The highly proliferating mass of tumor cells develops faster than the vasculature, resulting, in most malignant tumors, in hypoxic regions within the growing tumor. Tumor cells can survive under conditions in which nutrients and oxygen are limited. Tumor hypoxia is considered to be a potential therapeutic problem because it renders solid tumors more resistant to ionizing radiation and chemotherapeutic drugs. In contrast, the on-going development of hypoxic regions in growing tumors provides an opportunity for tumor-selective therapies.
Here we developed a Murine leukemia virus (MuLV)-based replication-competent retroviruses (RCR) delivery system that infects only growing cells, and efficiently infects, in vitro, two different tumor cell lines: human hepatocellular carcinoma (HepG2) and human uveal melanoma (C918). Additionally, we established persistence of the virus and knock-down of the expression of the regulators of hypoxia responding genes: CREB, HIF1, and HIF2, via shRNA targeting these genes separately and all together from a single polycistronic RNA. Knockdown of HIF1 directly or via the polycistronic RCR reduced VEGF expression in both cell lines, while knocking down CREB affected mostly HepG2 cells. In addition, these RCRs reduced tumor cell proliferation in hypoxia and increased caspase 3 activity. We found that CREB, more than HIF1 and HIF2, plays a pivotal role in the survival of HepG2 and C918 under hypoxia in vitro. However, the reduction in VEGF by shRNA targeting HIF1 may have a crucial effect on tumor survival in-vivo. Moreover, the recombinant RCRs that knocked-down these hypoxia responding regulators enhanced the toxicity of doxorubicin in HepG2 cells.
