Targeted polytherapy in small cell sarcoma and its association with doxorubicin

Abstract

A paradigm shift has occurred in the last decade from chemotherapy to targeted therapy for the management of many patients with advanced sarcoma. This work identifies a combination of targeted agents and doxorubicin that are effective against small cell sarcoma cell lines. Three small cell sarcoma cell lines were studied: RD18 (rhabdomyosarcoma), A204 (undifferentiated sarcoma) and TC 71 (Ewing's sarcoma). Each cell line was exposed to increasing concentrations of vorinostat (HDAC inhibitor), 17-DMAG (HSP90 inhibitor), abacavir (anti-telomerase) or sorafenib (tyrosine kinase inhibitor) alone, combined with one another, or combined with doxorubicin. Cell viability, cell cycle analysis and apoptosis were assessed by MTS assay, propidium iodide-Annexin V staining, and caspase 3/7 activity, respectively. The Chou and Talalay combination index (CI) was used to determine whether the effects were additive (CI = 1), synergistic (CI < 1) or antagonistic (CI > 1). In monotherapy, targeted agents achieved 30-90% reductions in viability, with the exception of abacavir. Dual-targeted combination therapies with vorinostat, sorafenib and 17-DMAG demonstrated synergy. Abacavir was antagonistic with every other drug and was not further studied. Both vorinostat and 17-DMAG synergized with doxorubicin, achieving 60% cell killing compared to 12% with doxorubicin alone. No synergy was observed for sorafenib with doxorubicin. The triple therapy vorinostat, 17-DMAG and doxorubicin did not show synergy, but increased the subG1 population at 24H, from 30% to 70% compared to monotherapies with an increase in apoptosis. This work provides evidence of synergy of combinations of vorinostat, 17-DMAG and sorafenib in small cell sarcoma. In addition to doxorubicin, these combinations enhance doxorubicin cytotoxicity at therapeutically relevant concentrations.

Keywords: Cell line; Combination; Doxorubicin; Small cell sarcoma; Targeted therapy.

Figure 1

Cell viability of cell lines with triple therapy (A) TC71 and (B) RD18. S = sorafenib, D = 17‐DMAG, V = vorinostat, T = triple therapy.

Figure 2

Cell viability of doxorubicin plus either (A) 17‐DMAG (RD18), (B) sorafenib (RD18) or (C) vorinostat (TC71).

Figure 3

Cell viability of doxorubicin plus 17‐DMAG and (A) vorinostat (TC71) or (B) sorafenib (RD18) and (C) drug sequence effect viability through sequential or concomitant administration of doxorubicin plus vorinostat‐17‐DMAG (RD18).

Figure 4

Cell cycle distribution of TC71 cells treated with vorinostat, doxorubicin and 17‐DMAG as (A) monotherapy and (B) in combination.

Figure 5

Measure of apoptosis by Caspase 3/7 activity of doxorubicin, vorinostat and 17‐DMAG combination in TC71 cell line.

Figure 6

Apoptosis measured with Alexa Fluor 488 Annexin V and PI staining of doxorubicin, vorinostat and 17‐DMAG combination in TC71 cell line.