*3.5. Apoptosis*

Apoptosis is a physiological programmed cell death mechanism which is essential for the flawless growth and development of organisms. Furthermore, it is a lively physiological course causing the self-destruction of cells that comprises lethal biochemical and morphological changes in the nucleus and cytoplasm. During cellular strain like oxidative stress and DNA damage, the process of apoptosis can arise, particularly in cells with high proliferation rates and a high expression of pro-apoptotic genes [93]. Intrinsic and extrinsic pathways regulate apoptosis, but both pathways are associated and the molecules involved in those pathways can influence one another.

Previous findings have demonstrated that an *M. koenigii* extract and its primary active compounds regulate multiple signaling pathways, including phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT), mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK). *M. koenigii* and its primary active compounds exert complementary effects on oxidative stress and the alteration of proteins [94,95]. They are associated with mitochondrial-mediated apoptotic pathways. Murrayazoline and O-methylmurrayamine were shown to induce the downregulation of Akt/mTOR, suggesting downstream targeting of the cell survival pathway and an ability to potentiate the antitumor activity of D.L. Dexter (DLD-1) colon cancer cells; interestingly, this inhibition of the Akt/mTOR pathway could possibly activate the intrinsic apoptotic program [86]. Mahanine and isomahanine derived from *M. koenigii* leaves exert anticancer effects on oral squamous cell carcinoma cells via the induction of microtubule-associated protein 1 light chain 3, type II (LC3B-II), and cleaved caspase-3, suggesting the inhibition of autophagic flux [96]. In human leukemic cells, Mahanine was reported to induce apoptosis by interrupting signal transfer between Apo-1/Fas signaling and the Bid protein and via mitochondrial pathways in humans [59]. Recently, it was reported that girinimbine, a carbazole alkaloid isolated from *M. koenigii*, inhibited the growth of and induced apoptosis in human hepatocellular carcinoma cells (HepG2) [97]. In addition, Xin et al. reported that girinimbine inhibited ovarian cancer cell proliferation in a dose-dependent manner. It also inducted apoptosis and cell cycle arrest due to inhibition of the PI3K/AKT/mTOR and Wnt/b-catenin signaling pathways [98].

The alkaloid koenimbin found in *M. koenigii* was shown to extend pro-apoptotic activities in MCF-7 cancer cells by inhibiting glycogen synthase kinase-3 beta (GSK-3β). Koenimbin induces apoptotic cell death, phosphorylation, the accumulation of β-catenin, and the activation of nuclear factor-κB (NF-kB) in cancers. Moreover, koenimbin suppresses the expression of various anti-apoptotic genes involved in the regulation of cell proliferation and apoptosis [99]. Koenimbine has also been reported to trigger caspase activation, induce the release of cytochrome c, decrease the anti-apoptotic proteins, and increase the pro-apoptotic proteins, and all of these events lead to intrinsic apoptotic pathway activation [99]. Another study demonstrated that pyrayafoline-D and murrafoline-I isolated from *M. koenigii* could induce apoptosis in HL-60 cells. The same study also induced the loss of mitochondrial membrane potential and the subsequent activation of caspase-9/caspase-3, leading to the activation of apoptotic pathways [100].
