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. 2012 May 1;83(9):1229-40.
doi: 10.1016/j.bcp.2012.01.027. Epub 2012 Feb 1.

Thiostrepton Is an Inducer of Oxidative and Proteotoxic Stress That Impairs Viability of Human Melanoma Cells but Not Primary Melanocytes

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Free PMC article

Thiostrepton Is an Inducer of Oxidative and Proteotoxic Stress That Impairs Viability of Human Melanoma Cells but Not Primary Melanocytes

Shuxi Qiao et al. Biochem Pharmacol. .
Free PMC article

Abstract

Pharmacological induction of oxidative and proteotoxic stress has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Guided by a differential phenotypic drug screen for novel lead compounds that selectively induce melanoma cell apoptosis without compromising viability of primary human melanocytes, we have focused on the cyclic pyridinyl-polythiazolyl peptide-antimicrobial thiostrepton. Using comparative gene expression-array analysis, the early cellular stress response induced by thiostrepton was examined in human A375 metastatic melanoma cells and primary melanocytes. Thiostrepton displayed selective antimelanoma activity causing early induction of proteotoxic stress with massive upregulation of heat shock (HSPA6, HSPA1A, DNAJB4, HSPB1, HSPH1, HSPA1L, CRYAB, HSPA5, DNAJA1), oxidative stress (HMOX1, GSR, SOD1), and ER stress response (DDIT3) gene expression, confirmed by immunodetection (Hsp70, Hsp70B', HO-1, phospho-eIF2α). Moreover, upregulation of p53, proapoptotic modulation of Bcl-2 family members (Bax, Noxa, Mcl-1, Bcl-2), and induction of apoptotic cell death were observed. Thiostrepton rapidly induced cellular oxidative stress followed by inactivation of chymotrypsin-like proteasomal activity and melanoma cell-directed accumulation of ubiquitinated proteins, not observed in melanocytes that were resistant to thiostrepton-induced apoptosis. Proteotoxic and apoptogenic effects were fully antagonized by antioxidant intervention. In RPMI 8226 multiple myeloma cells, known to be exquisitely sensitive to proteasome inhibition, early proteotoxic and apoptogenic effects of thiostrepton were confirmed by array analysis indicating pronounced upregulation of heat shock response gene expression. Our findings demonstrate that thiostrepton displays dual activity as a selective prooxidant and proteotoxic chemotherapeutic, suggesting feasibility of experimental intervention targeting metastatic melanoma and other malignancies including multiple myeloma.

