Immune Netw.  2012 Dec;12(6):253-260. 10.4110/in.2012.12.6.253.

alpha-Mangostin Reduced ER Stress-mediated Tumor Growth through Autophagy Activation

• Kim SJ
• Hong EH
• Lee BR
• Park MH
• Kim JW
• Pyun AR
• Kim YJ
• Chang SY
• Chin YW
• Ko HJ

• KMID: 2150757
• DOI: http://doi.org/10.4110/in.2012.12.6.253

Abstract

alpha-Mangostin is a xanthon derivative contained in the fruit hull of mangosteen (Garcinia mangostana L.), and the administration of alpha-Mangostin inhibited the growth of transplanted colon cancer, Her/CT26 cells which expressed Her-2/neu as tumor antigen. Although alpha-Mangostin was reported to have inhibitory activity against sarco/endoplasmic reticulum Ca2+ ATPase like thapsigargin, it showed different activity for autophagy regulation. In the current study, we found that alpha-Mangostin induced autophagy activation in mouse intestinal epithelial cells, as GFP-LC3 transgenic mice were orally administered with 20 mg/kg of alpha-Mangostin daily for three days. However, the activation of autophagy by alpha-Mangostin did not significantly increase OVA-specific T cell proliferation. As we assessed ER stress by using XBP-1 reporter system and phosphorylation of eIF2alpha, thapsigargin-induced ER stress was significantly reduced by alpha-Mangostin. However, coadministration of thapsigargin with alpha-Mangostin completely blocked the antitumor activity of alpha-Mangostin, suggesting ER stress with autophagy blockade accelerated tumor growth in mouse colon cancer model. Thus the antitumor activity of alpha-Mangostin can be ascribable to the autophagy activation rather than ER stress induction.

Keyword

Autophagy; alpha-Mangostin; Thapsigargin; ER stress; Antitumor activity; Colon cancer

MeSH Terms

Animals
Autophagy
Calcium-Transporting ATPases
Cell Proliferation
Colonic Neoplasms
Epithelial Cells
Fruit
Garcinia mangostana
Mice
Mice, Transgenic
Phosphorylation
Reticulum
Thapsigargin
Transplants
Xanthones
Calcium-Transporting ATPases
Thapsigargin
Xanthones

Figure

• 

  Figure 1 Administration of α-Mangostin induced antitumor effect. BALB/c mice were s.c. injected with of Her2/CT26 cells (2×105 cells/mouse). One day after tumor challenge, mice were orally administrated with 20 mg/kg of α-Mangostin daily. The growth of solid tumor was measured by caliper three times a week. *p<0.05 and **p<0.01 (Student's t-test; n=5/group). Tumor growth was monitored and mean values for tumor volume±SE are shown.

• 

  Figure 2 Autophagy activation of α-Mangostin in the murine intestine. (A) Transgenic GFP-LC3 mice were orally administrated with 20 mg/kg of α-Mangostin (b and d) or DMSO (a and c) daily for three days. To analyze the conversion of LC3-I to LC-3-II, GFP puncta formation in small intestinal villi (a and b) and crypt epithelial cells (c and d), were detected by confocal microscopy at 18 hrs after final administration with α-Mangostin. Scale bar: 10µm. (B) Quantification of autophagy dots/cells shown in (A). The value of mean and standard error is indicated. **p<0.01 (Student's t-test).

• 

  Figure 3 Ag-specific CD8+ T cell proliferation was not increase according to α-Mangostin administration. (A) Bone-marrow derived DCs were mixed with OVA protein (0.1 mg/ml). The DCs plus OVA protein were treated with α-Mangostin (1 uM) or control DMSO for 4 hrs. One day after adoptive transfer with CFSElabeled OT-I CD8+ T cells, mice were intravenously given with 5×105 cells/mouse of DC/OVA-DMSO or DC/OVA-α-Mangostin. Three days after DC vaccination, the CFSE dilution in OT-I cells was detected by flow cytometer. (B) The percentage of divided cells in total CFSE+ OT-I T cells shown in (A). The value of mean and standard error is indicated (n=3/group). n.s., not significant.

