Chelerythrine chloride induces apoptosis in renal cancer HEK-293 and SW-839 cell lines.

Previous studies have demonstrated that the benzo[c]phenanthridine alkaloid chelerythrine chloride (CC) has inhibitory effects on various tumors. However, the anticancer activity of CC and its underlying mechanisms have not been elucidated in renal cancer cells. The present study examined the effects of CC on growth inhibition and apoptosis of renal cancer cells in vitro and in vivo. Flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays revealed that CC markedly suppressed the growth of HEK-293 and human renal cancer SW-839 cells in a time- and dose-dependent manner. The xenograft mouse model, which was performed in nude mice, exhibited a reduced tumor growth following CC treatment. In addition, the present study revealed that CC significantly decreased the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, which was accompanied by upregulation of p53, B-cell lymphoma 2 (Bcl-2)-associated X protein, cleaved caspase-3 and cleaved poly (adenosine diphosphate-ribose) polymerase (PARP), and downregulation of Bcl-2, caspase-3 and PARP. Furthermore, the use of PD98059, a specific mitogen-activated protein kinase kinase inhibitor, potentiated the proapoptotic effects of CC, which indicated that CC may induce apoptosis in renal cancer cells partly via inhibition of ERK activity. Overall, the results of the present study demonstrated that CC may be developed as a potential anticancer treatment for patients with renal cancer.

Introduction

Renal cancer is one of the ten most common types of cancer in humans, and is often resistant to chemotherapy (1). Clear cell renal cell carcinoma (RCC) accounts for ~70% of cases of renal cancer (1). RCC is a common urological cancer, which accounts for ~3% of all adult malignancies (2) and 5% of all types of epithelial cancer that are diagnosed in the USA every year, the majority of which are clear cell RCC (3,4). In total, 20–30% of patients with RCC present metastases at diagnosis, and 20–40% of patients with localized disease who undergo nephrectomy subsequently develop metastases (5). Although it accounts for a small proportion of visceral malignancies, RCC constitutes a significant health problem, due to the unpredictable clinical course and poor prognosis of patients with distant metastasis (6). Generally, surgery is the only curative treatment for patients with RCC, since the response of patients to chemotherapy and radiotherapy is poor (7). RCC may be treated surgically if it is diagnosed in the early stage of the disease, and patients without metastasis may achieve a 5-year survival rate of ~85% (8). However, patients with distant metastasis present a poor prognosis, with a 5-year survival rate of <10% (9). The current recommended treatment for RCC consists of radical resection of the tumor mass and immunotherapy with cytokines, including interferon and interleukin-2 (10). The development of diagnostic technologies has led to an increase in the number of patients who are diagnosed with RCC in the early stages of the disease. Previous studies have indicated that the development and progression of RCC are closely associated with the tumor microenvironment (11).

In the past recent years, clinical trials using plant-derived drugs for the prevention and treatment of tumors have become increasingly popular in cancer therapy, and there has been an increase in the number of studies concerning novel drugs that induce cell cycle arrest and apoptosis of cancer cells (12). Chelerythrine chloride (CC) is a natural benzo[c]phenanthridine alkaloid that is present in numerous plant species (13,14), and is known to exert various biological activities, including antimicrobial, antifungal, anti-inflammatory and anticancer activities (15,16). Several studies have previously investigated the effects of CC as a cancer treatment (13,17–19). CC was observed to exhibit antiproliferative and apoptotic properties on various human cancer cell lines, including squamous cell carcinoma, human leukemia, human breast cancer, human colon carcinoma, human uveal melanoma and human neuroblastoma, in addition to neonatal rat cardiac myocytes (20–24). CC affects various signaling pathways via the inhibition of protein kinase C and mitogen-activated protein kinase (MAPK) phosphatase-1 (25,26). However, CC is cytotoxic, which results in controversy over its use (27). In addition, CC was revealed to induce cell death in normal cells, including human hepatocytes (28) and rat cardiac myocytes, and in cancer cells, including human primary uveal melanoma OCM-1 cells and human promyelocytic leukemia HL-60 cells. Notably, CC mediates its antitumor activity via different mechanisms, which may be promising targets for anticancer therapy (24,28,29). In addition, CC induces a cytotoxic effect against radio and chemotherapy-resistant squamous carcinoma cells, which resulted in delayed tumor growth and mild toxicity in an animal model (13). CC is considered to be a potential candidate for cancer therapy due to its apoptotic effect on cancer cells (30,31). However, there are limited studies regarding the mechanism by which CC induces apoptosis in renal cancer cells. Therefore, the present study investigated the effect of CC on cell proliferation, cycle progression and apoptosis in renal cancer cells.

