In vitro and in silico anticancer activities of Annona muricata linn leaves extracts on lung cancer cells

Lung cancer specifically non-small cell lung cancer (NSCLC) is one of the most devastating cancers. Despite having diverse treatment methods such as surgery, chemotherapy, radiation and targeted therapies, the overall 5-year survival rate for NSCLC is accounted for only 18.2 %. The high mortality rates for NSCLC are partially due to the lack of effective prognostic factors such as biomarkers. Nowadays, plantderived bioactive substances have been making way as potential anticancer agents. One such plant is Annona muricata Linn, which is also known as soursop or graviola. This plant has been widely reported to contain valuable phytochemical substances that could be developed as chemopreventive agents. The antiproliferative and anticancer activities of this tropical plant have been demonstrated in in vitro cell culture studies as well as in in vivo studies of animals. It has been discovered that A. muricata L. extract exerts inhibition against various number of cancer cells, involving multiple mechanism of actions. Nonetheless, the mode of action and the molecular interactions of the plant have not yet been unveiled for most of these mechanisms. In the current study, response surface methodology was utilized to optimize the ultrasonic-assisted extraction (UAE) and maceration extraction (ME) of A. muricata L. leaves. Central composite design was applied to optimize the antioxidant activity of both extracts. It has been found that ME extract showed high antioxidant activity as compared to UAE extract with 83.3 % and 31.6 % respectively. However, through cytotoxicity study that was done by using M TT assay, UAE extract exhibited significant cytotoxicity effects in NSCLC cell line (HLFa) with IC50 of 139.6 p,g/mL as compared to ME extract with IC30 of 108.4 p,g/mL. The effect of the extracts on nitric oxide (NO) production in HLFa cells was also evaluated using Griess reagent system assay. Both extracts reduced the release of nitrite in the cell supernatant which indicated the reduction in NO production. Caspase 3/7 apoptosis assay was used to detect the presence of apoptotic machinery in HLFa cells after incubation. The mRNA expression of several genes namely HMGB1, BCL2 and BAX were quantified. The mechanistic evalution of the results showed the possibility of involvement of these genes in A. muricata L. anticancer effects. At the end of this study, in silico molecular docking interaction of several phytoconstituents of A. muricata L. was analysed against Bcl-2 antiapoptotic proteins namely Bcl-2, Bcl-w and Mcl-1. The stability of complexes formed was evaluated using molecular dynamic simulation. Anonaine was also detected in UAE and ME extracts through HPLC screening process with 10.6 ppm and 10.7 ppm, respectively