Telmisartan-Induced Cytotoxicity via G₂/M Phase Arrest in Renal Cell Carcinoma Cell Lines

Abstract

Renal cell carcinoma (RCC) is the most common type of kidney cancer. Given that stage IV RCC is intractable, there is a need for a novel treatment strategy. We investigated the antitumor effects of telmisartan (TEL) and their underlying mechanisms in RCC, including their impact on apoptosis, Akt/mammalian target of rapamycin (mTOR) pathways, and the cell cycle using two human RCC cell lines: 786-O and Caki-2. Cell viability was detected via fluorescence-based assays. Cells were stained with Hoechst 33342 to observe chromatin condensation, and Western blotting was performed to analyze protein expression. The cell cycle was assessed using flow cytometry. Invasion and migration assays were performed using 24-well chambers. TEL induced cell death in a dose-dependent manner and increased the percentage of cells with high chromatin condensation and Bax/Bcl-2 ratio in both cell lines. TEL-induced cell death was attenuated by neither peroxisome proliferator-activated receptor-γ nor -δ inhibitors. Although TEL elevated c-Jun N-terminal kinase levels and p38 phosphorylation rates in Caki-2 cells, as well as extracellular signal-regulated kinase phosphorylation rates in 786-O cells, their inhibitors did not suppress TEL-induced cell death. TEL decreased Akt phosphorylation in 786-O cells and mTOR phosphorylation in both cell lines, increased the population of cells in the G₂/M phase, and altered G₂/M-related proteins in both cell lines. TEL moderately suppressed cell invasion and migration in 786-O and Caki-2 cells, respectively, and increased cell invasion in Caki-2 cells, suggesting a potential therapeutic role of TEL in RCC.

INTRODUCTION

Kidney cancer accounts for 2.2% of all cancer cases.¹⁾ Renal cell carcinoma (RCC) is the most common type of kidney cancer, and its incidence continues to increase.²⁾ The 5-year survival rate for stage IV RCC is ≤10%, and the disease is often intractable,³⁾ although surgical resection may be effective for early-stage RCC. Advanced RCC tends to be resistant to cytotoxic anticancer agents.⁴⁾ Sunitinib, the most widely prescribed drug for the treatment of metastatic RCC, significantly prolongs overall survival and progression-free survival compared to interferon-alpha. However, the effect of sunitinib has not been sufficient given the overall and progression-free survival times of only 26 and 11 months, respectively.⁵⁾ Recently, molecularly targeted drugs such as vascular endothelial growth factor, its receptor mammalian target of rapamycin (mTOR), and immune checkpoint inhibitors have been introduced in the treatment of RCC; however, their effects have been insufficient,⁴⁾ suggesting a need for a novel strategy for RCC treatment.

Angiotensin II receptor blockers (ARBs) are a type of antihypertensive drug used worldwide, and previous studies have highlighted their cardioprotective and renal protective effects.⁶⁾ ARBs have been reported to be involved in the development and progression of cancer and have been suggested to reduce the risk of cancer, improve progression-free survival, and reduce the risk of recurrence in a variety of cancer types, including kidney cancer.^7,8) Furthermore, ARBs such as telmisartan (TEL), irbesartan (IRB), candesartan, and losartan have shown antitumor effects such as inhibition of cell growth and induction of apoptosis in various cancer cells (e.g., lung,^9,10) prostate,^11,12) colon,¹³⁾ esophageal,^14,15) gastric,¹⁶⁾ ovarian,¹⁷⁾ endometrial,¹⁸⁾ and breast¹⁹⁾ cancers, cholangiocarcinoma,^20,21) hepatocellular carcinoma,²²⁾ melanoma,²³⁾ and osteosarcoma).²⁴⁾ Similarly, TEL has been reported to exert antitumor effects in RCC; however, the underlying mechanism remains to be elucidated.^25–27)

