Identification and Characterization of Anticancer Compounds Targeting Apoptosis and Autophagy from Chinese Native Garcinia Species

Abstract

Natural compounds from medicinal plants are important resources for drug development. Active compounds targeting apoptosis and autophagy are candidates for anti-cancer drugs. In this study, we collected Garcinia species from China and extracted them into water or ethanol fractions. Then, we performed a functional screen in search of novel apoptosis and autophagy regulators. We first characterized the anti-proliferation activity of the crude extracts on multiple cell lines. HeLa cells expressing GFP-LC3 were used to examine the effects of the crude extracts on autophagy. Their activities were confirmed by Western blots of A549 and HeLa cells. By using bioassay guided fractionation, we found that two caged prenylxanthones from Garcinia bracteata, neobractatin and isobractatin, can significantly induce apoptosis and inhibit autophagy. Our results suggest that different Garcinia species displayed various degrees of toxicity on different cancer cell lines. Furthermore, the use of a high content screening assay to screen natural products was an essential method to identify novel autophagy regulators.

Key words

Garcinia - Clusiaceae - autophagy - apoptosis - natural compound - caged prenylxanthones

^* Authors contributed equally to this work.

• References

• 1 Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144: 646-674
• 2 Llambi F, Green DR. Apoptosis and oncogenesis: give and take in the BCL-2 family. Curr Opin Genet Dev 2011; 21: 12-20
• 3 Tait SW, Green DR. Mitochondria and cell signalling. J Cell Sci 2012; 125: 807-815
• 4 Xie Z, Klionsky DJ. Autophagosome formation: core machinery and adaptations. Nat Cell Biol 2007; 9: 1102-1109
• 5 Rosenfeldt MT, Ryan KM. The multiple roles of autophagy in cancer. Carcinogenesis 2011; 32: 955-963
• 6 Levine B. Cell biology: autophagy and cancer. Nature 2007; 446: 745-747
• 7 Mathew R, Karantza-Wadsworth V, White E. Role of autophagy in cancer. Nat Rev Cancer 2007; 7: 961-967
• 8 Yang ZJ, Chee CE, Huang S, Sinicrope FA. The role of autophagy in cancer: therapeutic implications. Mol Cancer Ther 2011; 10: 1533-1541
• 9 Carew JS, Kelly KR, Nawrocki ST. Autophagy as a target for cancer therapy: new developments. Cancer Manag Res 2012; 4: 357-365
• 10 Kimmelman AC. The dynamic nature of autophagy in cancer. Genes Dev 2011; 25: 1999-2010
• 11 Mizushima N, Klionsky DJ. Protein turnover via autophagy: implications for metabolism. Annu Rev Nutr 2007; 27: 19-40
• 12 Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G. Cell death assays for drug discovery. Nat Rev Drug Discov 2011; 10: 221-237
• 13 Tyas L, Brophy VA, Pope A, Rivett AJ, Tavare JM. Rapid caspase-3 activation during apoptosis revealed using fluorescence-resonance energy transfer. EMBO Rep 2000; 1: 266-270
• 14 Luo KQ, Yu VC, Pu Y, Chang DC. Application of the fluorescence resonance energy transfer method for studying the dynamics of caspase-3 activation during UV-induced apoptosis in living HeLa cells. Biochem Biophys Res Commun 2001; 283: 1054-1060
• 15 Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 2004; 15: 1101-1111
• 16 Zhang L, Yu J, Pan H, Hu P, Hao Y, Cai W, Zhu H, Yu AD, Xie X, Ma D, Yuan J. Small molecule regulators of autophagy identified by an image-based high-throughput screen. Proc Natl Acad Sci U S A 2007; 104: 19023-19028
• 17 Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell 2010; 140: 313-326
• 18 Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A, Stenmark H, Johansen T. p 62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol 2005; 171: 603-614
• 19 Shen S, Niso-Santano M, Adjemian S, Takehara T, Malik SA, Minoux H, Souquere S, Marino G, Lachkar S, Senovilla L, Galluzzi L, Kepp O, Pierron G, Maiuri MC, Hikita H, Kroemer R, Kroemer G. Cytoplasmic STAT3 represses autophagy by inhibiting PKR activity. Mol Cell 2012; 48: 667-680
• 20 Han QB, Xu HX. Caged Garcinia xanthones: development since 1937. Curr Med Chem 2009; 16: 3775-3796
• 21 Anantachoke N, Tuchinda P, Kuhakarn C, Pohmakotr M, Reutrakul V. Prenylated caged xanthones: chemistry and biology. Pharm Biol 2012; 50: 78-91
• 22 Yi T, Yi Z, Cho SG, Luo J, Pandey MK, Aggarwal BB, Liu M. Gambogic acid inhibits angiogenesis and prostate tumor growth by suppressing vascular endothelial growth factor receptor 2 signaling. Cancer Res 2008; 68: 1843-1850
