Our current study establishes the anti-cancer oncolytic activity of RSV. In an in vitro prostate cancer cellular model and in an in vivo xenograft prostate tumor model, we show that the RSV infection rate is markedly enhanced in the cancer cells, but not in the non-cancerous cells. The selective increase of viral burden in the infected cancer cells led to the loss of cell viability, whereas the non-cancerous cells were protected from the virus-induced apoptosis. The in vitro results were validated in a human prostate cancer xenograft model in nude mice, which showed significant tumor regression in response to I.T or I.P administration of RSV. We further demonstrated that RSV-mediated oncolysis is due to apoptosis, induced primarily by the mitochondria-mediated activation of the intrinsic pathway involving caspase-3 activation, in association with impaired NF-κB activity.
Clinical trials show promising results with several oncolytic viruses. Oncolytic paramyxoviruses (RSV is a paramyxovirus) such as NDV and measles are currently undergoing successful clinical trials (7
). Ability of RSV to replicate slowly in normal (non-transformed cells) cells, without causing cell death has been reported (41
). Nevertheless, our study is the first demonstration that RSV possesses oncolytic activity. RSV is especially advantageous as an oncolytic virus based on the following rationale: 1)
RSV confers mild respiratory illness in infants and children, while infection is asymptomatic in adults (5
). The asymptomatic nature of RSV is borne out by routine intra-nasal infection of live (wild-type) RSV to human subjects enrolled in clinical studies. 2)
RSV does not cause systemic infection due to its respiratory tract-specific entrance into lung epithelial cells via the apical domain of the airway lumen (5
) and to date; RSV has not been detected in the serum samples from infected individuals. This is a desirable property for cancer treatment, because systemic delivery of RSV is expected to destroy only tumor cells while keeping the normal cells intact, hence causing limited toxicity to normal tissues. 3)
Since RSV replication occurs in the cytoplasm of the infected cells (5
), its transforming potential due to genetic recombination is avoided. 4)
Immune response against RSV is not robust accounting for non-symptomatic re-infection throughout the viral life cycle (5
The RSV genetic makeup of only ten genes facilitates its manipulation by reverse genetics (43
), which would allow us to engineer attenuated RSV-based efficient and safe
vectors for anti-cancer therapy. 6)
Systemic delivery of RSV should be a viable strategy for targeting tumor cell-specific apoptosis, since our results show that I.P injection of RSV can cause prostate tumor regression in mice (). Even with systemic administration, RSV specifically targeted the tumor mass and conferred its oncolytic activity (). Our I.P result suggests that administration of RSV via I.P route results in specific targeting of RSV to the tumor, where high viral multiplicity results in cell death. In contrast, RSV fails to infect organs such as lungs, liver etc, since the normal cells of these organs may launch an effective anti-viral response to clear virus infection rapidly. Therefore, we failed to detect RSV in these organs; whereas high viral titer was observed in the tumor following I.P injection (,3c,3d).
Advanced-stage cancer cells including androgen-independent prostate cancer cells (like PC-3) are resistant to apoptosis. Thus, development of therapeutic agents that would induce apoptosis in these cells to cause tumor regression has been a major challenge. Our results reveal that RSV confers its anti-tumor oncolytic activity to PC-3 prostate cancer cells by inducing apoptosis. RSV-mediated apoptosis of PC-3 cells utilized both the mitochondria-mediated intrinsic pathway and the death-receptor-mediated extrinsic pathway, although the former is the major apoptotic trigger in our experimental model ().
Figure 9 A model depicting mechanism of apoptosis in RSV infected prostate cells. Model for the mechanism of apoptosis in RSV infected PC-3 prostate cancer cells – apoptosis occur in PC-3 cells due to high (↑) level of RSV infectivity and inhibition (more ...)
We show that RSV infection of prostate cancer PC-3 cells result in the suppression of NF-κB activity leading to the up-regulation of pro-apoptotic proteins and down-regulation of anti-apoptotic proteins. Cleavage of pro-caspase-3 also occurs in virus-infected cells. Studies with caspase-specific inhibitors confirmed these observations, since apoptosis was significantly reduced in the presence of caspase-3 and -9 inhibitors. Although the intrinsic pathway constitutes the major route to apoptosis in infected cells, a minor role of the extrinsic pathway in the induction of apoptosis was also observed, especially with regard to caspase-8, which was activated by TNF secreted from infected cells.
Earlier studies failed to notice robust apoptosis of PC-3 cells in response to TNF (44
). Our result () showing approximately 15% contribution of TNF to the apoptotic signaling is similar to previous reports that 15%–20% of PC-3 cells undergo TNF-mediated apoptosis (46
). In that context, previous studies (46
) have shown that various agents (e.g. cycloheximide) can sensitize TNF to induce apoptosis efficiently (by 50% - 60%) in PC-3 cells. Therefore, we envision similar scenario, whereby RSV infection may enhance the sensitivity of PC-3 cells to undergo TNF-mediated apoptosis. We speculate that RSV infected cells may modulate cellular factors that could enhance the apoptotic potential of TNF. Further studies are required to unravel the mechanism of TNF sensitization in infected cells. Although crosstalk can occur between the extrinsic and intrinsic pathways [via Bid (47
)], our results rule out such crosstalk, since the caspase-9 inhibitor reduced apoptosis by 75%, while reduction in the presence of the caspase-8 inhibitor was only 15%. Crosstalk involving two pathways should have caused a significant decline (at least 75%) in apoptosis due to the impaired caspase-9 activation resulting from caspase-8 inhibition. Moreover, so far the ability of caspase-9 to modulate caspase-8 activity has not been demonstrated and our studies demonstrate that such event may not occur since inhibition of caspase-9 did not abolish apoptosis completely (). If caspase-9 regulated caspase-8 activity, one would expect complete loss of apoptosis activity following caspase-9 inhibition.
Previous studies demonstrated that modulation of both NF-κB and Akt activity by RSV led to apoptosis of lung adenocarcinoma epithelial cells and granulocytes (36
). However, our results show that NF-κB activity in RSV-infected prostate cells is important in order to maintain the anti-apoptotic status, since loss of NF-κB activation led to apoptosis. RSV utilizes this mechanism to suppress NF-κB activity and induce apoptosis in PC-3 cells. Lack of NF-κB inhibition in the RSV-infected normal RWPE cells prevented apoptosis. In contrast to NF-κB, Akt did not play a role in the RSV-mediated apoptosis of prostate cancer cells.
In summary, our study identifies RSV as an oncolytic virus, since the virus can induce apoptosis of cancerous but not non-cancerous prostate epithelial cells in culture, and in prostate tumor xenografts leading to tumor regression. Insights from our findings should lay the foundation for future work aimed at developing RSV-based virotherapy targeted to the clinical management of metastatic prostate cancer.