2011年台灣衛生福利部公告國人死因第一名為惡性腫瘤，男性第一名癌症死因為肝癌；女性則為乳腺癌，目前治療方式多以手術、放射性、化療、標靶治療等方式，雖然治癒率與五年存活率逐漸升高，但治療期間為病人帶來的副作用極其不舒服，近年來有學者提出：使用天然物併用臨床用藥可減輕藥物的副作用， 黃酮類化合物為植物次生代謝產物具有非常良好的抗發炎、抗氧化和干擾癌細胞週期生長等功能。為了開發天然藥物於癌症治療上的應用，我們藉由併用兩種天然物黃酮類化合物以低濃度達到比單一用藥更好的效果，我們以廣泛存在於食物當中的芹菜素 (Apigenin)和白楊素 (Chrysin)應用在肝癌與乳癌中，其中三陰性乳腺癌目前尚無有適合的標靶藥物。最近臨床研究表示在許多癌症中SKP2過度表達或Wnt/β-catenin不正常的被活化，然而目前並無針對此兩個蛋白的標靶藥物，因此開發SKP2及Wnt/β-catenin蛋白的抑制劑是很重要的。我們以Apigenin 併用Chrysin處理肝癌細胞HepG2與三陰性乳癌細胞MDA-MB-231在細胞存活率試驗中發現能有效減少癌細胞存活率；由西方墨點法結果得知Apigenin 併用Chrysin能明顯減少Wnt/β-catenin 訊號途徑中LRP6、phosph-LRP6、β-catenin的蛋白質表現量，並抑制調控細胞週期S期SKP2表現量，且近期研究指出Wnt/β-catenin及SKP2與細胞凋亡相關，在細胞實驗中發現Apigenin 併用Chrysin可藉由Caspase Pathways達到促進HepG2和MDA-MB-231凋亡的效果，由此可證SKP2不只可以調控細胞週期亦能促進細胞凋亡。為了進一步佐證藥物的效果，我們將MDA-MB-231細胞異體移植至免疫缺陷鼠體內產生腫瘤進行動物實驗，結果顯示併用兩種天然物可明顯使腫瘤縮小。由本實驗證明Apigenin併用Chrysin能有效抑制HepG2與MDA-MB-231的增生並抑制腫瘤生長，因此我們認為Apigenin併用Chrysin具有成為治療藥物的潛力，在未來癌症研究上，值得進一步研究其作用機制。

According to the Ministry of Health and Welfare, cancer is first leading cause death in Taiwan in 2011. Hepatocellular carcinoma (HCC, also called malignant hepatoma) is one of the most common tumors worldwide, and males usually are more affected than females. Breast cancer is the most universal cancer in women. Some patients may be given chemotherapy or radiation therapy after surgery to kill any cancer cells. Although multivariate analysis demonstrated that the overall five-year cancer survival rate for HCC and breast cancer has been increasing, but the drug treatments often cause uncomfortable side effects in patients. Recent clinical studies have shown that overexpression of S-phase kinase protein 2 (SKP2) and Wnt/β-catenin have been observed in many cancers. Unfortunately, specific drugs that target SKP2 and Wnt/β-catenin are unavailable at present.
Therefore, it is important to develop SKP2 and Wnt/β-catenin inhibitors as chemopreventive agents. Some scholars have proposed that the use of phytochemicals with clinical trials reduce the side effects of the drug treatments. Many studies have shown that the combined use of several drugs with different mechanisms of action can exhibit synergistic effects. In order to develop medicine in cancer treatment, we use two phytochemicals at low concentrations to achieve better results than a single drug.
Apigenin and chrysin (5,7 - dihydroxyflavone) are flavonoids, which have been reported in previous studies of anti-inflammatory, antioxidant, and can promote cell death or slow growth in many human cancers cells. However, one death signal transduction mechanisms apigenin inhibit the growth of liver cancer cells and breast cancer cells are still poorly understood. Although the anti-cancer activities of Apigenin and chrysin have attracted scientific attention, the detailed mechanisms of Apigenin and chrysin in HCC and breast cancer prevention have not been fully deciphered.
