EF24 induces G2/M arrest and apoptosis in cisplatin-resistant human ovarian cancer cells by increasing PTEN expression

Abstract

We report that EF24, a synthetic compound 3,5-bis(2-flurobenzylidene)piperidin-4-one, greatly inhibits cisplatin-resistant (CR) human ovarian cancer cell proliferation. The inhibitory effect of EF24 on cell proliferation is associated with G(2)/M phase cell cycle arrest and increased G(2)/M checkpoint protein (pp53, p53, and p21) levels. Within 24 h following treatment, EF24 induced apoptosis in CR cells. The apoptosis was partially blocked by the general caspase inhibitor z-VAD. Within 12 h, EF24 induced a membranous FasL expression, consistent with a substantial decrease in the Ser(473) and Thr(308) phosphorylation of Akt, a known negative regulator of FasL transcription. Also, EF24 activated the phosphorylated PTEN and marginally up-regulated total PTEN expression through the inhibition of ubiquitin-mediated PTEN degradation. Suppression of PTEN expression with siRNA significantly reduced the p53 and p21 levels and activated Akt phosphorylation at Ser(473) and Thr(308), resulting in decreased apoptosis and increased cell survival. On the other hand, overexpression of PTEN markedly induced apoptosis. Our results clearly suggested that EF24 induced significant increase in PTEN expression. The up-regulation of PTEN inhibited Akt and MDM2, which enhanced the level of p53, thereby inducing G(2)/M arrest and apoptosis. Therefore, EF24 appears to have a potential therapeutic role in human ovarian cancer through the activation of PTEN.

FIGURE 1. Effect of EF24 on cell viability

A, molecular structure of EF24 (3,5-bis(2-flurobenzylidene) piperidin-4-one). Effects of EF24 dose and incubation time on the cell viability, expressed as a fraction of untreated control are shown for CR and CS ovarian cancer cells (B and C) and CHO cells (D). Cell viability assays were performed using MTT as described under “Experimental Procedures.” The graph displays the mean ± S.E. of three independent experiments. The data indicate that EF24 is more effective in inhibiting the growth of ovarian cancer cells compared with normal cells.

FIGURE 2. Effect of EF24 on cell cycle progression and cell cycle regulatory molecules in CR cells

Cells were cultured in complete medium, and treated with either Me₂SO vehicle control or 2 µmEF24. After 3, 6, or 12 h of treatment, cells were collected, washed with PBS, digested with RNase, and then the cellular DNA was stained with propidium iodide. Flow cytometric analysis was then performed for cell cycle distribution. A, representative flow cytometry graph for each treatment group. B, G₂/M cell cycle distribution as a function of treatment period. C, immunoblot images of cell cycle regulatory molecules pp53, p53, MDM2, p21, p27, CycB1, CycD1, CycA, Cdc2, Cdc25C, and Wee1in CR cells treated with 2 µm EF24. D, quantitative results of phosphorylated p53 and p53 bands by densitometric analysis. Data represent mean ± S.E. of three independent experiments. *, p < 0.05 as compared with control group.

FIGURE 3. EF24-induced apoptosis in CR ovarian cancer cells

Cells were treated with 2 µm EF24 for 24 h, harvested, and the sub-G₁ population for 20,000 events within a fixed gate was analyzed. A, representative flow-cytometry profile for control (vehicle-treated) and EF24-treated group. B, immunoblot analysis for apoptosis. Cleavages in caspase-8, caspase-3, caspase-7, and PARP are shown in cells treated with 2 µm EF24 for 6, 12, or 24 h. C, CR cells were pretreated with 20 or 50 µm z-VAD-fmk for 1 h and then treated with EF24 for 24 h. Cell lysates were used for immunoblot assay for cleaved caspase-3, -7, and PARP. D, percentage of cells in sub-G₁ phase as determined by flow cytometry. Data represent mean ± S.E. of three independent experiments. *, p < 0.05 compared with EF24 group.

FIGURE 4. FasL-induced apoptosis

A, immunoblot analysis for the expression levels of caspase-8 associated death receptors, FasL, TNFR1, and Fas/ CD95 in CR cells treated with EF24 for 6, 12, or 24 h. A clear increase in the FasL expression, but not in the TNFR1 or Fas/CD95 expression, is observed. B, densitometric analyses of visualized bands for FasL expression. Data represent mean ± S.E. of three independent experiments. *, p < 0.05 as compared with control group.

