The Experimental Studies Of Effects Of Mithramycin A On The Expression Of Osteopontin,Sp1 In The Colonrectal Carcinoma Cells

Objective: To explore the expression of transcription factor Sp1 and osteopontin (OPN) in the colorectal carcinoma cell lines (SW480 and SW620) with different metastatic potential, We treat the lines with the mithramycin A(MIT , a Sp1 inhibitor),and observe cell cultures'growth , explore the influences of OPN expression. The aim of this study is to detect the effects of Sp1 and OPN on proliferation and metastasis of the colorectal cancer, whether transcription factor Spl could be an effective therapeutic target for human colorectal cancer.Methods:Taken established human colorectal cancer cell lines SW480 and SW620 in vitro as research object, Real-time PCR, Western-blot methods and cellular immunofluorescence technique were used to detect the Sp1,OPN expression at gene level and protein expression level before and after treatment with Mithramycin A , separately. Cell growth curve was used to assess the inhibitory effect of treatment with Mithramycin A on cell proliferation. The migration potential of cancer cells with different treatments was analyzed by the wound-healing assay respectively. The production of matrix metalloproteinases (MMPs) of SW480 and SW620 cell lines were measured by gelatin zymogram before and after treatment with Mithramycin A.Results: Real-time PCR results showed SW480(Dukes B stage) and SW620(Dukes C stage)had Sp1 and OPN expression;Western-blot also showed the expression of Sp1 and OPN protein existed in both cell lines.The level of Sp1 and OPN expression in cell line SW620 was higher than in SW480(P<0.05); after treated with Mithramycin A (0.10μM,) , the low level of Sp1 and OPN expression in both lines were validated by Real-time PCR , western blotting analysis and cellular immunofluorescence technique(P<0.05); Cell growth curve showed the proliferation decreased after treatment with Mithramycin A;Wound-healing assay showed the invasion capability of the two lines treated with Mithramycin A significantly weakened. As the expression of Sp1 suppressed; Gelatin zymography showed that the level of activated MMPs was significantly decreased.Conclusion: The expression of Sp1 , OPN and MMPs were observed in the colorectal carcinoma cell lines (SW480 and SW620) , and were significantly correlated to the tumor invasion and Dukes stages; Mithramycin A , a Sp1 inhibitor , could cause the down-regulation of OPN and MMPs (its downstream target gene) expression ; Maybe the activation of Sp1-OPN-MMPs molecular pathway was the cause of the migration and invasion in the colorectal cancer cells. Transcription factor Spl could be a new useful molecular target for the therapeutic development of human colorectal cancer.