In this study, we want to investigate the combined effects of co-treatment with Apigenin and chrysin on HCC and breast cancer. MTT assay indicated that apigenin combined with chrysin can reduced HepG2 and MDA-MB-231 cell viability. The effect of chrysin combined apigenin on cell cycle phase distribution in breast cancer cells and liver cancer were analyzed by flow cytometry. After treatment of each cell line with these compounds, we found that Apigenin combined with chrysin synergisticallyinduced apoptosis in HepG2 and HMDA-BM-231 cells through downregulation of SKP2 and low-density lipoprotein receptor-related protein 6 (LRP6) expression. Furthermore, the combination of Apigenin and chrysin also suppressed tumor growth in nude mice with xenografted MDA-MB-231 cells through the down-regulation of SKP2 protein. To further evidence the effect of the drug, we MDA-MB-231 cell xenograft in immune-deficient nude mice to produce tumors 28 days after injection, tumors were observed changes. The experimental results show that apigenin combined with chrysin can reduce the HepG2 and MDA-MB-231 proliferation and induce apoptosis. It also offers opportunities for exploring new drug targets, and further investigations are underway in this regard.

1. 衛生福利部http://www.mohw.gov.tw/CHT/Ministry/Index.aspx
2. 國泰綜合醫院 http://www.cgh.org.tw/index.html
3. 和信治癌中心醫院 http://www.kfsyscc.org/
4. Mathew, S.,et al.,Biomarkers for Virus-Induced Hepatocellular Carcinoma (HCC).Infect Genet Evol,2014.10.1016/j.meegid.2014.1567-7257 (Electronic)
5. 臺大醫院 http://www.ntuh.gov.tw/ntuh_chinese.aspx
6. Sorlie, T., How to personalise treatment in early breast cancer. Eur J Cancer, 2011. 47 Suppl 3: p. S310-1.
7. Zhao, X., et al., Combining gene signatures improves prediction of breast cancer survival. PLoS One, 2011. 6(3): p. e17845.
8. Flores, A. and J.A. Marrero, Emerging trends in hepatocellular carcinoma: focus on diagnosis and therapeutics. Clin Med Insights Oncol, 2014. 8: p. 71-6.
9. Goh, L.Y., A.H. Leow, and K.L. Goh, Observations on the epidemiology of gastrointestinal and liver cancers in the Asian-Pacific region. J Dig Dis, 2014.
10. Schweizer L1, V.H., Wnt/Wingless signaling through beta-catenin requires the function of both LRP/Arrow and frizzled classes of receptors. BMC Cell Biol., 2003 2;4:4.
11. Liu, G., et al., A novel mechanism for Wnt activation of canonical signaling through the LRP6 receptor. Mol Cell Biol, 2003. 23(16): p. 5825-35.
12. Li, Y., et al., LRP6 expression promotes cancer cell proliferation and tumorigenesis by altering beta-catenin subcellular distribution. Oncogene, 2004. 23(56): p. 9129-35.
13. Suraweera N1, R.J., Volikos E, Guenther T, Talbot I, Tomlinson I, Silver A., Mutations within Wnt pathway genes in sporadic colorectal cancers and cell lines. Int J Cancer. , 2006. 119(8):1837-42.
14. Clevers, H., Wnt breakers in colon cancer. Cancer Cell, 2004. 5(1): p. 5-6.
15. Nakamura, T., et al., Axin, an inhibitor of the Wnt signalling pathway, interacts with beta-catenin, GSK-3beta and APC and reduces the beta-catenin level. Genes Cells, 1998. 3(6): p. 395-403.
16. Lu, W., C. Lin, and Y. Li, Rottlerin induces Wnt co-receptor LRP6 degradation and suppresses both Wnt/beta-catenin and mTORC1 signaling in prostate and breast cancer cells. Cell Signal, 2014. 26(6): p. 1303-9.
17. Lin, S.Y., et al., Beta-catenin, a novel prognostic marker for breast cancer: its roles in cyclin D1 expression and cancer progression. Proc Natl Acad Sci U S A, 2000. 97(8): p. 4262-6.
18. Liu, C.C., et al., LRP6 overexpression defines a class of breast cancer subtype and is a target for therapy. Proc Natl Acad Sci U S A, 2010. 107(11): p. 5136-41.
19. Traub, F., et al., Prognostic impact of Skp2 and p27 in human breast cancer. Breast Cancer Res
83
Treat, 2006. 99(2): p. 185-91.