Materials and methods

Cell lines and reagents

The cell lines HEK-293 and human renal cancer SW-839 were obtained from the American Type Culture Collection (Manassas, VA, USA), and cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) - high glucose supplemented with 10% fetal bovine serum in an atmosphere containing 5% CO2 at 37°C. CC was purchased from Shanghai Tauto Biotech Co., Ltd. (Shanghai, China), and dimethylsulfoxide (DMSO) was purchased from Sigma-Aldrich, (St. Louis, MO, USA). Anti-extracellular signal-regulated kinase (ERK)1/2 (catalog no., 9102; dilution, 1:1,000), anti-phospho (p)-ERK1/2 (catalog no., 4370; dilution, 1:2,000), anti-p38 (catalog no., 8690; dilution, 1:1,000), anti-p-p38 (catalog no., 4511; dilution, 1:1,000), anti-c-Jun N-terminal kinase (JNK; catalog no., 9252; dilution, 1:1,000), anti-p-JNK (catalog no., 9251; dilution, 1:1,000), anti-poly (adenosine diphosphate-ribose) polymerase (PARP; catalog no., 9242; dilution, 1:1,000), anti-glyceraldehyde 3-phosphate dehydrogenase (catalog no., 2118; dilution, 1:1,000), horseradish peroxidase (HRP)-conjugated goat anti-rabbit (catalog no., 7074; dilution, 1:2,000) and anti-mouse immunoglobulin G (catalog no., 7076; dilution, 1:2,000) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against p53 (polyclonal; catalog no., YT0024; dilution, 1:1,000), caspase-3 (monoclonal; catalog no., YM3431; dilution, 1:1,000), B-cell lymphoma 2 (Bcl-2; polyclonal; catalog no., YT0433; dilution, 1:1,000) and Bcl-2-associated X protein (Bax; polyclonal; catalog no., YT0459; dilution, 1:1,000) antibodies were obtained from ImmunoWay Biotechnology Company (Newark, DE, USA).

Cell viability assay

Cell viability was evaluated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells (2×103 HEK-293 cells/well and 3×103 SW-839 cells/well) in 100 µl medium were seeded into Corning® Carbo-BIND™ 96-well plates, and incubated for 12 h. Next, the medium in each well was replaced with medium containing various concentrations of CC, and the cells were incubated at 37°C for an additional 24 and 48 h. Subsequently, 20 µl MTT (5 mg/ml; Sigma-Aldrich) was added to each well. Following an additional incubation at 37°C for 4 h, the supernatant was removed, and 100 µl DMSO was added to each well. The absorbance values (read at 540 nm) were determined using the iMark™ Microplate Absorbance Reader (Bio-Rad Laboratories, Inc., Hercules CA, USA). The data were analyzed using Microplate Manager software (ver. 6.3; 1689520; Bio-Rad Laboratories, Inc.)

Apoptosis assay

Detection of apoptotic cells was performed using an annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay. In brief, harvested cells were resuspended in 100 µl binding buffer to achieve a concentration of 1×106 cells/ml. Subsequently, 5 µl annexin V-FITC (Sigma-Aldrich) and 5 µl PI (20 µg/ml; Sigma-Aldrich) were added to the cells, which were incubated for an additional 15 min. A total of 400 µl binding buffer was then added to each tube, and the cells were analyzed using the BD ACCURI C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed using WinMDI version 2.9 software (The Scripps Research Institute, San Diego, CA, USA).

Western blot analysis

Cell protein preparation and western blot analysis were conducted as previously described (32). Proteins (25 µg) were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (40% acrylamide solution; catalog no., 1610140; Bio-Rad Laboratories, Inc.), and transferred to polyvinylidene fluoride membranes (pore size, 0.22 µm; EMD Millipore, Billerica, MA, USA) using Mini Trans-Blot® Electrophoretic Transfer Cell (catalog no., 170–3930; Bio-Rad Laboratories, Inc) at 30 V. The membranes were blocked with 5% skimmed milk, and subsequently probed with the corresponding primary antibodies at 4°C overnight. The membranes were washed with phosphate-buffered saline (PBS) with Tween 20 (0.05%) (Sigma-Aldrich), followed by incubation at room temperature with the HRP-labeled secondary antibodies for 1 h. The protein bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore). Protein expression was detected using ImageQuant™ LAS 4000 chemiluminescence reader (GE Healthcare Life Sciences, Chalfont, UK). The densitometry analysis was performed using the ImageQuant TL software (28-9175-41; v.7.2; GE Healthcare Life Sciences).