Peroxisome proliferator-activated receptor (PPAR) is a member of the nuclear hormone receptor superfamily and plays an important role in the regulation of glucose and lipid metabolism. In addition, PPAR is involved in cell proliferation, and PPARγ and PPARδ have both been reported to exhibit antitumor properties.²⁸⁾ Additional studies have indicated that PPARγ and PPARδ may be activated by TEL.^29,30)

The present study aimed to investigate the antitumor effects of TEL and the mechanisms underlying these effects in RCC cell lines. We examined the involvement of apoptosis and for the first time investigated PPARγ and PPARδ involvement, the activity of the mitogen-activated protein kinase (MAPK) and Akt/mTOR pathways, and the cell cycle in RCC cells. In addition, we examined whether TEL affects cell invasion and cell migration in RCC cells.

MATERIALS AND METHODS

Chemicals and Antibodies

TEL and IRB were obtained from Wako Pure Chemical Corporation (Osaka, Japan) and LKT Laboratories (St. Paul, MN, U.S.A.), respectively, and dissolved in dimethyl sulfoxide (Nacalai Tesque, Kyoto, Japan) immediately before use. In addition, GW9662 (PPARγ inhibitor), SP600125 (c-Jun N-terminal kinase (JNK) inhibitor), and SB202190 (p38 inhibitor) were provided by Sigma (St. Louis, MO, U.S.A.). GSK3787 (PPARδ inhibitor) and U0126 (extracellular signal-regulated kinase (ERK) inhibitor) were received from Merck (Darmstadt, Germany). Rabbit monoclonal anti-human antibodies against phospho-stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185, Product No. 4668), phospho-p38 MAPK (Thr180/Tyr182, D3F9, Product No. 4511), phospho-ERK1/2 (Thr202/Tyr204; D13.14.4E, Product No. 4370), phospho-Akt (Ser473, D9E, Product No. 4060), phospho-mTOR (Ser2448, D9C2, product No. 5536), and β-actin (13E5, Product No. 4970), as well as mouse monoclonal anti-human antibodies against cyclin A2 (BF683, product No. 9869) and cdc2 (POH1, Product No. 9868) and rabbit poly-clonal anti-human antibodies against cyclin B1 (Product No. 9869), were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.).

Cells and Cell Culture

In this study, 786-O and Caki-2 cells were used as human RCC cell lines and were received from Summit Pharmaceuticals International (Tokyo, Japan). In addition, 786-O and Caki-2 cell lines were cultured in RPMI1640 (Nacalai Tesque) and McCoy’s 5A Medium (Life Technologies, Carlsbad, CA, U.S.A.), respectively, including 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, U.S.A.) and 50 U/mL penicillin-50 µg/mL streptomycin (Nacalai Tesque). Cells were sub-cultured every 3 or 4 d and incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO₂.

Cell Viability

Cells (5.0 × 10³/well) were cultured in 96-well plates (Asahi Glass, Tokyo, Japan) for 24 h and then exposed to TEL with or without other chemicals, using an FBS-free medium. After 24 h, cell viability was measured as previously described.^31,32) The IC₅₀ were calculated according to the sigmoid inhibitory effect model. In the co-treatment experiments, TEL was used at approximately the IC₅₀ value of each cell line.

Detection of Chromatin Condensation via Fluorescence Microscopy

Chromatin condensation was detected as previously described.^31,33) Briefly, cells (3.3 × 10⁵) were plated in 60-mm dishes for 24 h, following which 786-O and Caki-2 cells were treated with 100 and 150 µM TEL, respectively. After 24 h, the cells were stained with Hoechst 33342 (Dojindo Molecular Technologies, Kumamoto, Japan) and observed under UV excitation. Cells were photographed at 20-fold magnification.

