臺灣博碩士論文加值系統

皂素是一種從台灣蚤休萃取出來的天然化合物並具有抗癌效果。先前文獻指出皂素可以誘使人類肺癌細胞進行細胞自噬並造成細胞凋亡，而在大腸癌細胞中發現會透過造成粒線體膜電位下降而造成細胞凋亡。膜電位下降是粒線體受損的一個訊號，因此這類的粒腺體會經由有選擇性的細胞自噬清除，這個過程又稱粒線體自噬。目前尚未知道皂素是否可以在細胞中引起粒線體自噬。我們發現細胞經過皂素處理後，LC3 II的表現量在七十二小時內持穩定增高，這代表細胞自噬的過程是異常的。因此，本篇的研究目的是釐清在細胞中皂素對於細胞自噬、粒線體自噬的效果和他們之間的關係，以及他們在皂素相關的細胞死亡中所扮演的角色。本篇所使用的細胞株為人類肺癌細胞A549。首先，我們驗證隨著處理的濃度增高、時間增強，皂素抑制癌細胞生長的效果越好。在施藥二十四小時後使一半的細胞死亡的濃度為4µM，接著我們在整個實驗中皆以這個濃度對細胞施藥。我們發現皂素和CQ (自噬小體和溶體融合的抑制劑)具有相似的效果，都可阻斷amiodarone(細胞自噬反應誘導劑)所引起的細胞自噬的過程。因此，我們可以證明皂素是細胞自噬的阻斷劑。此外，我們發現粒腺體的蛋白質存在於純化的自噬小體中，同樣地，LC3 II也存在於純化的粒腺體當中。我們進一步發現粒腺體和自噬小體是透過粒線體外膜上的BNIP3連接，BNIP3是已被報導的粒腺體自噬受器。PINK1會透過磷酸化Parkin去促進的粒線體自噬反應。皂素處理後，細胞中Parkin磷酸化的程度增加，這意味著皂素能夠誘使粒線體自噬發生。然而，皂素卻對細胞中粒腺體蛋白質的含量沒有影響，因此我們認為皂素引起的粒腺體自噬也是受到阻斷的。接著我們發現將細胞以皂素和自噬反應誘導劑或粒腺體自噬反應誘導劑一同處理後，細胞的生長速度相較於皂素單一處理組別明顯被大幅抑制。最後，我們探討在皂素以及CQ的存在下粒線體自噬對於細胞的影響，我們發現不管是皂素或CQ的組別，粒腺體自噬都可以加強抑制細胞生長。因此我們可以知道，細胞自噬反應和粒腺體自噬反應對於A549細胞株都是有細胞毒性的。綜上所述，我們是第一個證實皂素是細胞自噬及粒線體自噬阻斷劑的實驗室。促進細胞自噬和粒線體自噬可以加強皂素的抗癌效果，因此我們認為將皂素與細胞自噬或粒線體自噬誘導劑合併使用能有效地提升癌症治療的效果。

Formosanin C (FC) is a natural compound extracted from Paris formosana Hayata with anti-cancer activity. Zhang et al. reported that FC induces both autophagy and apoptosis in human lung cancer cells. Furthermore, Won et al. reported that FC‐induced apoptosis requires depolarization of mitochondrial membrane potential (MMP). Depolarized mitochondria engulfed by the autophagosome for degradation is defined as mitophagy. However, it is not known whether FC can induce mitophagy. We found that FC treatment of A549 cells leads o steady increase of LC3 II levels until the end of investigation (72 hr), indicating aberration of autophagic flux. Therefore, we aimed to clarify the effect of FC on autophagy, mitophagy, the relationship between these two events and the roles of autophagy and mitophagy in FC-related cell death. Human lung cancer cell line A549 was used as our cell model. Initially, we verified that FC inhibits A549 cell growth in a dose- and time-dependent manner by MTT assay and 4 µM FC (IC50 at 24 hr) was used in this entire study. We reveal that amiodarone (an autophagy inducer)-induced autophagic flux was blocked by FC as well as chloroquine (CQ, a known fusion blocker of autophagosome and lysosome) demonstrated by Western blotting and IFA analysis, indicating that FC is a blocker not an inducer of autophagy. We further found mitochondrial protein COX IV in the purified autophagosome and autophagosome protein LC3 II in the purified mitochondrion. Furthermore, we detected the binding of BNIP3, a mitophagy receptor, and LC3 II by immunoprecipitation after FC treatment for 24 hr. These data indicate BNIP3 links mitochondrion and autophagosome. PTEN-induced kinase (PINK1) phosphorylates Parkin to initiate mitophagy. FC also increased the phosphorylation of Parkin (p-Parkin) on Ser65. However, FC shows no effect on mitochondrial protein COX IV expression, indicates mitophagy is impaired. Altogether, FC seems to be able to initiate mitophagy, however, mitophagy progression was abrogated possibly by FC blockage of fusion of autophagosome and lysosome. Finally, we reveal both inducers of autophagy and mitophagy increased FC-related cell death. Furthermore, we also reveal that mitophagy enhanced the anti-tumor effect in the presence of FC or CQ. These data imply that autophagy and mitophagy are cytotoxic to FC-treated A549 cells. In summary, we are the first to reveal that FC blocks both autophagic and mitophagic flux in A549 cells. Autophagy and mitophagy play a promoting role in FC-related cell death. Combination of FC and autophagy or mitophagy inducers may enhance the anti-cancer activity. Therefore, it has the potential to become an effective treatment strategy.

