Anticancer, anti-inflammatory, and immune modulatory activity of ferulic acid fructo-oligosaccharide conjugated microparticle

Background and purposeFerulic acid exhibit anticancer activity but almost most of the free ferulic acid taken orally are absorbed in the stomach and extensively metabolised by the liver and hence hardly any free ferulic acid reach the large intestine to exert its beneficial activity. Fructo-oligosaccharide (dietary fibre) are resistant to gastro-intestinal enzymes and are poorly absorbed by the stomach but bioavailable in the large intestine where they are digested by gut microbiota. Ferulic acid fructo-oligosaccharide conjugate was synthesized which could self-assemble in to disc shaped amorphous microparticles, it was found to be resistant to gastro-intestinal enzymes and digestion by gut microbiota. The synthesized microparticles could be used for targeted delivery to the colon and accessed for its ability to ameliorate colo-rectal cancer and inflammation. Experimental approachThe anti-cancer activity of the FA FOS microparticle (FA FOS I) was tested in human colon cancer cell lines HT29, LoVo and compared with the toxicity to normal human colon fibroblast CCD18-Co, relative to that of conventional chemotherapeutic colon cancer drug oxaliplatin. The apoptosis induction by FA FOS I was assessed by TUNNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick-end Labelling) and FACS. The ability of the FA FOS microparticle to induce cell cycle arrest was determined. The gene expression profiling of both apoptosis related genes and cell cycle arrest related genes were analysed by using RT-PCR analysis of an array of apoptosis related genes and cell cycle related genes. In-vivo pre-clinical anti-colorectal cancer studies of FA FOS I microparticle were carried out in AOM-DSS mediated colitis associated colon cancer mice model (AOM DSS CAC) to determine its anti-cancer efficacy in the physiological, immunological and innate host microbiota setting. Key resultsThe in-vitro studies in colon cancer and normal colon cells exhibited selective cytotoxicity and apoptosis induction in colon cancer cells. The microparticle arrested the cell cycle in the G0-G1 phase. There was a reduction in 60.83% of tumour lesions in FA FOS I treated group compared to control group. The H&E histochemistry of the colon tissue revealed that there was 48.27% reduction in the malignant cell or tumour cells in the colon tissue on treatment with FA FOS I. The FA FOS conjugate treatment enhanced the gut barrier function and tight junction with the intestinal barrier guarded by the mucosal lining. The immunohistochemistry (IHC) and the immunofluorescence of the mouse colon tissue revealed the suppression of inflammation and related inflammatory cytokines in the colon. The inhibition of cell proliferation, up-regulation of tumour suppressor protein and apoptosis of the malignant or tumour cells were detected and quantified by IHC and TUNEL staining. The evaluation of immune status of the AOM DSS CAC mouse treated with FA FOS I microparticle was determined using haematological analysis of the blood lymphocytes which revealed a 9% increase in WBC count and the multiplex immunofluorescence of the colon tissue revealed an increase in the infiltration of T-helper cells and cytotoxic T-cells into the tumour microenvironment followed by the cells of the innate immune system. There was a considerable decrease in the expression of tumour suppressing PD-L1 by the tumour cells on four weeks treatment with FA FOS I microparticle. Conclusion and implicationsAll these data implicate better efficacy of the FA FOS I microparticle delivery to colon and amelioration of colo-rectal cancer, inflammation, and positive immune modulation of tumour microenvironment against tumour proliferation.