Western Blot Analysis

To examine the involvement of apoptosis, the MAPK signaling pathway, and the Akt/mTOR pathway, cells (8.7 × 10⁵) were plated in 100-mm dishes 24 h before treatment, following which 786-O and Caki-2 cells were treated with 120 and 200 µM TEL, respectively, for 24 h. To examine the involvement of the cell cycle, cells (5.2 × 10⁵) were plated in 100-mm dishes 24 h before treatment, following which 786-O and Caki-2 cells were treated with 80 and 70 µM TEL, respectively, for 48 h. Western blot analysis was performed as previously described.³³⁾ Briefly, a total of 10 µg of protein (per lane) was loaded onto 6.5 or 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and electrophoresed. The protein samples were then transferred to a polyvinylidene difluoride membrane (GE Healthcare, U.S.A.) and blocked for 1 h. The blocked membranes were incubated with primary antibodies (1 : 10000 dilution) at 4 °C overnight. The membranes were then incubated with the secondary antibody (1 : 25000 dilution) for 1 h at 20 °C, and chemiluminescence was detected.

Cell Cycle Analysis

Cells (8.7 × 10⁵) were plated in 100-mm dishes for 24 h, following which 786-O and Caki-2 cells were treated with 80 and 70 µM TEL, respectively, for 48 h. The cell cycle was examined as previously described.³¹⁾

Invasion and Migration Assay

FBS was used as a chemoattractant; 10% FBS-containing medium was added into the lower chambers, and the membranes were placed. Cells were seeded on the membranes at a density of 2.5 × 10⁴ (786-O) or 5 × 10⁴ (Caki-2) cells/well with FBS-free medium. After being cultured with or without TEL (maximum concentration without cytotoxicity: 10 and 70 µM in 786-O and Caki-2 cells, respectively) for 24 h, invasion and migration assays were performed as previously described.³³⁾

Statistical Analysis

Estimates were compared between groups using Student’s or Welch’s t-test and one-way ANOVA, followed by Dunnett’s post hoc test for multiple comparisons. p-Values <0.05 were considered statistically significant. Data were expressed as the mean ± standard deviation (S.D.). The Student’s and Welch’s t-tests were performed using Microsoft Excel 97-2003; the ANOVA and Dunnett’s post hoc analyses were performed using GraphPad InStat Demo (GraphPad Software, CA, U.S.A.).

RESULTS

Effects of TEL and IRB on Cell Viability

TEL exhibited cytotoxicity in a dose-dependent manner, with IC₅₀ values of 110 ± 11 and 190 ± 18 µM in 786-O and Caki-2 cells, respectively. Meanwhile, IRB did not show cytotoxicity in the concentration range examined (Fig. 1).

Fig. 1. Cytotoxic Effects of TEL and IRB in Two RCC Cell Lines (786-O and Caki-2)

Cell viability was assessed using a fluorescence-based assay. Data are represented as mean ± S.D. (n = 4). Statistical significance was assessed using Dunnett’s test (control vs. each concentration of TEL or IRB). ** p < 0.01 vs. control (for TEL). ^† p < 0.05 and ^†† p < 0.01 vs. control (for IRB). TEL, telmisartan; IRB, irbesartan; RCC, renal cell carcinoma.

Involvement of Apoptosis in TEL-Induced Cell Death

To investigate the effects of TEL on nuclear morphology, we observed the chromatin condensation level, which is an indicator of apoptosis. TEL significantly elevated the percentage of cells with chromatin condensation, from 5 to 13 and from 5 to 37% in 786-O (p < 0.01) and Caki-2 (p < 0.05) cells, respectively (Fig. 2A). We analyzed levels of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2. TEL upregulated Bax expression in 786-O cells and downregulated Bcl-2 expression in 786-O and Caki-2 cells. TEL also increased the Bax/Bcl-2 expression ratio (Fig. 2B). These findings suggest that TEL induces apoptosis via the mitochondrial pathway.

Fig. 2. Involvement of Apoptosis in TEL-Induced Cell Death

(A) Induction of chromatin condensation by TEL. Representative fluorescence microscopy images of cells stained with Hoechst 33342 and the percentage of cells with chromatin condensation. Data are represented as mean ± S.D. from three independent experiments. Statistical significance was assessed using a t-test. (B) Bax and Bcl-2 protein expression. Bax and Bcl-2 expression levels were corrected using β-actin and analyzed from three independent preparations. * p < 0.05 and ** p < 0.01 vs. control (t-test). TEL, telmisartan. (Color figure can be accessed in the online version.)

