Thymoquinone inhibits proliferation, induces apoptosis and chemosensitizes human multiple myeloma cells through suppression of signal transducer and activator of transcription 3 activation pathway - PubMed (original) (raw)
Thymoquinone inhibits proliferation, induces apoptosis and chemosensitizes human multiple myeloma cells through suppression of signal transducer and activator of transcription 3 activation pathway
Feng Li et al. Br J Pharmacol. 2010 Oct.
Abstract
Background and purpose: Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) pathway is frequently encountered in several human cancers including multiple myeloma (MM). Thus, agents that suppress STAT3 phosphorylation have a potential for treatment of MM. In the present report, we investigated whether thymoquinone (TQ), the main component isolated from the medicinal plant Nigella sativa, modulated the STAT3 signalling pathway in MM cells.
Experimental approach: The effect of TQ on both constitutive and IL-6-induced STAT3 activation, associated protein kinases, STAT3-regulated gene products involved in proliferation, survival and angiogenesis, cellular proliferation and apoptosis in MM cells, was investigated.
Key results: We found that TQ inhibited both constitutive and IL-6-inducible STAT3 phosphorylation which correlated with the inhibition of c-Src and JAK2 activation. Vanadate reversed the TQ-induced down-regulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that TQ can induce the expression of Src homology-2 phosphatase 2 that correlated with suppression of STAT3 activation. TQ also down-regulated the expression of STAT3-regulated gene products, such as cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1 and vascular endothelial growth factor. Finally, TQ induced the accumulation of cells in sub-G1 phase, inhibited proliferation and induced apoptosis, as indicated by poly ADP ribose polymerase cleavage. TQ also significantly potentiated the apoptotic effects of thalidomide and bortezomib in MM cells.
Conclusions and implications: Our study has identified STAT3 signalling as a target of TQ and has thus raised its potential application in the prevention and treatment of MM and other cancers.
Figures
Figure 1
TQ inhibits constitutively active STAT3 in U266 cells. (A) The structure of TQ. (B) TQ suppressed phospho-STAT3 levels in a dose-dependent manner. U266 cells (2 × 106 mL−1) were treated with the indicated concentrations of TQ for 4 h, after which whole-cell extracts were prepared, and 30 µg of protein was resolved by 7.5% SDS–PAGE gel, electrotransferred onto nitrocellulose membranes and probed for phospho-STAT3. (C) TQ suppresses phospho-STAT3 levels in a time-dependent manner. U266 cells (2 × 106 mL−1) were treated with 15 µM TQ for the indicated times, after which Western blotting was performed as described for (B). The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. (D) TQ has no effect on phospho-STAT5 and STAT5 protein expression. U266 cells (2 × 106 mL−1) were treated with 15 µM TQ for the indicated times. Whole-cell extracts were prepared, fractionated on SDS–PAGE and examined by Western blotting using antibodies against phospho-STAT5 and STAT5. (E) TQ causes inhibition of translocation of STAT3 to the nucleus. U266 cells (1 × 105 mL−1) were incubated with or without 15 µM TQ for 4 h, and then analysed for the intracellular distribution of STAT3 by immunocytochemistry. The same slides were counterstained for nuclei with Hoechst 33342 (50 ng·mL−1) for 5 min.
Figure 2
TQ down-regulates IL-6-induced phospho-STAT3. (A) RPMI 8266 cells (2 × 106 mL−1) were treated with indicated concentrations of IL-6 for 15 min, whole-cell extracts were prepared and phospho-STAT3 was detected by Western blot. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. (B) RPMI 8266 cells (2 × 106 mL−1) were treated with IL-6 (10 ng·mL−1) for the indicated times, whole-cell extracts were prepared and phospho-STAT3 was detected by Western blot. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. (C) RPMI 8266 cells (2 × 106 mL−1) were treated with 15 µM TQ for the indicated times, and then stimulated with IL-6 (10 ng·mL−1) for 15 min. Whole-cell extracts were then prepared and analysed for phospho-STAT3 by Western blotting. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. The results shown are representative of three independent experiments. (D) RPMI 8266 cells (2 × 106 mL−1) were treated with 15 µM TQ for the indicated times, and then stimulated with IL-6 (10 ng·mL−1) for 15 min. Whole-cell extracts were then prepared and analysed for phospho-Akt by Western blotting. The same blots were stripped and reprobed with Akt antibody to verify equal protein loading. The results shown are representative of three independent experiments. (E) A293 cells (5 × 105 mL−1) were transfected with STAT3-luciferase (STAT3-Luc) plasmid, incubated for 24 h and treated with 5 and 15 µM TQ for 4 h and then stimulated with IL-6 (10 ng·mL−1) for 24 h. Whole-cell extracts were then prepared and analysed for luciferase activity. The results shown are representative of three independent experiments. *P < 0.05.
