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Calcitriol enhances gemcitabine antitumor activity in vitro and in vivo by promoting apoptosis in a human pancreatic carcinoma model system Wei-Dong Yu,1 Yingyu Ma,1 Geraldine Flynn,1 Josephia R. Muindi,2 Rui-Xian Kong,1 Donald L. Trump2 and Candace S. Johnson1,* Department of Pharmacology and Therapeutics and 2Medicine; Roswell Park Cancer Institute; Buffalo, NY USA
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Key words: calcitriol, gemcitabine, pancreatic carcinoma, apoptosis, Akt, ERK1/2 Abbreviations: CDDase, cytidine deaminase; CI, combination index; dFdC, difluorodeoxycytidine; EGFR, epidermal growth factor receptor; ERK1/2, extracellular signal regulated kinase 1/2; ETOH, ethanol; MMPIs, matrix-metalloproteinase inhibitors; MTT, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NFκB, nuclear factor-kB; VEGFR, vascular endothelial growth factor receptor; PDGFR, platelet-derived growth factor receptor; SCC, squamous cell carcinoma
Gemcitabine is the standard care chemotherapeutic agent to treat pancreatic cancer. Previously we demonstrated that calcitriol (1, 25-dihydroxycholecalciferol) has significant anti-proliferative effects in vitro and in vivo in multiple tumor models and enhances the activity of a variety of chemotherapeutic agents. We therefore investigated whether calcitriol could potentiate the cytotoxic activity of gemcitabine in the human pancreatic cancer Capan-1 model system. Isobologram analysis revealed that calcitriol and gemcitabine had synergistic antiproliferative effect over a wide range of drug concentrations. Calcitriol did not reduce the cytidine deaminase activity in Capan-1 tumors nor in the livers of Capan-1 tumor bearing mice. Calcitriol and gemcitabine combination promoted apoptosis in Capan-1 cells compared with either agent alone. The combination treatment also increased the activation of caspases-8, -9, -6 and -3 in Capan-1 cells. This result was confirmed by substrate-based caspase activity assay. Akt phosphorylation was reduced by calcitriol and gemcitabine combination treatment compared to single agent treatment. However, ERK1/2 phosphorylation was not modulated by either agent alone or by the combination. Tumor regrowth delay studies showed that calcitriol in combination with gemcitabine resulted in a significant reduction of Capan-1 tumor volume compared to single agent treatment. Our study suggests that calcitriol and gemcitabine in combination promotes caspase-dependent apoptosis, which may contribute to increased anti-tumor activity compared to either agent alone.
Introduction Calcitriol (1, 25-dihydroxycholecalciferol) is the active metabolite of the secosteroid hormone vitamin D and is well-known for its important role in bone and mineral metabolism. Vitamin D can be synthesized in skin or obtained from the diet.1,2 Calcitriol causes anti-proliferative effects through multiple mechanisms including the induction of cell cycle arrest, apoptosis and differentiation in vitro and in vivo in a variety of cancer cell types including prostate, breast, colon, skin and leukemic cells.3-14 Calcitriol has also been shown to reduce angiogenesis in a number of cancer models, which also contributes to the antitumor effect of calcitriol in vivo.2 Clinical trials have demonstrated
that sufficient doses can be administered in patients without the development of hypercalcemia.15-17 Pancreatic cancer is the fourth leading cause of cancer death in the United States even though it only accounts for 2% of all new cancers diagnosed. The overall five year survival rate is 4% for advanced or metastatic carcinoma and 17% for localized, resectable tumors of the pancreas.18 Gemcitabine (difluorodeoxycytidine, dFdC, Gemzar®) is a nucleoside analogue that exhibits antitumor activity and is the standard of care to treat locally advanced and metastatic pancreatic carcinoma. Gemcitabine exhibits cell phase specificity, primarily killing cells undergoing DNA synthesis (S-phase) and also blocking the progression of cells through the cell cycle, specifically from G1 to S-phase.
