Objective To explore the effect of Latexin (Lxn) gene transfection on proliferation of CD13;MIAPaca-2 pancreatic cancer stem-like cells.Methods CD133+ MIAPaca-2 cells were isolated and sorted by magnetic activated cell sorting from pancreatic cancer MIAPaca-2 celt line.CD133+ MIAPaca-2 cells were cultured in serum-free medium and the capacity for proliferation,and tumorigenicity of CD133+ MIAPaca-2 cells was determined by the floating spheres test and tumor xenograft assays.The CD133+ MIAPaca-2 cells were transfected with Lxn plasmid (1,3,5 μg).After transfection,the protein and mRNA expression of Lxn in CD133+ and CD133+-MIAPaca-2 cells were detected by Western blotting and RT-PCR,respectively.Cell proliferation was assayed by CCK-8.Results CD133+ MIAPaca-2 cells were successfully isolated,and it grew into a ball-suspended way,the tumorigenicity rate in nude mice with subcutaneous injection 1 × 105 cancer cells was 100％.After Lxn plasmid transfection,the expression of Lxn in CD133+ MIAPaca-2 cells was increased in a dose dependent manner,the Lxn protein and mRNA expression of tumor cells transfected with 5 μg plasmid was 20.80 ±0.98,16.80± 2.73,which was significantly higher than that in non-transfected cells (1.02 ± 0.01,1.01 ± 0.01),and the difference between the two groups was statistically significant (P ＜ 0.05).After transfection,cellular proliferation activity also showed a transfection dose and culture time-dependent decrease,the inhibition rate of tumor cells transfected with 0.4 μg plasmid was 36.2％,which was significantly different from that in non-transfected cells (P ＜ 0.05).Conclusions CD133+ MIAPaca-2 pancreatic cancer cells have some characteristics of cancer stem cells.Lxn gene transfection can inhibit the proliferation of CD133+ MIAPaca-2 cells.

Objective To observe the effect of Latexin treatment on the chemoresistance in gemcitabine-resistant pancreatic cancer cell line SW1990, and explore the potential mechanism.Methods Gemcitabine-resistant pancreatic cancer cell line SWl990/GZ was induced and established by increasing gemcitabine dosage intermittently.IC50 of gemcitabine in SW1990 cells and SWl990/GZ cells pre and post Latexin treatment at the dosage of 10, 20 and 40 ng/μl for 48 h was evaluated using CCK-8 assay.The mRNA and protein expression of Latexin gene in SW1990 and SW1990/GZ cells were evaluated using qRT-PCR and Western blot, and the expression of Shh and Gli1 in 40 ng/μl Latexin treated SW1990 and SW1990/GZ cells for 48 h.Results A gemcitabine-resistant pancreatic cancer cell line SWl990/GZ was obtained successfully, which can grow stably and passage in the media containing 150 μmol/L gemcitabine.The IC50 values of gemcitabine in SW1990 cells and SWl990/GZ cells were (3.8±0.4)μmol/L and(226.52±13.61)μmol/L, respectively, and the later was greatly higher than the former, which was statistically different (P=0.000).The drug resistance indexes (RI) was 59.6.After treated with different concentrations of Latexin(10,20,40 ng/μl), the IC50 of SW1990 cells was (3.0±0.4)μmol/L, (2.5±0.3)μmol/L and (1.8±0.3)μmol/L, respectively,and the IC50 of SW1990/GZ cells was(113.08±5.01)μmol/L,(70.26±2.31)μmol/L and (42.12±1.31)μmol/L, respectively.Compared with the untreated cells,the IC50 of gemcitabine in 20,40 ng/μl Latexin treated cells was obviously decreased, and the differences were statistically significant (P<0.05).Compared with the SW1990 cells,the expression of Latexin in SW1990/GZ cells was obviously decreased.RI were 37.7, 28.1 and 23.1,respectively.mRNA relative expression of Latexin in SW1990 and SW1990/GZ cells were 0.85±0.08 and 0.31±0.07, and protein relative expression were 0.49±0.09 and 0.13±0.05, and Latexin expression in SW1990/GZ was obviously lower than that in SW1990 cells and the difference was statistically significant (P<0.05).After being treated by 40 ng/μl Latexin, SHH mRNA in SW1990/GZ cells decreased from 0.89±0.09 (control cells) to 0.53±0.06, Gli1 mRNA decreased from 0.58±0.06 to 0.35±0.05, Shh protein decreased from 0.72±0.09 to 0.35±0.06,Gli1 protein level decreased from 0.78±0.08 to 0.28±0.03, and all the differences were statistically significant (P<0.05 or 0.01).Conclusions Latexin can significantly improve the chemosensitivity in gemcitabine-resistant pancreatic cancer cells, and the potential mechanism may be related to the inhibition of sonic hedgehog pathway activation.

