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To investigate the expression and function of classical protein kinase C (PKC) isoenzymes in inducing MDR phenotype in gastric cancer cells.Two cell lines were used in the study: gastric cancer cell SGC7901 and its drug-resistant cell SGC7901/VCR stepwise-selected by vincristine 0.3, 0.7 and 1.0 mg.L(-1), respectively. The expression of classical PKC (cPKC) isoenzymes in SGC7901 cells and SGC7901/VCR cells were detected using immunofluorescent cytochemistry, laser confocal scanning microscope and Western blot. The effects of anti-PKC isoenzymes antibody on adriamycin accumulation in SGC7901/VCR cells were determined using flow cytometric analysis.(1)SGC7901 cells exhibited positive staining of PKC-alpha. SGC7901/VCR cells exhibited stronger staining of PKC-alpha than SGC7901 cells. The higher dosage vincristine selected, the much stronger staining of PKC-alpha was observed on SGC7901/VCR cells. (2)Both SGC7901 and SGC7901/VCR cells exhibited positive staining of PKC-beta I and PKC-beta II with no significant difference. (3) Compared with SGC7901, SGC7901/VCR cells had decreased adriamycin accumulation and retention. Accumulation of adriamycin in SGC7901 was 5.21+/-2.56 mg.L(-1),in SGC7901/VCR 0.3 was 0.85+/-0.29 mg.L(-1), in SGC7901/VCR 0.7 was 0.81+/-0.32 mg.L(-1), and in SGC7901/VCR 1.0 was 0.80+/-0.33 mg.L(-1); Retention of adriamycin in SGC 7901 was 2.51+/-1.23 mg.L(-1), in SGC7901/VCR 0.3 was 0.47+/-0.14 mg.L(-1), in SGC7901/VCR 0.7 was 0.44+/-0.15 mg.L(-1), and in SGC 7901/VCR 1.0 was 0.41+/-0.11 mg.L(-1). (4) Fluorescence intensity presented adriamycin accumulation in SGC7901/VCR cells was increased from 1.14+/-0.36 to 2.71+/-0.94 when cells were co-incubated with anti-PKC-alpha but not with anti-PKC-beta I PKC-beta II and PKCgamma antibodies.PKC-alpha, but not PKC-beta I, PKC-beta II or PKCgamma, may play a role in multidrug resistance of gastric cancer cells SGC7901/VCR.

To investigate the effect of L-NAME on nitric oxide and gastrointestinal motility alterations in cirrhotic rats.Rats with cirrhosis induced by carbon tetrachloride were randomly divided into two groups, one n =13 receiving 0.5mg.kg(-1) per day of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, for 10 days, whereas the other group (n =13) and control (n =10) rats were administrated the same volume of 9g.L(-1) saline. Half gastric emptying time and 2h residual rate were measured by SPECT, using (99m)Tc-DTPA-labeled barium sulfate as test meal. Gastrointestinal transition time was recorded simultaneously. Serum concentration of nitric oxide (NO) was determined by the kinetic cadmium reduction and colorimetric methods. Immunohistochemical SABC method was used to observe the expression and distribution of three types of nitric oxide synthase (NOS) isoforms in the rat gastrointestinal tract. Western blot was used to detect expression of gastrointestinal NOS isoforms.Half gastric emptying time and trans-gastrointestinal time were significantly prolonged(124.0 +/- 26.4 min; 33.7 +/- 8.9 min; 72.1 +/- 15.3 min; P0.01), (12.4 +/- 0.5h; 9.5 +/- 0.3h; 8.2 +/- 0.8h; P0.01), 2h residual rate was raised in cirrhotic rats than in controls and cirrhotic rats treated with L-NAME (54.9 +/- 7.6%,13.7 +/- 3.2%, 34.9 +/- 10.3%, P0.01). Serum concentration of NO was significantly increased in cirrhotic rats than in the other groups (8.20 +/- 2.48) micromol.L(-1), (5.94 +/-1.07) micromol.L(-1) and control (5.66 +/- 1.60 micromol.L(-1), P0.01. NOS staining intensities which were mainly located in the gastrointestinal tissues were markedly lower in cirrhotic rats than in the controls and cirrhotic rats after treated with L-NAME.Gastrointestinal motility was remarkably inhibited in cirrhotic rats, which could be alleviated by L-NAME. Nitric oxide may play an important role in the inhibition of gastrointestinal motility in cirrhotic rats.

AIM:To observe the drug sensitizing effect and related mechanisms of fas gene transduction on human drug-resistant gastric cancer cell SGC7901/VCR (resistant to Vincristine).METHODS:The cell cycle alteration was observed by FACS. The sensitivity of gastric cancer cells to apoptosis was determined by in vitro apoptosis assay. The drug sensitization of cells to several anti-tumor drugs was observed by MTT assay. Immunochemical method was used to show expression of P-gp and Topo II in gastric cancer cells.RESULTS:Comparing to SGC7901 and pBK-SGC7901/VCR, fas-SGC7901/VCR showed decreasing G2 cells and increasing S cells, the G2 phase fraction of pBK-SGC7901/VCR was about 3.0 times that of fas -SGC7901/VCR but S phase fraction of fas -SGC7901/VCR was about 1.9 times that of pBK-SGC7901/VCR, indicating S phase arrest of fas-SGC7901/VCR. FACS also suggested apoptosis of fas-SGC7901/VCR.fas-SGC7901/VCR was more sensitive to apoptosis inducing agent VM-26 than pBK-SGC7901/VCR. MTT assay showed increased sensitization of fas-SGC7901/VCR to DDP, MMC and 5-FU, but same sensitization to VCR according to pBK-SGC7901/VCR. SGC7901, PBK-SGC7901/VCR and fas -SGC7901/VCR had positively stained Topo II equally. P-gp staining in pBK-SGC7901/VCR was stronger than in SGC7901, but there was little staining of P-gp in fas-SGC7901/VCR.CONCLUSION:fas gene transduction could reverse the MDR of human drug-resistant gastric cancer cell SGC7901/VCR to a degree, possibly because of higher sensitization to apoptosis and decreased expression of P-gp.