Cell Biochem Biophys DOI 10.1007/s12013-014-0360-3
ORIGINAL PAPER
Synergistic Anti-Cancer Effects of Icariin and Temozolomide in Glioblastoma Lijuan Yang • Yuexun Wang • Hua Guo Meiling Guo
•
Ó Springer Science+Business Media New York 2014
Abstract Glioblastoma is an aggressive malignancy, which is associated with poor prognosis. Temozolomide (TMZ) has been showed to be an effective chemotherapeutic agent for glioblastoma treatment; however, the response rate is not satisfactory. Icariin is a natural compound with anti-cancer activity against a variety of cancers. This study is designed to determine whether icariin could potentiate the antitumor activity of TMZ in glioblastoma. Cell proliferation and apoptosis were measured using MTT assay and flow cytometry, respectively. Expression of apoptosis and proliferation-related molecules was detected by Western blotting while NF-jB activity was detected by ELISA. Icariin dose-dependently inhibited proliferation and induced apoptosis in tested glioblastoma cell lines. Icariin enhanced the anti-tumor activity of TMZ in vitro. The anti-tumor activity of icariin and the enhanced antitumor activity of TMZ by icariin correlated with suppression of NF-jB activity. Our results showed that icariin exhibited anti-tumor activity and potentiated the anti-tumor activity of TMZ in glioblastoma, at least in part, by inhibiting NF-jB activity. Although more studies including clinical trials are needed, this study provides insight for using icariin as a chemosensitizing agent in clinic settings.
Dr. Lijuan Yang and Dr. Yuexun Wang have contributed equally to this study. L. Yang H. Guo Shandong Provincial Hospital Affiliated to Shandong University, Shandong University, No.324, JingWu Road, Ji’nan 250021, China Y. Wang M. Guo (&) CT Department, First People’s Hospital of Jining, Shandong 272011, China e-mail: [email protected]
Keywords
GBM TMZ Icariin AMPK
Introduction Glioblastoma multiforme (GBM), accounting for approximately 60 % of all brain tumors, is the most common type of primary brain tumors in adults [1, 2]. GBMs are one of the most lethal, highly invasive, and least effectively treated solid tumors. Despite aggressive therapy regimen including maximal surgical resection, combined radiation/ chemotherapy, the recurrence of GBM is still quite common [3, 4]. Due to the ability to infiltrate diffusely into the normal brain parenchyma, the prognosis of patients with the highest grade malignant glioma, GBM, remains poor with a median survival of at 12–15 months and a 5-year survival rate of 2 % [4]. Therefore, improvement of treatment options for patients with GBM is imperative. Mounting evidence has showed that the inability of many chemotherapeutic agents to pass through the blood– brain barrier, and their low efficacy for induction of apoptosis have largely compromised their value in treatment of glioblastoma. Among the chemotherapeutic agents used to treat GBM, alkylating agent temozolomide (TMZ), (8-carbamoyl-3-methylimidazo [5,1-d]-1,2,3,5-tetrazin4(3H)-one) belonging to imidazotertrazines has been considered a promising one due to its ability of crossing the blood–brain barrier effectively with few myelocytotoxic effects [5]. Clinical data showed that the average survival of patients with glioblastoma after TMZ treatment is 22 months [6], which is still not satisfactory. Different regimens to attenuate resistance and increase the efficacy of TMZ have been investigated. Among the potential chemosensitizers, the natural compounds, such as icariin, may be an ideal agent for GBM therapy. Icariin, a
123
Cell Biochem Biophys
flavonoid isolated from Epimedi herba, is considered to be the major active ingredient of traditional Chinese medicine E. herba. Icariin has a variety of pharmacological activities including anti-inflammatory, anti-depressant, male sexual function improvement, cardiovascular protection, enhancing bone healing and neuroprotective while it has a low toxicity and can be given in relatively high doses without adverse effects in humans [7, 8]. Emerged evidence suggested that icariin might be a potential chemopreventive and/or chemotherapeutic in human cancer. Icariin exerted anti-proliferative effect in vitro on cell growth in mouse Leydig tumor cells, human lung cancer cells, human gastric cancer cells, human leukemia cells, human breast cancer cells, and human hepatoma cells [9–13]. More importantly, icariin can pass through blood–brain barrier into brain tissues [14]. Therefore, the present study was designed to investigate whether icariin can potentiate the antitumor effect of TMZ on the human GBM in U87MG cells and to demonstrate the involved mechanisms in the additive antitumor effects exerted by TMZ and icariin.
Materials and Methods Cell Culture In this study, a human GBM cell line U87MG was used as model cell line. Cells were cultured in DMEM supplemented with 10 % FBS, 100 U/mL penicillin and 100 lg/ mL streptomycin, and maintained in a humidified atmosphere of 95 % air and 5 % CO2 at 37 °C. MTT Assay For the cytotoxicity assay, 1.0 9 104 glioblastoma cells per well were seeded onto 96-well plates. After overnight incubation, the cells adhered to the plate. Following treatment with icariin or/and TMZ, 20 lL of MTT solution (5 mg/ml in PBS) was added to each well and incubated for 2 h. MTT formazan was dissolved in 150 lL of isopropanol and the absorbance was measured at 595 nm with an ELISA reader (Tecan Group Ltd, Ma¨nnedorf, Switzerland). H3-Thymidine Incorporation U87MG cells were plated in 24-well plates and exposed to a medium containing DMSO (Vehicle), TMZ, icariin, or icariin with TMZ. Cells were cultured 48 h prior to the addition of 0.5 lCi of H3-thymidine per well. After 4-h of incubation, the medium was removed and cells were washed twice with cold 0.05 M Tris–HCl and 5 % trichloroacetic acid, scrapped, and transferred to a scintillation cocktail. The level of
123
incorporated H3-thymidine was assessed using Beckman liquid scintillation counter. Apoptosis Analysis Cell apoptosis was determined using a FITC Annexin V apoptosis kit (BD Pharmingen, Franklin Lakes, NJ) according to the manufacturer’s instructions. Following drug treatment, the cell suspension was prepared using 0.125 % trypsin and was rinsed and centrifuged with icecold PBS at 1,000 rpm for 5 min. Cells were then resuspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2) at a concentration of 1 9 106 cells/ml. Cells were stained with annexin V-FITC and propidium (PI) for 20 min away from the light before analysis by a flow cytometer (Beckman Coulter Inc, Miami, Florida, USA). Cell Migration and Invasion Assays For cell invasion assay, Boyden chambers containing Transwell (Corning Costar Corp., Cambridge, MA) membrane filters with a pore size of 8 lm were used. Matrigel (60 ll) diluted in optimum was coated in invasion chambers rehydrated by adding 0.5 ml of RPMI-1640 medium to the upper chambers at 37 °C overnight. After rehydration, the medium was carefully removed, and 2 9 104 cells were added to the upper chambers in triplicate. The lower compartments of the migration and invasion chambers were filled with DMEM-F12 medium containing 1 % bovine serum albumin (Sigma) at concentrations of 5 and 20 %, respectively. After 48 h, nonmigrating cells on the upper surface of the filters were swept by cotton swabs. The migrated and invaded cells on the lower surface of the membrane were fixed and then stained with methanol mixed crystal violet. The number of cells was counted under a microscope. All assays were run independently three times. Western Blot Analysis Following treatment, the expression levels of signaling molecules in the glioblastoma cells were detected using Western blot analysis. Western blot analysis was performed using a standard protocol. The primary antibodies used in this study were as follows: rabbit anti-caspase-3 (Cell Signaling Technology, Inc., Danvers, MA, USA) and rabbit anti-PARP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, 1:2000). The secondary antibodies used in this study were alkaline phosphatase peroxidase-conjugated anti-rabbit IgG. Detection was performed by the BCIP/NBT Alkaline Phosphatase Color Development Kit
Cell Biochem Biophys Fig. 1 Effect of TMZ or/and icariin on cell viability and H3thymidine incorporation. Cells were treated with indicated concentration of icariin, TMZ or both (TMZ 200 lM, icariin 10 lM) for 48 h. *P \ 0.05 versus vehicle; **P \ 0.01 versus vehicle; ^P \ 0.05 versus TMZ only
(Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s instructions. NF-jB Activity Analysis Following treatment, nuclear extract was prepared using a nuclear extract kit (Active Motif, Carlsbad, CA) and activity of NF-jB p65 was examined using an ELISA kit (Active Motif, Carlsbad, CA). Statistical Analysis All data were expressed as Mean ± SD and represented the results of three separated experiments each performed in quadruplicate unless otherwise stated. The Student’s test was used to evaluate the statistical significance. P \ 0.05 was considered as statistical significant.
