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Purpose. To elucidate the apoptosis induction of Epigallocatechin-3-gallate (EGCG) on nasopharyngeal carcinoma (NPC) cells via mitochondrial signal transduction pathway regulated by EB-virus-encoded latent membrane protein 1 (LMP1). Methods. The survival rates of pTet-on-LMP1 HNE2 cells after the EGCG treatment were determined by MTT assay. Induction of apoptosis in pTet-on-LMP1 HNE2 cells after the EGCG treatment was analyzed by agarose gel electrophoresis. The activity of caspase-9 was determined by ApoAlert Caspase-9 Fluorescent Assay kit after the EGCG treatment. The protein expressions of cytochrome c and Bcl-2 were analyzed by Western blotting after the EGCG treatment. Results. EGCG inhibited the survival rates of pTet-on-LMP1 HNE2 cells and induced apoptosis of pTet-on-LMP1 HNE2 cells. EGCG raised the activities of caspase-9, enhanced the releasing of cytochrome c from the mitochondria, and suppressed the protein expression of Bcl-2. These are the key targets on the mitochondrial signal transduction pathway in apoptosis. Conclusions. EGCG inhibited the survival rate of NPC cells and induced apoptosis of NPC cells via the mitochondrial signal transduction pathway. This study suggests that the interference effect of EGCG on targets of the mitochondrial signal transduction pathway plays an important role in the anticancer function.

To determine the effect of activation of specific anti-tumor cytotoxic T lymphocytes (CTL) and the ability of cross-presentation in vitro by fusion of HLA-A2+ human dendritic cells (DCs) with HLA-A2- melanoma cells.The HLA-A2+ human dendritic cells and HLA-A2- melanoma cells were fused by PEG and were cultivated in complete RPMI1640 media containing FCS (10%) and GM-CSF for 24-48 h, and then co-cultured fusion cells with Melan-A specific T cells. HLA-A2- melanoma cells were negative control,While T2 cells and DC+Pts were positive control. The activation of anti-tumor CTL elicited by the fusion cells was detected by intracellular cytokine staining.The immature DC could express CD80, CD83, CD86, HLA-DR, and HLA-ABC,but the mature DC induced by TNF-alpha, PGE-2, and CD40L further highly expressed above molecules. The rate of specific CTL cells primed by the fusion cells was 16.72%+/-4.26%, negative control was 0.21%+/-1.84%,and positive control was 28.60%+/-5.67%. The CTL from vaccine by fusing DC and LAR6 induced lysis of HLA-A2+ LAR1 cells.The HLA-A2 restricted specific anti-tumor CTL can be induced in vitro by fusion of HLA-A2+ human dendritic cells with HLA-A2- melanoma cells.

To establish a melanoma cell line and identify its characteristics in vitro.The tissues from biopsies of melanoma were performed primary culture. After growing to 90% confluence, the cells were detached and transferred to another flask for subculture, and then we identified their characteristics including growth kinetic, morphology and tumorigenecity.This cell line was cultivated for more than 90 times. Its characteristics were as follows:The pathological morphology of the tumor transplanted in nude mice were similar to that of the original lesion;The growth curves of the 20th passage were determined, and the population doubling time calculated was 56.9 h; The cloning efficiency in soft agar was 19.1%; Karyo-type analysis showed aneuploidy with the modal chromosomal number 85-102; Electronmicroscopical observation showed that there were rich microvilli on the surface of the cells, abundant ribosomes and melanoid grain; SP immunohistochemical staining showed that the cells expressed HMB-45.The Melanoma cells were immortalized after being cultured in vitro and a new melanoma cell line was established.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) plays a vital role in cell transformation and the nasopharygeal carcinoma (NPC) is highly related to EBV infection. To elucidate whether LMP1 exerts oncogenic effect in NPC and its possible mechanism, a dual-stable LMP1 integrated NPC cell line with Tet-on regulating system were established. Results revealed that expression of LMP-1 could be regulated by Tet system when NPC cell line HNE(2) served as receipt cell, and doxycycline could increase this expression by about 10 fold. Therefore, it is possible that different level expression of LMP-1 results in its different biological functions and various efforts on nasopharyngeal carcinogenesis.

Previous work of the authors established the protein expression spectra of D-type cyclins, and found that there were differential expression in nasopharyngeal carcinoma (NPC) biopsies. Using Western Blotting, aberrant overexpression of the cyclinD1 protein in NPC cell lines is reported. CyclinD2 and cyclinD3, the other members of D-type cyclins, were also detected in NPC cell lines. A comparison of the expression patterns in the cell lines, positive or negative in the latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus, revealed that the expression of D-Type cyclin proteins might be increased by EBV-LMP1. The abundance of these proteins showed characteristic variation consistent with a cell- cycle-dependent oscillation and the peak levels expressed in G1 as analyzed by double-parameter flow cytometry. These data suggest that cyclinD1 is essential for cell cycle progression in G1 and may play a role in NPC carcinogenesis.