Oxidative stress triggered by naturally occurring flavone apigenin results in senescence and chemotherapeutic effect in human colorectal cancer cells

Recent studies involving phytochemical polyphenolic compounds have suggested flavones often exert pro-oxidative effect in vitro against wide array of cancer cell lines. The aim of this study was to evaluate the in-vitro pro-oxidative activity of apigenin, a plant based flavone against colorectal cancer cell lines and investigate cumulative effect on long term exposure. In the present study, treatment of colorectal cell lines HT-29 and HCT-15 with apigenin resulted in anti-proliferative and apoptotic effects characterized by biochemical and morphological changes, including loss of mitochondrial membrane potential which aided in reversing the impaired apoptotic machinery leading to negative implications in cancer pathogenesis. Apigenin induces rapid free radical species production and the level of oxidative damage was assessed by qualitative and quantitative estimation of biochemical markers of oxidative stress. Increased level of mitochondrial superoxide suggested dose dependent mitochondrial oxidative damage which was generated by disruption in anti-apoptotic and pro-apoptotic protein balance. Continuous and persistent oxidative stress induced by apigenin at growth suppressive doses over extended treatment time period was observed to induce senescence which is a natural cellular mechanism to attenuate tumor formation. Senescence phenotype inducted by apigenin was attributed to changes in key molecules involved in p16-Rb and p53 independent p21 signaling pathways. Phosphorylation of retinoblastoma was inhibited and significant up-regulation of p21 led to simultaneous suppression of cyclins D1 and E which indicated the onset of senescence. Pro-oxidative stress induced premature senescence mediated by apigenin makes this treatment regimen a potential chemopreventive strategy and an in vitro model for aging research. Keywords: Apigenin, Oxidative stress, Reactive oxygen species, Pro-oxidation, Senescenc

Probing the potential of apigenin liposomes in enhancing bacterial membrane perturbation and integrity loss

Along with discovery of new antibacterial agents, it is important to develop novel drug delivery systems to effectively deliver drugs within bacterial cells for enhanced therapeutic activity. Liposomes have been extensively investigated as pharmaceutical carriers for improvement of therapeutic index of antimicrobial agents. The aim of this present study was to evaluate the antibacterial activity of free and liposomal formulation of apigenin, a plant based isoflavone and elucidate the mode of action. Distearoylphosphatidylcholine liposomes were prepared having nano-range particle size (104.3 ± 1.8 nm), narrow particle distribution (0.204) and high encapsulation efficiency of apigenin (89.9 ± 2.31%). Antibacterial activity of apigenin and efficacy of liposome-mediated apigenin delivery were determined from minimum inhibitory concentration values. Interaction studies using electron microscopy revealed adherence and fusion of liposomal apigenin with the bacteria causing membrane perturbation through reactive oxygen species generation which was evaluated by epi-fluorescence microscopy and fluorescence activated cell sorting. The interaction of apigenin liposomes with bacterial membrane increased intracellular drug concentration and thus, can be employed to deliver apigenin within cells to augment its antibacterial activity. Increased efficacy and hemocompatibility of this formulation paves way for future evaluation of underlying molecular mechanisms and in vivo testing for enhanced therapeutic effects

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

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