Curcumin inhibits growth and triggers apoptosis in human castration-resistant prostate cancer cells via IGF-1/PI3K/Akt pathway

Abstract

Objective

This study aimed to investigate the possible mechanism by which curcumin inhibits human prostate cancer (PCa) and castration-resistant prostate cancer (CRPC).

Methods

CRPC cells were treated with curcumin and their viability was assessed by MTT assay and apoptosis was detected by annexinV/propidium iodide double-staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling assays. Expression levels of insulin-like growth factor 1 receptor (IGF-1R) were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. Phosphoinositide 3-kinase (PI3K), Akt, and forkhead box protein O1 (FOXO1) expression and phosphorylation were assessed by western blotting.

Results

The highly expressed PCa-related molecule IGF-1R was down-regulated in CRPC cells after curcumin treatment, as determined by RT-qPCR and western blotting. In addition, curcumin inhibited the tumor-related PI3K/Akt signaling pathway in CRPC cells. Moreover curcumin down-regulated the IGF-1/PI3K/Akt signaling pathway in tumors derived from CRPC cells.

Conclusions

These results demonstrated that curcumin inhibits growth and triggers apoptosis of human CRPC cells via the IGF-1/PI3K/Akt pathway, thus providing potential new therapeutic strategies for PCa and CRPC.

Introduction

Prostate cancer (PCa) is a malignant tumor originating from the prostate epithelium, with high morbidity and mortality. According to the Global Cancer Statistics 2020, PCa has become the second most frequently diagnosed male-related cancer and the main cause of cancer-related deaths among men in 48 countries. ¹ Androgen-deprivation therapy (ADT) and radiotherapy currently remain the mainstay treatments for PCa; however, although tumors initially respond to ADT for an average of up 14 to 20 months, hormone-refractory disease, termed castration-resistant PCa (CRPC), invariably occurs, with poor outcomes. ² In addition, ADT has been shown to increase the risks of diabetes, heart disease, and stroke. ³ There is thus an urgent need to identify safe and more effective therapies for PCa and CRPC.

Curcumin, derived from the roots of Curcuma longa, possesses anti-oxidative, anti-inflammatory, anti-microbial, and anti-cancer properties. ⁴ It has been shown to inhibit the growth and metastasis of cancer cells by inducing cell apoptosis and autophagy,^5–7 and to retard the progression of PCa through a variety of mechanisms, such as c-Jun N-terminal kinase, microRNA, and Notch1 signaling.^8–11 In addition, curcumin induced apoptosis and protective autophagy in CRPC cells through iron chelation. ¹² Notably however, curcumin may exert its anti-CRPC effects via multiple mechanisms, which still need to be identified.

High levels of circulating insulin-like growth factor (IGF) have been correlated with some types of cancer, including PCa and breast cancer.^13,14 IGF-1 receptor (IGF-1R) was shown to be overexpressed in about 30% of human PCas, potentially increasing the risk of tumor recurrence and metastasis, ¹⁵ and silencing the IGF-1R gene enhanced the sensitivity of PCa to chemotherapy, ionizing radiation, and targeted drugs. ¹⁶ Moreover, curcumin reduced the expression of IGF-1R and down-regulated the IGF-1 signaling pathway in cancer cells; however, whether curcumin affects IGF-1R or the IGF-1-related signaling pathway in CRPC cells remains unknown.^17,18 In addition, studies have revealed activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway in CRPC,^19,20 and this pathway was shown to be related to the anti-cancer effects of curcumin. ²¹ Based on these collective studies, we surmised that the IGF-1/PI3K/Akt signaling pathway might participate in the anti-CRPC effect of curcumin.

This study aimed to determine the effect of curcumin in CRPC and to identify its underlying mechanisms. Our results showed that curcumin induced apoptosis and reduced the expression of IGF-1R in CRPC cells and in tumors derived from CRPC cells. Meanwhile, the IGF-1/PI3K/Akt pathway was down-regulated in CRPC cells and related tumors after curcumin treatment, which might account for its apoptosis and tumor-inhibitory effects. Overall, these findings provide insights into the possible mechanism of curcumin’s inhibitory effect against CRPC.

