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Tanshinone IIA inhibits constitutive STAT3 activation, suppresses proliferation, and induces apoptosis in rat C6 glioma cells
Neuroscience Letters 470 (2010) 126–129
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Tanshinone IIA inhibits constitutive STAT3 activation, suppresses proliferation, and induces apoptosis in rat C6 glioma cells Chao Tang a,∗ , Hong-li Xue a , Hai-bo Huang b , Xiao-gang Wang a a b
Department of Neurosurgery, The General Hospital of Shenyang Military Region, Shen Yang 110016, Liaoning Province, PR China Liaoning University of Traditional Chinese Medicine, Shen Yang 110016, Liaoning Province, PR China
a r t i c l e
i n f o
Article history: Received 11 October 2009 Received in revised form 18 December 2009 Accepted 21 December 2009 Keywords: Tanshinone IIA STAT3 Apoptosis Glioma
a b s t r a c t Signal transducer and activator of transcription 3 (STAT3) is usually constitutively activated in a variety of malignancies. Thus, STAT3 may be a promising target for treatment of tumor cells. Recently, Tanshinone IIA (Tan IIA), a major active constituent from the root of Salvia miltiorrhiza Bunge, was reported to have apoptosis inducing effects on a large variety of cancer cells. In this study, we evaluate the anti-proliferation and apoptosis inducing effects of Tan IIA on C6 glioma cells. Cell growth and proliferation were measured by MTT assay, cell apoptosis was observed by flow cytometry and DNA-fragmentation analysis. Further more, we investigated inhibitory effects of Tan IIA on STAT3 activity and its downstream targets: BclXL, cyclin D1. Alteration of STAT3 activity was examined by measuring their DNA binding activity and tyrosine phosphorylation. Changes in the expression levels of Bcl-XL and cyclin D1 were examined by Western blot analysis. We found that the cellular growth were inhibited and cell apoptosis were observed after the treatment with Tan IIA. The STAT3 activity was signifcantly reduced by Tan IIA parallel with a significant attenuation of expression of Bcl-XL and cyclin D1. These results suggest that Tan IIA may serve as an effective adjunctive reagent in the treatment of glioma for its targeting of constitutive STAT3 signaling. © 2009 Elsevier Ireland Ltd. All rights reserved.
Gliomas are the most common primary tumors of the brain that arise from astrocytes and their precursors, which are consided to be advanced invasive, metastatic, malignant. Constitutive activation of STAT3 has been demonstrated in a variety of diverse human tumor cell lines, which are involved in the development and progression of cancers [1,2]. STAT3 is also constitutively activated in gliomas cells. Inhibition of STAT3 function leads to growth inhibition of gliomas cells [8,12]. Numerous cancer research studies have been conducted using traditional medicinal plants in an effort to discover new therapeutic agents that lack the toxic side effects associated with current chemotherapeutic agents. Salvia miltiorrhiza Bung (Danshen) has been widely used to treat cardiovascular diseases in traditional Chinese medical practice, which is considered to have the property of activating blood circulation and removing stasis. The malignant tumor is viewed as being associated with stagnation of blood in traditional chinese medicine, therefore, Danshen has been also used in anti-cancer therapy [3,13]. As one of the most abundant extracts of Danshen, Tan IIA is known to have anti-inflammatory, anti-oxidative and cytotoxic
∗ Corresponding author. E-mail address: [email protected] (C. Tang). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.12.069
activities, neuron-protective effects [11,17,20,22]. In addition, Tan IIA has been shown to relieve oxidative stress and immune dysfunction associated with the onset and progress of cancer [19]. Studies have also demonstrated that Tan IIA have the ability to arrest the cell cycle of tumor cell lines that are resistant to multiple chemotherapeutic drugs and act as inhibitors of key steps necessary for the progression of tumor angiogenesis [10,21]. Recently, it has been shown that Tan II A can suppress the proliferation of human glioma cells [18]. However, the mechanisms underlying the therapeutic action of Tan II A are not well understood. There are no reports, on whether Tan IIA affects STAT3 pathway in glioma cells. In the present report, we set out to establish whether Tan IIA can also suppress constitutive activation of STAT3 in C6 glioma cells. Tan IIA was isolated from the roots of Salvia miltiorrhiza Bunge,which is based on the method described previously [7]. The purity of Tan IIA was proven by high performance liquid chromatography (HPLC) as >95%. Rat C6 glioma cells were obtained from the American Type Culture Collection. C6 glioma cells were cultured in Dulbecco’s modified Eagle’s medium (Grand Island, NY, USA) supplemented with 7.5% fetal calf serum, penicillin (10 units/ml) and streptomycin (10 mg/ml) (Sigma, St. Louis, MO, USA). Cultures were maintained in a humidified atmosphere of 5% CO2 at 37 ◦ C.
