Diseases of the Esophagus (2015) ••, ••–•• DOI: 10.1111/dote.12370
Original article
Effects of bisphosphonates on human esophageal squamous cell carcinoma cell survival T. Minegaki, S. Fukushima, C. Morioka, H. Takanashi, J. Uno, S. Tsuji, S. Yamamoto, A. Watanabe, M. Tsujimoto, K. Nishiguchi Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Kyoto Pharmaceutical University, Kyoto, Japan
Summary. Esophageal squamous cell carcinoma (ESCC) is one of the most malignant cancers in Japan. Anticancer chemotherapy has been useful for ESCC treatment. However, therapeutic options are limited. Recently, bisphosphonates (BPs), which are osteoporosis drugs, have shown anticancer effects in several cancer cell lines, but the effects against ESCC cell lines are unknown. In this study, we examined the cytotoxic effects of BPs and their mechanisms of cytotoxicity in human ESCC cell lines. A first-generation BP (etidronate), two second-generation BPs (alendronate and pamidronate), and two third-generation BPs (risedronate and zoledronate) were used in this study. All BPs, except etidronate, were cytotoxic, as indicated by increased caspase-3/7 activity and numbers of Annexin-fluorescein isothiocyanate positive cells in ESCC cell lines. From cell cycle analysis, G0/G1-phase arrest was observed upon treatment with second- and third-generation BPs. In addition, Cyclin D1 protein expression levels were decreased by second- and third-generation BP treatment. Although squalene and trans, trans-farnesol minimally affected BP cytotoxicity, treatment with geranylgeraniol inhibited BP cytotoxicity almost completely. We concluded that second- and third-generation BPs are cytotoxic to ESCC cell lines as they induce apoptosis and inhibit the cell cycle through mevalonate pathway inhibition. Therefore, BP treatment may be a beneficial therapy in ESCC patients. KEY WORDS: bisphosphonate, esophageal cancer, mevalonate pathway.
INTRODUCTION In 2008, an estimated 482 300 new cases and 406 800 deaths attributed to esophageal cancer was reported worldwide.1 In Japan, over 10 000 patients died from esophageal cancer and the 5-year survival rate of this disease was about 40%.2 Esophageal squamous cell carcinoma (ESCC) is a major histological type of esophageal cancer in East Asian countries, including Japan. Surgical resection has been a good treatment option for early-stage ESCC. In unresectable ESCC, Address correspondence to: Mr Tetsuya Minegaki, Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan. Email: [email protected] Author contributions: TM and KN conceived and designed the study. SF, CM, HT, JU, ST, SY, and AW performed the experiments and statistical analysis. TM, MT, and KN performed data management and prepared the manuscript. Conflicts of interest: The authors have no conflicts of interest to disclose. © 2015 International Society for Diseases of the Esophagus
cisplatin and 5-fluorouracil-based chemotherapy is the first-line treatment. However, no standard second-line chemotherapy has been established.3 Therefore, to improve the therapeutic outcomes of ESCC patients, novel treatments are required. Bisphosphonates (BPs) have been widely used to treat osteoporosis, Paget’s disease, and tumor-related hypercalcemia. BPs can be divided into two groups by structure, based on the presence of the nitrogen atom. BPs lacking the nitrogen atom, namely first-generation BPs, are metabolized into nonhydrolyzable adenosine triphosphate analogs that induce osteoclast apoptosis and subsequently prevent bone resorption. On the other hand, nitrogencontaining BPs (second- or third-generation BPs, or N-BPs) more potently prevent bone resorption than first-generation BPs by inhibiting farnesyl diphosphate (FPP) synthase, a key enzyme in the mevalonate pathway, and decreasing synthesis of the isoprenoids FPP or geranylgeranyl diphosphate (GGPP).4 These isoprenoids engage in cellular signal 1
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transduction, leading to cell survival, proliferation, differentiation, and motility. Recently, many studies have shown that BPs, especially second- and third-generation BPs, can inhibit the growth of some cancer cell lines, including breast,5–7 lung,8,9 colorectal,10 renal,11 and prostate12 cancer cells, by preventing the prenylation of Ras and Rho in in vitro and in vivo assays. However, no studies have shown that BPs have cytotoxic effects against ESCC cell lines and the mechanism by which they exert their toxic effects. Therefore, if the efficacy of BPs against ESCC cell lines can be shown, there could be more therapeutic options for ESCC patients. The purpose of this study was to evaluate the cytotoxic effects of first-, second-, and thirdgeneration BPs and to clarify the mechanisms of BP cytotoxicity in ESCC cell lines in vitro.
METHODS Chemicals Alendronate (ALE) monosodium trihydrate, etidronate (ETI) disodium, and risedronate (RIS) sodium hydrate were purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Pamidronic acid (PAM) sodium salt hydrate and zoledronic acid (ZOL) disodium salt tetrahydrate were purchased from Toronto Research Chemicals Inc. (Toronto, ON, Canada). Trans, trans-farnesol (FOH) and geranylgeraniol (GGOH) were obtained from SigmaAldrich Chemical Co. (St. Louis, MO, USA). Squalene (SQ) was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan).
The culture medium was changed to medium containing BP at various concentrations. After 72 hours, the medium was exchanged for 110 μL of medium containing CellQuanti-Blue solution (10 μL CellQuantiBlue solution and 100 μL culture media) and 4 hours later, the fluorescence was quantified at an excitation wavelength of 535 nm and an emission wavelength of 590 nm using a microplate reader (GENios, Tecan, Seestrasse, Switzerland). The fluorescence intensity of the CellQuanti-Blue solution was linear between 1 and 5 hours of incubation. The effects of SQ, FOH, or GGOH on BP sensitivity were also evaluated using the CellQuanti-Blue kit. In this study, we used FOH and GGOH instead of FPP and GGPP, respectively. FOH and GGOH are cell permeable, while FPP and GGPP are not, and they are converted to FPP and GGPP, respectively.14 Cells were cultured for 24 hours and the culture medium was changed to media containing BP at various concentrations with or without SQ (20 μM), FOH (10 μM), or GGOH (10 μM). After incubation for 72 hours, the medium was replaced with a medium containing CellQuantiBlue solution and fluorescence was measured. The 50% growth inhibitory concentration (IC50) was calculated according to the sigmoid inhibitory effect model (1) using the non-linear least-squares fitting method (Solver, Microsoft® Excel, Microsoft Co., Redmond, WA, USA).
