Annexin V-FITC and propidium iodide staining solution, and incubated for 20 min at room temperature and protected from light, and then immediately detected by flow cytometry.

siRNA transfection

After incubating cells (2 × 10⁵/well) in 6-well plates for 24 h, jetPRIME in vitro siRNA transfection reagent (Yeasen, Shanghai, China) and complete media were added, along with small interfering RNAs (siRNAs) targeting Nrf2 (siNrf2) or negative control (NC) (50 nmol/l). The transfected cells were incubated at 37 °C in a 5% CO2 incubator for 24 h for subsequent experiments. The source of the siRNAs was GenePharma in Shanghai, China. The sequences of siNrf2 are enumerated in Supplement Table 1.

Measurement of ROS

To find the ROS level, DCFH-DA (Beyotime, Shanghai, China) was employed. Briefly, the cells were inoculated in 6-well plates. Following a 24-h Cur treatment, the cells were harvested and tagged with 10 µM DCFH-DA for 20 min in an incubator. Immediately after removing the DCFH-DA solution, the fluorescence value of DCF was detected using a fluorescence enzyme marker (Thermo Fisher Scientific, USA).

Measurement of GSH

A glutathione assay kit (Solarbio, Beijing, China) was used to assess the relative glutathione (GSH) concentration in cells. The enzyme meter was used to measure the absorbance at 412 nm.

Real-time quantitative PCR (rt-qPCR)

GC cells’ total RNA was extracted using the RNA-easy isolation reagent (Vazyme, Nanjing, China), and HiScript III RT SuperMix (Vazyme, Nanjing, China) was used to reverse transcribe the extracted RNA into cDNA. On a LightCycler 480 (Roche) system, rt-qPCR was carried out using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The 2^−ΔΔCt technique was utilized to ascertain the relative expression of target genes, with GAPDH serving as an internal control. Supplementary Table 1 contained the list of PCR primers that were utilized.

Western blotting

Following cell collection, the cells were lysed in RIPA buffer (Solarbio, Beijing, China) containing protease inhibitors for 30 min on ice. After centrifugation, the supernatant was collected, and the BCA assay (Elabscience, Wuhan, China) was used to measure the protein content. Using a continuous current of 300 mA, proteins were transferred to PVDF membranes after being separated into equal amounts by SDS-PAGE (10%). The PVDF membrane was sealed with 5% skim milk powder and let to stand at room temperature for 2 h. Then, it was incubated with the primary antibody overnight at 4 °C. The secondary antibody attached to HRP was incubated on the membrane for 1 h at room temperature the next day. Protein bands were identified under an infrared imaging system (ChemiDoc XRS + with Bio-Rad gel imager) using a chemiluminescence kit (Affinity Biosciences, Jiangsu, China). An internal control was employed: GAPDH. Supplementary Table 1 contained a list of the used antibodies.

Animals and treatment

Beijing Vital River Laboratory Animal Technology Co., Ltd. provided male BALB/c nude mice that were six weeks old. AGS cell lines were transfected using empty vector or Nrf2 interfering lentiviral vector. The transfected cells were adjusted to a cell concentration of approximately 1 × 10⁸ cells/ml with PBS and inoculated into the axilla of the left anterior upper limb of nude mice at 0.1 ml/only. Groupings included Nrf2-NC (solvent only); Nrf2-NC + Cur (20 mg/kg intraperitoneal injection); Nrf2-KD (solvent only); Nrf2-KD + Cur (20 mg/kg intraperitoneal injection). All groups were administered every 2 days and nude mice were monitored for body weight and tumor size. After 30 days, all mice were deeply anesthetized with isoflurane and then executed by cervical dislocation. Axillary tumor tissues and visceral organs were collected from nude mice in each group. Every animal experiment was approved by the Animal Care and Welfare Committee of the Affiliated Hospital of Qingdao University (ethics number AHQU-MAL20230825) and was conducted in accordance with the ARRIVE guidelines related to animal handling.

Statistical analysis

Graphpad Prism 8 software was used to do t-test and one-way ANOVA to compare the statistical differences between the groups. Every experimental result was shown as mean ± SD. Statistical significance was defined as p < 0.05.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

YW, SSW, and WSQ conceived and designed the study; YW, SSW, CCM, WWQ, and JL conducted the study; MQZ, SBW, RW, and YYL analyzed and interpreted the data; YW and SSW did most of the experiments and wrote the manuscript; WSQ and CCM revised the manuscript; WWQ and JL directed the experiments. All authors contributed to the article and approved the final manuscript.

Data availability

The mass spectrometry proteomics data generated during the current study are available in the PRIDE repository with the dataset identifier PXD050978.

Declarations

Competing interests

The authors declare no competing interests.

Statements of accordance and arrive guidelines

Accordance: We confirmed that all experiments in this study were performed in accordance with the relevant guidelines and regulations. Arrive: All the procedure of the study is followed by the ARRIVE guidelines.

Ethics statement

Animal experiments were reviewed and approved by the Animal Care and Welfare Committee of the Affiliated Hospital of Qingdao University (ethics number AHQU-MAL20230825).