*Correspondence to: Candace S. Johnson; Email:
[email protected] Submitted: 05/17/10; Accepted: 05/17/10 Previously published online: www.landesbioscience.com/journals/cc/article/12381 DOI: 10.4161/cc.9.15.12381 3022
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Figure 2. Calcitriol modulates CDDase activity in Capan-1 tumor. Capan-1 tumor-bearing nude mice were treated with a single dose 0.75 µg/mouse calcitriol for 4 to 48 h. Tumors and liver tissues were obtained and subjected to CDDase activity assay measured by monitoring the rate of cytidine deamination to uridine. The results shown are representative of three independent experiments.
pathway and the activation of several key members of the caspase family. Results
Figure 1. Calcitriol promotes gemcitabine antiproliferative effect in Capan-1 cells. (A) Capan-1 cells were pretreated with various doses of calcitriol for 24 h followed by various doses of gemcitabine for 48 h and subjected to MTT assay. Fraction affected (Fa) was calculated as 1-(MTT value of the treatment cells)/(MTT value of control ETOH-treated cells). (B) Mutually exclusive CI plots for gemcitabine/calcitriol combination in Capan-1 cells as determined by MTT assay. Each CI was calculated from the fraction affected at each drug ratio. The results shown are representative of three independent experiments.
Gemcitabine also induces apoptosis.19 Cytidine deaminase (CDDase) can deaminate gemcitabine which results in its inactive metabolite dFd-U.20 Gemcitabine can also be inactivated by dephosphorylation of dFd-CMP by 59-NU.20 It has been reported that calcitriol works synergistically with chemotherapeutic drugs such as cisplatin and paclitaxel.6,13,21,22 In this study, we investigated the effects of calcitriol alone and in combination with gemcitabine on the Capan-1 pancreatic carcinoma model system in vivo and in vitro. We observed a significant antitumor effect in vivo following the combination treatment which was greater than either agent alone. The interaction of calcitriol and gemcitabine is synergistic over a range of concentrations as assessed by the cytotoxicity assay. The in vitro studies postulate that calcitriol and gemcitabine when used in combination increase apoptosis in Capan-1 cells which might be mediated via inhibition of the Akt survival signaling
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Calcitriol promotes gemcitabine antiproliferative effect. To examine whether calcitriol and gemcitabine combination treatment affects cell growth in the human pancreatic cancer model system Capan-1 and the nature of the interaction, their effects on cell growth were examined by MTT assay. Capan-1 cells were pretreated with various doses of calcitriol for 24 h followed by various doses of gemcitabine for 48 h. The results showed that pretreatment with calcitriol enhanced the gemcitabine-mediated growth inhibition in a dose-dependent manner (Fig. 1A). Standard median-dose effect isobologram analysis revealed that the interaction between calcitriol and gemcitabine was synergistic (CI < 1) over the combination of calcitriol (0.093–6 µm) with gemcitabine (1.6–200 nM) (Fig. 1B). These data indicate that calcitriol promotes the antiproliferative effect of gemcitabine in vitro. Modulation of CDDase activity in tumor and liver tissues by calcitriol. Calcitriol modulates the expression and activity of CDDase, the key gemcitabine degrading enzymes in both normal and tumor tissues. Therefore, we investigated if calcitriol-induced changes in CDDase activity contribute to the enhanced antitumor activity of the calcitriol + gemcitabine combination. Time course of the changes in CDDase activity in tumor and liver tissues obtained from Capan-1 tumorbearing nude mice after a single dose 0.75 µg/mouse calcitriol were shown (Fig. 2). The results show significant (p < 0.05, ANOVA) transient increase in tumor CDDase 24 h post treatment; and no significant (p > 0.632, ANOVA) changes in liver CDDase activity. Calcitriol promotes gemcitabine-mediated apoptosis. Calcitriol induces apoptosis in a number of cancer cells. To
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examine whether apoptosis was involved in calcitriol and gemcitabine induced growth inhibition in pancreatic cancer cells, Capan-1 cells were pretreated with calcitriol for 24 h followed by gemcitabine for 24 h and apoptosis was assessed by annexin V staining. Calcitriol or gemcitabine alone induced similar level of apoptosis in Capan-1 cells (Fig. 3A and B). The pretreatment with calcitriol markedly enhanced gemcitabine-induced apoptosis (Fig. 3A and B), suggesting that calcitriol and gemcitabine may inhibit Capan-1 cell growth, at least in part, through the induction of apoptosis. Calcitriol and gemcitabine induce the activation of caspases. To examine the involvement of caspases in calcitriol and gemcitabine-induced apoptosis, the cleavage of caspases was assessed by Western blot analysis. Calcitriol induced modest activation of caspase-9 but not that of caspases-8, -6 or -3 in Capan-1 cells (Fig. 4A). In contrast, gemcitabine induced the activation of all these caspases (Fig. 4A). The pretreatment with calcitriol enhanced the activation of caspases-8, -6, -9 and -3 (Fig. 4A). These results were confirmed using a substrate-based caspase activity assay, which showed that calcitriol alone had miniFigure 3. Induction of apoptosis by calcitriol and gemcitabine. Capan-1 cells were pretreated with either mal