E = E max ×
1−Cγ C γ + IC50γ
(1)
E and Emax represent the surviving fraction (% of control) and the maximum, respectively. C and γ are the BP concentration in the culture medium and the sigmoidicity factor, respectively.15
Cell culture The human ESCC cell lines KYSE30, KYSE150, and KYSE170 cells were obtained from Health Science Research Resources Bank (Osaka, Japan).13 These cell lines were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY, USA) supplemented with 100 U/mL penicillin G and 100 μg/mL streptomycin sulfate (Nacalai Tesque, Kyoto, Japan) and 10% fetal bovine serum (lot. no. AWD12933, Thermo Fisher Scientific, Waltham, MA, USA). Cells (2 × 106 cells) were seeded on 100-mm culture dishes, grown in a 5% CO2 humidified atmosphere at 37°C, and subcultured two times per week. Drug sensitivity assay Drug sensitivity was measured using the CellQuantiBlue Cell Viability Assay Kit (Bio Assay Systems, Hayward, CA, USA) according to the manufacturer’s instructions. In brief, cells (5 × 103/well) were seeded in 96-well culture plates and cultured for 24 h.
Caspase-3/7 activity The caspase-3/7 activities of KYSE150 cells treated with BPs were determined using the Caspase-Glo 3/7 Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, KYSE150 cells (1 × 105/well) were seeded in 96-well culture plates and incubated for 24 hours at 5% CO2 and 37°C. The culture medium was exchanged for medium containing ALE (50 μM), ETI (1 mM), PAM (50 μM), RIS (50 μM), or ZOL (50 μM) and cells were cultured for 24 hours. Caspase-Glo reagent (100 μL) was added to each well and 2 hours later, luminescence was measured using a microplate reader (GENios). Annexin V assay KYSE150 cells were seeded at a density of 1.5 × 105 cells/well into 6-well culture plates and cultured for 24 hours and then treated with ALE (50 μM), © 2015 International Society for Diseases of the Esophagus
Bisphosphonate in esophageal cancer cell
ETI (1 mM), PAM (50 μM), RIS (50 μM), or ZOL (50 μM) for 48 hours. Then, floating and adherent cells were harvested and labeled with fluorescein isothiocyanate (FITC)-conjugated Annexin V (BioLegend, San Diego, CA, USA) and propidium iodide (PI, 50 μg/mL, Wako Pure Chemical, Osaka, Japan) for early and late apoptosis detection. FITC and PI fluorescence signals were detected using the FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed using CELLQuest Ver. 3.3 (Becton Dickinson) software. At least 10 000 cells were used in the analysis. Cell cycle analysis KYSE150 cells (3 × 105 cells/60 mm dish) were seeded in 60-mm culture dishes. After 24-hour culture, cells were treated with ALE (50 μM), ETI (1 mM), PAM (50 μM), RIS (50 μM), or ZOL (50 μM) for 48 hours and then fixed with 70% ethanol at −30°C. Fixed cells were stained with PI/RNase buffer (BD Pharmingen, San Diego, CA, USA) at room temperature for 15 minutes. PI fluorescence was measured by FACSCalibur (Becton Dickinson) and analyzed using CELLQuest Ver. 3.3 (Becton Dickinson) software. At least 20 000 cells were analyzed for each sample.
groups were performed with Student’s t-test and repeated one-way analysis of variance followed by post hoc Tukey’s honest significant difference tests. A P value of less than 0.05 (two-tailed) was considered statistically significant. RESULTS Cytotoxic effects of BPs on ESCC cell lines ETI, a first-generation BP, did not show any cytotoxic effects on the three ESCC cell lines. Concentration-dependent cytotoxicity was seen with ALE, PAM, RIS, and ZOL, which are second- and third-generation BPs, in ESCC cell lines (Fig. 1). BP cytotoxicity varied and the IC50 for ZOL was the lowest among those for the five BPs (Table 1). In addition, BP cytotoxic effects differed among the cell lines and KYSE150 was the most sensitive to BPs among the three ESCC cell lines. Apoptosis induction in KYSE150 cells In KYSE150 cells, caspase-3/7 activity was significantly potentiated by all investigated BPs except ETI.
Western blot analysis KYSE150 cells treated with BPs were harvested and protein was extracted with CelLytic-M (SigmaAldrich) according to the manufacturer’s instructions. The protein concentration was determined according to the Lowry method16 and 10 μg of protein was loaded in each lane to detect the expression levels of Cyclin D1 and β-actin and electrophoresed using 10% sodium dodecyl sulfatepolyacrylamide gel according to the Laemmli method.17 Then, protein was transferred to a polyvinylidene difluoride (PVDF) membrane (ClearTrans® SP PVDF membrane, Wako) and the membrane was blocked with 1% skim milk (Wako) in phosphate buffered saline containing 0.1% Tween 20. The membranes were incubated at 4°C overnight with anti-Cyclin D1 antibody (1 : 2000, Cell Signaling Technology Japan, Tokyo, Japan) and anti-β-actin antibody (1 : 1000, Wako) and then membranes were incubated with horseradish peroxidase (HRP)conjugated anti-mouse IgG antibody (GE Healthcare Japan, Tokyo, Japan) for 1 hour at room temperature. The secondary antibody HRP activity was detected with the VersaDoc 5000 MP imaging system (Bio-Rad, Hercules, CA, USA) using Immuno Star® LD reagent (Wako). Statistical analyses Data were shown as the mean ± standard deviation. Comparisons between two and among three or more © 2015 International Society for Diseases of the Esophagus
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Fig. 1 Bisphosphonate-mediated inhibitory effects on cell proliferation in esophageal squamous cell carcinoma cell lines. Cells were treated with culture media containing various concentrations of alendronate (ALE), etidronate (ETI), pamidronate (PAM), risedronate (RIS), or zoledronate (ZOL) for 72 hours. Cell viability was determined by CellQuanti-Blue assay. Data are presented as the mean ± standard deviation (n = 4).
