ONCOLOGY REPORTS 32: 821-828, 2014
Thymoquinone induces apoptosis in human colon cancer HCT116 cells through inactivation of STAT3 by blocking JAK2- and Src‑mediated phosphorylation of EGF receptor tyrosine kinase JUTHIKA KUNDU1, BU YOUNG CHOI2, CHUL-HO JEONG1, JOYDEB KUMAR KUNDU1 and KYUNG-SOO CHUN1 1
College of Pharmacy, Keimyung University, Dalseo-Gu, Daegu 704-701; 2Department of Pharmaceutical Science and Engineering, Seowon University, Cheongju, Chungbuk 361-7472, Republic of Korea Received February 13, 2014; Accepted April 22, 2014 DOI: 10.3892/or.2014.3223
Abstract. Thymoquinone (TQ), a compound isolated from black seed oil (Nigella sativa), has been reported to possess anti-inflammatory and anticancer activities. However, the molecular mechanisms underlying the anticancer effects of TQ remain poorly understood. In the present study, we found that TQ significantly reduced the viability of human colon cancer HCT116 cells in a concentration- and time-dependent manner. Treatment of cells with TQ induced apoptosis, which was associated with the upregulation of Bax and inhibition of Bcl-2 and Bcl-xl expression. TQ also activated caspase-9,-7, and -3, and induced the cleavage of poly-(ADP-ribose) polymerase (PARP). Pretreatment with a pan-caspase inhibitor, z-VAD-fmk, abrogated TQ-induced apoptosis by blocking the cleavage of caspase-3 and PARP. Treatment of cells with TQ also diminished the constitutive phosphorylation, nuclear localization and the reporter gene activity of signal transducer and activator of transcription-3 (STAT3). TQ attenuated the expression of STAT3 target gene products, such as survivin, c-Myc, and cyclin-D1, -D2, and enhanced the expression of cell cycle inhibitory proteins p27 and p21. Treatment with TQ attenuated the phosphorylation of upstream kinases, such as Janus-activated kinase-2 (JAK2), Src kinase and epidermal growth factor receptor (EGFR) tyrosine kinase. Pharmacological inhibition of JAK2 and Src blunted tyrosine phosphorylation of EGFR and STAT3, while treatment with an EGFR tyrosine kinase inhibitor gefitinib inhibited phosphorylation of STAT3 without affecting that of JAK2 and Src in HCT116 cells. Collectively, our study revealed that TQ induced apoptosis in HCT116 cells by blocking STAT3 signaling via
Correspondence to: Professor Kyung-Soo Chun, College of
Pharmacy, Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu 704-701, Republic of Korea E-mail: [email protected]
Key words: thymoquinone, HCT116 cells, apoptosis, STAT3, EGFR
inhibition of JAK2- and Src-mediated phosphorylation of EGFR tyrosine kinase. Introduction Colorectal cancer is one of the leading causes of mortality and ranks among the three most common cancers in developed countries (1,2). More than one million new cases of colorectal cancer are diagnosed every year (3). Multiple lines of evidence suggest that bioactive compounds present in various edible and medicinal plants can prevent colon carcinogenesis (4). Thymoquinone (TQ; Fig. 1A), a dietary phytochemical, is the major bioactive constituent present in black seed oil (Nigella sativa), which is widely consumed as a condiment and has long been used in Ayurvedic medicine. TQ has been reported to exert antioxidative, anti-inflammatory and anticancer effects (5-7). Several studies have reported that TQ inhibits cell proliferation, induces apoptosis and impedes the in vivo growth of xenograft tumors of various human cancer cells including those of the stomach (8,9), colorectal (10,11), lungs (12), breast (5) and prostate (13). In a recent study, TQ was shown to inhibit 1,2-dimethylhydrazine-induced initiation and promotion of colon carcinogenesis in Wister rats (14). However, the molecular basis of the anticancer effects of TQ in colon cancer has yet to be fully elucidated. Evasion from apoptosis is one of the hallmarks of cancer (15). Tumor cells bypass the apoptotic process following two biochemical pathways (16). The intrinsic or mitochondriamediated pathway of apoptosis involves the depolarization of mitochondrial membrane, release of cytochrome c, sequential activation of caspase-9, -7 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP). The extrinsic pathway, on the other hand, is mediated through the activation of cell membrane-bound death receptors, followed by the activation of pro-caspase-8, which then execute cell death by triggering the activity of caspase-3 (16,17). According to the intrinsic mechanism of apoptosis induction, the mitochondrial membrane potential is largely regulated by relative abundance of different B-cell lymphoma (Bcl) family proteins. Whereas localization of Bcl-2 and Bcl-xl proteins stabilizes mitochondrial membrane integrity, downregulation of Bcl-2 and concomitant over
