OBJECTIVE: To explore the effect of hypoxia on the chemosensitivity of B-acute lymphoblastic leukemia （B-ALL） cells to Vincristine (VCR) and the mechanisms. METHODS: B-ALL cells SUP-B15, Nalm-6 and RS4;11 were selected as the research objects. The cells were divided into the control group and the hypoxia mimic group (CoCl₂ pretreatment). The two groups were treated with VCR at different concentrations for 24 hours, CCK-8 was used to detect cell viability, flow cytometry was used to detect cell apoptosis, and Western bolt method was used to detect hypoxia inducible factor (HIF-1α), BAX, Bcl-2 and β-actin protein expression. Quantitative real-time fluorescent PCR (qRT-PCR) was used to detect BAX and β-actin mRNA levels. RESULTS: CoCl₂ could simulate hypoxic environment to induce the expression of HIF-1α. The cells SUP-B15 and RS4;11 of the hypoxia mimic group were lower sensitivity to VCR as compared with the control group; the apoptosis rate of the hypoxia mimic group was lower than that of the control group after 80 nmol/L VCR treatment. The expression levels of BAX protein and mRNA in the hypoxia mimic group were lower than those of the control group, and there was no significant difference in the expression levels of Bcl-2 protein between two groups. CONCLUSION Under hypoxic conditions, HIF-1α may mediate VCR resistance in B-ALL cells by downregulating the pro-apoptotic protein BAX.
Actins/pharmacology* ; Apoptosis ; Cell Hypoxia ; Humans ; Hypoxia ; Hypoxia-Inducible Factor 1, alpha Subunit ; Proto-Oncogene Proteins c-bcl-2 ; RNA, Messenger ; Vincristine/pharmacology* ; bcl-2-Associated X Protein/pharmacology*

The aim of this study was to investigate the effect of Wnt5a on the vincristine (VCR) resistance in human ovarian carcinoma SKOV3 cells and its possible mechanism. The drug-resistant SKOV3/VCR cells were established by stepwise exposure to VCR, and then the SKOV3/VCR cells were stably transfected with specific shRNA interference plasmid vector targeting for Wnt5a. The mRNA expression level of Wnt5a was measured by RT-PCR. CCK-8 assay was used to detect the cell viability of SKOV3/VCR cells. The apoptosis was analyzed by flow cytometry. The protein expression levels of Wnt5a, MDR1, Survivin, β-catenin, Akt, p-Akt(S473), GSK3β and p-GSK3β(Ser9) were detected by Western blot. The result showed that SKOV3/VCR cells had significantly higher protein expression levels of Wnt5a, MDR1, Survivin and β-catenin, phosphorylation levels of Akt and GSK3β, and mRNA expression level of Wnt5a, compared with SKOV3 cells (P < 0.05). WNT5A gene silencing significantly increased the sensitivity of SKOV3/VCR cells to VCR, the IC of VCR being decreased from 38.412 to 9.283 mg/L (P < 0.05), synergistically enhanced VCR-induced apoptosis of SKOV3/VCR cells (P < 0.05), down-regulated the protein expression levels of MDR1, β-catenin and Survivin (P < 0.05), and inhibited phosphorylation of Akt and GSK3β (P < 0.05). Meanwhile, LY294002 (PI3K inhibitor) decreased the protein expression levels of MDR1, β-catenin and Survivin, as well as the phosphorylation levels of Akt and GSK3β in SKOV3/VCR cells (P < 0.05). These results suggest that WNT5A gene silencing reverses VCR resistance in SKOV3/VCR cells possibly through blocking the PI3K/Akt/GSK3β/β-catenin signaling pathway, and thus down-regulating the protein expression levels of MDR1 and Survivin.
ATP Binding Cassette Transporter, Subfamily B ; metabolism ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Female ; Gene Silencing ; Glycogen Synthase Kinase 3 beta ; metabolism ; Humans ; Ovarian Neoplasms ; pathology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; Survivin ; metabolism ; Vincristine ; pharmacology ; Wnt-5a Protein ; metabolism