FIGURE 5. Inhibition of pAkt and pERK1/2 by EF24

A, expression levels of Akt and ERK1/2 and their phosphorylated counterparts by immunoblot assay. The CR cells were treated with 2 µm EF24 for 3, 6, or 12 h. The data show a decrease in the expression of pAkt (Ser⁴⁷³ and Thr³⁰⁸) while the expressions of ERK1/2 and pERK1/2 are unchanged. The expression levels of both Ser- and Thr-phosphorylated Akt are decreased by the PI3K inhibitor, LY-294002 (10 µm). B, cell lysates were subjected to immunoprecipitation with anti-Akt, followed by immunoblotting analysis. C, expression of ERK1/2 and pERK1/2 at 12, 24, and 48 h. The pERK1/2 levels are clearly down-regulated while the total ERK levels are unchanged.

FIGURE 6. Up-regulation of PTEN expression by EF24

PTEN and pPTEN were determined using PTEN and pPTEN antibodies. Densitometric analysis of visualized bands was performed to quantify PTEN and pPTEN as a function of EF24 dose (A) and treatment period (B). The data show mean ± S.E. of three independent experiments. *, p < 0.05 as compared with respective controls. C, IP-IB of PTEN ubiquitination. CR cells were incubated with EF24 for 0.5, 1, or 2 h prior to protein extraction. Cell lysates were immunoprecipitated with agarose-conjugated ubiquitin antibody. The precipitates were subjected to SDS-PAGE and immunoblotting using PTEN, pPTEN, and p53 antibodies. Ubiquitinated PTEN bands were detected as described under “Experimental Procedures.” MG-132 was used as a positive control. PTEN, but not p53, shows a clear increase in ubiquitination. D, CK2 activity in CR cells treated with EF24 showing no change in the pCK2 levels. E, IP-IB of PTEN and p53 interaction showing no significant interaction between PTEN and p53 in EF24-treated CR cells.

FIGURE 7. PTEN siRNA inhibits EF24-induced cell death

A, effect of PTEN siRNA on EF24-induced overexpression of PTEN. Control siRNA was used a negative control. B, PTEN siRNA-transfected cells treated with EF24 for 6 h resulted in a significant decrease in G₂/M cell-cycle arrest as compared with non-transfected cells treated with EF24 (**, p < 0.05). *, p < 0.05 versus untreated control cells. Data represent mean ± S.E. of three independent experiments. C, Western blots of cleaved caspases-3, -7, and PARP in PTEN siRNA- or control siRNA-transfected cells treated with EF24 for 24 h. Control represents cells untreated with EF24. D, densitometry analysis of visualized bands for cleaved caspase-3, -7, and PARP. Suppression of PTEN shows significant inhibition of cleaved caspase-3, -7, and PARP activation as compared with non-transfected or control siRNA-transfected cells treated with EF24 (*, p < 0.05; n = 3). E, quantification of apoptotic cells (cell death) determined by flow cytometry. Data represent mean ± S.E. of three independent experiments. *, p < 0.05 versus untreated control; **, p < 0.05 versus non-transfected cells treated with EF24.

FIGURE 8. Effect of PTEN overexpression on EF24-induced apoptosis

The PTEN/FLAG gene was transfected into cells and then treated with 2 µm EF24 for 24 h. The effect of PTEN overexpression and EF24 on the viability of (A) CHO and (B) CR cells. Cell viability assays were performed using MTT assay. Data represent mean ± S.E. of three independent experiments. *, p < 0.05 versus Control; **, p < 0.05 versus PTEN cDNA. C, effect of overexpression of PTEN and EF24 treatment on the apoptotic markers in CR cells. Expression of PTEN, p53, p21, pAkt, Akt, cleaved caspase-3, PARP, and FLAG expression in untransfected cells treated without (Control) and with EF24 treatment (EF24), and cDNA-transfected cells without (cDNA) and with EF24 treatment (EF24 + cDNA) were determined by Western blot. D, densitometry analysis of the bands shown in C. Data represent mean ± S.E. of three independent experiments.

FIGURE 9. A schematic presentation for the anticancer mechanisms of EF24 shown in the present study

EF24 up-regulates PTEN expression through the inhibition of proteasomal degradation of PTEN. Activation of PTEN down-regulates pAkt and MDM2, which enhances the level of p53. The up-regulation of PTEN by EF24 leads to the PTEN-mediated cell cycle progression, and increase in sensitivity to apoptosis through the activation of FasL.