Materials and methods

Cell and culture conditions

PC-3 and DU145 CRPC cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco, MA, USA). The cells were inoculated in six-well culture plates at 37°C and cultured in a humidified atmosphere with 5% CO₂.

Chemical reagents and antibodies

Curcumin ((E,E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) (C1386, Sigma-Aldrich, MA, USA) was dissolved in dimethylsulfoxide (D2650, Sigma-Aldrich). Human recombinant IGF-I protein was purchased from Thermo Fisher Scientific (PHG0078, Gibco). The primary antibodies used included anti-β-actin rabbit monoclonal antibody (mAb) (4970, Cell Signaling Technology, MA, USA), anti-IGF-1R rabbit mAb (ab182408, Abcam, MA, USA), anti-PI3K mouse mAb (ab86714, Abcam), anti-phospho (p)-PI3K rabbit polyclonal antibody (pAb) (ab182651, Abcam), anti-Akt rabbit pAb (ab8805, Abcam), anti-p-Akt rabbit pAb (ab8933, Abcam), anti-FOXO1 rabbit mAb (ab52857, Abcam), and anti-p-FOXO1 rabbit pAb (ab131339, Abcam). The secondary antibodies were horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (115-035-003, Jackson ImmunoResearch, PA, USA) and HRP-conjugated anti-rabbit IgG antibody (111-035-003, Jackson ImmunoResearch).

Cell viability

Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (M2003, Sigma-Aldrich). PC-3 and DU145 cells were cultured in 96-well plates and treated with different concentrations of curcumin for 48 hours, or incubated with curcumin for different durations. The absorbance of each well was measured at 570 nm. Cell viability was expressed as the relative optical density (OD)₅₇₀, representing the ratio of OD as follows: (OD₅₇₀ experimental condition − OD₅₇₀ medium alone)/(OD₅₇₀ mock − OD₅₇₀ medium alone). There were at least three replicates per treatment and the experiment was repeated three times.

Western blotting

For western blotting, whole cells and isolated tumors were lysed in lysis buffer using a rotary mixer at 4°C, and the total amounts of sample protein were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. The samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (IPFL00010, Millipore, MA, USA). The membranes were blocked with 5% (w/v) skim milk and then incubated with the primary antibody and corresponding secondary antibodies for 2 hours at room temperature. Protein bands were detected using a QuickGel 6100 Imager (Monad).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted with TRIzol reagent (10296010, Invitrogen, CA, USA) and RT-qPCR was performed with iTaq Universal SYBR Green Supermix (1725124, BioRad Laboratories, CA, USA) on a Bio-Rad CFX96 Real-Time PCR System, after reverse transcription. Relative gene expression was calculated using the 2^−ΔΔCt method, with U6 as an endogenous control. The following primers were used to amplify the corresponding genes: IGF-1R-F: 5ʹ-TCGACATCCGCAACGACTATC-3ʹ and IGF-1R-R: 5ʹ-CCAGGGCGTAGTTGTAGAAGAG-3ʹ; U6-F: 5ʹ-GCTTCGGCAGCACATATACTAAAAT-3ʹ and U6-R: 5ʹ-CGCTTCACGAATTTGCGTGTCAT-3ʹ.

Apoptosis assay

The annexin V/propidium iodide (PI) protocol was used for apoptosis detection. PC-3 and DU145 cells were plated into 12-well plates and treated with curcumin (50 μM) for 24 hours. The cells were then washed with ice-cold phosphate-buffered saline, followed by annexin V/PI staining (V13245, Invitrogen) and flow cytometry detection (BD Biosciences, NY, USA). Tissue cell apoptosis was monitored by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL, Roche Diagnostics, Basel, Switzerland).

Effect of curcumin on tumor growth

Twenty 4- to 6-week-old BALB/c nude mice (Shanghai Slack Laboratory Animal Co., Ltd., Shanghai, China) were divided randomly into four groups (n = 5 mice per group). PC-3 or DU145 cells were injected subcutaneously into the nude mice, which were treated or untreated with 100 mg/kg curcumin (three times a week for 4 weeks), and the tumor volume was measured every 3 days. All procedures were conducted under isoflurane anesthesia to minimize animal suffering. All the mice were sacrificed after 30 days and the tumor tissues were collected and analyzed accordingly. All animal handling procedures were performed in compliance with the Chinese Animal Protection Act and the National Research Council criteria. The experiments and protocols were approved by the Committee on the Ethics of Animal Experiments of The General Hospital of Western Theater Command PLA (permit no. 2021EC3-53).