Results Icariin Enhances the Growth Inhibition Effect of TMZ in GBM Cells To explore whether icariin could sensitize glioblastoma cells to TMZ, we examined the effect of treatment with TMZ alone or in combination with icariin (ICA) on the growth U87MG cells. As shown in Fig. 1a, TMZ treatment resulted in a dose-dependent loss of cell viability. Relative to the control, U87MG cell viability was reduced to 49.1 ± 6.2 % relative to control after 200 lM TMZ treatment for 48 h. The cytotoxicity of icariin was also
examined. As shown in Fig. 1b, treatment with icariin alone also dose-dependent inhibited the growth of U87MG cells. Since icariin at 10 lM significantly inhibited cell growth in U87MG cells, we chose 10 lM icariin in the combinational treatment with TMZ. As shown in Fig. 1c, combination treatment with TMZ and icariin dramatically reduced viable U87MG cells to approximately 26.5 ± 8.1 %. To evaluate the synergism, we calculated the combination index value according to Chou’s method [15]. Results showed that icariin exerted synergistically antitumor effect on GBM cell line U87MG when combined with TMZ (CI = 0.694).
The Effect of Icariin and Icariin with TMZ on H3Thymidine Incorporation in the Glioblastoma Cell Line (U87MG) The effects of different treatments on proliferation were also examined with H3-thymidine incorporation assay. As shown in Fig. 1d, our results revealed a strong inhibitory potential on U87MG cells proliferation after a 48-h exposition demonstrated by almost 30 % decrease of H3-thymidine incorporation compared to control. In contrast, icariin at a dose of 10 lM lowered DNA synthesis by approximately 50 % in relation to control. Moreover, we found that a combination of TMZ and icariin was significantly more powerful to arrest cell division (about 36 % of control) than TMZ or icariin alone (about 70 % of control). Taken together, our results demonstrated that a combination of icariin with TMZ exhibited the synergistic effect decreasing proliferation in U87MG cells.
123
Cell Biochem Biophys Fig. 2 Cell apoptosis induced by TMZ, icariin and combination of icariin and TMZ in U87MG cells. Cells were treated with indicated concentration of icariin (10 lM), TMZ (200 lM) or both for 48 h. Cell apoptosis was analyzed by flow cytometry while levels of capase-3 and PARP were determined by Western blots. Flow cytometric graphs and Western blots shown were representative of three experiments. *P \ 0.05 versus vehicle; ^P \ 0.05 versus TMZ only
Icariin Sensitizes GBM to TMZ-Induced Apoptosis Our before mentioned results showed that icariin could enhance the inhibitory effect of TMZ on U87MG cell proliferation. Therefore, we examined whether the potentiated anti-proliferative effect was mediated by apoptosis. Compared with treatment with TMZ alone, combination treatment with icariin and TMZ induced significantly more apoptosis in tested U87MG cells (Fig. 2a). In addition, the level of activated caspase-3 and cleaved PARP were also examined. As shown in Fig. 2b, joint treatment of icariin and TMZ caused a more prominent elevation in activated caspase-3 and cleaved PARP compare to monotherapy. Taken together, our results suggested that the pro-apoptotic effect of TMZ combined with icariin is superior to either one as a solo treatment, indicating that icariin potentiates the cytotoxic effect of TMZ by inducing apoptosis. Combination Treatment with Icariin and TMZ Inhibits Cell Migration and Invasion Next, the effect of combination treatment on cell migration and invasion was examined. As shown in Fig. 3a, both icariin and TMZ exerted inhibitory effect on cell migration. However, icariin cooperated with TMZ was significantly more effective in inhibiting cell migration compared to TMZ or icariin used as single agent. In addition, as shown
123
in Fig. 3b, TMZ combined with icariin resulted in significantly greater inhibitory effects on cell invasion. Taken together, our results showed that combination of TMZ and icariin significantly altered the invasion and migration capacities of U87MG cells, as shown by the markedly decreased cell membrane-penetrating capacity. The Effect of Icariin and Icariin with TMZ on NF-jB Activity in Glioblastoma Cell Line (U87MG) Emerging evidences showed that NF-jB play a crucial role in cell proliferation, invasion, apoptosis inhibition, chemoresistance and radioresistance [16]. Therefore, we examined the NF-jB activity to investigate whether icariin potentiated the anti-cancer effect of TMZ in U87MG cells by modulating NF-jB activity. The U87MG cells were treated with TMZ (200 lM) or icariin (10 lM) alone or a combination of TMZ and icariin (200 and 10 lM, respectively) for 48 h. As shown in Fig. 4, we found that TMZ did not markedly alter the NF-jB activity in U87MG cells since nuclear localization of both p65 almost remained at the level similar to vehicle. In contrast, icariin alone led to a remarkable decrease of nuclear content of p65 subunits. Moreover, the incubation of cells with both of TMZ and icariin resulted in a more profound reduction of NF-jB activity, which clearly suggested a synergic effect of icariin and TMZ. Based on our findings, we suggested that the
Cell Biochem Biophys Fig. 3 Cell migration and invasion caused by TMZ, icariin and combination of icariin and TMZ in U87MG cells. Cells were treated with indicated concentration of icariin (10 lM), TMZ (200 lM) or both for 48 h. Cell migration and invasion were assessed by transwell assay. *P \ 0.05 versus vehicle; ^P \ 0.05 versus TMZ only
Fig. 4 NF-jB activity suppressed by TMZ, icariin and combination of icariin and TMZ in U87MG cells. Cells were treated with indicated concentration of icariin (10 lM), TMZ (200 lM) or both for 48 h. NF-jB activity was determined by ELISA kit. *P \ 0.05 versus vehicle; ^P \ 0.05 versus TMZ only
sensitization of GBM cells to TMZ by Icariin correlated with suppressed NF-jB activity.