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KUNDU et al: THYMOQUINONE INDUCES APOPTOSIS IN HCT116 CELLS
expression and subsequent translocation of proapoptotic protein Bax to the mitochondria lead to mitochondrial membrane depolarization and release of cytochrome c, which triggers the activation caspase cascade, thereby inducing cell death (16,17). Bcl-2 and Bcl-xl are oncoproteins that are frequently overexpressed in many cancers (16). One of the transcriptional regulators of Bcl-2 family proteins is signal transducer and activator of transcription-3 (STAT3), which is a latent transcription factor that normally resides in the cytoplasm (18). In response to diverse growth stimulatory signals, STAT3 gets activated through phosphorylation by upstream kinases, such as Janus-activated kinase-2 (JAK2) (19) and Src tyrosine kinase (20) followed by STAT3 dimerization and nuclear localization. The oncogenic signal transduction pathway mediated through phosphorylation of epidermal growth factor receptor (EGFR) tyrosine kinases also transmits activating signals to STAT3 (21,22). While transient activation of STAT3 is associated with the growth and development of various organs, constitutive activation of STAT3 has been implicated in carcinogenesis (23). The activation of STAT3 not only assists tumor cells to evade apoptosis but also promotes cell proliferation by transactivating the genes encoding various cell survival proteins, such as cyclins, c-Myc and survivin (24,25). The blockade of inappropriate activation of STAT3 signaling inhibits cell proliferation and induces apoptosis. Thus, STAT3 appears as a molecular target of many anticancer agents. Herein, we report that TQ induces apoptosis in HCT116 cells through the inhibition of STAT3 signaling pathway by blocking the phosphorylation of EGFR tyrosine kinase via modulation of JAK2 and Src kinases. Materials and methods Materials. TQ (purity 99%), z-VAD-fmk and β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). AG490 and PP2 were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Gefitinib was purchased from LC Laboratories (Woburn, MA, USA) and antibodies against cleaved caspase-9, -3, and -7, PARP, Bcl-2, Bcl-xl, Bax, STAT3, p-STAT3 (Y705), JAK2, p-JAK2, Src, p-Src, EGFR, p-EGFR (Y1173), cyclin-D1, -D2, and survivin were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). Primary antibodies against each of c-Myc, p27, p21, lamin A and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were of analytical or highest purity grade available. Cell culture and treatment. HCT116 cells were obtained from American Type Culture Collection (ATCC) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) at 37˚C in a humidified incubator containing 5% CO2 and 95% air. In all the experiments, cells were seeded at 2x105 cells/ml and treated with TQ at 50-60% confluence. All chemicals were dissolved in ethanol. Cell proliferation assay. The anti-proliferative effect of TQ against HCT116 cells was measured by using a solution
of tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) (Promega, Madison, WI, USA). Briefly, cells (2x103) were incubated in triplicate in a 96-well plate in the presence or absence of TQ in a final volume of 0.1 ml for different time intervals at 37˚C. Thereafter, 20 µl of MTS solution was added to each well and incubated for 60 min. The number of viable cells was measured in a 96-well plate at an optical density of 492 nm on a microplate reader (Tecan Trading AG, Männedorf, Switzerland). Cell viability was described as the relative percentage of control. Annexin V staining. Annexin V staining was performed using FITC-Annexin V staining kit (BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. Briefly, TQ-treated cells were washed with PBS and resuspended in binding buffer containing Annexin V and propidium iodide (PI). Fluorescence intensity was measured using flow cyto metry (BD Biosciences). Western blot analysis. Cells were harvested and lysed with RIPA buffer to obtain whole cell lysate. Collected protein samples were quantified by using bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The cytosolic and nuclear extracts were prepared by using NE-PER Nuclear and Cytoplasmic Extraction Reagent kit (Thermo Scientific, Rockford, IL, USA) according to the procedure described previously (6). Nuclear proteins were collected and stored at -70˚C after determination of protein concentration by using Bradford Reagent (Bio-Rad Laboratories, Hercules, CA, USA). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis was performed according to the protocol described previously (6). Immunoblot membranes were incubated with SuperSignal Pico Chemiluminescent substrate or Dura Luminol substrate (Thermo Scientific), according to the manufacturer's instructions, and visualized with ImageQuant™ LAS 4000 (Fujifilm Life Science, Tokyo, Japan). Caspase-3 activity assay. The activity of caspase-3 was detected using the Caspase-3 Colorimetric Activity Assay kit (Millipore, Billerica, MA, USA). The assay was performed in 96-well plates by incubating cell lysates (50 µg) in 100 µl reaction buffer containing caspase-3 substrate Ac-DEVD-pNA at 37˚C for 2 h and 30 min according to the manufacturer's protocol. STAT3-Luciferase reporter gene assay. Cells were seeded into 12-well plates at a density of 5x104 cells/well prior to transfection. Cells were transfected with p-STAT3-TA-luc (Clontech, Palo Alto, CA, USA) or control vector using Genefectin transfection reagent (Genetrone Biotech, Seoul, Korea). After 24 h of transfection, cells were treated with TQ for an additional 24 h and cell lysis was carried out with 1X reporter lysis buffer. After mixing the cell lysates with luciferase substrate (Promega), the luciferase activity was measured by using luminometer (Tecan Trading AG). The β-galactosidase assay was carried out according to the supplier's instructions (Promega Enzyme Assay System) for normalizing the luciferase activity and the results were expressed as fold transactivation.
ONCOLOGY REPORTS 32: 821-828, 2014
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Figure 1. Cytotoxic effect of thymoquinone (TQ) in HCT116 cells. (A) Chemical structure of TQ. (B) HCT116 cells were treated with the indicated concentrations of TQ for 24, 48 or 72 h. Cell viability was determined by the MTS assay. Values are expressed as means ± SD. *p
Thymoquinone induces apoptosis in human colon cancer HCT116 cells through inactivation of STAT3 by blocking JAK2- and Src‑mediated phosphorylation of EGF receptor tyrosine kinase JUTHIKA KUNDU1, BU YOUNG CHOI2, CHUL-HO JEONG1, JOYDEB KUMAR KUNDU1 and KYUNG-SOO CHUN1 1
College of Pharmacy, Keimyung University, Dalseo-Gu, Daegu 704-701; 2Department of Pharmaceutical Science and Engineering, Seowon University, Cheongju, Chungbuk 361-7472, Republic of Korea Received February 13, 2014; Accepted April 22, 2014 DOI: 10.3892/or.2014.3223
Abstract. Thymoquinone (TQ), a compound isolated from black seed oil (Nigella sativa), has been reported to possess anti-inflammatory and anticancer activities. However, the molecular mechanisms underlying the anticancer effects of TQ remain poorly understood. In the present study, we found that TQ significantly reduced the viability of human colon cancer HCT116 cells in a concentration- and time-dependent manner. Treatment of cells with TQ induced apoptosis, which was associated with the upregulation of Bax and inhibition of Bcl-2 and Bcl-xl expression. TQ also activated caspase-9,-7, and -3, and induced the cleavage of poly-(ADP-ribose) polymerase (PARP). Pretreatment with a pan-caspase inhibitor, z-VAD-fmk, abrogated TQ-induced apoptosis by blocking the cleavage of caspase-3 and PARP. Treatment of cells with TQ also diminished the constitutive phosphorylation, nuclear localization and the reporter gene activity of signal transducer and activator of transcription-3 (STAT3). TQ attenuated the expression of STAT3 target gene products, such as survivin, c-Myc, and cyclin-D1, -D2, and enhanced the expression of cell cycle inhibitory proteins p27 and p21. Treatment with TQ attenuated the phosphorylation of upstream kinases, such as Janus-activated kinase-2 (JAK2), Src kinase and epidermal growth factor receptor (EGFR) tyrosine kinase. Pharmacological inhibition of JAK2 and Src blunted tyrosine phosphorylation of EGFR and STAT3, while treatment with an EGFR tyrosine kinase inhibitor gefitinib inhibited phosphorylation of STAT3 without affecting that of JAK2 and Src in HCT116 cells. Collectively, our study revealed that TQ induced apoptosis in HCT116 cells by blocking STAT3 signaling via