OBJECTIVE: This study aimed to explore the relationship among expression of Survivin and MRP and drug resistance in NPC. METHOD: Expression of Survivin were detected by immunohistochemistry method in 45 cases of NPC and 24 cases of normal mucous membrane of nasopharynx (NMMN). The relationship between expression of Survivin and pathological factors in NPC were analysized. Expression of Survivin and MRP were detected in 31 patients of NPC with paclitaxel resistance and 20 patients of NPC without paclitaxel resistance. The relation- ship among the expression of Survivin or MRP and paclitaxel resistance in NPC were analysized. The paclitaxel resistance cell line, 5-8F-PTX(+); was established by a step-increased method. The expression of Survivin and MRP were detected by western blot in 5-8F-PTX(+) and 5-8F. RESULT: The positive were 71. 1% (32/45) in NPC and 8.33% (2/24) in NMMN. And there were significantly differences between them (P < .05). There were relationship among expression of Survivin and differentiation degree, lymph node metastasis, distant metastasis, and clinic stages of NPC. The positive were 75.9% (31/39) in moderately differentiated NPC and 16.7% (1/6) in lowly differentiated NPC, respectively. There were significantly differences between them (P < 0.05). The positive of Survivin were 83.9% (26/31) in NPC patients with paclitaxel resistance and 45.0% (9/20) in NPC patients without Paclitaxel resistance, respectively. There were significantly differences between them (P < 0.05). The positive of MRP were 87.1% (27/31) in NPC patients with paclitaxel resistance and 40.0% (8/20) in NPC patients without paclitaxel resistance, respectively. There were significantly differences between them (P < 0.05). There were positive correlation between the expression of Survivin and MRP in NPC patients with Paclitaxel resistance. The expression of Survivin and MRP were higher in 5-8F-PTX(+) than in 5-8F. The IC50 of paclitaxel, cDDP, 5-FU and Vincristine were significantly higher in 5-8F-PTX(+) than in 5-8F. CONCLUSION There were relationship among the expression of Survivin and difference, metastasis and TNM stages of NPC. Survivin may serves as a molecular marker for development and progress in NPC. There were relationship among the high expression of Survivin and MRP and increasing of drug resistance in NPC.
Antineoplastic Agents ; pharmacology ; Carcinoma ; Cisplatin ; Drug Resistance, Neoplasm ; Fluorouracil ; Humans ; Immunohistochemistry ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Lymphatic Metastasis ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; Nasopharynx ; metabolism ; Paclitaxel ; pharmacology ; Survivin ; Vincristine

OBJECTIVETo explore the reversal effect of multidrug resistance of Curcuma Wenyujin (CW) and its possible mechanism by establishing Vincristine-resistant gastric cancer SGC-7901 cells (SGC-7901/VCR) induced subcutaneous transplanted tumor in nude mice.

METHODSFirst we identified the resistance of SGC-7901/VCR by using methyl thiazolyl tetrazolium (MTT). The SGC-7901/VCR induced subcutaneous transplanted tumor model was established in 50 BALB/c nude mice by tissue block method. After 2 -3 weeks 36 mice with similar tumor size were selected and divided into 6 groups by random digit table, i.e., the model group, the Vincristine (VCR) group, the low dose CW group, the high dose CW group, the low dose CW combined VCR group, and the high dose CW combined VCR group, 6 in each group. Normal saline was intraperitoneally injected to mice in the model group at 10 mL/kg, once per 2 days. VCR was intraperitoneally injected to mice in the VCR group at 0.28 mg/kg once per 2 days. CW at 1.4 and 2.8 g/kg was administered to mice in the low and high dose CW groups by gastrogavage, 0.2 mL each time, once daily. CW at 1.4 and 2.8 g/kg was administered by gastrogavage and VCR was intraperitoneally injected at 0.28 mg/kg, once per 2 days to mice in the low dose CW combined VCR group and the high dose CW combined VCR group. All medication lasted for 14 days. The tumor growth was observed. The inhibition rate was calculated. Meanwhile, the positioning and expression of P-glycoprotein (P-gp) were detected by immunohistochemistry and Western blot.

