A Small Molecule SMAC Mimic LBW242 Potentiates TRAIL- and Anticancer Drug-Mediated Cell Death of Ovarian Cancer Cells

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

A Catalytic Approach to the Metal-Free Reaction of Epoxides with Ketene Silyl Acetals for Accessing γ‑Lactones

The first catalytic approach to the nucleophilic addition of silyl ketene acetals <b>2</b> to epoxides <b>1</b> is reported. The defined protocol is metal-free using tetrabutylammonioum fluoride as the catalyst. It works in a very efficient manner under solvent-free conditions (SolFC) allowing γ-lactones <b>3</b> to be directly obtained with high regioselectivities and yields

Feasibility of using a dose-area product ratio as beam quality specifier for photon beams with small field sizes

International audiencePurpose: To investigate the feasibility of using the ratio of dose-area product at 20 cm and 10 cm water depths (DAPR(20,10)) as a beam quality specifier for radiotherapy photon beams with field diameter below 2 cm.Methods: Dose-area product was determined as the integral of absorbed dose to water (D-w) over a surface larger than the beam size. 6 MV and 10 MV photon beams with field diameters from 0.75 cm to 2 cm were considered. Monte Carlo (MC) simulations were performed to calculate energy-dependent dosimetric parameters and to study the DAPR(20,10) properties. Aspects relevant to DAPR(20,10) measurement were explored using large-area plane-parallel ionization chambers with different diameters.Results: DAPR(20,10) was nearly independent of field size in line with the small differences among the corresponding mean beam energies. Both MC and experimental results showed a dependence of DAPR(20,10) on the measurement setup and the surface over which D-w is integrated. For a given setup, DAPR(20,10) values obtained using ionization chambers with different air-cavity diameters agreed with one another within 0.4%, after the application of MC correction factors accounting for effects due to the chamber size. DAPR(20,10) differences among the small field sizes were within 1% and sensitivity to the beam energy resulted similar to that of established beam quality specifiers based on the point measurement of D-w.Conclusions: For a specific measurement setup and integration area, DAPR(20,10) proved suitable to specify the beam quality of small photon beams for the selection of energy-dependent dosimetric parameters

c-FLIP_L overexpression protects A2780WT, A2780ADR and SKOV3 cells from the pro-apoptotic effects induced by LBW242.

<p>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.</p

Effect of LBW242, TRAIL or a combination of both agents on cell growth (<i>left upper panel</i>) and induction of apoptosis (<i>right upper panel</i>) of ovarian cancer cells derived from 3 ovarian cancer at presentation (black bars) and 6 relapsing ovarian cancers (white bars).

<p>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. <i>Lower panel on the right</i>: 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). <i>Lower panel on the left</i>: The cell number was significantly lower in LBW242+TRAIL than in TRAIL or LBW242-treated cells (for both p = <0.05).</p

Immunoblotting analysis of XIAP, c-IAP1, c-IAP2, Survivin, Casp-9, Casp-8, c-FLIP_L, 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.

<p>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.</p

Clinical characteristics of the nine ovarian cancer patients whose tumor samples were Investigated for sensitivity to LBW242.

<p>Clinical characteristics of the nine ovarian cancer patients whose tumor samples were Investigated for sensitivity to LBW242.</p

LBW242 potentiates the proapoptotic effects of some anticancer drugs, including Cisplatin, Paclitaxel, Topotecan, Etoposide and Doxorubicin.

<p>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.</p

Figure 6

<p><b>A</b>- LBW242 potentiates the proapoptotic effect of Topotecan Hydrochloride, a topoisomerase I inhibitor. A2780WT PINCO and FLIP (<i>top panels</i>) and SKOV3 PINCO and FLIP (<i>bottom panels</i>) 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>B</b> and <b>C</b> – 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).</p