Objective To investigate the effect and mechanism of CRM197, the heparin binding-epidermal growth factor-like growth factor (HB-EGF) inhibitor, on the reverse of the resistance ofovarian cancer to paclitaxel. Methods (1)The effect of CRM197 on the 50% inhibitory concentrations (IC50) of human ovarian carcinoma cell line A2780 and paclitaxel-resistant ovarian carcinoma cell line A2780/Taxol was tested by methyl thiazolyl tetrazolium (MTT) assay. Western blot was used to detect the effect of CRM197 on the expression of HB-EGF,epidermal growth factor receptor (EGFR) and plasma membrane glycoprotein (P-gp) protein in A2780 and A2780/Taxol cells. Real-time PCR was used to examine the MDR1 mRNA expression in these cells. (2) A2780/Taxol cells were divided into 4 groups, including the cells transfected with empty vector and saline treatment (empty vector group), MDR1 small interference RNA (siRNA)vector and saline treatment (MDR1 siRNA group),empty vector and CRM197 treatment (empty vector+CRM197 group) and MDR1 siRNA vector and CRM197 treatment (MDR1 siRNA+CRM197 group), respectively. Flow cytometry was used to detecte the effect of intracellular rhodomine 123 (Rh123) accumulation, and caspase-3 activity assay was used to test the effect of apoptosis in four groups of A2780/Taxol cells. (3) In experiments in vivo, A2780/Taxol cells were inoculated to nude mouse subcutaneously to determine the EGFR and P-gp protein expression following CRM197 treatment by immunohistochemistry. Results (1)In vitro,MTT examination showed that the IC50 of A2780/Taxol cells to paclitaxel in A2780/Taxol+CRM197 group [(6.4±0.3)μmol/L] was significantly lower than the IC50 in A2780/Taxol group [(34.1± 0.5)μmol/L,P<0.01], and the reveral fold of CRM197 was 5.3. The expression level of HB-EGF protein in A2780/Taxol+CRM197 group (1.44 ± 0.29) was significantly lower than HB-EGF protein in A2780/Taxol group (2.72 ± 0.32),respectively (P<0.05). The expression level of EGFR protein (0.71 ± 0.25) and P-gp protein (0.82±0.19) in A2780/Taxol+CRM197 group was significantly lower than EGFR protein (1.87±0.31) and P-gp protein (1.84 ± 0.27) of A2780/Taxol group (P<0.05). Compared with A2780/Taxol group(1.78 ± 0.27), MDR1 mRNA was significantly down-regulated in A2780/Taxol+CRM197 group (0.79 ± 0.13,P<0.05). (2) The fluorescence intensity of Rh123 of the A2780/Taxol cells in empty vector group, MDR1 siRNA group,empty vector+CRM197 group, MDR1 siRNA+CRM197 group was 33.4±1.6, 56.3±3.3, 43.5± 3.1,100.4 ± 7.4, and the pNA of the A2780/Taxol cells was(11.4 ± 1.2),(52.8 ± 0.9),(71.2 ± 3.6),(82.7 ± 3.8)μmol/L. The expression levels in MDR1 siRNA+CRM197 group were both higher than the expression levels in empty vector+CRM197 group, and the expression levels in empty vector+CRM197 group, MDR1 siRNA group were both higher than the expression levels in empty vector group (P<0.05). (3) In vivo, the expression scores of EGFR protein in A2780/Taxol+CRM197 tumors (4.4 ± 1.4) were lower than that in A2780/Taxol tumors (10.2 ± 3.1,P<0.05). The expression scores of P-gp protein in A2780/Taxol+CRM197 tumors (3.8 ± 1.1) were lower than that in A2780/Taxol tumors (8.8 ± 2.7,P<0.05). Conclusion CRM197 reverses the resistance of ovarian cancer to paclitaxel by increasing caspase-3 activity to advance apoptosis via EGFR/MDR1/P-gp pathway.

To explore the role and possible mechanism of apoptosis and caspase-3 activity in the development of multicellular drug resistance of ovary cancer. Ovarian cancer cell A2780 multicellular spheroids (MCS) were obtained from three-dimensional culture. Drug sensitivity of monolayer cells (MC) and MCS were respectively tested by MTT staining and cytometry. The apoptosis of MC and MCS were determined by the flow cytometry (FCM). The expression of bcl-2 and caspase-3 in A2780/MC and A2780/MCS were detected by using Western blot and caspase-3 assay kit. A2780/MC was compacted into mass after 2 days in three-dimensional cell culture model, and MCS had more than two layers of cells growing within 5 days. Compared with A2780/MC, A2780/MCS were more resistant to the anticancer drug, and the apoptosis rate was significantly lower than those of A2780/MC. The activity of caspase-3 in A2780/MCS was significantly lower than the A2780/MC. But the expression of bcl-2 in A2780/MCS was significantly higher than that in A2780/MC. It was suggested that the drug resistance of MCS might be associated with the overexpression of anti-apoptosis protein bcl-2 and the down-regulation of caspase-3 activity.