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Table 1 IC50 values (μM) for bisphosphonates in esophageal squamous cell carcinoma cell lines IC50 value (μM) Bisphosphonates
KYSE30
KYSE150
KYSE170
Alendronate (μM) Etidronate (μM) Pamidronate (μM) Risedronate (μM) Zoledronate (μM)
5.30 ± 1.62 NC 5.97 ± 1.52 163 ± 16.4 2.98 ± 0.615
1.54 ± 0.0613 NC 3.33 ± 0.636 0.461 ± 0.178 0.364 ± 0.0499
8.36 ± 1.15 NC 35.0 ± 4.83 234 ± 38.5 6.15 ± 1.82
Mean ± standard deviation (n = 4). NC, not calculated. IC50, 50% growth inhibitory concentration.
Although the numbers of Annexin V-positive cells did not increase with ETI and RIS treatment, the other second- and third-generation BPs increased the numbers of apoptotic cells after 48 hours of treatment (Fig. 2). After 72 hours of treatment, RIS significantly increased the number of Annexin V-positive KYSE150 cells (Fig. 2B inset). Effects of BPs on KYSE150 cell cycle distribution The second-generation BPs, namely, ALE and PAM, significantly increased the numbers of cells in G0/G1 phase and decreased that of cells in G2/M phase after 24-hour treatment. After 48-hour treatment with PAM, RIS, or ZOL, the numbers of cells in G0/G1 phase increased and those in S and G2/M phase decreased. ALE slightly increased the number of cells in G0/G1 phase and decreased that of cells in G2/M phase with 48-hour treatment. ETI did not affect KYSE150 cell cycle with either 24 or 48 hours of treatment (Fig. 3). Effects of BPs on Cyclin D1 protein expression Cyclin D1 protein expression levels significantly decreased when KYSE150 cells were treated with ALE, PAM, or ZOL. ETI and RIS did not affect Cyclin D1 protein levels (Fig. 4). Effects of FOH, GGOH, or SQ on the BP sensitivity of KYSE150 cells SQ did not affect BP cytotoxicity, but FOH slightly decreased the cytotoxicity of PAM and RIS. GGOH, a precursor of GGPP, attenuated the cytotoxic effects of ALE and PAM and almost completely blocked the cytotoxic effects of RIS and ZOL, which are thirdgeneration BPs (Fig. 5).
DISCUSSION In this study, we showed the N-BPs (ALE, PAM, RIS, and ZOL) exhibited concentration-dependent
Fig. 2 Evaluation of apoptosis after bisphosphonate treatment in KYSE150 cells. (A) Cells were incubated with alendronate (ALE, 50 μM), etidronate (ETI, 1 mM), pamidronate (PAM, 50 μM), risedronate (RIS, 50 μM), or zoledronate (ZOL, 50 μM). After 24 hours, caspase-3/7 activities were measured by Caspase-Glo 3/7 assay kit. (B) Cells were treated with ALE (50 μM), ETI (1 mM), PAM (50 μM), RIS (50 μM), or ZOL (50 μM) for 48 hours, or RIS (50 μM) for 72 hours (inset). Then, floating and adherent cells were harvested and stained with Annexin V-fluorescein isothiocyanate and propidium iodide (PI). Apoptotic cells were detected using flow cytometry. Data are presented as the mean ± standard deviation (n = 3). Significant differences between mean values were determined by Student’s t-test (B inset, ††P < 0.01) or analysis of variance followed by the Tukey’s honest significant difference test (A and B, **P < 0.01 compared with control).
cytotoxic effects in the KYSE30, KYSE150, or KYSE170 ESCC cell lines (Fig. 1). In addition, BP treatment induced apoptosis in KYSE150 cells (Fig. 2), the most sensitive cell line to N-BPs (Fig. 1 and Table 1). However, the first-generation BP, ETI, did not exhibit cytotoxic effects or induce apoptosis. Therefore, these data suggest that ESCC cell lines, as well as cell lines originating from other kinds of cancer,5–12 can be killed by second- and thirdgeneration BPs, but not first-generation BPs. From the data of all three ESCC cell lines, ZOL was the most potently cytotoxic among these five BPs (Table 1). Verdijk et al.6 and Senaratne et al.7 showed that ZOL was the most potent BP that inhibited cell growth in breast cancer cell lines. These results and reports suggest that ZOL is the most cytotoxic BP in cancer cell lines. © 2015 International Society for Diseases of the Esophagus
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Fig. 4 Cyclin D1 protein expression levels detected by Western blot after bisphosphonate treatment in KYSE150 cells. Cells were treated with alendronate (ALE, 50 μM), etidronate (ETI, 1 mM), pamidronate (PAM, 50 μM), risedronate (RIS, 50 μM), or zoledronate (ZOL, 50 μM) for 24 hours. Then, total protein was extracted from the cell and Western blots were performed. β-actin was used as an internal standard protein. The data are presented as mean ± standard deviation (n = 4). A representative blot from four independent experiments is shown. Significant differences between the mean values were determined by analysis of variance followed by the Tukey’s honest significant difference test (*P < 0.05, **P < 0.01 compared with control). Fig. 3 Cell cycle distribution after bisphosphonate treatment in KYSE150 cells. Cells were treated with alendronate (ALE, 50 μM), etidronate (ETI, 1 mM), pamidronate (PAM, 50 μM), risedronate (RIS, 50 μM), or zoledronate (ZOL, 50 μM) for 24 or 48 hours. Then, cells were fixed with 70% ethanol and stained with propidium iodide. DNA content was detected by flow cytometry. The data are presented as the mean ± standard deviation (n = 3–4). Significant differences between the mean values were determined by analysis of variance followed by the Tukey’s honest significant difference test (**P < 0.01 compared with control).
