Figure 12. Shows intracellular staining for pBad, Bax and Bcl-XL in MiaPaCa-2 cells were either pretreated with AT-101 for 24 h or left untreated followed by incubation with ATC or aATC before staining. Top panel show histogram overlays representing isotype control, phosphor-Bad, Bax and Bcl-XL positive cells. Proportion of phosphor-Bad, Bax and Bcl-XL positive cells and MFI were gated on tumor cells. Bottom panel shows the graphic representation of MFI under the indicated experimental conditions. presence of ATC or aATC regardless of AT-101 sensitization, suggesting that ATC and aATC alone are potent inhibitors of Stat3 phosphorylation in the tumor cells. Interestingly, pS727-Stat1 expression and percent positive cells both increased significantly in AT-101 sensitized tumor cells in the presence of aATC. In addition, a significant correlation was found between IFN-c and Stat1 expression. Since Stat1 and Stat3 show opposing patterns that promote or inhibit apoptosis, respectively, we examined the expression of antiapoptotic Bcl-XL and pro-apoptotic pBad and Bax proteins in AT101 sensitized tumor cells in the presence of ATC or aATC. Studies have shown that IFN-c-induced dephosphorylation of
Stat3 could induce apoptosis [33] through down-regulation of Bcl2, Bcl-XL, and Mcl-1, and up-regulation of Bax [47]. AT-101 treated L3.6pl and MiaPaCa-2 cells showed reduced MFI for BclXL and increased MFI for pS112-Bad and Bax compared to untreated L3.6pl and MiaPaCa-2 cells in the presence of ATC or aATC; however, the difference was not statistically significant. Intriguingly, the ratio of pBad:Bcl-XL and Bax:Bcl-XL under the same conditions showed a significant difference between AT-101 treated and untreated L3.6pl and MiaPaCa-2 cells. Constitutively high levels of Bcl-2 or Bcl-XL have been associated with drug resistance and more aggressive phenotype in both liquid and solid tumors [48,49].
Figure 13. Shows the ratio of p-Bad: Bcl-XL and Bax: Bcl-XL under the indicated experimental conditions. Ratio of p-Bad: BclXL and Bax: Bcl-XL showed a significant difference between AT-101 treated and untreated L3.6pl and MiaPaCa-2 cells in the presence of aATC. Representative data from two experiments with the same results are shown. Figure 14. Schematic diagram showing the mechanism of EGFRBi armed ATC-mediated killing of AT-101 sensitized tumor cells. AT-101 a Pan-Bcl-2 inhibitor binds to the BH3 binding domain of Bcl-2 and disrupts its interaction with pro-apoptotic family members such as Bax and Bad. Free Bax and Bad re-localizes into the mitochondria where they initiate the mitochondrial apoptosis. Our results demonstrate that tumor cell stimulated aATC can produce IFN-c that in turn can phosphorylate Stat1. pStat activation results in Bad/Bax mitochondrial localization and induction of apoptosis. IFN-c itself is a suppressor of anti-apoptotic Bcl-XL. strongly correlated with resistance to most chemotherapy agents [50]. Cellular fate between apoptosis and survival depends upon the balance between proapoptotic and antiapoptotic protein levels. High levels of Bcl-XL and low levels of Bax provide a survival advantage and vice versa. Del Poeta et al. (2003) showed that the ratio of Bax/Bcl-2 MFI predicted the outcome in acute myeloid leukemia (AML) [51]. Lower Bax/Bcl-2 levels were correlated with poor-risk cytogenetics; and a longer overall survival (OS) and disease-free survival (DFS) observed in patients with higher Bax/ Bcl-2 levels. Another study in B-CLL showed that enhanced Bcl2/Bax ratio contributes to B-CLL survival [52]. These results are consistent with our current findings. While combination of targeted therapies with chemotherapeutic drugs has been tested against PC, this is the first study, to the bestof our knowledge, to report that priming of tumor cells with highly specific Bcl-2 inhibitor enhances cytotoxic activity armed ATC. In summary, we have shown that the combination of Bcl-2 inhibitor AT-101 (conditioning of PC cells) with targeted immunotherapy using EGFRBi armed ATC offers an attractive therapeutic approach that can lead to reduced toxicity but potent anti-tumor activity. Priming of tumor cells with suboptimal concentration for short duration can significantly enhance anti-tumor activity of EGFRBi armed ATC through the inhibition of Stat3 and activation of Stat1. These proof-of-concept studies provide rationale to design future strategies for the treatment of pancreatic cancer and warrant further clinical investigations against deadly PC.
Abstract
Telomerase is a specialized reverse transcriptase responsible for the de novo synthesis of telomeric DNA repeats. In addition to its established reverse transcriptase and terminal transferase activities, recent reports have revealed unexpected cellular activities of telomerase, including RNA-dependent RNA polymerization. This telomerase characteristic, distinct from other reverse transcriptases, indicates that clinically relevant reverse transcriptase inhibitors might have unexpected telomerase inhibition profiles. This is particularly important for the newer generation of RT inhibitors designed for anti-HIV therapy, which have reported higher safety margins than older agents. Using an in vitro primer extension assay, we tested the effects of clinically relevant HIV reverse transcriptase inhibitors on cellular telomerase activity. We observed that all commonly used nucleoside reverse transcriptase inhibitors (NRTIs), including zidovudine, stavudine, tenofovir, didanosine and abacavir, inhibit telomerase effectively in vitro. Truncated telomere synthesis was consistent with the expected mode of inhibition by all tested NRTIs. Through dose-response experiments, we established relative inhibitory potencies of NRTIs on in vitro telomerase activity as compared to the inhibitory potencies of the corresponding dideoxynucleotide triphosphates. In contrast to NRTIs, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and efavirenz did not inhibit the primer extension activity of telomerase, even at millimolar concentrations. Long-term, continuous treatment of human HT29 cells with select NRTIs resulted in an accelerated loss of telomere repeats. All tested NRTIs exhibited the same rank order of inhibitory potencies on telomerase and HIV RT, which, according to published data, were orders-of-magnitude more sensitive than other DNA polymerases, including the susceptible mitochondria-specific DNA polymerase gamma. We concluded that telomerase activity could be inhibited by common NRTIs, including currently recommended RTI agents tenofovir and abacavir, which warrants large-scale clinical and epidemiological investigation of the off-target effects of longterm highly active antiretroviral therapy (HAART) with these agents.