To investigate the relationship between the expression of early growth response gene 1 (EGR-1) and p38MAPK pathway in the paclitaxel resistance of ovarian carcinoma cells, the effect of p38MAPK inhibitor SB203580 on cell apoptosis was examined by using Hoechst 33258 staining. The intracellular Rh123 (Rhodamine 123) accumulation was detected by the flow cytometry (FCM). The 50% inhibition concentration (IC50) of paclitaxel for A2780/Taxol cells was determined by MTT method. Electrophoretic motility shift assay (EMSA) was employed to examine the EGR-1DNA binding activity. MDR1 and EGR-1 mRNA were assessed by RT-PCR. The expressed of p-gp, phosphorylated p53 and p38 were detected by Western blotting. SB203580 could remarkably promote the apoptosis of A2780/Taxol cells, and the cell apoptosis was in a time-dependent manner. Cellular Rh123 accumulation was increased, and the IC50 of paclitaxel for A2780/Taxol cells was decreased significantly. A2780/Taxol cell line after SB203580 treatment was shown to have a significantly higher level of EGR-1 DNA binding activity. SB203580 down-regulated the activity of p38MAPK pathway, but up-regulated EGR-1 expression. SB203580 significantly increased the level of cellular phosphorylated p53 protein, but decreased the p-gp protein level and MDR1 mRNA level in A2780/Taxol cells. There existed a close relationship between p38MAPK pathway and the paclitaxel resistance of ovarian carcinoma cells. The expression of EGR-1 mediated by p38MAPK pathway plays a critical role in paclitaxel resistance of ovarian carcinoma cells.

Objective To examine the expression of heparin binding-epidermal growth factor-like growth factor (HB-EGF) in paclitaxel-resistant ovarian cancer and elucidate the relationship between HB-EGF and the resistance of ovarian cancer to paclitaxel. Methods The human ovarian carcinoma cell line A2780 and the paclitaxel-resistant human ovarian carcinoma cell line A2780/Taxol were cultured in vitro. Western blot was used to dectect the expression of HB-EGF protein in A2780 and A2780/Taxol groups. The A2780 cells were treated with cross-reacting material 197 (CRM197 and A2780 + CRM197 group) or dimethyl sulphoxide (DMSO;A2780 group), while the A2780/Taxol cells were treated with CRM197 (A2780/Taxol+CRM197 group) or DMSO (A2780/Taxol group). The effects of CRM197 on growth and proliferation was tested by methyl thiazolyl tetrazolium( MTT) and the results were showed as absorbance (A).The effects of CRM197 on cell cycles was tested by flow cytometry, while the effects of CRM197 on apoptosis was examined by caspase-3 activity assay and the results were showed as p-nitroaniline(pNa). In animal experiment, four groups of cells were inoculated to BALB/c nude mouse subcutaneously to observe tumor formation ability following CRM197 treatment. Immunohistochemistry was used to determine the expression of HB-EGF protein in A2780 and A2780/Taxol group. Results The expression level of HB-EGF protein in A2780/Taxol group (2.11±0.41) was significantly higher than that of A2780 group (0.75±0.20;P<0.01). The inhibition effect of CRM197 on the cell growth of A2780+CRM197 and A2780/Taxol+CRM197 group was accompanied by the acceleration of CRM197 concentration(P<0.01). When CRM197≥1 μg/ml, the inhibition effect of CRM197 on the cell growth of A2780/Taxol+CRM197 group was significantly higher than that in A2780/Taxol group(P<0.05). In cell cycle experiment, CRM197 induced the cell-cycle arrest at the G0/G1 phase in A2780+CRM197 cells[(67 ± 4)%] compared with A2780 cells[(54 ± 6)%;P<0.01], while CRM197 significantly induced the cell-cycle arrest at the G0/G1 phase in A2780/Taxol+CRM197 cells [(72± 4)%] compared with A2780/Taxol cells [(24±8)%;P<0.01]. CRM197 treatment in A2780+CRM197 group [(40 ± 6)μmol/L] led to the acceleration of the caspase-3 activity when compared to A2780 group [(6 ± 6)μmol/L;P<0.01], while CRM197 treatment in A2780/Taxol+CRM197 group [(66 ± 12)μmol/L] led to significant acceleration of the caspase-3 activity when compared to A2780 group [(9 ± 6)μmol/L;P<0.01]. In experiments in vivo, the expression scores of HB-EGF protein in A2780/Taxol tumors(10.8 ± 3.3) were higher than that in A2780 tumors (5.0±2.2;P<0.01). The tumor size and tumor weight of the A2780/Taxol+CRM197 group were both higher than those of the A2780+CRM197 group [(546 ± 85) mm3 vs (1 355 ± 119) mm3,(0.56 ± 0.09) g vs (1.31 ± 0.27) g; all P<0.01]. The CRM197 inhibition rate of the A2780+CRM197 and A2780/Taxol + CRM197 group were 43% and 68% respectively, showed that CRM197 significantly suppressed the growth of A2780/Taxol xenografts in vivo(P<0.01). Conclusions HB-EGF is over-expressed in paclitaxel-resistant ovarian cancer and may be contributes to drug resistance. Inhibition of HB-EGF expression potently enhances apoptosis and inhibit the growth of paclitaxel-resistant ovarian cancer, shedding light on the HB-EGF-targeted therapy options for chemoresistant ovarian cancer patients.