ALE, PAM, RIS, and ZOL potentiated caspase 3/7 activities (Fig. 2A) and ALE, PAM, and ZOL increased the numbers of Annexin V-positive KYSE150 cells (Fig. 2B). Some studies have shown that N-BPs have cytotoxic effects by inducing apoptosis in several cancer cell lines.5,10,18,19 However, in cholangiocarcinoma cells, ZOL inhibited cell growth without inducing apoptosis.20 Although the mechanism of N-BP cytotoxicity is thought to be dependent on cell type, our results indicated that the cytotoxic mechanism in ESCC cell lines is induction of apoptosis. Treatment with second- and third-generation BPs increased the numbers of cells in G0/G1 phase (Fig. 3) and decreased the protein expression of Cyclin D1, a cell cycle regulator (Fig. 4). In nonsmall-cell lung cancer cell lines, ZOL induces G1 phase cell cycle arrest.9 However, some reports have shown that BPs such as ZOL induce S-phase arrest in several cancer cell lines.18,19,21,22 Iguchi et al.19 reported that ZOL induced S-phase arrest accompanied by DNA damage and activation of the ataxiatelangiectasia mutated kinase and check-point kinase © 2015 International Society for Diseases of the Esophagus
1 pathway in osteosarcoma cells. Ohnuki et al.23 also reported that in normal human oral keratinocytes, ZOL-mediated S-phase arrest was accompanied by a significant increase in the marker of DNA damage γH2AX. Therefore, detailed mechanisms remain unknown; previous studies showed that BP-mediated DNA damage induced S-phase arrest. In this study, N-BPs induced G0/G1 phase arrest in ESCC cell lines. DNA damage may not have occurred, but there was decreased Cyclin D1 protein expression. Further investigations are needed to clarify the reasons for non-damage of DNA in ESCC cell lines. RIS potentiated caspase 3/7 activity, but the number of Annexin V-positive cells did not increase after 48 hours of treatment (Fig. 2). For 72-hour RIS treatment, the number of Annexin V-positive KYSE150 cells significantly increased (Fig. 2B inset). In addition, Cyclin D1 expression did not change after 24 hours of RIS treatment, but the number of cells in G0/G1 phase increased after 48 hours of treatment. Similarly, DNA fragmentation, an apoptotic index, was not observed with 48-hour RIS treatment, but it was observed with 72-hour treatment in osteosarcoma cell lines.24 Therefore, the cytotoxic effect of RIS results from apoptosis induction and cell cycle inhibition, as with the other BPs. N-BPs inhibit FPP synthase4 and GGPP synthase,25 key enzymes in the mevalonate pathway, thereby depleting intracellular FPP and GGPP. FPP and GGPP depletion decreases prenylation of small G-proteins such as Ras, Rac, Rap1, and Rho.26,27
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Fig. 5 Effects of mevalonate pathway intermediate on bisphosphonate cytotoxicity in KYSE150 cells. Cells were incubated with culture media containing 2.5, 10, and 40 μM alendronate (ALE), etidronate (ETI), pamidronate (PAM), risedronate (RIS), or zoledronate (ZOL) in the absence or presence of squalene (SQ, 20 μM), farnesol (FOH, 10 μM), or geranylgeraniol (GGOH, 10 μM) for 72 hours. Cell viability was evaluated with the CellQuanti-Blue assay. The data are presented as the mean ± standard deviation (n = 3). Significant differences between the mean values were determined by analysis of variance followed by the Tukey’s honest significant difference test (**P < 0.01 compared with control).
In this study, N-BP-mediated cytotoxicity was hardly influenced by SQ or FOH treatment (Fig. 3). However, treatment with GGOH inhibited N-BP cytotoxicity almost completely (Fig. 3). Although the cell types were different, these results are consistent with those from previous reports.26,28 Therefore, N-BP cytotoxicity in ESCC cell lines is suggested to work via cellular GGPP depletion. Rap1, a small G-protein in the Ras superfamily, is geranylgeranylated and it activates mitogen-activated protein kinase (MAPK)/extracellular signalregulated kinase (ERK) signaling, which plays an important role in cell proliferation.29,30 Tsubaki et al.31 showed that N-BPs, such as ALE, induced apoptosis in hematopoietic tumor cell lines by suppressing ERK and mammalian target of rapamycin (mTOR) signaling, and this apoptosis induction was abolished with GGPP addition. Moreover, Cyclin D1 is positively regulated by the MAPK/ERK and mTOR signaling pathways.32,33 Therefore, in ESCC cell lines, N-BPs may induce apoptosis and cell cycle inhibition by inhibiting the MAPK/ERK and mTOR pathways. In conclusion, the second- and third-generation BPs are cytotoxic in ESCC cell lines. The cytotoxic mechanisms of these BPs are apoptosis induction and accompanying cell cycle arrest through mevalonate pathway inhibition, especially GGPP production. Although further in vivo studies are needed, our data suggest that BPs may be promising therapeutic agents for ESCC.
Acknowledgments This study was supported in part by a Grant-aided Project for Private Universities from Ministry of Education, Culture, Sport, Science, and Technology of Japan and JSPS KAKENHI grant number 25460230.
