Tumor Biology

, Volume 37, Issue 2, pp 1853–1862 | Cite as

Gambogic acid enhances the radiosensitivity of human esophageal cancer cells by inducing reactive oxygen species via targeting Akt/mTOR pathway

  • Yan Yang
  • Xiangdong Sun
  • Yuehua Yang
  • Xi Yang
  • Hongcheng Zhu
  • Shengbin Dai
  • Xiaochen Chen
  • Hao Zhang
  • Qing Guo
  • Yaqi Song
  • Feng Wang
  • Hongyan Cheng
  • Xinchen Sun
Original Article

Abstract

Radiotherapy is a widespread treatment in human solid tumors. However, therapy resistance and poor prognosis are still problems. Gambogic acid (GA), extracted from the dried yellow resin of gamboges, has an anticancer effect against various types of cancer cells. To explore the radiosensitivity of GA on esophageal cancer cell line TE13, cell viability was tested by Cell Counting Kit-8 (CCK-8) assay, colony formation assay was used to assess the effects of GA on the radiosensitivity of TE13, and flow cytometry was performed to meter the percentage of apoptosis. The protein levels of microtubule-associated protein 1 light chain 3 (LC3), caspase3, caspase8, casepase9, pAkt, and p-mammalian target of rapamycin (p-mTOR) were tested using Western blot. The distribution of LC3 was detected by immunofluorescence. Additionally, we also examined reactive oxygen species (ROS) expression by laser scanning confocal microscope (LSCM). The cells were transfected with adenovial vector to monitor the autophagy through the expression of green fluorescent protein (GFP–red fluroscent protein (RFP)–LC3. The rates of apoptotic cells in combined-treated TE13 increased significantly compared with the control groups in accordance with the results of Western blot. The clonogenic survival assay showed that GA enhances radiosensitivity with a sensitizing enhancement ratio (SER) of 1.217 and 1.436 at different concentrations. The LC3-II protein level increased in the combined group indicating the increase of autophagy incidence, and the results of GFP–RFP–LC3 experiment showed that GA may block the process of autophagic flux in TE13 cells. Moreover, we successfully demonstrated that ROS is involved in the induction of autophagy. ROS-mediated autophagy depends on the inhibition of the Akt/mTOR pathway. Taken together, GA induced radiosensitivity involves autophagy and apoptosis which are regulated by ROS hypergeneration and Akt/mTOR inhibition.

Keywords

Gambogic acid Esophageal cancer Radiosensitivity GFP–RFP LC3 Autophagy 

Abbreviation

DCFH-DA, 29

79-dichlorofluorescein diacetate

DMSO

Dimethyl sulfoxide

ESCC

Esophageal squamous cell carcinoma

FITC

Fluorescein isothiocyanate

GA

Gambogic acid

GFP

Green fluorescent protein

LC3

Microtubule-associated protein 1 light chain 3

LSCM

Laser scanning confocal microscope

mTOR

Mammalian target of rapamycin

NAC

Scavenger N-acetylcysteine

PI

Propidium iodide

RFP

Red fluorescent protein

ROS

Reactive oxygen species

SE

Standard error of the mean

SER

Sensitizing enhancement ratio

μM

μmol/L

Notes

Acknowledgments

Our study was supported by the Natural Science Foundation of China (no. 81272504, 81472809), the Innovation Team (no. LJ201123-EH11), Jiangsu Provincial Science and Technology Projects BK2011854 (DA11), and the Six Major Talent Peak Project of Jiangsu Province. The priority academic program development of Jiangsu Higher Education Institution (JX10231801), grants from the Key Academic Discipline of Jiangsu Province “Medical Aspects of Specific Environments,” and “333” Project of Jiangsu Province BRA2012210 (RS12) funded our study.