Tumor xenograft model

A total of 5×106 SW-839 cells were mixed with Matrigel® (Corning Life Sciences, Corning, NY, USA), and injected subcutaneously into the flanks of 14 5-week-old male BALB/c nude mice. The mice were purchased from the Institute of Laboratory Animal Sciences of the Chinese Academy of Medical Sciences (Beijing, China), and were maintained in 18×30-cm cages containing three mice each, at a temperature of 22°C using a 12 h light/dark cycle. Food and water was available ad libitum. The mice were randomly divided into two groups (n=7). As previously described, the mice were administrated with CC at a dose of 5 mg/kg/day via intraperitoneal injection for 5 weeks, with the first injection occurring 24 h after injection with the SW-839 cells. The control mice were administered with the same volume of PBS containing 1% DMSO. The volume and weight of the mouse tumors were measured once a week. All the mice were sacrificed 36 days subsequent to inoculation of the cancer cells, when the tumors were resected. All the animal experiments were approved by The Ethics Review Board of Henan Provincial People's Hospital (Zhengzhou, China).

Immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assays

All the xenograft tumors were formalin (Sigma-Aldrich)-fixed and paraffin (Sigma-Aldrich)-embedded, prior to be sliced into 6-µm sections for IHC and TUNEL assays. For the IHC assay, the sections were washed with PBS, treated with 3% hydrogen peroxide (Sigma-Aldrich) at room temperature, blocked with normal goat serum in PBS (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 20 min, and incubated overnight at room temperature with human monoclonal anti-Bax and anti-Bcl-2 antibodies. Upon incubation with the secondary antibody, the expression of intracellular Bax and Bcl-2 was detected using 3,3′-diaminobenzidine (Sigma-Aldrich) staining. One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Haimen, China) was used to stain the apoptotic tumor cells. The cells were visualized with red fluorescence under a fluorescence microscope (IX83; Olympus Corporation, Tokyo, Japan) with excitation and emission wavelengths of 488 nm and 588 nm, respectively. The images were analyzed using cellSens Standard software (Olympus Corporation).

Statistical analysis

Student's t-test was used to determine statistical differences between treatment and control values. P<0.05 was considered to indicate a statistically significant difference. All the data are presented as the mean ± standard deviation of three independent experiments.

Results

CC inhibits the proliferation of renal cancer cells

To study the effects of CC on the proliferation of RCC cells, HEK-293 and SW-839 cells were exposed to various concentrations of CC for 24 and 48 h. The results demonstrated that CC significantly inhibited the proliferation of HEK-293 and SW-839 cells (Fig. 1A and B, respectively) in a time- and dose-dependent manner. The cell viability assay also indicated that HEK-293 cells were more sensitive to CC-induced proliferation inhibition, compared with SW-839 cells exposed to CC for 24 h.

Figure 1.

Cell viability analysis by MTT assay following treatment with CC. (A) HEK-293 and (B) SW-839 cells were treated with various concentrations of CC (0, 5, 10, 20 and 40 µM) for 24 and 48 h. CC significantly inhibited the proliferation of HEK-293 and SW-839 cells in a time- and dose-dependent manner. *P<0.05 vs. control cells; **P<0.01 vs. control cells. CC, chelerythrine chloride.

