| Objective To study the function and mechanism of ubenimex in human renal cell carcinoma cell lines786-0and OS-RC-2.Methods1.The renal cell carcinoma cell lines786-0and OS-RC-2was used to research the function of ubenimex.The methods are as follows:Cell was cultured with ubenimex of different concentration. WST-8cell proliferation assay and cell growth curve was used to measure the effect of ubenimex on cell proliferation.The scratch motility assay was used to describe renal cell carcinoma cell786-O and OS-RC-2movement. Matrigel invasion assay was used to test the invasion ability of renal cell carcinoma cell786-O and OS-RC-2.2.The mechanism of ubenimex on rcc cells. The activity of CD13/APN in renal cell carcinoma cell lines786-0and OS-RC-2was detected by the substrate of CD13/APN, that is alanine-p-nitroanilido. The CD13/aminopeptidaseN(APN), Caspase3, LC-3B protein expression of renal cell carcinoma cell786-0and OS-RC-2after treated with different concentration of ubenimex was tested by western blot analysis.3.The pathological kidney tissue was collected from patients with malignant or benign renal tumor.Western blot analysis was used to test the CD13/APN expression in renal carcinoma tissue ans adjacent normal tissue of each patient.The kidney tissue micro-array was constructed to reflect the expression intensity of CD13/APN in different lesion by immunohistochemistry.Results The cell growth curve and WST-8assay demonstrated that O.lmg/ml ubenimex could significantly inhibit growth of renal carcinoma cell lines786-O and OS-RC-2in vitro (P<0.05), as the concentration of ubenimex increased, the inhibiting effect become more apparent(P<0.05). Scratch wound-healing migration assay showed that0.5mg/ml ubenimex could inhibit the migration of renal carcinoma cell lines786-O and OS-RC-2,(P<0.05),and the effect is more significant as the drug concentration increased. APN activity assay shows that0.25mg/ml ubenimex could decrease APN activity in786-0and OS-RC-2cell line(P<0.05). The western blot analysis showed that ubenimex had no effect on CD13/APN expression in786-0and OS-RC-2cell line (P>0.05). The expression of LC-3B was up-regulated after treated with ubenimex, which could be blocked by3-MA or enhanced by rapamycin. The CD13/APN expression in renal carcinoma tissue was higher than in adjacent normal tissue in each patient by western blot analysis(P<0.05).Immunohistochemistry shows that the CD13/APN expression had no relationship with tumor grade and stage (P>0.05).Conclusion1.ubenemex can inhibit the proliferation, migration, invasion of renal carcinoma cell lines786-O and OS-RC-2in vivo.2. The mechanism maybe related to the inhibition of aminopeptidaseN activity. The cytotoxicity of ubenimex can be enhanced by rapamycin or blocked by3-methyladenine, which indicated that the antitumor activity maybe related to its effect on autophagy. |
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