Cephalomannine as in vitro and in vivo anticancer agent in mesothelioma cells: mechanisms of action

Figures

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  Fig. 1. NOD-SCID female mice were injected i.p. with REN cells. Mice had administrations of solvent or fludarabine at (A) 200 mg/Kg bw, (B) 100 mg/Kg bw, (C) 40 mg/kg bw, (D) Crenolanib 15 mg/kg bw, (E) Fludarabine 40 mg/Kg bw + Crenolanib 15 mg/Kg bw, (F) Cephalomannine 2 mg/Kg bw. Bw = body weight

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  Fig. 2. Tumour dimension and localization before and after cephalomannine treatment, acquired by the in vivo imaging system IVIS. A Baseline (pre-treatment, 12 days after i.p. injection) IVIS imaging results of mice i.p. injected with luciferase transduced REN cells and assigned to the control (n = 10) and treatment (n = 10) groups. Imaging confirmed that all mice in both groups harbor intra-abdominal growing tumors. B Post-treatment IVIS imaging of mice treated with the vehicle (PBS/2%BSA) or cephalomannine (2 mg/Kg). The imaging was acquired 2 days after the end of the treatment with CPM

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  Fig. 3. Assessment of susceptibility to oxidative stress in MeT-5A and REN cells. CPM treatment at 0.1 µM revealed that REN cells produced a significantly higher amount of ROS compared to the vehicle medium (DMSO), indicating increased susceptibility to oxidative stress. The data were analysed in triplicate. The values are expressed as mean ± standard deviation (SD). Statistical analysis was conducted using an unpaired t test with Welch’s correction. * p < 0.05, ** p < 0.005

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  Fig. 4. Mitochondrial respiratory analysis of MeT-5A and REN cell lines after CPM treatment. Micro-oxygraphy track showed a reduced OCR in REN cell line treated with CPM at 0.1 µM, particularly evident after FCCP injection, reflecting a deficient SRC and a reduced ATP production. Data represent mean ± SEM. Three independent experiments were run with four technical replicates for each condition. Statistical analysis was assessed by multiple unpaired t tests with Bonferroni’s correction. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

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  Fig. 5. Representative western blots of BAX, Bcl2 and IL-18 expression in MeT-5A and REN cell lines following CPM treatment at 0.1 µM for 72 h. (A) Blot and the corresponding quantification (B) of BAX protein expression levels showed a statistically significant activation of the protein in both cell lines after CPM treatment. (C) Blot and corresponding quantification (D) of Bcl2 showed a statistically significant reduction of protein expression levels, in both cell lines, after CPM treatment. (E) Blot and corresponding quantification (F) of IL-18 showed that CPM treatment does not appear to alter the expression levels of this protein. β-Tubulin was used as loading control. Three independent experiments were performed, here represented in the histograms as mean ± SD. Statistic comparison between the mean levels of each treated line and the control mean (vehicle, DMSO) were assessed using multiple Mann–Whitney tests with Bonferroni-Dunn correction for multiple comparison. * p < 0.05, ** p < 0.005. NT = not treated cells