We studied the methodology of 3-(4, 5-dimethyl-2-lhiazoly) )2, 5-diphenyl-2H-tetrazolium bromide(MTT) colorimetric assay for anticancer drug sensitivity test of human solid tumors. Results showed that the optimal cell concentration per well is 0.5-1 ? 10~5, the optimal incubation period for the action of anticancer drugs on tumor cells is three days. The concentration of anticancer drugs used should be varied with different drugs. We observed 167 samples of human solid tumors. The applicability of this test is concerned with the growth characteristics of tumor cells, histological types of malignancy, and the resected portion of the tumor specimen. The MTT colorimetric assay is a simple, rapid and reliable method for anticancer drug sensitivity test of human solid tumors

Objective To observe the effects of oxaliplatin on proliferation in human colon carcinoma cell lines HT29 in vitro and investigate the mechanism. Methods The inhibition of proliferation in HT29 cell was estimated by MTT test. Morphologic changes were observed under electron microscope. Distribution of cell cycle and apoptosis was analyzed by using flow cytometry. The expression of cell cycle protein and apoptosis-associated gene protein was detected with immunohistochemical technique. Results Oxaliplatin could inhibit the proliferation of HT29 cells and the inhibition depended on the exposure dose and time. When treated with oxaliplatin for 72 h, apoptotic cells and apoptotic body could be found under the transmission electronic microscope. The analysis of cell cycle indicated that oxaliplatin blocked cells at G2/M phases and cells of G0/G1 and S phase reduced. The expression of Fas and Bax was up-regulated; Bcl-2, CyclinB1, and PCNA down-regulated. Conclusion Oxaliplatin could inhibit proliferation of the colon carcinoma cell lines HT29. The mechanism of inhibited proliferation is related to the induced apoptosis and the blocked cells.

ObjectiveTo construct the eukaryotic expression plasmids of tumor susceptibitity gene(TSG)101-siRNA and investigate the effect of RNA interference targeting tsg101 gene and cisplatin on liver cancer resistant strain QGY/CDDP.MethodsThe targeting fragments specifically against TSG101 were designed according to the principle of small interfering RNA designation using computer software.The sequences were cloned into siRNA expression plasmids through DNA recombinant technology.The expressions of tsg101mRNA and TSG101 after TSG101-siRNA transfection was detected by RT-PCR and Western blot respectively.MTT test were applied to measure the proliferation of QGY/CDDP strain.ResultsThe fragments of interest were obtained by digestion and further confirmed by DNA sequencing.TSG101-siRNA inhibited the expressions of tsg101mRNA and TSG101 and the proliferation of QGY/CDDP,especially in combination with cisplatin.ConclusionThe eukaryotic expression vector of TSG101-siRNA combined with cisplatin inhibits the proliferation of QGY/CDDP and increases the sensitivity of liver cancer resistant strain QGY/CDDP to chemotherapy.

Objective To construct an siRNA expression plasmid against tumor susceptibility gene 101(tsg101) and investigate its effect of RNA interference on oxaliplatin(L-OHP)-resistant human colon cancer cell line HT29/L-OHP.Methods The targeting fragments specifically against tsg101 were designed according to the principle of small interfering RNA designation using an online software.The sequences were cloned into siRNA expression plasmids,and the plasmids were transfected into HT29/L-OHP cells.The expression of tsg101 mRNA after the transfection of TSG101-siRNA plasmid was detected by RT-PCR.The expression of TSG101 after TSG101-siRNA transfection was detected by Western blotting.MTT test was applied to measure the inhibition combined with L-OHP on the proliferation of HT29/L-OHP cells.Distribution of cell cycle was analyzed using flow cytometry after RNA interference to HT29/L-OHP cells.Results The expected fragments were designed,and the TSG101-siRNA plasmid was confirmed by DNA sequencing.The TSG101-siRNA plasmid inhibited the expression of tsg101 mRNA,and the expression of TSG101 protein,and suppressed the proliferation of HT29/L-OHP cells obviously when combined with oxaliplatin.The analysis of cell cycle indicated that the TSG101-siRNA plasmid reduced the cells in G2/M phases and increased the cells in G0/G1 and S phases.Conclusion The expression vector of TSG101-siRNA combined with oxaliplatin inhibits the proliferation of HT29/L-OHP cells and increases the sensitivity to chemotherapy.