Involvement of PPARγ and PPARδ in TEL-Induced Cell Death

To evaluate whether PPARγ or PPARδ activation by TEL is involved in cytotoxicity, we treated RCC cells with TEL and GW9662 (PPARγ antagonist) or GSK3787 (PPARδ antagonist). Neither GW9662 nor GSK3787 attenuated TEL-induced cell death (Fig. 3), suggesting that TEL cytotoxicity is independent of PPARγ and PPARδ stimulation.

Fig. 3. The Effect of a PPARγ Inhibitor (GW9662) and a PPARδ Inhibitor (GSK3787) on TEL-Induced Cell Death

Cell viability was assessed using a fluorescence-based assay (n = 4). Statistical significance was assessed using Dunnett’s test (TEL vs. TEL + inhibitors). TEL, telmisartan.

Involvement of MAPK Signaling Pathway in TEL-Induced Cell Death

To elucidate the involvement of the MAPK pathway in TEL cytotoxicity, expression levels of the MAPK subfamily proteins (JNK, p38, and ERK) were examined. Although TEL did not affect JNK or p38 phosphorylation in 786-O cells, it did increase JNK and p38 phosphorylation in Caki-2 cells (Fig. 4A). Next, we examined the effects of the JNK inhibitor SP600125 and the p38 inhibitor SB202190 on TEL cytotoxicity in Caki-2 cells. Neither SP600125 nor SB202190 attenuated TEL cytotoxicity (Figs. 4B, C). TEL increased the phosphorylation levels of ERK in 786-O cells (Fig. 4A). We also treated cells with TEL and the ERK inhibitor U0126. The combination of U0126 and TEL did not recover cell viability in 786-O cells (Fig. 4D). These findings suggest that the involvement of the MAPK pathway in TEL cytotoxicity is limited.

Fig. 4. Involvement of the MAPK Signaling Pathway in TEL-Induced Cell Death

(A) JNK, p38, and ERK expression in cells treated with TEL. Typical bands showing phosphorylation of JNK, p38, and ERK were observed on the western blots. β-Actin was used as a loading control. Data are represented as the mean ± S.D. from three independent experiments. Statistical significance was assessed using a t-test. * p < 0.05 vs. control. (B) Effect of a JNK inhibitor (SP600125) on TEL-induced cell death in Caki-2 cells. (C) Effect of a p38 inhibitor (SB202190) on TEL-induced cell death in Caki-2 cells. (D) Effect of an ERK inhibitor (U0126) on TEL-induced cell death in 786-O cells. Cell viability was assessed using a fluorescence-based assay (n = 4). Statistical significance was assessed using Dunnett’s test (TEL vs. TEL + inhibitors). MAPK: mitogen-activated protein kinase; TEL, telmisartan; JNK: c-Jun N-terminal kinase; ERK: extracellular signal-regulated kinase.

Involvement of Akt/mTOR Pathway in TEL-Induced Cell Death

To investigate whether the Akt/mTOR pathway is involved in TEL cytotoxicity, we examined the expression levels of Akt and mTOR proteins. TEL decreased the phosphorylation of mTOR in 786-O and Caki-2 cells. A decrease in Akt phosphorylation was observed in 786-O cells but not in Caki-2 cells (Fig. 5). These findings suggest that TEL exerts cytotoxicity by downregulating the activity of the Akt/mTOR pathway in 786-O cells.

Fig. 5. Akt and mTOR Expressions in Cells Treated with TEL

Typical bands showing phosphorylation of Akt and mTOR were observed on the western blots. β-Actin was used as a loading control. Data are represented as the mean ± S.D. from three independent experiments. Statistical significance was assessed using a t-test. * p < 0.05 and ** p < 0.01 vs. control. mTOR, mechanistic target of rapamycin; TEL, telmisartan.

