Insulin enhances apoptosis induced by cisplatin in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy.
BACKGROUND
Chemoresistance is common among patients with esophageal squamous cell carcinoma (ESCC). We investigated the effect and mechanism of insulin on enhancing anticancer functions of cisplatin in human esophageal cancer cell line EC9706.


METHODS
The viability of EC9706 cells exposed to cisplatin was assessed using MTT assay. The times T1, when the number of living cells reached a plateau and T2, when the number of living cells reached a new plateau after the addition of insulin were found. T1 and T2 plateau cells were stained by Annexin V-FITC/PI and monodansylcadaverin (MDC). Fluorescent microscopy was used to observe the expression of apoptosis and autophagy intuitively. Apoptotic ratio and fluorescent intensity were analysed by flow cytometry (FCM) quantitatively. Western blotting analysis was used to estimate the protein expression levels of AKT, mTOR, PI3K, PTEN, autophage related indicator LC3-II and autophage related protein Beclin1 changes that occurred in the course of treatment.


RESULTS
A larger number of typical autophagosomes were detected in EC9706 cells exposed to cisplatin. Insulin can increase the apoptosis induced by cisplatin. Apoptotic ratio of T1 plateau cells ((32.6 ± 4.3)%) is significantly less than T2 plateau ((47.5 ± 5.6)%). MDC fluorescent intensity at T1 plateau (104.9 ± 13.2) was significantly higher than intensity at T2 plateau (82.6 ± 10.3). After cotreatment with insulin, the expression level of LC3-II, Beclin1 and PTEN in T2 plateau cells were significantly downregulated, but AKT, mTOR and PI3K expressions significantly upregulated compared with T1 plateau.


CONCLUSIONS
Insulin could enhance cisplatin-induced apoptosis in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy. The activation of PI3K/Akt/mTOR signaling pathway induced by insulin resulted in the suppression of autophagy in EC9706 cells, which may be attributed to the anticancer effects of cisplatin.
Department of Thoracic Surgery, First Affiliated Hospital, Zhengzhou University, Open Key Clinical Medical Experimental Laboratory Institute of Henan Province, Institute of Molecular Cancer Surgery of Zhengzhou University, the Key Thoracic Tumour Experimental Laboratory of Zhengzhou, Zhengzhou, Henan 450052, China.
Yang
Y
Yang
Department of Thoracic Surgery, First Affiliated Hospital, Zhengzhou University, Open Key Clinical Medical Experimental Laboratory Institute of Henan Province, Institute of Molecular Cancer Surgery of Zhengzhou University, the Key Thoracic Tumour Experimental Laboratory of Zhengzhou, Zhengzhou, Henan 450052, China.
Fengbiao
F
Wen
Physical Examination Centre, First Affiliated Hospital, Zhengzhou University, Open Key Clinical Medical Experimental Laboratory Institute of Henan Province, Institute of Molecular Cancer Surgery of Zhengzhou University, the Key Thoracic Tumour Experimental Laboratory of Zhengzhou, Zhengzhou, Henan 450052, China.
Lifeng
L
Dang
Department of Thyroid Surgery, First Affiliated Hospital, Zhengzhou University, Open Key Clinical Medical Experimental Laboratory Institute of Henan Province, Institute of Molecular Cancer Surgery of Zhengzhou University, the Key Thoracic Tumour Experimental Laboratory of Zhengzhou, Zhengzhou, Henan 450052, China.
Yuxia
Y
Fan
Department of Thoracic Surgery, First Affiliated Hospital, Zhengzhou University, Open Key Clinical Medical Experimental Laboratory Institute of Henan Province, Institute of Molecular Cancer Surgery of Zhengzhou University, the Key Thoracic Tumour Experimental Laboratory of Zhengzhou, Zhengzhou, Henan 450052, China.
Donglei
D
Liu
Department of Thoracic Surgery, First Affiliated Hospital, Zhengzhou University, Open Key Clinical Medical Experimental Laboratory Institute of Henan Province, Institute of Molecular Cancer Surgery of Zhengzhou University, the Key Thoracic Tumour Experimental Laboratory of Zhengzhou, Zhengzhou, Henan 450052, China.
Kai
K
Wu
Department of Thoracic Surgery, First Affiliated Hospital, Zhengzhou University, Open Key Clinical Medical Experimental Laboratory Institute of Henan Province, Institute of Molecular Cancer Surgery of Zhengzhou University, the Key Thoracic Tumour Experimental Laboratory of Zhengzhou, Zhengzhou, Henan 450052, China. Email: zhaosong@zzu.edu.cn.
Song
S
Zhao
D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D004938:Esophageal Neoplasms / Q000378:metabolism*; D000077277:Esophageal Squamous Cell Carcinoma; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D002945:Cisplatin