Conflicts of interest

None

References

  1. 1.
    Liesenklas W, Auterhoff H. The constitution of gambogic acid and its isomerization. 4. Chemistry of gum-resin. Arch Pharm Ber Dtsch Pharm Ges. 1966;299:797–8.CrossRefPubMedGoogle Scholar
  2. 2.
    Wang X, Lu N, Yang Q, Gong D, Lin C, Zhang S, et al. Studies on chemical modification and biology of a natural product, gambogic acid (iii): determination of the essential pharmacophore for biological activity. Eur J Med Chem. 2011;46:1280–90.CrossRefPubMedGoogle Scholar
  3. 3.
    Panthong A, Norkaew P, Kanjanapothi D, Taesotikul T, Anantachoke N, Reutrakul V. Anti-inflammatory, analgesic and antipyretic activities of the extract of gamboge from Garcinia hanburyi Hook f. J Ethnopharmacol. 2007;111:335–40.CrossRefPubMedGoogle Scholar
  4. 4.
    Zhao L, Guo QL, You QD, Wu ZQ, Gu HY. Gambogic acid induces apoptosis and regulates expressions of Bax and Bcl-2 protein in human gastric carcinoma MGC-803 cells. Biol Pharm Bull. 2004;27:998–1003.CrossRefPubMedGoogle Scholar
  5. 5.
    Zhao Q, Yang Y, Yu J, You QD, Zeng S, Gu HY, et al. Posttranscriptional regulation of the telomerase hTERT by gambogic acid in human gastric carcinoma 823 cells. Cancer Lett. 2008;262:223–31.CrossRefPubMedGoogle Scholar
  6. 6.
    Guizzunti G, Theodorakis EA, Yu AL, Zurzolo C, Batova A. Cluvenone induces apoptosis via a direct target in mitochondria: a possible mechanism to circumvent chemo-resistance? Investig New Drugs. 2012;30:1841–8.CrossRefGoogle Scholar
  7. 7.
    Yu J, Guo QL, You QD, Zhao L, Gu HY, Yang Y, et al. Gambogic acid-induced G2/M phase cell-cycle arrest via disturbing CDK7-mediated phosphorylation of CDC2/p34 in human gastric carcinoma BGC-823 cells. Carcinogenesis. 2007;28:632–8.CrossRefPubMedGoogle Scholar
  8. 8.
    Yang Z, Klionsky DJ. Eaten alive: a history of macroautophagy. Nat Cell Biol. 2010;12:814–22.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, et al. Molecular definitions of cell death subroutines: recommendations of the nomenclature committee on cell death 2012. Cell Death Differ. 2012;19:107–20.CrossRefPubMedGoogle Scholar
  10. 10.
    O Farrell F, Rusten TE, Stenmark H. Phosphoinositide 3-kinases as accelerators and brakes of autophagy. FEBS J. 2013;280:6322–37.CrossRefPubMedGoogle Scholar
  11. 11.
    Huang J, Lam GY, Brumell JH. Autophagy signaling through reactive oxygen species. Antioxid Redox Signal. 2011;14:2215–31.CrossRefPubMedGoogle Scholar
  12. 12.
    Liu Z, Lenardo MJ. Reactive oxygen species regulate autophagy through redox-sensitive proteases. Dev Cell. 2007;12:484–5.CrossRefPubMedGoogle Scholar
  13. 13.
    Chang L, Graham PH, Hao J, Ni J, Bucci J, Cozzi PJ, et al. PI3K/Akt/mTOR pathway inhibitors enhance radiosensitivity in radioresistant prostate cancer cells through inducing apoptosis, reducing autophagy, suppressing NHEJ and HR repair pathways. Cell Death Dis. 2014;5, e1437.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Fujiwara K, Iwado E, Mills GB, Sawaya R, Kondo S, Kondo Y. Akt inhibitor shows anticancer and radiosensitizing effects in malignant glioma cells by inducing autophagy. Int J Oncol. 2007;31:753–60.PubMedGoogle Scholar
  15. 15.
    Mehta M, Khan A, Danish S, Haffty BG, Sabaawy HE. Radiosensitization of primary human glioblastoma stem-like cells with low-dose Akt inhibition. Mol Cancer Ther. 2015;14:1171–80.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Ge Y, Liu J, Yang X, Zhu H, Yang B, Zhao K, et al. Fenofibrate enhances radiosensitivity of esophageal squamous cell carcinoma by suppressing hypoxia-inducible factor-1alpha expression. Tumour Biol J Int Soc Oncodev Biol Med. 2014;35:10765–71.CrossRefGoogle Scholar
  17. 17.
    Hoshikawa H, Indo K, Mori T, Mori N. Enhancement of the radiation effects by d-allose in head and neck cancer cells. Cancer Lett. 2011;306:60–6.CrossRefPubMedGoogle Scholar
  18. 18.
    Nie F, Zhang X, Qi Q, Yang L, Yang Y, Liu W, et al. Reactive oxygen species accumulation contributes to gambogic acid-induced apoptosis in human hepatoma smmc-7721 cells. Toxicology. 2009;260:60–7.CrossRefPubMedGoogle Scholar
  19. 19.
    Wang J, Yuan Z. Gambogic acid sensitizes ovarian cancer cells to doxorubicin through ROS-mediated apoptosis. Cell Biochem Biophys. 2013;67:199–206.CrossRefPubMedGoogle Scholar
  20. 20.
    Yang LJ, Chen Y, He J, Yi S, Wen L, Zhao S, et al. Effects of gambogic acid on the activation of caspase-3 and downregulation of SIRT1 in RPMI-8226 multiple myeloma cells via the accumulation of ROS. Oncol Lett. 2012;3:1159–65.PubMedPubMedCentralGoogle Scholar
  21. 21.
    Tanida I, Ueno T, Kominami E. Lc3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol. 2004;36:2503–18.CrossRefPubMedGoogle Scholar
  22. 22.