Objective To investigate the effect of phosphatidylinositol 3-kinase(PI3K) inhibitor wortmannin on migration and invasion of human thyroid follicular carcinoma cell line CGTHW-1 and its mechanism.Methods MTT colorimetric assay was used to select the optimal concentration and time of giving wortmannin.CGTHW-1 cells used in present study were divided into control group and experimental group(cultured in the medium with or without blood serum).Cells of experimental group were treated with wortmannin,and that of control group were given no treatment.The migration and invasion ability of CGTHW-1 cells was detected by scratch test and Transwell chamber assay respectively.Microfilament fluorescence staining with fluorescein isothiocyanate(FITC)-labeled phalloidin was performed to observe the morphologic changes in CGTHW-1 cells.The expressions of Rac1 protein and mRNA in CGTHW-1 cells were examined by immunocytochemistry staining and semiquantitative RT-PCR.Results When CGTHW-1 cells were cultured in serum medium,the migrating speed of the cells slowed down,and the number of cells passing through the Transwell chamber reduced significantly after being treated with wortmannin in experimental group(13.8?3.03) than in control group(41.6?6.95,P0.05).Conclusions Wortmannin can effectively depress the migration and invasion of human thyroid follicular carcinoma cells,and the depression effect may be due to cytoskeleton rearrangement induced by inhibiting the expression of Rac1(the downstream molecule of PI3K).PI3K and Rac1 are probably potential targets for human thyroid cancer treatment,which may provide a theoretical evidence for the drug treatment of thyroid cancer.

Objective To investigated the effect of somatostatin(SS)on cholecystokinin-induced alterations of exocrine function and the redistribution of the F-actin in the pancreatic acinar cells.MethodsIn vitro,isolated rat pancreatic acini were divided into 12 groups:normal control group,different concentrations ofcholecystokinin-octapeptide groups(CCK-8)of 10-12,10-11,10-10,10-9 or 10-8 mol/L,SS group(10-7mol/L),SS and different dose CCK-8 groups.The acinar cells were stimulated with different concentrations of CCK-8 for 30 min in the presence or absence of 10-7 mol/L SS.Amylase and trypsin activity were measured using corresponding assay kits,and F-actin distribution in pancreatic acinar cells were detected by laser confocal microscopy.ResultsSS stabilized the structure of cytoskeleton in acinar cells,and remarkably increased the ratio of subapical/basolateral F-actin(P

Objective To investigate the antitumor mechanism of sodium arsenite on human gastric carcinoma cell line SGC-7901 in vitro. Methods MTT assay, light microscopy, electron microscopy, flow cytometry, and immunocytochemical staining were used to analyze the effect of sodium arsenite on biologic behavior of SGC-7901 cells. Results Sodium arsenite (2.50 ～ 40.00 ?mol/L) could inhibit the growth of gastric carcinoma cells, it depended on the duxation and concentration, and its 50% inhibitory concentration(IC50) was 8.69 ?mol/L after 72 hours' treatment. SGC-7901 cells were arrested significantly in G2/M phase treated with sodium arsenite for 48 and 72 hours. SGC-7901 cells presented typical morphologic feature of apoptosis and necrosis after exposure to sodium arsenite. Sodium arsenite up-regulated Caspase-3 protein expression in SGC-7901. Conclusion Sodium arsenite could obviously inhibit the proliferation of SGC-7901 cells, induce cell cycle arrest and apoptosis and necrosis of the cells. its mechanism is possibly associated with inhibition of elimination of ROS and the up-regulated expression of Caspase-3 protein.

