膀胱癌在台灣癌症死因排名第十四位，全球癌症死因排名第九位。目前美國食品藥品監督管理局（US FDA）認可的膀胱癌第一線臨床用藥cisplatin及gemcitabine合併使用，對人體具有副作用及高復發的問題，因此需要尋找新藥物。爵床素A（JA）由傳統中草藥爵床全草萃取的純化物，本實驗室先前已發現，JA對人類正常周邊血單細胞毒性非常低，但對許多人類癌細胞株具毒殺能力、影響粒線體膜電位改變、引起細胞凋亡、誘導細胞自噬，JA也可以抑制腫瘤生長。因此，本研究將探討JA對於膀胱癌細胞T24及TSGH8301是否具毒殺能力、影響粒線體膜電位改變、誘導細胞自噬，及誘發選擇性粒線體細胞自噬（mitophagy），並結合臨床用藥，觀察是否能提高臨床用藥敏感性。以SRB assay檢測毒殺能力，發現毒殺能力與劑量呈正相關。利用acridine orange（AO）染劑及西方墨點法檢測，發現可以誘導細胞自噬的產生。利用染劑rhodamine123染劑檢測粒線體膜電位，發現可以誘導T24粒線體膜電位改變，JA並可誘發PINK1-Parkin及Bnip3，產生mitophagy。經NAO及MitoTracker deep red染劑及西方墨點法發現，T24的粒線體會有生合成及膨脹的現象。JA與cisplatin及gemcitabine合併使用，在T24細胞呈現加成效果。整體而言，本研究結果顯示，JA可誘發膀胱癌細胞T24產生毒殺及提升藥物敏感性，可能與JA誘導細胞自噬及mitophagy現象有關。

Bladder cancer is the 14th death of cancer in Taiwan, and is the 9th of death of cancer in the world. The US FDA-approved first line clinical drugs, cisplatin combined with gemcitabine, exhibit side effects and relapse problems. Thus, to develop new drugs for bladder cancer is urgent. Justicidine A (JA) is a purified compound isolated from whole Justicia procumbens. Our previous published reports indicate that JA showed low cytotoxicity to human normal peripheral blood mononuclear cells, whereas JA induced cytotoxicity, disrupted mitochondrial membrane potential, and induced apoptosis and autophagy in many human cancer cell lines. JA also suppressed tumor growth. The present study is proposed to investigate if JA can exhibit cytotoxicity, disrupt mitochondrial membrane potential, induce autophagy and mitophagy, as well as enhance the sensitivity of clinical drugs against human bladder cancer cell lines T24 and TSGH8301. Here we show that JA inhibited the population growth of both cells using SRB assay. Flow cytometric and Western analysis indicate that JA induced autophagy in both cell lines. JA also disrupted mitochondrial membrane potential in T24 cells after staining with rhodamin123. The JA-induced mitophagy was via PINK1-Parkin and Bnip3 pathways in T24 cells. By staining with NAO and MitoTracker deep red, JA promoted mitochondrial biogenesis and swelling phenomena. Synergistic effect was found when T24 cells were treated with JA in combination with cisplatin and gemcitabine. Taken together, our data suggest that JA-induced cytotoxicity and enhanced sensitivity of clinical drugs may be associated with the induction of autophagy and mitophagy in T24 cells.

Akita M, Suzuki-Karasaki M, Fujiwara K, Nakagawa C, Soma M,
Yoshida Y, Ochiai T, Tokuhashi Y, Suzuki-Karasaki Y. Mitochondrial division inhibitor-1 induces mitochondrial hyperfusion and sensitizes human cancer cells to TRAIL-induced apoptosis. (2014). Int J Oncol. 45(5):1901-12. doi: 10.3892/ijo.2014.2608.
Allen G. F, Toth R, James J, Ganley I. G. (2013). Loss of iron
triggers PINK1/Parkin-independent mitophagy.EMBO Rep. 14(12):1127-35. doi: 10.1038/embor.2013.168.
Aragon-Ching J, Trump D. L. (2016). Systemic therapy in muscle-
invasive and metastatic bladder cancer: current trends and future promises. Future Oncol, 12(17):2049-58. doi: 10.2217/fon-2016-0155.