effect of caspase activation; ETOH or 0.75 µM calcitriol for 24 h followed by gemcitabine (6.25 µM or 12.5 µM) for 24 h. Flow cytometric however, it enhanced the caspase analysis of annexin V-PE and 7-AAD binding in Capan-1 cells was performed. The results shown are repreactivity when used in combination sentative of three independent experiments. with gemcitabine (Fig. 4B–E), supporting the observations that of gemcitabine in vivo, Capan-1 pancreatic tumor model system calcitriol promoted gemcitabine-mediated apoptosis induction. Calcitriol and gemcitabine inhibit Akt activation. We next was employed. Capan-1 tumor-bearing nude mice were treated in investigated the effect of calcitriol and gemcitabine on prosur- 6 groups for 4 weeks: saline, calcitriol alone (2.5 µg/mouse once vival molecules Akt and ERK1/2. Calcitriol slightly decreased or twice a week), gemcitabine alone (6 mg/mouse once a week) or the level of phosphorylated Akt, which was further reduced fol- the combination of calcitriol (2.5 µg/mouse once or twice a week) lowing the addition of gemcitabine (Fig. 5). Gemcitabine alone and gemcitabine. Calcitriol or gemcitabine alone resulted in tumor did not suppress the activation of Akt (Fig. 5). In contrast, regrowth delay compared with saline control (Fig. 6A). Calcitriol ERK1/2 phosphorylation was not reduced by either agent alone administered twice a week reduced tumor growth even further or in combination (Fig. 5). These results suggest that Akt may be than administered once a week (Fig. 6A). The combination treatinvolved in calcitriol and gemcitabine-mediated growth inhibi- ment with calcitriol and gemcitabine further inhibited tumor growth with a 10-fold reduction in tumor size (p < 0.01) (Fig. 6A). tory effects in Capan-1 cells. Calcitriol enhances gemcitabine antitumor activity in vivo. The gross images of the tumors removed were shown in Figure 6B. To investigate whether calcitriol enhances the antitumor activity These data indicate that the combined administration of calcitriol
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Figure 4. Effects of calcitriol and gemcitabine on caspase activation. (A) Capan-1 cells were pre-treated with either ETOH or 0.75 µM calcitriol for 24 h followed by varying concentrations of gemcitabine for 24 h. Cell lysates were prepared and analyzed by Western blot analysis for caspases-8, -6, -9 and -3. Actin was the loading control. (B–E) Capan-1 cells were treated with ETOH, calcitriol (0.75 µM), gemcitabine (12.5 µM) or pretreatment with calcitriol for 24 h followed by gemcitabine for 24 h. Activities of caspases-8 (B), -6 (C), -9 (D) and -3 (E) were measured by substrate-based caspase activity assay. Absorbance at 400 nm was determined and the caspase activity was expressed as absorbance per mg of protein per reaction. The results shown are representative of three independent experiments.
and gemcitabine resulted in a greater antitumor effect than either agent alone in the Capan-1 pancreatic cancer model system. Discussion Pancreatic cancer is one of the most deadly solid malignancies worldwide. Gemcitabine is used as the first line therapy in most cases but is only moderately efficacious. The overall 5-year survival rate is 1 indicates synergistic, additive and anti-caspase 9 (9504), anti-caspase 6 (9762) and anti-phos- antagonistic effects, respectively. pho-Akt (Ser473, 9271) were from Cell Signaling Technology CDDase activity assay. Capan-1 tumor-bearing nude mice (Beverly, MA). Anti-phospho-ERK1/2 (sc-7383) was from Santa were treated with a single dose 0.75 µg/mouse calcitriol for 4 to
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48 h. Tumors and liver tissues were harvested and rinsed twice with 5 ml of cold normal saline, 5 mM Tris-HCL buffer, pH 7.4 and stored at -80oC until enzyme activity assay. Tissues were sonicated in 0.1 ml of cold 5 mM Tris-HCL buffer, pH 7.4 containing 5 mM DTT and centrifuged at 14,000 rpm for 10 min at 4oC. The clear supernatant was assayed for CDDase activity as previously described.32,33 Briefly, CDDase activity was measured spectrophotometrically by monitoring the rate of cytidine deamination to uridine at 286 nm. The assay contained 50–100 µg of protein of tumor or liver tissue in 1 ml of 20 mM Tris HCL buffer pH 7.4 containing 100 mM KCL. Absorbance change at 286 nm was recorded for 3 min prior to and after the addition of 100 µM cytidine. The difference in absorbance change/min the presence and absence of cytidine was used to calculate CDDase activity. CDDase activity was expressed as nmol uridine formed/min/ mg protein. (uridine extinction coefficient = 3,000 cm2 /mmol). Substrate-based caspase activity assay. Caspase-3, 6, 8 and 9 activity was measured using the caspase-family Colorimetric Assay kit from BioVision Research Products (Mountain View, CA) according to the manufacturer’s protocol. Briefly, the cells were pre-treated for 24 h with calcitriol (0.75 µM) and/or gemcitabine (12.5 µM) was added the following day for another 24 h. The cells were trypsinized and centrifuged at 1,000 rpm for 10 min. The cell pellets (1–5 x 106 cells) were resuspended in 50 µl of ice cold cell lysis buffer and placed on ice for 10 min. The lysates were centrifuged for 1 min at 11,000 rpm and the supernatant collected to assay protein concentration. 50–200 µg protein was diluted in 50 µl cell lysis buffer for each assay. 50 µl of 2x reaction buffer containing 10 mM DTT was added to each References 1.
