Silymarin selectively protects human renal cells from cisplatin-induced cell death

Abstract

Context: Cisplatin-induced nephrotoxicity has been accepted as an important obstacle for efficient cisplatin-based chemotherapy. Silymarin from seeds of milk thistle [Silybum marianum L. (Asteraceae)] has been shown to possess various potential pharmacological properties; however, whether or not this agent selectively protects renal cells from cisplatin-induced cell death with no interfering effect on cancer cells is not clear.

Objective: Potential of silymarin in protection of cisplatin-induced renal cell death without compromising effect on anticancer activity of cisplatin was demonstrated in this study.

Materials and methods: Cisplatin-induced cell death was evaluated in human proximal tubular HK-2, lung carcinoma H460, and melanoma G361 cells using MTT, Hoechst 33342, and propidium iodide assays.

Results: Cisplatin induced both apoptosis and necrosis in HK-2 cells and caused a decrease in cell viability by ~40% and 60% at the doses of 25 and 100 µM, respectively. Pretreatment with 25–200 µM of silymarin significantly protected against cisplatin-induced cell death in a dose-dependent manner. In contrast, pretreatment of silymarin (25–100 µM) caused no significant change on cisplatin-induced cell death in H460 cells but significantly potentiated cisplatin-induced apoptosis in G361 cells.

Discussion and conclusion: These findings reveal the selectivity of silymarin in protection of renal cells from cisplatin-induced cell death and could be beneficial for the development of this considerately safe compound as a renoprotective agent.

Keywords::

• Silymarin
• cisplatin
• nephrotoxicity
• renoprotection
• selectivity

Introduction

Cisplatin [cis-diamminedichloroplatinum (II)] is accepted as one of the most prescribed drugs for the treatment of many solid tumors such as testicular, head and neck, lung, melanoma, and ovarian cancers (Patrick & Lawrence, 1984; Atallah & Flaherty, 2005; Kelland, 2007). However, nephrotoxicity-induced by cisplatin has been shown to be an important obstacle for efficient cisplatin-based chemotherapy in many patients (Patrick & Lawrence, 1984; Launay-Vacher et al., 2008; Pabla & Dong, 2008). Co-administration of cisplatin and effective nephroprotective agents protecting renal cells from cisplatin-induced damage while having minimal effect on cisplatin actions against cancer cells should be a recommended part of the treatment regimen of cancer. Silymarin, a mixture of flavonolignans extracted from seeds of milk thistle [Silybum marianum L. (Asteraceae)], has long been used for the treatment of various liver diseases and the hepatoprotection against several toxic substances (Muriel et al., 1992; Flora et al., 1998; Saller et al., 2001; Mansour et al., 2006). Silymarin has been shown to possess various potential pharmacological properties including the following: scavenge reactive oxygen species (Mira et al., 1994; Svobodová et al., 2006; Asghar and Masood, 2008), inhibit lipid peroxidation (Bosisio et al., 1992; Muriel et al., 1992), increase glutathione and superoxide dismutase levels (Fehér et al., 1987; Valenzuela et al., 1989), induce tissue regeneration (Sonnenbichler et al., 1999), and promote DNA, RNA, and protein synthesis (Sonnenbichler & Zetl, 1986; Sonnenbichler et al., 1999). Moreover, silymarin exerts cancer-suppressing activity in many kinds of tumor cells such as lung, prostate, cervical, leukemia, and breast cancers (Zi et al., 1998; Sharma et al., 2003; Huang et al., 2005; Deep et al., 2006; Zhong et al., 2006). Although silymarin has potential activities, namely, antioxidant and tissue-stimulating effects, whether or not this agent selectively protects renal cell damage without interfering effect on cancer cells is not known. The present study aims to investigate the effects of silymarin for its potential to be developed for further use in benefiting cisplatin-based chemotherapy.

Materials and methods

Cells and reagents

Human proximal tubular epithelial HK-2, human melanoma G361, and human lung cancer epithelial H460 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). HK-2 and G361 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), whereas H460 cell was cultured in RPMI 1640 medium in a 5% CO₂ environment at 37°C. All media were supplemented with 2 mM l-glutamine, 10% fetal bovine serum, and 100 units/mL of penicillin/streptomycin (Gibco, Gaithersburg, MA). Cisplatin, silymarin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst 33342, and propidium iodide (PI) were obtained from Sigma Chemical, Inc. (St. Louis, MO).

