ENHANCING DE NOVO SYNTHESIS OF CERAMIDE CAN OVERCOME THE CrmA INHIBITION OF TNF-⍺- INDUCED APOPTOSIS

Abstract

Cowpox virus encodes for the anti-apoptotic protein cytokine response modifier A (CrmA), an inhibitor of Interleukin 1-ß (IL-1) converting enzyme (ICE) also known as Caspase-1, which, in addition to inhibiting the generation of IL-1 and decreasing the inflammatory response to viral infection, it will inhibit TNF-⍺-induced apoptosis of viral infected cells. Ceramide, a signaling sphingolipid, responds to cellular stress and death signals by activating “downstream” caspases that cleave PARP and drive apoptosis. We previously showed that CrmA inhibited ceramide generation and prevented TNF-α–induced apoptosis in MCF-7 breast cancer cell line. In turn, exogenous ceramide administration bypassed cell death inhibition by CrmA. These studies demonstrated an upstream action of CrmA on ceramide-mediated apoptosis in response to TNF-α treatment.

Thus, in our current work we aim to study whether modulating the de novo synthesis pathway of ceramide or ceramide catabolism would sensitize CrmA overexpressing MCF-7 cells to TNF-⍺-induced response.

We measured by qRT-PCR, the CrmA effect on the transcription of de novo enzymes of ceramide synthesis pathway upon TNF-⍺ treatment and showed that the mRNA expression of DEGS1, CerS2 and CerS4, but not CerS6, was significantly decreased after 8h. We also measured the mRNA expression of SphK1, an enzyme of the salvage pathway of ceramide, and showed no significant change in its transcription; suggesting that CrmA selectively regulates de novo enzymes responsible for generating ceramide at the transcriptional level upon TNF-α treatment. We also modulated the de novo synthesis pathway of ceramide or one of the ceramide catabolism pathways using three modulators: 4-HPR, ST1926, and SK1-I to examine whether we would sensitize CrmA-Overexpressing MCF-7 cells to TNF-α-induced cytotoxicity using MTT. Treatment with 4-HPR or SK1-I alone had no effect on the viability of MCF-7 CrmA cells; however, 4-HPR treatment, which increases de novo synthesis of dihydroceramide but not ceramide, increased the viability of MCF-7 Vector cells in response to TNF-α at 48h. On the other hand, upon treatment with the synthetic retinoid ST1926, which increases the de novo synthesis of ceramide, and TNF-α, there was a significant enhancement of the cytotoxicity on both the MCF-7 Vector and MCF-7 CrmA cells at 48h, which might indicate that ST1926 treatment may sensitize MCF-7 CrmA cells to TNF-α induced cytotoxicity. Also, we measured the expression of cleaved PARP-1, an indicator of apoptosis, and the tumor suppressor p53. We found that neither was expressed in MCF-7 CrmA cells upon TNF-α treatment or in response to TNF-α and ST1926. We also found that TNF-α-induced expression of p53 might play a role in mediating apoptosis (increase in PARP-1 cleavage) of MCF-7 Vector cells and that ST1926 was able to slightly synergize with TNF-α to induce the apoptosis of MCF-7 Vector cells. ST1926 was not likely to function through p53.

We conclude that ST1926 might sensitize resistant MCF-7 CrmA cells to TNF-α-induced cytotoxicity, however not through apoptosis. Further work is needed to determine the potential therapy that can overcome CrmA-mediated block of apoptosis in viral infections.