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Hypoxia microenvironment regulates immunotherapy effect of TSST-1 on CEA positive colon cancer LoVo cells
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Abstract:
Objective : To evaluate the regulation of hypoxia microenvironment on cytotoxicity effect of peripheral blood lymphocyte (PBL) activated by the superantigen of toxic shock syndrome toxin-1 (TSST-1) against CEA+ human colon cancer cell line LoVo. Methods: The recombinant retroviral vector pLEGFP-N1-5HRE-CEAp-TSST-1-linker-CD80TM (pLEGFP-N1-5HCTC) containing a CEA promoter and 5 copies of the hypoxia-response elements (HRE) enhancer, which was constructed in our previous study, was packaged and collected. pLEGFP-N1-5HCTC was then transducted into CEA+LoVo cells or CEA- human cervical carcinoma HeLa cells, and LoVo or HeLa cells with stable expression of transmembrane superantigen fusion gene TSST-1-linker-CD80TM protein (TC fusion protein) were obtained. Hypoxia microenvironment was simulated by CoCl2, and RT-PCR and Western blotting were employed to examine the expression of TSST-1 regulated by hypoxia. Human PBL was extracted and co-cultured with transgene tumor cells infected with pLEGFP-N1-5HCTC retrovirus; 3H-TdR assay was employed to detect the proliferation of PBL regulated by hypoxia; and the cytotocixity effect of PBL regulated by hypoxia against the transgene tumor cells was detected by MTT assay. Results: CEA+LoVo cells infected with recombinant retrovirus pLEGFP-N1-5HCTC were obtained (5HCTC/LoVo). The expression of TSST-1 mRNA and protein in 5HCTC/LoVo cells was further increased with hypoxia, which was confirmed by RT-PCR and Western blotting. There was no TSST-1 expression in CEA-HeLa cells under either hypoxic condition or normoxic condition. The proliferation of PBL activated by 5HCTC/LoVo cells was increased with hypoxia (7.3×103 vs 3.1×103 cpm, P<005); and the cytotocixity of PBL on 5HCTC/LoVo cells was more effective in hypoxia condition than in normoxic condition (82.69% vs 53.50%, P＜0.01). There was no proliferation of PBL activated by CEA-HeLa cells, and PBL also could not inhibit the proliferation of CEA-HeLa cells. Conclusion: Hypoxia microenvironment can increase the cytotocixity effect of targeting expressed superantigen TSST-1 on CEA+LoVo cells.
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Project Supported:
Project supported by the National Natural Science Foundation of China (No.30672070, No.30400430), and the Science and Technology Foundation of Shaanxi Province (No.2009K01-70)
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