Abstract
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 CDDase 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.Citation1,Citation2 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.Citation3–Citation14 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.Citation2 Clinical trials have demonstrated that sufficient doses can be administered in patients without the development of hypercalcemia.Citation15–Citation17
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.Citation18 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. Gemcitabine also induces apoptosis.Citation19 Cytidine deaminase (CDDase) can deaminate gemcitabine which results in its inactive metabolite dFd-U.Citation20 Gemcitabine can also be inactivated by dephosphorylation of dFd-CMP by 59-NU.Citation20
It has been reported that calcitriol works synergistically with chemotherapeutic drugs such as cisplatin and paclitaxel.Citation6,Citation13,Citation21,Citation22 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 pathway and the activation of several key members of the caspase family.
Results
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 (). 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) (). 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 tumor-bearing nude mice after a single dose 0.75 µg/mouse calcitriol were shown (). 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 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 ( and B). The pretreatment with calcitriol markedly enhanced gemcitabine-induced apoptosis ( 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 (). In contrast, gemcitabine induced the activation of all these caspases (). The pretreatment with calcitriol enhanced the activation of caspases-8, -6, -9 and -3 (). These results were confirmed using a substrate-based caspase activity assay, which showed that calcitriol alone had minimal effect of caspase activation; however, it enhanced the caspase activity when used in combination with gemcitabine (), supporting the observations that calcitriol promoted gemcitabine-mediated apoptosis induction.
Calcitriol and gemcitabine inhibit Akt activation.
We next investigated the effect of calcitriol and gemcitabine on prosurvival molecules Akt and ERK1/2. Calcitriol slightly decreased the level of phosphorylated Akt, which was further reduced following the addition of gemcitabine (). Gemcitabine alone did not suppress the activation of Akt (). In contrast, ERK1/2 phosphorylation was not reduced by either agent alone or in combination (). These results suggest that Akt may be involved in calcitriol and gemcitabine-mediated growth inhibitory effects in Capan-1 cells.
Calcitriol enhances gemcitabine antitumor activity in vivo.
To investigate whether calcitriol enhances the antitumor activity of gemcitabine in vivo, Capan-1 pancreatic tumor model system was employed. Capan-1 tumor-bearing nude mice were treated in 6 groups for 4 weeks: saline, calcitriol alone (2.5 µg/mouse once or twice a week), gemcitabine alone (6 mg/mouse once a week) or the combination of calcitriol (2.5 µg/mouse once or twice a week) and gemcitabine. Calcitriol or gemcitabine alone resulted in tumor regrowth delay compared with saline control (). Calcitriol administered twice a week reduced tumor growth even further than administered once a week (). The combination treatment with calcitriol and gemcitabine further inhibited tumor growth with a 10-fold reduction in tumor size (p < 0.01) (). The gross images of the tumors removed were shown in . These data indicate that the combined administration of calcitriol 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 <5% even with the treatment. Research is ongoing in the hope of improving the antitumor effect of gemcitabine without increasing the toxicity. Several clinical trials have been conducted using gemcitabine in combination with a number of cytotoxic agents; most of which had disappointing results. Using innovative targeted therapies in conjunction with gemcitabine may be more effective.Citation23,Citation24 Pancreatic carcinoma cells pre-treated with the soy isoflavone genistein before treatment with gemcitabine were more susceptible to apoptosis than either agent alone. It was noted that genistein enhanced the effects of gemcitabine through the downregulation of NFκB and Akt. The antitumor effects of the combination treatment were also observed in vivo with inactivation of NFκB in the tumor.Citation25
Targeting the epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) via inhibition of phosphorylation along with gemcitabine treatment had a significant antitumor effect in vivo, with greater than 80% reduction in tumor size and improved survival rates. Increased apoptosis was observed in tumor cells and tumor-associated endothelial cells, along with decreased proliferation. There was also a decrease in mean vessel density observed in the treated tumor samples. Gemcitabine inhibits angiogenesis observed in several animal models of pancreatic carcinoma.Citation26,Citation27 Gemcitabine in combination with antiangiogenic agents is under investigation for potential therapeutic intervention in pancreatic carcinoma. Matrix-metalloproteinase inhibitors (MMPIs), anti-VEGF, COX-2 inhibitors and thalidomide are some of those agents being studied.Citation28
Calcitriol has antitumor effects over a wide range of tumors.Citation2 Preclinical studies have shown that calcitriol has additive or synergistical effects when used in combination with chemotherapeutic agents including cisplatin and paclitaxel. Calcitriol enhances doxorubicin-caused oxidative damage by reducing the expression and activity of cytoplasmic antioxidant enzyme Cu/Zn superoxide dismutase, and thus sensitizes breast cancer cells to doxorubicin treatment.Citation29 Calcitriol and cisplatin combination treatment enhances the antitumor activity of cisplatin in squamous cell carcinoma (SCC) cells, possibly through enhancing the expression of mitogen-activated protein kinase kinase kinase and induces caspase 3 activation.Citation21 Calcitriol promotes the cisplatin antiproliferative effects in SCC cells by the induction of p73 expression and enhanced apoptosis.Citation22 Calcitriol has also been shown to promote the growth inhibitory effects of paclitaxel, and the mechanisms may include enhanced Bcl-2 phosphorylation in breast cancer cells and reduced expression of p21 in prostate cancer cells.Citation6,Citation30 Clinical trials demonstrated that calcitriol can be safely administered to achieve sufficient doses in patients as compared to the doses given in the in vitro model systems.Citation15–Citation17,Citation31 Therefore, we investigated whether calcitriol would enhance the antitumor activity of gemcitabine in a pancreatic adenocarcinoma model system Capan-1.