RESULTSSGC-7901/VCR had strong resistance to VCR, Adramycin (ADM), fluorouracil (5-FU), and Cisplatin (DDP), especially to VCR. Proliferation activities of SGC-7901/VCR were significantly enhanced after drug elution. The tumor volume gradually increased as time went by. The tumor volume was the minimum in the high dose CW combined VCR group. The tumor volume was obviously reduced in the high dose CW combined VCR group with obviously reduced with increased inhibition rate of 51.56%, when compared with that of the model group and the VCR group (P < 0.05). Western blot test showed that, when compared with the model group, the gray level of P-gp in the VCR group increased (P < 0.05), and the relative expression of P-gp in the high dose CW group, the low dose CW combined VCR group, and the high dose CW combined VCR group significantly decreased (P < 0.05). Compared with the VCR group, the gray level of the P-gp decreased in the low dose CW group, the high dose CW group, the low dose CW combined VCR group, and the high dose CW combined VCR group (P < 0.05). Results of immunohistochemistry showed that, when compared with the model group, expression scores of P-gp in the high dose CW group, the low dose CW combined VCR group, and the high dose CW combined VCR group decreased with statistical difference (P < 0.05). Compared with the VCR group, expression scores of P-gp were obviously lowered in the low dose CW group, the high dose CW group, the low dose CW combined VCR group, and the high dose CW combined VCR group (P < 0.05).

CONCLUSIONSCW could reverse the drug resistance of SGC-7901/VCR subcutaneous transplanted tumor. And its mechanism might be related to down-regulating the expression of P-gp, suggesting that CW could be used as a kind of multidrug resistance reversal agent based on P-gp.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Animals ; Cell Line, Tumor ; Cisplatin ; therapeutic use ; Curcuma ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Fluorouracil ; therapeutic use ; Guanylate Cyclase ; Mice ; Mice, Nude ; Receptors, Cytoplasmic and Nuclear ; Soluble Guanylyl Cyclase ; Stomach Neoplasms ; Vincristine ; therapeutic use

More than 95% of the thyroid carcinomas are well differentiated types showing favorable prognosis. However, only a few therapeutic options are available to treat the patients with undifferentiated thyroid carcinomas, especially with refractory thyroid carcinomas that are not amenable to surgery or radioiodine ablation. We investigated the anticancer effects of 20 chemotherapy and hormonal therapy drugs on 8 thyroid carcinoma cell lines. In vitro chemosensitivity was tested using the adenosine-triphosphate-based chemotherapy response assay (ATP-CRA). The tumor inhibition rate (TIR; or cell death rate) or half maximal inhibitory concentration (IC50) was analyzed to interpret the results. Of the 12 chemotherapy drugs, etoposide (178.9 index value in follicular carcinoma cell line) and vincristine (211.7 in Hurthle cell carcinoma cell line) were the most active drugs showing the highest chemosensitivity, and of the 8 additional drugs, trichostatin A (0.03 microg/mL IC50 in follicular carcinoma cell line) showed favorable outcome having the anticancer effect. In our study, the result of etoposide and vincristine show evidence as active anticancer drugs in thyroid carcinoma cell lines and trichostatin A seems be the next promising drug. These drugs may become an innovative therapy for refractory thyroid carcinomas in near future.
Adenosine Triphosphate/chemistry/pharmacology/therapeutic use ; Antineoplastic Agents/chemistry/*pharmacology/therapeutic use ; Apoptosis/drug effects ; Cell Line, Tumor ; Etoposide/chemistry/pharmacology/therapeutic use ; Humans ; Hydroxamic Acids/chemistry/pharmacology/therapeutic use ; Thyroid Neoplasms/drug therapy ; Vincristine/chemistry/pharmacology/therapeutic use

OBJECTIVETo investigate the changes of drug sensitivity of spindle poison-induced polyploid tumor cells to chemotherapeutic agents and its possible mechanism.