Discussions Currently, surgical resection followed by radiotherapy and chemotherapy has been considered as standard therapy.
TMZ, an alkylating agent, is the only drug shown to improve survival when administered with concomitant radiotherapy [17]. However, still more than 90 % of patients die within 3 years of diagnosis despite the employment of this standard therapy incorporating TMZ [17]. Therefore, novel therapeutic strategies are imperative to improve chemotherapy response. The goal of this study was to investigate therapeutic effect of sensitization of GBM to TMZ using a flavonoid compound, icariin. Here, we showed that icariin presented cytotoxic effect and enhanced the antitumor effect of TMZ in GBM cell line U87MG, which may at least partially mediated by a reduced activity of NF-jB. The antitumor activities of icariin has been examined in a variety of human cancer cell lines and various mechanisms have been proposed to explain the anti-tumor effects of icariin, including GPER1-mediated modulation of the EGFR-MAPK signaling pathway [18], activating ROS/ JNK-dependent mitochondrial pathway [10], suppressing piwil4 expression [13], and downregulating Rac1 and vasodilator-stimulated phosphoprotein (VASP) [12]. However, the effect of Icariin against human glioma cells is not known. Recently, emerging evidences demonstrated that icariin might pass through the blood–brain barrier [19], which highlights the potential of icariin to be used as a chemotherapeutic agent in the treatment of glioma. In this
123
Cell Biochem Biophys
study, our results showed that icariin inhibited proliferation, induced apoptosis, prevented migration and invasion in U87MG cells, which for the first time demonstrated the antitumor activities of icariin against GBM. In the past decade, a number of flavonoid compounds have been found to be able to enhance the antitumor effect of TMZ in glioblastoma cells. It has reported that resveratrol enhanced the antitumor of TMZ in glioblastoma via multiple mechanisms including activating ROS-dependent AMPK-TSC-mTOR signaling pathway, abrogating the TMZ-induced G2 arrest leading to mitotic catastrophe and reinforcing the TMZ-induced senescence in glioma cells, as well as Resveratrol reverses TMZ resistance by downregulating MGMT by the NF-jB-dependent pathway [20– 22]. Another flavonoid compound, curcumin, has also been found to sensitize glioblastoma to TMZ by simultaneously generating ROS and disrupting Akt/mTOR signaling [23]. In line with these previously studies, our results showed that icariin, by inhibiting NF-jB activity, potentiated the in vitro anti-tumor activity of TMZ in GBM. The role of NF-jB as one of the major transcription factors associated with cancer has been well established with evidence showing the involvement of NF-jB in many hallmarks of cancer development, including growth factorindependent proliferation, inhibition of apoptosis, limitless replicative potential, and tissue invasion and metastasis [24]. In the context of GBM, the association between NFjB and GBM is known for more than 15 years and a number of studies have reported that that the NF-jB pathway is constitutively activated or is upregulated in response to different stimuli, mainly cytokines, in GBM cells [25]. Although little is known about the precise mechanism how NF-jB is activated in GBMs, there are numerous proteins and pathways dysregulated in GBMs that may cause NF-jB activation [26] and activation of NF-jB by several conventional chemotherapeutics also resulted in unfavorable clinical outcomes [26]. Very recently, it has been reported that activation of NF-jB is upregulated during differentiation of glioblastoma initiating cells (GICs) [24]. Taken together, all these results highlight the potential of NF-jB to be a novel target for GBM therapy. Indeed, a number of drugs with inhibitory activities on NF-jB signaling pathway have been investigated for the efficacy against GBM. For example, the antiinflammatory drug sulfasalazine, one of the more promising preclinical drugs for glioma in the last 5 year which has showed marked therapeutic efficacy on mice model, has been suggested to work via inhibition of NF-jB activity. Consistent with previous studies, our results here also showed that the antitumor effect of icariin against GBM cells was, at least in part, mediated by targeting NF-jB signaling pathway. On the other hand, Caporali et al. have reported that NF-jB is activated in response to TMZ in an
123
AKT-dependent manner and confers protection against the growth suppressive effect of the drug [27]. Furthermore, a number of studies have showed that the ant-tumor effect of TMZ was enhanced by different agents via inhibiting NFjB. In GBM, the efficacy of TMZ was augmented when NF-jB activity was inhibited by propolis [28], triptolide [29], dehydroxymethylepoxyquinomicin [30], or resveratrol [22]. Besides GBM, enhanced chemosensitivity to TMZ was also reported to be associated with decreased NF-jB activity in colorectal cancer and melanoma [27, 31]. In agreement with these studies, we found that the sensitization of GBM to TMZ by Icariin correlated with suppressed NF-jB activity. Taken together, these results indicated that the regulation of NF-jB activity could be a possible new treatment for the chemosensitization of GBM to TMZ. In summary, the present study found that icariin potentiated the antitumor effects of TMZ in GBM in vitro and the synergistic anti-tumor activities was associated with suppression of NF-jB activity. Given the low toxicity of icariin to normal tissue and its ability of passing cross blood–brain barrier, our preset results suggest that icariin may be used as chemosensitizer of TMZ in GBM.
References 1. Surawicz, T. S., Davis, F., Freels, S., Laws, E. R, Jr., & Menck, H. R. (1998). Brain tumor survival: Results from the National Cancer Data Base. Journal of Neuro-oncology, 40, 151–160. 2. Furnari, F. B., Fenton, T., Bachoo, R. M., Mukasa, A., Stommel, J. M., Stegh, A., et al. (2007). Malignant astrocytic glioma: Genetics, biology, and paths to treatment. Genes & Development, 21, 2683–2710. 3. Grossman, S. A., & Batara, J. F. (2004). Current management of glioblastoma multiforme. Seminars in Oncology, 31, 635–644. 4. Quick, A., Patel, D., Hadziahmetovic, M., Chakravarti, A., & Mehta, M. (2010). Current therapeutic paradigms in glioblastoma. Reviews on Recent Clinical Trials, 5, 14–27. 5. Uzzaman, M., Keller, G., & Germano, I. M. (2007). Enhanced proapoptotic effects of tumor necrosis factor-related apoptosisinducing ligand on temozolomide-resistant glioma cells. Journal of Neurosurgery, 106, 646–651. 6. Aoki, T., Mizutani, T., Ishikawa, M., Sugiyama, K., & Hashimoto, N. (2003). A first feasibility study of temozolomide for Japanese patients with recurrent anaplastic astrocytoma and glioblastoma multiforme. International Journal of Clinical Oncology, 8, 301–304. 7. Chen, Y., Huang, J. H., Ning, Y., & Shen, Z. Y. (2011). Icariin and its pharmaceutical efficacy: Research progress of molecular mechanism. Zhong Xi Yi Jie He Xue Bao, 9, 1179–1184. 8. Zhang, D. C., Liu, J. L., Ding, Y. B., Xia, J. G., & Chen, G. Y. (2013). Icariin potentiates the antitumor activity of gemcitabine in gallbladder cancer by suppressing NF-kappaB. Acta Pharmacologica Sinica, 34, 301–308. 9. Huang, X., Zhu, D., & Lou, Y. (2007). A novel anticancer agent, icaritin, induced cell growth inhibition, G1 arrest and mitochondrial transmembrane potential drop in human prostate
Cell Biochem Biophys
10.