ObjectiveTo discuss the expression of tumor shift suppressor gene KAI1,matrix metal proteinase 9 (MMP-9) in endometriosis sickness in dystopia internal membrane and its significance.MethodsIn 53 patients and 30 healthy controls,the expression of KAI1,MMP-9 in the endometriosis was detected with immunohistochemistry SP method,and the relationship was analyzed.ResultsKAI1 had low expression in EMS patients,while MMP-9 was highly expressed.In the control group,KAI1 was highly expressed while MMP-9 lowly expressed,and the differences between two groups were remarkably significant (P ＜0.05).KAI1 and the MMP-9 expression showed negative correlation between two groups.Conclusions KAI1 and MMP-9 was negatively expressed in EMS,which may play a role in the EMS prognosis.KAI1 and MMP-9 may be served as the prognosis and monitor markers for EMS.

Objective: To investigate the relationship of National Institute of Health Stroke Scale(NIHSS)with heart rate variability(HRV)and cardiac complication in elderly patients with acute cerebral infarction, and to clarify the effect of early drug intervention on the regulation of autonomic nerve function in patients with high NIHSS score. Methods: One hundred twenty-six inpatients with first-onset acute cerebral infarction(ACI)(the cerebral infarction group)and 40 healthy subjects with no history of stroke(the control group)were retrospectively enrolled.All subjects underwent examinations of NIHSS and 24 h dynamic electrocardiogram.According to NIHSS score, patients in the cerebral infarction group were divided into 3 subgroups: NIHSS score 0-4 group(n=32), NIHSS score 5-15 group(n=66)and NIHSS score ≥ 15 group(n=28). The difference in HRV parameters were compared between ACI patients and the controls.Ninety-four ACI patients with NIHSS score ≥5 were randomly divided into 2 groups: (1)the traditional treatment group(n=44), taking routine drugs for cerebral infarction; (2)the special treatment group(n=42), taking metoprolol sustained release tablet and Shensonyangxin capsule as add-on to the routine drugs for cerebral infarction.The 24 h dynamic electrocardiogram examination were conducted 30 days after treatment.The differences in HRV parameters and cardiac complications were compared between the two treatment groups. Results: In patients with acute cerebral infarction, the time-domain parameters of normal-to-normal intervals(NNI), standard deviation of normal-to-normal intervals(SDNN), square root of the mean squared successive differences between normal-to-normaI RR intervals(RMSSD), percentage of adjacent normal-to-normal intervals that differed more than 50 ms(PNN50), frequency domain parameters low-frequency(LF)power and high-frequency(HF)power were significantly reduced as compared with those in the control group(P<0.05). While, pulse rate and frequency domain parameters of sympathetic vagus balance index(LF/HF)were significantly increased(P<0.05). The time-domain parameters(NNI, SDNN, RMSSD and PNN50)and frequency domain parameters(LF, HF)were significantly reduced in the NIHSS score 5-15 group and NIHSS score ≥15 group as compared with the control group and the NIHSS score 0-4 group(P<0.05). The time-domain parameters(RMSSD and PNN50)and frequency domain parameters(LF and HF)were reduced(P<0.05), and the frequency domain parameters(LF/HF)were increased(P<0.05)in NIHSS score ≥15 group compared with in NIHSS score 5-15 group(P<0.05). Compared with pre-treatment, the special treatment group after treatment showed that the time domain parameters(NNI, SDNN, RMSSD and PNN50)and frequency domain parameters(LF and HF)were significantly increased(P<0.05), the frequency domain parameters(LF/HF)were significantly reduced(P<0.05)and the incidence of cardiac complication(total incidence of atrial arrhythmias and ventricular arrhythmias and cardiac complication)were significantly reduced(P<0.05). As compared with the traditional treatment group after treatment, the special treatment group after treatment showed that the time domain parameters(NNI, SDNN, RMSSD and PNN50)and frequency domain parameters(LF and HF)were significantly increased(P<0.05), frequency domain parameters(LF/HF)were significantly reduced(P<0.05)and the total incidence of cardiac complication were significantly reduced(18.4% or 7 cases vs.40.0% or 16 cases, P<0.05). Conclusions The HRV is decreased in patients with acute cerebral infarction.The reduction degree of HRV is more significant along with the higher NIHSS scores, which suggests that patients with high NIHSS scores are prone to an impairment of autonomic nerve function.For patients with high NIHSS scores after stroke, early drug intervention for regulating autonomic nerve function can effectively prevent the occurrence of cardiac emergencies and improve the prognosis of stroke.

