Vitamin D analogs enhance the anticancer activity of 5-fluorouracil in an in vivo mouse colon cancer model

Magdalena Milczarek 0 Mateusz Psurski 0 2 Andrzej Kutner 1 Joanna Wietrzyk 0 0 Department of Experimental Oncology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences , R. Weigla St. 12, Wroclaw 53-114 , Poland 1 Pharmaceutical Research Institute , L. Rydygiera St. 8, Warsaw 01-793 , Poland 2 Division of Medicinal Chemistry and Microbiology, Wroclaw University of Technology , Wroclaw , Poland Background: Active vitamin D analogs that are less toxic than calcitriol can be useful in the combined treatment of patients suffering from colon cancer. In the present study we demonstrate, for the first time in an in vivo model system, the biological effect of combined therapy using 5-fluorouracil (5-FU) along with vitamin D analog PRI-2191 (tacalcitol, 1,24-dihydroxyvitamin D3) or PRI-2205 (5,6-trans-isomer of calcipotriol) on colon cancer. Methods: We investigated the influence of vitamin D analogs on the anticancer activity of 5-FU or capecitabine in the treatment of mice bearing MC38 mouse colon tumors implanted subcutaneously or orthotopically. The cell cycle distribution, E-cadherin expression and caspase 3/7 activity in vitro were also evaluated. Results: We observed that both PRI-2191 and PRI-2205 significantly enhanced the antitumor activity of 5-FU; but these results depend on the treatment regimen. Applying the optimal schedule of combined therapy we observed a significant decrease in tumor growth, metastasis and also a prolongation of the survival time of mice, in comparison with the administrations of 5-FU given alone. Both combinations indicated a synergistic effect and did not cause toxicity. Moreover, analogs applied after completed course of administration of 5-FU, prolonged the antitumor effect of the drug. Furthermore, when the prodrug of 5-FU, capecitabine, was used, potentiation of its activity was also observed. Conclusions: Our data suggest that vitamin D analogs (especially PRI-2191) might be potentially applied to clinical use in order to enhance the anticancer effect of 5-FU and also prolong its activity against colon cancer. The activity of PRI-2191 is realized through stopping the cells in the G0/G1 cell cycle phase and increasing the expression of E-cadherin. - Background According to the World Health Organizations International Agency for Research on Cancer, colorectal cancer is the third most frequent malignancy and the fourth leading cause of deaths from cancer worldwide [1]. Despite significant progress in the treatment of patients suffering from colorectal cancer in the last decade, there is a constant need for new therapies. One of the directions is the development of novel combined treatment strategies. The benefit of such an approach is the possibility of enhancing the therapeutic effect of a drug, which is the basis of a standard therapy. Promising candidates for this strategy are vitamin D analogs. Epidemiological and clinical data and also research on animals suggest a protective role for the active form of vitamin D (calcitriol, 1,25-dihydroxyvitamin D3) in the development of colon cancer [2-4]. Data from the in vivo studies have shown that a diet supplemented with vitamin D significantly delayed MC-26 colon cancer tumor growth compared to a diet deficient in this vitamin [5]. Calcitriol affects proliferation, differentiation and apoptosis of human colon cancer cells. It exerts a biological effect mainly through the vitamin D receptor (VDR) [6]. It has been shown that the expression of VDR increases from normal colon epithelial cells through precancerous lesions to well-differentiated tumors and then decreases in advanced stages of cancer [6,7]. The antitumor activity of calcitriol is observed only when it is applied in hyper-physiological doses, which can cause the side effect of hypercalcemia and hypercalciuria [8-10]. For this reason, the synthesis of analogs has been initiated in order to dissociate the calcemic effect from the anticancer activity of calcitriol. In our previous studies, we have examined the biological activity of a series of side-chain modified analogs of vitamin D and a series of diastereometric and geometric ones against various cancer and normal cell lines [11,12]. We also evaluated the influence of vitamin D analogs on the activity of a range of anticancer drugs in vitro and in vivo against the human and murine cancer cells [13-18]. We observed that vitamin D analogs increased the antitumor effect of cyclophosphamide and cisplatin compared to the cytostatic drug applied alone. Based on our results, we selected two analogs for further research: PRI-2191 (tacalcitol, 1,24-dihydroxyvitamin D3) and PRI-2205 (5, 6-trans calcipotriol), which reveal higher antitumor activity and lower calcemic activity, as well as lower toxicity than calcitriol [12,19]. These two analogs, used in combined HT-29 colon cancer treatment with irinotecan or oxaliplatin showed, in selected schedules of treatment, improvement in mice survival and tumor growth delay [20]. 5-Fluorouracil (5-FU) is one of the oldest anticancer drugs and is still used in the treatment of colorectal cancer [21,22]. Two recent reports of in vitro studies demonstrate that calcitriol and calcipotriol promote the sensitivity of human colon carcinoma cells to 5-FU and enhance the cytotoxicity of the FOLFIRI anticancer regimen. These results also indicate that the mechanism of calcitriol and calcipotriol action is dependent on the calcium sensing receptor (CaSR). Protein expression and the gene transcriptional activity of survivin and thymidylate synthase are suppressed by inducing the expression and activation of CaSR by calcitriol or calcipotriol. This leads to an increase in the sensitivity of colon carcinoma cells to 5-FU [23,24]. Therefore, the aim of our present studies was to examine the biological effect (antitumor activity, influence on the life span of mice, toxicity and antimetastatic activity) of combined therapy with the use of 5-FU along with PRI-2191 or PRI-2205 against MC38 mouse colon cancer in vivo. In addition, we examined whether vitamin D analogs would prolong the antitumor activity of 5-FU (application of analogs was initiated after administration of 5-FU ended). Methods Compounds Calcitriol and its analogs: PRI-2191, PRI-2201 and PRI-2205 are certified synthetic materials obtained from the Pharmaceutical Research Institute, Warsaw, Poland. Samples of the compounds were stored, under argon, in amber ampoules at 20C. Prior to usage, in the case of in vitro studies, compounds were dissolved in 99.8% ethanol to the concentration of 10-4 M and subsequently diluted in culture medium in order to reach the concentration of 100 nM. For animal experiments, compounds were dissolved in 99.8% ethanol, then diluted in 80% propylene glycol in order to reach the required concentrations and administered subcutaneously (s.c.) or orally (p.o.) to mice in a volume of 5 l per 1 g of body weight. 5-Fluorouracil (5-FU) (...truncated)