AIM:To investigate the effects of fucoidan on the angiogenesis of multiple myeloma cells in vitro, and its related mechanisms .METHODS:The human multiple myeloma RPMI 8226 cells and human endothelial cells were cultured in vitro.The growth inhibition rate of RPMI 8226 cells was examined by MTT assay .The cell cycle and apoptosis rate were measured by flow cytometry .RPMI 8226 cells were treated with fucoidan for 72 h, and the cell culture superna-tant was collected .The VEGF concentration was examined by ELISA , and the tube formation assay was applied to assess the angiogenic activity .After treatment with fucoidan for 72 h at different concentrations , the protein levels of HIF-1α, VEGF, p-AKT and p-ERK1/2 were detected by Western blot .RESULTS: Fucoidan inhibited the growth of RPMI 8226 cells in a dose-and time-dependent manner .After treatment with fucoidan for 72 h, the cell cycle was arrested at G 1 phase , and the apoptotic rate of RPMI 8226 cells was increased with the increasing concentration of fucoidan , which was much higher than that in control group (P<0.05).The VEGF concentration was significantly decreased with the increa-sing concentration of fucoidan .The numbers and areas of the capillary-like structures decreased while the concentration of fucoidan increased, and those at 100 mg/L were less than those in the control (P<0.05).The protein levels of HIF-1α, VEGF, p-AKT and p-ERK1/2 in fucoidan group were significantly lower than those in control group (P<0.05).CON-CLUSION:Fucoidan inhibits the secretion of VEGF in multiple myeloma cells , and reduces angiogenesis induced by mul-tiple myeloma cells .It inhibits the protein expression of HIF-1αand VEGF , which may be related to inhibiting the phos-phorylation of AKT and ERK1/2.

Objective To establish a drug-resistant human colon cancer cell line to oxaliplatin(L-OHP)based on cell line HT29.Methods A resistant cell line-HT29/L-OHP was established by gradually increasing the dose of L-OHP and intermittent administration.The growth curves,multidrug resistance and resistance index of HT29/L-OHP cell line to anticancer agents were detected by MTT assay.The changes of its biological characteristics were determined by light microscopy,electron microscopy,and flow cytometry.Results HT29/L-OHP cell line was established after 3 months,which had stable resistance to L-OHP and a resistance index of 10.64.HT29/L-OHP cells exhibited cross resistance to many other chemotherapeutic agents.As compared with parental cells,the morphological and chromatosome number of HT29/L-OHP were changed;its doubling time was prolonged;and the number of cells in S phase and G0/G1 phase were decreased while in G2/M phase increased.Conclusion HT29/L-OHP cell line shows the typical multidrug-resistant phenotype.

Objective To investigate the effects of curcumin on the proliferation, cell cycle distribution, and apoptosis of the acute myeloid leukemic cell line HL 60 and the hepatocarcinoma cell line QGY. Methods MTT method was used to detect the biological activities of curcumin at different concentrations and at different time. The cell cycle distribution was analyzed by flow cytometry, and changes of the cellular ultrastructure were observed by electronic microscopy. Results Curcumin could inhabit the growth of HL 60 and QGY in dose and time dependent manners. IC50 values of curcumin at 72 h to HL 60 and QGY were 24.8 ?mol/L and 49.5 ?mol/L, respectively. The cell growth of HL 60 was arrested at S and G 2/M stages (apoptosis peak: 8.65%), and that of QGY was arrested at S stage (apoptosis peak: 10.84%). Curcumin could lead to fat degeneration in HL 60 cells and cell degeneration and necrosis of QGY cells, resulting in apoptosis of QGY cells. Conclusion Curcumin has inhibitory effect on the growth of HL 60 cells through its anti proliferation and on QGY cells through the induction of apoptosis of QGY cells.