20. Gustavo A. Miranda-Carboni, S.A.K., A functional link between Wnt signaling and SKP2-independent p27 turnover in mammary tumors. gene and development, 2008. 22(22):3121-3134.
21. Tang, Y., et al., WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells. Mol Cancer Ther, 2009. 8(2): p. 458-68.
22. Huang, C.S., et al., Protection by chrysin, apigenin, and luteolin against oxidative stress is mediated by the Nrf2-dependent up-regulation of heme oxygenase 1 and glutamate cysteine ligase in rat primary hepatocytes. Arch Toxicol, 2013. 87(1): p. 167-78.
23. Hwang, Y.J., et al., Molecular mechanisms of luteolin-7-O-glucoside-induced growth inhibition on human liver cancer cells: G2/M cell cycle arrest and caspase-independent apoptotic signaling pathways. BMB Rep, 2013. 46(12): p. 611-6.
24. Yang, M.Y., et al., Luteolin enhances paclitaxel-induced apoptosis in human breast cancer MDA-MB-231 cells by blocking STAT3. Chem Biol Interact, 2014. 213: p. 60-8.
25. Kritas, S.K., et al., Luteolin inhibits mast cell-mediated allergic inflammation. J Biol Regul Homeost Agents, 2013. 27(4): p. 955-9.
26. Spoerlein, C., et al., Effects of chrysin, apigenin, genistein and their homoleptic copper(II) complexes on the growth and metastatic potential of cancer cells. J Inorg Biochem, 2013. 127: p. 107-15.
27. Polier, G., et al., Targeting CDK9 by wogonin and related natural flavones potentiates the anti-cancer efficacy of the Bcl-2 family inhibitor ABT-263. Int J Cancer, 2014.
28. Farag, M.A., et al., Metabolic profiling and systematic identification of flavonoids and isoflavonoids in roots and cell suspension cultures of Medicago truncatula using HPLC-UV-ESI-MS and GC-MS. Phytochemistry, 2007. 68(3): p. 342-54.
29. Chong, F.W., et al., Expression of transforming growth factor-beta and determination of apoptotic index in histopathological sections for assessment of the effects of Apigenin (4', 5', 7'- Trihydroxyflavone) on Cyclosporine A induced renal damage. Malays J Pathol, 2009. 31(1): p. 35-43.
30. Wang W1, H.L., Chung CS, Pelling JC, Koehler KJ, Birt DF., Cell-cycle arrest at G2/M and growth inhibition by apigenin in human colon carcinoma cell lines. Mol Carcinog. , 2000. ;28(2):102-10.
31. Wang, W., et al., Cell-cycle arrest at G2/M and growth inhibition by apigenin in human colon carcinoma cell lines. Mol Carcinog, 2000. 28(2): p. 102-10.
32. Khoo, B.Y., S.L. Chua, and P. Balaram, Apoptotic effects of chrysin in human cancer cell lines. Int J Mol Sci, 2010. 11(5): p. 2188-99.
33. Li, X., et al., Chrysin sensitizes tumor necrosis factor-alpha-induced apoptosis in human tumor cells via suppression of nuclear factor-kappaB. Cancer Lett, 2010. 293(1): p. 109-16.
84
34. XIAO-ZHENG CAO, H.-L.X., Inhibition of cell growth by BrMC through inactivation of Akt in HER-2/neu-overexpressing breast cancer cells. Oncology Letters, 2014. 7(5): : p. 1632–1638
35. Yang, B., et al., Chrysin inhibits metastatic potential of human triple-negative breast cancer cells by modulating matrix metalloproteinase-10, epithelial to mesenchymal transition, and PI3K/Akt signaling pathway. J Appl Toxicol, 2014. 34(1): p. 105-12.
36. M., P.-R., Drug Therapy for Advanced-Stage Liver Cancer. Liver Cancer., 2014 May. 3(2):125-131.
37. Meng-Shih Weng, Y.-S.H., Jen-Kun Lin, Chrysin induces G1 phase cell cycle arrest in C6 glioma cells through inducing p21Waf1/Cip1 expression: involvement of p38 mitogen-activated protein kinase. Biochem Pharmacol 2005. 69(12):1815-27
38. Mantawy EM1, E.-B.W., Esmat A1, Badr AM1, El-Demerdash E3., Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis. Eur J Pharmacol., 2014. 728:107-18.