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Original article
Effects of bisphosphonates on human esophageal squamous cell carcinoma cell survival T. Minegaki, S. Fukushima, C. Morioka, H. Takanashi, J. Uno, S. Tsuji, S. Yamamoto, A. Watanabe, M. Tsujimoto, K. Nishiguchi Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Kyoto Pharmaceutical University, Kyoto, Japan
Summary. Esophageal squamous cell carcinoma (ESCC) is one of the most malignant cancers in Japan. Anticancer chemotherapy has been useful for ESCC treatment. However, therapeutic options are limited. Recently, bisphosphonates (BPs), which are osteoporosis drugs, have shown anticancer effects in several cancer cell lines, but the effects against ESCC cell lines are unknown. In this study, we examined the cytotoxic effects of BPs and their mechanisms of cytotoxicity in human ESCC cell lines. A first-generation BP (etidronate), two second-generation BPs (alendronate and pamidronate), and two third-generation BPs (risedronate and zoledronate) were used in this study. All BPs, except etidronate, were cytotoxic, as indicated by increased caspase-3/7 activity and numbers of Annexin-fluorescein isothiocyanate positive cells in ESCC cell lines. From cell cycle analysis, G0/G1-phase arrest was observed upon treatment with second- and third-generation BPs. In addition, Cyclin D1 protein expression levels were decreased by second- and third-generation BP treatment. Although squalene and trans, trans-farnesol minimally affected BP cytotoxicity, treatment with geranylgeraniol inhibited BP cytotoxicity almost completely. We concluded that second- and third-generation BPs are cytotoxic to ESCC cell lines as they induce apoptosis and inhibit the cell cycle through mevalonate pathway inhibition. Therefore, BP treatment may be a beneficial therapy in ESCC patients. KEY WORDS: bisphosphonate, esophageal cancer, mevalonate pathway.
INTRODUCTION In 2008, an estimated 482 300 new cases and 406 800 deaths attributed to esophageal cancer was reported worldwide.1 In Japan, over 10 000 patients died from esophageal cancer and the 5-year survival rate of this disease was about 40%.2 Esophageal squamous cell carcinoma (ESCC) is a major histological type of esophageal cancer in East Asian countries, including Japan. Surgical resection has been a good treatment option for early-stage ESCC. In unresectable ESCC, Address correspondence to: Mr Tetsuya Minegaki, Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan. Email: [email protected] Author contributions: TM and KN conceived and designed the study. SF, CM, HT, JU, ST, SY, and AW performed the experiments and statistical analysis. TM, MT, and KN performed data management and prepared the manuscript. Conflicts of interest: The authors have no conflicts of interest to disclose. © 2015 International Society for Diseases of the Esophagus
cisplatin and 5-fluorouracil-based chemotherapy is the first-line treatment. However, no standard second-line chemotherapy has been established.3 Therefore, to improve the therapeutic outcomes of ESCC patients, novel treatments are required. Bisphosphonates (BPs) have been widely used to treat osteoporosis, Paget’s disease, and tumor-related hypercalcemia. BPs can be divided into two groups by structure, based on the presence of the nitrogen atom. BPs lacking the nitrogen atom, namely first-generation BPs, are metabolized into nonhydrolyzable adenosine triphosphate analogs that induce osteoclast apoptosis and subsequently prevent bone resorption. On the other hand, nitrogencontaining BPs (second- or third-generation BPs, or N-BPs) more potently prevent bone resorption than first-generation BPs by inhibiting farnesyl diphosphate (FPP) synthase, a key enzyme in the mevalonate pathway, and decreasing synthesis of the isoprenoids FPP or geranylgeranyl diphosphate (GGPP).4 These isoprenoids engage in cellular signal 1
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transduction, leading to cell survival, proliferation, differentiation, and motility. Recently, many studies have shown that BPs, especially second- and third-generation BPs, can inhibit the growth of some cancer cell lines, including breast,5–7 lung,8,9 colorectal,10 renal,11 and prostate12 cancer cells, by preventing the prenylation of Ras and Rho in in vitro and in vivo assays. However, no studies have shown that BPs have cytotoxic effects against ESCC cell lines and the mechanism by which they exert their toxic effects. Therefore, if the efficacy of BPs against ESCC cell lines can be shown, there could be more therapeutic options for ESCC patients. The purpose of this study was to evaluate the cytotoxic effects of first-, second-, and thirdgeneration BPs and to clarify the mechanisms of BP cytotoxicity in ESCC cell lines in vitro.
METHODS Chemicals Alendronate (ALE) monosodium trihydrate, etidronate (ETI) disodium, and risedronate (RIS) sodium hydrate were purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Pamidronic acid (PAM) sodium salt hydrate and zoledronic acid (ZOL) disodium salt tetrahydrate were purchased from Toronto Research Chemicals Inc. (Toronto, ON, Canada). Trans, trans-farnesol (FOH) and geranylgeraniol (GGOH) were obtained from SigmaAldrich Chemical Co. (St. Louis, MO, USA). Squalene (SQ) was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan).
The culture medium was changed to medium containing BP at various concentrations. After 72 hours, the medium was exchanged for 110 μL of medium containing CellQuanti-Blue solution (10 μL CellQuantiBlue solution and 100 μL culture media) and 4 hours later, the fluorescence was quantified at an excitation wavelength of 535 nm and an emission wavelength of 590 nm using a microplate reader (GENios, Tecan, Seestrasse, Switzerland). The fluorescence intensity of the CellQuanti-Blue solution was linear between 1 and 5 hours of incubation. The effects of SQ, FOH, or GGOH on BP sensitivity were also evaluated using the CellQuanti-Blue kit. In this study, we used FOH and GGOH instead of FPP and GGPP, respectively. FOH and GGOH are cell permeable, while FPP and GGPP are not, and they are converted to FPP and GGPP, respectively.14 Cells were cultured for 24 hours and the culture medium was changed to media containing BP at various concentrations with or without SQ (20 μM), FOH (10 μM), or GGOH (10 μM). After incubation for 72 hours, the medium was replaced with a medium containing CellQuantiBlue solution and fluorescence was measured. The 50% growth inhibitory concentration (IC50) was calculated according to the sigmoid inhibitory effect model (1) using the non-linear least-squares fitting method (Solver, Microsoft® Excel, Microsoft Co., Redmond, WA, USA).
