[Relationship between the Akt-regulated direct p53 mitochondrial translocation and the resistance to cisplatin of ovarian cancer cells].

OBJECTIVE: To explore Akt-regulated direct p53 mitochondrial translocation in cisplatin-induced apoptosis in ovarian cancer cells and the relationship between this and chemoresistance in ovarian cancer. METHODS: Chemosensitive ovarian cancer cell lines (OV2008 and A2780s) and chemoresistant cells (C13(*) and A2780cp) were treated with cisplatin and whole cell and mitochondrial p53 contents were determined by Western blot. The p53 accumulation in mitochondria was determined in purified mitochondrial fractions in cisplatin-sensitive and -resistant ovarian cancer cells. Akt1/2 siRNA were transfected into C13(*) cells. Cisplatin-induced apoptosis was measured by Hoechst staining and p53 translocation was determined by Western blot. RESULTS: Cisplatin induced mitochondrial p53 accumulation and apoptosis in chemosensitive cells (P < 0.05), but not in resistant cells (P > 0.05). Over-expression of active Akt2 inhibited p53 directly translocate to mitochondria, and downregulation of Akt by Akt1/2 siRNA increased p53 mitochondrial accumulation and sensitize C13(*) cells to cisplatin treatment. CONCLUSIONS: Cisplatin induces direct p53 mitochondrial accumulation in chemosensitive cells, and Akt confers resistance in ovarian cancer cells, in part, by regulating the direct action of p53 in mitochondrial death pathway.

[Inhibition of mitochondrial Smac release by Akt in chemoresistant ovarian cancer cells].

OBJECTIVE: To investigate the relationship between Akt and second mitochondria-derived activator of caspases (Smac) in cisplatin (CDDP)-induced apoptosis in human ovarian cancer cells and the role of Akt in the molecular mechanism of chemoresistance in ovarian cancer. METHODS: Chemosensitive (OV2008 and A2780s) and chemoresistant (C13* and A2780cp) ovarian cancer cell lines were treated with CDDP and subcellular Smac contents were determined by Western blot. Smac siRNA and Smac N7 peptide were transfected into OV2008 and C13* cells, respectively. CDDP-induced apoptosis was measured by flow cytometry. A2780s cells stably transfected with Akt2 (A2780s-AAkt2) and C13* cells transfected with Aktl/2 siRNA were treated with CDDP, and Smac content and apoptosis in the cells were determined to detect the changes of their chemoresistance to CDDP. RESULTS: CDDP induced mitochondrial Smac release and apoptosis in chemosensitive cells (P < 0.05), but not resistant cells (P > 0.05). Downregulation of Smac by Smac siRNA confer resistance in OV2008 cells and Smac N7 peptide sensitized C13* cells to CDDP treatment. Overexpression of Akt2 inhibited mitochondrial Smac release and downregulation of Akt by siRNA sensitized C13* cells to CDDP treatment. CONCLUSION: Smac is required in CDDP-induced apoptosis in ovarian cancer cells and overexpression of Akt inhibits mitochondrial Smac release. Akt is closely related to the chemoresistance of ovarian cancer.

[Mdr-1 ribozyme in the reversal of multidrug resistance in human ovarian cancer].

OBJECTIVE: To study the mechanism of multidrug resistance and its reversal by mdr-1 ribozyme in human ovarian cancer. METHODS: The expression of mdr-1 and p-glycoprotein (p-gp) was studied by confocal laser microscope (Confocal), RT-PCR and Western blot analysis in adriamycin-resistant human ovarian cancer cell line (A2780/ADM) and adriamycin-sensitive one (A2780). The mdr-1 ribozyme was transfected into the A2780/ADM by Lipofectamine 2000 to overcome the multidrug resistance in ovarian cancer. RESULTS: The expression of mdr-1 mRNA and p-gp in A2780/ADM was significantly higher than that in A2780. The expression of mdr-1 mRNA and p-gp in A2780/ADM was lowered after being transfected by mdr-1 ribozyme. CONCLUSION: Multidrug resistance of A2780/ADM, possibly being caused by overexpression of mdr-1 gene, can be partially reversed by mdr-1 ribozyme.

[Resistance of multicellular spheroids to taxol in human ovarian cancer and its mechanism].

