Metformin and Calcitriol Enhance 5-Fluorouracil Tumoricidal Effects in Colon Cancer By Modulating The PI3K/Akt/PTEN/mTOR Network

Abstract

Purpose: Chemoresistance to 5-Fluorouracil (5-FU) is common during colorectal cancer (CRC) treatment. This study measured the chemotherapeutic effects of 5-FU, calcitriol, and/or metformin single/dual/triple regimens as complementary/alternative therapies. Methods: Ninety male mice were divided into: negative and positive (PC) controls, 5-FU, Cal, Met, 5-FU/Cal, 5-FU/Met, Cal/Met, and 5-FU/Cal/Met groups. Treatments lasted four weeks following CRC induction by azoxymethane. The therapeutic regimens were also applied in the SW480 and SW620 CRC cell lines. Results: The PC mice had abundant tumours, markedly elevated proliferation markers (survivin/CCND1) and PI3K/Akt/mTOR alongside reduced p21/PTEN/Cytochrome-C/Caspase-3 and apoptosis. All therapies reduced tumour numbers, with 5-FU/Cal/Met most prominent regimen. All protocols also decreased cell proliferation markers, inhibited PI3K/Akt/mTOR molecules, increased pro-apoptotic molecules with apoptosis index, and 5-FU/Cal/Met revealed the strongest anti-cancer effects. In vitro, all therapies equally induced G1-phase arrest in SW480 cells, whereas metformin-alone showed maximal SW620 cell numbers in G0/G1-phase. 5-FU/Met co-therapy also showed the highest apoptotic SW480 cell numbers (13%), whilst 5-FU/Cal/Met disclosed the lowest percentage (81%) of viable SW620 cells. Moreover, 5-FU/Cal/Met revealed maximal inhibitions of cell cycle inducers (CCND1/CCND3), cell survival (BCL2) and the PI3K/Akt/mTOR molecules alongside highest expression of cell cycle inhibitors (p21/p27), pro-apoptotic markers (BAX/Cytochrome-C/Caspase-3), and PTEN in both cell lines. Conclusions: Metformin monotherapy was superior to calcitriol, whereas the 5-FU/metformin protocol showed better anti-cancer effects relative to the other dual therapies. However, the 5-FU/Cal/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.

Profiling estrogen, progesterone, and androgen receptors in colorectal cancer in relation to gender, menopausal status, clinical stage, and tumour sidedness

“…All cells were sub-cultured, and growth maintained at 37°C in a humified incubator with 5% CO 2 . Following seeding in 96-well plates, both cell lines were treated with each drug alone for 48h to determine the concentrations associated with 50% inhibition (IC50) or 10% increase (EC10) of cell viability using the MTT cytotoxicity assay, as described earlier (42,43).…”

“…Propidium iodide (PI; 2 μg/ml; Thermo Fisher) was used to stain cellular DNA immediately prior to cell cycle analysis with an Acea Novocyte 3000 flow cytometer (Agilent Technologies, CA, USA). The percentage of cells in each phase of cell cycle were determined for 20,000 events (n = 3/group) by the NovoExpress software cell cycle algorithm, as reported earlier (42,43).…”