Fig 1: KOR agonist inhibits proliferation in HCC cells. (A) Hep3B and Huh7 cells were treated with the KOR agonist U50488h (0,0.1,1, and 10µM), the mixed agonist oxycodone (0,0.1,1, and10µM) and the selective MOR agonist morphine (0,0.1,1, and10 µM) for 24 h and 48 h, and CCK-8 assay was performed to assess cell viability. (B, C) Hep3B and Huh7 cells were treated with U50488h (10 µM), Oxycodone (10 µM) and Morphine (10 µM) for 48 h for colony formation assay. Values are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; #P < 0.05 vs. Morphine group.
Fig 2: KOR agonist induces apoptosis in HCC cells. (A) Hep3B and Huh7 cells were exposed to 10 µM U50488h, 10 µM oxycodone and 10 µM morphine for 48 h using PI/Annexin V-FITC flow cytometry. (B) Histogram indicates the rate of apoptosis of cells. (C–E) Cells were cultured with 50 µM U50488h, 10 µM oxycodone and 10 µM morphine for 48 h, and the expression of apoptosis-related proteins such as Bax and Cleaved caspase-3 were analyzed by western blotting. ß-actin was used as an internal control. Values are reported as the mean ± standard deviation (n = 3). **P < 0.01, ***P < 0.001 vs. control group; ##P < 0.01, ###P < 0.001 vs. U50488h group; &P < 0.1, &&P < 0.01 vs. oxycodone group. PI, propidium iodide; FITC, fluorescein isothiocyanate.
Fig 3: KOR agonist U50488h and oxycodone decreases the migration of HCC cells. (A) Hep3B and Huh7 cells were treated with 10 µM U50488h, 10 µM oxycodone and 10µM morphine for 48 h to detected the motility of cells, and wound healing assay was performed, scale bar=100 um. (B) Histogram represents the wound closure of cells. (C-F) Western blotting was used to detect the expression of E-cadherin, N-cadherin, Vimentin and ß-actin in Hep3B and Huh7 cells treated with U50488h (10 µM), oxycodone (10 µM) and morphine (10 µM). ß-actin was used as an internal control. Values are reported as the mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; ##P < 0.01, ###P < 0.001vs. U50488h group.
Fig 4: KOR agonist increased expression level of PERK and Caspase 3 in vivo. (A) Representative IHC staining in tumor tissue for PERK and Caspase 3 in the control and OR agonists treated mice (n = 5) (scaled bar= 25 um); (B) Histogram represent the mean of IOD. (C) Levels of PERK and Caspase 3 determined by western blot (n = 3). Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001 vs. control group; ###P < 0.001 vs. U50488h group.
Fig 5: The expression of KOR and MOR in HCC cell lines and human HCC tissues. (A) The expression of KOR and MOR were detected in LO2, Hep3B and Huh7 cells by immunofluorescent staining, scale bar, 25um. The expression of KOR and MOR was evaluated in 4 types of HCC cell lines (HepG2, Bel-7402, Hep3B and Huh-7) by RT-qPCR (B) and western blotting (C). (D) In human HCC tissues, KOR and MOR were detected by western blotting, GADPH was used as an internal control. N, Non-tumor; T, Tumor. Values are presented as the mean ± standard deviation (n = 3). KOR, kappa opioid receptor; MOR, mu opioid receptor; GADPH, glyceraldehyde-3-phosphate dehydrogenase.
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