| Cancer is a complex disease involving aberrations in multiple cellular pathways. In addition, tumorigenic cells are constantly evolving and developing resistance to treatment, particularly radio- and chemotherapy which are additionally hampered by toxic side effects. The alternative means of treatment entail ing use of specific eel lular genes that distinguish between normal and transformed states and that ultimately induce apoptosis in tumor cells, is being increasing explored. Melanoma differentiation associated gene-7 (mda-7) is such a specifically targeting tumor cells gene, which was screened and identified in terminally differentiated human melanoma cells based on "differntiation therapy" via substraction hybridization in 1995. Mda-7 was assigned to human chromosome 1q, at 1q32.2 to 1q41, and the open reading frame of its cDNA encoded a protein with a predicted size of 23. 8 kDa. This molecular was subsequently confirmed to have cytokine properties, being to IL-10 cytokine family, and has been renamed IL-24.Human IL-24 is stably expressed in cells having immune functions. As a cytokine, IL-24 can stimulate production of inflammatory factors, and participate in immunity activation. Besides this, IL-24 has anti-tumor potential, and this property has attracted more and more interest. Plenty of study data indicated that ectopic expression of IL-24 mediated by replication defective adenovirus could result in growth suppression and apoptosis induction of adverse cancer cells without no apparent effect on normal cells. Ad. IL-24 is currently undergoing phase II clinical trials in American.However the explicit mechanism of apoptosis induction by IL-24 is still unclear. IL-24 is a secreted cytokine, but all present studies about anti-tumor function of IL-24 are not based on this fact. Therefore many issues about IL-24 need to be comprehended afresh. In addition, liver cancer is one of malignant diseases which severely menace people's health. What effect does IL-24 have on liver cancer? But so far, to our knowledge there is no report concerning inhibitory effect of IL-24 on human liver cancers. In this report, we aimed to demenstrate the tumor suppression activity of IL-24 on liver cancers, to investigate the relations between the anti-tumor efficiency and IL-24's concentration, and to find whether or not IL-24 receptors transduct anti-tumor signal of IL-24.MethodsThe whole experiment was divided into several parts. At first, we cloned human IL-24 cDNA from spleen tissue, then to recombine it with Igk leading peptide squence and myc-tag. 293 cells were stably transfected with pcDNAS. l-Igk7m, constructed by cloning confluent gene IgkVm into the expression vector pcDNA3.1 (+), and rhIL-24 (IL-24-MYC) protein was secreted into cultured media. Secondly, following treatment with rhIL-24 ptotein in different constrations in vitro, viable cells counting were used to analyze growth-suppressive effect of rhIL-24 on liver cancer cells (BEL-7402, Hep3B, Huh-7, PLC/PRF/5, SMMC-7721) and normal liver cells (WRL-68), apoptosis-inductive effect of rhIL-24 on liver cancer cells and normal liver cells was analyzed by TUNEL detection. Finally, SMMC-7721 cells were inoculated in nude mice after pretreatment with rhIL-24 in vitro by hypodermic injection, as well as SMMC-7721 cells untreated, and effect of rhIL-24 on tumor formation of liver cancer cells was investigated.Results1. We successfully cloned IL-24 cDNA from human spleen tissue and recombined Igk7m gene composed of Igk leading peptide sequence, IL-24 cDNA and myc-tag. Sequence detection indicated nucleotide sequence of each partwas identical with that in literatures.2. After stable transfection with pcDNA3.1-Igk7m, IL-24 mRNA was detectable inside 293 cells by northern blot. In the same time, rhIL-24 protein was detected to be expressed inside 293 cells and secreted in cultured media by western blot.3. After treatment with rhIL-24 protein in vitro, growth and proliferation of liver cancer cells (BEL-7402 Hep3B Huh-7 PLC/PRF/5 SMMC-7721) were suppressed, and...
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