Asano J, Chiba K, Tada M, Yoshii T. (1996). Antiviral activity of
lignans and their glycosides from Justicia procumbens. Phytochemistry. 42(3):713-7. doi: 10.1016/0031-9422(96)00024-6
Basit F, van Oppen LM, Schöckel L, Bossenbroek HM, van Emst-de
Vries SE, Hermeling JC, Grefte S, Kopitz C, Heroult M, Hgm Willems P, Koopman WJ. (2017). Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells. Cell Death Dis. 8(3):e2716. doi: 10.1038/cddis.2017.133.
Babjuk ., Böhle A, Burger M, Capoun O, Cohen D, Compérat
E.M, Hernández V, Kaasinen E, Palou J, Rouprêt M, van Rhijn B.W, Shariat S.F, Soukup V, Sylvester R.J, Zigeuner R. (2016). EAU Guidelines on Non-Muscle-invasive Urothelial Carcinoma of the Bladder: Update 2016. Eur Urol. (3):447-461. doi: 10.1016/j.eururo.2016.05.041.
Burger M, Catto J. W. F, Dalbagni G, Grossman H. B, Herr H,
Karakiewicz K, Kassouf W, Kiemeney L. A, Vecchia C. L, Shariat S, Lotan Y.(2013). Epidemiology and Risk Factors of Urothelial Bladder Cancer. Eur Urol, 63(2):234-41. doi: 10.1016/j.eururo.2012.07.033.
Chang C. M, Lan K. L, Huang W. S, Lee Y. J, Lee T. W, Chang C. H,
Chuang C. M. (2017). 188Re-Liposome Can Induce Mitochondrial Autophagy and Reverse Drug Resistance for Ovarian Cancer: From Bench Evidence to Preliminary Clinical Proof-of-Concept. Int J Mol Sci. 18(5). doi: 10.3390/ijms18050903.
Chen Y, Dorn II GW. (2013). PINK1-phosphorylated mitofusin 2 is a
Parkin receptor for culling damaged mitochondria. Science. 340(6131):471-5. doi: 10.1126/science.1231031.
Chou TC. (2006). Theoretical basis, experimental design, and
computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev. 58(3):621-81. doi: 10.1124/pr.58.3.10
Chourasia A. H, Boland M. L, Macleod K. F. (2015). Mitophagy and
cancer. Cancer Metab. 26;3:4. doi: 10.1186/s40170-015-0130-8.
Cook SJ, Stuart K, Gilley R, Sale MJ. (2017). Control of cell death and
mitochondrial fission by ERK1/2 MAP kinase signalling. FEBS J. 284(24):4177-4195. doi: 10.1111/febs.14122.
da Silva GN, de Castro Marcondes JP, de Camargo EA, da Silva Passos
Júnior GA, Sakamoto-Hojo ET, Salvadori DM. (2010). Cell cycle arrest and apoptosis in TP53 subtypes of bladder carcinoma cell lines treated with cisplatin and gemcitabine. Exp Biol Med (Maywood). 235(7):814-24. doi: 10.1258/ebm.2010.009322.
Dasari S, Tchounwou PB. (2014). Cisplatin in cancer therapy: molecular
mechanisms of action. Eur J Pharmacol. 740:364-78. doi: 10.1016/j.ejphar.2014.07.025.
Day S. H, Chiu N. Y, Won S. J, Lin C. N. (1999). Cytotoxic lignans
of Justicia ciliata. J Nat Prod. 62(7):1056-8. doi: 10.1021/np9900167
de Oliveira MR, Jardim FR, Setzer WN, Nabavi SM, Nabavi SF. (2016).
Curcumin, mitochondrial biogenesis, and mitophagy: Exploring recent data and indicating future needs. Biotechnol Adv. 34(5):813-826. doi: 10.1016/j.biotechadv.2016.04.004.
Ding W. X, Yin X. M. (2012). Mitophagy: mechanisms,
pathophysiological roles, and analysis. Biol Chem. 393(7):547-64. doi: 10.1515/hsz-2012-0119.
Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason
CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, Morrison B 3rd, Stockwell BR. (2012). Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 149(5):1060-72. doi: 10.1016/j.cell.2012.03.042.
Dorn II GW, Vega RB, Kelly DP. (2015). Mitochondrial biogenesis and
dynamics in the developing and. Genes Dev. 29(19):1981-91. doi: 10.1101/gad.269894.115. diseased heart.