To explore the role and possible mechanism of apoptosis and caspase-3 activity in the development of multicellular drug resistance of ovary cancer. Ovarian cancer cell A2780 multicellular spheroids (MCS) were obtained from three-dimensional culture. Drug sensitivity of monolayer cells (MC) and MCS were respectively tested by MTT staining and cytometry. The apoptosis of MC and MCS were determined by the flow cytometry (FCM). The expression of bcl-2 and caspase-3 in A2780/MC and A2780/MCS were detected by using Western blot and caspase-3 assay kit. A2780/MC was compacted into mass after 2 days in three-dimensional cell culture model, and MCS had more than two layers of cells growing within 5 days. Compared with A2780/MC, A2780/MCS were more resistant to the anticancer drug, and the apoptosis rate was significantly lower than those of A2780/MC. The activity of caspase-3 in A2780/MCS was significantly lower than the A2780/MC.But the expression of bcl-2 in A2780/MCS was significantly higher than that in A2780/MC. It was suggested that the drug resistance of MCS might be associated with the overexpression of anti-apoptosis protein bcl-2 and the down-regulation of caspase-3 activity.

To investigate the relationship between p38 mitogen-activated protein kinase (p38MAPK)and cell apoptosis during the paclitaxel resistance of ovarian carcinoma cell lines, flow cytometry(FCM) and PI staining were employed to determine the effect of p38MAPK inhibitor SB203580 onthe apoptosis of A2780/Taxol cells, a drug-resistant human ovarian carcinoma cell line. p38MAPKprotein expression in SB203580-treated cells was immunochemically measured. The 50% inhibitionconcentration (IC<,50) of paclitaxel on A2780/Taxol cells was determined by MTT assay. MDR-1mRNA, and expression of p38MAPK and phospho-p53 protein were detected by RT-PCR and West-ern blotting, respectively. The apoptosis rate of A2780/Taxol cells was (19.7±1.04)% 24 h afterSB203580 treatment. A significant difference in apoptosis rate was found among experiment group,control group and untreated group (P<0.05). The relative reversal rate of A2780/Taxol cells to pacli-taxel was (57.18±2.01)%. As compared with the control group and the untreated group, p38MAPKprotein and MDR-1 mRNA in SB203580-treated cells was substantially decreased. The expression ofp53 protein was significantly increased. It is concluded that p38MAPK pathway is related to pacli-taxel resistance of ovarian carcinoma, and blockade of this pathway can promote the apoptosis of thedrug-resistant cells and reverse the drug-resistance. Moreover, p38MAPK-mediated apoptosis in pa-clitaxel-resistant ovarian carcinoma cells depends on the activation of p53.