中文摘要 2
Abstract 4
致謝 6
Index 7
Abbreviation 8
Introduction 9
I. Lung cancer 9
II. Formosanin C 9
III. Autophagy 9
IV. Autophagy and cancer 10
V. Formosanin C and autophagy 11
VI. Mitophagy 12
VII. Mitophagy and anti-cancer strategy 13
VIII. Specific aims 13
Materials and methods 14
I. Cell lines and Media 14
II. Cell proliferation 14
III. Cell death analysis 14
IV. Western blotting 14
V. Immunofluorescent staining 15
VI. Mitochondrial isolation 15
VII. Autophagosome purification 15
VIII. Immunoprecipitation (IP) 16
Results 17
Discussion 21
References 24

1Herbst, R. S., Morgensztern, D. ＆ Boshoff, C. The biology and management of non-small cell lung cancer. Nature 553, 446 (2018).
2Zappa, C. ＆ Mousa, S. A. Non-small cell lung cancer: current treatment and future advances. Transl. Lung Cancer Res. 5, 288 (2016).
3Malhotra, J., Malvezzi, M., Negri, E., La Vecchia, C. ＆ Boffetta, P. Risk factors for lung cancer worldwide. Eur Respir J 48, 889-902 (2016).
4Wu, R.-T., Chiang, H.-C., Fu, W.-C., Chien, K.-Y., Chung, Y.-M. ＆ Horng, L.-Y. Formosanin-C, an immunomodulator with antitumor activity. Int J Immunopharmacol. 12, 777-786 (1990).
5Lee, J. C., Su, C. L., Chen, L. L. ＆ Won, S. J. Formosanin C‐induced apoptosis requires activation of caspase‐2 and change of mitochondrial membrane potential. Cancer Sci. 100, 503-513 (2009).
6Xiao, X., Zou, J., Bui-Nguyen, T. M., Bai, P., Gao, L., Liu, J., Liu, S., Xiao, J., Chen, X. ＆ Zhang, X. Paris saponin II of Rhizoma Paridis–a novel inducer of apoptosis in human ovarian cancer cells. Biosci Trends 6, 201-211 (2012).
7Zhang, L., Man, S., Wang, Y., Liu, J., Liu, Z., Yu, P. ＆ Gao, W. Paris Saponin II induced apoptosis via activation of autophagy in human lung cancer cells. Chem.-Biol. Interact. 253, 125-133 (2016).
8Man, S., Gao, W., Zhang, Y., Liu, Z., Yan, L., Huang, L. ＆ Liu, C. Formosanin C-inhibited pulmonary metastasis through repression of matrix metalloproteinases on mouse lung adenocarcinoma. Cancer Biol Ther 11, 592-598 (2011).
9Wu, S. Y., Lan, S. H., Wu, S. R., Chiu, Y. C., Lin, X. Z., Su, I. J., Tsai, T. F., Yen, C. J., Lu, T. H., Liang, F. W., Li, C. Y., Su, H. J., Su, C. L. ＆ Liu, H. S. Hepatocellular carcinoma–related cyclin D1 is selectively regulated by autophagy degradation system. Hepatology (Baltimore, Md.) 68, 141-154, doi:10.1002/hep.29781 (2018).
10Wu, S. Y., Lan, S. H. ＆ Liu, H. S. Degradative autophagy selectively regulates CCND1 (cyclin D1) and MIR224, two oncogenic factors involved in hepatocellular carcinoma tumorigenesis. Autophagy 15, 729-730, doi:10.1080/15548627.2019.1569918 (2019).
