MPM is a very aggressive malignancy with an extreme resistance to current treatments. Evasion of apoptosis induction due to the inactivated apoptosis machinery and/or up-regulated anti-apoptotic mechanisms, is largely responsible for its resistance to treatments . A new treatment with ability to selectively activate the apoptotic machinery while inhibiting multiple antiapoptotic mechanisms in MPM cells will be a promising candidate treatment for MPM. This study reveals that the TRAIL and PI combinatorial treatment can induce a robust proapoptotic activity in MPM cells of all three major pathological types, but not in normal mesothelial cells. The robust activity is achieved through the PFM-governed caspase activation and subsequent protein cleavage of both Akt and Mcl-1, the two important anti-apoptotic proteins in MPM cells, therefore suggesting that the TRAIL and PI combination may serve as a promising new treatment for MPMs.
The PFM was characterized in a previous study as a highly efficient apoptosis induction mechanism, [23, 24]. It conveys apoptosis signaling from the initially activated intrinsic pathway to the extrinsic pathway leading to cleavage/activation of caspase 10/8. The activated extrinsic pathway then forms an apoptosis signaling loop with the intrinsic pathway through the Bid protein and the mitochondria. Continuous signaling flow along this loop results in the amplified caspase activation and subsequent cleavage of the anti-apotpotic protein Mcl-1, thus ensuring a quick complete apoptotic cell death . The protein cleavage for both caspase 10 and Mcl-1 can thus serve as a surrogate indicator of the PFM activation. Given the observation of a significant elevation in Mcl-1 protein level without caspase 10 cleavage in all three MPM cells following PI alone treatment, we conclude that PI alone treatment is insufficient to activate the PFM and can induce only a limited apoptosis in MPM cells.
The TRAIL protein induces apoptosis through binding death receptor DR4/DR5 and then recruiting/activating caspase 8/10. The TRAIL protein or death receptor activating antibodies have demonstrated a strong proapoptotic activity in various human cancer cells [25, 26]. However, similar to PI alone, TRAIL alone can induce only a limited apoptosis in MPM cells probably due to its insufficiency to activate the PFM.
Although TRAIL and PI share a limited apoptotic activity in MPM cells, the sensitivity profiles of the MPM cells are different to these two agents: NCI-H28 cells are resistant to PIs but mildly sensitive to TRAIL, whereas NCI-H2052 cells are resistant to TRAIL but slightly sensitive to PI. Such difference reflects a mechanistic discrepancy in apoptosis induction between the two agents and encourages their combination for achieving an enhanced cell death. Indeed, the compensation can be seen during the activation of the PFM. Following the PFM model, it is believed that TRAIL can help PI induce sufficient activation of caspase 3/7 for initiating the PFM, resulting in caspase 10 activation while reducing Mcl-1 protein level through protein cleavage .
The Mcl-1 protein, among the antiapoptotic Bcl-2 and IAP family proteins, has been characterized to be a major protein regulating the PI-induced apoptosis in MPM cells . This specificity is determined largely by the fact that Mcl-1 is a degradation target of the proteasome [34, 35]. The Mcl-1 protein can also be cleaved by the activated caspase 3 . It is thus not surprising to observe that proteasome inhibition in the absence of sufficient caspase 3 activation, as seen in PI alone treatment, elevates Mcl-1 protein level. However, the proteasome inhibition with sufficient caspase 3 activation, as seen in the combinatorial treatment, reduces Mcl-1 protein level.
Whereas Mcl-1 regulates the PI-induced apoptosis, the PI3K/Akt signaling regulates both PIs- and TRAIL-induced apoptosis in MPM cells. Hyperactive PI3K/Akt signaling is commonly seen in MPMs due to frequent loss of PTEN expression  and is considered to be a negative prognosis marker for MPM . This study reveals that the hyperactive Akt is associated with low sensitivity of MPM cells to PI-induced apoptosis. In addition, the sustained Akt activation, as seen in myr-Akt-transfected MPM cells, significantly reduces the sensitivity of tumor cells to PI alone, TRAIL alone, or even to the combinatorial treatment.
It has been well established that the Akt activity is most commonly regulated via phosphorylation and dephosphorylation. One consequence of the increased Akt phosphorylation/activation is the inhibition of its downstream protein GSK3β, which in turn can stabilize Mcl-1 protein from degradation by the proteasome. However, the present study did not suggest that Akt-GSK3β pathway is involved in regulating Mcl-1 stability in MPM cells. More likely, Akt and Mcl-1 are separately regulated in MPM cells and represent two independent resistance mechanisms to the PI-induced apoptosis, as demonstrated in the experiment using PI3K inhibitor LY294002 and Mcl-1 silencing in PI-treated NCI-H28 cells. It is possible that, during the regulation of the PI-induced apoptosis, the elevated Mcl-1 protein level blocks pro-apoptotic Bak protein , whereas the active Akt phosphorylate/sequester pro-apoptotic Bad protein from interacting with Bcl-XL.
However, the Akt protein and the Mcl-1 protein share the same feature of the PFM-directed protein cleavage in MPM cells following the combinatorial treatment. As seen in this study, the endogenous Akt protein was reduced through the caspase-dependent protein cleavage rather than the reduction of protein phosphorylation or gene transcription. The involvement of caspase 9 in the reduction of P-Akt has been seen previously in UVA-induced apoptosis , suggesting that cleavage-dependent mechanism may be an alternative mechanism to the phosphorylation mechanism, especially when the apoptotic machinery is fully activated by the PFM. Since several Akt isoforms exist in MPM cells including at least Akt1 and Akt3 and the antibodies used in the present study could not distinguish different isoforms , it is of great interest to determine which isoform is more involved in caspase-dependent regulation.
Consequently, following the PFM model, the increased caspase activities with the decreased resistance mechanisms of both Akt and Mcl-1 together ensure quick ultimate apoptotic cell death. More importantly, such robust proapoptotic activity exhibits a relative selectivity in MPM cells than in non-tumorigenic mesothelial cells. However, definitely, the underlying mechanism for the selectivity warrants further investigation.