E = E max ×
1−Cγ C γ + IC50γ
(1)
E and Emax represent the surviving fraction (% of control) and the maximum, respectively. C and γ are the BP concentration in the culture medium and the sigmoidicity factor, respectively.15
Cell culture The human ESCC cell lines KYSE30, KYSE150, and KYSE170 cells were obtained from Health Science Research Resources Bank (Osaka, Japan).13 These cell lines were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY, USA) supplemented with 100 U/mL penicillin G and 100 μg/mL streptomycin sulfate (Nacalai Tesque, Kyoto, Japan) and 10% fetal bovine serum (lot. no. AWD12933, Thermo Fisher Scientific, Waltham, MA, USA). Cells (2 × 106 cells) were seeded on 100-mm culture dishes, grown in a 5% CO2 humidified atmosphere at 37°C, and subcultured two times per week. Drug sensitivity assay Drug sensitivity was measured using the CellQuantiBlue Cell Viability Assay Kit (Bio Assay Systems, Hayward, CA, USA) according to the manufacturer’s instructions. In brief, cells (5 × 103/well) were seeded in 96-well culture plates and cultured for 24 h.
Caspase-3/7 activity The caspase-3/7 activities of KYSE150 cells treated with BPs were determined using the Caspase-Glo 3/7 Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, KYSE150 cells (1 × 105/well) were seeded in 96-well culture plates and incubated for 24 hours at 5% CO2 and 37°C. The culture medium was exchanged for medium containing ALE (50 μM), ETI (1 mM), PAM (50 μM), RIS (50 μM), or ZOL (50 μM) and cells were cultured for 24 hours. Caspase-Glo reagent (100 μL) was added to each well and 2 hours later, luminescence was measured using a microplate reader (GENios). Annexin V assay KYSE150 cells were seeded at a density of 1.5 × 105 cells/well into 6-well culture plates and cultured for 24 hours and then treated with ALE (50 μM), © 2015 International Society for Diseases of the Esophagus
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ETI (1 mM), PAM (50 μM), RIS (50 μM), or ZOL (50 μM) for 48 hours. Then, floating and adherent cells were harvested and labeled with fluorescein isothiocyanate (FITC)-conjugated Annexin V (BioLegend, San Diego, CA, USA) and propidium iodide (PI, 50 μg/mL, Wako Pure Chemical, Osaka, Japan) for early and late apoptosis detection. FITC and PI fluorescence signals were detected using the FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed using CELLQuest Ver. 3.3 (Becton Dickinson) software. At least 10 000 cells were used in the analysis. Cell cycle analysis KYSE150 cells (3 × 105 cells/60 mm dish) were seeded in 60-mm culture dishes. After 24-hour culture, cells were treated with ALE (50 μM), ETI (1 mM), PAM (50 μM), RIS (50 μM), or ZOL (50 μM) for 48 hours and then fixed with 70% ethanol at −30°C. Fixed cells were stained with PI/RNase buffer (BD Pharmingen, San Diego, CA, USA) at room temperature for 15 minutes. PI fluorescence was measured by FACSCalibur (Becton Dickinson) and analyzed using CELLQuest Ver. 3.3 (Becton Dickinson) software. At least 20 000 cells were analyzed for each sample.
groups were performed with Student’s t-test and repeated one-way analysis of variance followed by post hoc Tukey’s honest significant difference tests. A P value of less than 0.05 (two-tailed) was considered statistically significant. RESULTS Cytotoxic effects of BPs on ESCC cell lines ETI, a first-generation BP, did not show any cytotoxic effects on the three ESCC cell lines. Concentration-dependent cytotoxicity was seen with ALE, PAM, RIS, and ZOL, which are second- and third-generation BPs, in ESCC cell lines (Fig. 1). BP cytotoxicity varied and the IC50 for ZOL was the lowest among those for the five BPs (Table 1). In addition, BP cytotoxic effects differed among the cell lines and KYSE150 was the most sensitive to BPs among the three ESCC cell lines. Apoptosis induction in KYSE150 cells In KYSE150 cells, caspase-3/7 activity was significantly potentiated by all investigated BPs except ETI.
Western blot analysis KYSE150 cells treated with BPs were harvested and protein was extracted with CelLytic-M (SigmaAldrich) according to the manufacturer’s instructions. The protein concentration was determined according to the Lowry method16 and 10 μg of protein was loaded in each lane to detect the expression levels of Cyclin D1 and β-actin and electrophoresed using 10% sodium dodecyl sulfatepolyacrylamide gel according to the Laemmli method.17 Then, protein was transferred to a polyvinylidene difluoride (PVDF) membrane (ClearTrans® SP PVDF membrane, Wako) and the membrane was blocked with 1% skim milk (Wako) in phosphate buffered saline containing 0.1% Tween 20. The membranes were incubated at 4°C overnight with anti-Cyclin D1 antibody (1 : 2000, Cell Signaling Technology Japan, Tokyo, Japan) and anti-β-actin antibody (1 : 1000, Wako) and then membranes were incubated with horseradish peroxidase (HRP)conjugated anti-mouse IgG antibody (GE Healthcare Japan, Tokyo, Japan) for 1 hour at room temperature. The secondary antibody HRP activity was detected with the VersaDoc 5000 MP imaging system (Bio-Rad, Hercules, CA, USA) using Immuno Star® LD reagent (Wako). Statistical analyses Data were shown as the mean ± standard deviation. Comparisons between two and among three or more © 2015 International Society for Diseases of the Esophagus
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Fig. 1 Bisphosphonate-mediated inhibitory effects on cell proliferation in esophageal squamous cell carcinoma cell lines. Cells were treated with culture media containing various concentrations of alendronate (ALE), etidronate (ETI), pamidronate (PAM), risedronate (RIS), or zoledronate (ZOL) for 72 hours. Cell viability was determined by CellQuanti-Blue assay. Data are presented as the mean ± standard deviation (n = 4).