Figures

Figure 1
Figure 1. Thiostrepton induces apoptotic death in human metastatic A375 and G361 melanoma cells but not in primary epidermal melanocytes
(A–B) Induction of A375 and human epidermal melanocyte (HEMa) cell death in reponse to thiostrepton exposure (T; 1, 5, 10 μM, 24 h) performed in the absence or presence of zVADfmk (40μM) as assessed by flow cytometric analysis. The numbers (panel A) indicate viable (AV-, PI-) in percent of total gated cells (mean ± SD, n=3). The bar graph (panel B) represents data obtained from three independent repeats involving metastatic melanoma cell lines (A375, G361) and primary melanocytes (HEMa). (C) T-induced (10 μM, 0–24 h) caspase-3 activation was examined in A375 cells by flow cytometric detection using an Alexa488-conjugated monoclonal antibody directed against proteolytically activated caspase 3. A representative experiment taken from three similar repeats is shown. (D) T-induced (10 μM, 0–12 h) caspase 9 activation was examined in A375 cells using a luminescent assay (based on conversion of a luminogenic caspase 9 substrate, Caspase-Glo 9 Assay; mean ± SD, n=3). (E) Time course analysis (0–18 h continuous exposure, 10 μM T) of cell death performed in A375 cells. A representative experiment taken from three similar repeats is shown. (F) A375 cells [control (panel I) and T-treated (10 μM, 12 and 24h; panels II and III, respectively)] were examined by transmission electron microscopy (direct magnification: 8,800 ×; N, nucleus; PM, plasma membrane).
Figure 2
Figure 2. Comparative gene expression array analysis of thiostrepton-treated human A375 metastatic melanoma and primary melanocytes
(A–B) The scatter blots depict differential gene expression as detected by the RT2 Human Stress and Toxicity Profiler PCR Expression Array technology [T: 10 μM, 6 h) in A375 melanoma cells (panel A) and melanocytes (panel B). Upper and lower lines: cut-off indicating fourfold up- or down-regulated expression, respectively. Arrays were performed in three independent repeats and analyzed using the two-sided Student’s t test. (C) The table summarize expression changes by at least twofold (p < 0.05). (D) T-modulation (10 μM, 1–6 h) of HSPA1A and HSPA6 mRNA levels in A375 cells by independent real time RT-PCR analysis. (E) T-modulation (10 μM, 6 h) of Hsp70B′ protein levels in A375 cells as assessed by ELISA and analyzed using the Student’s t test. (F) T-modulation (10 μM, 1–6 h) of protein expression in A375 cells as assessed by immunoblot analysis. (G) Comparative analysis of heat shock- and ER stress-related gene expression changes at the mRNA level induced by T-treatment in A375 cells and melanocytes (as observed by expression array analysis performed in panels A and B); [n=3, mean ± SD; (p<0.05; T-treated versus untreated); * denotes statistically significant differences between HEMa and A375 cells (*p<0.05; **p<0.01; ***p<0.001)]. (H) T-modulation (10 μM, 6 h) of protein expression in melanocytes as assessed by immunoblot analysis.
Figure 2
Figure 2. Comparative gene expression array analysis of thiostrepton-treated human A375 metastatic melanoma and primary melanocytes
(A–B) The scatter blots depict differential gene expression as detected by the RT2 Human Stress and Toxicity Profiler PCR Expression Array technology [T: 10 μM, 6 h) in A375 melanoma cells (panel A) and melanocytes (panel B). Upper and lower lines: cut-off indicating fourfold up- or down-regulated expression, respectively. Arrays were performed in three independent repeats and analyzed using the two-sided Student’s t test. (C) The table summarize expression changes by at least twofold (p < 0.05). (D) T-modulation (10 μM, 1–6 h) of HSPA1A and HSPA6 mRNA levels in A375 cells by independent real time RT-PCR analysis. (E) T-modulation (10 μM, 6 h) of Hsp70B′ protein levels in A375 cells as assessed by ELISA and analyzed using the Student’s t test. (F) T-modulation (10 μM, 1–6 h) of protein expression in A375 cells as assessed by immunoblot analysis. (G) Comparative analysis of heat shock- and ER stress-related gene expression changes at the mRNA level induced by T-treatment in A375 cells and melanocytes (as observed by expression array analysis performed in panels A and B); [n=3, mean ± SD; (p<0.05; T-treated versus untreated); * denotes statistically significant differences between HEMa and A375 cells (*p<0.05; **p<0.01; ***p<0.001)]. (H) T-modulation (10 μM, 6 h) of protein expression in melanocytes as assessed by immunoblot analysis.
Figure 2
Figure 2. Comparative gene expression array analysis of thiostrepton-treated human A375 metastatic melanoma and primary melanocytes
(A–B) The scatter blots depict differential gene expression as detected by the RT2 Human Stress and Toxicity Profiler PCR Expression Array technology [T: 10 μM, 6 h) in A375 melanoma cells (panel A) and melanocytes (panel B). Upper and lower lines: cut-off indicating fourfold up- or down-regulated expression, respectively. Arrays were performed in three independent repeats and analyzed using the two-sided Student’s t test. (C) The table summarize expression changes by at least twofold (p < 0.05). (D) T-modulation (10 μM, 1–6 h) of HSPA1A and HSPA6 mRNA levels in A375 cells by independent real time RT-PCR analysis. (E) T-modulation (10 μM, 6 h) of Hsp70B′ protein levels in A375 cells as assessed by ELISA and analyzed using the Student’s t test. (F) T-modulation (10 μM, 1–6 h) of protein expression in A375 cells as assessed by immunoblot analysis. (G) Comparative analysis of heat shock- and ER stress-related gene expression changes at the mRNA level induced by T-treatment in A375 cells and melanocytes (as observed by expression array analysis performed in panels A and B); [n=3, mean ± SD; (p<0.05; T-treated versus untreated); * denotes statistically significant differences between HEMa and A375 cells (*p<0.05; **p<0.01; ***p<0.001)]. (H) T-modulation (10 μM, 6 h) of protein expression in melanocytes as assessed by immunoblot analysis.
Figure 2
Figure 2. Comparative gene expression array analysis of thiostrepton-treated human A375 metastatic melanoma and primary melanocytes