11.
12.
13.
14. 15.
16.
17.
18.
19.
20.
carcinoma PC-3 cells. European Journal of Pharmacology, 564, 26–36. Li, S., Dong, P., Wang, J., Zhang, J., Gu, J., Wu, X., et al. (2010). Icariin, a natural flavonol glycoside, induces apoptosis in human hepatoma SMMC-7721 cells via a ROS/JNK-dependent mitochondrial pathway. Cancer Letters, 298, 222–230. Lin, C. C., Ng, L. T., Hsu, F. F., Shieh, D. E., & Chiang, L. C. (2004). Cytotoxic effects of Coptis chinensis and Epimedium sagittatum extracts and their major constituents (berberine, coptisine and icariin) on hepatoma and leukaemia cell growth. Clinical and Experimental Pharmacology and Physiology, 31, 65–69. Wang, Y., Dong, H., Zhu, M., Ou, Y., Zhang, J., Luo, H., et al. (2010). Icariin exterts negative effects on human gastric cancer cell invasion and migration by vasodilator-stimulated phosphoprotein via Rac1 pathway. European Journal of Pharmacology, 635, 40–48. Wang, Q., Hao, J., Pu, J., Zhao, L., Lu, Z., Hu, J., et al. (2011). Icariin induces apoptosis in mouse MLTC-10 Leydig tumor cells through activation of the mitochondrial pathway and down-regulation of the expression of piwil4. International Journal of Oncology, 39, 973–980. Li, L., & Wang, X. M. (2008). Progress of pharmacological research on icariin. Zhongguo Zhong yao za zhi, 33, 2727–2732. Chou, T. C., & Talalay, P. (1984). Quantitative analysis of dose– effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Advances in Enzyme Regulation, 22, 27–55. Wang, Y. W., Wang, S. J., Zhou, Y. N., Pan, S. H., & Sun, B. (2012). Escin augments the efficacy of gemcitabine through down-regulation of nuclear factor-kappaB and nuclear factorkappaB-regulated gene products in pancreatic cancer both in vitro and in vivo. Journal of Cancer Research and Clinical Oncology, 138, 785–797. Stupp, R., Mason, W. P., van den Bent, M. J., Weller, M., Fisher, B., Taphoorn, M. J., et al. (2005). Organisation for, T. Treatment of Cancer Brain, G. Radiotherapy, G. National Cancer Institute of Canada Clinical Trials, Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. The New England journal of medicine, 352, 987–996. Ma, H. R., Wang, J., Chen, Y. F., Chen, H., Wang, W. S., & Aisa, H. A. (2014). Icariin and icaritin stimulate the proliferation of SKBr 3 cells through the GPER1-mediated modulation of the EGFR-MAPK signaling pathway. International Journal of Molecular Medicine, 33, 1627–1634. Li, F., Gong, Q. H., Wu, Q., Lu, Y. F., & Shi, J. S. (2010). Icariin isolated from Epimedium brevicornum Maxim attenuates learning and memory deficits induced by d-galactose in rats. Pharmacology, Biochemistry and Behavior, 96, 301–305. Yuan, Y., Xue, X., Guo, R. B., Sun, X. L., & Hu, G. (2012). Resveratrol enhances the antitumor effects of temozolomide in
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
glioblastoma via ROS-dependent AMPK-TSC-mTOR signaling pathway. CNS Neuroscience & Therapeutics, 18, 536–546. Filippi-Chiela, E. C., Thome, M. P., Bueno e Silva, M. M., Pelegrini, A. L., Ledur, P. F., Garicochea, B., et al. (2013). Resveratrol abrogates the temozolomide-induced G2 arrest leading to mitotic catastrophe and reinforces the temozolomide-induced senescence in glioma cells. BMC cancer, 13, 147. Huang, H., Lin, H., Zhang, X., & Li, J. (2012). Resveratrol reverses temozolomide resistance by downregulation of MGMT in T98G glioblastoma cells by the NF-kappaB-dependent pathway. Oncology Reports, 27, 2050–2056. Kunnumakkara, A. B., Diagaradjane, P., Guha, S., Deorukhkar, A., Shentu, S., Aggarwal, B. B., et al. (2008). Curcumin sensitizes human colorectal cancer xenografts in nude mice to gammaradiation by targeting nuclear factor-kappaB-regulated gene products. Clinical Cancer Research, 14, 2128–2136. Naugler, W. E., & Karin, M. (2008). NF-kappaB and canceridentifying targets and mechanisms. Current Opinion in Genetics & Development, 18, 19–26. Nogueira, L., Ruiz-Ontanon, P., Vazquez-Barquero, A., Moris, F., & Fernandez-Luna, J. L. (2011). The NFkappaB pathway: A therapeutic target in glioblastoma. Oncotarget, 2, 646–653. Atkinson, G. P., Nozell, S. E., & Benveniste, E. T. (2010). NFkappaB and STAT3 signaling in glioma: targets for future therapies. Expert Review of Neurotherapeutics, 10, 575–586. Caporali, S., Levati, L., Graziani, G., Muzi, A., Atzori, M. G., Bonmassar, E., et al. (2012). NF-kappaB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drug. Journal of Translational Medicine, 10, 252. Markiewicz-Zukowska, R., Borawska, M. H., Fiedorowicz, A., Naliwajko, S. K., Sawicka, D., & Car, H. (2013). Propolis changes the anticancer activity of temozolomide in U87MG human glioblastoma cell line. BMC Complementary and Alternative Medicine, 13, 50. Sai, K., Li, W. Y., Chen, Y. S., Wang, J., Guan, S., Yang, Q. Y., et al. (2014). Triptolide synergistically enhances temozolomideinduced apoptosis and potentiates inhibition of NF-kappaB signaling in glioma initiating cells. The American Journal of Chinese Medicine, 42, 485–503. Brassesco, M. S., Roberto, G. M., Morales, A. G., Oliveira, J. C., Delsin, L. E., Pezuk, J. A., et al. (2013). Inhibition of NF-kappa B by Dehydroxymethylepoxyquinomicin Suppresses Invasion and Synergistically Potentiates Temozolomide and gamma-radiation cytotoxicity in glioblastoma Cells. Chemotherapy Research and Practice, 2013, 593020. Chen, M., Rose, A. E., Doudican, N., Osman, I., & Orlow, S. J. (2009). Celastrol synergistically enhances temozolomide cytotoxicity in melanoma cells. Molecular Cancer Research, 7, 1946–1953.