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Table 1 IC50 values (μM) for bisphosphonates in esophageal squamous cell carcinoma cell lines IC50 value (μM) Bisphosphonates
KYSE30
KYSE150
KYSE170
Alendronate (μM) Etidronate (μM) Pamidronate (μM) Risedronate (μM) Zoledronate (μM)
5.30 ± 1.62 NC 5.97 ± 1.52 163 ± 16.4 2.98 ± 0.615
1.54 ± 0.0613 NC 3.33 ± 0.636 0.461 ± 0.178 0.364 ± 0.0499
8.36 ± 1.15 NC 35.0 ± 4.83 234 ± 38.5 6.15 ± 1.82
Mean ± standard deviation (n = 4). NC, not calculated. IC50, 50% growth inhibitory concentration.
Although the numbers of Annexin V-positive cells did not increase with ETI and RIS treatment, the other second- and third-generation BPs increased the numbers of apoptotic cells after 48 hours of treatment (Fig. 2). After 72 hours of treatment, RIS significantly increased the number of Annexin V-positive KYSE150 cells (Fig. 2B inset). Effects of BPs on KYSE150 cell cycle distribution The second-generation BPs, namely, ALE and PAM, significantly increased the numbers of cells in G0/G1 phase and decreased that of cells in G2/M phase after 24-hour treatment. After 48-hour treatment with PAM, RIS, or ZOL, the numbers of cells in G0/G1 phase increased and those in S and G2/M phase decreased. ALE slightly increased the number of cells in G0/G1 phase and decreased that of cells in G2/M phase with 48-hour treatment. ETI did not affect KYSE150 cell cycle with either 24 or 48 hours of treatment (Fig. 3). Effects of BPs on Cyclin D1 protein expression Cyclin D1 protein expression levels significantly decreased when KYSE150 cells were treated with ALE, PAM, or ZOL. ETI and RIS did not affect Cyclin D1 protein levels (Fig. 4). Effects of FOH, GGOH, or SQ on the BP sensitivity of KYSE150 cells SQ did not affect BP cytotoxicity, but FOH slightly decreased the cytotoxicity of PAM and RIS. GGOH, a precursor of GGPP, attenuated the cytotoxic effects of ALE and PAM and almost completely blocked the cytotoxic effects of RIS and ZOL, which are thirdgeneration BPs (Fig. 5).
DISCUSSION In this study, we showed the N-BPs (ALE, PAM, RIS, and ZOL) exhibited concentration-dependent
Fig. 2 Evaluation of apoptosis after bisphosphonate treatment in KYSE150 cells. (A) Cells were incubated with alendronate (ALE, 50 μM), etidronate (ETI, 1 mM), pamidronate (PAM, 50 μM), risedronate (RIS, 50 μM), or zoledronate (ZOL, 50 μM). After 24 hours, caspase-3/7 activities were measured by Caspase-Glo 3/7 assay kit. (B) Cells were treated with ALE (50 μM), ETI (1 mM), PAM (50 μM), RIS (50 μM), or ZOL (50 μM) for 48 hours, or RIS (50 μM) for 72 hours (inset). Then, floating and adherent cells were harvested and stained with Annexin V-fluorescein isothiocyanate and propidium iodide (PI). Apoptotic cells were detected using flow cytometry. Data are presented as the mean ± standard deviation (n = 3). Significant differences between mean values were determined by Student’s t-test (B inset, ††P < 0.01) or analysis of variance followed by the Tukey’s honest significant difference test (A and B, **P < 0.01 compared with control).
cytotoxic effects in the KYSE30, KYSE150, or KYSE170 ESCC cell lines (Fig. 1). In addition, BP treatment induced apoptosis in KYSE150 cells (Fig. 2), the most sensitive cell line to N-BPs (Fig. 1 and Table 1). However, the first-generation BP, ETI, did not exhibit cytotoxic effects or induce apoptosis. Therefore, these data suggest that ESCC cell lines, as well as cell lines originating from other kinds of cancer,5–12 can be killed by second- and thirdgeneration BPs, but not first-generation BPs. From the data of all three ESCC cell lines, ZOL was the most potently cytotoxic among these five BPs (Table 1). Verdijk et al.6 and Senaratne et al.7 showed that ZOL was the most potent BP that inhibited cell growth in breast cancer cell lines. These results and reports suggest that ZOL is the most cytotoxic BP in cancer cell lines. © 2015 International Society for Diseases of the Esophagus
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Fig. 4 Cyclin D1 protein expression levels detected by Western blot after bisphosphonate treatment in KYSE150 cells. Cells were treated with alendronate (ALE, 50 μM), etidronate (ETI, 1 mM), pamidronate (PAM, 50 μM), risedronate (RIS, 50 μM), or zoledronate (ZOL, 50 μM) for 24 hours. Then, total protein was extracted from the cell and Western blots were performed. β-actin was used as an internal standard protein. The data are presented as mean ± standard deviation (n = 4). A representative blot from four independent experiments is shown. Significant differences between the mean values were determined by analysis of variance followed by the Tukey’s honest significant difference test (*P < 0.05, **P < 0.01 compared with control). Fig. 3 Cell cycle distribution after bisphosphonate treatment in KYSE150 cells. Cells were treated with alendronate (ALE, 50 μM), etidronate (ETI, 1 mM), pamidronate (PAM, 50 μM), risedronate (RIS, 50 μM), or zoledronate (ZOL, 50 μM) for 24 or 48 hours. Then, cells were fixed with 70% ethanol and stained with propidium iodide. DNA content was detected by flow cytometry. The data are presented as the mean ± standard deviation (n = 3–4). Significant differences between the mean values were determined by analysis of variance followed by the Tukey’s honest significant difference test (**P < 0.01 compared with control).