• 

  Figure 4 ER stress was significantly reduced by α-Mangostin treatment. (A) CT26 cells transfected with XBP1-venus reporter were treated with serially diluted α-Mangostin in the absence or presence of 200 nM of thapsigargin for 24 hrs. As negative control, DMSO was used. After incubation, the fluorescence of the spliced XBP1 in the treated cells was measured by fluorescence microplate reader (emission at 520 nm; excitation at 485 nm). (B) Western blot analysis of phosphorylated eIF2α in CT26 cells treated with 200 nM of thapsigargin with or without 250 ng/ml of α-Mangostin. (C) Quantification of phosphorylated eIF2α band density shown in (B).

• 

  Figure 5 Antitumor effect of α-Mangostin was abrogated by ER stress induction by thapsigargin treatment. BALB/c mice were s.c. injected with 2×105 cells of Her2/CT26 cells. One day before tumor challenge, thapsigargin alone or thapsigargin together with α-Mangostin were administrated via oral route daily starting one day before tumor challenge. The growth of tumor was measured by caliper. *p<0.05 as compared with DMSO-treated group (Student's t-test; n=5/group).

Reference

1. Chao AC, Hsu YL, Liu CK, Kuo PL. α-Mangostin, a dietary xanthone, induces autophagic cell death by activating the AMP-activated protein kinase pathway in glioblastoma cells. J Agric Food Chem. 2011. 59:2086–2096.

2. Furukawa K, Shibusawa K, Chairungsrilerd N, Ohta T, Nozoe S, Ohizumi Y. The mode of inhibitory action of alpha-mangostin, a novel inhibitor, on the sarcoplasmic reticulum Ca(2+)-pumping ATPase from rabbit skeletal muscle. Jpn J Pharmacol. 1996. 71:337–340.

3. Ganley IG, Wong PM, Gammoh N, Jiang X. Distinct autophagosomal-lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest. Mol Cell. 2011. 42:731–743.

4. Thastrup O, Cullen PJ, Drøbak BK, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc Natl Acad Sci USA. 1990. 87:2466–2470.

5. Rabinowitz JD, White E. Autophagy and metabolism. Science. 2010. 330:1344–1348.

6. Ogata M, Hino S, Saito A, Morikawa K, Kondo S, Kanemoto S, Murakami T, Taniguchi M, Tanii I, Yoshinaga K, Shiosaka S, Hammarback JA, Urano F, Imaizumi K. Autophagy is activated for cell survival after endoplasmic reticulum stress. Mol Cell Biol. 2006. 26:9220–9231.

7. Kouroku Y, Fujita E, Tanida I, Ueno T, Isoai A, Kumagai H, Ogawa S, Kaufman RJ, Kominami E, Momoi T. ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation. Cell Death Differ. 2007. 14:230–239.

8. Bi M, Naczki C, Koritzinsky M, Fels D, Blais J, Hu N, Harding H, Novoa I, Varia M, Raleigh J, Scheuner D, Kaufman RJ, Bell J, Ron D, Wouters BG, Koumenis C. ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth. EMBO J. 2005. 24:3470–3481.

9. Blais JD, Addison CL, Edge R, Falls T, Zhao H, Wary K, Koumenis C, Harding HP, Ron D, Holcik M, Bell JC. Perk-dependent translational regulation promotes tumor cell adaptation and angiogenesis in response to hypoxic stress. Mol Cell Biol. 2006. 26:9517–9532.

10. 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.

11. Kim YJ, Ko HJ, Kim YS, Kim DH, Kang S, Kim JM, Chung Y, Kang CY. alpha-Galactosylcer-amide-loaded, antigen-expressing B cells prime a wide spectrum of antitumor immunity. Int J Cancer. 2008. 122:2774–2783.