123
ORIGINAL PAPER
Synergistic Anti-Cancer Effects of Icariin and Temozolomide in Glioblastoma Lijuan Yang • Yuexun Wang • Hua Guo Meiling Guo
•
Ó Springer Science+Business Media New York 2014
Abstract Glioblastoma is an aggressive malignancy, which is associated with poor prognosis. Temozolomide (TMZ) has been showed to be an effective chemotherapeutic agent for glioblastoma treatment; however, the response rate is not satisfactory. Icariin is a natural compound with anti-cancer activity against a variety of cancers. This study is designed to determine whether icariin could potentiate the antitumor activity of TMZ in glioblastoma. Cell proliferation and apoptosis were measured using MTT assay and flow cytometry, respectively. Expression of apoptosis and proliferation-related molecules was detected by Western blotting while NF-jB activity was detected by ELISA. Icariin dose-dependently inhibited proliferation and induced apoptosis in tested glioblastoma cell lines. Icariin enhanced the anti-tumor activity of TMZ in vitro. The anti-tumor activity of icariin and the enhanced antitumor activity of TMZ by icariin correlated with suppression of NF-jB activity. Our results showed that icariin exhibited anti-tumor activity and potentiated the anti-tumor activity of TMZ in glioblastoma, at least in part, by inhibiting NF-jB activity. Although more studies including clinical trials are needed, this study provides insight for using icariin as a chemosensitizing agent in clinic settings.
Dr. Lijuan Yang and Dr. Yuexun Wang have contributed equally to this study. L. Yang H. Guo Shandong Provincial Hospital Affiliated to Shandong University, Shandong University, No.324, JingWu Road, Ji’nan 250021, China Y. Wang M. Guo (&) CT Department, First People’s Hospital of Jining, Shandong 272011, China e-mail: [email protected]
Keywords
GBM TMZ Icariin AMPK
Introduction Glioblastoma multiforme (GBM), accounting for approximately 60 % of all brain tumors, is the most common type of primary brain tumors in adults [1, 2]. GBMs are one of the most lethal, highly invasive, and least effectively treated solid tumors. Despite aggressive therapy regimen including maximal surgical resection, combined radiation/ chemotherapy, the recurrence of GBM is still quite common [3, 4]. Due to the ability to infiltrate diffusely into the normal brain parenchyma, the prognosis of patients with the highest grade malignant glioma, GBM, remains poor with a median survival of at 12–15 months and a 5-year survival rate of 2 % [4]. Therefore, improvement of treatment options for patients with GBM is imperative. Mounting evidence has showed that the inability of many chemotherapeutic agents to pass through the blood– brain barrier, and their low efficacy for induction of apoptosis have largely compromised their value in treatment of glioblastoma. Among the chemotherapeutic agents used to treat GBM, alkylating agent temozolomide (TMZ), (8-carbamoyl-3-methylimidazo [5,1-d]-1,2,3,5-tetrazin4(3H)-one) belonging to imidazotertrazines has been considered a promising one due to its ability of crossing the blood–brain barrier effectively with few myelocytotoxic effects [5]. Clinical data showed that the average survival of patients with glioblastoma after TMZ treatment is 22 months [6], which is still not satisfactory. Different regimens to attenuate resistance and increase the efficacy of TMZ have been investigated. Among the potential chemosensitizers, the natural compounds, such as icariin, may be an ideal agent for GBM therapy. Icariin, a
123
Cell Biochem Biophys
flavonoid isolated from Epimedi herba, is considered to be the major active ingredient of traditional Chinese medicine E. herba. Icariin has a variety of pharmacological activities including anti-inflammatory, anti-depressant, male sexual function improvement, cardiovascular protection, enhancing bone healing and neuroprotective while it has a low toxicity and can be given in relatively high doses without adverse effects in humans [7, 8]. Emerged evidence suggested that icariin might be a potential chemopreventive and/or chemotherapeutic in human cancer. Icariin exerted anti-proliferative effect in vitro on cell growth in mouse Leydig tumor cells, human lung cancer cells, human gastric cancer cells, human leukemia cells, human breast cancer cells, and human hepatoma cells [9–13]. More importantly, icariin can pass through blood–brain barrier into brain tissues [14]. Therefore, the present study was designed to investigate whether icariin can potentiate the antitumor effect of TMZ on the human GBM in U87MG cells and to demonstrate the involved mechanisms in the additive antitumor effects exerted by TMZ and icariin.
Materials and Methods Cell Culture In this study, a human GBM cell line U87MG was used as model cell line. Cells were cultured in DMEM supplemented with 10 % FBS, 100 U/mL penicillin and 100 lg/ mL streptomycin, and maintained in a humidified atmosphere of 95 % air and 5 % CO2 at 37 °C. MTT Assay For the cytotoxicity assay, 1.0 9 104 glioblastoma cells per well were seeded onto 96-well plates. After overnight incubation, the cells adhered to the plate. Following treatment with icariin or/and TMZ, 20 lL of MTT solution (5 mg/ml in PBS) was added to each well and incubated for 2 h. MTT formazan was dissolved in 150 lL of isopropanol and the absorbance was measured at 595 nm with an ELISA reader (Tecan Group Ltd, Ma¨nnedorf, Switzerland). H3-Thymidine Incorporation U87MG cells were plated in 24-well plates and exposed to a medium containing DMSO (Vehicle), TMZ, icariin, or icariin with TMZ. Cells were cultured 48 h prior to the addition of 0.5 lCi of H3-thymidine per well. After 4-h of incubation, the medium was removed and cells were washed twice with cold 0.05 M Tris–HCl and 5 % trichloroacetic acid, scrapped, and transferred to a scintillation cocktail. The level of
123
incorporated H3-thymidine was assessed using Beckman liquid scintillation counter. Apoptosis Analysis Cell apoptosis was determined using a FITC Annexin V apoptosis kit (BD Pharmingen, Franklin Lakes, NJ) according to the manufacturer’s instructions. Following drug treatment, the cell suspension was prepared using 0.125 % trypsin and was rinsed and centrifuged with icecold PBS at 1,000 rpm for 5 min. Cells were then resuspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2) at a concentration of 1 9 106 cells/ml. Cells were stained with annexin V-FITC and propidium (PI) for 20 min away from the light before analysis by a flow cytometer (Beckman Coulter Inc, Miami, Florida, USA). Cell Migration and Invasion Assays For cell invasion assay, Boyden chambers containing Transwell (Corning Costar Corp., Cambridge, MA) membrane filters with a pore size of 8 lm were used. Matrigel (60 ll) diluted in optimum was coated in invasion chambers rehydrated by adding 0.5 ml of RPMI-1640 medium to the upper chambers at 37 °C overnight. After rehydration, the medium was carefully removed, and 2 9 104 cells were added to the upper chambers in triplicate. The lower compartments of the migration and invasion chambers were filled with DMEM-F12 medium containing 1 % bovine serum albumin (Sigma) at concentrations of 5 and 20 %, respectively. After 48 h, nonmigrating cells on the upper surface of the filters were swept by cotton swabs. The migrated and invaded cells on the lower surface of the membrane were fixed and then stained with methanol mixed crystal violet. The number of cells was counted under a microscope. All assays were run independently three times. Western Blot Analysis Following treatment, the expression levels of signaling molecules in the glioblastoma cells were detected using Western blot analysis. Western blot analysis was performed using a standard protocol. The primary antibodies used in this study were as follows: rabbit anti-caspase-3 (Cell Signaling Technology, Inc., Danvers, MA, USA) and rabbit anti-PARP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, 1:2000). The secondary antibodies used in this study were alkaline phosphatase peroxidase-conjugated anti-rabbit IgG. Detection was performed by the BCIP/NBT Alkaline Phosphatase Color Development Kit
Cell Biochem Biophys Fig. 1 Effect of TMZ or/and icariin on cell viability and H3thymidine incorporation. Cells were treated with indicated concentration of icariin, TMZ or both (TMZ 200 lM, icariin 10 lM) for 48 h. *P \ 0.05 versus vehicle; **P \ 0.01 versus vehicle; ^P \ 0.05 versus TMZ only
(Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s instructions. NF-jB Activity Analysis Following treatment, nuclear extract was prepared using a nuclear extract kit (Active Motif, Carlsbad, CA) and activity of NF-jB p65 was examined using an ELISA kit (Active Motif, Carlsbad, CA). Statistical Analysis All data were expressed as Mean ± SD and represented the results of three separated experiments each performed in quadruplicate unless otherwise stated. The Student’s test was used to evaluate the statistical significance. P \ 0.05 was considered as statistical significant.
Results Icariin Enhances the Growth Inhibition Effect of TMZ in GBM Cells To explore whether icariin could sensitize glioblastoma cells to TMZ, we examined the effect of treatment with TMZ alone or in combination with icariin (ICA) on the growth U87MG cells. As shown in Fig. 1a, TMZ treatment resulted in a dose-dependent loss of cell viability. Relative to the control, U87MG cell viability was reduced to 49.1 ± 6.2 % relative to control after 200 lM TMZ treatment for 48 h. The cytotoxicity of icariin was also
examined. As shown in Fig. 1b, treatment with icariin alone also dose-dependent inhibited the growth of U87MG cells. Since icariin at 10 lM significantly inhibited cell growth in U87MG cells, we chose 10 lM icariin in the combinational treatment with TMZ. As shown in Fig. 1c, combination treatment with TMZ and icariin dramatically reduced viable U87MG cells to approximately 26.5 ± 8.1 %. To evaluate the synergism, we calculated the combination index value according to Chou’s method [15]. Results showed that icariin exerted synergistically antitumor effect on GBM cell line U87MG when combined with TMZ (CI = 0.694).
The Effect of Icariin and Icariin with TMZ on H3Thymidine Incorporation in the Glioblastoma Cell Line (U87MG) The effects of different treatments on proliferation were also examined with H3-thymidine incorporation assay. As shown in Fig. 1d, our results revealed a strong inhibitory potential on U87MG cells proliferation after a 48-h exposition demonstrated by almost 30 % decrease of H3-thymidine incorporation compared to control. In contrast, icariin at a dose of 10 lM lowered DNA synthesis by approximately 50 % in relation to control. Moreover, we found that a combination of TMZ and icariin was significantly more powerful to arrest cell division (about 36 % of control) than TMZ or icariin alone (about 70 % of control). Taken together, our results demonstrated that a combination of icariin with TMZ exhibited the synergistic effect decreasing proliferation in U87MG cells.
123
Cell Biochem Biophys Fig. 2 Cell apoptosis induced by TMZ, icariin and combination of icariin and TMZ in U87MG cells. Cells were treated with indicated concentration of icariin (10 lM), TMZ (200 lM) or both for 48 h. Cell apoptosis was analyzed by flow cytometry while levels of capase-3 and PARP were determined by Western blots. Flow cytometric graphs and Western blots shown were representative of three experiments. *P \ 0.05 versus vehicle; ^P \ 0.05 versus TMZ only
Icariin Sensitizes GBM to TMZ-Induced Apoptosis Our before mentioned results showed that icariin could enhance the inhibitory effect of TMZ on U87MG cell proliferation. Therefore, we examined whether the potentiated anti-proliferative effect was mediated by apoptosis. Compared with treatment with TMZ alone, combination treatment with icariin and TMZ induced significantly more apoptosis in tested U87MG cells (Fig. 2a). In addition, the level of activated caspase-3 and cleaved PARP were also examined. As shown in Fig. 2b, joint treatment of icariin and TMZ caused a more prominent elevation in activated caspase-3 and cleaved PARP compare to monotherapy. Taken together, our results suggested that the pro-apoptotic effect of TMZ combined with icariin is superior to either one as a solo treatment, indicating that icariin potentiates the cytotoxic effect of TMZ by inducing apoptosis. Combination Treatment with Icariin and TMZ Inhibits Cell Migration and Invasion Next, the effect of combination treatment on cell migration and invasion was examined. As shown in Fig. 3a, both icariin and TMZ exerted inhibitory effect on cell migration. However, icariin cooperated with TMZ was significantly more effective in inhibiting cell migration compared to TMZ or icariin used as single agent. In addition, as shown
123
in Fig. 3b, TMZ combined with icariin resulted in significantly greater inhibitory effects on cell invasion. Taken together, our results showed that combination of TMZ and icariin significantly altered the invasion and migration capacities of U87MG cells, as shown by the markedly decreased cell membrane-penetrating capacity. The Effect of Icariin and Icariin with TMZ on NF-jB Activity in Glioblastoma Cell Line (U87MG) Emerging evidences showed that NF-jB play a crucial role in cell proliferation, invasion, apoptosis inhibition, chemoresistance and radioresistance [16]. Therefore, we examined the NF-jB activity to investigate whether icariin potentiated the anti-cancer effect of TMZ in U87MG cells by modulating NF-jB activity. The U87MG cells were treated with TMZ (200 lM) or icariin (10 lM) alone or a combination of TMZ and icariin (200 and 10 lM, respectively) for 48 h. As shown in Fig. 4, we found that TMZ did not markedly alter the NF-jB activity in U87MG cells since nuclear localization of both p65 almost remained at the level similar to vehicle. In contrast, icariin alone led to a remarkable decrease of nuclear content of p65 subunits. Moreover, the incubation of cells with both of TMZ and icariin resulted in a more profound reduction of NF-jB activity, which clearly suggested a synergic effect of icariin and TMZ. Based on our findings, we suggested that the
Cell Biochem Biophys Fig. 3 Cell migration and invasion caused by TMZ, icariin and combination of icariin and TMZ in U87MG cells. Cells were treated with indicated concentration of icariin (10 lM), TMZ (200 lM) or both for 48 h. Cell migration and invasion were assessed by transwell assay. *P \ 0.05 versus vehicle; ^P \ 0.05 versus TMZ only
Fig. 4 NF-jB activity suppressed by TMZ, icariin and combination of icariin and TMZ in U87MG cells. Cells were treated with indicated concentration of icariin (10 lM), TMZ (200 lM) or both for 48 h. NF-jB activity was determined by ELISA kit. *P \ 0.05 versus vehicle; ^P \ 0.05 versus TMZ only
sensitization of GBM cells to TMZ by Icariin correlated with suppressed NF-jB activity.
Discussions Currently, surgical resection followed by radiotherapy and chemotherapy has been considered as standard therapy.
TMZ, an alkylating agent, is the only drug shown to improve survival when administered with concomitant radiotherapy [17]. However, still more than 90 % of patients die within 3 years of diagnosis despite the employment of this standard therapy incorporating TMZ [17]. Therefore, novel therapeutic strategies are imperative to improve chemotherapy response. The goal of this study was to investigate therapeutic effect of sensitization of GBM to TMZ using a flavonoid compound, icariin. Here, we showed that icariin presented cytotoxic effect and enhanced the antitumor effect of TMZ in GBM cell line U87MG, which may at least partially mediated by a reduced activity of NF-jB. The antitumor activities of icariin has been examined in a variety of human cancer cell lines and various mechanisms have been proposed to explain the anti-tumor effects of icariin, including GPER1-mediated modulation of the EGFR-MAPK signaling pathway [18], activating ROS/ JNK-dependent mitochondrial pathway [10], suppressing piwil4 expression [13], and downregulating Rac1 and vasodilator-stimulated phosphoprotein (VASP) [12]. However, the effect of Icariin against human glioma cells is not known. Recently, emerging evidences demonstrated that icariin might pass through the blood–brain barrier [19], which highlights the potential of icariin to be used as a chemotherapeutic agent in the treatment of glioma. In this
123
Cell Biochem Biophys
study, our results showed that icariin inhibited proliferation, induced apoptosis, prevented migration and invasion in U87MG cells, which for the first time demonstrated the antitumor activities of icariin against GBM. In the past decade, a number of flavonoid compounds have been found to be able to enhance the antitumor effect of TMZ in glioblastoma cells. It has reported that resveratrol enhanced the antitumor of TMZ in glioblastoma via multiple mechanisms including activating ROS-dependent AMPK-TSC-mTOR signaling pathway, abrogating the TMZ-induced G2 arrest leading to mitotic catastrophe and reinforcing the TMZ-induced senescence in glioma cells, as well as Resveratrol reverses TMZ resistance by downregulating MGMT by the NF-jB-dependent pathway [20– 22]. Another flavonoid compound, curcumin, has also been found to sensitize glioblastoma to TMZ by simultaneously generating ROS and disrupting Akt/mTOR signaling [23]. In line with these previously studies, our results showed that icariin, by inhibiting NF-jB activity, potentiated the in vitro anti-tumor activity of TMZ in GBM. The role of NF-jB as one of the major transcription factors associated with cancer has been well established with evidence showing the involvement of NF-jB in many hallmarks of cancer development, including growth factorindependent proliferation, inhibition of apoptosis, limitless replicative potential, and tissue invasion and metastasis [24]. In the context of GBM, the association between NFjB and GBM is known for more than 15 years and a number of studies have reported that that the NF-jB pathway is constitutively activated or is upregulated in response to different stimuli, mainly cytokines, in GBM cells [25]. Although little is known about the precise mechanism how NF-jB is activated in GBMs, there are numerous proteins and pathways dysregulated in GBMs that may cause NF-jB activation [26] and activation of NF-jB by several conventional chemotherapeutics also resulted in unfavorable clinical outcomes [26]. Very recently, it has been reported that activation of NF-jB is upregulated during differentiation of glioblastoma initiating cells (GICs) [24]. Taken together, all these results highlight the potential of NF-jB to be a novel target for GBM therapy. Indeed, a number of drugs with inhibitory activities on NF-jB signaling pathway have been investigated for the efficacy against GBM. For example, the antiinflammatory drug sulfasalazine, one of the more promising preclinical drugs for glioma in the last 5 year which has showed marked therapeutic efficacy on mice model, has been suggested to work via inhibition of NF-jB activity. Consistent with previous studies, our results here also showed that the antitumor effect of icariin against GBM cells was, at least in part, mediated by targeting NF-jB signaling pathway. On the other hand, Caporali et al. have reported that NF-jB is activated in response to TMZ in an
123
AKT-dependent manner and confers protection against the growth suppressive effect of the drug [27]. Furthermore, a number of studies have showed that the ant-tumor effect of TMZ was enhanced by different agents via inhibiting NFjB. In GBM, the efficacy of TMZ was augmented when NF-jB activity was inhibited by propolis [28], triptolide [29], dehydroxymethylepoxyquinomicin [30], or resveratrol [22]. Besides GBM, enhanced chemosensitivity to TMZ was also reported to be associated with decreased NF-jB activity in colorectal cancer and melanoma [27, 31]. In agreement with these studies, we found that the sensitization of GBM to TMZ by Icariin correlated with suppressed NF-jB activity. Taken together, these results indicated that the regulation of NF-jB activity could be a possible new treatment for the chemosensitization of GBM to TMZ. In summary, the present study found that icariin potentiated the antitumor effects of TMZ in GBM in vitro and the synergistic anti-tumor activities was associated with suppression of NF-jB activity. Given the low toxicity of icariin to normal tissue and its ability of passing cross blood–brain barrier, our preset results suggest that icariin may be used as chemosensitizer of TMZ in GBM.
References 1. Surawicz, T. S., Davis, F., Freels, S., Laws, E. R, Jr., & Menck, H. R. (1998). Brain tumor survival: Results from the National Cancer Data Base. Journal of Neuro-oncology, 40, 151–160. 2. Furnari, F. B., Fenton, T., Bachoo, R. M., Mukasa, A., Stommel, J. M., Stegh, A., et al. (2007). Malignant astrocytic glioma: Genetics, biology, and paths to treatment. Genes & Development, 21, 2683–2710. 3. Grossman, S. A., & Batara, J. F. (2004). Current management of glioblastoma multiforme. Seminars in Oncology, 31, 635–644. 4. Quick, A., Patel, D., Hadziahmetovic, M., Chakravarti, A., & Mehta, M. (2010). Current therapeutic paradigms in glioblastoma. Reviews on Recent Clinical Trials, 5, 14–27. 5. Uzzaman, M., Keller, G., & Germano, I. M. (2007). Enhanced proapoptotic effects of tumor necrosis factor-related apoptosisinducing ligand on temozolomide-resistant glioma cells. Journal of Neurosurgery, 106, 646–651. 6. Aoki, T., Mizutani, T., Ishikawa, M., Sugiyama, K., & Hashimoto, N. (2003). A first feasibility study of temozolomide for Japanese patients with recurrent anaplastic astrocytoma and glioblastoma multiforme. International Journal of Clinical Oncology, 8, 301–304. 7. Chen, Y., Huang, J. H., Ning, Y., & Shen, Z. Y. (2011). Icariin and its pharmaceutical efficacy: Research progress of molecular mechanism. Zhong Xi Yi Jie He Xue Bao, 9, 1179–1184. 8. Zhang, D. C., Liu, J. L., Ding, Y. B., Xia, J. G., & Chen, G. Y. (2013). Icariin potentiates the antitumor activity of gemcitabine in gallbladder cancer by suppressing NF-kappaB. Acta Pharmacologica Sinica, 34, 301–308. 9. Huang, X., Zhu, D., & Lou, Y. (2007). A novel anticancer agent, icaritin, induced cell growth inhibition, G1 arrest and mitochondrial transmembrane potential drop in human prostate
Cell Biochem Biophys
10.