39. Lee Y, S.B., Kang YJ, Kim DH, Jang JY, Hwang SY, Kim M, Lim HS, Yoon JH, Chung HY, Kim ND., Apigenin-induced apoptosis is enhanced by inhibition of autophagy formation in HCT116 human colon cancer cells. Int J Oncol. , 2014 May;. 44(5):1599-606. doi: 10.3892/ijo.2014.2339. .
40. Fagan-Solis KD1, P.B., Gozgit JM, Bentley BA, Marconi SM, Otis CN, Anderton DL, Schneider SS, Arcaro KF, SKP2 Overexpression Is Associated With Increased Serine 10 Phosphorylation of p27 (pSer10p27) in Triple-Negative Breast Cancer. J Cell Physiol., 2014 Sep. 229(9):1160-9. doi: 10.1002/jcp.24545.
41. Yang Q1, H.J., Wu Q3, Cai Y2, Zhu L2, Lu X2, Chen S1, Chen C1, Wang Z4., Acquisition of epithelial-mesenchymal transition is associated with Skp2 expression in paclitaxel-resistant breast cancer cells. Br J Cancer. , 2014. 110(8):1958-67. doi: 10.1038/bjc.2014.136. .
42. Tung, E.K., et al., Upregulation of the Wnt co-receptor LRP6 promotes hepatocarcinogenesis and enhances cell invasion. PLoS One, 2012. 7(5): p. e36565.
43. Fagan-Solis KD1, P.B., Gozgit JM, Bentley BA, Marconi SM, Otis CN, Anderton DL, Schneider SS, Arcaro KF., SKP2 Overexpression Is Associated With Increased Serine 10 Phosphorylation of p27 (pSer10p27) in Triple-Negative Breast Cancer. J Cell Physiol. , 2014. 229(9):1160-9. doi: 10.1002/jcp.24545.
44. Park, S.Y., et al., Frondoside A has an anti-invasive effect by inhibiting TPA-induced MMP-9 activation via NF-kappaB and AP-1 signaling in human breast cancer cells. Int J Oncol, 2012. 41(3): p. 933-40.
45. Tsuji, P.A. and T. Walle, Cytotoxic effects of the dietary flavones chrysin and apigenin in a normal trout liver cell line. Chem Biol Interact, 2008. 171(1): p. 37-44.
46. Brechbuhl, H.M., et al., Chrysin enhances doxorubicin-induced cytotoxicity in human lung epithelial cancer cell lines: the role of glutathione. Toxicol Appl Pharmacol, 2012. 258(1): p. 1-9.
85
47. Seo, H.S., et al., Induction of Caspase-dependent Apoptosis by Apigenin by Inhibiting STAT3 Signaling in HER2-overexpressing MDA-MB-453 Breast Cancer Cells. Anticancer Res, 2014. 34(6): p. 2869-82.
48. Kim, K., et al., Overexpression of beta-catenin induces apoptosis independent of its transactivation function with LEF-1 or the involvement of major G1 cell cycle regulators. Mol Biol Cell, 2000. 11(10): p. 3509-23.
49. Gedaly R1, G.R., Daily MF1, Shah M1, Maynard E1, Chen C1, Zhang X2, Esser KA2, Cohen DA3, Evers BM4, Jiang J3, Spear BT3, Targeting the Wnt/β-Catenin Signaling Pathway in Liver Cancer Stem Cells and Hepatocellular Carcinoma Cell Lines with FH535. PLoS One., 2014 Jun. 18;9(6):e99272.
50. Lu W1, L.Y., Salinomycin suppresses LRP6 expression and inhibits both Wnt/β-catenin and mTORC1 signaling in breast and prostate cancer cells. J Cell Biochem. , 2014 Jun. doi: 10.1002/jcb.24850.
51. Smith, M.R., F. Jin, and I. Joshi, Bortezomib sensitizes non-Hodgkin's lymphoma cells to apoptosis induced by antibodies to tumor necrosis factor related apoptosis-inducing ligand (TRAIL) receptors TRAIL-R1 and TRAIL-R2. Clin Cancer Res, 2007. 13(18 Pt 2): p. 5528s-5534s.