Apoptosis

To investigate if the CC-induced growth inhibitory effect on RCC cells was due to cell apoptosis, a cytometric apoptosis assay was performed. Annexin V-conjugated FITC and PI staining was used to verify and quantify the percentage of apoptotic cells induced by CC. The percentage of early and late apoptotic cells were represented in the lower right (LR) and upper right (UR) quadrant of the flow cytometry histograms, respectively (Fig. 2A and B). The total percentage of apoptotic HEK-293 cells (UR + LR) increased in CC-treated cells (5 µM CC, 21.80%; 10 µM CC, 31.93%), compared with non-treated cells (2.47%) for 24 h (*P<0.05 vs. controls; and **P<0.01 vs. controls, respectively; Fig. 2C). This was similar to the results observed in SW-839 cells, where the total percentage of apoptotic cells increased from 2.75% in non-CC treated cells to 8.02 and 17.00% in cells treated with 5 and 10 µM CC, respectively (*P<0.05 vs. controls; and **P<0.01 vs. controls, respectively; Fig. 2C). Treatment of SW-839 and HEK-293 cells with 5 and 10 µM CC for 24 h induced apoptosis in the two cell lines in a dose-dependent manner. The significant induction of apoptosis following CC treatment indicates that CC exerts an anticancer effect on renal cancer cells.

Figure 2.

CC-induced dose-dependent apoptosis of HEK-293 and human renal cancer SW-839 cells. (A) HEK-293 and (B) SW-839 cells were treated with various concentrations of CC (0, 5 and 10 µM) for 24 h, and stained with annexin V-fluorescein isothiocyanate/propidium iodide. The percentage of early-stage apoptotic cells is shown in the lower right quadrant, while the percentage of late-stage apoptotic cells is shown in the upper right quadrant. (C) Treatment of HEK-293 and SW-839 cells with 5 and 10 µM CC for 24 h induced cell apoptosis. CC, chelerythrine chloride; FL-H, fluorescence line height.

Tumor growth inhibition in a xenograft model

To determine whether CC inhibits tumor growth in vivo, the present study subcutaneously injected 5×106 SW-839 cells into the flanks of 14 nude mice. The inhibition of tumor growth in mice treated with CC at 5 mg/kg/day was significant, compared with mice treated with PBS, as observed by tumor volume (Fig. 3A and B) and weight (Fig. 3C) measurements. Furthermore, no significant toxicity to mice was observed following treatment with CC, as deduced by assessing the body weight of the mice in the two groups (Fig. 3D). These results suggest that weight loss does not indicate toxicity. To evaluate if CC induced apoptosis of renal cancer cells in vivo, paraffin sections of the SW-839 tumor xenografts from the nude mice were used in a TUNEL assay. The increased number of TUNEL+ cells in the CC-treated mice compared with the PBS-treated mice confirmed that CC induced apoptosis of RCC cells in vivo (**P<0.01 vs. controls; Fig. 3E and F).

Figure 3.

Tumor growth suppression in a human renal cancer SW-839 xenograft nude mouse model. (A and B) Tumor volume of CC and control tumors. (C) Tumor weight of CC and control tumors. (D) No significant toxicity to mice was observed following treatment with CC (5 mg/kg/day), according to the body weight of the mice. (E) Representative TUNEL staining (red fluorescence) of SW-839 renal cancer xenografts. (F) Quantification of TUNEL+ SW-839 cells in tumor xenografts was performed using cellSens Standard software. The data are presented as the mean ± standard deviation of three experiments. **P<0.01 vs. control cells. (G) IHC staining for Bcl-2 and Bcl-2-associated X protein on tumor sections, and (H) quantification of IHC staining using cellSens Standard software. CC, chelerythrine chloride; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; IHC, immunohistochemistry.

Expression of cell apoptosis-associated proteins in vitro and in vivo

Previous studies have demonstrated that the expression of the proapoptotic protein Bax was associated with increased cell apoptosis, while the antiapoptotic protein Bcl-2 was associated with the inhibition of apoptosis in HeLa cells and the basal cell carcinoma ASZ001 cell line (33). The present study investigated the alteration in the expression levels of Bax and Bcl-2 in SW-839 mouse tumor xenografts following treatment with CC by analyzing paraffin sections of the above SW-839 tumor xenografts via IHC. The results shown in Fig. 3G demonstrate that Bax expression was increased, while Bcl-2 expression was decreased, in the xenograft tumors of mice treated with CC, suggesting that the tumor growth inhibition induced by CC was due to an increased rate of cell apoptosis. To identify the mechanism of activation of the apoptotic pathway, the present study examined the expression of apoptosis-associated proteins in HEK-293 and SW-839 cells following treatment with increasing concentrations of CC for 48 h. Since the activation of p53 may lead to cell cycle arrest, DNA repair or apoptosis (34), the present study evaluated the expression of p53 in HEK-293 and SW-839 cells in response to CC-treatment. The results suggested that CC treatment led to a dose-dependent accumulation of p53 (Fig. 4A). Although an increase in apoptosis was observed in the SW-839 and HEK-293 cells, following CC treatment the expression levels of Bax were only slightly increased and the expression levels of Bcl-2 were slightly decreased (Fig. 4A). In addition, the expression levels of pro-caspase-3 were decreased, whereas the expression levels of cleaved caspase-3 and cleaved PARP were increased.