11Nazarko, T. Y. Pexophagy is responsible for 65% of cases of peroxisome biogenesis disorders. Autophagy 13, 991-994, doi:10.1080/15548627.2017.1291480 (2017).
12Eskelinen, E.-L. Autophagy: supporting cellular and organismal homeostasis by self-eating. ‎Int. J. Biochem. Cell Biol (2019).
13Mathew, R., Karantza-Wadsworth, V. ＆ White, E. Role of autophagy in cancer. Nat Rev Cancer 7, 961 (2007).
14Wu, Y.-W. ＆ Li, F. Bacterial interaction with host autophagy. Virulence 10, 352-362 (2019).
15Gatica, D. ＆ Klionsky, D. J. Towards understanding mRNA-binding protein specificity: lessons from post-transcriptional regulation of ATG mRNA during nitrogen starvation-induced autophagy. Curr Genet, 1-3 (2019).
16Rodríguez-Vargas, J. M., Oliver-Pozo, F. J. ＆ Dantzer, F. PARP1 and Poly (ADP-ribosyl) ation Signaling during Autophagy in Response to Nutrient Deprivation. Oxid Med Cell Longev. 2019 (2019).
17Izumi, M., Nakamura, S. ＆ Li, N. Autophagic Turnover of Chloroplasts: Its Roles and Regulatory Mechanisms in Response to Sugar Starvation. Front Plant Sci. 10 (2019).
18Kim, C., Park, J. M., Song, Y., Kim, S. ＆ Moon, J. HIF1α‐mediated AIMP3 suppression delays stem cell aging via the induction of autophagy. Aging cell 18, e12909 (2019).
19Wang, L., Howell, M., Sparks-Wallace, A., Hawkins, C., Nicksic, C., Kohne, C., Hall, K., Moorman, J., Yao, Z. Q. ＆ Ning, S. p62-mediated Selective Autophagy Endows Virus-transformed Cells with Insusceptibility to DNA Damage under Oxidative Stress. bioRxiv, 502823 (2019).
20Chakraborty, D., Felzen, V., Hiebel, C., Stürner, E., Perumal, N., Manicam, C., Sehn, E., Grus, F., Wolfrum, U. ＆ Behl, C. Enhanced autophagic-lysosomal activity and increased BAG3-mediated selective macroautophagy as adaptive response of neuronal cells to chronic oxidative stress. bioRxiv, 580977 (2019).
21Dikic, I. ＆ Elazar, Z. Mechanism and medical implications of mammalian autophagy. Nat Rev Mol Cell Biol 19, 349-364, doi:10.1038/s41580-018-0003-4 (2018).
22New, J. ＆ Thomas, S. M. Autophagy-dependent secretion: mechanism, factors secreted, and disease implications. Autophagy, 1-12 (2019).
23Ponpuak, M., Mandell, M. A., Kimura, T., Chauhan, S., Cleyrat, C. ＆ Deretic, V. Secretory autophagy. Curr Opin Cell Biol 35, 106-116, doi:10.1016/j.ceb.2015.04.016 (2015).
24Delorme-Axford, E. ＆ Klionsky, D. J. Secretory autophagy holds the key to lysozyme secretion during bacterial infection of the intestine. Autophagy 14, 365-367, doi:10.1080/15548627.2017.1401425 (2018).
25Münch, C. ＆ Dikic, I. Hitchhiking on selective autophagy. Nat Cell Biol. 20, 122-124, doi:10.1038/s41556-018-0036-0 (2018).
26Lan, S. H., Wu, S. Y., Zuchini, R., Lin, X. Z., Su, I. J., Tsai, T. F., Lin, Y. J., Wu, C. T. ＆ Liu, H. S. Autophagy-preferential degradation of MIR224 participates in hepatocellular carcinoma tumorigenesis. Autophagy 10, 1687-1689, doi:10.4161/auto.29959 (2014).
27Chu, C. T. Mechanisms of selective autophagy and mitophagy: Implications for neurodegenerative diseases. Neurobiol Dis 122, 23-34, doi:10.1016/j.nbd.2018.07.015 (2019).
28Deretic, V., Jiang, S. ＆ Dupont, N. Autophagy intersections with conventional and unconventional secretion in tissue development, remodeling and inflammation. Trends Cell Biol 22, 397-406, doi:10.1016/j.tcb.2012.04.008 (2012).
29Jiang, S., Dupont, N., Castillo, E. F. ＆ Deretic, V. Secretory versus degradative autophagy: unconventional secretion of inflammatory mediators. Journal of innate immunity 5, 471-479, doi:10.1159/000346707 (2013).