Statistical analysis

Statistical analysis was performed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). Data are presented as mean ± standard deviation. Differences among treatment groups were compared using Student’s t-test, one-way ANOVA, or two-way ANOVA. A P-value <0.05 was considered statistically significant. All experiments were repeated at least three times.

Results

Curcumin induced apoptosis in CRPC cells

We confirmed the effects of curcumin on CRPC cell viability by MTT assay. The viabilities of PC-3 and DU145 cells treated with 0, 2, 5, 10, 25, 50 μM curcumin for 48 hours were 100.0%, 87.0%, 78.4%, 54.6%, 28.7%, and 21.9%, and 100.0%, 89.6%, 79.4%, 59.0%, 31.1%, and 23.7%, respectively (Figure 1a), indicating that curcumin reduced the viability of PC-3 and DU145 cells in a dose-dependent manner (P < 0.05). The viabilities of PC-3 and DU145 cells treated with 25 μM curcumin for 0, 12, 24, 36, and 48 hours were 100.0%, 75.0%, 46.8%, 35.3%, and 27.3%, and 100.0%, 78.2%, 48.7%, 39.5%, and 30.1%, respectively (Figure 1b). Moreover, annexinV/PI double-staining was used to detect cell apoptosis in CRPC cells treated with 0 or 25 μM curcumin for 24 hours. Curcumin 25 μM increased the apoptosis rates of CRPC cells two-fold (Figure 1c), showing that curcumin induced apoptosis in CRPC cells.

Figure 1.

Curcumin induced apoptosis in castration-resistant prostate cancer (CRPC) cells. (a, b) CRPC cells were treated with curcumin at 0, 2, 5, 10, 25, or 50 μM for 48 hours, or incubated with 25 μM curcumin for different times. Cell viability was measured by MTT assay and expressed relative to control (0 μM: dimethylsulfoxide (DMSO)-treated cells), the results are representative of three separate experiments. Each value represents the mean ± standard deviation of three separate replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant (one-way ANOVA). (c) CRPC cells were treated with 25 μM curcumin for 24 hours and the percentage of apoptotic cells was analyzed by flow cytometry after double-staining. Control (0 μM): DMSO-treated cells. **P < 0.01 (one-way ANOVA).

Curcumin reduced the expression of IGF-1R in CRPC cells

IGF-1R is overexpressed in about 30% of human PCas, increasing the risk of tumor recurrence and metastasis. ¹⁵ We therefore explored the effect of curcumin on the expression of IGF-1R in CRPC cells. PC-3 and DU145 cells were incubated with 0, 10, 25, and 50 μM curcumin for 24 hours and then examined by RT-qPCR and western blot. Curcumin decreased the expression of IGF-1R at both the mRNA and protein levels (Figure 2), suggesting that curcumin may inhibit human CRPC via an IGF-1R-related signaling pathway (P < 0.05).

Figure 2.

Curcumin reduced the expression of insulin-like growth factor-1 receptor (IGF-1R) in castration-resistant prostate cancer (CRPC) cells. (a) CRPC cells were treated with 0, 10, 25, or 50 μM curcumin for 24 hours and the mRNA expression levels of IGF-1R were measured by real-time quantitative polymerase chain reaction. The expression of IGF-1R/β-actin was expressed relative to control (0 μM). Results represent three separate experiments. Each value represents the mean ± standard deviation of three separate replicates. **P < 0.01; ***P < 0.001; ns, not significant (one-way ANOVA). (b) CRPC cells were treated with 0, 10, 25, or 50 μM curcumin for 24 hours and the protein expression level of IGF-1R was measured by western blot.

