Combinations of vitamins A, D2 and D3 have synergistic effects in gastric and colon cancer cells

Abstract

Background: Vitamin D was first hypothesized to play a role in cancer chemoprevention in 1980 when it was observed that there was a higher rate of colon cancer in the Northern USA as compared with populations living in the South. While cholecalciferol (vitamin D3) has been tested in many cancer cell lines, published results for ergocalciferol (vitamin D2) are lacking for most epithelial cell cancers, and combination studies of both D2 and D3 and vitamin A (retinoic acid) are lacking in general. The goal of the study was to investigate the effects of vitamins D2, D3, and A, and combinations on the growth of all gastric and colon cancer cell lines in vitro.Methods: Cell viability and cytotoxicity were determined using the CellTiter-Glo® 2.0, caspase and cytotoxicity activities were determined with Caspase-Glo® 3/7, Caspase 8, ApoTox-Glo™ Triplex Assay. Autophagy was determined using the CYTO-ID® Autophagy Detection Kit 2.0. Gene expression studies were performed using qPCR.Results: Both vitamin D2 and D3 inhibited the growth of all cell lines tested. The IC50 ranged from 19-56 μM. However, when combined (1:1), the IC50 for the combination of D2 and D3 was significantly reduced to a range of 5-6.0 μM indicating synergism. Vitamin A in the form of all trans-retinoic acid (ATRA) also inhibited the growth of all cell lines tested with an IC50 range of 2.6 to 5.6 μM. When ATRA was combined with D2 and D3, the IC50s were again significantly reduced to 0.65 to 1.4 μM, indicating synergistic effects. In gastric and colon cancer cell lines, the combination induced apoptosis via caspase 3/7 and 8, increased the Bax/Bcl-2 ratio; induced autophagy in SW480 cells, increased p53 expression and inhibited the expression of HDACs.Conclusion: Our data demonstrate that vitamins D2, D3 and ATRA reduce the proliferation of colon and gastric cancer cells by increasing Bax expression and the Bax/Bcl-2 ratio, and by increasing caspase activities in favor of apoptosis. The ATRA+D2+D3 combination had synergistic effects in all cell lines tested. In HCT 116 colon cancer and AGS gastric cancer cells, the combination also inhibited HDAC 1/3 and SIRT1/3 and increased p53 expression. While in SW480 colon cancer cells the combination also induced autophagy.Key words: apoptosis, autophagy cholcalciferol, ergocalciferol, gastric cancer, colon cancer, caspase, HDAC inhibitors, SIRT1, synergism