13 1.3 Conclusion and Discussion

Discussion

PI3K/Akt/mTOR Signalling Pathway

Within the A549 cell, PI3K/Akt/mTOR signalling plays a regulatory role in cancer activity as a result of over activation. Vitexin suppresses the mTOR pathway, however in this study the researchers did not examine possible molecular mechanisms that might be responsible. The authors confirmed vitexin had a downregulating effect on mTOR by pretreating the Akt activator SC79, which resulted in blocking the apoptotic effect that was observed; however, the downstream effects were not clearly defined.

Bcl-2/Bax Ratio

The other signalling pathway observed is the regulation of the Bcl-2/Bax protein ratios. Bcl-2 is an anti-apoptotic protein whereas Bax is a pro-apoptotic protein. Vitexin reduced the Bcl-2/Bax ratio (Bcl-2 down regulated, while Bax was up-regulated). This caused the release of cytochrome c from the mitochondria into the cytosol of the cell. Cytochrome c is a heme protein, crucial to the process of respiration^[1]. Cytochrome c is also considered a death-promoting protein.^[2] As stated in Chapter 1.2 , the release of cytochrome c indicates mitochondrial distress since cytochrome c is originally harbored within the intermembrane space of the mitochondria.^[3] This signifies that vitexin might target the mitochondria of A549 cells. Once the cytochrome c was in the cytosol, caspase 3 was cleaved. The significance of this, though not explicitly mentioned in this study, is that upon cytochrome c entering the cytosol, apoptosomes are formed. This is followed by the cleavage of caspase 3, which activates the protein. Caspase 3 is known as the executioner caspase because of its several roles leading to apoptosis, such as: DNA fragmentation, cytoskeletal protein degradation, as well as cleaving over 1000 other proteins resulting in degradation.^[4][5][6].

Considering the presence of a low Bax/Bcl-2 ratio in the study done by Liu et al (2019), a proposed mechanism in which apoptosis was successfully induced, was the simultaneous completion of MOMP (mitochondrial outer membrane permeabilization). MOMP is controlled by a series of events that chooses between promoting either the survival or death of the cell. For successful apoptosis through a mitochondrial-dependant/ intrinsic mechanism, MOMP is required.^[7] MOMP allows for the translation of cellular stress into apoptotic initiation.^[8] MOMP is directly controlled by the Bcl-2 protein family; particularly, the pro-apoptotic domain of Bcl-2, known as Bax. When Bax is constitutively expressed, it is suggested that MOMP is underwent following apoptotic stimuli.^[9] Bax is inactive in non-apoptotic cells, and only inserts into the mitochondrial membrane upon activation by an apoptotic stimuli.^[10] Bax is then oligomerized, creating a pore within the outer mitochondrial membrane which enables the release of cytochrome c and other intramembrane proteins into the cytosol.^[11] The reliability of Bax is shown in another study, which presented the effects of mice lacking Bax. This resulted in the resistance against apoptosis-inducing insults, in hopes of maintaining cellular viability.^[12] This signifies the effect that Bax/Bcl-2 balance has on promoting either cell survival or cell death, as well as the possible effect that vitexin might have on the A549 cells. Understanding more pathways that are implicated may be useful for researchers as they can research more specific targets to prevent carcinoma proliferation.

For further knowledge on apoptosis, please review this video on the mechanism of apoptosis:

LDH Release

Lastly, vitexin induced the release of LDH ( lactase dehydrogenase), which indicates cell membrane damage as a result of cell death by apoptosis.^[13] This signifies that vitexin induced cell death, which is important to the resulting effect of vitexin.