Cytotoxicity assay

Cell viability was determined by the MTT colorimetric assay. In brief, cells in 96-well plate were incubated with 500 μg/mL of MTT for 4 h at 37°C. The supernatant was then removed and dimethyl sulfoxide (DMSO) was added to dissolve the formazan product. The intensity was spectrophotometrically measured at 570 nm using an ELISA reader (Anthros, Durham, NC). All analyses were performed in at least three independent replicate cultures. The absorbance ratio of treated to non-treated control cells was calculated and presented in terms of relative cell viability.

Apoptosis and necrosis assay

Apoptotic and necrotic cell death was detected by Hoechst 33342 and PI co-staining. After specific treatments, cells were stained with 10 µM of the Hoechst and 5 µg/mL PI dye for 30 min at 37°C. The apoptotic cells having condensed chromatin and/or fragmented nuclei stained by Hoechst 33342 and PI-positive necrotic cells were visualized and scored under a fluorescence microscope (Olympus IX51 with DP70).

Statistical analysis

Data were presented as the means ± SD from three or more independent experiments. Statistical analysis was performed by Student’s t-test at a significance level of P < 0.05.

Results

Cisplatin induces cell death

According to clinical evidence, cisplatin-induced nephrotoxicity is a severe side effect, which frequently limits the clinical use of cisplatin in patients (Patrick & Lawrence, 1984; Launay-Vacher et al., 2008; Pabla & Dong, 2008). Cisplatin was recommended for the treatment of many tumors including lung cancer and melanoma (Patrick & Lawrence, 1984; Atallah & Flaherty, 2005; Kelland, 2007). We thus demonstrated cisplatin-induced toxicity and selectivity in protection of silymarin in human proximal tubular epithelial HK-2, human lung cancer epithelial H460, and human melanoma G361. First, we characterized cisplatin-induced cell death in these cells by leaving the cells untreated or treated with various concentrations of cisplatin (5–300 µM). Cell viability was then measured after 24 h by the MTT assay. Figure 1A–1C demonstrates that cisplatin treatment caused a dose-dependent decrease in cell viability with ~50% of the cells remaining viable at cisplatin concentrations of 50, 100, and 100 µM in HK-2, H460, and G361 cells, respectively. As apoptosis and necrosis have been shown to be two major modes of cisplatin-induced cell death, we further identified mode of cell death in response to cisplatin treatment in these cells. A Hoechst 33342 assay was performed for apoptosis detection, and propidium iodide staining assay was used for necrosis detection. Figure 1D–1F shows that the number of apoptotic cells exhibiting intense nuclear fluorescence and DNA condensation and necrotic cells with positive PI staining were increased in response to cisplatin treatment in HK-2 and H460 cells, whereas only Hoechst-positive cells were observed in G361 cells. These results suggest that cisplatin-induced cytotoxicity in HK-2 and H460 cells was due to apoptosis and necrosis cell death, although cisplatin induced G361 cell damage mainly through the apoptosis mechanism.

Figure 1.  Cytotoxic effect of cisplatin on HK-2, H460, and G361 cells. (A–C) Cells were treated with various concentrations of cisplatin (0–300 µM) for 24 h and cell viability was measured by MTT assay. Values are means ± SD of three independent experiments, *P < 0.05 versus non-treated control. (D–F) Apoptotic and necrotic cells were detected by Hoechst 33342 and propidium iodide co-staining assay and examined under a fluorescence microscope.

Silymarin exhibits anticancer activity against lung cancer H460 and melanoma G361 cells

To determine whether silymarin has direct anticancer activity, HK-2, H460, and G361 cells were incubated with various concentrations of silymarin (25–200 µM) for 24 h, and cell viability and mode of cell death were analyzed by MTT, Hoechst 33342 staining, and PI staining assays. Figure 2A indicates that silymarin at low concentrations (25–50 µM) has neither cytotoxic nor proliferative effects on HK-2 cells. However, at increased concentrations of up to 100 and 200 µM, silymarin caused a significant increase in renal epithelial HK-2 cell proliferation. Treatment of the H460 cells with low concentrations of silymarin showed only a minimal effect on cell viability, but at concentrations of 200 µM, silymarin caused a significant toxic effect on these cells with ~30% reduction of cell viability (Figure 2B). In melanoma cells, silymarin exhibited similar dose-dependent cytotoxic effects with ~50% reduction in cell viability in response to 200 µM of silymarin treatment (Figure 2C). Accordingly, results of Hoechst 33342 and PI co-staining assays in Figure 2D–2F show that treatment with silymarin alone caused no significant apoptosis and necrosis in HK-2 cells, whereas silymarin treatment caused a dose-dependent increase in the number of apoptotic cells in H460 and G361 cells. Notably, at a high concentration (200 µM), significant necrosis was detected in H460 cells, whereas only minimal necrosis was detected in G361 cells.

