Thymoquinone up-regulates PTEN expression and induces apoptosis in doxorubicin-resistant human breast cancer cells

Abstract

The use of innocuous naturally occurring compounds to overcome drug resistance and cancer recalcitrance is now in the forefront of cancer research. Thymoquinone (TQ) is a bioactive constituent of the volatile oil derived from seeds of Nigella sativa Linn. TQ has shown promising anti-carcinogenic and anti-tumor activities through different mechanisms. However, the effect of TQ on cell signaling and survival pathways in resistant cancer cells has not been fully delineated. Here, we report that TQ greatly inhibits doxorubicin-resistant human breast cancer MCF-7/DOX cell proliferation. TQ treatment increased cellular levels of PTEN proteins, resulting in a substantial decrease of phosphorylated Akt, a known regulator of cell survival. The PTEN expression was accompanied with elevation of PTEN mRNA. TQ arrested MCF-7/DOX cells at G2/M phase and increased cellular levels of p53 and p21 proteins. Flow cytometric analysis and agarose gel electrophoresis revealed a significant increase in Sub-G1 cell population and appearance of DNA ladders following TQ treatment, indicating cellular apoptosis. TQ-induced apoptosis was associated with disrupted mitochondrial membrane potential and activation of caspases and PARP cleavage in MCF-7/DOX cells. Moreover, TQ treatment increased Bax/Bcl2 ratio via up-regulating Bax and down-regulating Bcl2 proteins. More importantly, PTEN silencing by target specific siRNA enabled the suppression of TQ-induced apoptosis resulting in increased cell survival. Our results reveal that up-regulation of the key upstream signaling factor, PTEN, in MCF-7/DOX cells inhibited Akt phosphorylation, which ultimately causes increase in their regulatory p53 levels affecting the induction of G2/M cell cycle arrest and apoptosis. Overall results provide mechanistic insights for understanding the molecular basis and utility of the anti-tumor activity of TQ.

Fig. 1. Structure of TQ (2-isopropyl-5-methyl-1,4 benzoquinone)

Fig. 2. Anti-proliferative effect of TQ on MCF-7/DOX cells

Exponentially growing MCF-7/Dox cells were seeded in 96-well plates. The cells were treated with indicated concentrations of TQ, maintained in culture for 12, 24 or 48 hours and incubated in medium containing MTT for additional 2 hours. Cell growth was determined by MTT assay, assessing the ability of cells to convert the soluble MTT into an insoluble formazan precipitates in cells. Values represent mean ±SE of n=3. *: Denotes significance at p<0.05

Fig. 3. TQ induces apoptosis in MCF-7/DOX cells

(A) Gel analysis of DNA fragmentation patterns of TQ-treated or untreated cells. (B) Morphologic examination of TQ-treated and untreated cells by Hoechst 33342 staining. (C) Flow cytometric analysis of cell populations at sub-G0/G1 (sub-G1) phase.

Fig. 4. TQ alters mitochondrial membrane potential and activates caspase pathway

(A) MCF-7/DOX cells were incubated with 100 µM TQ for 48 hours and incubated with MitoCapture reagent at 37°C. In healthy cells, MitoCapture accumulates and aggregates in mitochondria, giving off a bright red fluorescence; in apoptotic cells, MitoCapture does not aggregate in mitochondria and thus remains in its monomeric form, generating green fluorescence. (B) MCF-7/Dox cells were pre-treated with Z-VAD-FMK and then incubated with 100 µM TQ for the indicated time points. DNA fragmentation was analyzed using agarose gel electrophoresis. (C) MCF-7/Dox cells were treated with or without general caspase inhibitor z-VAD-FMK for 1 hour and then incubated with100 µM TQ for 24 or 48 hours. PARP, Caspase-3, -7, -8 and 9 were examined by Western blot analysis. (D) Cells were treated with 100 µM TQ for 24 or 48 hours. Cellular Bax and Bcl2 were analyzed by Western blotting and the Bcl2/Bax ratio was calculated. Values represent mean ±SE of n=3. *: Denotes significance at p<0.05

Fig. 5. TQ induces DNA damage and G2/M arrest in MCF-7/DOX cells

Cells were treated with TQ or its vehicle for 24 hours. (A) Cell cycle distribution was analyzed using flow cytometry; (B) cellular levels of Cyclin B1, Cdc25C and p21 were determined by Western blot analysis. (C) γH2AX foci formation in TQ-treated cells compared to control untreated cells. Slides were immuno-stained with γH2AX antibody and nuclei were counter stained with DAPI (blue fluorescence) and examined by fluorescence microscopy. (D) γH2AX level was determined using Western blot analysis.

Fig. 6. TQ up-regulates PTEN in MCF-7/DOX cells

Cells were treated with 50 µM TQ or its vehicle for different time periods. (A) PTEN protein expression was determined by Western blot analysis. (B) Western blot analysis of the downstream targets of PTEN, p-Akt, p53 and Fas L in the100 µM TQ treatment and untreated cells. (C) Total RNA was extracted from the cells after TQ treatment, and, the PTEN mRNA was detected by real-time RT–PCR assay using gene-specific primers as described in ‘Materials and methods’. The levels of mRNA transcripts were expressed in relative (fold) to DMSO-treated unirradiated cells as control.

Fig. 7. Silencing PTEN by siRNA hinders the ability of TQ to induce apoptosis in MCF-7/DOX cells

Cells were transfected with 50 nM PTEN siRNA for 24 hours and then treated with TQ for 48 hours. (A) Cellular levels of PTEN and PARP proteins were determined by Western blotting. (B) Cell survival after siRNA-mediated PTEN Silencing and TQ treatment was determined using MTT assay. Values represent mean ±SE of n=3. *: Denotes significance at p<0.05