Objective Previous studies have found that micheliolide(MCL) could improve the sensitivity of breast cancer cells to chemotherapeutic drugs such as cisplatin and induce the apoptosis of breast cancer cells.This article aims to study the proliferation inhibition effect of micheliolide on lung cancer cells H460 and its underlying mechanism.Methods Human lung cancer cell line H460 was treated with different concentrations of micheliolide(30,60,90μmol/L).Then the cell proliferation was measured by-CCK8 and plate colony formation assays.The apoptosis and the cell cycle were detected by flow cytometry.Western blot was used to determine the mechanism of how MCL affecting cancer cell H460.Results Compared with the control (275.00±7.21), the clone numbers after 30μmol/L,60μmol/L and 90μmol/L MCL treatment(199.00±5.66,166.00±1.41, 90.00±7.81) were significantly decreased (P<0.05).Meanwhile, the CCK-8 results showed that compared to the control group, A value was significantly increased after 30μmol/L MCL treatment for 72h and 96h, and 60μmol/L or 90μmol/L MCL treatment for 48h,72h and 96 h (P<0.05).Compared with the control apoptotic ratio [(2.90±0.03)％], the ratio of early and late apoptotic cells after 30μmol/L, 60μmol/L and 90μmol/L MCL treatment [(5.23±0.76)％, (9.06±0.47)％, (19.00±0.64)％] were significantly increased (P<0.05).Compared with the control group, the ratio of G2/M phase cells[(12.52±0.88) ％ ,(17.22±0.43)％, (19.84±0.31)％] was gradually increased after 30μmol/L, 60μmol/L and 90μmol/L MCL treatment, and there was statistically significant difference after 60μmol/L and 90μmol/L MCL treatment (P<0.05).The ratio of S+G1/G0 phase cells[(87.53±1.06)％ ,(82.94±0.67)％ ,(79.79±0.21)％] was gradually decreased after 30μmol/L, 60μmol/L and 90μmol/L MCL treatment, and there was statistically significant difference after 60μmol/L and 90μmol/L MCL treatment (P<0.05).The expression level of notch4 was significantly decreased after 30μmol/L, 60μmol/L and 90μmol/L MCL treatment (P<0.05), while the expression level of cleaved caspase3 was significantly upregulated (P<0.05).Conclusion MCL exerted an inhibitory effect on lung cancer cell H460.

Objective:To explore whether tumor-inducing agent 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and bromodeoxyuridine (BrdU) affect CD133 expression in nasopharyngeal carcinoma (NPC) 5-8F cells. Methods:NPC cell line 5-8F was treated with single TPA, single BrdU, or TPA plus BrdU, respectively. CD133 mRNA and protein expression levels were detected by real-time fluorogentic quantitative PCR and Western blotting, respectively. Flow cytometry was used to separate CD133-positive cells and determine their levels. Boyden chamber test was used to measure the invasion capability of the cells. Results:Compared with untreated group, CD133 mRNA levels were increased in single BrdU group and BrdU plus TPA group (P=0.037 and 0.003, respectively), and decreased in single TPA group. Western blotting indicated that the expressions of CD133 protein was increased in all the three treated groups, and FCM showed that the quantity of CD133-positive cells also increased. The invasion capability was enhanced, especially in BrdU plus TPA group. Conclusion:Both TPA and BrdU increased CD133 expression in NPC.The effects of TPA and BrdU are synergestic.

Objective To construct a recombinant lentiviral expression vector for RNA interference (RNAi) of human Snail gene and to study its effects on the proliferation and invasion of nasopharyngeal carcinoma cell line 5-8F. Methods The effective sequence of short hairpin RNAs (shRNA) targeting Snail gene was designed and cloned into the linear pLVTHM vector after enzyme digestion. After confirmation by DNA sequencing, 5-8F cells were infected with the viral supernatants. The cells with stable Snail gene knock-down were separated by fluorescence activated cell sorter (FASC). The expression of Snail mRNA was detected by real time RT-PCR. MTT and cell invasion assay were used to detect the proliferation and invasion of 5-8F cells after plVTHM-siSnail transfection. Results The lentivirus vector plVTHM-siSnail was constructed successfully. The separated 5-8F-plVTHM-siSnail exhibited significant knock-down of Snail mRNA expression. Slower proliferation and decreased cells to permeate through the Matrigel were found after plVTHM-siSnail transfection (P

AIM:Based on comparative genomic hybridization(CGH) data,to construct tree model of esophageal carcinoma and to explore mechanism of multigene involved,multistep development and multipathway progression during esophageal carcinogenesis.METHODS:Using the software developed by Desper et al,tree models of esophageal carcinoma were constructed according to the CGH data of 78 esophageal carcinoma patients.RESULTS:Tree models for esophageal carcinoma suggested that there were-4p,-9p,-18q,+7p,+8q,+17p,+17q,+20p,+20q nine nonrandom genetic events,and +7p、+8q and +20q might be important early events in esophageal carcinogenesis,indicating that there might be cancer-related genes in these chromosomal arms.CONCLUSION:Tree models based on CGH data of esophageal carcinoma imply the process of multigene involved,multistep and multipathway progression.The tree models also give the direction to search for esophageal cancer-related genes.