Correspondence to: Professor Kyung-Soo Chun, College of
Pharmacy, Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu 704-701, Republic of Korea E-mail: [email protected]
Key words: thymoquinone, HCT116 cells, apoptosis, STAT3, EGFR
inhibition of JAK2- and Src-mediated phosphorylation of EGFR tyrosine kinase. Introduction Colorectal cancer is one of the leading causes of mortality and ranks among the three most common cancers in developed countries (1,2). More than one million new cases of colorectal cancer are diagnosed every year (3). Multiple lines of evidence suggest that bioactive compounds present in various edible and medicinal plants can prevent colon carcinogenesis (4). Thymoquinone (TQ; Fig. 1A), a dietary phytochemical, is the major bioactive constituent present in black seed oil (Nigella sativa), which is widely consumed as a condiment and has long been used in Ayurvedic medicine. TQ has been reported to exert antioxidative, anti-inflammatory and anticancer effects (5-7). Several studies have reported that TQ inhibits cell proliferation, induces apoptosis and impedes the in vivo growth of xenograft tumors of various human cancer cells including those of the stomach (8,9), colorectal (10,11), lungs (12), breast (5) and prostate (13). In a recent study, TQ was shown to inhibit 1,2-dimethylhydrazine-induced initiation and promotion of colon carcinogenesis in Wister rats (14). However, the molecular basis of the anticancer effects of TQ in colon cancer has yet to be fully elucidated. Evasion from apoptosis is one of the hallmarks of cancer (15). Tumor cells bypass the apoptotic process following two biochemical pathways (16). The intrinsic or mitochondriamediated pathway of apoptosis involves the depolarization of mitochondrial membrane, release of cytochrome c, sequential activation of caspase-9, -7 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP). The extrinsic pathway, on the other hand, is mediated through the activation of cell membrane-bound death receptors, followed by the activation of pro-caspase-8, which then execute cell death by triggering the activity of caspase-3 (16,17). According to the intrinsic mechanism of apoptosis induction, the mitochondrial membrane potential is largely regulated by relative abundance of different B-cell lymphoma (Bcl) family proteins. Whereas localization of Bcl-2 and Bcl-xl proteins stabilizes mitochondrial membrane integrity, downregulation of Bcl-2 and concomitant over
822
KUNDU et al: THYMOQUINONE INDUCES APOPTOSIS IN HCT116 CELLS
expression and subsequent translocation of proapoptotic protein Bax to the mitochondria lead to mitochondrial membrane depolarization and release of cytochrome c, which triggers the activation caspase cascade, thereby inducing cell death (16,17). Bcl-2 and Bcl-xl are oncoproteins that are frequently overexpressed in many cancers (16). One of the transcriptional regulators of Bcl-2 family proteins is signal transducer and activator of transcription-3 (STAT3), which is a latent transcription factor that normally resides in the cytoplasm (18). In response to diverse growth stimulatory signals, STAT3 gets activated through phosphorylation by upstream kinases, such as Janus-activated kinase-2 (JAK2) (19) and Src tyrosine kinase (20) followed by STAT3 dimerization and nuclear localization. The oncogenic signal transduction pathway mediated through phosphorylation of epidermal growth factor receptor (EGFR) tyrosine kinases also transmits activating signals to STAT3 (21,22). While transient activation of STAT3 is associated with the growth and development of various organs, constitutive activation of STAT3 has been implicated in carcinogenesis (23). The activation of STAT3 not only assists tumor cells to evade apoptosis but also promotes cell proliferation by transactivating the genes encoding various cell survival proteins, such as cyclins, c-Myc and survivin (24,25). The blockade of inappropriate activation of STAT3 signaling inhibits cell proliferation and induces apoptosis. Thus, STAT3 appears as a molecular target of many anticancer agents. Herein, we report that TQ induces apoptosis in HCT116 cells through the inhibition of STAT3 signaling pathway by blocking the phosphorylation of EGFR tyrosine kinase via modulation of JAK2 and Src kinases. Materials and methods Materials. TQ (purity 