Figure 4.

(Aa) Western blot analysis of the expression levels of apoptosis-associated proteins in HEK-293 and human renal cancer SW-839 cells following treatment with CC. Quantification of the expression of various proteins in HEK-293 and SW-839 cells, such as (Ab) p53, (Ac) Bax, (Ad) Bcl-2, (Ae) pro-caspase-3, (Af) cleaved caspase 3 and (Ag) PARP using GAPDH as a control. Multiple bands were observed in the cleaved caspase-3 lane due to non-specific binding of antibodies. (Ab-Ag) Quantification of western blotting (*P<0.05 and **P<0.01 vs. controls). (Ba) Western blot analysis of MAPK and Akt pathways after CC treatment in HEK-293 and SW-839 cells. The quantification of protein expression was performed for (Bb) pERK, (Bc) p-p38, (Bd) p-JNK and (Be) p-AKT, with GADPH as a control. The results are representative of ≥3 independent experiments. Multiple bands were observed in the p-ERK, p-JNK and JNK lanes due to non-specific binding of antibodies. *P<0.05 and **P<0.01 vs. controls. CC, chelerythrine chloride; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; PARP, poly (adenosine diphosphate-ribose) polymerase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p-, phospho-; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.

Inhibition of ERK pathway enhanced the antiproliferative effect of CC

The present study investigated whether the CC-induced apoptosis of HEK-293 and SW-839 cells was associated with the modulation of intracellular signaling pathways, including MAPK and Akt pathways. The present study evaluated the effects of CC treatment on the activation of ERK, p38 and JNK in the two aforementioned cell lines (Fig. 4B). The results demonstrated that CC significantly enhanced the phosphorylation of ERK1/2 in a dose-dependent manner. In addition, CC inhibited the phosphorylation of p38. However, there was not a clear alteration in the activation of JNK (Fig. 4B). The phosphorylation of the kinase Akt was increased by CC treatment in a dose-dependent manner, but the total levels of Akt were not altered. The proliferation and growth of cancer cells has been revealed to be dependent on the activation of ERKs (34,35). To examine whether a blockade of ERK signaling using the MAPK kinase inhibitor PD98059 may potentiate the ability of CC to inhibit cell proliferation of renal cancer cells, HEK-293 and SW-839 cells were cultured in the presence of CC (5 µM), PD98059 (50 µM) or a combination of the two. The protein levels of ERK1/2, p-ERK1/2, Bax and Bcl-2 were detected using western blot analysis. The results revealed that inhibition of ERK activity with PD98059 enhanced the upregulation of Bax expression and the downregulation of Bcl-2 expression induced by CC (Fig. 5A–F). Similarly, the cell viability assay demonstrated that PD98059 potentiated the proapoptotic effects of CC (Fig. 5G and H). In addition, the present study observed that treatment with PD98059 alone exerted moderate effects, whereas PD98059 significantly enhanced the antiproliferative effect of CC in HEK-293 and SW-839 cells. This suggests that an inhibition of the ERK signaling pathway may enhance the antitumor effect of CC.

Figure 5.

Treatment with CC and with the mitogen-activated protein kinase kinase inhibitor PD98059, alone or in combination, inhibited the proliferation of renal cancer cells. (A) HEK-293 and (B) human renal cancer SW-839 cells were cultured with 8 µM CC, 50 µM PD98059, or a combination of the two compounds for 24 h, and the levels of ERK1/2, phospho-ERK1/2, Bcl-2 and Bcl-2-associated X protein were analyzed by western blotting, using glyceraldehyde 3-phosphate dehydrogenase as a control. Multiple bands were observed in the lanes corresponding to ERK and p-ERK due to non-specific binding of antibodies. (C-F) Quantification of western blotting (*P<0.05 and **P<0.01 vs. controls). (G) HEK-293 and (H) SW-839 cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results are represented as the mean ± standard deviation of three independent experiments *P<0.05 and **P<0.01 vs. cells treated with dimethylsulfoxide (control). CC, chelerythrine chloride; ERK, extracellular signal-regulated kinase; PD, PD98059; p-, phospho-; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Discussion

