Research Papers:

Chinese herb cinobufagin-reduced cancer pain is associated with increased peripheral opioids by invaded CD3/4/8 lymphocytes

Tao Chen, Shenjun Yuan, Xin-nian Wan, Ling Zhan, Xue-qin Yu, Jian-hong Zeng, Hong Li, Wen Zhang, Xiang-yang Hu, Yi-fei Ye and Wei Hu _

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Oncotarget. 2017; 8:11425-11441. https://doi.org/10.18632/oncotarget.14005

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Tao Chen1,2,4,*, Shenjun Yuan1,3,*, Xin-nian Wan1,3, Ling Zhan1,3, Xue-qin Yu1,3, Jian-hong Zeng1,2, Hong Li1,3, Wen Zhang1,3, Xiang-yang Hu1,3, Yi-fei Ye1,3, Wei Hu1,2

1Third-Grade Pharmacological Laboratory on Chinese Medicine Approved by State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang 443002, China

2Department of Pharmacology, China Three Gorges University, Yichang 443002, China

3College of Medical Science, China Three Gorges University, Yichang 443002, China

4Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang 443002, China

*These authors have contributed equally to this work

Correspondence to:

Wei Hu, email: [email protected]

Tao Chen, email: [email protected]

Keywords: cinobufagin, cancer pain, β-endorphin, peripheral opioid receptor, lymphocytes

Received: July 13, 2016     Accepted: November 14, 2016     Published: December 17, 2016


Objectives: To investigate the mechanism of cinobufagin-reduced cancer pain in mouse cancer pain model and in vitro cell co-culture system.

Methods: Female Kunming mice were randomly divided into 4 groups. One group of animals was set as normal control without any treatment. Other three groups of animals received H22 hepatoma cell inoculation in right hind paw. At day 9 after inoculation, mice in other three groups were injected intraperitoneally once a day for 8 days with the solvent, morphine or cinobufagin, respectively. The pain behavior was recorded daily. On the last day, all mice were sacrificed and xenograft tissues homogenate and plasma levels of β-endorphin (β-END), corticotropin-releasing factor (CRF) and interleukin-1β (IL-1β) were assessed by ELISA assay. Immunohistochemistry was performed to determine the expression of β-END, pro-opiomelanocortin (POMC) and the μ-opioid receptor (μ-OR) in the xenograft tissues. Immunofluorescence was used to localize lymphocytes with expression of CD3+, CD4+ and CD8+ in xenograft tumors and adjacent tissues. Mice splenic lymphocytes and H22 hepatoma carcinoma ascites cells were prepared for co-culture. β-END and CRF were detected in co-culture supernatants. The MTT assay and cytometry were used to assess cell proliferation. RT-PCR was conducted to determine the gene expression of POMC and Cathepsin L (CTSL). Chemotaxis was examined using a transwell-based migration assay.

Results: Compared to the model group, the thermal and mechanical pain thresholds were increased in mice after cinobufagin treatment. The expression of β-END and CRF in the plasma and tumor tissues of cinobufagin group were much higher than that of the model group mice, but the expression of IL-1β in the plasma and tumor tissues was much lower than that in the model group mice. Meanwhile, the expression of β-END, POMC and μ-OR proteins was significantly increased in the xenograft tissues from cinobufagin group. Lymphocyte population of CD3+, CD4+, CD8+ were also elevated in xenograft tumors and adjacent tissues. In the cell co-culture assays, the content of β-END in the supernatant was significantly increased by cinobufagin in a dose-dependent manner. Cinobufagin also largely increased the proliferation of immune cells and inhibited H22 hepatoma carcinoma cell proliferation in single or co-culture cell assays. Gene expression of POMC and CTSL in cinobufagin group was significantly up-regulated comparing to the control group. Finally, cinobufagin addition enhanced the migration of immune cells in transwell assay.

Conclusions: Cinobufagin-induced local analgesic effect might be associated with increased activity of POMC/β-END/μ-OR pathway released from invaded CD3/4/8 lymphocytes in cancer tissues.

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