METHODSNocodazole in a dose of 100 ng/ml was used to induce polyploidization in a breast cancer cell line MDA-MB-231 cells. The polyploid cells (T-MDA-MB-231) were sorted by flow cytometry. The morphological changes and proliferation of T-MDA-MB-231 cells were compared with that of MDA-MB-231 cells. The cell growth inhibition was assessed by MTT assay. The cells were treated with paclitaxel, docetaxel, vincristine, epirubicin, 5-Fu, VP16 and oxaliplatin, respectively. Those cells were labeled with annexin V-FITC/PI and analyzed by flow cytometry. Bcl-2 was knocked down in T-MDA-MB-231 cells using SiRNA and their growth inhibition was evaluated by MTT assay to evaluate the reversing effect of Bcl-2-silencing on drug resistance.

RESULTSThe polyploid T-MDA-MB-231 cells grew in vitro continuously and maintained constant DNA content. They had a larger cell size, and grew more slowly than MDA-MB-231 cells. The IC(50(s)) of T-MDA-MB-231 cells were significantly higher than that of the MDA-MB-231 cells: paclitaxel: (6.37 ± 0.07) vs. (2.05 ± 0.83) µmol/L; docetaxel: (32.98 ± 1.48) vs. (11.95 ± 0.98) µmol/L; vincristine: (35.28 ± 1.66) vs. (14.58 ± 0.94) µmol/L; oxaliplatin: (19.07 ± 0.45) vs. (9.75 ± 1.05) µmol/L; 5-Fu: (85.49 ± 3.21) vs. (31.35 ± 1.51) µmol/L; and epirubicin: (0.53 ± 0.06) vs. (0.15 ± 0.01) µmol/L, (all P < 0.05). The IC(50(s)) of VP16 in T-MDA-MB-231 cells was (2.85 ± 0.50)µmol/L, significantly lower than the (12.20 ± 1.55) µmol/L in MDA-MB-231 cells (P < 0.05), and that of T-MDA-MB-231 cells after Bcl-2-knocked down by siRNA was (19.59 ± 0.48) µmol/L, significantly higher than the (12.20 ± 1.55) µmol/L in the MDA-MB-231 cells (P < 0.05). The IC(50(s)) of docetaxel of T-MDA-MB-231 cells after Bcl-2-knocked down by siRNA was (21.52 ± 0.68) µmol/L, significantly decreased and lower than that before Bcl-2 silencing (32.98 ± 1.48) µmol/L.

CONCLUSIONSOur results indicate that polyploid tumor cells induced by spindle poison Nocodazole are more resistant to most of chemotherapeutic drugs. Downregulation of Bcl-2 increases the sensitivity of polyploid cells to docetaxel. The high expression of Bcl-2 may be one of the drug resistance mechanisms of polyploid tumor cells. The polyploid tumor cells are relatively sensitive to VP16, suggesting that VP16 might be an effective candidate drug for treatment of chemoresistant polyploid tumors.

Antineoplastic Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Down-Regulation ; Drug Resistance, Neoplasm ; Epirubicin ; pharmacology ; Etoposide ; pharmacology ; Female ; Fluorouracil ; pharmacology ; Gene Knockdown Techniques ; Humans ; Inhibitory Concentration 50 ; Nocodazole ; pharmacology ; Organoplatinum Compounds ; pharmacology ; Paclitaxel ; pharmacology ; Polyploidy ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Taxoids ; pharmacology ; Vincristine ; pharmacology