Figure 3
(A) TQ suppresses phospho-Src levels in a time-dependent manner. U266 cells (2 × 106 mL−1) were treated with 15 µM TQ, after which whole-cell extracts were prepared and 30 µg aliquots of those extracts were resolved by 10% SDS–PAGE, electrotransferred onto nitrocellulose membranes and probed for phospho-Src antibody. The same blots were stripped and reprobed with Src antibody to verify equal protein loading. (B) TQ suppresses phospho-JAK2 levels in a time-dependent manner. U266 cells (2 × 106 mL−1) were treated with 15 µM TQ for indicated time intervals, after which whole-cell extracts were prepared and 30 µg portions of those extracts were resolved by 10% SDS–PAGE, electrotransferred onto nitrocellulose membranes and probed for JAK2 antibody. The same blots were stripped and reprobed with JAK2 antibody to verify equal protein loading. (C) U266 cells (2 × 106 mL−1) were treated with 15 µM TQ for indicated time intervals, after which whole-cell extracts were prepared and 30 µg portions of those extracts were resolved by 10% SDS–PAGE, electrotransferred onto nitrocellulose membranes and probed for phospho-ERK1/2 antibody. The same blots were stripped and reprobed with ERK2 antibody to verify equal protein loading. (D) Pervanadate reverses the phospho-STAT3 inhibitory effect of TQ. U266 cells (2 × 106 mL−1) were treated with the indicated concentrations of pervanadate and 15 µM TQ for 4 h, after which whole-cell extracts were prepared and 30 µg portions of those extracts were resolved by 7.5% SDS–PAGE gel, electrotransferred onto nitrocellulose membranes and probed for phospho-STAT3 and STAT3. (E) TQ induces the expression of SH-PTP2 protein in a dose-dependent manner in U266 cells. U266 cells (2 × 106 mL−1) were treated with indicated concentrations of TQ for 4 h, after which whole-cell extracts were prepared and 30 µg portions of those extracts were resolved by 10% SDS–PAGE, electrotransferred onto nitrocellulose membranes and probed for SH-PTP2 antibody. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading.
Figure 4
TQ suppresses STAT3-regulated gene products involved in proliferation, survival and angiogenesis. U266 cells (2 × 106 mL−1) were treated with 15 µM TQ for indicated time intervals, after which whole-cell extracts were prepared and 30 µg portions of those extracts were resolved by 10% SDS–PAGE; membrane sliced according to molecular weight; and probed against cyclin D1, Bcl-2, Bcl-XL, survivin, Mcl-1 and VEGF antibodies. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading.
Figure 5
TQ suppresses proliferation, causes accumulation of cells in sub-G1 phase and activates caspase-3. (A) U266 and RPMI 8226 cells (5 × 103 mL−1) were plated in triplicate; treated with indicated concentrations of TQ; and then subjected to MTT assay after 12, 24, 48 and 72 h to analyse proliferation of cells. Standard deviations between the triplicates are indicated. (B) U266 cells (2 × 106 mL−1) were synchronized by incubation overnight in the absence of serum, and then treated with 15 µM TQ in the presence of serum for the indicated times, after which the cells were washed, fixed, stained with PI and analysed for DNA content by flow cytometry. (C) U266 cells were treated with 15 µM TQ for the indicated times; whole-cell extracts were prepared, separated by SDS–PAGE and subjected to Western blotting against pro-caspase-3 antibody. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. (D) U266 cells were treated with 15 µM TQ for the indicated times, and whole-cell extracts were prepared, separated by SDS–PAGE and subjected to Western blot against PARP antibody. The same blot was stripped and reprobed with β-actin antibody to show equal protein loading. The results shown are representative of three independent experiments.
Figure 6
(A) Over-expression of constitutive STAT3 rescues A293 cells from TQ-induced apoptosis. First, A293 cells were transfected with constitutive STAT3 plasmid. After 24 h of transfection, the cells were treated with 15 µM TQ for 24 h, and then the apoptosis was determined by LIVE/DEAD assay and 20 random fields were counted. (B) TQ potentiates the apoptotic effect of thalidomide and bortezomib. U266 cells (1 × 106 mL−1) were treated with 5 µM TQ and 10 µg·mL−1 thalidomide or 20 nM bortezomib alone or in combination for 24 h at 37°C. The cells were stained with a LIVE/DEAD assay reagent for 30 min and then analysed under a fluorescence microscope. The % apoptosis has been plotted, and results shown are representative of three independent experiments. *P < 0.05. (C) Deletion of STAT3 inhibited the apoptotic effect of TQ. Wild-type and STAT3 deleted fibroblasts were treated with 15 µM TQ for 48 h and analysed for the percentage of apoptosis by LIVE/DEAD assay. The cells were stained with a LIVE/DEAD assay reagent for 30 min and then analysed under a fluorescence microscope.
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