30Zahoor, M. ＆ Farhan, H. Crosstalk of Autophagy and the Secretory Pathway and Its Role in Diseases. Int Rev Cell Mol Biol 337, 153-184, doi:10.1016/bs.ircmb.2017.12.004 (2018).
31Udristioiu, A. ＆ Nica-Badea, D. Autophagy dysfunctions associated with cancer cells and their therapeutic implications. Biomed Pharmacother. 115, 108892 (2019).
32Amaravadi, R., Kimmelman, A. C. ＆ White, E. Recent insights into the function of autophagy in cancer. Genes Dev 30, 1913-1930, doi:10.1101/gad.287524.116 (2016).
33White, E. The role for autophagy in cancer. J Clin Invest. 125, 42-46 (2015).
34Folkerts, H., Hilgendorf, S., Vellenga, E., Bremer, E. ＆ Wiersma, V. R. The multifaceted role of autophagy in cancer and the microenvironment. MED RES REV (2018).
35Yue, Z., Jin, S., Yang, C., Levine, A. J. ＆ Heintz, N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci U S A. 100, 15077-15082 (2003).
36Ning, L., Guo-Chun, Z., Sheng-Li, A., Xue-Rui, L., Kun, W., Jian, Z., Chong-Yang, R., Ling-Zhu, W. ＆ Hai-Tong, L. Inhibition of autophagy induced by PTEN loss promotes intrinsic breast cancer resistance to trastuzumab therapy. Tumour Biol 37, 5445-5454, doi:10.1007/s13277-015-4392-0 (2016).
37Lan, S. H., Wu, S. Y., Zuchini, R., Lin, X. Z., Su, I. J., Tsai, T. F., Lin, Y. J., Wu, C. T. ＆ Liu, H. S. Autophagy suppresses tumorigenesis of hepatitis B virus‐associated hepatocellular carcinoma through degradation of microRNA‐224. Hepatology (Baltimore, Md.) 59, 505-517 (2014).
38Guo, J. Y., Chen, H.-Y., Mathew, R., Fan, J., Strohecker, A. M., Karsli-Uzunbas, G., Kamphorst, J. J., Chen, G., Lemmons, J. M. ＆ Karantza, V. Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. GENE DEV (2011).
39Lock, R., Roy, S., Kenific, C. M., Su, J. S., Salas, E., Ronen, S. M. ＆ Debnath, J. Autophagy facilitates glycolysis during Ras-mediated oncogenic transformation. Mol Biol Cell. 22, 165-178 (2011).
40Yang, S., Wang, X., Contino, G., Liesa, M., Sahin, E., Ying, H., Bause, A., Li, Y., Stommel, J. M. ＆ Dell'Antonio, G. Pancreatic cancers require autophagy for tumor growth. GENE DEV (2011).
41Mortensen, M., Soilleux, E. J., Djordjevic, G., Tripp, R., Lutteropp, M., Sadighi-Akha, E., Stranks, A. J., Glanville, J., Knight, S. ＆ Jacobsen, S.-E. W. The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance. J Exp Med. 208, 455-467 (2011).
42Mowers, E. E., Sharifi, M. N. ＆ Macleod, K. F. Autophagy in cancer metastasis. Oncogene 36, 1619 (2017).
43Dyczynski, M., Yu, Y., Otrocka, M., Parpal, S., Braga, T., Henley, A. B., Zazzi, H., Lerner, M., Wennerberg, K., Viklund, J., Martinsson, J., Grander, D., De Milito, A. ＆ Pokrovskaja Tamm, K. Targeting autophagy by small molecule inhibitors of vacuolar protein sorting 34 (Vps34) improves the sensitivity of breast cancer cells to Sunitinib. Cancer Lett 435, 32-43, doi:10.1016/j.canlet.2018.07.028 (2018).
44Fu, Y., Hong, L., Xu, J., Zhong, G., Gu, Q., Gu, Q., Guan, Y., Zheng, X., Dai, Q., Luo, X., Liu, C., Huang, Z., Yin, X.-M., Liu, P. ＆ Li, M. Discovery of a small molecule targeting autophagy via ATG4B inhibition and cell death of colorectal cancer cells in vitro and in vivo. Autophagy 15, 295-311, doi:10.1080/15548627.2018.1517073 (2019).
45Li, X., Xu, H.-l., Liu, Y.-x., An, N., Zhao, S. ＆ Bao, J.-k. Autophagy modulation as a target for anticancer drug discovery. Acta Pharmacol. Sin. 34, 612 (2013).