Objective To investigate the expression of stathmin and evaluate its influence to anti-microtubule adjuvant chemotherapy in non-small-cell lung cancer(NSCLC).Methods The clinical data and survival status of 78 NSCLC patients were collected,and their paraffin-embedded tissue were detected immunohistochemically with a rabbit anti-human stathmin polyclonal antibody.The clinical significance of stathmin expression and its influence to overall survival rate were analyzed statistically between patients who received paclitaxel or vinblastine adjuvant chemotherapy.Results The positive expression of stathmin could only be observed in the cytoplasm of cancer cells.Among 78 patients,40 (51.3 ％ ) patients were stained stathmin-positive.The positive rate of stathmin was significantly higher in male than female,in central type than peripheral type,in pleura-involved than non-involved,and in dead patients than survival patients ( P ＜ 0.05 ),but showed no significant differences in patients with different age,differentiated grade,pathological type,clinical stage,or lymph-node metastasis status.The expression of stathmin had a significant influence to overall survival rate(x2 =4.348,P ＜0.05 ),and those stathmin-negative patients showed a longer survival time.In stathmin-negative patients,those who received adjuvant chemotherapy with vinblastine exhibited a shorter survival time than those with paclitaxel,but the P =0.06.In stathmin-positive patients,the survival rate or time showed no difference between groups with paclitaxel and vinblastine.The differentiated grade,metastasis to lymph node and expression of stathmin were independent risk factors influencing survival rate.The positive expression of stathmin could only be observed in the cytoplasm of cancer cells.Among 78 patients,40 (51.3 ％ )patients were stained stathminpositive.The positive rate of stathmin was significantly higher in male than female,in central type than peripheral type,in pleura-involved than non-involved,and in dead patients than survival patients ( P ＜ 0.05 ),but showed no significant differences in patients with different age,differentiated grade,pathological type,clinical stage,or lymph-node metastasis status.The expression of stathmin had a significant influence to overall survival rate(x2 =4.348,P ＜ 0.05 ),and those stathmin-negative patients showed a longer survival time.In stathmin-negative patients,those who received adjuvant chemotherapy with vinblastine exhibited a shorter survival time than those with paclitaxel,but the P =0.06.In stathmin-positive patients,the survival rate or time showed no difference between groups with paclitaxel and vinblastine.The differentiated grade,metastasis to lymph node and expression of stathmin were independent risk factors influencing survival rate.Conclusion Our study suggested that the detection of stathmin in resected NSCLC tumor tissues may be helpful for prediction of prognosis,but helpless for making a choice between paclitaxel and vinblastine.NSCLC patients with stathmin-negative,no metastasis to lymph node or good-differentiated grade may have a better prognosis.

Objective To study the effects of Lxn on CD133 + PANC-1 pancreatic cancer cells.Methods CD133 + PANC-1 cell were isolated by magnetic activated cell sorting (MACS).The properties of the CD133 + PANC-1 cells and Lxn effects on CD133 + PANC-1 cell proliferation in transplanted tumor in nude mice were determined by floating spheres test and tumor xenograft assays.Cell proliferation was assayed by Cell Counting Kit-8 (CCK-8).The Bcl-2,Bax protein and mRNA expression of CD133 + PANC-1 cells treated by Lxn were analyzed by Western blot and Quantitative real-time PCR (qRT-PCR).Results We successfully isolated the CD133 + PANC-1 cells and cultured in serum free medium,CD133 + PANC-1 cells formed sphere,while CD133-PANC-1 cells grew with adherence slowly and then underwent apoptotic process.CD133 + PANC-1 cells showed high tumorigenic in athymic BALB/c mice.Lxn suppressed the growth of transplanted tumor obviously.Compared with control group [(225.52 ± 34.09) mm3],tumor volume decreased significantly (P ＜ 0.05).Significant reduction in cell proliferation was observed in response to Lxn in PANC-1 CD133 + cells by CCK-8 assay with concentration and time dependent manners (P ＜ 0.05).Treated by Lxn,Bcl-2 expression decreased,Bax expression increased.Conclusions Lxn inhibits the proliferation of CD133 + PANC-1 cells probably through a mechanism down-regualting Bcl-2 and up-regulating Bax.