Drayton RM, Catto JW. (2012). Molecular mechanisms of cisplatin
resistance in bladder cancer. Expert Rev Anticancer Ther. 12(2):271-81. doi: 10.1586/era.11.201.
Elmore S. (2007). Apoptosis: a review of programmed cell death. Toxicol
Pathol. 35(4):495-516. doi: 10.1080/01926230701320337
Filadi R, Pendin D, Pizzo P. (2018). Mitofusin 2: from functions to
disease. Cell Death Dis. 9(3):330. doi: 10.1038/s41419-017-0023-6.
Fukamiya N, LeeK. H. (1986). Antitumor Agents, 81. Justicidin-A and
Diphyllin, Two Cytotoxic Principles from Justicia procumbens. J. Nat. Prod, 1986, 49 (2), pp 348–350. doi: 10.1021/np50044a030
Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O, Castedo
M, Kroemer G. (2012). Molecular mechanisms of cisplatin resistance. Oncogene. 31(15):1869-83. doi: 10.1038/onc.2011.384.
Georgakopoulos N. D, Wells G, Campanella M. (2017). Nat Chem
Biol. 13(2):136-146. doi: 10.1038/nchembio.2287.
Gewirtz D. A. (2014). The four faces of autophagy: implications for
cancer therapy. Cancer Res. 74(3):647-51. doi: 10.1158/0008-5472.CAN-13-2966.
GLOBOCAN 2012: Estimated Cancer Incidance, Motality and
Prevalance Worldwide in 2012. International Agency for Research on Cancer, World Health Organization. [Internet]. Retrieved Available from: http://globocan.iarc.fr/Pages/fact_sheets_population.aspx#
Gondo T, Ohori M, Hamada R, Tanaka A, Satake N, Takeuchi
H, Nakashima J, Hatano T, Tachibana M. (2011). The efficacy
and safety of gemcitabine plus cisplatin regimen for patients with advanced urothelial carcinoma after failure of M-VAC regimen. Int J Clin Oncol, 16(4):345-51. doi: 10.1007/s10147-011-0188-4.
Graves JA, Wang Y, Sims-Lucas S, Cherok E, Rothermund K, Branca
MF, Elster J, Beer-Stolz D, Van Houten B, Vockley J, Prochownik EV. (2012). Mitochondrial structure, function and dynamics are temporally controlled by c-Myc. PLoS One. 7(5):e37699. doi: 10.1371/journal.pone.0037699.
Gu M. Y, Kim J, Yang H.O. (2016). The Neuroprotective Effects of
Justicidin A on Amyloid Beta25-35-Induced Neuronal Cell Death Through Inhibition of Tau Hyperphosphorylation and Induction of Autophagy in SH-SY5Y Cells. Neurochem Res. 41(6):1458-67. doi: 10.1007/s11064-016-1857-5.
Guerra F, Arbini AA, Moro L. (2017). Mitochondria and cancer
chemoresistance. Biochim Biophys Acta. s1858(8):686-699. doi: 10.1016/j.bbabio.2017.01.012.
Hamacher-Brady A, Brady N.R. (2016).Mitophagy programs:
mechanisms and physiological implications of mitochondrial targeting by autophagy. Cell Mol Life Sci.73(4):775-95. doi: 10.1007/s00018-015-2087-8.
Horbay R, Bilyy R. (2016). Mitochondrial dynamics during cell cycling.
Apoptosis. 2016 Dec;21(12):1327-1335. doi: 10.1007/s10495-016-1295-5
Kondo Y, Kanzawa T, Sawaya R, Kondo S. (2005). The role of
autophagy in cancer development and response to therapy. Nat Rev Cancer. 5(9):726-34. doi: 10.1038/nrc1692
Kulikov A. V, Luchkina E. A, Gogvadze V, Zhivotovsky B. (2017).
Mitophagy: Link to cancer development and therapy. Biochem Biophys Res Commun. 15;482(3):432-439. doi: 10.1016/j.bbrc.2016.10.088.
Lázaro M, Gallardo E, Doménech M, Pinto Á, Del Alba
A.G, Puente J, Fernández O, Font A, Lainez N, Vázquez S. (2016). SEOM Clinical Guideline for treatment of muscle-invasiveand metastatic urothelial bladder cancer. Clin Transl Oncol, 18(12):1197-1205. DOI: 10.1007/s12094-016-1584-z
Lee J. C, Lee C. H, Su C. L, Huang C. W, Liu H. S, Lin C. N, Won S.