C. Tang et al. / Neuroscience Letters 470 (2010) 126–129
The cytotoxicity of Tan IIA was determined by the MTT assay. Cells were plated in 100 l of medium/well in 96-well plates. After incubation overnight, Tan IIA was added in various concentrations; After treatment with Tan IIA for 12–36 h, 20 l of 5 mg/ml MTT (pH 4.7) were added to each well and cultivated for another 4 h, the supernatant fluid was removed, 100 l/well DMSO were added .Absorbance at 570 nm was measured with a microplate reader (Bio-Rad, Richmond, CA, USA), using wells without cells as blanks. Date was expressed as the percent cytoviability (%cytoviability = A570 of treated cells/A570 of control cells × 100%). The stock solutions of Tan IIA were prepared in DMSO. The concentration of DMSO in experimental media was 0.1% (v/v). The effect of Tan IIA on the induction of apoptosis of C6 glioma cells was investigated by flow cytometry analysis. After Tan IIA treatment for 12 h,cells was washed with cold PBS and resuspended with binding buffer (10 m HEPES NaOH, pH7.4, 140 mM NaCl, 2.5 mM CaCl2 ). Cells were stained for 15 min at room temperature in the dark with annexin V-FITC and propidium iodide and then added to each tube and analyzed by a Becton Dickinson FACScan (excitation light, 488 nm) equipped with CELLQUEST software (Becton Dickinson). Cell apoptosis was further observed by DNA-fragmentation analysis. After incubation with Tan IIA for 24 h, the cells were washed with PBS and lysed in a solution containing 400 mM NaCl, 10 mM. Tris–HCl (pH 7.5), 1% sodium dodecyl sulfate, and 0.15 mg/ml of proteinase K. The lysate was incubated for 4 h at 37 ◦ C, followed by the addition of 5 M NaCl, to a final concentration of 1 M. After centrifugation at 7500 × g for 30 min, the supernatant was collected and DNA was precipitated with 70% isopropanol. After digestion for 1 h with ribonuclease A, these samples were electrophoresed in 2% agarose gel and stained with ethidium bromide. The DNA binding activity of dimeric-activated STAT molecules were measured by electrophoretic mobility shift assay (EMSA). Nuclear extracts were prepared as described previously [6] with Na3 VO4 (1 mM), and complete protease inhibitor. The reactions were performed in a total volume of 24 l in buffer consisting of 10 mM HEPES (N[2-hydroxeth yl]piperazine-N-[2-ethane-sulfonic acid]) (pH 7.8), 50 mM KCl, 1 mM EDTA, 5 mM MgCl2 , 10% glycerol, 5 mM dithiothreitol, 1 mg of bovine serum albumin per ml, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM Na3 VO4 with 1 g of poly (dI-dC) and 0.3 ng of 32 P-labelled high affinity sis-inducible element (hSIE). Following incubation for 15 min at room temperature, the reactions were electrophoresed on 4% native polyacrylamide gels. For supershift experiments, the appropriate antibody was added to the nuclear extract in buffer and incubated for 60 min at room temperature prior to the addition of the labelled-hSIE/dI-dC mixture. Changes in the expression levels of P-STAT3, Bcl-XL and cyclin D1 were examined by Western blot analysis. Whole cell extracts were mixed with 2× SDS sample buffer [125 mmol/l Tris–HCl (pH 6.8), 4% SDS, 20% glycerol, and 10% 2 mercaptoethanol] at 1:1 ratio and were heated for 5 min at 100 ◦ C. Proteins (50 g/lane) were separated by 12.5% SDS-PAGE and transferred onto a nitrocellulose membrane. Prestained molecular weight markers were included in each gel. Membranes were blocked for 30 min in TBST and 5% BSA. After blocking, membranes were incubated with primary antibodies (1:1000 dilution) according to the manufacturer’s instructions (Santa Cruz, CA, USA). After washing the membranes three times with TBST (5 min each), they were incubated with horseradish peroxidase-conjugated secondary antibody in TBST and 1% BSA for 30 min. The proteins were detected by the enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL, USA) using X-ray film.Statistical analysis data were expressed as mean ± SD. The statistical differences among groups were evaluated using one-way ANOVA with Fisher’s PLSD test. P < 0.05 was considered significant. Results were analyzed using SPSS13.0 software (Spss Inc., Chicago, TL, USA).
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Fig. 1. Effects of Tan IIA on the viability of C6 glioma cells. Cells were treated with the indicated concentrations of Tan IIA (×103 mug ml−1 ) for 12, 24, 36 h. The cell survival was determined using the MTT assay. Data are expressed as mean ± SD in triplicate. * P < 0.05 vs untreated group.
