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