In order to investigate the effect of rapamycin on the proliferation of human acute myeloid leukemia (AML) cells, the sensitive cells HL-60 and multidrug-resistant HL-60/VCR cells were chosen as research objects. The proliferation of cells was detected by growth curve method. The flow cytometer was used to analyze cell cycle. The expression of P-glycoprotein (Pgp) was determined by Western blot. The results demonstrated that there was a significant difference of cell growth inhibition rate between control group and rapamycin group (p < 0.05). The cell growth inhibition rate was dose- and time- dependent (p < 0.05). Flow cytometry detection showed that the cell percentage of G(1) phase in rapamycin group was higher than that in group without rapamycin, and that of S phase was lower. The cell growth inhibition rate in 50 nmol/L and 100 nmol/L rapamycin plus daunorubicin (DNR) group was more than that in DNR alone group (p < 0.05), especially when DNR was added at 24 hours interval after RAP. The expression of Pgp of HL-60/VCR cells was inhibited by rapamycin. It is concluded that the rapamycin can inhibit the proliferation of sensitive HL-60 and multidrug resistant HL-60/VCR cells. It can also increase sensitivity of HL-60 and HL-60/VCR cells to DNR, which provides new strategy for the therapy of refractory AML.
ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antibiotics, Antineoplastic ; pharmacology ; Cell Proliferation ; drug effects ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; pathology ; Sirolimus ; pharmacology ; Vincristine ; pharmacology

OBJECTIVETo evaluate the effects and molecular mechanism of action of Changweiqing (CWQ) in reversing multidrug resistance by observing its impacts on nuclear translocation of Y-box binding protein-1 (YB-1), multi-drug resistance gene (MDR1) expression and P-glycoprotein (P-gp) expression in human colon cancer cell line HCT8/V with drug-resistance induced by vincristine.

METHODSCultured HCT8/V cells were exposed to the CWQ-containing rat serum prepared by drug perfusion. YB-1 expressions in cell plasma and nuclei were examined by Western blot; the binding activity of YB-1 to MDR1 gene promoter sequences was detected by electrophoretic mobility shift assay (EMSA); the mRNA transcription levels of MDR1, YB-1 and multi-resistance related protein (MRP) were examined by RT-PCR; the expression of P-gp on cell membrane was determined by flow cytometry. Results Along with the increasing drug's concentration of CWQ-containing serum from 1.25% up to 2.5% and 5%, the expressions of YB-1 decreased in HCT8/V cell nuclear and increased in cytoplasm gradually; the binding activity of YB-1 to MDR1 gene promoter weakened (P < 0.01), MDR1 mRNA expression and fluorescence intensity of P-gp on cell membrane attenuated (P < 0.05 or P < 0.01), while YB-1 and MRP mRNA unchanged (P > 0.05).

CONCLUSIONCWQ could reverse the drug-resistance of colon cancer cells by influencing nuclear translocation of YB-1 and reducing the expression of MDR1/P-gp.

ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Active Transport, Cell Nucleus ; drug effects ; Animals ; Cell Line, Tumor ; Colonic Neoplasms ; pathology ; Drug Resistance, Neoplasm ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Rats ; Vincristine ; pharmacology ; Y-Box-Binding Protein 1 ; metabolism

BACKGROUND AND OBJECTIVEMultidrug resistance (MDR) is a major obstacle in the chemotherapy of cancer patients. The aim of this study was to establish a mutidrug-resistant cell line SK-Hep1/DDP and explore its molecular mechanism of the MDR.

METHODSSK-Hep1/DDP cell line was induced by pulse treatment using a high concentration of cisplatin (DDP) in vitro. The chemoresistance indexes of cells were evaluated by CCK-8 assays. The protein of MDR1 (ABCB1), MRP1 (ABCC1), MRP2 (ABCC2) and Bax were detected by Western blotting, and the effect of MDR1 inhibitor cyclosporine A (CsA) on expression of MDR1 proteins in SK-Hep1 and SK-Hep1/DDP cell lines. Flow cytometry was performed to determine the distribution of the cell cycle and cell apoptosis ratio.