46Liu, Z., He, K., Ma, Q., Yu, Q., Liu, C., Ndege, I., Wang, X. ＆ Yu, Z. Autophagy inhibitor facilitates gefitinib sensitivity in vitro and in vivo by activating mitochondrial apoptosis in triple negative breast cancer. PLOS ONE 12, e0177694, doi:10.1371/journal.pone.0177694 (2017).
47Chude, C. I. ＆ Amaravadi, R. K. Targeting Autophagy in Cancer: Update on Clinical Trials and Novel Inhibitors. Int J Mol Sci 18, 1279, doi:10.3390/ijms18061279 (2017).
48Fulda, S. Autophagy in Cancer Therapy. Front Oncol. 7, 128-128, doi:10.3389/fonc.2017.00128 (2017).
49Chang, Y. L., Liu, S. T., Wang, Y. W., Lin, W. S. ＆ Huang, S. M. Amiodarone promotes cancer cell death through elevated truncated SRSF3 and downregulation of miR-224. Oncotarget 9, 13390-13406, doi:10.18632/oncotarget.24385 (2018).
50Liang, Q. P., Xu, T. Q., Liu, B. L., Lei, X. P., Hambrook, J. R., Zhang, D. M. ＆ Zhou, G. X. Sasanquasaponin ΙΙΙ from Schima crenata Korth induces autophagy through Akt/mTOR/p70S6K pathway and promotes apoptosis in human melanoma A375 cells. Phytomedicine 58, doi:10.1016/j.phymed.2018.11.029 (2019).
51Sun, J., Feng, Y., Wang, Y., Ji, Q., Cai, G., Shi, L., Wang, Y., Huang, Y., Zhang, J. ＆ Li, Q. α-hederin induces autophagic cell death in colorectal cancer cells through reactive oxygen species dependent AMPK/mTOR signaling pathway activation. Int J Oncol. 54, 1601-1612, doi:10.3892/ijo.2019.4757 (2019).
52Wang, L., Yun, L., Wang, X., Sha, L., Wang, L., Sui, Y. ＆ Zhang, H. Endoplasmic reticulum stress triggered by Soyasapogenol B promotes apoptosis and autophagy in colorectal cancer. Life Sci. 218, 16-24, doi:10.1016/j.lfs.2018.12.023 (2019).
53Li, B., Wu, G. L., Dai, W., Wang, G., Su, H. Y., Shen, X. P., Zhan, R., Xie, J. M., Wang, Z., Qin, Z. H., Gao, Q. G. ＆ Shen, G. H. Aescin-induced reactive oxygen species play a pro-survival role in human cancer cells via ATM/AMPK/ULK1-mediated autophagy. Acta Pharmacol. Sin. 39, 1874-1884, doi:10.1038/s41401-018-0047-1 (2018).
54Chung, Y., Jeong, S., Choi, H. S., Ro, S., Lee, J. S. ＆ Park, J. K. Upregulation of autophagy by Ginsenoside Rg2 in MCF-7 cells. ANIM CELLS SYST. 22, 382-389, doi:10.1080/19768354.2018.1545696 (2018).
55Yoo, H. S., Kim, J. M., Jo, E., Cho, C. K., Lee, S. Y., Kang, H. S., Lee, M. G., Yang, P. Y. ＆ Jang, I. S. Modified Panax ginseng extract regulates autophagy by AMPK signaling in A549 human lung cancer cells. Oncol. Rep. 37, 3287-3296, doi:10.3892/or.2017.5590 (2017).
56Sachan, R., Kundu, A., Jeon, Y., Choi, W. S., Yoon, K., Kim, I. S., Kwak, J. H. ＆ Kim, H. S. Afrocyclamin A, a triterpene saponin, induces apoptosis and autophagic cell death via the PI3K/Akt/mTOR pathway in human prostate cancer cells. Phytomedicine 51, 139-150, doi:10.1016/j.phymed.2018.10.012 (2018).
57Wu, Q., Deng, J., Fan, D., Duan, Z., Zhu, C., Fu, R. ＆ Wang, S. Ginsenoside Rh4 induces apoptosis and autophagic cell death through activation of the ROS/JNK/p53 pathway in colorectal cancer cells. Biomed Pharmacother. 148, 64-74, doi:10.1016/j.bcp.2017.12.004 (2018).