The main aim of the present study was to investigate the effect of CC on RCC cells. The present study used HEK-293 and SW-839 cells to study the effects of CC. Apoptosis, also known as programmed cell death, is closely associated with the initiation, progression and metastasis of tumors, and the induction of apoptosis has been used in the treatment of malignant tumors (36,37). The present study aimed to investigate the inhibition of migration and invasion of RCC cells induced by treatment with CC, including if CC induces RCC cells to undergo apoptosis, which has not been previously elucidated. To the best of our knowledge, the present study demonstrated for the first time that CC was able to effectively inhibit the proliferation of RCC cells by inducing apoptosis. In addition, the current study evaluated the molecular mechanisms through which CC induces apoptosis, and revealed that ERK activation was required for the induction of apoptosis by CC. The present results reveal a novel mechanism by which CC exhibits its proapoptotic effect on RCC cells.

The two major kinases that are key in numerous signaling pathways are ERK and Akt, which are often aberrantly activated in cancer cells (38,39). Akt is an important cell survival kinase, which also controls other cellular functions, including migration and integrin activation (40,41). The ERK pathway has been widely studied as a potential pharmacological target for targeted tumor therapy (42) and is important in tumor initiation and progression, since it promotes cell survival and proliferation (43). It has been previously demonstrated that CC induces the apoptosis of cells in association with reactive oxidative species, which subsequently activates JNK and p38 (44). JNK and p38 are members of the MAPK family, which also includes ERK (45). Previous studies have demonstrated that the activation of the ERK pathway promotes cell survival, while inhibition of the ERK pathway increases the sensitivity of cancer cells to apoptosis (46,47). These studies indicate that the activation of ERK has an antiapoptotic effect on cells. The present study investigated the activity of ERK in renal cancer cells that were treated with CC, and observed that the activity of ERK was decreased in a time-dependent manner. A similar result was revealed in osteosarcoma cancer cells following treatment with CC (17). In addition, the present study revealed that the inhibition of ERK activity using PD98059 for 24 h significantly increases the sensitivity of renal cancer cells to CC-induced apoptosis. p53 is a tumor suppressor protein that induces the death of abnormal cells by activating cell growth arrest or apoptosis, and has been associated with several members of the Bcl-2 family (48). The present study demonstrated that CC increased the protein expression levels of p53 in RCC cells in a dose-dependent manner, which suggests that p53 is activated during CC-induced apoptosis. In a previous study, CC was reported to be an inhibitor of Bcl-extra large, a member of the antiapoptotic Bcl-2 family, which is involved in stabilizing mitochondrial membrane integrity (49). Additional studies have elucidated that Bcl-2 preserves the mitochondrial membrane and inhibits the release of internal calcium stores into the cytoplasm, while Bax is processed on the outer mitochondrial membrane and regulates the release of cytochrome c (21,50). Cell apoptosis is induced by caspases, a family of cysteine aspartyl-specific proteases (21,50). Initiating caspases, including caspase-8 and caspase-9, cleave and activate downstream effector caspases such as caspase-3 and caspase-7, which in turn cleave a large number of cellular substrates associated with apoptosis, including PARP (21,50). Therefore, the present study investigated the alterations in the expression levels of Bcl-2 and Bax in RCC cells treated with CC, and observed that Bax expression was increased, while Bcl-2 expression was decreased, in vitro and in vivo. The present results indicate that CC-induced upregulation of Bax expression and downregulation of Bcl-2 expression may lead to the induction of apoptosis in RCC cells. Overall, the present results suggest that there is an association between the decreased activity of ERK and altered expression of Bcl-2 and Bax in the CC-induced apoptosis of RCC cells. Inhibition of ERK activity enhanced the upregulation of Bax expression and the downregulation of Bcl-2 expression induced by CC, which suggests that ERK may be the initiator of CC-induced apoptosis in RCC cells.

In conclusion, the present results demonstrate that CC inhibits the proliferation of HEK-293 and SW-839 RCC cells in vitro and in vivo. In addition, the present results revealed that suppression of the ERK pathway contributes to CC-induced apoptosis in RCC cells. Therefore, the present study provides evidence for the therapeutic potential of CC for the treatment of RCC.