Effects of TEL on the Cell Cycle

Figure 6 shows the effects of TEL on the cell cycle. TEL increased the proportion of cells in the G₂/M phase from 28.1 to 36.8 and from 16.6 to 27.7% in 786-O and Caki-2 cells, respectively. Furthermore, in both 786-O and Caki-2 cells, upregulated expression of cyclin B1 and downregulated expression of cdc2 were observed following TEL treatment (Fig. 7). These findings suggest that TEL induces G₂/M phase arrest.

Fig. 6. Flow Cytometric Analysis of Cells Treated with TEL

Cell number was assessed using flow cytometry. Statistical significance was assessed using a t-test. * p < 0.05 and ** p < 0.01 vs. control. TEL, telmisartan.

Fig. 7. Levels of G₂/M Phase-related Protein Expression in Cells Treated with TEL

Typical bands showing cyclin A2, cyclin B1, and cdc2 were observed on the western blots. β-Actin was used as a loading control. Data are represented as the mean ± S.D. from three independent experiments. Statistical significance was assessed using a t-test. * p < 0.05 and ** p < 0.01 vs. control. TEL, telmisartan.

Effects of TEL on Cell Invasion and Migration

In 786-O cells, cell migration rates were not affected by exposure to TEL; however, cell invasion rates decreased after exposure to TEL (p < 0.01). Meanwhile, in Caki-2 cells, TEL increased cell invasion (p < 0.01) and decreased migration (p < 0.01) rates (Fig. 8).

Fig. 8. Effects of TEL on Cell Invasion and Cell Migration

(A) Effect of TEL on cell invasion. The cell invasion assay was performed using 24-well BD BioCoat™ Matrigel^® invasion chambers with 8.0-µm polycarbonate membrane filters. (B) Effect of TEL on cell migration. The cell migration assay was conducted in the same manner as the cell invasion assay, except for the use of non-coated chambers. Data are represented as the mean + S.D. from three or four independent preparations. ** p < 0.01 vs. control (t-test). TEL, telmisartan.

DISCUSSION

We investigated the antitumor effects of TEL and their underlying mechanisms in RCC cell lines. TEL treatment resulted in cytotoxicity with apoptosis via the mitochondrial pathway as well as PPARγ-, PPARδ-, and MAPK-independent downregulation of mTOR and cell cycle arrest.

TEL has been reported to exert cytotoxicity in RCC cells,^25–27) suggesting that TEL can aid in the treatment of RCC. One meta-analysis suggested that ARBs can increase the risk of cancer; however, another meta-analysis did not confirm this finding.³⁴⁾ In addition, the relative binding affinity of angiotensin II to the type 1 receptor is reported to be IRB > TEL³⁵⁾; however, in our results, IRB did not show antitumor effects. Therefore, different types of ARBs may have different effects on cancer.

To investigate the mechanisms underlying the antitumor effects of TEL, we first examined its impact on apoptosis (Fig. 2). Increased Bax/Bcl-2 ratio affects mitochondrial function and induces apoptosis.³⁶⁾ Our findings indicated that TEL induced apoptosis at least partially via the mitochondrial pathway. This finding is consistent with those of previous studies.^25–27)

To elucidate the involvement of PPARγ or PPARδ, we examined the combined effects of GW9662 or GSK3787 (Fig. 3). However, our analyses suggested that TEL cytotoxicity was independent of PPARγ and PPARδ in the present cell lines. TEL may exhibit antitumor effects via PPARγ and PPARδ receptors in lung³⁷⁾ and prostate cancer cells.¹²⁾ Given this discrepancy, the involvement of PPAR in TEL cytotoxicity may differ among cancer cell types. This study is the first to show that TEL cytotoxicity is independent of PPARγ and PPARδ activity in RCC cell lines.

We investigated the involvement of the MAPK signaling pathway, which regulates cell survival, growth, and proliferation, and which has been reported to be affected by TEL.^38–40) We examined TEL-related changes in the expression of three classical MAPK proteins (ERK, JNK, and p38) (Fig. 4A). The phosphorylated form of the three classical MAPK proteins is the active form. To examine the involvement of the MAPK signaling pathway in TEL cytotoxicity, we co-exposed RCC cells to TEL and their inhibitors (Figs. 4B–D). Our results suggest that the associated signaling was not involved in TEL cytotoxicity in RCC cells; nevertheless, TEL may activate JNK, p38, and ERK proteins.