Figure 2.  Effect of silymarin on HK-2, H460, and G361 cells. (A–C) Cells were treated with various concentrations of silymarin (0–200 µM) for 24 h and cell viability was measured by MTT assay. Values are means ± SD of three independent experiments, *P < 0.05 versus non-treated control. (E–F) Nuclear morphology of apoptosis and necrosis detected by Hoechst 33342 and propidium iodide assay.

Silymarin protects HK-2 cells from cisplatin-induced cytotoxicity

Cells were pre-incubated with various concentrations of silymarin (25–200 µM) for 1 h and treated with various concentrations of cisplatin (25–100 µM). After 24-h incubation, cell viability and cell death detection were performed. Treatment of the HK-2 cells with cisplatin alone at concentrations of 25, 50, and 100 µM caused a reduction in cell survival by approximately 40, 50, and 60%, respectively. Figure 3A shows that pretreatment of HK-2 cells with silymarin (25–200 µM) significantly prevented the loss of cell viability induced by cisplatin treatment in the concentration range of 25–100 µM. In addition, the results of Hoechst 33342 and PI assays show that silymarin succeeded in decreasing both apoptosis and necrosis induced by cisplatin in HK-2 cells (Figure 3B–3D). These results indicate that silymarin treatment was able to ameliorate cisplatin-induced HK-2 cell damage.

Figure 3.  Protective effect of silymarin on cisplatin-induced HK-2 cell death. (A) Cells were pretreated with various concentrations of silymarin (25–200 µM) for 1 h prior to 24-h cisplatin exposure. Cell viability was measured by MTT assay. Values are means ± SD of three independent experiments, *P < 0.05 versus non-treated control, and ^#P < 0.05 versus cisplatin-treated control. (B–D) Nuclear morphology of apoptosis and necrosis detected by Hoechst 33342 and propidium iodide assay.

Silymarin potentiates cisplatin-induced melanoma cell damages while causing minimal effect on cisplatin-induced lung cancer cell damages

A limitation of using antioxidants for the protection of normal cell toxicities in chemotherapy is that antioxidants frequently attenuated anticancer activities of drugs on tumor cells (Eastman, 1987; Roller & Weller, 1998; Miyajima et al., 1999). We further investigated whether silymarin could reduce efficacy of cisplatin in the induction of cancer cell death. Cells were left untreated or pretreated with various concentrations of silymarin (25–200 µM) for 1 h and treated with various concentrations of cisplatin (25–100 µM). After 24-h incubation, cell viability, apoptosis, and necrosis were determined. Figure 4A shows that silymarin at the concentrations of 100 µM and lower caused no significant change on lung cancer cell viability after cisplatin treatment. As the dose of silymarin was increased up to 200 µM, silymarin dramatically enhanced the cytotoxic effect of cisplatin with ~2-fold reduction as compared with the cisplatin-treated control. In melanoma, treatment with cisplatin alone caused cell survival reduction by approximately 40–50% at the concentration range of 25–100 µM. Addition of silymarin in cisplatin-treated melanoma cells significantly potentiated cisplatin-induced toxicity in a concentration-dependent manner (Figure 4B).

Figure 4.  Effect of silymarin on cisplatin-induced H460 and G361 cell death. (A and B) Cells were pretreated with various concentrations of silymarin (25–200 µM) for 1 h prior to 24-h cisplatin treatment. Cell viability was measured by the MTT assay. Values are means ± SD of three independent experiments, *P < 0.05 versus non-treated control, and ^#P < 0.05 versus cisplatin-treated control. (C–H) Nuclear morphology of apoptosis and necrosis detected by Hoechst 33342 and propidium iodide assay.

The result of Hoechst 33342 and PI staining assays confirmed that in H460 cells, pretreatment of low concentrations silymarin caused no significant change in mode and quantity of cell death in response to cisplatin treatment. Consistently, at 200 µM silymarin, the number of both apoptotic and necrotic cells significantly increased (Figure 4C, 4E, and 4G). Unlike H460 cells, co-treating the G361 cells with silymarin and cisplatin mainly caused an increase in apoptotic cell death. Our results suggest that the addition of silymarin in cisplatin treatment not only protects against human renal cell damage, but also, at least, either does not interfere with the cytotoxic mechanisms of cisplatin or enhances the response of cancer cells to cisplatin-induced cell death.

