拓樸異構酶抑制劑ellipticine是具有抗癌效果的化學治療藥劑之ㄧ，但它在肺癌化學治療效應報導不多。本研究發現ellipticine可抑制人類非小細胞肺癌(NSCLC)A549細胞株的生長。但轉殖突變Akt-Ser473質體至細胞後，由ellipticine所誘導產生的細胞凋亡會被抑制。更多證據顯示ellipticine可調節細胞內部生長存活相關因子Akt與p53共同移動至細胞核，而當轉殖突變Akt-Ser473質體後，p53與Akt的核移動則會被抑制。此外，轉殖突變Akt-Thr308質體雖會抑制細胞凋亡的發生，但其效應較Akt-Ser473不明顯。此外，轉殖野生型p53進入p53缺失的H1299細胞株後，也會有類似的效果﹔但是若轉殖突變p53，則不會有這種效果。因此，可以看出ellipticine可促進細胞p53與Akt移動至細胞核及Akt於Ser-473位點的磷酸化，得以調控ellipticine所引發的細胞凋亡。有更多的證據顯示A549細胞株經由ellipticine所引發的細胞凋亡與細胞自噬的形成有關，這點可使用acridine orange染色來證明。當轉殖突變Akt質體後，ellipticine所誘導的細胞自噬體聚集也會降低，此外，使用細胞自噬抑制劑也有相同的效果。因此本研究顯示ellipticine所誘導產生的細胞凋亡是與Akt及p53移動至細胞核及細胞自噬形成有關。

Topoisomerase II inhibitor ellipticine and its analogues were reported as promising antitumor agent. In this work, we showed that the growth of human non-small-cell-lung-cancer (NSCLC) epithelial cells A549 was inhibited by ellipticine. The induced apoptotic cell death disappeared upon transfection with dominant-negative Akt-Ser473 construct and the phenotype reversed. More evidence indicated that ellipticine regulated through nucleus translocalization of endogenous survival signals Akt and p53, and the effects were blocked by dominant-negative Akt-Ser473 and the growth of suppression reverted. On the other hand, the apoptotic phenotype was also be reverted by Akt-Thr308 with less dominant effect. The same effects took place by transfecting into p53-null H1299 cells with wild-typed p53, but not in those with mutated p53. Thus, the apoptotic death caused by ellipticine in A549 cells was determined by modulating subcellular distribution of Akt and its phosphorylation at serine-473 , which was assisted by p53. The effect is distinctive among the topoisomerase II inhibitors. More evidence indicated that the onset of apoptotic death in A549 cells is associated with autophagy, that was confirmed by acridine orange staining. Further more, the autophagesome aggregation caused by ellipticine was blocked by dominant-negative Akt-Ser473 as well as autophagy inhibitor. Altogether, our work corroborates that nuclear translocalization of Akt and p53 are essential in bringing the onset of autophagy in ellipticine-induced apoptosis in A549 cells.

Acton E. M., Narayanan V. L., Risbood P. A., Shoemaker R. H., Vistica D. T., and Boyd M. R. Anticancer specificity of some ellipticinium salts against human brain tumors in vitro. J. Med. Chem. 37: 2185–9 (1994).

Altomare D. A., Wang H. Q., Skele K. L., Rienzo A. D., Klein-Szanto A. J., Godwin A. K., and Testa J. R. Akt and mTOR phosphorylation is frequently detected in ovarian cancer and can be targeted to disrupt ovarian tumor cell growth. Oncogene. 23:5853-5857 (2004).

Andrews N. W. Regulated secretion of conventional lysosomes. Trends. Cell Biol. 10:316-321 (2000).

Ayala G., Thompson T., Yang G., Frolov A., Li R., Scardino P., Ohori M., Wheeler T., and Harper W. High levels of phosphorylated form of Akt-1 in prostate cancer and non-neoplastic prostate tissues are strong predictors of biochemical recurrence. Cancer Res. 10:6572-6578 (2004).

Balsara B. R., Pei J., Mitsuuchi Y., Page R., Klein-Szanto A., Wang H., Unger M. and Testa J. R. Frequent activation of Akt in non-small cell lung carcinomas and preneoplastic bronchial lesions. Carcinogenesis. 25:2053-2059 (2004).

Braybrooke J. P., Levitt N. C., Joel S., Davis T., Madhusudan S., Turley H., Wilner S., Harris A. L., and Talbot D. C. Pharmacokinetic study of cisplatin and infusional etoposide phosphate in advanced breast cancer with correlation of response to topoisomerase IIalpha expression. Clin. Cancer Res. 9: 4682-4688 (2003).

