Abstract
ObjectivesTo investigate the mechanism of cinobufagin-reduced cancer pain in mouse cancer pain model and in vitro cell co-culture system.MethodsFemale 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.ResultsCompared 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.ConclusionsCinobufagin-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.
Highlights
Cancer pain is a complicated syndrome in cancer patient via distinct causes from cancer mass itself or by its treatment such as surgery, chemotherapy, radiotherapy
The expression of β-END, POMC and μ-opioid receptor (μ-OR) proteins was significantly increased in the xenograft tissues from cinobufagin group
Cinobufagin largely increased the proliferation of immune cells and inhibited H22 hepatoma carcinoma cell proliferation www.impactjournals.com/oncotarget in single or co-culture cell assays
Summary
Cancer pain is a complicated syndrome in cancer patient via distinct causes from cancer mass itself or by its treatment such as surgery, chemotherapy, radiotherapy. Cancer pain often times leads to mental, psychological and even ‘social pain’. It produces anxiety, depression and negative feelings of worth. There are a variety of methods applying to treat cancer pain, such as bisphosphonates, chemotherapy, surgery, nerve block, adoptive tumor immunotherapy, and gene knockout, the clinic treatment of cancer pain is still to focus on the three-step program. Many patients tortured by cancer pain could still not been controlled appropriately, and there are many problems needed to be solved such as “mirror pain”, morphine tolerance, constipation, respiratory depression for opioid drugs, and stomach ulcers and kidney toxicity for nonsteroidal antiinflammatory analgesics. The clinical use of these drugs could be limited by these side effects [2]
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