Ten ATP binding cassette (ABC) transporters are confirmed to be associated with resistance against anticancer drugs. To investigate the relationship between these ten ABC transporters and the nasopharyngeal carcinoma cell line CNE2 resistant to cisplatin, cisplatin and cisplatin with 5-fluorouracil were used to induce the CNE2 cell to acquire the drug-resistance for 1 year. After these cells were cultured without drugs for 2 months, the MTT assay method was used to determine the dose-effect relationship of cisplatin and resistant index. Quantitative real time polymerase chain reaction was used to detect the mRNA expression of ten ABC transporters in CNE2 and the drug-resistant CNE2 cells, and the result was confirmed by immunocytochemical method. The results of MTT method showed that two cell lines resistant to cisplatin (named as CNE2/DDP) and cisplatin with 5-fluorouracil (named as CNE2/DDP+5Fu) were established, with resistant index 2.58 and 5.31, respectively. Of ten ABC transporters, only ABCC2 was found to be up-regulated both in CNE2/DDP and CNE2/DDP+5Fu cells, for increasing about 2.50 and 4.08 folds, respectively. The results of immunocytochemical method also confirmedthat the expression of ABCC2 in CNE2/DDP and CNE2/DDP+5Fu cells were stronger that that in CNE2 cell. Furthermore, ABCC2 protein was found to be located at nuclear membrane of CNE2/DDP +5Fu cell but not at nuclear membrane of CNE2 cell. The results suggest that ABCC2 may play an important role in cisplatinresistance of nasopharyngeal carcinoma cell line CNE2.

Objective To screen potential genes associated with drug resistance and multidrug resistance. Methods Microarray with 8000 genes was used to detect the different expression of 5-8F cells and 6-10B cells. Subsquently, genes of drug resistance and multidrug resistance were screened by MILANO online programme. Semiquantitative RT-PCR was utilized to confirmed the reliability of differentially expressed genes. Results 283 genes were identified the differential expression. Of these, 85 genes were shown to be upregulated and 98 downregulated. After the analysis of MILANO, 4 genes including UGT1A9 (15.85 folds),MVP(6.77 folds), CAV1(2.49 folds) and HIF1A(2.67 folds) with higher expression in 5-8F cells were found to be likely associated with drug resistance and multidrug resistance. Subsquently, semiquantitative RT-PCR confirmed the reliability of differential expression of these 4 genes. Conclusion Differentially expressed genes shown in NPC 5-8F cells compared to 6-10B cells with the identification of online MILANO program analysis are likely associated with drug resistance and mnltidrug resistance of NPC cells with the ability of metastasis.

AIM: To investigate the expression map of two p53 binding proteins 53BP1 and 53BP2 in nasopharyngeal carcinoma (NPC) tissue.METHODS: The expression of 53BP1 and 53BP2 mRNA in NPC biopsy and control group are tested by RT-PCR. The expression of two mRNA in NPC paraffin section are examined by in situ hybridization. RESULTS: No expression of 53BP1 mRNA was found in NPC tissue and control group. However, expression of 53BP2 was detected in NPC biopsy and control group by RT-PCR, specific expressoin found cancerous nest in NPC paraffin section by in situ hybridization.CONCLUSION: The high expression of 53BP2 may be related to the development of NPC.

Site-directed mutagenesis method was used to introduce two desired mutations, which were confirmed by DNA sequencing, into mouse complement receptor Type II gene(MCR2). Then the constructed eukaryotic expression vectors containing wild type mouse CR2/1(wtMCR2/1), mutant type mouse CR2/1 (mtMCR2/1) and human CR2 (hCR2) cDNA were transferred into mouse SP2/0 cells by electroporation. After two-week screening by G418, the stably transfected clones were obtained. Several ways including PCR, RT-PCR, and immunohistochemistry were utilized to screen those clones with interesting genes integrated and expressed. Then Epstein-Barr virus(EBV) was used to infect these transfected cells and EBER-1 (EBV encoded RNAs) hybridization results showed that only hCR2 and mtMCR2 transfected SP2/0 cells could be infected by EBV, but positive rate of the former was much higher than the latter. This study sets groundwork for elucidating the mechanism by which EBV enters the cells and for establishing the animal model of EBV-related nasopharyngeal carcinoma (NPC).

AIM:To examine the latent membrane protein 1(LMP1)-DNA sequence in nasopharyngeal carcinoma(NPC) and detect mRNA expression of LMP1, EBNA1, EBNA2, and to explore the relationship between EBV infectious status, expression products and NPC carcinogenesis.METHODS: LMP1 DNA was detected in NPC by PCR. Direct sequence was applied to analyze the difference between NPC-LMP1-DNA and B95-8-LMP1-DNA. mRNA expressions of LMP1, EBNA1, EBNA2 in NPC were detected by nested RT-PCR.RESULTS: LMP1 DNA existed in all 47 NPC tissues. Several single nucleotide variations were found between NPC-LMP1-DNA and B95-8-LMP1-DNA. The notable variation was the lost of XhoⅠrestriction site in NPC. Direct sequence showed 30 bp deletion in NPC. The mRNA expressions of LMP1, EBNA1 and EBNA2 in NPC were 76.6%, 80.0% and 74.5% respectively by nested RT-PCR. The expression of EBNA1 in NPC was promoted by Q promoter while the expression of EBNA1 in B95-8 was promoted by C promoter.CONCLUSION: The way of EBV involved in NPC is complex. Latent genes such as LMP1, EBNA1 and EBNA2 as well as early lytic gene BARF1 may all play certain roles in NPC carcinogenesis.

AIM: Our study focused on investigation of tissue specific regulatory activity of the newly cloned promoter: PLUNC-p with driving enhanced green fluorescent protein ( EGFP ). METHODS: Transgenic Xenopus Laevis system was applied.RESULTS: The green fluorescence protein directed under PLUNC-p was expressed strictly in branchial arches and epidermis of Xenopus Laevis embryos while CMV promoter showed ubiquitous regulation characteristic.CONCLUSION: PLUNC-p is able to direct epithelia specific expression of EGFP . This property of PLUNC-p might raise the possibility that lead target genes to express tissue-specifically.