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
First Online:
Received:
Accepted:

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

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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

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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

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