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[Expression and significance of heparin binding-epidermal growth factor-like growth factor in paclitaxel-resistant ovarian cancer].

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.