Curcumin down-regulated the IGF-1/PI3K/Akt pathway in CRPC cells

Previous studies^19,20 have indicated that dysregulation of the PI3K signaling pathway may be associated with cancer progression, and curcumin’s anti-cancer effect has been connected with the PI3K/Akt signaling pathway.^19,20 We therefore determined if curcumin inhibited CRPC by interfering with the PI3K/Akt signaling pathway. PC-3 and DU145 cells were stimulated with IGF-1 (10 nM) and curcumin (5 mM), alone or together, for 0, 5, 15, and 30 minutes, and protein was extracted and measured by western blot. Phosphorylation levels of PI3K, Akt, and FOXO1 increased with increasing IGF-1 treatment time but this effect was inhibited by curcumin, and protein expression levels remained unchanged (P < 0.05) (Figure 3). However, phosphorylation levels of the proteins remained the same in cells treated with IGF-1 (10 nM) and curcumin (5 mM) together. These results showed that curcumin down-regulated the IGF-1/PI3K/Akt pathway in CRPC cells, and suggested that curcumin inhibited human CRPC via the IGF-1/PI3K/Akt pathway.

Figure 3.

Curcumin down-regulated the insulin-like growth factor-1 (IGF-1)/phosphoinositide 3-kinase (PI3K)/Akt pathway in castration-resistant prostate cancer (CRPC) cells. CRPC cells were stimulated with IGF-1 (10 nM) and curcumin (5 mM), alone or together, for 0, 5, 15 and 30 minutes, and the protein was then extracted and measured by western blot.

p-: phosphorylated; FOXO1: forkhead box protein O1.

Curcumin down-regulated the IGF-1/PI3K/Akt pathway in tumors derived from CRPC cells

We further investigated the effects of curcumin in vivo. CRPC cells were injected subcutaneously into BALB/c nude mice and the resulting tumors were evaluated. Curcumin (100 mg/kg) retarded tumor growth (Figure 4a, b): tumor volume was significantly smaller in curcumin-treated compared with untreated control mice at 30 days post-inoculation, and the tumor weight was correspondingly reduced (P < 0.05) (Figure 4c). Curcumin also induced tumor apoptosis, as shown by TUNEL assay (Figure 4d). In addition, levels of IGF-1R and phosphorylation of IGF-1/PI3K/Akt pathway-related factors decreased in response to curcumin treatment (P < 0.05) (Figure 4e, f), consistent with the results of in vitro cell experiments. These observations strongly suggested that curcumin inhibited growth and triggered apoptosis in tumors derived from CRPC cells via the IGF-1/PI3K/Akt pathway.

Figure 4.

Curcumin down-regulated the insulin-like growth factor-1 (IGF-1)/phosphoinositide 3-kinase (PI3K)/Akt pathway in tumors derived from castration-resistant prostate cancer (CRPC) cells. (a) Difference between curcumin treated and untreated tumors on day 30. (b) Volumes of curcumin-treated and -untreated tumors at different time points. Data represent results of three experiments (n = 5 mice/group). (c) Weights of curcumin-treated and -untreated tumors on day 30. Data represent results of three experiments (n = 5 mice/group). ****P < 0.0001 (two-way ANOVA). (d) Apoptosis rates of curcumin-treated and -untreated tumors on day 30. Data represent results of three experiments (n = 3 mice/group). ****P < 0.0001 (two-way ANOVA). (e) mRNA and protein expression levels of IGF-1R in curcumin-treated and -untreated tumors on day 30. Data represent results of three experiments (n = 3 mice/group). ****P < 0.0001 (two-way ANOVA). (f) Expression and phosphorylation of PI3K, Akt, and forkhead box protein O1 (FOXO1) in curcumin-treated and -untreated tumors on day 30.

p-: phosphorylated; FOXO1: forkhead box protein O1.

