Experimental Research Of NF-kappa B Specific Inhibitor PDTC On Mouse Melanoma Cell Line B16in Vitro

Melanoma, one of the common human malignant tumor, is often found in later period and quickly developed. Chemotherapy is one of the important and common treatments of melanoma, which is often one of the vital means of adjunctive therapy and recurrence therapy of post-operation and inoperableness therapy. At present, there has been an agreement that the induction of apoptosis by chemotherapeutic drugs is the main mechanism of killing tumor cells. However, it may cause the resistance of tumor cells to chemotherapeutic drugs, then reduces the effect of chemotherapy. It is obvious that chemotherapeutic drugs activate a series of biological signals, which keep the balance with the apoptosis induced by chemotherapeutic drugs. The interaction between promotive and inhibitive factors of apoptosis determines the cell fate.Nuclear factor-KB (NF-κB) is one kind of transcription factor, which is widely found in eukaryotic cells and acts diversely. It has been reported that NF-κB plays a key role in cell proliferation, apoptosis, invasiveness, metastasis, tumor-genesis and angiogenesis. Recently, it has been reported that the resistance of tumor chemotherapy is related with NF-κB. It is activated by diverse stimuli, which include proinflammatory cytokines, cellular stress as well as growth factors; and also known to be activated by chemotherapeutic drugs, including daunorubicin, adriamycin, as well as cisplatin. NF-κB is also a key regulator of apoptosis, which suppresses apoptotic cell death by inducing the expression of several anti-apoptotic factors such as Survivin and Bcl-2. Its activation is tightly regulated by inhibitor of NF-κB (IκB). Hence, optimal inhibitor of NF-κB combined with traditional drugs, which is blocking NF-κB pathway and suppressing of related-proteins, may decrease local recurrence and improve patient survival.Pyrrolidine dithioearbamate (PDTC) is a low molecular-weight thiol compound with a variety of biochemical activities, such as redox state alternation, heavy metal chelation, and enzyme inhibition. PDTC, initially regarded as an antioxidant compound to counteract the toxic effeets of free radicals and interfere with the generation of pro-inflammatory cytokines, has been used as potent inhibitor of NF-κB recently. It has been found that PDTC can promote apoptosis and inhibit proliferation of a variety of tumor cells in vitro, and may increase the sensitivity of tumor cells to chemotherapy and radiotherapy. No studies have been reported about effects of PDTC on melanoma cells. In our study, the impact of PDTC on the proliferation of melanoma cell B16and its apoptosis was studied in vitro. Furthermore, the effect of PDTC on the expression of vascular endothelial growth factor (VEGF), which is correlated with tumor angiogenesis and metastasis, was studied in our work.Objective:To explore the effect of PDTC on the proliferation, apoptosis and expression of VEGF in mouse melanoma cell line B16, and then understand the effect and mechanism of PDTC anti-melanoma in vitro, providing an important laboratory and theoretical basis for the PDTC treatment of melanoma.Methods:B16cells were respectively treated in vitro with PDTC at different concentrations (25,50,100μmol/L) and different time (12,24,36h)(1) The morphology of B16cells were observed by inversion microscope.(2)The inhibition rates of cell growth were assayed by MTT method.(3)Caspase-3activity was detected by colorimetric method.(4)The expressions of proliferating cell nuclear antigen(PCNA)、Bcl-2and Survivin were detected by immunohistochemical staining. (5) The expression of VEGF was detected by ELISA method.Restults:(1) The B16melanoma cells adhered to the bottom of the flask, growing actively with longer pseudopodia and spindle-like shape. After treated with PDTC, the B16melanoma cells were converted to the oval shape and spindle-like cells were absent. Intracellular conjunction was decreased, and a large number of floating cells were observed.(2)The inhibition rates of B16cell growth were increased in a time-dependent and dose-dependent manner after treated with PDTC. Compared with the control group, the difference was significant (P<0.01)(3)The Caspase-3activity was obviously increased with the increased concentration of PDTC. The difference was significant (P<0.01)(4)The results of immunohistochemical assessment showed that following24hours of treatment with PDTC, the expression level of PCNA、Bcl-2and Survivin were significantly decreased in B16cell, compared with the control group (P<0.01)(5) The results of ELISA showed that following24hours of treatment with PDTC, the expression of VEGF was downregulated in B16cell compared with the control group (P<0.01), and in a dose-dependent manner.Conclusion:(1) PDTC can inhibit the growth and induce apoptosis of B16cells in a manner of time-and-dose-dependent manner.(2)PDTC can induce apoptosis of B16cells significantly. The most possible anti-tumor mechanism of PDTC suppressing tumor growth and inducing apoptosis is correlated with down-regulation of PCNA、 Bcl-2and Survivin and up-regulation of Caspase-3.(3)PDTC can inhibit the expression of VEGF in B16cells. (4)PDTC may provide novel prospect for treatment of melanoma.