Results of the effect of Tan IIA on C6 glioma cells growth by MTT assay are shown in Fig. 1. Tan II A exhibited a statistically significant growth inhibitory effect on cells. It was observed for low drug concentrations and lessened the cell viability in a dose- and time-dependent manner. Quantitative estimation of apoptosis was accomplished by flow cytometry analyse of annexin V and propidium iodide (PI) labeling in C6 glioma cells. Spontaneous apoptosis (annexin V labeling) was low in C6 glioma cells cultured under control conditions. Treatment of cells with Tan IIA led to a dose-dependent increase in the fraction of annexin V-positive labeling (Fig. 2). One of the features distinguishing apoptosis from necrosis is the early onset of specific endonuclease-mediated cleavage of cellular DNA into nucleosome ladders. In our study, Tan IIA can elicit a similar pattern of DNA fragmentation in C6 glioma cells. As shown in Fig. 3, cells exposed to 2.0 × 103 mug ml−1 and more Tan IIA for 24 h showed the typical apoptosis DNA cleavage, whereas no such pattern was seen in untreated cells. To investigate the inhibitory effect of Tan IIA on the STAT3 activation, we measured the DNA binding activity of dimeric-activated STAT molecules by EMSA. After incubation of C6 glioma cells in different concentrations of Tan IIA for a fixed duration of 24 h, an obvious decline of STAT3 binding activity was observed and the STAT3 binding activity was reduced to almost undetectable levels when treated with 8.0 × 103 mug ml−1 Tan IIA (Fig. 4). As another independent means to determining STAT3 activation, we observed the amount of STAT3 phosphorylation in C6 glioma cells. In the present study, Tan IIA inhibited the tyrosine phosphorylation of STAT3 in the C6 glioma cells in a dosedependent manner, as shown in Fig. 5. The inhibition appeared as early at a concentration as low as 1.0 × 103 mug ml−1 . To investigate the molecular mechanisms for the cell growth inhibition and apoptosis by Tan IIA, we examined the expression of the known Stat3 target genes in the C6 glioma cells, which harbor constitutively active Stat3. In our study, Tan IIA caused a decrease in cyclin D1, Bcl-XL proteins expression in a dose-dependent manner (Fig. 5). Tan IIA 1 × 103 mug ml−1 downregulated proteins expression, but the difference was not significant, the significant inhibition appeared at aconcentration as low as 2.0 × 103 mug ml−1 . These results indicated that Tan IIA represses induction of the cell cycle and anti-apoptotic regulatory genes in malignant cells.
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C. Tang et al. / Neuroscience Letters 470 (2010) 126–129
Fig. 3. DNA-fragmentation analysis of Tan IIA treated C6 glioma cells. C6 glioma cells cells were treated with indicated concentrations of Tan IIA for 24 h. DNA from C6 glioma cells was analyzed on 1.8% agarose gel-electrophoresis. Lane 1: Marker, Lane 2: Control, Lane 3: 1.0 × 103 mug ml−1 , Lane 4: 2.0 × 103 mug ml−1 , Lane 5: 4.0 × 103 mug ml−1 , Lane 6: 8.0 × 103 mug ml−1 .
Fig. 2. Flow cytometric analysis of apoptosis in Tan IIA treated C6 glioma cells. C6 glioma cells were treated with indicated concentrations of Tan IIA (×103 mug ml−1 ) for 12 h and stained with annexin V-FITC/PI. The percentage of cells positive for only annexin V is indicated in the lower right quadrants and that of cells positive for both annexin V and PI is indicated in the top right quadrants. Fig. 4. Alteration of STAT3 DNA binding by Tan IIA. C6 glioma cells were treated with Tan IIA in different concentrations for 24 h. Nuclear extracts were prepared and used in EMSA experiments with the hSIE probe. Lane 1: Control, Lane 2: 1.0 × 103 mug ml−1 , Lane 3: 2.0 × 103 mug ml−1 , Lane 4: 4.0 × 103 mug ml−1 , Lane 5: 8.0 × 103 mug ml−1 .
Fig. 5. Alteration of levels of p-STAT3, cyclin D1, and Bcl-XL proteins in C6 glioma cells. Cells were treated with Tan IIA in indicated concentrations for 24 and extracts were prepared, levels of p-STAT3, cyclin D1, and Bcl-XL proteins were examined in whole-cell extracts by Western blotting, as shown in (A). Lane 1: Control, Lane 2: 1.0 × 103 mug ml−1 , Lane 3: 2.0 × 103 mug ml−1 , Lane 4: 4.0 × 103 mug ml−1 , Lane 5: 8.0 × 103 mug ml−1 . Histogram presented the relative expression level of Bcl-XL (B), cyclin D1 (C), p-STAT3 (D). Data are expressed as mean ± SD in triplicate, * P < 0.05 vs Control group.