Calcitriol treatment upregulates CDDase expression in some cells but not others.Citation32 In our previous study, we observed decrease in CDDase activity in cancer patient peripheral blood monocytes during calcitriol treatment.Citation33 Therefore, we suggested a pharmacokinetic approach for enhancing antitumor activity of drugs such as gemcitabine that are catabolized by CDDase if used in combination with calcitriol. The results of present study show no calcitriol-mediated decrease in CDDase activity and thus the modulation of gemcitabine PK plays no role in the antitumor activity synergy of the calcitriol + gemcitabine in the Capan-1 pancreatic tumor model system.
Treatment of Capan-1 cells with calcitriol and gemcitabine resulted in increased apoptosis as assessed by annexin V staining, which is associated with an increase in caspase-8 cleavage and enhanced activation of caspase-6. This suggests that enhanced cytotoxicity may be mediated by increased activation of the caspase-8/caspase-6/nuclear lamin pathway.
Caspase-9 was modestly increased in Capan-1 cells after treatment with calcitriol or gemcitabine alone and strongly increased by the combination. Caspase-3 was not activated by calcitriol but by gemcitabine and the combination treatment. The pro-survival signaling molecule Akt phosphorylation was strongly decreased by the combination of gemcitabine and calcitriol, but the phosphorylation of ERK1/2 was not markedly affected by the two agent combination. These data indicate that gemcitabine and calcitriol-enhanced cytotoxicity may result, at least in part, from the inhibition of the activation of Akt survival signaling pathway.
Median dose effect and isobologram analyses reveal that the combination of calcitriol with gemcitabine is strongly synergistic in vitro. Results from tumor regrowth delay experiments showed that calcitriol alone had antitumor effect in pancreatic cancer in vivo, in a dose-dependent manner. When it was administered twice a week, it delayed tumor growth to a similar extent as gemcitabine. The combination therapy with calcitriol and gemcitabine for 4 weeks significantly inhibited tumor growth as compared to either agent alone.
In summary, we investigated the in vitro and in vivo effects of treatment with gemcitabine in combination with calcitriol. Our results show that the combination treatment of calcitriol and gemcitabine in human pancreatic cancer model system Capan-1 is synergistic and preferable to either agent alone. Induction of apoptosis mediated via the activation of key caspase family members and inhibition of Akt survival signaling pathway are vital to the success of the combination treatment. It is also possible that the combination treatment is targeting the tumor vasculature since previous studies have shown the anti-proliferative effects of calcitriol on tumor-derived endothelial cells.Citation34 Understanding the effects of calcitriol in enhancing gemcitabine treatment of pancreatic carcinoma cells in vitro and in vivo is essential in designing more effective targeted therapies for pancreatic cancer using gemcitabine.
Material and Methods
Materials.
Calcitriol was from Hoffmann-LaRoche (Nutley, NJ). Gemcitabine (Gemzar®) was from Eli Lilly and Company (Indianapolis, IN). Anti-caspase 3 (9662), anti-caspase 8 (4927), anti-caspase 9 (9504), anti-caspase 6 (9762) and anti-phospho-Akt (Ser473, 9271) were from Cell Signaling Technology (Beverly, MA). Anti-phospho-ERK1/2 (sc-7383) was from Santa Cruz (Santa Cruz, CA). Anti-actin (CP-01) was from Calbiochem (San Diego, CA).
