Reversal of Warburg effect by Apigenin and 5-Fluorouracil loaded dual drug liposomes result in enhanced colorectal chemotherapy

Abstract

Recent combinatorial approaches have demonstrated multidrug-based chemotherapy which target multiple pathways simultaneously leading to enhanced efficacy and reduced toxicity. Our study reports simultaneous delivery of 5-Fluorouracil (a clinically approved drug) and Apigenin (a naturally occurring isoflavonoid abundantly present in fruit and vegetables) through a single liposome to combat and control colon cancer effectively in-vitro and in-vivo based on synergy studies after screening natural product library. Uniform homogeneous suspension of dual drug-loaded large unilamellar vesicles with substantial entrapment of both drugs was obtained by modified thin film hydration method. The vesicles exhibited excellent hemocompatibility and cytocompatibility against normal fibroblasts. The cytotoxicity and cellular effects of individual and combinatorial free drugs, neat vesicles and single or dual drug-loaded liposomes tested against different human colon cancer cell lines revealed significantly higher cytotoxicity of dual-drug liposomes in comparison to other formulations. The dual drug loaded liposomal formulation was observed to be effectively internalized by endocytosis and demonstrated enhanced inhibition of angiogenesis, better reduction in cell proliferation, increased apoptotic potential and significantly decreased cellular migration and invasion capacity. Cell signaling studies indicating a significant upregulation of AMPK and activity against downstream targets by dual drug liposomes resulted in apoptosis, autophagy and senescence, suggesting its role in the reversal of Warburg effect. Enhanced apoptotic potential of the formulation was traced to its ability to induce greater oxidative stress through the generation of reactive oxygen and nitrogen species. The formulation was tested in a preclinical setting in nude mice tumor xenograft model and was found to have greater anti-neoplastic and anti-tumorigenic effect. The study indicated the potential of the synergistic effect of Apigenin in 5-Fluorouracil treatment of colon cancer which effectively circumvents the problem of drug resistance and 5-Fluorouracil associated toxicity. This dual drug loaded liposomal formulation thus, offers a new attractive alternative to enhance the therapeutic potency of drugs and paves way for potential clinical applications.