C. Tang et al. / Neuroscience Letters 470 (2010) 126–129
In the present study, we found that Tan IIA effectively inhibited the STAT3 pathway and downregulated Bcl-XL and cyclin D1 which are targets of STAT3, induced apoptosis in C6 glioma cells. Constitutive activation of STAT3 recently has been observed in many tumor cells. C6 glioma cells involved in this study were proven to express the STAT3. This is in line with previous studies showing that STAT3 are expressed in glioma cell lines [5,15]. Moreover, in our study, it showed a significant response to Tan IIA treatment by inhibited phosphorylation of STAT3. The inhibition of constitutive activation of the Jak/Stat pathway in this cell line was also seen in terms of a basal STAT3 binding activity in EMSA. The anti-phosphorylation of STAT3 following Tan IIA exposure may serve as part of initial protective mechanisms against STAT3-induced glioma. The oncogenic significance of activated STAT3 molecules is due to their effects on numerous parameters of the development and progression of malignancy. Studies have demonstrated that activation of STAT3 signaling regulates the expression of numerous genes involved in growth control and survival. For example, STAT3 regulates the expression of cyclin D1, which are involved in cell-cycle progression. In primary breast tumors and breast cancer-derived cell lines, cyclin D1 mRNA expression was increased by constitutively activated STAT3 [9,14]. The downregulation of cyclin D1 by Tan IIA in cultured vascular smooth muscle cells has been reported [16]. Our findings also show that Tan IIA can decrease expression of cyclin D1 in C6 glioma cells. In addition to cell proliferation, STAT3 regulates anti-apoptotic genes and contributes to tumor cell survival. Studies have shown that the anti-apoptotic gene encoding Bcl-XL protein is a downstream target of STAT3. Blocking of STAT3 activity inhibits Bcl-XL expression accompanied by induction of apoptosis in head and neck squamous cell carcinoma [4]. Similarly, in our study, inhibition of STAT3 activity leads to a decrease in Bcl-XL expression as well as induction of C6 glioma cells death. This cell death is strongly suggested to be apoptosis, because tanshinone IIA treated cells was characterized by annexin V labeling, moreover, DNA and nuclear fragmentation were also observed. We therefore conclude that Tan IIA has significant antiproliferation effect by induction of apoptosis via inhibition of STAT3 activity. STAT3 may be a promising target for treatment of tumor cells,which provide a point of convergence for tyrosine kinase signaling. In our study, it has been demonstrated that blocking Stat3 function by Tan IIA is sufficient to inhibit tumor cell growth and induce apoptosis. Other studies have also demonstrated that Tan IIA have the ability to arrest the cell cycle of tumor cell lines that are resistant to multiple chemotherapeutic drugs and act as inhibitors of key steps necessary for the progression of tumor angiogenesis [10,21]. In summary, our data suggest that Tan IIA may serve as an effective adjunctive reagent for the treatment of glioma, and that in vivo anti-cancer effects as well as its potential clinical effectiveness need further investigation.
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References [1] T. Bowman, R. Garcia, J. Turkson, R. Jove, STATs in oncogenesis, Oncogene 19 (2000) 2474–2488. [2] J.F. Bromberg, M.H. Wrzeszczynska, G. Devgan, Y. Zhao, R.G. Pestell, C. Albanese, J.E. Darnell, Stat3 as an oncogene, Cell 98 (1999) 295–303. [3] T. Efferth, P.C. Li, V.S. Konkimalla, B. Kaina, From traditional Chinese medicine to rational cancer therapy, Trends Mol. Med. 13 (2007) 353–361. [4] J.R. Grandis, S.D. Drenning, Q. Zeng, S.C. Watkins, M.F. Melhem, S. Endo, D.E. Johnson, L. Huang, Y. He, J.D. Kim, Constitutive activation of Stat3 signaling abrogates apoptosis in squamous cell carcinogenesis in vivo, Proc. Natl. Acad. Sci. 97 (2000) 4227–4232. [5] S. Jenab, V. Quinones-Jenab, The effects of interleukin-6, leukemia inhibitory factor and interferon-gamma on STAT DNA binding and c-fos mRNA levels in cortical astrocytes and C6 glioma cells, Neuro. Endocrinol. Lett. 23 (2002) 325–328. [6] W.K. Kim, S.Y. Huang, E.S. Oh, H.Z. Piao, K.W. Kim, I.O. Han, TGF-beta1 represses activation and resultant death of microglia via inhibition of phosphatidylinositol 3-kinase activity, J. Immunol. 172 (2004) 7015–7023. [7] H.H. Kim, J.H. Kim, H.B. Kwak, H. Huang, S.H. Han, H. Ha, Inhibition of osteoclast differentiation and bone resorption by tanshinone IIA isolated from Salvia miltiorrhiza Bunge, Biochem. Pharmacol. 