Mounika Aare
Florida A&M University
Co-Authors: Jassy Lazarte1, Breana Boirie1, Mandip Singh Sachdeva1
1Florida A&M University
Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with few treatment options and a high likelihood of developing drug resistance. Cannabinoids have emerged as potential anticancer agents, and their “entourage effect”—the synergistic interplay between different cannabinoids—may enhance therapeutic efficacy. Epigenetic modifications, particularly histone methylation and acetylation, are critical in driving drug resistance and altering gene expression profiles. Moreover, DNMT2, an RNA methyltransferase primarily targeting tRNA, plays a role in cellular stress responses and may contribute through altered RNA methylation. This study investigates the combined effects of cannabichromene (CBC) and cannabidiol (CBD) on overcoming drug resistance in TNBC by modulating epigenetic and epitranscriptomic regulators.
Objectives: This study evaluates the cytotoxicity of CBC, CBD, and their combination in drug resistant TNBC cells, examines their effects on cell cycle regulation and apoptosis, investigates histone methylation and acetylation changes, and validate in vitro findings using an in vivo xenograft model.
Methods: MTT assays determined IC50 values for CBC, CBD, and their combination in MDA-MB-468 RM RT cells. Flow cytometry analyzed cell cycle distribution, while western blotting assessed key signaling proteins. Epigenetic modifications were examined via histone profiling and DNMT2. In vivo studies used MCJB 20C PDX xenografts.
Results: Cytotoxicity studies in MDA-MB-468 Doxorubicin RT cells demonstrated that CBC+CBD exhibited a significantly lower IC50 (0.42 ± 0.022 μM) compared to individual treatments (CBD: 3.88 ± 0.54 μM, CBC: 5.06 ± 0.1 μM), and Doxorubicin suggesting strong synergy (CI: 0.34–0.64). Cell Cycle revealed that CBC+CBD induced significantly higher G1-phase arrest. Epigenetic Reprogramming studies revealed that CBC+CBD significantly reversed H3K27me3 and H3K4me3 modifications, suggesting chromatin remodeling and reactivation of tumor-suppressor genes. Notably, DNMT2 expression was downregulated, suggesting reduced RNA methylation linked to drug resistance. CBC+CBD reduced tumor volume by 65% (p<0.0001), compared to 27% with CBD and 22% with CBC alone respectively. Western blotting revealed significant reduction in vimentin (p<0.0001) and survivin, PARP (p<0.000), Nf-κB, mTOR (p<0.001) and an increase in cleaved PARP and cleaved caspases-9 and 3 in CBC+CBD-treated tumors. Current studies are ongoing to explore the impact of CBC+CBD on additional histone modifications, including acetylation (H3K5ac, H3K9ac, H3K27ac, H4K16ac) and phosphorylation (H3S10ph), as well as on chromatin remodeling proteins such as HDAC1, EZH2, and p300.
Conclusion: The combination of CBC and CBD exhibits a synergistic entourage effect, leading to enhanced cytotoxicity, cell cycle arrest, apoptosis induction, and epigenetic reprogramming in drug-resistant TNBC. These findings warrant further investigation to translate CBC+CBD therapy into clinical applications.