Alternative Pathways of Cancer Cell Death by Rottlerin: Apoptosis versus Autophagy

Abstract

Since the ability of cancer cells to evade apoptosis often limits the efficacy of radiotherapy and chemotherapy, autophagy is emerging as an alternative target to promote cell death. Therefore, we wondered whether Rottlerin, a natural polyphenolic compound with antiproliferative effects in several cell types, can induce cell death in MCF-7 breast cancer cells. The MCF-7 cell line is a good model of chemo/radio resistance, being both apoptosis and autophagy resistant, due to deletion of caspase 3 gene, high expression of the antiapoptotic protein Bcl-2, and low expression of the autophagic Beclin-1 protein. The contribution of autophagy and apoptosis to the cytotoxic effects of Rottlerin was examined by light, fluorescence, and electron microscopic examination and by western blotting analysis of apoptotic and autophagic markers. By comparing caspases-3-deficient (MCF-7(3def)) and caspases-3-transfected MCF-7 cells (MCF-7(3trans)), we found that Rottlerin induced a noncanonical, Bcl-2-, Beclin 1-, Akt-, and ERK-independent autophagic death in the former- and the caspases-mediated apoptosis in the latter, in not starved conditions and in the absence of any other treatment. These findings suggest that Rottlerin could be cytotoxic for different cancer cell types, both apoptosis competent and apoptosis resistant.

Figure 1

Rottlerin is cytotoxic for MCF-7^3def  cells. (a) Rottlerin treatment for 24 h induced cytotoxicity, evaluated by the SRB assay, in a dose-dependent manner, with a IC50 of approximately 20 μM. (b) Cytotoxicity after 24–72 h exposure to 20 μM Rottlerin. Controls (24–72 h) were exposed to DMSO alone. (c) Cell survival expressed as % of seeded cells (control). Results are the means ± SD of at least three independent experiments in quadruplicate. ((d) and (e)) Dose- and time-dependent cytotoxicity evaluated by direct cell counting (Trypan Blue). % cytotoxicity: 100× (cell control−experimental)/cell control. *P < 0.05 compared with the control.

Figure 2

Rottlerin did not induce apoptosis in MCF-7^3def  cells. (a) Western blot analysis of the antiapoptotic protein Bcl-2, caspase 9, caspase 3, and PARP after 1–24 h of 20 μM Rottlerin treatment; β-actin was used as loading control. (b) Nuclear staining with Hoechst 33342 after 18 and 24 h treatment; o.m.: original magnification. Representative of three independent experiments.

Figure 3

Rottlerin induced the cytoplasmic accumulation of multiple vacuole-like structures in MCF-7^3def cells. (a) Light microscopy images of Toluidine blue-stained cells; (b) phase contrast microscopy; (c) transmission electron microscopy images after a 24 h exposure to 20 μM Rottlerin; control was treated with DMSO alone; o.m.: original magnification. Representative of three independent experiments.

Figure 4

Vacuoles have reference to autophagy. (a) MDC staining of autophagosomes after 20 μM Rottlerin treatment for 6, 18, and 24 h; o.m.: original magnification; (b) western blotting analysis of the autophagosome marker LC3I-II after 20 μM Rottlerin treatment for 1–24 h and increased LC3-II/LC3-I ratio, calculated by densitometry; (c) autophagic clearance of SQSTM1/p62. β-actin was used as loading control. Representative of three independent experiments.

Figure 5

Rottlerin induced Bcl-2/Beclin-1-independent autophagic death in MCF-7^3def cells. (a) Western blotting analysis of Beclin-1 expression after 20 μM Rottlerin treatment for 1–24 h. (b) Western blotting analysis of LC3 after 1–10 μM of Rottlerin treatment for 24 h in Bcl-2-silenced and Bcl-2-overexpressing MCF-7^3def cells. (c) Western blotting analysis of LC3 after 20 μM of Rottlerin treatment for 6 h, in the presence and absence of 5 mM 3-MA. β-actin was used as loading control. Representative of two independent experiments.

Figure 6

Morphological and biochemical features of apoptosis in Rottlerin-treated MCF-7^3trans cells: (a) Cell survival expressed as % of seeded cells (control), evaluated by the SRB assay. (b) Phase-contrast microscopy and (c) Hoechst 33342 staining after 18 and 24 h of 20 μM Rottlerin exposure; o.m.: original magnification. (d) Western blotting analysis of Bcl-2, caspases 9, caspases 3,  PARP, and LC3-I-II after 20 μM of Rottlerin treatment for 1–24 h. β-actin was used as a loading control. Representative of three independent experiments.