67 (2004) 1647–1656. [8] L. Konnikova, M. Kotecki, M.M. Kruger, B.H. Cochran, Knockdown of STAT3 expression by RNAi induces apoptosis in astrocytoma cells, BMC Cancer 3 (2003) 23–24. [9] K. Leslie, C. Lang, G. Devgan, Cyclin D1 is transcriptionally regulated by and required for transformation by activated signal transducer and activator of transcription 3, Cancer Res. 66 (2006) 2544–2552. [10] M.A. Mosaddik, In vitro cytotoxicity of tanshinones isolated from Salvia miltiorrhiza Bunge against P388 lymphocytic leukemia cells, Phytomedicine 10 (2003) 682–685. [11] X.L. Niu, K. Ichimori, X. Yang, Y. Hirota, K. Hoshiai, M. Li, H. Nakazawa, Tanshinone IIA inhibits low-density lipoprotein oxidation in vitro, Free Radic. Res. 33 (2000) 305–312. [12] S.O. Rahaman, P.C. Harbor, O. Chernova, G.H. Barnett, M.A. Vogelbaum, S.J. Haque, Inhibition of constitutively active Stat3 suppresses proliferation and induces apoptosis in glioblasto-mamultiforme cells, Oncogene 55 (2002) 8404–8813. [13] S.M. Sagar, R. Wong, The scientific basis of chinese medicine and cancer care: a Western medicine perspective, Asia Pacific Biotech. News 11 (2007) 1168–1197. [14] D. Sinibaldi, W. Wharton, J. Turkson, T. Bowman, W.J. Pledger, R. Jove, Induction of p21WAF1/CIP1 and cyclin D1 expression by the Src oncoprotein in mouse fibroblasts: role of activated STAT3 signaling, Oncogene 19 (2000) 5419–5427. [15] H. Takanaga, T. Yoshitake, E. Yatabe, S. Hara, M. Kunimoto, Beta-naphthoflavone disturbs astrocytic differentiation of C6 glioma cells by inhibiting autocrine interleukin-6, J. Neurochem. 90 (2004) 750–757. [16] H. Wang, X. Gao, B. Zhang, Tanshinone: an inhibitor of proliferation of vascular smooth muscle cells, J. Ethnopharmacol. 99 (2005) 93–98. [17] A.M. Wang, S.H. Sha, W. Lesniak, J. Schacht, Tanshinone (Salviae miltiorrhizae extract) preparations attenuate aminoglycoside-induced free radical formation in vitro and ototoxicity in vivo, Antimicrob. Agents Chemother. 47 (2003) 1836–1841. [18] J. Wang, X. Wang, S. Jiang, S. Yuan, P. Lin, Z.J. hang, Y. Lu, Q. Wang, Z. Xiong, Y. Wu, J. Ren, H. Yang, Growth inhibition and induction of apoptosis and differentiation of tanshinone IIA in human glioma cells, J. Neurooncol. 82 (2006) 11–21. [19] W.L. Wu, W.L. Chang, C.F. Chen, Cytotoxic activities of tanshinones against human carcinoma cell lines, Am. J. Chin. Med. 19 (1991) 207–216. [20] X.Y. Yu, S.G. Lin, Z.W. Zhou, X. Chen, J. Liang, W. Duan, X.Q. Yu, J.Y. Wen, B. Chowbay, C.G. Li, F.S. Sheu, E. Chan, S.F. Zhou, Tanshinone IIB, a primary active constituent from Salvia miltiorrhza, exhibits neuro-protective activity in experimentally stroked rats, Neurosci. Lett. 417 (2007) 261–265. [21] S.L. Yuan, Y.Q. Wei, X.J. Wang, F. Xiao, S.F. Li, J. Zhang, Growth inhibition and apoptosis induction of tanshinone IIA on human hepatocellular carcinoma cells, World J. Gastroenterol. 10 (2004) 2024–2028. [22] G.Y Zhou, B.L. Zhao, J.W. Hou, G.E. Ma, W.J. Xin, Protective effects of sodium tanshinone IIA sulphonate against adriamycin-induced lipid peroxidation in mice hearts in vivo and in vitro, Pharmacol. Res. 40 (1999) 487–491.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Tanshinone IIA inhibits constitutive STAT3 activation, suppresses proliferation, and induces apoptosis in rat C6 glioma cells Chao Tang a,∗ , Hong-li Xue a , Hai-bo Huang b , Xiao-gang Wang a a b
Department of Neurosurgery, The General Hospital of Shenyang Military Region, Shen Yang 110016, Liaoning Province, PR China Liaoning University of Traditional Chinese Medicine, Shen Yang 110016, Liaoning Province, PR China
a r t i c l e
i n f o
Article history: Received 11 October 2009 Received in revised form 18 December 2009 Accepted 21 December 2009 Keywords: Tanshinone IIA STAT3 Apoptosis Glioma
a b s t r a c t Signal transducer and activator of transcription 3 (STAT3) is usually constitutively activated in a variety of malignancies. Thus, STAT3 may be a promising target for treatment of tumor cells. Recently, Tanshinone IIA (Tan IIA), a major active constituent from the root of Salvia miltiorrhiza Bunge, was reported to have apoptosis inducing effects on a large variety of cancer cells. In this study, we evaluate the anti-proliferation and apoptosis inducing effects of Tan IIA on C6 glioma cells. Cell growth and proliferation were measured by MTT assay, cell apoptosis was observed by flow cytometry and DNA-fragmentation analysis. Further more, we investigated inhibitory effects of Tan IIA on STAT3 activity and its downstream targets: BclXL, cyclin D1. Alteration of STAT3 activity was examined by measuring their DNA binding activity and tyrosine phosphorylation. Changes in the expression levels of Bcl-XL and cyclin D1 were examined by Western blot analysis. We found that the cellular growth were inhibited and cell apoptosis were observed after the treatment with Tan IIA. The STAT3 activity was signifcantly reduced by Tan IIA parallel with a significant attenuation of expression of Bcl-XL and cyclin D1. These results suggest that Tan IIA may serve as an effective adjunctive reagent in the treatment of glioma for its targeting of constitutive STAT3 signaling. © 2009 Elsevier Ireland Ltd. All rights reserved.