We evaluated the involvement of the Akt/mTOR pathway, which is associated with cell proliferation and anti-apoptotic mechanisms. Previous researches have indicated that TEL may regulate the Akt/mTOR pathway.^10,14,22,24) The phosphorylated form of Akt or mTOR protein is the active form. In the present study, TEL inhibited the phosphorylation of Akt and mTOR in 786-O cells (Fig. 5), suggesting that it induced cell death via Akt/mTOR suppression. In Caki-2 cells, phosphorylation of mTOR was downregulated by TEL; in contrast, phosphorylation of Akt was not (Fig. 5). These findings suggest that TEL exerts cytotoxicity by inactivating mTOR through another pathway that does not involve Akt. A previous study has shown that Akt inhibitors may lead to dose-dependent cytotoxicity.³²⁾ In addition, mTOR inhibitors have been used in the treatment of RCC. Although the detailed mechanism underlying the inactivation of the Akt/mTOR pathway requires further investigation, TEL may contribute to the treatment of RCC via this pathway.

Some studies have reported that TEL arrests the cell cycle in various cancer cells.^{12,14–16,21,22)} Thus, we investigated the effect of TEL on the cell cycle in RCC cells. TEL increased the population of cells in the G₂/M phase (Fig. 6). Cyclin A2 and cyclin B1 form a complex with cdc2 to promote the transition to the G₂/M phase,⁴¹⁾ and TEL affected these proteins (Fig. 7). The present findings suggest that TEL reduces the expression of cyclin A2/B1-cdc2 complexes, triggering G₂/M phase arrest. TEL has been shown to induce S-phase arrest in esophageal squamous cell carcinoma¹⁵⁾ and G₀/G₁ phase arrest in esophageal adenocarcinoma,¹⁴⁾ gastric cancer,¹⁶⁾ cholangiocarcinoma,²¹⁾ and hepatocellular carcinoma cells.²²⁾ TEL-induced cell cycle arrest may vary among cancer cell types. Further studies are required to elucidate the mechanism underlying TEL-induced G₂/M phase arrest.

As ARBs have been reported to inhibit metastasis,⁴²⁾ we investigated the effect of TEL on cell migration and cell invasion. The suppression of TEL migration and invasion was minimal overall (Fig. 8), indicating that TEL may be ineffective at suppressing metastasis of RCC cells.

The difference in results between 786-O and Caki-2 cells may be due to differences in cell characteristics, such as p53 mutations in 786-O cells and loss-of-function mutations in von Hippel-Lindau tumor suppressor protein in Caki-2 cells.

de Araújo Júnior et al.²⁵⁾ and Funao et al.²⁶⁾ reported that TEL induced apoptosis in RCC, which is consistent with our results. This is the first study to analyze the involvement of PPARγ, PPARδ, MAPK, Akt/mTOR, and the cell cycle in TEL-induced cell death in RCC cell lines.

In conclusion, the present results indicated that TEL exhibited cytotoxicity in two RCC cell lines and induced cell death via a mitochondria-mediated apoptosis pathway without PPARγ and PPARδ dependency. TEL cytotoxicity may be independent of the MAPK signaling pathway and may be caused by the inactivation of mTOR. In addition, TEL may induce G₂/M phase arrest. Therefore, although the detailed mechanisms underlying mTOR downregulation and cell cycle arrest remain to be elucidated, the present findings indicate that TEL may be effective in the treatment of RCC. The present findings may contribute to identifying candidates for drug repositioning or development in the treatment of kidney cancer. As we only examined the effects of TEL in vitro, further studies are required to investigate the detailed mechanisms of the antitumor effects of TEL, especially in vivo.

Conflict of Interest

The authors declare no conflict of interest.

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