J. (2005). Justicidin A decreases the level of cytosolic Ku70 leading to apoptosis in human colorectal cancer cells. Carcinogenesis. 26(10):1716-30. doi: 10.1093/carcin/bgi133
Levine JJ, Somer RA, Hosoya H, Squillante C. (2017). Atezolizumab-
induced Encephalitis in Metastatic. Bladder Cancer: A Case Report and Review of the Literature. Clin Genitourin Cancer. 15(5):e847-e849. doi: 10.1016/j.clgc.2017.03.001.
Li GB, Fu RQ, Shen HM, Zhou J, Hu XY, Liu YX, Li YN, Zhang HW,
Liu X, Zhang YH, Huang C, Zhang R, Gao N. (2017). Polyphyllin I induces mitophagic and apoptotic cell death in human breast cancer cells by increasing mitochondrial PINK1 levels. 8(6):10359-10374. doi: 10.18632/oncotarget.14413.
Liu J. J, Lin M, Yu J. Y, Liu B., Bao J. K. (2011). Targeting apoptotic
and autophagic pathways for cancer therapeutics. Cancer Lett. 28;300(2):105-14. doi: 10.1016/j.canlet.2010.10.001.
Massari F, Santoni M, Ciccarese C, Brunelli M, Conti A, Santini D,
Montironi R, Cascinu S, Tortora G. (2015). Emerging concepts on drug resistance in bladder cancer: Implications for future strategies. Crit Rev Oncol Hematol. 96(1):81-90. doi: 10.1016/j.critrevonc.2015.05.005.
Mauro-Lizcano M, Esteban-Martínez L, Seco E, Serrano-Puebla
A, Garcia-Ledo L, Figueiredo-Pereira C, Vieira H. L, Boya P. (2015). New method to assess mitophagy flux by flow cytometry. Autophagy. 11(5):833-43. doi: 10.1080/15548627.2015.1034403.
Mei Y, Glover K, Su M., Sinha S. C. (2016). Conformational flexibility
of BECN1: Essential to its key role in autophagy and beyond. Protein Sci. 25(10):1767-85. doi: 10.1002/pro.2984.
Ni C, Su CL. (2017). The saponin formosanin C-induced synthetic
lethalty on human colorectal cancer cells with oncogenic KRAS mutations.
Orogo A. M, Gustafsson Å. B. (2015). Therapeutic targeting of
autophagy: potential and concerns in treating cardiovascular disease. Circ Res. 116(3):489-503. doi: 10.1161/CIRCRESAHA.116.303791.
Ouyang L, Shi Z, Zhao S, Wang F. T, Zhou T. T, Liu B, Bao J.
K. (2012). Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif. 45(6):487-98. doi: 10.1111/j.1365-2184.2012.00845.x.
Pasquier B. (2016). Autophagy inhibitors. Cell Mol Life Sci. 73(5):985-
1001. doi: 10.1007/s00018-015-2104-y.
Qian W, Wang J, Roginskaya V, McDermott L. A, Edwards R. P, Stolz D.
B, Llambi F, Green D. R, Van Houten B. (2014). Novel combination of mitochondrial division inhibitor 1 (mdivi-1) and platinum agents produces synergistic pro-apoptotic effect in drug resistant tumor cells. Oncotarget. 5(12):4180-94. doi: 10.18632/oncotarget.1944
Shabalala S, Muller C. J. F, Louw J, Johnson R. (2017).
Polyphenols, autophagy and doxorubicin-induced cardiotoxicity. Life Sci. 1;180:160-170. doi: 10.1016/j.lfs.2017.05.003.
Springer M. Z, Macleod K, F. (2016). In Brief: Mitophagy: mechanisms
and role in human disease.J Pathol. 240(3):253-255. doi: 10.1002/path.4774.
Su CL, Huang LL, Huang LM, Lee JC, Lin CN, Won SJ. (2006).
Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells.FEBS Lett. 580(13):3185-91. doi: 10.1016/j.febslet.2006.04.085
Sun L, Li T, Wei Q, Zhang Y, Jia X, Wan Z, Han L. (2014). Upregulation
of BNIP3 mediated by ERK/HIF-1α pathway induces autophagy and contributes to anoikis resistance of hepatocellular carcinoma cells. Future Oncol. 10(8):1387-98. doi: 10.2217/fon.14.70.