Cell culture and tumor model system.
Capan-1 cells (human pancreas, adenocarcinoma, ATCC) were cultured in Iscove's Dulbecco medium/20% FBS/penicillin and streptomycin (100 U/ml). Capan-1 tumors were routinely produced by s.c. inoculation of 3 × 106 log-phase tissue culture cells in the right rear flank of nude mice. The mice protocols used in tumor regrowth delay were approved by the Institutional Animal Care and Use Committee at Roswell Park Cancer Institute.
Tumor growth assay.
Capan-1 tumor cells (3 × 106 cells) were inoculated s.c. into nude mice. At day 8–9 post implantation, when the tumors were palpable (6.5 × 5 mm), animals were treated with i.p. calcitriol or gemcitabine alone or in combination. Tumor growth was assessed by measuring tumor size with calipers three times/week. Tumor volumes were calculated by (length × width2)/2 and expressed as a fraction of pre-treatment size at the time of the first treatment.
In vitro cytotoxicity assay/CI index determination.
Capan-1 cells were suspended at 0.15 × 105 cells/ml and 100 µl/well dispensed into 96-well microtiter plates. The following day, various concentrations of each agent were added. Calcitriol was reconstituted in 100% ethanol (ETOH) and stored protected from light under a layer of nitrogen gas at −70°C. Dilutions of calcitriol were made in medium just prior to use. Gemcitabine was also diluted in medium just prior to use. The cells were harvested 48 h after treatment by adding 20 µl of a stock solution of 0.5% MTT (5 mg/ml) to each well. The plates were incubated for an additional 3–4 h at 37°C. Formazan crystals were dissolved with 100 µl of 10% SDS/10 mM HCl solution overnight at 37°C. The absorbance at 590 nm was measured using a SPECTRAmax340pc microplate reader. The CalcuSyn program (T.C. Chou and M.P. Hayball, Biosoft) was used to analyze the drug combinations. Constant ratios of drug concentrations were used in these studies, and mutually exclusive equations were used to determine the combination index (CI). CI < 1, = 1, > 1 indicates synergistic, additive and antagonistic effects, respectively.
CDDase activity assay.
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 harvested and rinsed twice with 5 ml of cold normal saline, 5 mM Tris-HCL buffer, pH 7.4 and stored at −80°C 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.Citation32,Citation33 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 × 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 sample and 5 µl of 4 mM pNA conjugated substrate was added to each sample and incubated at 37°C 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).Citation14 The data was analysed using Winlist™ 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.Citation14
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 <0.05 was considered significant. In all other analyses, statistical significances between groups were determined by two-tailed Student's t-test.
Grant support
This study was supported by NIH/NCI grants CA067267 and CA085142 to Dr. Candace S. Johnson and CA095045 to Dr. Donald L. Trump. It was also supported, in part, by the NCI Cancer Center Support Grant to the Roswell Park Cancer Institute (CA016056).
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-κB |
| VEGFR | = | vascular endothelial growth factor receptor |
| PDGFR | = | platelet-derived growth factor receptor |
| SCC | = | squamous cell carcinoma |
Figures and Tables
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.
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.
Figure 3 Induction of apoptosis by calcitriol and gemcitabine. Capan-1 cells were pretreated with either ETOH or 0.75 µM calcitriol for 24 h followed by gemcitabine (6.25 µM or 12.5 µM) for 24 h. Flow cytometric analysis of annexin V-PE and 7-AAD binding in Capan-1 cells was performed. The results shown are representative of three independent experiments.
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.
Figure 5 Calcitriol and gemcitabine inhibit Akt survival signaling pathway. Capan-1 cells were pretreated for 24 h with either ETOH or 0.75 µM calcitriol and then with varying concentrations of gemcitabine for another 24 h. Cells were lysed and the levels of phosphorylated Akt and ERK1/2 were assessed by Western blot analysis. Actin was the loading control. The results shown are representative of three independent experiments.
Figure 6 Calcitriol enhances gemcitabine antitumor activity in the Capan-1 pancreatic model system in vivo. Nude mice bearing Capan-1 tumors were treated with saline, calcitriol, gemcitabine or the combination of calcitriol and gemcitabine for 4 weeks. (A) Tumor measurements were taken three times a week. Differences in tumor volume were significant on day 21 (p < 0.05), day 24, 27, 30 (p < 0.01) observed for calcitriol treatment in combination with gemcitabine compared to saline or either agent alone. (B) Gross images of the Capan-1 tumors removed from each treatment group were presented.
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