3.3 Apoptosis Analysis Answers

2.3.3. Apoptosis analysis
The flow cytometry is one of the efficient and specific methods to investigate the cell death or apoptosis. The fractions of cell populations in different quadrants are analyzed using quadrant statistics such as lower left quadrant (R2-living cells), lower right quadrant (R3-early apoptosis cells), the upper right quadrant (R4- the late apoptosis cells) and the upper left quadrant (R5-dead cells). To examine the apoptosis effect of 3a, we treated HepG-2 cells with 25, 50, 75 and 100 µM concentrations of 3a for 24 h. Apoptotic cells were measured by annexin V-FITC/PI staining assay as per manufacturer’s guidelines. The results showed that after treatment with 3a for 24 h there was dramatically increase in the apoptosis
The expressions of Bcl-2, Bax, cytochrome c and caspase-3 proteins were analysed by western blotting for 24 h treatment. Bcl-2 is anti-apoptotic gene as it retards progression of the mitochondrial pathway in apoptosis. Bcl-2 is localized in the outer mitochondrial membrane and is known to maintain mitochondrial integrity. In addition, anti-apoptotic gene of Bcl-2 was involved in over expressions during all cancer diseases. Our results revealed that Bcl-2 expression was down regulated and Bax expression was increased after treatment with 3a against HepG-2 cells at 25 and 50 μM concentrations for 24 h (Fig. 8). The intrinsic apoptosis signal pathway is the release of cytochrome c in mitochondria and the extrinsic pathway is the activation of death receptors. Moreover, we evaluated the expression of apoptosis involving cytochrome c after treatment with 3a against HepG-2 cells. Our results showed that the level of cytochrome c was upregulated after treatment with 3a (Fig. 8). After expression cytochrome c binds with caspase-9 and activates caspase-3 to promote cell death or apoptosis. Caspases are a family of cysteine proteases and play pivotal role in the induction of apoptosis through cleavage of different substrates. In addition, caspase-3 is a key caspase that plays a critical role in apoptotic machinery. Our results showed that cleavage of caspase-3 was up
In 3a, O-H interacted with the N-H of Ser-936 and formed a hydrogen bond with the bond length of 2.8 Å. Also, C=O interacted with the N-H of Leu-932 and formed a hydrogen bond with the bond length of 1.8 Å. In addition to the hydrogen bonds, hydrophobic interaction was exhibited between phenyl ring of the 3a and Leu-855, Ala-880, Val-911, Leu-932, Leu-983 and Gly-993 amino acids. Binding interaction of the 3a with the JAK-2 is receptor shown in Figure 11B1 and 11B2. Compound 3a showed the binding energy value of -6.04 kcal/mol with the docked Bcl-2 receptor and it was interacted with the 14 active sites of amino acids namely Phe-63, Tyr-67, Asp-70, Phe-71, Met-74, Val-92, Leu-96, Gly-104, Arg-105, Ala-108, Glu-111, Phe-112 and Val-115. In 3a, O-H interacted with the C=O of Ala-108 and formed a hydrogen bond with the bond length of 1.9 Å. Furthermore, π- π interaction was exhibited between phenyl rings of the 3a and Phe-63, Tyr-67, Phe-71 and Phe-112 amino acids. In addition to the hydrogen bond and π-π interactions, hydrophobic interaction was exhibited between phenyl ring of the compound with