In Vivo and In Vitro Enhanced Tumoricidal Effects of Metformin, Active Vitamin D(3), and 5-Fluorouracil Triple Therapy against Colon Cancer by Modulating the PI3K/Akt/PTEN/mTOR Network

SIMPLE SUMMARY: Failure of chemotherapy is common during the treatment of colon cancer, and there is a compelling need to develop alternative therapeutic approaches against this common malignancy. Metformin, which is an oral hypoglycaemic agent used for treating diabetes mellitus, and vitamin D have shown promising anticancer activities, and both agents boosted the actions of chemotherapy against colon cancer. This study, therefore, measured the potential beneficial effects of adding metformin and/or active vitamin D to the main cytotoxic drug used for treating colon cancer. The results demonstrate that metformin had superior anticancer effects relative to active vitamin D and ameliorated the effects of chemotherapy in animals and in cells. To the best of our knowledge, this study is also the first to report that triple treatment with the drugs of interest showed the best inhibition of cancer progression, which could provide a better therapeutic strategy against colon cancer. ABSTRACT: Chemoresistance to 5-fluorouracil (5-FU) is common during colorectal cancer (CRC) treatment. This study measured the chemotherapeutic effects of 5-FU, active vitamin D(3) (VD(3)), and/or metformin single/dual/triple regimens as complementary/alternative therapies. Ninety male mice were divided into: negative and positive (PC) controls, and 5-FU, VD(3), Met, 5-FU/VD(3), 5-FU/Met, VD(3)/Met, and 5-FU/VD(3)/Met groups. Treatments lasted four weeks following CRC induction by azoxymethane. Similar regimens were also applied in the SW480 and SW620 CRC cell lines. The PC mice had abundant tumours, markedly elevated proliferation markers (survivin/CCND1) and PI3K/Akt/mTOR, and reduced p21/PTEN/cytochrome C/caspase-3 and apoptosis. All therapies reduced tumour numbers, with 5-FU/VD(3)/Met being the most efficacious regimen. All protocols decreased cell proliferation markers, inhibited PI3K/Akt/mTOR molecules, and increased proapoptotic molecules with an apoptosis index, and 5-FU/VD(3)/Met revealed the strongest effects. In vitro, all therapies equally induced G1 phase arrest in SW480 cells, whereas metformin-alone showed maximal SW620 cell numbers in the G0/G1 phase. 5-FU/Met co-therapy also showed the highest apoptotic SW480 cell numbers (13%), whilst 5-FU/VD(3)/Met disclosed the lowest viable SW620 cell percentages (81%). Moreover, 5-FU/VD(3)/Met revealed maximal inhibitions of cell cycle inducers (CCND1/CCND3), cell survival (BCL2), and the PI3K/Akt/mTOR molecules alongside the highest expression of cell cycle inhibitors (p21/p27), proapoptotic markers (BAX/cytochrome C/caspase-3), and PTEN in both cell lines. In conclusion, metformin monotherapy was superior to VD(3), whereas the 5-FU/Met protocol showed better anticancer effects relative to the other dual therapies. However, the 5-FU/VD(3)/Met approach displayed the best in vivo and in vitro tumoricidal effects related to cell cycle arrest and apoptosis, justifiably by enhanced modulations of the PI3K/PTEN/Akt/mTOR pathway.