(A–B) The scatter blots depict differential gene expression as detected by the RT2 Human Stress and Toxicity Profiler PCR Expression Array technology [T: 10 μM, 6 h) in A375 melanoma cells (panel A) and melanocytes (panel B). Upper and lower lines: cut-off indicating fourfold up- or down-regulated expression, respectively. Arrays were performed in three independent repeats and analyzed using the two-sided Student’s t test. (C) The table summarize expression changes by at least twofold (p < 0.05). (D) T-modulation (10 μM, 1–6 h) of HSPA1A and HSPA6 mRNA levels in A375 cells by independent real time RT-PCR analysis. (E) T-modulation (10 μM, 6 h) of Hsp70B′ protein levels in A375 cells as assessed by ELISA and analyzed using the Student’s t test. (F) T-modulation (10 μM, 1–6 h) of protein expression in A375 cells as assessed by immunoblot analysis. (G) Comparative analysis of heat shock- and ER stress-related gene expression changes at the mRNA level induced by T-treatment in A375 cells and melanocytes (as observed by expression array analysis performed in panels A and B); [n=3, mean ± SD; (p<0.05; T-treated versus untreated); * denotes statistically significant differences between HEMa and A375 cells (*p<0.05; **p<0.01; ***p<0.001)]. (H) T-modulation (10 μM, 6 h) of protein expression in melanocytes as assessed by immunoblot analysis.
Figure 3
Figure 3. Thiostrepton inhibits proteasome activity in A375 and HEMa cells, but causes accumulation of ubiquitinated proteins only in A375 cells
(A–D) A375 (panels A–B) and HEMa cells (panels C–D) were exposed to T (10 μM, up to 6 h). Cells were then harvested and processed for immunoblot analysis of protein ubiquitination (panels A and C) or luminescent analysis of proteasomal enzymatic activity (panels B and D). Treatment with MG132 (10 μM, 6 h; panels C and D) served as a positive control. Bar graphs represent data obtained from three independent repeats. (E) After MG132 exposure (10 μM) of A375 cells, immunoblot analysis of p-eIF2α and ubiquitination status was performed (6 h), and cell viability was determined (24 h exposure) by flow cytometric analysis. The numbers indicate viable (AV-, PI-) in percent of total gated cells. A representative experiment taken from three similar repeats is shown.
Figure 4
Figure 4. Antioxidant treatment antagonizes thiostrepton-induced proteasome inhibition and cytotoxicity in A375 melanoma cells
(A) Induction of cellular oxidative stress as assessed by flow cytometric determination of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) oxidation in response to T-exposure (10 μM, up to 6 h). Histograms depict one representative experiment out of three similar repeats. (B) Time course analysis of T-modulation of intracellular reduced glutathione content in A375 cells (10 μM, up to 6h) normalized to cell number (mean ± SD, n=3). (C) After preincubation (24 h) with BSO (1 mM) or NAC (10 mM), T-induced (1 and 10 μM, 24 h) A375 cell death was assessed by flow cytometric analysis of AV/PI-stained cells (mean ± SD, n=3). (D) T-induced inhibition (1–10 μM, 6h) of chymotrypsin-like proteasomal activity was examined with or without antioxidant treatment (NAC, 10 mM).
Figure 5
Figure 5. Thiostrepton inhibits proteasome activity with induction of proteotoxic stress and apoptosis in human RPMI 8226 multiple myeloma cells
(A) Induction of cell death by exposure to T (0.5 to 10 μM, 24 h) in the absence or presence of zVADfmk (40μM) or NAC (10 mM) was assessed by flow cytometric analysis. The numbers indicate viable (AV-, PI-) in percent of total gated cells. A representative experiment taken from three similar repeats is shown. (B) Gene expression array analysis of T-treated (10 μM, 6 h) RPMI 8226 cells. The scatter blot (left panel) depicts differential gene expression as detected by the RT2 Human Stress and Toxicity Profiler PCR Expression Array technology. Upper and lower lines: cut-off indicating fourfold up-or down-regulated expression, respectively. Arrays were performed in three independent repeats and analyzed using the two-sided Student’s t test. The table (right panel) summarizes expression changes by at least four-fold (p < 0.05). (C) T-modulation (10 μM, 6 h) of HMOX1, HSPA1A, and HSPA6 mRNA levels as assessed by independent real time RT-PCR analysis (mean ± SD, n=3). (D) Immunoblot analysis examining T-modulation of proteotoxic stress markers and ubiquitination (10 μM, up to 6 h). (E) Inhibition of proteasomal enzymatic activity [up to 10 μM T, 6 h; upper panel: chymotrypsin-like versus trypsin-like activity; lower panel: chymotrypsin-like activity, dose-response relationship; mean ± SD; n = 3; * denotes statistically significant differences (*p<0.05; **p<0.01; ***p<0.001)].
Figure 5
Figure 5. Thiostrepton inhibits proteasome activity with induction of proteotoxic stress and apoptosis in human RPMI 8226 multiple myeloma cells
(A) Induction of cell death by exposure to T (0.5 to 10 μM, 24 h) in the absence or presence of zVADfmk (40μM) or NAC (10 mM) was assessed by flow cytometric analysis. The numbers indicate viable (AV-, PI-) in percent of total gated cells. A representative experiment taken from three similar repeats is shown. (B) Gene expression array analysis of T-treated (10 μM, 6 h) RPMI 8226 cells. The scatter blot (left panel) depicts differential gene expression as detected by the RT2 Human Stress and Toxicity Profiler PCR Expression Array technology. Upper and lower lines: cut-off indicating fourfold up-or down-regulated expression, respectively. Arrays were performed in three independent repeats and analyzed using the two-sided Student’s t test. The table (right panel) summarizes expression changes by at least four-fold (p < 0.05). (C) T-modulation (10 μM, 6 h) of HMOX1, HSPA1A, and HSPA6 mRNA levels as assessed by independent real time RT-PCR analysis (mean ± SD, n=3). (D) Immunoblot analysis examining T-modulation of proteotoxic stress markers and ubiquitination (10 μM, up to 6 h). (E) Inhibition of proteasomal enzymatic activity [up to 10 μM T, 6 h; upper panel: chymotrypsin-like versus trypsin-like activity; lower panel: chymotrypsin-like activity, dose-response relationship; mean ± SD; n = 3; * denotes statistically significant differences (*p<0.05; **p<0.01; ***p<0.001)].

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