Discussion

PCa is the second most frequently diagnosed cancer among men worldwide, and the leading cause of cancer-related deaths among men in 48 countries. ¹ However, the emergence of CRPC has made the treatment of PCa more challenging, and current therapies are still unsatisfactory. Surgery, hormone therapy, radiation, chemotherapy, and chemical anticancer drugs are commonly used to treat PCa, but these are expensive, have limited efficacy, and are accompanied by serious side effects. There is thus an urgent need to develop more effective treatments for PCa and CRPC. Curcumin has been shown to be a safe, economical, and effective natural extract with activities against various cancers, including breast, liver, lung, and oral cancer.^22–24 Here, we aimed to identify the novel functions of curcumin against CRPC. Curcumin reduced the viabilities of PC-3 and DU145 cells, as shown by MTT and annexinV/PI double-staining assays. Curcumin regulates cell cycle progression and causes cancer cell death, and was shown to inhibit cell proliferation and increase apoptosis of human HepG2 and SK-Hep-1 hepatoma cells in time-dependent and concentration-dependent manners, and to block the cell cycle in G0/G1 phase. ²⁵ The present study also showed that curcumin induced apoptosis in CRPC cells in time-dependent and concentration-dependent manners. This anti-cancer effect suggests that curcumin may be a safe and effective treatment for CRPC.

Several recent studies have reported on the mechanisms behind the anti-PCa effects of curcumin. Sha et al. found that curcumin down-regulated Notch1 and the Notch ligand Jagged-1 gene expression in PCa cells, thus activating downstream signaling pathways to promote tumor cell apoptosis. ⁹ Several studies indicated that microRNAs, including miR-34a, miR-143, and miR-145, played an important role in the anti-proliferation effect of curcumin in PCa.^10,11 Meanwhile, Li et al. reported that curcumin inhibited the expression of p65 by regulating the c-Jun N-terminal kinase signaling pathway, thereby inhibiting the expression of the cancer-associated protein mucin 1 and ultimately inhibiting PCa. ⁸ Moreover, Yang et al. demonstrated that curcumin induced apoptosis and protective autophagy in CRPC cells through iron chelation. ⁸ Nevertheless, the possible mechanism responsible for curcumin’s anti-CRPC effect remains unclear.

IGF binds to its cell surface receptors, such as IGF-IR, and then initiates multiple signaling pathways including PI3K/Akt, mitogen-activated protein kinase, Janus kinase/signal transducer and activator of transcription, and Src. Activation of IGF signaling pathways promotes growth, metastasis, and drug resistance in many types of human tumors, including epithelial and mesenchymal cancers.^26–29 Notably, IGF-1R is overexpressed in about 30% of human PCas. ¹⁵ We accordingly investigated the effects of curcumin on the expression of IGF-1R in CRPC cells, and RT-qPCR and western blot showed that curcumin significantly decreased the expression of IGF-1R. Combined with the reported curcumin-induced down-regulation of the IGF signaling pathway in cancer cells,^17,18 we surmised that IGF-1R-related signaling pathways may participate in the anti-CRPC effects of curcumin. In addition, activation of the PI3K/Akt pathway has been demonstrated in CRPC, and the pathway was intimately involved in the anti-cancer effects of curcumin. We therefore examined the correlation between curcumin and the IGF-1/PI3K/Akt pathway in CRPC cells, and found that the expression levels of pathway-related proteins were significantly reduced in cells treated with curcumin (5 mM), while their expression levels were increased after combined treatment with IGF1 and curcumin. These results suggest that curcumin regulates the IGF-1/PI3K/Akt pathway in CRPC cells.

This study had some limitations. First, we concluded that curcumin inhibited the growth and triggered apoptosis of human CRPC cells via the IGF-1/PI3K/Akt pathway; however, further studies are needed to determine if curcumin directly affects the progression of prostate cancer and to clarify its overall molecular mechanism. Second, we found that curcumin's inhibitory effect on prostate cancer was related to regulation of the IGF-1/PI3K/Akt pathway; however, the precise molecular mechanism remains to be explored. In addition, more studies are needed to determine the optimal concentration of curcumin.

In conclusion, we identified a possible correlation between the IGF-1/PI3K/Akt pathway and apoptosis induced by curcumin in CRPC cells and in tumors derived from CRPC cells. Curcumin decreased the phosphorylation levels of PI3K, Akt, and FOXO1, a transcription factor that is the main target of insulin signaling, while IGF-1 treatment had an enhancing effect. We verified these results in vivo, and showed that curcumin induced apoptosis and down-regulated the IGF-1/PI3K/Akt pathway in tumors derived from CRPC cells. These phenomena may thus help to explain the mechanism underlying the anti-CRPC effect of curcumin.

Data availability statement

All data generated or analyzed during this study are available from the corresponding author on reasonable request.