Tan M. L, Ooi J. P, Ismail N, Moad A. I, Muhammad T.S. (2009).
Programmed cell death pathways and current antitumor targets. Pharm Res. 26(7):1547-60. doi: 10.1007/s11095-009-9895-1.
Tang K, Lin Y, Li LM. (2013). The role of phenethyl isothiocyanate on
bladder cancer ADM resistance reversal and its molecular mechanism. Anat Rec (Hoboken). 296(6):899-906. doi: 10.1002/ar.22677.
Twig G, Elorza A, Molina AJ, Mohamed H, Wikstrom JD, Walzer G,
Stiles L, Haigh SE, Katz S, Las G, Alroy J, Wu M, Py BF, Yuan J, Deeney JT, Corkey BE, Shirihai OS. (2008). Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. EMBO J. 27(2):433-46. doi: 10.1038/sj.emboj.7601963.
Vichai V, Kirtikara K. (2006). Sulforhodamine B colorimetric assay for
cytotoxicity screening. Nat Protoc. 1(3):1112-6. doi: 10.1038/nprot.2006.179
Vyas S, Zaganjor E, Haigis M. C. (2016). Mitochondria and Cancer.
Cell. 166(3):555-66. doi: 10.1016/j.cell.2016.07.002.
Wang Y. W, Chuang J. J, Chang T. Y, Won S. J, Tsai H. W, Lee C. T,
Cheng H. L, Tzai T. S, Liu H. S, Chow N. H. (2015). Antiangiogenesis as the novel mechanism for justicidin A in the anticancer effect on human bladder cancer. Anticancer Drugs. 26(4):428-36. doi: 10.1097/CAD.0000000000000203.
Weng J. R, Ko H. H, Yeh T. L, Lin H. C, Lin C. N. (2004). Two new
arylnaphthalide lignans and antiplatelet constituents from Justicia procumbens. Arch Pharm (Weinheim). 337(4):207-12. DOI: 10.1002/ardp.200300841
Westermann B. (2012). Bioenergetic role of mitochondrial fusion and
fission. Biochim Biophys Acta. 1817(10):1833-8. doi: 10.1016/j.bbabio.2012.02.033.
Won S. J, Yen C. H, Liu H. S, Wu S. Y, Lan S. H., Jiang-Shieh Y. F,
Lin C. N, Su C. L. (2015). Justicidin A-induced autophagy flux enhances apoptosis of human colorectal cancer cells via class III PI3K and Atg5 pathway. J Cell Physiol. 230(4):930-46. doi: 10.1002/jcp.24825.
Won S. J, Wu H. C, Lin K. T, Yu D. H, Chen Y. T, Wu C. S., Huang C.
Y. F. Liu H. S. Lin C. N, Su C. L. (2015). Discovery of molecular mechanisms of lignan justicidin A using L1000 gene expression profiles and the Library of Integrated Network-based Cellular Signatures database. Journal of Functional Foods. 16, 81 93. doi: 10.1016/j.jff.2015.04.025
Yang Y. P, Hu L. F, Zheng H. F, Mao C. J, Hu W. D, Xiong K.
P, Wang F, Liu C. F. (2013). Application and interpretation of current autophagy inhibitors and activators. Acta Pharmacol Sin. 34(5):625-35. doi: 10.1038/aps.2013.5.
Yeh H. L, Hsu S. W, Chang T. C, Chan T. C., Tsou H. C, Chang Y. C,
Chiang P. H. (2017). Spatial Analysis of Ambient PM2.5 Exposure and Bladder Cancer Mortality in Taiwan. Int J Environ Res Public Health, 10;14(5). doi: 10.3390/ijerph14050508.
Yoshii SR, Kishi C, Ishihara N, Mizushima N. (2011). Parkin mediates
proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane. J Biol Chem. 286(22):19630-40. doi: 10.1074/jbc.M110.209338.
Zhang X, Wang X, Wu T, Li B., Liu T, Wang R, Liu Q, Liu Z, Gong
Y, Shao C. (2015). Isoliensinine induces apoptosis in triple-negative human breast cancer cells through ROS generation and p38 MAPK/JNK activation. Sci Rep. 5:12579. doi: 10.1038/srep12579.