Biondi M. R. and Nebreda R. A. Signaling specificity of Ser/Thr protein kinases through docking-site-mediated interactions. Biochem. J. 372:1-13 (2003).

Datta S. R., Brunet A., and Greenberg M. E. Cellular survival：a play in three Akts. Genes Dev. 13:2905-27 (1999).

Dennis S., Frank E., Schulze-Osthoff K. and Reiner U. J. p21 blocks irradiation-induced apoptosis downstream of mitochondria by inhibition of cyclin-dependent kinase–mediated caspase-9 activation. Cancer Res. 66:11254-11262 (2006).

Degenhardt K., Mathew R., Beaudoin B., Bray K., Anderson D., Chen G., Mukherjee C., Shi Y., Gelinas C., Fan Y., Nelson D. A., Jin S., and White E. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer cell. 10:51-64 (2006).

Ewa C. S., Slawomir W., Milena D., Janusz D., and Tomasz A. The effect of doxorubicin and retinoids on proliferation, necrosis and apoptosis in MCF-7 breast cancer cells. Folia Histochem. Cytobiol. 42(4):221-227 (2004).

Franke T. F., Hornik C. P., Segev L., Shostak G. A., and Sugimoto C. PI3K/Akt and apoptosis: size matters. Oncogene. 22(56):8983-8998 (2003).

Giono L. E. and Manfredi J. J. Mdm2 is required for inhibition of Cdk2 activity by p21, thereby contributing to p53-dependent cell cycle arrest. Mol. Cell Biol. 27:4166-4178 (2007).

Goodwin S., Smith A. F., and Horning E. C. Alkaloids of Ochrosia elliptica Labill, J Am. Chem. Soc. 81: 1903–1908 (1959).

Greco F. A. Oral etoposide in lymphoma. Drugs. 3: 35-41(1999).

Ghobrial I. M., Witzig T. E., and Adjei A. A. Targeting apoptosis pathways in cancer therapy. CA. Cancer J. Clin. 55:178-194 (2005).

Hendrix M. J. De-mystifying the mechanism(s) of maspin. Nature Med. 6:374-376 (2000).

Gozuacik D. and Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene. 23:2891-2906 (2004).

Hsu J. H., Shi Y., and Frost P. Interleukin-6 activates phosphoinositol-3-kinase in multiple myeloma tumor cells by signaling through RAS-dependent and, separately, through p85-dependent pathways. Oncogene. 23(19):3368-3375 (2004).

Inoki K., Ouyang H., and Zhu T. TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell. 126(5):955-968 (2006).

Kamradt J. M. and Pienta K. J. Etoposide in prostate cancer. Expert Opin. Pharmacother. 1: 271-275 (2000).

Klionsky D. J. Autophagy: From phenomenology to molecular understanding in less than a decade. Nature Rev. Mol. Cell Biol. 8:931-937 (2007).

Koga F., Xu W., Karpova T. S., McNally J. G., Baron R., and Neckers L. Hsp90 inhibition transiently activates Src kinase and promotes Src-dependent Akt and Erk activation. Proc. Natl. Acad. Sci. 103:11318-11322 (2006).

Kuo P. L., Hsu Y. L., Chang C. H., and Lin C. H. The mechanism of ellipticine-induced apoptosis and cell cycle arrest in human breast MCF-7 cancer cells. Cancer letters. 223:293-301(2005).

Lahair M. M., Howe C. J., Rodriguez-Mora O., McCubrey J. A., and Franklin R. A. Antioxidants & Redox Signaling. 8(9-10):1749-1756 (2006).

Levine B. and Klionsky D. J., Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev. Cell, 6(4):463-77 (2004).

Li Y., Dowbenko D., and Lasky L. A. AKT/PKB phosphorylation of p21Cip/WAF1 enhances protein stability of p21Cip/WAF1 and promotes cell survival. J. Biol. Chem. 277(13):11352-11361 (2002).

Liang K., Jin W., Knuefermann C., Schmidt M., Mills G. B., Ang K. K., Milas L., and Fan Z. Targeting the phosphatidylinositol 3-kinase/Akt pathway for enhancing breast cancer cells to radiotherapy. Molecular Cancer Therapeutics. 2:353-360 (2003).

Lu C., Fu W., Zhao D., and Mattson M. The DNA damageing agent etoposide activates a cell survival pathway involving α-amono-3-hydroxyle-5-methylisoxazole-4-propionate receptors and mitogen-activated protein kinases in hippocampual neurons. J. Neurosci. 70:671-79 (2002).