Gliomas are the most common primary tumors of the brain that arise from astrocytes and their precursors, which are consided to be advanced invasive, metastatic, malignant. Constitutive activation of STAT3 has been demonstrated in a variety of diverse human tumor cell lines, which are involved in the development and progression of cancers [1,2]. STAT3 is also constitutively activated in gliomas cells. Inhibition of STAT3 function leads to growth inhibition of gliomas cells [8,12]. Numerous cancer research studies have been conducted using traditional medicinal plants in an effort to discover new therapeutic agents that lack the toxic side effects associated with current chemotherapeutic agents. Salvia miltiorrhiza Bung (Danshen) has been widely used to treat cardiovascular diseases in traditional Chinese medical practice, which is considered to have the property of activating blood circulation and removing stasis. The malignant tumor is viewed as being associated with stagnation of blood in traditional chinese medicine, therefore, Danshen has been also used in anti-cancer therapy [3,13]. As one of the most abundant extracts of Danshen, Tan IIA is known to have anti-inflammatory, anti-oxidative and cytotoxic
∗ Corresponding author. E-mail address: [email protected] (C. Tang). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.12.069
activities, neuron-protective effects [11,17,20,22]. In addition, Tan IIA has been shown to relieve oxidative stress and immune dysfunction associated with the onset and progress of cancer [19]. Studies have also demonstrated that Tan IIA have the ability to arrest the cell cycle of tumor cell lines that are resistant to multiple chemotherapeutic drugs and act as inhibitors of key steps necessary for the progression of tumor angiogenesis [10,21]. Recently, it has been shown that Tan II A can suppress the proliferation of human glioma cells [18]. However, the mechanisms underlying the therapeutic action of Tan II A are not well understood. There are no reports, on whether Tan IIA affects STAT3 pathway in glioma cells. In the present report, we set out to establish whether Tan IIA can also suppress constitutive activation of STAT3 in C6 glioma cells. Tan IIA was isolated from the roots of Salvia miltiorrhiza Bunge,which is based on the method described previously [7]. The purity of Tan IIA was proven by high performance liquid chromatography (HPLC) as >95%. Rat C6 glioma cells were obtained from the American Type Culture Collection. C6 glioma cells were cultured in Dulbecco’s modified Eagle’s medium (Grand Island, NY, USA) supplemented with 7.5% fetal calf serum, penicillin (10 units/ml) and streptomycin (10 mg/ml) (Sigma, St. Louis, MO, USA). Cultures were maintained in a humidified atmosphere of 5% CO2 at 37 ◦ C.
C. Tang et al. / Neuroscience Letters 470 (2010) 126–129
The cytotoxicity of Tan IIA was determined by the MTT assay. Cells were plated in 100 l of medium/well in 96-well plates. After incubation overnight, Tan IIA was added in various concentrations; After treatment with Tan IIA for 12–36 h, 20 l of 5 mg/ml MTT (pH 4.7) were added to each well and cultivated for another 4 h, the supernatant fluid was removed, 100 l/well DMSO were added .Absorbance at 570 nm was measured with a microplate reader (Bio-Rad, Richmond, CA, USA), using wells without cells as blanks. Date was expressed as the percent cytoviability (%cytoviability = A570 of treated cells/A570 of control cells × 100%). The stock solutions of Tan IIA were prepared in DMSO. The concentration of DMSO in experimental media was 0.1% (v/v). The effect of Tan IIA on the induction of apoptosis of C6 glioma cells was investigated by flow cytometry analysis. After Tan IIA treatment for 12 h,cells was washed with cold PBS and resuspended with binding buffer (10 m HEPES NaOH, pH7.4, 140 mM NaCl, 2.5 mM CaCl2 ). Cells were stained for 15 min at room temperature in the dark with annexin V-FITC and propidium iodide and then added to each tube and analyzed by a Becton Dickinson FACScan (excitation light, 488 nm) equipped with CELLQUEST software (Becton Dickinson). Cell apoptosis was further observed by DNA-fragmentation analysis. After incubation with Tan IIA for 24 h, the cells were washed with PBS and lysed in a solution containing 400 mM NaCl, 10 mM. Tris–HCl (pH 7.5), 1% sodium dodecyl sulfate, and 0.15 mg/ml of proteinase K. The lysate was incubated for 4 h at 37 ◦ C, followed by the addition of 5 M NaCl, to a final concentration of 1 M. After centrifugation at 7500 × g for 30 min, the supernatant was collected and DNA was precipitated with 70% isopropanol. After digestion for 1 h with ribonuclease A, these samples were electrophoresed in 2% agarose gel and stained with ethidium bromide. The DNA binding activity of dimeric-activated STAT molecules were measured by electrophoretic mobility shift assay (EMSA). Nuclear extracts were prepared as described previously [6] with Na3 VO4 (1 mM), and complete protease inhibitor. The reactions were performed in a total volume of 24 l in buffer consisting of 10 mM HEPES (N[2-hydroxeth yl]piperazine-N-[2-ethane-sulfonic acid]) (pH 7.8), 50 mM KCl, 1 mM EDTA, 5 mM MgCl2 , 10% glycerol, 5 mM dithiothreitol, 1 mg of bovine serum albumin per ml, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM Na3 VO4 with 1 g of poly (dI-dC) and 0.3 ng of 32 P-labelled high affinity sis-inducible element (hSIE). Following incubation for 15 min at room temperature, the reactions were electrophoresed on 4% native polyacrylamide gels. For supershift experiments, the appropriate antibody was added to the nuclear extract in buffer and incubated for 60 min at room temperature prior to the addition of the labelled-hSIE/dI-dC mixture. Changes in the expression levels of P-STAT3, Bcl-XL and cyclin D1 were examined by Western blot analysis. Whole cell extracts were mixed with 2× SDS sample buffer [125 mmol/l Tris–HCl (pH 6.8), 4% SDS, 20% glycerol, and 10% 2 mercaptoethanol] at 1:1 ratio and were heated for 5 min at 100 ◦ C. Proteins (50 g/lane) were separated by 12.5% SDS-PAGE and transferred onto a nitrocellulose membrane. Prestained molecular weight markers were included in each gel. Membranes were blocked for 30 min in TBST and 5% BSA. After blocking, membranes were incubated with primary antibodies (1:1000 dilution) according to the manufacturer’s instructions (Santa Cruz, CA, USA). After washing the membranes three times with TBST (5 min each), they were incubated with horseradish peroxidase-conjugated secondary antibody in TBST and 1% BSA for 30 min. The proteins were detected by the enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL, USA) using X-ray film.Statistical analysis data were expressed as mean ± SD. The statistical differences among groups were evaluated using one-way ANOVA with Fisher’s PLSD test. P < 0.05 was considered significant. Results were analyzed using SPSS13.0 software (Spss Inc., Chicago, TL, USA).
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Fig. 1. Effects of Tan IIA on the viability of C6 glioma cells. Cells were treated with the indicated concentrations of Tan IIA (×103 mug ml−1 ) for 12, 24, 36 h. The cell survival was determined using the MTT assay. Data are expressed as mean ± SD in triplicate. * P < 0.05 vs untreated group.
Results of the effect of Tan IIA on C6 glioma cells growth by MTT assay are shown in Fig. 1. Tan II A exhibited a statistically significant growth inhibitory effect on cells. It was observed for low drug concentrations and lessened the cell viability in a dose- and time-dependent manner. Quantitative estimation of apoptosis was accomplished by flow cytometry analyse of annexin V and propidium iodide (PI) labeling in C6 glioma cells. Spontaneous apoptosis (annexin V labeling) was low in C6 glioma cells cultured under control conditions. Treatment of cells with Tan IIA led to a dose-dependent increase in the fraction of annexin V-positive labeling (Fig. 2). One of the features distinguishing apoptosis from necrosis is the early onset of specific endonuclease-mediated cleavage of cellular DNA into nucleosome ladders. In our study, Tan IIA can elicit a similar pattern of DNA fragmentation in C6 glioma cells. As shown in Fig. 3, cells exposed to 2.0 × 103 mug ml−1 and more Tan IIA for 24 h showed the typical apoptosis DNA cleavage, whereas no such pattern was seen in untreated cells. To investigate the inhibitory effect of Tan IIA on the STAT3 activation, we measured the DNA binding activity of dimeric-activated STAT molecules by EMSA. After incubation of C6 glioma cells in different concentrations of Tan IIA for a fixed duration of 24 h, an obvious decline of STAT3 binding activity was observed and the STAT3 binding activity was reduced to almost undetectable levels when treated with 8.0 × 103 mug ml−1 Tan IIA (Fig. 4). As another independent means to determining STAT3 activation, we observed the amount of STAT3 phosphorylation in C6 glioma cells. In the present study, Tan IIA inhibited the tyrosine phosphorylation of STAT3 in the C6 glioma cells in a dosedependent manner, as shown in Fig. 5. The inhibition appeared as early at a concentration as low as 1.0 × 103 mug ml−1 . To investigate the molecular mechanisms for the cell growth inhibition and apoptosis by Tan IIA, we examined the expression of the known Stat3 target genes in the C6 glioma cells, which harbor constitutively active Stat3. In our study, Tan IIA caused a decrease in cyclin D1, Bcl-XL proteins expression in a dose-dependent manner (Fig. 5). Tan IIA 1 × 103 mug ml−1 downregulated proteins expression, but the difference was not significant, the significant inhibition appeared at aconcentration as low as 2.0 × 103 mug ml−1 . These results indicated that Tan IIA represses induction of the cell cycle and anti-apoptotic regulatory genes in malignant cells.
