Figure 9. Shows induction of pS727-Stat1 in L3.6pl cells incubated with 10 ng/ml recombinant human IFN-c protein with or without 10 mg/ml anti-IFN-c antibodies in the presence or absence of AT-101 (*p,0.05; **p,0.005; ***p,0.0005). aATC Immunotherapy Suppresses Anti-Apoptotic Bcl-XL and Induces Pro-Apoptotic Proteins Bax and Bad in AT101 Sensitized Tumor CellsSince ATC and aATC treated tumor cells showed significant dephosphorylation of pY705-Stat3 and increased phosphorylation of pS727-Stat1, we next examined the effect of Stat3 dephosphorylation on its target anti-apoptotic gene Bcl-XL, and Stat1 activation on its target pro-apoptotic genes Bax and Bad. We stained Bcl-XL, pBad and Bax proteins intracellularly and analyzed for percent positive cells and relative mean fluorescence intensities. Our data demonstrate a significant reduction in the MFI of Bcl-XL, (L3.6pl, p,0.04; MiaPaca-2, P,0.03) and an increase in pro-apoptotic pS112-Bad (L3.6pl, p,0.05; MiaPaca-2, P,0.03) and Bax (L3.6pl, p,0.04; MiaPaca-2, P,0.05) expression in both AT-101 sensitized L3.6pl cells (Figure 11) andMiaPaCa-2 cells (Figure 12) treated with EGFRBi armed ATC. Upper panels show histogram overlays representing isotype control, Bcl-XL, Bax and p-Bad positive cells gated on tumor cells (Figure 11 and 12). Interestingly, ratios of both pBad :BclXL and Bax:Bcl-XL MFI were also noticeably higher (more death) in both cell lines, L3.6pl (pBad :Bcl-XL, p,0.0001; Bax:Bcl-XL, p,0.0001) and MiaPaCa-2 (pBad :Bcl-XL, p,0.005; Bax:Bcl-XL, p,0.05) treated with EGFRBi armed ATC after 24 h AT-101 sensitization (Figure 13).
Discussion
PC is among the deadliest of all malignancies and urgently requires new therapeutic approaches. Pretreatment of tumor cells with small molecule inhibitors or chemotherapeutic drugs has been shown to sensitize tumor cells for enhanced CTL responsesFigure 10. Shows induction of pS727-Stat1 in MiaPaCa-2 cells incubated with 10 ng/ml recombinant human IFN-c protein with or without 10 mg/ml anti-IFN-c antibodies in the presence or absence of AT-101 (*p,0.05; **p,0.005; ***p,0.0005). by altering the expression levels of key apoptosis regulators [36?38]. Using this principle, we have for the first time investigated the effect of combination therapy where PC cells were pretreated with suboptimal concentrations of AT-101 followed by incubation with ATC and aATC-mediated cytotoxicity of tumor targets. We observed that 1) sensitization of tumor cells with AT-101 enhanced ATC and aATC-mediated cytotoxicity in L3.6pl, MiaPaCa-2 and CoLo-357 cells; 2) enhanced GrzB uptake of tumor cells; 3) enhanced IFN-c induced Stat1 phosphorylation and Stat3 dephosphorylation, which in turn inhibited Bcl-XL and induced expression of pBad and Bax (Figure 14). EGFRBi armed ATCmediated apoptosis in AT-101 sensitized L3.6pl and MiaPaCa-2 cells may be facilitated by increased expression of pBad and Bax and partially through repressing Bcl-XL expression. ATC are known to induce cytotoxicity via Fas/FasL or perforin/granzyme pathways; however, in previous studies we have shown that perforin/granzyme is the predominant pathwayof aATC-mediated killing [39]. During effector-target interaction, the pore forming protein perforin is released by activated T cell (ATC) and facilitates the delivery of serine proteases GrzA and GrzB to the target cell cytoplasm and nucleus, where they deliver the apoptotic hits through independent pathways [40?2]. First, we asked whether interaction of AT-101 sensitized PC cells with ATC or aATC can enhance perforin/granzymes-mediated cytotoxicity of tumor cells by ATC or aATC. Induction of early apoptosis was determined by Annexin V/7-AAD, which showed no difference between untreated tumor cells and tumor cells treated with 1 mM AT-101 (24 h). These data suggest that a suboptimal concentration of AT-101 could not induce apoptosis within 24 h exposure but was sufficient to cause rapid induction of apoptosis following 4 h incubation with ATC or aATC. We examined the changes in degranulation markers CD107a/b and cytolytic granule GrzB following a 4 h incubation of ATC or aATC with AT-101 treated tumor cells at 10:1 E/T. ATC andFigure 11. The combination of pan Bcl-2 inhibitor AT-101 with aATC immunotherapy induces pro-apoptotic proteins and inhibits the expression of anti-apoptotic proteins. L3.6pl cells were either pretreated with AT-101 for 24 h or left untreated followed by additional incubation with ATC or aATC for 4 h and before intracellular staining for pBad, Bax and Bcl-XL. 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. aATC incubated with AT-101 treated tumor cells showed increased numbers of GrzB positive cells as well as increased expression compared to ATC or aATC incubated with untreated tumor cells. CD107a/b did not show much change in percent positivity or expression in ATC and aATC incubated with AT-101 treated cells compared to untreated tumor cells. Interestingly, an increased percentage of GrzB positive tumor cells were seen after tumor cells were sensitized with AT-101 compared to untreated tumor cells in the presence of ATC and aATC, suggesting that AT-101 sensitization enhances penetration of cytolytic granules into the tumor cells. These results are consistent with other studies showing increased penetration of GrzB during cytotoxicity withantigen specific CTL activity in chemotherapy sensitized tumor cells [36]. Earlier, we have shown that stimulation of ATC or aATC with target cells induces production of IFN-c. IFN-c-induced phosphorylation of Stat1 and dephosphorylation of Stat3 play essential roles in anti-proliferative, pro-apoptotic effects of IFN-c [33,43,44]. Since constitutive phosphorylation of Stat3 has been detected in a variety of human cancers including pancreatic tumors and PC cell lines [33,45,46], we examined whether ATC or aATC can induce increased apoptosis of AT-101 sensitized tumor cells via IFN-c/Stat pathways.