RESULTSThe SK-Hep1/DDP cells were 13.76 times more resistant to DDP in comparison with SK-Hep1 cells, and SK-Hep1/DDP cells also exhibited cross-resistance to many other chemotherapeutic agents (adramycin and 5-fuorouracil). MDR1, MRP1, and MRP2 protein expressions were significantly higher in the SK-Hep1/DDP than in the SK-Hep1 (P < 0. 01), but Bax was lower in the SK-Hep1/DDP than in the SK-Hep1(P < 0. 01). There was no obvious influence between SK-Hep1 and SK-Hep1/DDP cells in the expression of MDR1 by MDR1 inhibtor CsA (P > 0. 05). The percentages of cells in G(2)/M and S phase were significantly increased in SK-Hep1/DDP in comparison with those in SK-Hep1 [(20.67 +/- 5.69)% vs. (12.14 +/- 3.36)%; (42.20 +/- 2.65)% vs. (27.91 +/- 2.16)%; P < 0. 01]. After the cells were exposed to 10 μg/mL DDP for 24 h, the cell apoptosis rate of SK-Hep1/DDP was decreased in comparison with SK-Hep1, but it was increased in those with pretreatment of MDR1 inhibitor CsA as compared with those without pretreatment.

CONCLUSIONSA reliable multi-drug resistant human hepatoma cell line SK-Hep1/DDP is successfully established. The MDR mechanisms of this cell lines are closely related to the over-expression of MDR1 MRP1 and MRP2, lower expression of Bax and the attenuated cell apoptosis induced by chemotherapeutic agents.

ATP-Binding Cassette, Sub-Family B, Member 1 ; antagonists & inhibitors ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Cyclosporine ; pharmacology ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Fluorouracil ; pharmacology ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Multidrug Resistance-Associated Proteins ; metabolism ; Vincristine ; pharmacology ; bcl-2-Associated X Protein ; metabolism

Liver cancer is one of the most common neoplastic diseases with high mortality in China. Currently, antimicrotubule drugs such as paclitaxel (PTX) and vincristine (VCR), are used as the common agents in the clinical chemotherapy for liver cancer. However, the responses of patients to these drugs vary markedly. Successful identification of intracellular factors influencing liver cancer's sensitivity to antimicrotubule drugs would be of great clinical importance. In this study, by engineering human hepatoma cell HepG2 to overexpress synuclein-gamma (SNCG), we investigated if SNCG is a molecular factor associated with the sensitivity to antimicrotubule drug treatment. Real-time RT-PCR and Western blotting assays showed SNCG was successfully overexpressed in HepG2/ SNCG cells compared with HepG2/Neo cells. The overexpressed SNCG altered the proliferation activity in HepG2 cells, which was 66% higher than that of HepG2/Neo cells through MTT method. The overexpressed SNCG also reduced sensitivity of HepG2 cells to antimicrotubule drugs: after PTX or VCR treatment, the proportion of HepG2/SNCG cells in G2/M arrest was significantly lower than that in HepG2/Neo cells. Correspondingly, HepG2/SNCG cells showed significantly lower mitotic index than HepG2/Neo cells. Meanwhile, HepG2/SNCG cells showed higher resistance to PTX and VCR than HepG2/Neo cells, with resistance index 21 and 15 respectively. Our studies suggested that the overexpression of SNCG could confer resistance to antimicrotubule drugs in hepatoma cells; and it indicated that SNCG may be as a potential response marker for antimicrotubule drugs in liver cancer chemotherapy.
Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Cycle ; Cell Proliferation ; Drug Resistance, Neoplasm ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Hep G2 Cells ; drug effects ; metabolism ; Humans ; Microtubules ; drug effects ; Mitosis ; drug effects ; Mitotic Index ; Paclitaxel ; pharmacology ; Plasmids ; RNA, Messenger ; metabolism ; Transfection ; Vincristine ; pharmacology ; gamma-Synuclein ; biosynthesis ; genetics ; physiology