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. 2012;7(4):e35073.
doi: 10.1371/journal.pone.0035073. Epub 2012 Apr 25.

A small molecule SMAC mimic LBW242 potentiates TRAIL- and anticancer drug-mediated cell death of ovarian cancer cells

Eleonora Petrucci  1 , Luca Pasquini, Manuela Bernabei, Ernestina Saulle, Mauro Biffoni, Fabio Accarpio, Simone Sibio, Angelo Di Giorgio, Violante Di Donato, Assunta Casorelli, Pierluigi Benedetti-Panici, Ugo Testa
Affiliations

A small molecule SMAC mimic LBW242 potentiates TRAIL- and anticancer drug-mediated cell death of ovarian cancer cells

Eleonora Petrucci et al. PLoS One. 2012.

Abstract

Background: Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (SMAC) has been described to sensitize for apoptosis. We have explored the pro-apoptotic activity of LBW242, a mimic of SMAC/DIABLO, on ovarian cancer cell lines (A2780 cells and its chemoresistant derivative A2780/ADR, SKOV3 and HEY cells) and in primary ovarian cancer cells. The effects of LBW242 on ovarian cancer cell lines and primary ovarian cancer cells was determined by cell proliferation, apoptosis and biochemical assays.

Principal findings: LBW242 added alone elicited only a moderate pro-apoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or anticancer drugs in inducing apoptosis of both ovarian cancer cell lines and primary ovarian cancer cells. Mechanistic studies show that LBW242-induced apoptosis in ovarian cancer cells is associated with activation of caspase-8. In line with this mechanism, c-FLIP overexpression inhibits LBW242-mediated apoptosis.

