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: