惡性神經膠質瘤為中樞神經系統中最常見好發於成人的原發性腦瘤，具有高度增生、威脅生命和侵略正常組織的能力。即使近年來在醫療科技的進步下，患有惡性神經膠質瘤的病患預後仍然非常差，大部分的病人經過治療後存活率只有12到15個月。我們實驗室在先前的研究發現合成的喹唑啉酮衍生物2-(naphthalene-1-yl)-6-pyrrolidinyl-4-quinazolinone (MJ-66) 在惡性神經膠質瘤細胞中會透過使細胞週期停止在G2/M 時期和引起有絲分裂災變，導致惡性神經膠質瘤細胞走向死亡，然而其中詳細的機制仍未釐清。已有許多文獻指出DNA損傷與有絲分裂災變有關聯性。此外，併用治療被認為是能增加治療效果以及增加病人存活率的一種治療方式。我們實驗室在先前的研究也發現米諾環素是透過引發自我吞噬作用造成惡性神經膠質瘤細胞死亡，可能是對抗惡性神經膠質瘤的治療藥物。因此本研究將探討MJ-66是否透過引發DNA損傷而導致神經膠質瘤細胞死亡，並且與米諾環素是否有協同作用。
首先，利用西方點墨法和免疫螢光染色法在神經膠質瘤細胞中發現MJ-66會引發γH2AX的活化，接著會造成Chk1和Cdc25C磷酸化的過度表現。此外，同時給予UCN-01(Chk1抑制劑)可以回復MJ-66所活化的Cdc25C和caspase 3。在原位神經膠質瘤動物模式中也發現MJ-66可以抑制腫瘤生長和延長存活時間。以上結果顯示MJ-66會引發DNA損傷，活化Chk1/Cdc25C路徑，並且在動物模式中有抗腫瘤的效果。我們進一步地利用藥理併用治療的方式去探討MJ-66和米諾環素對於惡性神經膠質瘤的作用。我們發現合併使用MJ-66與米諾環素的治療方式具有協同效果，並且我們也發現MJ-66合併米諾環素不會增加caspase 3的活性和影響自我吞噬作用。然而，我們發現MJ-66合併米諾環素的作用是透過引發更多DNA損傷去抑制腫瘤細胞生長和導致死亡。因此我們推測MJ-66合併米諾環素協同抑制惡性神經膠質瘤細胞生長和增加細胞毒性的作用是透過引發更多DNA損傷。最後，原位神經膠質瘤動物實驗結果顯示MJ-66合併米諾環素有更好的療效。綜合以上實驗可得知，合併使用MJ-66與米諾環素具有更好的抗癌效果，希望這樣的發現未來可以在對抗惡性神經膠質瘤提供一個新的治療方向。

Malignant gliomas are the most prevalent primary brain tumor in adults and are usually growing rapidly, life-threatening and invasive. Despite recent advances in treatment of malignant gliomas, the prognosis of patients remains very poor. Most patients with malignant gliomas succumb to disease within only 12 to 15 months. In the previous study, our lab found that 2-(Naphthalene-1-yl)-6-pyrrolidinyl- 4-quinazolinone (MJ-66), a synthetic quinazolinone analog, significantly induced glioma cell death by G2/M phase arrest and mitotic catastrophe. However, the detailed mechanisms of MJ-66 against malignant gliomas remain unclear. Several studies have shown that mitotic catastrophe is associated with DNA damage. Besides, combination therapy is believed that could increase therapeutic effects and may contribute to enhance the survival of patients. In previous studies, our lab also found that minocycline (Mino) induced glioma cell death through autophagy as a promising agent against malignant gliomas. Thus, the aim of this study is to investigate whether MJ-66 induces glioma cell death through DNA damage and has synergistic effects with minocycline.
We found that MJ-66 induced γH2AX activation after treated with MJ-66 in a time-dependent manner. Subsequently, MJ-66 interfered with G2/M DNA damage checkpoint through increasing phosphorylated levels of Chk1 and Cdc25C. Besides, UCN-01 (Chk1 inhibitor) reversed MJ-66-induced activations of Cdc25C and caspase 3. Intracranial glioma xenograft animal model showed that MJ-66 could inhibit tumor growth and prolong survival time. These results indicated that MJ-66 induced glioma cells DNA damage, Chk1/Cdc25C pathway activation and had antineoplastic effects in the animal model. Further, we used a pharmacological combination to investigate the effects of MJ-66 and Mino in gliomas. We found that the combined treatment of MJ-66 and Mino had synergistic effects on growth inhibition and cytotoxicity. We also found that the combination of MJ-66 and Mino did not increase the activity of caspase 3 and affect autophagy. However, we found that the combined effects of MJ-66 and Mino were through inducing enhanced DNA damage. Hence, we suggest that the combined effects of MJ-66 and Mino on growth inhibition and cytotoxicity were through inducing enhanced DNA damage. Finally, orthotopic glioma animal model indicated that the group of MJ-66 combined with Mino displayed synergistic effects compared to that treated with MJ-66 or Mino only. These findings provide the evidence that the combination of MJ-66 with Mino which may be developed as a new therapeutic strategy against malignant gliomas.

Abraham, R. T. (2001). Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes & development 15, 2177-2196.

Agnihotri, S., Burrell, K. E., Wolf, A., Jalali, S., Hawkins, C., Rutka, J. T., and Zadeh, G. (2013). Glioblastoma, a brief review of history, molecular genetics, animal models and novel therapeutic strategies. Archivum immunologiae et therapiae experimentalis 61, 25-41.

Andreassen, P. R., Lacroix, F. B., Lohez, O. D., and Margolis, R. L. (2001). Neither p21WAF1 nor 14-3-3sigma prevents G2 progression to mitotic catastrophe in human colon carcinoma cells after DNA damage, but p21WAF1 induces stable G1 arrest in resulting tetraploid cells. Cancer research 61, 7660-7668.

Bergnes, G., Brejc, K., and Belmont, L. (2005). Mitotic kinesins: prospects for antimitotic drug discovery. Current topics in medicinal chemistry 5, 127-145.

Bonner, W. M., Redon, C. E., Dickey, J. S., Nakamura, A. J., Sedelnikova, O. A., Solier, S., and Pommier, Y. (2008). GammaH2AX and cancer. Nature reviews Cancer 8, 957-967.

Butowski, N. A., and Berger, M. (2012a). Malignant Gliomas Part I: Epidemiology, Risk Factors, Prognostic Factors, and Imaging Findings. Contemporary Neurosurgery 34, 1-6.

Butowski, N. A., and Berger, M. (2012b). Malignant Gliomas Part II: Gliomagenesis and Glioblastoma Therapy. Contemporary Neurosurgery 34, 1-8.

Cao, S. L., Feng, Y. P., Zheng, X. L., Jiang, Y. Y., Zhang, M., Wang, Y., and Xu, M. (2006). Synthesis of substituted benzylamino- and heterocyclylmethylamino carbodithioate derivatives of 4-(3H)-quinazolinone and their cytotoxic activity. Archiv der Pharmazie 339, 250-254.

Castedo, M., Perfettini, J. L., Roumier, T., Andreau, K., Medema, R., and Kroemer, G. (2004). Cell death by mitotic catastrophe: a molecular definition. Oncogene 23, 2825-2837.

Chandana, S. R., Movva, S., Arora, M., and Singh, T. (2008). Primary brain tumors in adults. American family physician 77, 1423-1430.

Chinigo, G. M., Paige, M., Grindrod, S., Hamel, E., Dakshanamurthy, S., Chruszcz, M., Minor, W., and Brown, M. L. (2008). Asymmetric synthesis of 2,3-dihydro-2-arylquinazolin-4-ones: methodology and application to a potent fluorescent tubulin inhibitor with anticancer activity. Journal of medicinal chemistry 51, 4620-4631.

Chiu, Y. J., Hour, M. J., Lu, C. C., Chung, J. G., Kuo, S. C., Huang, W. W., Chen, H. J., Jin, Y. A., and Yang, J. S. (2011). Novel quinazoline HMJ-30 induces U-2 OS human osteogenic sarcoma cell apoptosis through induction of oxidative stress and up-regulation of ATM/p53 signaling pathway. Journal of orthopaedic research : official publication of the Orthopaedic Research Society 29, 1448-1456.

Clarke, J., Butowski, N., and Chang, S. (2010). Recent advances in therapy for glioblastoma. Archives of neurology 67, 279-283.

De Bont, R., and van Larebeke, N. (2004). Endogenous DNA damage in humans: a review of quantitative data. Mutagenesis 19, 169-185.

Dent, P., Tang, Y., Yacoub, A., Dai, Y., Fisher, P. B., and Grant, S. (2011). CHK1 inhibitors in combination chemotherapy: thinking beyond the cell cycle. Molecular interventions 11, 133-140.

Domercq, M., and Matute, C. (2004). Neuroprotection by tetracyclines. Trends in pharmacological sciences 25, 609-612.

Encalada, S. E., Willis, J., Lyczak, R., and Bowerman, B. (2005). A spindle checkpoint functions during mitosis in the early Caenorhabditis elegans embryo. Molecular biology of the cell 16, 1056-1070.

Fernandez-Capetillo, O., Celeste, A., and Nussenzweig, A. (2003). Focusing on foci: H2AX and the recruitment of DNA-damage response factors. Cell cycle 2, 426-427.

Fife, R. S., Sledge, G. W., Jr., Roth, B. J., and Proctor, C. (1998). Effects of doxycycline on human prostate cancer cells in vitro. Cancer letters 127, 37-41.

Fragkos, M., and Beard, P. (2011). Mitotic catastrophe occurs in the absence of apoptosis in p53-null cells with a defective G1 checkpoint. PloS one 6, e22946.

Freeman, C. D., Nightingale, C. H., and Quintiliani, R. (1994). Minocycline: old and new therapeutic uses. International journal of antimicrobial agents 4, 325-335.

Gascoigne, K. E., and Taylor, S. S. (2009). How do anti-mitotic drugs kill cancer cells? Journal of cell science 122, 2579-2585.

Giri, R. S., Thaker, H. M., Giordano, T., Chen, B., Nuthalapaty, S., Vasu, K. K., and Sudarsanam, V. (2010). Synthesis and evaluation of quinazolinone derivatives as inhibitors of NF-kappaB, AP-1 mediated transcription and eIF-4E mediated translational activation: inhibitors of multi-pathways involve in cancer. European journal of medicinal chemistry 45, 3558-3563.

Harvey, S. L., Charlet, A., Haas, W., Gygi, S. P., and Kellogg, D. R. (2005). Cdk1-dependent regulation of the mitotic inhibitor Wee1. Cell 122, 407-420.

Hochegger, H., Takeda, S., and Hunt, T. (2008). Cyclin-dependent kinases and cell-cycle transitions: does one fit all? Nature reviews Molecular cell biology 9, 910-916.

Hour, M. J., Huang, L. J., Kuo, S. C., Xia, Y., Bastow, K., Nakanishi, Y., Hamel, E., and Lee, K. H. (2000). 6-Alkylamino- and 2,3-dihydro-3'-methoxy-2-phenyl-4- quinazolinones and related compounds: their synthesis, cytotoxicity, and inhibition of tubulin polymerization. Journal of medicinal chemistry 43, 4479-4487.

Huang, X., Tran, T., Zhang, L., Hatcher, R., and Zhang, P. (2005). DNA damage-induced mitotic catastrophe is mediated by the Chk1-dependent mitotic exit DNA damage checkpoint. Proceedings of the National Academy of Sciences of the United States of America 102, 1065-1070.

Hwang, S. H., Rait, A., Pirollo, K. F., Zhou, Q., Yenugonda, V. M., Chinigo, G. M., Brown, M. L., and Chang, E. H. (2008). Tumor-targeting nanodelivery enhances the anticancer activity of a novel quinazolinone analogue. Molecular cancer therapeutics 7, 559-568.

Imreh, G., Norberg, H. V., Imreh, S., and Zhivotovsky, B. (2011). Chromosomal breaks during mitotic catastrophe trigger gammaH2AX-ATM-p53-mediated apoptosis. Journal of cell science 124, 2951-2963.

Jiang, N., Wang, X., Yang, Y., and Dai, W. (2006). Advances in mitotic inhibitors for cancer treatment. Mini reviews in medicinal chemistry 6, 885-895.

Karachitos, A., Garcia Del Pozo, J. S., de Groot, P. W., Kmita, H., and Jordan, J. (2013). Minocycline mediated mitochondrial cytoprotection: premises for therapy of cerebrovascular and neurodegenerative diseases. Current drug targets 14, 47-55.

Kim, H. S., and Suh, Y. H. (2009). Minocycline and neurodegenerative diseases. Behavioural brain research 196, 168-179.

Lapenna, S., and Giordano, A. (2009). Cell cycle kinases as therapeutic targets for cancer. Nature reviews Drug discovery 8, 547-566.

Li, H. Z., He, H. Y., Han, Y. Y., Gu, X., He, L., Qi, Q. R., Zhao, Y. L., and Yang, L. (2010). A general synthetic procedure for 2-chloromethyl-4(3H)-quinazolinone derivatives and their utilization in the preparation of novel anticancer agents with 4-anilinoquinazoline scaffolds. Molecules 15, 9473-9485.

Lim, S. K., Llaguno, S. R., McKay, R. M., and Parada, L. F. (2011). Glioblastoma multiforme: a perspective on recent findings in human cancer and mouse models. BMB reports 44, 158-164.

Liu, W. T., Huang, C. Y., Lu, I. C., and Gean, P. W. (2013). Inhibition of glioma growth by minocycline is mediated through endoplasmic reticulum stress-induced apoptosis and autophagic cell death. Neuro-oncology.

Liu, W. T., Lin, C. H., Hsiao, M., and Gean, P. W. (2011). Minocycline inhibits the growth of glioma by inducing autophagy. Autophagy 7, 166-175.

Mah, L. J., El-Osta, A., and Karagiannis, T. C. (2010). gammaH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, UK 24, 679-686.

Malhotra, J. D., and Kaufman, R. J. (2007). The endoplasmic reticulum and the unfolded protein response. Seminars in cell & developmental biology 18, 716-731.

Marzaro, G., Guiotto, A., and Chilin, A. (2012). Quinazoline derivatives as potential anticancer agents: a patent review (2007 - 2010). Expert opinion on therapeutic patents 22, 223-252.

Meng, Q., Xu, J., Goldberg, I. D., Rosen, E. M., Greenwald, R. A., and Fan, S. (2000). Influence of chemically modified tetracyclines on proliferation, invasion and migration properties of MDA-MB-468 human breast cancer cells. Clinical & experimental metastasis 18, 139-146.

Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immunological methods 65, 55-63.

Motoyama, N., and Naka, K. (2004). DNA damage tumor suppressor genes and genomic instability. Current opinion in genetics & development 14, 11-16.

Nyberg, K. A., Michelson, R. J., Putnam, C. W., and Weinert, T. A. (2002). Toward maintaining the genome: DNA damage and replication checkpoints. Annual review of genetics 36, 617-656.

Onoda, T., Ono, T., Dhar, D. K., Yamanoi, A., Fujii, T., and Nagasue, N. (2004). Doxycycline inhibits cell proliferation and invasive potential: combination therapy with cyclooxygenase-2 inhibitor in human colorectal cancer cells. The Journal of laboratory and clinical medicine 143, 207-216.

Perry, J., Okamoto, M., Guiou, M., Shirai, K., Errett, A., and Chakravarti, A. (2012). Novel therapies in glioblastoma. Neurology research international 2012, 428565.

Peters, J. M. (2006). The anaphase promoting complex/cyclosome: a machine designed to destroy. Nature reviews Molecular cell biology 7, 644-656.
Pietenpol, J. A., and Stewart, Z. A. (2002). Cell cycle checkpoint signaling: cell cycle arrest versus apoptosis. Toxicology 181-182, 475-481.

Plane, J. M., Shen, Y., Pleasure, D. E., and Deng, W. (2010). Prospects for minocycline neuroprotection. Archives of neurology 67, 1442-1448.

Pourgholami, M. H., Mekkawy, A. H., Badar, S., and Morris, D. L. (2012). Minocycline inhibits growth of epithelial ovarian cancer. Gynecologic oncology 125, 433-440.

Redon, C. E., Dickey, J. S., Bonner, W. M., and Sedelnikova, O. A. (2009). gamma-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin. Advances in space research : the official journal of the Committee on Space Research 43, 1171-1178.

Redon, C. E., Nakamura, A. J., Martin, O. A., Parekh, P. R., Weyemi, U. S., and Bonner, W. M. (2011). Recent developments in the use of gamma-H2AX as a quantitative DNA double-strand break biomarker. Aging 3, 168-174.

Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S., and Bonner, W. M. (1998). DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. The Journal of biological chemistry 273, 5858-5868.

Rutkowski, D. T., and Kaufman, R. J. (2004). A trip to the ER: coping with stress. Trends in cell biology 14, 20-28.

Schmit, T. L., and Ahmad, N. (2007). Regulation of mitosis via mitotic kinases: new opportunities for cancer management. Molecular cancer therapeutics 6, 1920-1931.

Scholey, J. M., Brust-Mascher, I., and Mogilner, A. (2003). Cell division. Nature 422, 746-752.

Schwartzbaum, J. A., Fisher, J. L., Aldape, K. D., and Wrensch, M. (2006). Epidemiology and molecular pathology of glioma. Nature clinical practice Neurology 2, 494-503; quiz 491 p following 516.

Soczynska, J. K., Mansur, R. B., Brietzke, E., Swardfager, W., Kennedy, S. H., Woldeyohannes, H. O., Powell, A. M., Manierka, M. S., and McIntyre, R. S. (2012). Novel therapeutic targets in depression: minocycline as a candidate treatment. Behavioural brain research 235, 302-317.

Son, K., Fujioka, S., Iida, T., Furukawa, K., Fujita, T., Yamada, H., Chiao, P. J., and Yanaga, K. (2009). Doxycycline induces apoptosis in PANC-1 pancreatic cancer cells. Anticancer research 29, 3995-4003.

Stock, M. L., Fiedler, K. J., Acharya, S., Lange, J. K., Mlynarczyk, G. S., Anderson, S. J., McCormack, G. R., Kanuri, S. H., Kondru, N. C., Brewer, M. T., and Carlson, S. A. (2013). Antibiotics acting as neuroprotectants via mechanisms independent of their anti-infective activities. Neuropharmacology 73C, 174-182.

Vakifahmetoglu, H., Olsson, M., and Zhivotovsky, B. (2008). Death through a tragedy: mitotic catastrophe. Cell death and differentiation 15, 1153-1162.

Vermeulen, K., Van Bockstaele, D. R., and Berneman, Z. N. (2003). The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell proliferation 36, 131-149.

Vitale, I., Galluzzi, L., Castedo, M., and Kroemer, G. (2011). Mitotic catastrophe: a mechanism for avoiding genomic instability. Nature reviews Molecular cell biology 12, 385-392.

Wang, Q., Fan, S., Eastman, A., Worland, P. J., Sausville, E. A., and O'Connor, P. M. (1996). UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53. Journal of the National Cancer Institute 88, 956-965.

Wen, P. Y., and Kesari, S. (2008). Malignant gliomas in adults. The New England journal of medicine 359, 492-507.

Wu, Y. C., Hour, M. J., Leung, W. C., Wu, C. Y., Liu, W. Z., Chang, Y. H., and Lee, H. Z. (2011). 2-(Naphthalene-1-yl)-6-pyrrolidinyl-4-quinazolinone inhibits skin cancer M21 cell proliferation through aberrant expression of microtubules and the cell cycle. The Journal of pharmacology and experimental therapeutics 338, 942-951.

Yamada, H. Y., and Gorbsky, G. J. (2006). Spindle checkpoint function and cellular sensitivity to antimitotic drugs. Molecular cancer therapeutics 5, 2963-2969.

Yong, V. W., Wells, J., Giuliani, F., Casha, S., Power, C., and Metz, L. M. (2004). The promise of minocycline in neurology. Lancet neurology 3, 744-751.

Yue, Q. X., Liu, X., and Guo, D. A. (2010). Microtubule-binding natural products for cancer therapy. Planta medica 76, 1037-1043.

Zhivotovsky, B., and Kroemer, G. (2004). Apoptosis and genomic instability. Nature reviews Molecular cell biology 5, 752-762.

Zhou, B. B., and Bartek, J. (2004). Targeting the checkpoint kinases: chemosensitization versus chemoprotection. Nature reviews Cancer 4, 216-225.

Zhou, B. B., and Elledge, S. J. (2000). The DNA damage response: putting checkpoints in perspective. Nature 408, 433-439.