Conditionally Replicating Adenoviruses Kill Tumor Cells via a Basic Apoptotic Machinery-Independent Mechanism That Resembles Necrosis-Like Programmed Cell Death

Abstract

Conditionally replicating adenoviruses (CRAds) represent a promising class of novel anticancer agents that are used for virotherapy. The E1A⌬24 mutation-based viruses, Ad5-⌬24 [CRAd(E3؊); E3 region deleted] and infectivity-enhanced Ad5-⌬24RGD [CRAd(E3؉)] have been shown to potently eradicate tumor cells. The presence of the E3 region in the latter virus is known to improve cell killing that can be attributed to the presence of the oncolysis-enhancing Ad death protein. The more precise mechanism by which CRAds kill tumor cells is unclear, and the role of the host cell apoptotic machinery in this process has been addressed only in a limited way. Here, we examine the role of several major apoptotic pathways in the CRAd-induced killing of nonsmall-cell lung cancer H460 cells. As expected, CRAd(E3؉) was more potent than CRAd(E3؊). No evidence for the involvement of the p53-Bax apoptotic pathway was found. Western blot analyses demonstrated strong suppression of p53 expression and unchanged Bax levels during viral replication, and stable overexpression of human papillomavirus type 16-E6 in H460 cells did not affect killing by both CRAds. CRAd activity was also not hampered by stable overexpression of anti-apoptotic Bcl2 or BclXL, and endogenous Bcl2/BclXL protein levels remained constant during the oncolytic cycle. Some evidence for caspase processing was obtained at late time points after infection; however, the inhibition of caspases by the X-linked inhibitor of apoptosis protein overexpression or cotreatment with zVAD-fmk did not inhibit CRAd-dependent cell death. Analyses of several apoptotic features revealed no evidence for nuclear fragmentation or DNA laddering, although phosphatidylserine externalization was detected. We conclude that despite the known apoptosis-modulating abilities of individual Ad proteins, Ad5-⌬24-based CRAds trigger necrosis-like cell death. In addition, we propose that deregulated apoptosis in cancer cells, a possible drug resistance mechanism, provides no barrier for CRAd efficacy.

Potent antitumor efficacy of XAF1 delivered by conditionally replicative adenovirus vector via caspase-independent apoptosis

“…Consistent with previous report that CRAd-induced oncolysis was not associated with apoptotic DNA fragmentation, 22 ONYX-015 and ZD55-EGFP could not induce DNA fragmentation in SW620 cells. However, obvious DNA ladder was observed in ZD55-XAF1-treated cells (Figure 3B), indicating that XAF1 played a crucial role in this effect.…”

Adenovirus’s last trick: You say lysis, we say autophagy

“…More recently, this type of cell death was further identified as autophagic by Kondo's group. 11,12 Consistent with the above studies, when we tested the therapeutic effect of the oncolytic adenovirus D-24-RGD in brain tumor stem cells, the virus induced prominent autophagy at the late stage of viral infection. [13][14][15] We were interested to notice a drastic increase in the level of the Atg12-Atg5 complex 48 hours after viral infection.…”

Paclitaxel and vincristine potentiate adenoviral oncolysis that is associated with cell cycle and apoptosis modulation, whereas they differentially affect the viral life cycle in non-small-cell lung cancer cells

“…Previously, we showed that Ad5 induces necrosis-like programmed cell death, which is triggered independent from the basic apoptotic machinery. 20 It is conceivable that drug-induced activation of other cell death route, such as caspase activation, will enhance cell killing and viral release.…”

“…20 The percentage survival (taking the blank as 100% survival) was plotted as a function of the MOI and the lethal concentration (LC) 50 values of Ad5 alone or in combination with PTX or VCR were determined being the MOI of Ad5 required to kill 50% of the cultured cells. Possible synergistic effects of the combined treatments were evaluated by calculating the combination index (CI) according to Chou Quantitative RT-PCR Ad5 gene expression assay H460 cells were harvested at 0, 6, 12, 24, 30, 36 and 48 h post-infection and were dissolved in 1 ml Trizol for the isolation of total RNA as described by the manufacturer (Invitrogen, Breda, The Netherlands).…”