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Fig. 3. DNA-fragmentation analysis of Tan IIA treated C6 glioma cells. C6 glioma cells cells were treated with indicated concentrations of Tan IIA for 24 h. DNA from C6 glioma cells was analyzed on 1.8% agarose gel-electrophoresis. Lane 1: Marker, Lane 2: Control, Lane 3: 1.0 × 103 mug ml−1 , Lane 4: 2.0 × 103 mug ml−1 , Lane 5: 4.0 × 103 mug ml−1 , Lane 6: 8.0 × 103 mug ml−1 .
Fig. 2. Flow cytometric analysis of apoptosis in Tan IIA treated C6 glioma cells. C6 glioma cells were treated with indicated concentrations of Tan IIA (×103 mug ml−1 ) for 12 h and stained with annexin V-FITC/PI. The percentage of cells positive for only annexin V is indicated in the lower right quadrants and that of cells positive for both annexin V and PI is indicated in the top right quadrants. Fig. 4. Alteration of STAT3 DNA binding by Tan IIA. C6 glioma cells were treated with Tan IIA in different concentrations for 24 h. Nuclear extracts were prepared and used in EMSA experiments with the hSIE probe. Lane 1: Control, Lane 2: 1.0 × 103 mug ml−1 , Lane 3: 2.0 × 103 mug ml−1 , Lane 4: 4.0 × 103 mug ml−1 , Lane 5: 8.0 × 103 mug ml−1 .
Fig. 5. Alteration of levels of p-STAT3, cyclin D1, and Bcl-XL proteins in C6 glioma cells. Cells were treated with Tan IIA in indicated concentrations for 24 and extracts were prepared, levels of p-STAT3, cyclin D1, and Bcl-XL proteins were examined in whole-cell extracts by Western blotting, as shown in (A). Lane 1: Control, Lane 2: 1.0 × 103 mug ml−1 , Lane 3: 2.0 × 103 mug ml−1 , Lane 4: 4.0 × 103 mug ml−1 , Lane 5: 8.0 × 103 mug ml−1 . Histogram presented the relative expression level of Bcl-XL (B), cyclin D1 (C), p-STAT3 (D). Data are expressed as mean ± SD in triplicate, * P < 0.05 vs Control group.
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In the present study, we found that Tan IIA effectively inhibited the STAT3 pathway and downregulated Bcl-XL and cyclin D1 which are targets of STAT3, induced apoptosis in C6 glioma cells. Constitutive activation of STAT3 recently has been observed in many tumor cells. C6 glioma cells involved in this study were proven to express the STAT3. This is in line with previous studies showing that STAT3 are expressed in glioma cell lines [5,15]. Moreover, in our study, it showed a significant response to Tan IIA treatment by inhibited phosphorylation of STAT3. The inhibition of constitutive activation of the Jak/Stat pathway in this cell line was also seen in terms of a basal STAT3 binding activity in EMSA. The anti-phosphorylation of STAT3 following Tan IIA exposure may serve as part of initial protective mechanisms against STAT3-induced glioma. The oncogenic significance of activated STAT3 molecules is due to their effects on numerous parameters of the development and progression of malignancy. Studies have demonstrated that activation of STAT3 signaling regulates the expression of numerous genes involved in growth control and survival. For example, STAT3 regulates the expression of cyclin D1, which are involved in cell-cycle progression. In primary breast tumors and breast cancer-derived cell lines, cyclin D1 mRNA expression was increased by constitutively activated STAT3 [9,14]. The downregulation of cyclin D1 by Tan IIA in cultured vascular smooth muscle cells has been reported [16]. Our findings also show that Tan IIA can decrease expression of cyclin D1 in C6 glioma cells. In addition to cell proliferation, STAT3 regulates anti-apoptotic genes and contributes to tumor cell survival. Studies have shown that the anti-apoptotic gene encoding Bcl-XL protein is a downstream target of STAT3. Blocking of STAT3 activity inhibits Bcl-XL expression accompanied by induction of apoptosis in head and neck squamous cell carcinoma [4]. Similarly, in our study, inhibition of STAT3 activity leads to a decrease in Bcl-XL expression as well as induction of C6 glioma cells death. This cell death is strongly suggested to be apoptosis, because tanshinone IIA treated cells was characterized by annexin V labeling, moreover, DNA and nuclear fragmentation were also observed. We therefore conclude that Tan IIA has significant antiproliferation effect by induction of apoptosis via inhibition of STAT3 activity. STAT3 may be a promising target for treatment of tumor cells,which provide a point of convergence for tyrosine kinase signaling. In our study, it has been demonstrated that blocking Stat3 function by Tan IIA is sufficient to inhibit tumor cell growth and induce apoptosis. Other studies have also demonstrated that Tan IIA have the ability to arrest the cell cycle of tumor cell lines that are resistant to multiple chemotherapeutic drugs and act as inhibitors of key steps necessary for the progression of tumor angiogenesis [10,21]. In summary, our data suggest that Tan IIA may serve as an effective adjunctive reagent for the treatment of glioma, and that in vivo anti-cancer effects as well as its potential clinical effectiveness need further investigation.
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