Characterization of butyrate resistant colorectal cancer spheroid cells and its response to anticancer drugs

Abstract

Colorectal cancer (CRC) is the most common cancer worldwide, including Thailand. There are several risk factors for CRC, especially microbiome. Gut-microbiota plays a critical role in homeostasis and carcinogenesis. Butyrate, a short-chain fatty acid-producing by gut-microbiota, plays a role in intestinal homeostasis. Butyrate acts as anti-cancer agent by growth inhibition and apoptosis induction. However, microbiota study reveals that butyrate-producing bacteria were found in CRC patients more than normal and correlated to chemoresistance feature. We characterized the butyrate resistance (BR) CRC cells by treating the HCT-116 and PMF-ko14 cells line with a maximum butyrate concentration of 3.2 mM. The butyrate 50% inhibitory concentration (IC50) were increase in BR cells. The butyrate resistance mechanism was investigated with butyrate influx, and drug efflux genes expression. The increasing of influx and efflux gene expression in BR cells were found comparing to parental (PT) cells. Proteomic analysis was used to distinguish the normal and butyrate-resistant phenotype. Cell migration was used to evaluate the aggressive behavior of BR cells. The analysis reveal that HCT-BR cell show lower migration rate; however, the PMF-BR cell show higher migration rate than their PT cell. The cross-resistance to anti-cancer drugs was elucidated. We found the cross-resistance of metformin (MET) and oxaliplatin in HCT cells, and 5-fluorouracil was cross-resistance in PMF cells. Our study suggests that acquisition of resistance to butyrate induces chemoresistance in CRC cells, which may play an important role in CRC development, treatment, and metastasis. Moreover, we would like to further investigate the cytotoxicity of MET, an anti-diabetic drug with an anti-cancer activity, on PMF-BR -CRC cells in a 3D spheroid culture model. The results demonstrated that MET decreases spheroid size, viability, migration. Meanwhile, MET increases spheroid death through caspase 3/7 activity. The molecular mechanism from western blotting revealed that AMP-activated protein kinase (AMPK) and AKT serine/threonine kinase 1(Akt) were significantly upregulated in MET treatment group, whereas Acetyl-CoA-carboxylase (ACC) and mammalian target of rapamycin (mTOR) were downregulated. This situation leads to caspase activation and apoptosis. Our results confirm that MET show a potential cytotoxicity especially on the BR cells. This finding suggest that MET is an effective strategy for drug-resistant CRC cells.