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Reichel H, Koeffler HP, Norman AW. The role of the vitamin D endocrine system in health and disease. N Engl J Med 1989; 320:980-91. Deeb KK, Trump DL, Johnson CS. Vitamin D signalling pathways in cancer: potential for anticancer therapeutics. Nat Rev Cancer 2007; 7:684-700. Getzenberg RH, Light BW, Lapco PE, Konety BR, Nangia AK, Acierno JS, et al. Vitamin D inhibition of prostate adenocarcinoma growth and metastasis in the Dunning rat prostate model system. Urology 1997; 50:999-1006. Peehl DM, Skowronski RJ, Leung GK, Wong ST, Stamey TA, Feldman D. Antiproliferative effects of 1,25-dihydroxyvitamin D3 on primary cultures of human prostatic cells. Cancer Res 1994; 54:805-10. McGuire TF, Trump DL, Johnson CS. Vitamin D(3)induced apoptosis of murine squamous cell carcinoma cells. Selective induction of caspase-dependent MEK cleavage and upregulation of MEKK-1. J Biol Chem 2001; 276:26365-73. Hershberger PA, Yu WD, Modzelewski RA, Rueger RM, Johnson CS, Trump DL. Calcitriol (1,25-dihydroxycholecalciferol) enhances paclitaxel antitumor activity in vitro and in vivo and accelerates paclitaxelinduced apoptosis. Clin Cancer Res 2001; 7:1043-51. Bernardi RJ, Trump DL, Yu WD, McGuire TF, Hershberger PA, Johnson CS. Combination of 1alpha,25-dihydroxyvitamin D(3) with dexamethasone enhances cell cycle arrest and apoptosis: role of nuclear receptor cross-talk and Erk/Akt signaling. Clin Cancer Res 2001; 7:4164-73. Kumagai T, O’Kelly J, Said JW, Koeffler HP. Vitamin D2 analog 19-nor-1,25-dihydroxyvitamin D2: antitumor activity against leukemia, myeloma and colon cancer cells. J Natl Cancer Inst 2003; 95:896-905.
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sample and 5 µl of 4 mM pNA conjugated substrate was added to each sample and incubated at 37oC for 1–2 h. The samples were read using a spectrophotometer (Absorbance 400 nm). Caspase activity was expressed as absorbance (O.D.) per milligram of protein per reaction. Apoptosis analysis—annexin V staining. Capan-1 cells were pre-treated for 24 h with calcitriol (0.75 µM) and/or gemcitabine (6.25 µM or 12.5 µM) was added the following day for a further 24 h. The cells were harvested by trypsinization following treatment. Cells were stained with Annexin V-PE and 7-AAD according to the manufacturer’s instructions (BD Pharmingen).14 The data was analysed using WinlistTM program (Verity House, Topsham, ME). Western blot analysis. Following pre-treatment for 24 h with calcitriol, the cells were treated with varying concentrations of gemcitabine for a further 24 h. Cell lysates were prepared and Western blot analysis performed as described previously.14 Statistical analysis. The data for calcitriol-induced changes in tissue CDDase activity was analyzed by One-way ANOVA using GraphPad Prism 5 software (La Jolla, CA). A p-value of