BACKGROUND & OBJECTIVE: Cytotoxic anticancer drugs are less effective in killing tumor cells grown as multicellular spheroids than monolayer cell cultures. The aim of this study was to investigate the molecular mechanism of chemoresistance. METHODS: Ovarian cancer A2780, CAOV3 multicellular spheroids were obtained from three-dimensional culture. Expression of P-glycoprotein (P-gp) was determined using Western blot analysis and flow cytometry (FCM). The subcellular distribution of P-gp was also determined using laser confocal microscope. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to detect the mdr1 mRNA of both monolayer cells and multicellular spheroids. Cell cycle profiles and apoptosis were also analyzed using FACS. The resistance was detected with trypan blue exclusion testing. RESULTS: Compared with control cells, no expression of P-gp was detected in monolayer cells, but expression of P-gp in aggregate cells was significantly elevated(P< 0.05). The mdr1 mRNA positive nodes were confirmed by RT-PCR in the aggregate cells. The percentage of cells increased in G(0)-G(1) phase and decreased in S and G(2)-M phase significantly in spheroids cells. Spheroids cells showed higher cell viability than monolayer cells (P(A2780)=0.003, P(CAOV3)=0.015). More apoptotic cells were induced by Taxol in MCS cells than in monolayer cells. CONCLUSION: Ovarian cancer A2780, CAOV3 multicellular spheroids cultures induced cell resistance to Taxol. High expression of P-gp was induced in ovarian multicellular spheroids and the cells were arrested in G(0)-G(1) phase.

[Role of apoptosis-associated genes and caspase-3 in cisplatin-resistant human ovarian cancer cell lines].

OBJECTIVE: To explore the role of apoptosis-associated genes and caspase-3 activity in cisplatin (DDP)-resistant human ovarian cancer cell lines. METHODS: The expressions of apoptosis-associated genes (bcl-2, bax, bcl-X(L) and bcl-X(S)) and the activity of caspase-3 were studied by reverse transcription-polymerase chain reaction (RT-PCR) and western blot in the cisplatin-resistant (A2780/DDP, COC1/DDP) and sensitive human ovarian cancer cells (A2780 and COC1). The apoptotic rates of A2780, COC1, A2780/DDP and COC1/DDP were measured with flow cytometry when treated with cisplatin. RESULTS: The mRNA expressions of bcl-2 and bcl-X(L) in A2780/DDP cells were 1.87 +/- 0.25 and 1.73 +/- 0.15, and significantly higher than those in A2780 cells (P < 0.05), which were 1.48 +/- 0.14 and 1.41 +/- 0.19 respectively. The protein expressions of bcl-2 and bcl-X(L) in A2780/DDP cells were 1.99 +/- 0.11 and 1.69 +/- 0.16, and significantly higher than those in A2780 cells (P < 0.05), which were 1.51 +/- 0.17 and 1.28 +/- 0.11 respectively. The expressions of bcl-2 and bcl-X(L) in COC1/DDP cells were also significantly higher than those in COC1 cells (P < 0.05). While there was no significant difference in expression of bax between A2780/DDP and A2780 as well as between COC1/DDP and COC1. No expression of bcl-X(S) was detected. When cells treated with different concentration of DDP, the caspase-3 activities, apoptotic rates and PARP expressions in A2780/DDP and COC1/DDP were significantly lower than those in A2780 and COC1 (P < 0.05), which showed dose-dependent (P < 0.05). CONCLUSION: Overexpression of anti-apoptotic genes and decrease of caspase-3 activity may relate to cisplatin resistance in human ovarian cancer cell lines.

[Relationship between expression of apoptosis-associated proteins and caspase-3 activity in cisplatin-resistant human ovarian cancer cell line].

BACKGROUND & OBJECTIVE: Cisplatin-based chemotherapy is an important way for treatment of ovarian cancer, but resistance to cisplatin is one of the reasons of treatment failure of ovarian cancer. This study was designed to investigate the relationship between apoptosis-associated proteins and caspase-3 activity as well as their effects on chemoresistance in human ovarian cancer cell lines. METHODS: The expression of apoptosis-associated proteins (bcl-2, bcl-xL, bax, bcl-xs), the activity of caspase-3 and cleavage of poly ADP-ribose polymerase (PARP) were determined with Western blot analysis in the cisplatin-resistant cell (COC1/DDP) and cisplatin-sensitive human ovarian cancer cell (COC1). The apoptotic ratios of COC1 and COC1/DDP were measured with flow cytometry after treated with different concentration of cisplatin. RESULTS: The expression of bcl-2 and bcl-XL in COC1/DDP cell was significantly higher than that in COC1 cell, whereas the expression of bax showed no change in COC1/DDP and COC1. There was no bcl-xs expression in both COC1 and COC1/DDP cells. The activity of caspase-3, the amount of PARP fragments, and apoptotic ratio in COC1/DDP reduced much more than COC1 did after treated with cisplatin. CONCLUSIONS: Cisplatin-resistance in human ovarian cancer cell lines may associated with the overexpression of anti-apoptotic protein bcl-2 and downregulation of caspase-3 activity, but not associated with the expression of bax and bcl-xs.