11.
12.
13.
14. 15.
16.
17.
18.
19.
20.
carcinoma PC-3 cells. European Journal of Pharmacology, 564, 26–36. Li, S., Dong, P., Wang, J., Zhang, J., Gu, J., Wu, X., et al. (2010). Icariin, a natural flavonol glycoside, induces apoptosis in human hepatoma SMMC-7721 cells via a ROS/JNK-dependent mitochondrial pathway. Cancer Letters, 298, 222–230. Lin, C. C., Ng, L. T., Hsu, F. F., Shieh, D. E., & Chiang, L. C. (2004). Cytotoxic effects of Coptis chinensis and Epimedium sagittatum extracts and their major constituents (berberine, coptisine and icariin) on hepatoma and leukaemia cell growth. Clinical and Experimental Pharmacology and Physiology, 31, 65–69. Wang, Y., Dong, H., Zhu, M., Ou, Y., Zhang, J., Luo, H., et al. (2010). Icariin exterts negative effects on human gastric cancer cell invasion and migration by vasodilator-stimulated phosphoprotein via Rac1 pathway. European Journal of Pharmacology, 635, 40–48. Wang, Q., Hao, J., Pu, J., Zhao, L., Lu, Z., Hu, J., et al. (2011). Icariin induces apoptosis in mouse MLTC-10 Leydig tumor cells through activation of the mitochondrial pathway and down-regulation of the expression of piwil4. International Journal of Oncology, 39, 973–980. Li, L., & Wang, X. M. (2008). Progress of pharmacological research on icariin. Zhongguo Zhong yao za zhi, 33, 2727–2732. Chou, T. C., & Talalay, P. (1984). Quantitative analysis of dose– effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Advances in Enzyme Regulation, 22, 27–55. Wang, Y. W., Wang, S. J., Zhou, Y. N., Pan, S. H., & Sun, B. (2012). Escin augments the efficacy of gemcitabine through down-regulation of nuclear factor-kappaB and nuclear factorkappaB-regulated gene products in pancreatic cancer both in vitro and in vivo. Journal of Cancer Research and Clinical Oncology, 138, 785–797. Stupp, R., Mason, W. P., van den Bent, M. J., Weller, M., Fisher, B., Taphoorn, M. J., et al. (2005). Organisation for, T. Treatment of Cancer Brain, G. Radiotherapy, G. National Cancer Institute of Canada Clinical Trials, Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. The New England journal of medicine, 352, 987–996. Ma, H. R., Wang, J., Chen, Y. F., Chen, H., Wang, W. S., & Aisa, H. A. (2014). Icariin and icaritin stimulate the proliferation of SKBr 3 cells through the GPER1-mediated modulation of the EGFR-MAPK signaling pathway. International Journal of Molecular Medicine, 33, 1627–1634. Li, F., Gong, Q. H., Wu, Q., Lu, Y. F., & Shi, J. S. (2010). Icariin isolated from Epimedium brevicornum Maxim attenuates learning and memory deficits induced by d-galactose in rats. Pharmacology, Biochemistry and Behavior, 96, 301–305. Yuan, Y., Xue, X., Guo, R. B., Sun, X. L., & Hu, G. (2012). Resveratrol enhances the antitumor effects of temozolomide in
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
glioblastoma via ROS-dependent AMPK-TSC-mTOR signaling pathway. CNS Neuroscience & Therapeutics, 18, 536–546. Filippi-Chiela, E. C., Thome, M. P., Bueno e Silva, M. M., Pelegrini, A. L., Ledur, P. F., Garicochea, B., et al. (2013). Resveratrol abrogates the temozolomide-induced G2 arrest leading to mitotic catastrophe and reinforces the temozolomide-induced senescence in glioma cells. BMC cancer, 13, 147. Huang, H., Lin, H., Zhang, X., & Li, J. (2012). Resveratrol reverses temozolomide resistance by downregulation of MGMT in T98G glioblastoma cells by the NF-kappaB-dependent pathway. Oncology Reports, 27, 2050–2056. Kunnumakkara, A. B., Diagaradjane, P., Guha, S., Deorukhkar, A., Shentu, S., Aggarwal, B. B., et al. (2008). Curcumin sensitizes human colorectal cancer xenografts in nude mice to gammaradiation by targeting nuclear factor-kappaB-regulated gene products. Clinical Cancer Research, 14, 2128–2136. Naugler, W. E., & Karin, M. (2008). NF-kappaB and canceridentifying targets and mechanisms. Current Opinion in Genetics & Development, 18, 19–26. Nogueira, L., Ruiz-Ontanon, P., Vazquez-Barquero, A., Moris, F., & Fernandez-Luna, J. L. (2011). The NFkappaB pathway: A therapeutic target in glioblastoma. Oncotarget, 2, 646–653. Atkinson, G. P., Nozell, S. E., & Benveniste, E. T. (2010). NFkappaB and STAT3 signaling in glioma: targets for future therapies. Expert Review of Neurotherapeutics, 10, 575–586. Caporali, S., Levati, L., Graziani, G., Muzi, A., Atzori, M. G., Bonmassar, E., et al. (2012). NF-kappaB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drug. Journal of Translational Medicine, 10, 252. Markiewicz-Zukowska, R., Borawska, M. H., Fiedorowicz, A., Naliwajko, S. K., Sawicka, D., & Car, H. (2013). Propolis changes the anticancer activity of temozolomide in U87MG human glioblastoma cell line. BMC Complementary and Alternative Medicine, 13, 50. Sai, K., Li, W. Y., Chen, Y. S., Wang, J., Guan, S., Yang, Q. Y., et al. (2014). Triptolide synergistically enhances temozolomideinduced apoptosis and potentiates inhibition of NF-kappaB signaling in glioma initiating cells. The American Journal of Chinese Medicine, 42, 485–503. Brassesco, M. S., Roberto, G. M., Morales, A. G., Oliveira, J. C., Delsin, L. E., Pezuk, J. A., et al. (2013). Inhibition of NF-kappa B by Dehydroxymethylepoxyquinomicin Suppresses Invasion and Synergistically Potentiates Temozolomide and gamma-radiation cytotoxicity in glioblastoma Cells. Chemotherapy Research and Practice, 2013, 593020. Chen, M., Rose, A. E., Doudican, N., Osman, I., & Orlow, S. J. (2009). Celastrol synergistically enhances temozolomide cytotoxicity in melanoma cells. Molecular Cancer Research, 7, 1946–1953.
123
