Marine Actinomycetes-Derived Secondary Metabolites Overcome TRAIL-Resistance via the Intrinsic Pathway through Downregulation of Survivin and XIAP

Abstract

Resistance of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis represents the major hurdle to the clinical use of TRAIL or its derivatives. The discovery and development of lead compounds able to sensitize tumor cells to TRAIL-induced cell death is thus likely to overcome this limitation. We recently reported that marine actinomycetes' crude extracts could restore TRAIL sensitivity of the MDA-MB-231 resistant triple negative breast cancer cell line. We demonstrate in this study, that purified secondary metabolites originating from distinct marine actinomycetes (sharkquinone (1), resistomycin (2), undecylprodigiosin (3), butylcyclopentylprodigiosin (4), elloxizanone A (5) and B (6), carboxyexfoliazone (7), and exfoliazone (8)), alone, and in a concentration-dependent manner, induce killing in both MDA-MB-231 and HCT116 cell lines. Combined with TRAIL, these compounds displayed additive to synergistic apoptotic activity in the Jurkat, HCT116 and MDA-MB-231 cell lines. Mechanistically, these secondary metabolites induced and enhanced procaspase-10, -8, -9 and -3 activation leading to an increase in PARP and lamin A/C cleavage. Apoptosis induced by these compounds was blocked by the pan-caspase inhibitor QvD, but not by a deficiency in caspase-8, FADD or TRAIL agonist receptors. Activation of the intrinsic pathway, on the other hand, is likely to explain both their ability to trigger cell death and to restore sensitivity to TRAIL, as it was evidenced that these compounds could induce the downregulation of XIAP and survivin. Our data further highlight that compounds derived from marine sources may lead to novel anti-cancer drug discovery.

Keywords: TRAIL; apoptosis; marine actinomycetes; therapy.

Figure 1

Purified marine actinomycetes-derived secondary metabolites. (A) Source of compounds. Sharkquinone (1), and resistomycin (2) were purified from Streptomyces sp. EGY1 and EGY34 isolated from marine sediment of the Red Sea, Egypt. Undecylprodigiosin (3) and butylcycloheptylprodigiosin (4) were purified from the actinomycetes strain RA2 which was isolated from a marine sponge Spheciospongia mastoidea collected in Ras Mohammed, South of Sinai, Egypt. The marine compounds elloxazinone A (5), elloxazinone B (6), carboxyexfoliazone (7), and exfoliazone (8) were purified from the actinomycetes strain EGY25, isolated from a sea sand sample obtained from Marsa Matruh city, Mediterranean Sea, Egypt. (B) Chemical structures of the corresponding purified marine-derived compounds.

Figure 2

Effect of marine compounds on the viability of HCT116 and MDA-MB-231 cells. (A,B) The human colorectal and breast cancer cell lines were treated with various concentrations of the marine compounds for 24 h and viability was determined by methylene blue assay. Results correspond to three independent experiments. Error bars represent the SD values. (1) Sharkquinone, (2) resistomycin, (3) undecylprodigiosin, (4) butylcycloheptylprodigiosin, (5) elloxazinone A, (6) elloxazinone B, (7) carboxyexfoliazone, and (8) exfoliazone.

Figure 3

Monitoring apoptosis induced by marine compounds using flow cytometry in Jurkat and HCT116 cells. (A) Jurkat WT, Cas-8-/- and FADD-/- cells were treated with the indicated compounds for 24 h then stained with 7AAD and Annexin V and fluorescence was analyzed by flow cytometry. (B) Flow cytometry and Western blot analysis to determine the sensitivity of HCT116-Cas8-/- toward TRAIL-and FasL-induced cell death. Cells were treated with and without TRAIL and FasL for 6 h then subjected to dual staining as above and apoptotic percentage was determined by flow cytometry. For Western blot, cell lysates were prepared and loaded on SDS-PAGE (12%) followed by immunoblotting using the corresponding antibodies. HSC70 was used as loading control. (C) Colon HCT116 (WT, Cas8-/-, and TRAIL-R1/TRAIL-R2-/-) cancer cell lines were treated as previously mentioned. All values are presented here as ±SD (n = 3). Significance was evaluated by two-way ANOVA. *p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 4

Characterization of apoptosis induced by marine-derived compounds with and without TRAIL in Jurkat and HCT116 cells. (A,B) Cells were treated with 50 µM of the indicated compounds for 6 h in the absence and in the presence of TRAIL (250 ng) and stained with the fluorescent DNA-binding dye Hoechst 33342. Morphological changes (A,B) were detected under fluorescent microscope using a blue filter. Corresponding quantifications are shown in (C,D). All values are presented here as ±SD (n = 3). Significance was evaluated by two-way ANOVA. **** p < 0.0001.

Figure 5

Western blot analysis of pro-apoptotic and anti-apoptotic proteins in cancer cells treated with marine compounds alone or combined with TRAIL. (A) Jurkat and (B) HCT116 cells were treated or not with marine-derived compounds (50 µM) in the presence or absence of TRAIL (250 ng/mL). Following incubation, cell lysates were prepared and loaded on SDS-PAGE (12%) and the indicated proteins were detected by immunoblotting. (C) MDA-MB-231 cells were stimulated as above and apoptosis-induced by marine-derived compounds in the presence or absence of TRAIL was analyzed by Hoechst staining. All values are presented here as ±SD (n = 3). Significance was evaluated by two-way ANOVA. * p < 0.05 and **** p < 0.0001. (D) Corresponding MDA-MB-231 cell lysates were prepared, loaded on SDS-PAGE (12%) and relevant proteins were detected by immunoblot. In all cases, HSC70 was used as a loading control.

Figure 6

Western blot analysis of pro- and anti-apoptotic proteins in HCT116 cells stimulated with TRAIL or marine compounds. (A) Cells were treated with the identified compounds (50 µM) or TRAIL (0.25 µg/mL) for 24 h. Following incubation, cell lysates were prepared and loaded on SDS-PAGE (12%) and the indicated proteins were detected by immunoblotting. GAPDH or HSC70 were used as loading controls. (B) Cells were pre-incubated for 30 min in the presence or absence of the pan-caspase inhibitor QvD (10 µM) then treated as above with marine compounds (50 µM) or TRAIL (0.25 and 1 µg/mL) for 24 h. Cell lysates were prepared as described above and analyzed for Lamin A/C cleavage by immunoblot.