Fig. 2: Cannabichromene (CBC) treatment induces cell death via a ferroptosis-related signaling pathway. | Cell Death Discovery

Fig. 2: Cannabichromene (CBC) treatment induces cell death via a ferroptosis-related signaling pathway.

From: Cannabichromene: integrative modulation of apoptosis, ferroptosis, and endocannabinoid signaling in pancreatic cancer therapy

Fig. 2

A Heatmap of ferroptosis-related genes, obtained from mRNA-seq data. B Quantitative PCR analysis of ferroptosis-related genes, performed using CBC-treated pancreatic cancer cells. HMOX1, CHAC1, SLC3A2, SLC7A11, and FTH1 levels were quickly increased by CBC treatment at the transcriptional level. C Western blot analysis showed that HMOX1 protein expression was upregulated by CBC treatment. The graphs were obtained from densitometric data for western blot images. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s multiple comparison test, with P < 0.01 or P < 0.001 vs. 0 h. D Increased HMOX1 protein expression examined by performing an immunostaining experiment. Statistical significance was determined using Student’s t test, with P ≤ 0.05. E We performed flow cytometric analysis using H2DCFDA to measure the reactive oxygen species (ROS) level after CBC treatment. The ROS level was increased in CBC-treated pancreatic cancer cells but slightly restored after combined treatment with CBC and ferrostatin, a ferroptosis inhibitor. However, the ROS level was severely increased after combination treatment with CBC and erastin, a ferroptosis inducer. F Lipid peroxidation in pancreatic cancer cells was examined after CBC treatment. The malondialdehyde (MDA) level was increased in CBC-treated cancer cells but downregulated after combined treatment with CBC and ferrostatin. However, the MDA level was severely increased after combination treatment with CBC and erastin. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s multiple comparison test, with P < 0.01 or P < 0.001 vs. no treatment. G The GSH/GSSG ratio was examined using a GSH kit. All data are similar to the data obtained from the ROS and lipid peroxidation analysis. Statistical significance was determined using Student’s t tests, with p-values ≤ 0.05 indicating statistical significance. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s multiple comparison test, with P < 0.01 or P < 0.001 vs. no treatment. MIA PaCa-2 and PANC-1 cells were treated with 35 μM and 30 μM CBC, respectively. Additional treatments included 10 μM ferrostatin-1 (Fer-1) and erastin.

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