ALE, PAM, RIS, and ZOL potentiated caspase 3/7 activities (Fig. 2A) and ALE, PAM, and ZOL increased the numbers of Annexin V-positive KYSE150 cells (Fig. 2B). Some studies have shown that N-BPs have cytotoxic effects by inducing apoptosis in several cancer cell lines.5,10,18,19 However, in cholangiocarcinoma cells, ZOL inhibited cell growth without inducing apoptosis.20 Although the mechanism of N-BP cytotoxicity is thought to be dependent on cell type, our results indicated that the cytotoxic mechanism in ESCC cell lines is induction of apoptosis. Treatment with second- and third-generation BPs increased the numbers of cells in G0/G1 phase (Fig. 3) and decreased the protein expression of Cyclin D1, a cell cycle regulator (Fig. 4). In nonsmall-cell lung cancer cell lines, ZOL induces G1 phase cell cycle arrest.9 However, some reports have shown that BPs such as ZOL induce S-phase arrest in several cancer cell lines.18,19,21,22 Iguchi et al.19 reported that ZOL induced S-phase arrest accompanied by DNA damage and activation of the ataxiatelangiectasia mutated kinase and check-point kinase © 2015 International Society for Diseases of the Esophagus
1 pathway in osteosarcoma cells. Ohnuki et al.23 also reported that in normal human oral keratinocytes, ZOL-mediated S-phase arrest was accompanied by a significant increase in the marker of DNA damage γH2AX. Therefore, detailed mechanisms remain unknown; previous studies showed that BP-mediated DNA damage induced S-phase arrest. In this study, N-BPs induced G0/G1 phase arrest in ESCC cell lines. DNA damage may not have occurred, but there was decreased Cyclin D1 protein expression. Further investigations are needed to clarify the reasons for non-damage of DNA in ESCC cell lines. RIS potentiated caspase 3/7 activity, but the number of Annexin V-positive cells did not increase after 48 hours of treatment (Fig. 2). For 72-hour RIS treatment, the number of Annexin V-positive KYSE150 cells significantly increased (Fig. 2B inset). In addition, Cyclin D1 expression did not change after 24 hours of RIS treatment, but the number of cells in G0/G1 phase increased after 48 hours of treatment. Similarly, DNA fragmentation, an apoptotic index, was not observed with 48-hour RIS treatment, but it was observed with 72-hour treatment in osteosarcoma cell lines.24 Therefore, the cytotoxic effect of RIS results from apoptosis induction and cell cycle inhibition, as with the other BPs. N-BPs inhibit FPP synthase4 and GGPP synthase,25 key enzymes in the mevalonate pathway, thereby depleting intracellular FPP and GGPP. FPP and GGPP depletion decreases prenylation of small G-proteins such as Ras, Rac, Rap1, and Rho.26,27
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Fig. 5 Effects of mevalonate pathway intermediate on bisphosphonate cytotoxicity in KYSE150 cells. Cells were incubated with culture media containing 2.5, 10, and 40 μM alendronate (ALE), etidronate (ETI), pamidronate (PAM), risedronate (RIS), or zoledronate (ZOL) in the absence or presence of squalene (SQ, 20 μM), farnesol (FOH, 10 μM), or geranylgeraniol (GGOH, 10 μM) for 72 hours. Cell viability was evaluated with the CellQuanti-Blue assay. The data are presented as the mean ± standard deviation (n = 3). Significant differences between the mean values were determined by analysis of variance followed by the Tukey’s honest significant difference test (**P < 0.01 compared with control).
In this study, N-BP-mediated cytotoxicity was hardly influenced by SQ or FOH treatment (Fig. 3). However, treatment with GGOH inhibited N-BP cytotoxicity almost completely (Fig. 3). Although the cell types were different, these results are consistent with those from previous reports.26,28 Therefore, N-BP cytotoxicity in ESCC cell lines is suggested to work via cellular GGPP depletion. Rap1, a small G-protein in the Ras superfamily, is geranylgeranylated and it activates mitogen-activated protein kinase (MAPK)/extracellular signalregulated kinase (ERK) signaling, which plays an important role in cell proliferation.29,30 Tsubaki et al.31 showed that N-BPs, such as ALE, induced apoptosis in hematopoietic tumor cell lines by suppressing ERK and mammalian target of rapamycin (mTOR) signaling, and this apoptosis induction was abolished with GGPP addition. Moreover, Cyclin D1 is positively regulated by the MAPK/ERK and mTOR signaling pathways.32,33 Therefore, in ESCC cell lines, N-BPs may induce apoptosis and cell cycle inhibition by inhibiting the MAPK/ERK and mTOR pathways. In conclusion, the second- and third-generation BPs are cytotoxic in ESCC cell lines. The cytotoxic mechanisms of these BPs are apoptosis induction and accompanying cell cycle arrest through mevalonate pathway inhibition, especially GGPP production. Although further in vivo studies are needed, our data suggest that BPs may be promising therapeutic agents for ESCC.
Acknowledgments This study was supported in part by a Grant-aided Project for Private Universities from Ministry of Education, Culture, Sport, Science, and Technology of Japan and JSPS KAKENHI grant number 25460230.
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