Conclusion: LBW242 sensitizes ovarian cancer cells to the antitumor effects of TRAIL and anticancer drugs commonly used in clinic. These observations suggest that the SMAC/DIABLO mimic LBW242 could be of value for the development of experimental strategies for treatment of ovarian cancer.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of LBW242 on the cell growth (A) and apoptosis (B) of A2780WT, A2780ADR, HEY and SKOV3 cells.
1A- A2780WT, A2780ADR, HEY and SKOV3 cells have been grown for 48h either in the absence or in the presence of TRAIL (50 ng/ml) and either in the absence or in the presence of growing concentrations of LBW242 (from 1 to 10 μM) and at the end of the culture the number of living cells was determined. The difference between Control and TRAIL was statistically significant: p = <0.001 for A2780WT and HEY; p = <0.01 for SKOV3; p = <0.05 for A2780ADR. 1B- A2780WT and SKOV3 cells have been grown as above and after 48 h of culture the proportion of apoptotic cells was determined by Annexin-V binding assay and propide iodide staining. The percentage of apoptotic cells was determined by flow cytometry. The results represent the mean values observed in three separate experiments. The difference between Control and TRAIL was statistically significant: p = <0.01 for both A2780WT and SKOV3 cells.
Figure 2
Figure 2. c-FLIPL overexpression protects A2780WT, A2780ADR and SKOV3 cells from the pro-apoptotic effects induced by LBW242.
A2780WT, A2780ADR and SKOV3 cells have been stably transfected with either an empty vector (PINCO) or with the vector containing the cDNA of c-FLIP (FLIP) and the resulting cells were grown either in the absence ( Control ) or in the presence of LBW242 (10 μM) or TRAIL (50 ng/ml) or both these agents at the above concentrations. The percentage of apoptotic cells was determined by flow cytometry using the Annexin-V/PI binding assay. The data represent the mean values ± SEM observed in three separate experiments. Statistical analysis: * p = <0.05; ** p = <0.01; *** p = <0.001.
Figure 3
Figure 3. Effect of a pan-caspase inhibitor (zVAD-fmk) and a specific caspase-8 inhibitor (zIETD-fmk) on the induction of apoptosis by LBW242 (LBW) or TRAIL or both agents.
A2780WT, A2780FLIP, A2780ADR, A2780ADR FLIP, SKOV3 and SKOV3 FLIP cells have been grown as reported in Fig. 2 either in the absence or in the presence of 40 μM zVAD-fmk or zIETD-fmk and after 48 h of incubation the percentage of apoptotic cells was determined by Annexin-V/PI binding assay. The results represent mean values ± SEM observed in three separate experiments.
Figure 4
Figure 4. Immunoblotting analysis of XIAP, c-IAP1, c-IAP2, Survivin, Casp-9, Casp-8, c-FLIPL, FADD, PARP, Casp-3 and BID in A2780WT PINCO and FLIP (A), A2780ADR PINCO and FLIP (B), SKOV3 PINCO and SKOV3 FLIP (C) cells grown for 24 h either in the absence ( Control ) or in the presence of LBW242 10 μM, or TRAIL 50 ng/ml or both agents at the above concentrations.
In the panel C both the full length (FL) and the cleaved form (CF) of caspase-3 are shown. The PARP immunoblotting shows two bands, of which the upper corresponds to a full-length form of PARP and the lower band to a cleaved form of PARP. In the Figure are reported the representative data observed in one of the three experiments carried out.
Figure 5
Figure 5. LBW242 potentiates the proapoptotic effects of some anticancer drugs, including Cisplatin, Paclitaxel, Topotecan, Etoposide and Doxorubicin.
A- HEY cells have been incubated for 24 h either in the absence (control) or in the presence of LBW242 (30 µM) or of either Cisplatin (CPLAT at 1.6 or 8 µM) or Paclitaxel (TAXOL at 2.5 or 12.5 µM) or Topotecan (TOPO at 0.2 or 1 µM) or Etoposide (ETOPO at 8 or 40 µM) or Doxorubicin (DOXO at 0.17 or 0.85 µM) alone or in combination with LBW242 and analysed for induction of cell death by flow cytometry. The data represent the mean values ± SEM observed in three separate experiments. Statistical analysis: * p = <0.05; ** p = <0.01; *** p = <0.001. B – HEY cells have been incubated for 24 h in the presence of increasing concentrations of LBW242either in the absence (Control) or in the presence of Cisplatin (1.6 µM) or Paclitaxel (2.5 µM) or Topotecan (0.2 µM) and analysed for induction of cell death by flow cytometry. The data represent the mean value ± SEM observed in three separate experiments. The differences between control and Topotecan (p = <0.001), control and Paclitaxel (p = <0.001) and control and Cisplatin (p = <0.05) were all significant.
Figure 6
Figure 6
A- LBW242 potentiates the proapoptotic effect of Topotecan Hydrochloride, a topoisomerase I inhibitor. A2780WT PINCO and FLIP (top panels) and SKOV3 PINCO and FLIP (bottom panels) cells have been incubated for 24 h either in the absence (Control) or in the presence of either DMSO 0.01% or zVAD-fmk 40 μM, Topotecan Hydrochloride 1 μM, TRAIL 50 ng/ml or LBW242+zVAD-fmk, LBW242+Topotecan Hydrochloride, TRAIL+zVAD-fmk, TRAIL+Topotecan Hydrochloride, Topotecan Hydrochloride +zVAD-fmk, LBW242+TRAIL, LBW242+TRAIL+zVAD-fmk, LBW242+Topotecan Hydrochloride+zVAD-fmk and analysed for induction of apoptosis by flow cytometry. The data represent the mean values ±SEM observed in three separate experiments. In A7280 WT PINCO and in SKOV3 PINCO cells LBW242+TOPO induced a proportion of dead cells higher than that observed with either LBW242 (p = <0.05) or TOPO (p = <0.05). B and C – Evaluation of caspases-8 activation in A2780 and A2780ADR cells incubated either in the absence (Control) or in the presence of LBW242 or of Topotecan or of both LBW242 and Topotecan. Caspase-8 activity in intact cells was measured using a caspases-8 specific fluorigenic substrate. Original results from one representative analysis are reported in B, while the mean values±SEM of the percentages of cells displaying caspase-8 activation are reported in C. The percentage of cells exhibiting activated caspases-8 was higher both for WT and ADR cells in LBW242 than in NT cells (p = <0.05) and in LBW242+TOPO than in LBW242 or TOPO-treated cells (p = <0.05).
Figure 7
Figure 7. Effect of LBW242, TRAIL or a combination of both agents on cell growth (left upper panel) and induction of apoptosis (right upper panel) of ovarian cancer cells derived from 3 ovarian cancer at presentation (black bars) and 6 relapsing ovarian cancers (white bars).
The gray bars represent mean values ± SEM. Ovarian cancer cells isolated from tumor biopsies have been grown for 24 hours either in the absence or in the presence of LBW242 (10 μM) or of TRAIL (50 ng/ml) or both agents at the above concentrations. Lower panel on the right: The proportion of dead cells was higher in LBW242 or TRAIL or LBW242+TRAIL-treated cells than in control not-treated cells (p = <0.01); furthermore, the percentage of dead cells was lower in LBW242+TRAIL than in TRAIL or LBW242-treated cells (for both p = <0.05). Lower panel on the left: The cell number was significantly lower in LBW242+TRAIL than in TRAIL or LBW242-treated cells (for both p = <0.05).
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