58Peng, F., Wang, X., Shu, M., Yang, M., Wang, L., Ouyang, Z., Shen, C., Hou, X., Zhao, B., Wang, X., Zhang, L. W., Liu, Y. ＆ Zhao, S. Raddeanin a suppresses glioblastoma growth by inducing ROS generation and subsequent JNK activation to promote cell apoptosis. Cell Physiol Biochem 47, 1108-1121, doi:10.1159/000490187 (2018).
59Gao, G. Y., Ma, J., Lu, P., Jiang, X. ＆ Chang, C. Ophiopogonin B induces the autophagy and apoptosis of colon cancer cells by activating JNK/c-Jun signaling pathway. Biomed Pharmacother. 108, 1208-1215, doi:10.1016/j.biopha.2018.06.172 (2018).
60Li, B., Wang, Z., Xie, J. M., Wang, G., Qian, L. Q., Guan, X. M., Shen, X. P., Qin, Z. H., Shen, G. H., Li, X. Q. ＆ Gao, Q. G. TIGAR knockdown enhanced the anticancer effect of aescin via regulating autophagy and apoptosis in colorectal cancer cells. Acta Pharmacol. Sin. 40, 111-121, doi:10.1038/s41401-018-0001-2 (2019).
61Zhan, Y., Wang, K., Li, Q., Zou, Y., Chen, B., Gong, Q., Ho, H. I., Yin, T., Zhang, F., Lu, Y., Wu, W., Zhang, Y., Tan, Y., Du, B., Liu, X. ＆ Xiao, J. The novel autophagy inhibitor alpha-hederin promoted paclitaxel cytotoxicity by increasing reactive oxygen species accumulation in non-small cell lung cancer cells. Int J Mol Sci 19, doi:10.3390/ijms19103221 (2018).
62Yu, S., Wang, L., Cao, Z., Gong, D., Liang, Q., Chen, H., Fu, H., Wang, W., Tang, X., Xie, Z., He, Y., Peng, C. ＆ Li, Y. Anticancer effect of polyphyllin I in colorectal cancer cells through ROS-dependent autophagy and G2/M arrest mechanisms. Nat. Prod. Res. 32, 1489-1492, doi:10.1080/14786419.2017.1353512 (2018).
63Park, H. H., Choi, S. W., Lee, G. J., Kim, Y. D., Noh, H. J., Oh, S. J., Yoo, I., Ha, Y. J., Koo, G. B., Hong, S. S., Kwon, S. W. ＆ Kim, Y. S. A formulated red ginseng extract inhibits autophagic flux and sensitizes to doxorubicin-induced cell death. J Ginseng Res. 43, 86-94, doi:10.1016/j.jgr.2017.08.006 (2019).
64Cvetanova, B., Shen, Y. C. ＆ Shyur, L. F. Cumingianoside A, a phyto-triterpenoid saponin inhibits acquired BRAF inhibitor resistant melanoma growth via programmed cell death. Front Pharmacol. 9, doi:10.3389/fphar.2019.00030 (2019).
65Jiang, S. L., Guan, Y. D., Chen, X. S., Ge, P., Wang, X. L., Lao, Y. Z., Xiao, S. S., Zhang, Y., Yang, J. M., Xu, X. J., Cao, D. S. ＆ Cheng, Y. Tubeimoside-1, a triterpenoid saponin, induces cytoprotective autophagy in human breast cancer cells in vitro via Akt-mediated pathway. Acta Pharmacol. Sin., doi:10.1038/s41401-018-0165-9 (2018).
66Cui, J., Man, S., Cui, N., Yang, L., Guo, Q., Ma, L. ＆ Gao, W. The synergistic anticancer effect of formosanin C and polyphyllin VII based on caspase-mediated cleavage of Beclin1 inhibiting autophagy and promoting apoptosis. Cell Proliferation 52, doi:10.1111/cpr.12520 (2019).
67Bai, T., Wang, F., Zheng, Y., Liang, Q., Wang, Y., Kong, J. ＆ Cai, L. Myocardial redox status, mitophagy and cardioprotection: a potential way to amend diabetic heart? Clinical Science 130, 1511-1521 (2016).
68Chourasia, A. H., Boland, M. L. ＆ Macleod, K. F. Mitophagy and cancer. Cancer ＆ metabolism 3, 4 (2015).
69Liu, K., Lee, J., Kim, J. Y., Wang, L., Tian, Y., Chan, S. T., Cho, C., Machida, K., Chen, D. ＆ Ou, J. J. Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells. Mol Cell 68, 281-292.e285, doi:10.1016/j.molcel.2017.09.022 (2017).