    Huang H, Chen D, Li S, Li X, Liu N, Lu X, et al. Gambogic acid enhances proteasome inhibitor-induced anticancer activity. Cancer Lett. 2011;301:221–8.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Pandey MK, Sung B, Ahn KS, Kunnumakkara AB, Chaturvedi MM, Aggarwal BB. Gambogic acid, a novel ligand for transferrin receptor, potentiates TNF-induced apoptosis through modulation of the nuclear factor-kappaB signaling pathway. Blood. 2007;110:3517–25.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Luo GX, Cai J, Lin JZ, Luo WS, Luo HS, Jiang YY, et al. Autophagy inhibition promotes gambogic acid-induced suppression of growth and apoptosis in glioblastoma cells. Asian Pac J Cancer Prev APJCP. 2012;13:6211–6.CrossRefPubMedGoogle Scholar
  25. 25.
    Shi Y, Tang B, Yu PW, Tang B, Hao YX, Lei X, et al. Autophagy protects against oxaliplatin-induced cell death via ER stress and ROS in Caco-2 cells. PLoS One. 2012;7, e51076.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Rubinstein AD, Kimchi A. Life in the balance—a mechanistic view of the crosstalk between autophagy and apoptosis. J Cell Sci. 2012;125:5259–68.CrossRefPubMedGoogle Scholar
  27. 27.
    Yang Y, Yang Y, Yang X, Zhu H, Guo Q, Chen X, Zhang H, Cheng H, Sun X. Autophagy and its function in radiosensitivity. Tumour Biol J Int Soc Oncodev Biol Med. 2015.Google Scholar
  28. 28.
    Thorburn A. Apoptosis and autophagy: regulatory connections between two supposedly different processes. Apoptosis Int J Program Cell Death. 2008;13:1–9.CrossRefGoogle Scholar
  29. 29.
    Dewaele M, Maes H, Agostinis P. Ros-mediated mechanisms of autophagy stimulation and their relevance in cancer therapy. Autophagy. 2010;6:838–54.CrossRefPubMedGoogle Scholar
  30. 30.
    Scherz-Shouval R, Elazar Z. Regulation of autophagy by ROS: physiology and pathology. Trends Biochem Sci. 2011;36:30–8.CrossRefPubMedGoogle Scholar
  31. 31.
    Deorukhkar A, Ahuja N, Mercado AL, Diagaradjane P, Raju U, Patel N, et al. Zerumbone increases oxidative stress in a thiol-dependent ROS-independent manner to increase DNA damage and sensitize colorectal cancer cells to radiation. Cancer Med. 2015;4:278–92.CrossRefPubMedGoogle Scholar
  32. 32.
    Powell S, McMillan TJ. DNA damage and repair following treatment with ionizing radiation. Radiother Oncol J Eur Soc Ther Radiol Oncol. 1990;19:95–108.CrossRefGoogle Scholar
  33. 33.
    Zhang H, Lei Y, Yuan P, Li L, Luo C, Gao R, et al. ROS-mediated autophagy induced by dysregulation of lipid metabolism plays a protective role in colorectal cancer cells treated with gambogic acid. PLoS One. 2014;9, e96418.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Ishaq M, Khan MA, Sharma K, Sharma G, Dutta RK, Majumdar S. Gambogic acid induced oxidative stress dependent caspase activation regulates both apoptosis and autophagy by targeting various key molecules (NF-kappaB, Beclin-1, p62 and NBR1) in human bladder cancer cells. Biochim Biophys Acta. 1840;2014:3374–84.Google Scholar
  35. 35.
    Zhen YZ, Lin YJ, Li KJ, Yang XS, Zhao YF, Wei J, et al. Gambogic acid lysinate induces apoptosis in breast cancer MCF-7 cells by increasing reactive oxygen species. Evid Based Complement Alternat Med eCAM. 2015;2015:842091.CrossRefPubMedGoogle Scholar
  36. 36.
    Lee SH, Park DW, Park SC, Park YK, Hong SY, Kim JR, et al. Calcium-independent phospholipase A2beta-Akt signaling is involved in lipopolysaccharide-induced NADPH oxidase 1 expression and foam cell formation. J Immunol. 2009;183:7497–504.CrossRefPubMedGoogle Scholar
  37. 37.
    Saunders JA, Rogers LC, Klomsiri C, Poole LB, Daniel LW. Reactive oxygen species mediate lysophosphatidic acid induced signaling in ovarian cancer cells. Free Radic Biol Med. 2010;49:2058–67.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Yan Yang
    • 1
  • Xiangdong Sun
    • 2
  • Yuehua Yang
    • 1
  • Xi Yang
    • 1
  • Hongcheng Zhu
    • 1
  • Shengbin Dai
    • 3
  • Xiaochen Chen
    • 1
  • Hao Zhang
    • 1
  • Qing Guo
    • 1
    • 3
  • Yaqi Song
    • 5
  • Feng Wang
    • 6
  • Hongyan Cheng
    • 4
  • Xinchen Sun
    • 1
  1. 1.Department of RadiotherapyThe First Affiliated Hospital of Nanjing Medical UniversityNanjingChina
  2. 2.Department of RadiotherapyThe 81st Hospital of PLANanjingChina
  3. 3.Department of OncologyPeople’s Hospital of TaizhouTaizhouChina
  4. 4.Department of Synthetic Internal MedicineThe First Affiliated Hospital of Nanjing Medical UniversityNanjingChina
  5. 5.Department of Radiation Oncology, Huai’an First People’s HospitalNanjing Medical UniversityNanjingChina
  6. 6.Department of Radiation Oncology, Nantong Tumor HospitalAffiliated Tumor Hospital of Nantong UniversityNantongChina

Personalised recommendations