Marino G. and Lopez-Otin C. Autophagy: molecular mechanisms, physiological functions and relevance in human pathology. Cell Mol. Life Sci. 61(12):1439-54 (2004).

Martin A. C., Facchiano A. M., Cuff A. L., Hernandez-Boussard T., Olivier M., Hainaut P., and Thornton J. M. Integrating mutation data and structural analysis of the TP53 tumor-suppressor protein. Hum. Mutat. 19:149-64 (2002).

Mayo L. D. and Donner D. B. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc. Natl Acad. Sci. 98(20):11598-11603 (2001).

McCubrey J. A., Steelman L. S., Franklin R. A., Abrams S. L., Chappell W. H., Wong E. W., Lehmann B. D., Terrian D. M., Basecke J., Stivala F., Libra M., Evangelisti C., and Martelli A. M. Targeting the Raf/MEK/ERK, PI3K/Akt and p53 pathways in hematopoietic drug resistance. Adv. Enzyme Reg. 40:305-337 (2007).

Meng Q., Xia C., Fang J., Rojanasakul Y., and Jiang B. H. Role of PI3K and Akt specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway. Cell Signal. 18(12):2262-2271 (2006).

Minna J.D. Harrison's Principles of Internal Medicine. McGraw-Hill, 506–516 (2004).

Mitsui H., Takuwa N., Maruyama T., Maekawa H., Hirayama M., Sawatari T., Hashimoto N., Takuwa Y., Kimura S. The MEK1-ERK map kinase pathway and the PI 3-kinase-Akt pathway independently mediate anti-apoptotic signals in HepG2 liver cancer cells. Int. J. Cancer, 92(1):55-62 (2001).

Monnot M., Mauffret O., Simon V., Lescot E., Psaume B., and Saucier J. M. DNA-drug recognition and effects on topoisomerase II-mediated cytotoxicity: A three-mode binding model for ellipticine derivatives. J. Biol. Chem. 266:1820-1829 (1991).

Ogawa M., Yoshimori T., Suzuki T., Sagara H., Mizushima N., and Sasakawa C. Escape of intracellular Shigella from autophagy. Science. 307: 727-731 (2005).

Pene F., Claessens Y. E., and Muller O. Role of the phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma. Oncogene. 21(43):6587-6597 (2002).

Reggiori, F. and Klionsky D. J., Autophagosomes: biogenesis from scratch? Curr. Opin. Cell Biol. 17(4):415-22 (2005).

Rho J. K., Choi Y. J., Ryoo B-Y., Na I. I., Yang S. H., Kim C. H., and Lee J. C. p53 enhances gefitinib-induced growth inhibition and apoptosis by regulation of Fas in non-small cell lung cancer. Cancer Res. 67:1163-1169 (2007).

Tian B., Lessan K., Kahm J., Kleidon J., and Henke C. Integrin regulates fibroblast viability during collagen matrix contraction through a phosphatidylinositol 3-kinase/Akt/protein kinase B signaling pathway. J. Biol. Chem. 277:24667-24675 (2002).

Vaporciyan A.A., Nesbitt J.C., and Lee J.S. Cancer Medicine. B C Decker Inc., 1227–1292 (2000).

Verheul H. M. and Pinedo H. M. The role of vascular endothelial growth factor (VEGF) in tumor angiogenesis and early clinical development of VEGF-receptor kinase inhibitors. Clin. Breast Cancer. 1:S80-4 (2000).

Vivanco I. and Sawyers C. L. The phosphatidylinositol 3-kinase Akt pathway in human cancer. Nat. Rev. Cancer. 2:489-501 (2002).

Xue L., Fletcher G. C., and Tolkovsky A. M. Autophagy is activated by apoptotic signalling in sympathetic neurons: An alternative mechanism of death execution. Mol. Cell. Neurosci. 14:180-198 (1999).

Yi H. K., Kim S. Y., and Hwang P. H. Impact of PTEN on the expression of insulin-like growth factors (IGFs) and IGF-binding proteins in human gastric adenocarcinoma cells. Biochem. Biophys. Res. Commun. 330(3):760-767 (2005).

Zhang Y, Fujita N and Tsuruo T. p21Waf1/Cip1 acts in synergy with bcl-2 to confer multidrug resistance in a camptothecin-selected human lung-cancer cell line. Int. J. Cancer. 83:790-797 (1999).

Zhou B. P., Liao Y., Xia W., Zou Y., Spohn B., and Hung M-C. HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nature Cell Biol. 3:973-982 (2001).