12. Chang SY, Cha HR, Chang JH, Ko HJ, Malissen B, Iwata M, Kweon MN. Lack of retinoic acid leads to increased langerin-expressing dendritic cells in gut-associated lymphoid tissues. Gastroenterology. 2010. 138:1468–1478.

13. Ko HJ, Yang H, Yang JY, Seo SU, Chang SY, Seong JK, Kweon MN. Expansion of Tfh-like cells during chronic Salmonella exposure mediates the generation of autoimmune hypergammaglobulinemia in MyD88-deficient mice. Eur J Immunol. 2012. 42:618–628.

14. Iwawaki T, Akai R, Kohno K, Miura M. A transgenic mouse model for monitoring endoplasmic reticulum stress. Nat Med. 2004. 10:98–102.

15. Quan GH, Oh SR, Kim JH, Lee HK, Kinghorn AD, Chin YW. Xanthone constituents of the fruits of Garcinia mangostana with anticomplement activity. Phytother Res. 2010. 24:1575–1577.

16. Sato A, Fujiwara H, Oku H, Ishiguro K, Ohizumi Y. Alpha-mangostin induces Ca2+-ATPase-dependent apoptosis via mitochondrial pathway in PC12 cells. J Pharmacol Sci. 2004. 95:33–40.

17. Watanapokasin R, Jarinthanan F, Nakamura Y, Sawasjirakij N, Jaratrungtawee A, Suksamrarn S. Effects of α-mangostin on apoptosis induction of human colon cancer. World J Gastroenterol. 2011. 17:2086–2095.

18. Nakagawa Y, Iinuma M, Naoe T, Nozawa Y, Akao Y. Characterized mechanism of alpha-mangostin-induced cell death: caspase-independent apoptosis with release of endonuclease-G from mitochondria and increased miR-143 expression in human colorectal cancer DLD-1 cells. Bioorg Med Chem. 2007. 15:5620–5628.

19. Matsumoto K, Akao Y, Yi H, Ohguchi K, Ito T, Tanaka T, Kobayashi E, Iinuma M, Nozawa Y. Preferential target is mitochondria in alpha-mangostin-induced apoptosis in human leukemia HL60 cells. Bioorg Med Chem. 2004. 12:5799–5806.

20. Lee HK, Mattei LM, Steinberg BE, Alberts P, Lee YH, Chervonsky A, Mizushima N, Grinstein S, Iwasaki A. In vivo requirement for Atg5 in antigen presentation by dendritic cells. Immunity. 2010. 32:227–239.

21. Li Y, Hahn T, Garrison K, Cui ZH, Thorburn A, Thorburn J, Hu HM, Akporiaye ET. The vitamin E analogue α-TEA stimulates tumor autophagy and enhances antigen cross-presentation. Cancer Res. 2012. 72:3535–3545.

22. Kim I, Xu W, Reed JC. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov. 2008. 7:1013–1030.

23. Hetz C, Thielen P, Matus S, Nassif M, Court F, Kiffin R, Martinez G, Cuervo AM, Brown RH, Glimcher LH. XBP-1 deficiency in the nervous system protects against amyotrophic lateral sclerosis by increasing autophagy. Genes Dev. 2009. 23:2294–2306.

24. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature. 2008. 451:1069–1075.

25. Deretic V. Autophagy in innate and adaptive immunity. Trends Immunol. 2005. 26:523–528.

26. Kaser A, Blumberg RS. Autophagy, microbial sensing, endoplasmic reticulum stress, and epithelial function in inflammatory bowel disease. Gastroenterology. 2011. 140:1738–1747.

27. Kaser A, Blumberg RS. Endoplasmic reticulum stress in the intestinal epithelium and inflammatory bowel disease. Semin Immunol. 2009. 21:156–163.