99%), z-VAD-fmk and β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). AG490 and PP2 were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Gefitinib was purchased from LC Laboratories (Woburn, MA, USA) and antibodies against cleaved caspase-9, -3, and -7, PARP, Bcl-2, Bcl-xl, Bax, STAT3, p-STAT3 (Y705), JAK2, p-JAK2, Src, p-Src, EGFR, p-EGFR (Y1173), cyclin-D1, -D2, and survivin were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). Primary antibodies against each of c-Myc, p27, p21, lamin A and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were of analytical or highest purity grade available. Cell culture and treatment. HCT116 cells were obtained from American Type Culture Collection (ATCC) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) at 37˚C in a humidified incubator containing 5% CO2 and 95% air. In all the experiments, cells were seeded at 2x105 cells/ml and treated with TQ at 50-60% confluence. All chemicals were dissolved in ethanol. Cell proliferation assay. The anti-proliferative effect of TQ against HCT116 cells was measured by using a solution
of tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) (Promega, Madison, WI, USA). Briefly, cells (2x103) were incubated in triplicate in a 96-well plate in the presence or absence of TQ in a final volume of 0.1 ml for different time intervals at 37˚C. Thereafter, 20 µl of MTS solution was added to each well and incubated for 60 min. The number of viable cells was measured in a 96-well plate at an optical density of 492 nm on a microplate reader (Tecan Trading AG, Männedorf, Switzerland). Cell viability was described as the relative percentage of control. Annexin V staining. Annexin V staining was performed using FITC-Annexin V staining kit (BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. Briefly, TQ-treated cells were washed with PBS and resuspended in binding buffer containing Annexin V and propidium iodide (PI). Fluorescence intensity was measured using flow cyto metry (BD Biosciences). Western blot analysis. Cells were harvested and lysed with RIPA buffer to obtain whole cell lysate. Collected protein samples were quantified by using bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The cytosolic and nuclear extracts were prepared by using NE-PER Nuclear and Cytoplasmic Extraction Reagent kit (Thermo Scientific, Rockford, IL, USA) according to the procedure described previously (6). Nuclear proteins were collected and stored at -70˚C after determination of protein concentration by using Bradford Reagent (Bio-Rad Laboratories, Hercules, CA, USA). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis was performed according to the protocol described previously (6). Immunoblot membranes were incubated with SuperSignal Pico Chemiluminescent substrate or Dura Luminol substrate (Thermo Scientific), according to the manufacturer's instructions, and visualized with ImageQuant™ LAS 4000 (Fujifilm Life Science, Tokyo, Japan). Caspase-3 activity assay. The activity of caspase-3 was detected using the Caspase-3 Colorimetric Activity Assay kit (Millipore, Billerica, MA, USA). The assay was performed in 96-well plates by incubating cell lysates (50 µg) in 100 µl reaction buffer containing caspase-3 substrate Ac-DEVD-pNA at 37˚C for 2 h and 30 min according to the manufacturer's protocol. STAT3-Luciferase reporter gene assay. Cells were seeded into 12-well plates at a density of 5x104 cells/well prior to transfection. Cells were transfected with p-STAT3-TA-luc (Clontech, Palo Alto, CA, USA) or control vector using Genefectin transfection reagent (Genetrone Biotech, Seoul, Korea). After 24 h of transfection, cells were treated with TQ for an additional 24 h and cell lysis was carried out with 1X reporter lysis buffer. After mixing the cell lysates with luciferase substrate (Promega), the luciferase activity was measured by using luminometer (Tecan Trading AG). The β-galactosidase assay was carried out according to the supplier's instructions (Promega Enzyme Assay System) for normalizing the luciferase activity and the results were expressed as fold transactivation.
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Figure 1. Cytotoxic effect of thymoquinone (TQ) in HCT116 cells. (A) Chemical structure of TQ. (B) HCT116 cells were treated with the indicated concentrations of TQ for 24, 48 or 72 h. Cell viability was determined by the MTS assay. Values are expressed as means ± SD. *p