Discussion

The development of nephrotoxicity during cisplatin treatment has been shown to be a major limitation of efficient therapy (Patrick & Lawrence, 1984; Launay-Vacher et al., 2008; Pabla & Dong, 2008). Cisplatin induces cytotoxicity by the mechanisms involved in DNA-adduct formation and/or intracellular reactive oxygen species (ROS) induction (Reedijk & Lohman, 1985; Miyajima et al., 1997; Wang & Lippard, 2005; Bragado et al., 2007). In cancer cells, DNA-adduct formation seems to be a major mechanism of cisplatin-mediated cell death since the cells have evolved mechanisms such as increase of superoxide dismutase or glutathione levels to protect themselves from intrinsic high oxidative stress resulted from abnormal metabolism (Valko et al., 2007; Gibellini et al., 2010). Although a sufficient number of studies have indicated that cisplatin-induced renal cell damage is mainly due to its ability to generate oxidative stress (Matsushima et al., 1998; Yang et al., 2002; Baek et al., 2003), introducing an antioxidant could be beneficial in preventing renal cell damage during cisplatin therapy (Ajith et al., 2007; Sung et al., 2008). Unfortunately, many antioxidants fail to be further developed for the use in clinical treatment since their antioxidant activities frequently attenuate the cytotoxic mechanisms of cisplatin in cancerous cells (Eastman, 1987; Roller & Weller, 1998; Miyajima et al., 1999). Silymarin has been shown to have antioxidant activity and capability to protect normal cells including liver and renal cells from several death stimuli (Muriel et al., 1992; Sonnenbichler et al., 1999; Mansour et al., 2006). However, whether or not this compound could be able to inhibit cisplatin-induced renal cell death without interfering with the effects of cisplatin on the cancer cells is still largely unknown.

Our results indicate that silymarin selectively protected only human renal tubular HK-2 cells from cisplatin-induced apoptosis without compromising the anticancer activity of cisplatin as demonstrated in lung carcinoma and melanoma cells. The mechanism by which silymarin protects HK-2 cells from cisplatin-induced damage is not fully understood. Our results indicate that treatment with cisplatin resulted in renal cell death, and necrosis seemed to be the main mode of cell death in this condition (Figure 1D). Previous studies reported that hydroxyl radicals could induce lipid peroxidation of the cell membrane, finally resulting in cell necrosis (Gutteridge, 1984; Salahudeen, 1995; Matsushima et al., 1998; Yang et al., 2002). Silymarin has been reported to possess potent scavenging activity against hydroxyl radicals (Mira et al., 1994), and consequently protect lipid peroxidation of the cell membrane (Bosisio et al., 1992; Muriel et al., 1992). These data could be a possible explanation of our finding that treatment with silymarin inhibited cisplatin-induced necrosis in HK-2 cells. Moreover, silymarin has been shown to increase glutathione level in liver, stomach, and intestine of rat (Valenzuela et al., 1989) and superoxide dismutase level in lymphocytes (Fehér et al., 1987). Therefore, silymarin may protect against renal cell damage via the mechanism involving its antioxidant properties against intracellular ROS induced by cisplatin. Some studies have reported that silymarin exerts antitumor activities in some cancer cells (Zi et al., 1998; Sharma et al., 2003; Huang et al., 2005; Deep et al., 2006; Zhong et al., 2006) and we further confirmed that silymarin has a direct anticancer activity against melanoma G361 cells. However, silymarin seemed to have only minimal anticancer activity on non-small-lung cancer H460 cells.

The possible reasons for silymarin selectivity in the protection of renal cells while not interfering with the mode of cisplatin action in cancer cells include the following: (i) silymarin possesses direct anticancer activity; (ii) renal cells were shown to be highly susceptible to oxidative stress-induced cell damage, whereas many cancer cells have been shown to resist ROS-induced cell death (Andreoli, 1991; Nath & Norby, 2000; Galle, 2001; Djamali, 2007), and the main mechanism of cisplatin-induced cancer cell death is through DNA-adduct formation (Reedijk & Lohman, 1985; Wang & Lippard, 2005).

In summary, we reported herein for the first time that silymarin exhibited a strong protective effect against cisplatin-induced human renal cells damage, has no significant effect on cisplatin-induced lung carcinoma H460 cells death, and enhanced cisplatin-induced melanoma G361 cell death. These findings may advocate the necessity of developing silymarin, a considerately safe compound, for its potential use in clinical cancer therapy in combination with anticancer agents.

Acknowledgement

The authors are grateful to Chulalongkorn University Centenary Academic Development project for facility support.

Declaration of interest

The authors declare that they have no conflicts of interest.