70Singh, K., Roy, M., Prajapati, P., Lipatova, A., Sripada, L., Gohel, D., Singh, A., Mane, M., Godbole, M. M., Chumakov, P. M. ＆ Singh, R. NLRX1 regulates TNF-α-induced mitochondria-lysosomal crosstalk to maintain the invasive and metastatic potential of breast cancer cells. Biochim Biophys Acta Mol Basis Dis. 1865, 1460-1476, doi:10.1016/j.bbadis.2019.02.018 (2019).
71Kang, X., Wang, H., Li, Y., Xiao, Y., Zhao, L., Zhang, T., Zhou, S., Zhou, X., Li, Y., Shou, Z., Chen, C. ＆ Li, B. Alantolactone induces apoptosis through ROS-mediated AKT pathway and inhibition of PINK1-mediated mitophagy in human HepG2 cells. Artif Cells Nanomed Biotechnol. 47, 1961-1970, doi:10.1080/21691401.2019.1593854 (2019).
72Rodríguez-Enríquez, S., Pacheco-Velázquez, S. C., Marín-Hernández, Á., Gallardo-Pérez, J. C., Robledo-Cadena, D. X., Hernández-Reséndiz, I., García-García, J. D., Belmont-Díaz, J., López-Marure, R., Hernández-Esquivel, L., Sánchez-Thomas, R. ＆ Moreno-Sánchez, R. Resveratrol inhibits cancer cell proliferation by impairing oxidative phosphorylation and inducing oxidative stress. Toxicology and Applied Pharmacology 370, 65-77, doi:10.1016/j.taap.2019.03.008 (2019).
73Yan, C., Luo, L., Guo, C. Y., Goto, S., Urata, Y., Shao, J. H. ＆ Li, T. S. Doxorubicin-induced mitophagy contributes to drug resistance in cancer stem cells from HCT8 human colorectal cancer cells. Cancer Lett 388, 34-42, doi:10.1016/j.canlet.2016.11.018 (2017).
74Zhang, J., Sun, X., Wang, L., Wong, Y. K., Lee, Y. M., Zhou, C., Wu, G., Zhao, T., Yang, L. ＆ Lu, L. Artesunate-induced mitophagy alters cellular redox status. Redox biology 19, 263-273 (2018).
75Seglen, P. O. ＆ Brinchmann, M. F. Purification of autophagosomes from rat hepatocytes. Autophagy 6, 542-547, doi:10.4161/auto.6.4.11272 (2010).
76Yoshii, S. R. ＆ Mizushima, N. Monitoring and Measuring Autophagy. Int J Mol Sci 18, doi:10.3390/ijms18091865 (2017).
77Di Rita, A., Peschiaroli, A., D′Acunzo, P., Strobbe, D., Hu, Z., Gruber, J., Nygaard, M., Lambrughi, M., Melino, G., Papaleo, E., Dengjel, J., El Alaoui, S., Campanella, M., Dötsch, V., Rogov, V. V., Strappazzon, F. ＆ Cecconi, F. HUWE1 E3 ligase promotes PINK1/PARKIN-independent mitophagy by regulating AMBRA1 activation via IKKα. Nat. Commun. 9, 3755, doi:10.1038/s41467-018-05722-3 (2018).
78Song, Y., Du, Y., Zou, W., Luo, Y., Zhang, X. ＆ Fu, J. Involvement of impaired autophagy and mitophagy in Neuro-2a cell damage under hypoxic and/or high-glucose conditions. Sci. Rep. 8, 3301, doi:10.1038/s41598-018-20162-1 (2018).
79Wu, M.-Y., Wang, S.-F., Cai, C.-Z., Tan, J.-Q., Li, M., Lu, J.-J., Chen, X.-P., Wang, Y.-T., Zheng, W. ＆ Lu, J.-H. Natural autophagy blockers, dauricine (DAC) and daurisoline (DAS), sensitize cancer cells to camptothecin-induced toxicity. Oncotarget 8, 77673 (2017).
80Lao, Y., Wan, G., Liu, Z., Wang, X., Ruan, P., Xu, W., Xu, D., Xie, W., Zhang, Y. ＆ Xu, H. The natural compound oblongifolin C inhibits autophagic flux and enhances antitumor efficacy of nutrient deprivation. Autophagy 10, 736-749 (2014).
81Yao, N., Wang, C., Hu, N., Li, Y., Liu, M., Lei, Y., Chen, M., Chen, L., Chen, C. ＆ Lan, P. Inhibition of PINK1/Parkin-dependent mitophagy sensitizes multidrug-resistant cancer cells to B5G1, a new betulinic acid analog. Cell Death Dis. 10, 232 (2019).
