Recombinant lentivirus with enhanced expression of caudal-related homeobox protein 2 inhibits human colorectal cancer cell proliferation in vitro

Caudal-related homeobox protein 2 (CDX2), a tumor suppressor in the adult colon, is overexpressed under a non-cancer specific cytomegalovirus promoter in certain tumor cells; furthermore, non-specific expression of CDX2 may result in aberrant side effects in normal cells. The human telomerase reverse transcriptase (hTERT) promoter is active in the majority of cancer cells but not in normal cells. Hypoxia is a key feature of solid tumors, and targeted genes may be significantly upregulated by five copies of hypoxia-response elements (HREs) under hypoxic conditions. However, the effect of CDX2 overexpression, as controlled by five copies of HREs and the hTERT promoter, on human colorectal cancer (CRC) cell proliferation in vitro remains to be fully elucidated. In the current study, a recombinant lentivirus containing the CDX2 gene under the control of five HREs and the hTERT promoter was generated. An immunofluorescence assay was used to detect CDX2 expression by the 5 HhC lentivirus, whereas an MTT assay was used to detect the effects of CoCl2 on the viability of LoVo cells. Western blot analysis was conducted in order to determine the relative ratios of recombinant CDX2 protein to the internal control β-actin, following 5 HhC/LoVo cell culture under normoxic and hypoxic conditions (100, 200, 300, 400 or 500 µmol/l CoCl2) for 24 h, then for 12, 24 or 36 h with the optimal concentration (300 µmol/l) of CoCl2. Reverse transcription polymerase chain reaction analysis was used to determine the transcription of recombinant CDX2 mRNA following culture of 5 HhC/LoVo cells under normoxic or hypoxic conditions. Finally, a cloning assay was used to detect the proliferative ability of 5 HhC/LoVo and 5 Hh cells. High CDX2 expression was observed in hTERT-positive LoVo cells under hypoxic conditions, an effect which was mimicked by treatment with CoCl2 to inhibit LoVo cell proliferation in vitro. High expression of CDX2 therefore provides a promising strategy for the development of novel targeted treatments and gene therapy for CRC.