In this study, after several trials, it was found that the percentage of cell death in NDV-infected Bax-knockout cells was lower compared to wt cells. This showed that the presence of Bax (and its subsequent interaction with the viral proteins such as the M protein) speeds up the apoptosis event. Since the Bax-knockout cells die following infection with the virus, it can be concluded that NDV is able to interact with apoptotic proteins other than Bax as well. The release of cytochrome c into cytosol proves that the mitochondrial pathway is activated through some other interaction(s) that directly or indirectly affected the proteins of the surface of mitochondria, allowing formation of pores that release mitochondrial factors.
Many Bcl-2 family members interact with each other via the BH domains
. For instance, Bax binds to several of these proteins by its BH3 domain
[23, 24] resulting in a series of complex interactions. Since the M protein of NDV contains a BH3 domain that interacts with Bax, it could be possible that in the absence of Bax, the M protein activates other pro-apoptotic members. However, so far this viral protein has shown no interaction with Bad, Bcl-XL and BimL
. Fusion (F) protein also failed to interact with the mentioned Bcl-2 members. Moreover, the BH3-like regions on the large (L) protein of NDV have not yet been examined for their ability to bind to Bcl-2 family proteins.
Bcl-2 normally blocks Bax oligomerization to inhibit its insertion into the mitochondria
. Since NDV infection also leads to apoptosis in absence of Bax, the classic Bax/Bcl-2 ratio
[10–12] may not be a major determinant in NDV-induced apoptosis. This statement is in agreement with our previous findings that there was very little alteration on mRNA and protein levels of Bax and Bcl-2 following infection with NDV
[9, 17]. Moreover, little is known about the role of anti-apoptotic members of the Bcl-2 family during NDV infection. Since HT29 cells are Bcl-2-free, it will be interesting to check if the absence of Bcl-2 has a significant role in NDV-induced apoptosis at all, or its overexpression has any effect on the apoptosis rate.
Bak and Bax have almost the same protein structure, share similar BH domains and are the only two pro-apoptotic Bcl-2 family proteins discovered so far that form oligomeric pores on the mitochondria surface. Bak can independently release cytochrome c from mitochondria in absence of Bax
, and therefore these characteristics make it an excellent candidate for future studies. Although Bax is a cytosolic protein and Bak localizes to mitochondria, they both facilitate the release of cytochrome c from mitochondria through MPTP pores
BH3-only members such as Bid and Bad are located upstream of mitochondria that upon activation move towards mitochondria to, respectively, activate Bak and Bax
 and inhibit Bcl-2 and Bcl-XL function
 to facilitate release of mitochondrial factors. Therefore, if NDV proteins directly or indirectly (through activation of BH3-only protein) activate Bak in HCT116 Bax−/− cells then perhaps this could be a possible reason behind the release of cytochrome c. Use of Bak−/− cells in future studies will definitely let us investigate this hypothesis further. On the other hand, it has been previously shown that NDV kills Bcl-XL-overexpressing cells that are resistant to Bak overexpression
 and also overcomes the anti-apoptotic function of Livin
, a novel member of the inhibitor of apoptosis protein (IAP) family frequently overexpressed in melanoma. Moreover, it has been found that during infection with RNA viruses a BH3-like domain of IRF-3 mediates binding to cytosolic Bax (but not Bcl-2, Bcl-XL or Bak) to induce Bax activation, culminating in cytochrome c release and apoptosome formation
. Together it could be further suggested that proteins located upstream of mitochondria are strong candidates for such activities.
Overall, it was concluded that Bax is not the only cellular protein that NDV proteins interact with to trigger apoptosis. However, Bax plays a role in the apoptosis of HCT116 cells, since the lack of Bax contributes to slower cell death. This further suggested that the interaction between Bax and NDV proteins, such as that with the M protein
, is crucial and acts as determinant during NDV-induced apoptosis. Further study is needed to find other possible interactions between NDV proteins and cellular apoptotic proteins.