To investigate the relationship between the expression of early growth response gene 1(EGR-1) and p38MAPK pathway in the paclitaxel resistance of ovarian carcinoma cells, the effect of p38MAPK inhibitor SB203580 on cell apoptosis was examined by using Hoechst 33258 staining.The intracellular Rh123 (Rhodamine 123) accumulation was detected by the flow cytometry (FCM).The 50% inhibition concentration (IC50) of paclitaxel for A2780/Taxol cells was determined by MTT method. Electrophoretic motility shift assay (EMSA) was employed to examine the EGR-1DNA binding activity. MDR1 and EGR-1 mRNA were assessed by RT-PCR. The expressed of p-gp, phosphorylated p53 and p38 were detected by Western blotting. SB203580 could remarkably promote the apoptosis of A2780/Taxol cells, and the cell apoptosis was in a time-dependent manner. Cellular Rh123 accumulation was increased, and the IC50 of paclitaxel for A2780/Taxol cells was decreased significantly. A2780/Taxol cell line after SB203580 treatment was shown to have a significantly higher level of EGR-1 DNA binding activity. SB203580 down-regulated the activity of p38MAPK pathway, but up-regulated EGR-1 expression. SB203580 significantly increased the level of cellular phosphorylated p53 protein, but decreased the p-gp protein level and MDR1 mRNA level in A2780/Taxol cells. There existed a close relationship between p38MAPK pathway and the paclitaxel resistance of ovarian carcinoma cells. The expression of EGR-1 mediated by p38MAPK pathway plays a critical role in paclitaxel resistance of ovarian carcinoma cells.

Objective To investigate the role and mechanism of the regulation of nuclear factor-κB (NF-κB) by heparin binding-epidermal growth factor-like growth factor (HB-EGF) in paclitaxel resistance of ovarian cancer in vitro and in vivo. Methods (1) The detection of NF-κB expression: parental (A2780) and paclitaxel-resistant (A2780/Taxol) ovarian carcinoma cells were divided into four groups, named A2780 group, A2780+cross-reacting material 197 (CRM197, HB-EGF inhibitor) group, A2780/Taxol group and A2780/Taxol+CRM197 group. Among four groups, the expression level HB-EGF and epidermal growth factor receptor (EGFR) were examined by immunofluorescence double staining on confocal microscopy. Western blot was used to detect the expression level of NF-κB. In vivo, A2780 and A2780/Taxol cells were injected intraperitoneally to nude mouse to determine the expression level of NF-κB of the tumors from these four groups by immunohistochemistry method. (2) The detection on the function of NF-κB: A2780/Taxol cells were divided into four groups, named transfected with empty vector+saline group, NF-κB small interference RNA (siRNA)+saline group, empty vector+CRM197 group and NF-κB siRNA+CRM197 group respectively. Among four groups, the 50% inhibitory concentrations (IC50) of A2780/Taxol cells to paclitaxel, the expression level of plasma membrane glycoprotein (P-gp) and the effect of intracellular rhodomine123 (Rh123) accumulation were detected. Results (1) The detection of NF-κB expression: the expression scores of HB-EGF protein among four groups were 5.6±1.3, 2.1±1.2, 11.7±3.5 and 6.2±1.4; the expression scores of EGFR protein were 5.1±1.6, 2.8±0.6, 10.4±3.1 and 5.6±1.9, respectively. The expression levels of NF-κB protein in the cells of the group named A2780, A2780+CRM197, A2780/Taxol and A2780/Taxol+CRM197 group were 1.89±0.23, 0.74±0.12, 3.45±0.16 and 1.31±0.08, respectively; the expression scores of NF-κB protein in the tissue tumors from four groups were 3.3±1.1, 1.4±0.4, 8.7±2.3 and 3.6±1.2, respectively. The expression level of HB-EGF, EGFR and NF-κB protein between A2780 and A2780/Taxol groups in vivo and in vitro were higher than these in A2780+CRM197 and A2780/Taxol+CRM197 group, while the expression level of HB-EGF, EGFR and NF-κB protein in A2780 group were lower than those in A2780/Taxol groups in vivo and in vitro (P＜0.05). (2) The examination of NF-κB function: the IC50 of A2780/Taxol cells to paclitaxel in groups transfected with empty vector+saline, NF-κB siRNA+saline, empty vector+CRM197 and NF-κB siRNA+CRM197 group were respectively (39.4±0.8), (7.6±0.6), (6.7±0.5) and (4.2±0.4) μmol/L, while the expression levels of P-gp protein among four groups were respectively 3.11±0.23,1.45±0.16, 1.73± 0.21 and 0.68±0.14, the cellular Rh123 accumulation among four groups were respectively 110±15, 246±19, 231 ± 22 and 296 ± 24. The expression levels of IC50 and P-gp protein in groups transfected with NF-κB siRNA+saline, empty vector+CRM197 and NF-κB siRNA+CRM197 group were significantly higher than those in group transfected with empty vector+saline group (P<0.01), while the cellular Rh123 accumulation among three groups were significantly lower than that in group transfected with empty vector+saline (P<0.01). Conclusions The expression of NF-κB may contributes to the paclitaxel resistance to ovarian cancer. HB-EGF may induce the paclitaxel resistance of ovarian cancer by the regulation of EGFR/NF-κB/P-gp pathway.