82Han, H., Chou, C. C., Li, R., Liu, J., Zhang, L., Zhu, W., Hu, J., Yang, B. ＆ Tian, J. Chalcomoracin is a potent anticancer agent acting through triggering Oxidative stress via a mitophagy- and paraptosis-dependent mechanism. Sci Rep 8, 9566, doi:10.1038/s41598-018-27724-3 (2018).
83Zorova, L. D., Popkov, V. A., Plotnikov, E. Y., Silachev, D. N., Pevzner, I. B., Jankauskas, S. S., Babenko, V. A., Zorov, S. D., Balakireva, A. V., Juhaszova, M., Sollott, S. J. ＆ Zorov, D. B. Mitochondrial membrane potential. Anal Biochem 552, 50-59, doi:10.1016/j.ab.2017.07.009 (2018).
84Pyatrikas, D. V., Fedoseeva, I. V., Varakina, N. N., Rusaleva, T. M., Stepanov, A. V., Fedyaeva, A. V., Borovskii, G. B. ＆ Rikhvanov, E. G. Relation between cell death progression, reactive oxygen species production and mitochondrial membrane potential in fermenting Saccharomyces cerevisiae cells under heat-shock conditions. FEMS Microbiol Lett 362, doi:10.1093/femsle/fnv082 (2015).
85Drake, L. E., Springer, M. Z., Poole, L. P., Kim, C. J. ＆ Macleod, K. F. Expanding perspectives on the significance of mitophagy in cancer. Semin Cancer Biol 47, 110-124, doi:10.1016/j.semcancer.2017.04.008 (2017).
86Zhang, T., Xue, L., Li, L., Tang, C., Wan, Z., Wang, R., Tan, J., Tan, Y., Han, H. ＆ Tian, R. BNIP3 protein suppresses PINK1 kinase proteolytic cleavage to promote mitophagy. J Biol Chem 291, 21616-21629 (2016).
87Kuwana, T., Bouchier-Hayes, L., Chipuk, J. E., Bonzon, C., Sullivan, B. A., Green, D. R. ＆ Newmeyer, D. D. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol. Cell. 17, 525-535 (2005).
88Hanna, R. A., Quinsay, M. N., Orogo, A. M., Giang, K., Rikka, S. ＆ Gustafsson, A. B. Microtubule-associated protein 1 light chain 3 (LC3) interacts with Bnip3 protein to selectively remove endoplasmic reticulum and mitochondria via autophagy. J Biol Chem 287, 19094-19104, doi:10.1074/jbc.M111.322933 (2012).
89Shi, R. Y., Zhu, S. H., Li, V., Gibson, S. B., Xu, X. S. ＆ Kong, J. M. BNIP3 interacting with LC3 triggers excessive mitophagy in delayed neuronal death in stroke. CNS Neurosci Ther. 20, 1045-1055 (2014).
90Su, S.-H., Wu, Y.-F., Wang, D.-P. ＆ Hai, J. Inhibition of excessive autophagy and mitophagy mediates neuroprotective effects of URB597 against chronic cerebral hypoperfusion. Cell Death Dis. 9, 733, doi:10.1038/s41419-018-0755-y (2018).
91Ly, J. D., Grubb, D. R. ＆ Lawen, A. The mitochondrial membrane potential (Δψ m) in apoptosis; an update. Apoptosis 8, 115-128 (2003).
92Wu, M. Y., Wang, S. F., Cai, C. Z., Tan, J. Q., Li, M., Lu, J. J., Chen, X. P., Wang, Y. T., Zheng, W. ＆ Lu, J. H. Natural autophagy blockers, dauricine (DAC) and daurisoline (DAS), sensitize cancer cells to camptothecin-induced toxicity. Oncotarget 8, 77673-77684, doi:10.18632/oncotarget.20767 (2017).
93Zheng, K., Li, Y., Wang, S., Wang, X., Liao, C., Hu, X., Fan, L., Kang, Q., Zeng, Y., Wu, X., Wu, H., Zhang, J., Wang, Y. ＆ He, Z. Inhibition of autophagosome-lysosome fusion by ginsenoside Ro via the ESR2-NCF1-ROS pathway sensitizes esophageal cancer cells to 5-fluorouracil-induced cell death via the CHEK1-mediated DNA damage checkpoint. Autophagy 12, 1593-1613, doi:10.1080/15548627.2016.1192751 (2016).