• 23 Wang X, Chen W. Gambogic acid is a novel anti-cancer agent that inhibits cell proliferation, angiogenesis and metastasis. Anticancer Agents Med Chem 2012; 12: 994-1000
• 24 Chantarasriwong O, Batova A, Chavasiri W, Theodorakis EA. Chemistry and biology of the caged Garcinia xanthones. Chemistry 2010; 16: 9944-9962
• 25 Han QB, Tian HL, Yang NY, Qiao CF, Song JZ, Chang DC, Luo KQ, Xu HX. Polyprenylated xanthones from Garcinia lancilimba showing apoptotic effects against HeLa-C3 cells. Chem Biodiversity 2008; 5: 2710-2717
• 26 Huang SX, Feng C, Zhou Y, Xu G, Han QB, Qiao CF, Chang DC, Luo KQ, Xu HX. Bioassay-guided isolation of xanthones and polycyclic prenylated acylphloroglucinols from Garcinia oblongifolia . J Nat Prod 2009; 72: 130-135
• 27 Liu X, Yu T, Gao XM, Zhou Y, Qiao CF, Peng Y, Chen SL, Luo KQ, Xu HX. Apoptotic effects of polyprenylated benzoylphloroglucinol derivatives from the twigs of Garcinia multiflora . J Nat Prod 2010; 73: 1355-1359
• 28 Xu G, Kan WL, Zhou Y, Song JZ, Han QB, Qiao CF, Cho CH, Rudd JA, Lin G, Xu HX. Cytotoxic acylphloroglucinol derivatives from the twigs of Garcinia cowa . J Nat Prod 2010; 73: 104-108
• 29 Gao XM, Yu T, Cui MZ, Pu JX, Du X, Han QB, Hu QF, Liu TC, Luo KQ, Xu HX. Identification and evaluation of apoptotic compounds from Garcinia oligantha . Bioorg Med Chem Lett 2012; 22: 2350-2353
• 30 Feng C, Zhou LY, Yu T, Xu G, Tian HL, Xu JJ, Xu HX, Luo KQ. A new anticancer compound, oblongifolin C, inhibits tumor growth and promotes apoptosis in HeLa cells through Bax activation. Int J Cancer 2012; 131: 1445-1454
• 31 Fu WM, Zhang JF, Wang H, Tan HS, Wang WM, Chen SC, Zhu X, Chan TM, Tse CM, Leung KS, Lu G, Xu HX, Kung HF. Apoptosis induced by 1, 3, 6, 7-tetrahydroxyxanthone in hepatocellular carcinoma and proteomic analysis. Apoptosis 2012; 17: 842-851
• 32 Fu WM, Zhang JF, Wang H, Xi ZC, Wang WM, Zhuang P, Zhu X, Chen SC, Chan TM, Leung KS, Lu G, Xu HX, Kung HF. Heat shock protein 27 mediates the effect of 1, 3, 5-trihydroxy-13, 13-dimethyl-2H-pyran [7, 6-b] xanthone on mitochondrial apoptosis in hepatocellular carcinoma. J Proteomics 2012; 75: 4833-4843
• 33 Kan WL, Yin C, Xu HX, Xu G, To KK, Cho CH, Rudd JA, Lin G. Antitumor effects of novel compound, guttiferone K, on colon cancer by p 21Waf1/Cip1-mediated G(0)/G(1) cell cycle arrest and apoptosis. Int J Cancer 2013; 132: 707-716
• 34 Wan G, Xie W, Liu Z, Xu W, Lao Y, Huang N, Cui K, Liao M, He J, Jiang Y, Yang BB, Xu H, Xu N, Zhang Y. Hypoxia-induced MIR155 is a potent autophagy inducer by targeting multiple players in the MTOR pathway. Autophagy 2014; 10: 70-79
• 35 Lao Y, Wan G, Liu Z, Wang X, Ruan P, Xu W, Xu D, Xie W, Zhang Y, Xu H, Xu N. The natural compound oblongifolin C inhibits autophagic flux and enhances antitumor efficacy of nutrient deprivation. Autophagy 2014; 10: 736-749
• 36 Thoison O, Fahy J, Dumontet V, Chiaroni A, Riche C, Tri MV, Sevenet T. Cytotoxic prenylxanthones from Garcinia bracteata . J Nat Prod 2000; 63: 441-446
• 37 Shen T, Li W, Wang YY, Zhong QQ, Wang SQ, Wang XN, Ren DM, Lou HX. Antiproliferative activities of Garcinia bracteata extract and its active ingredient, isobractatin, against human tumor cell lines. Arch Pharmacal Res 2014; 37: 412-420
• 38 Inglese J, Johnson RL, Simeonov A, Xia M, Zheng W, Austin CP, Auld DS. High-throughput screening assays for the identification of chemical probes. Nat Chem Biol 2007; 3: 466-479
• 39 Shu CW, Madiraju C, Zhai D, Welsh K, Diaz P, Sergienko E, Sano R, Reed JC. High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4. J Biomol Screen 2011; 16: 174-182
• 40 Na Z, Hu HB, Fan QF. A novel caged-prenylxanthone from Garcinia bracteata . Chin Chem Lett 2010; 21: 443-445
• 41 Zhang C, Wu X, Zhang M, Zhu L, Zhao R, Xu D, Lin Z, Liang C, Chen T, Chen L, Ren Y, Zhang J, Qin N, Zhang X. Small molecule R1498 as a well-tolerated and orally active kinase inhibitor for hepatocellular carcinoma and gastric cancer treatment via targeting angiogenesis and mitosis pathways. PloS One 2013; 8: e65264
• 42 Xu D, Cao J, Qian S, Li L, Hu C, Weng Q, Lou J, Zhu D, Zhu H, Hu Y, He Q, Yang B. 5 k, a novel beta-O-demethyl-epipodophyllotoxin analogue, inhibits the proliferation of cancer cells in vitro and in vivo via the induction of G2 arrest and apoptosis. Invest New Drugs 2011; 29: 786-799