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Synergistic Anticancer Activity of Cannabinoids and Terpenes Against Doxorubicin Resistance Breast Cancer

Mounika Aare
Florida A&M University

Co-Authors: Breana Boirie, Jassy Lazarte, Aakash Nathani, Mandip Singh Sachdeva
Florida A&M University

Objective: The objective of this study was to evaluate the effect of combinations of various cannabinoids and terpenes against drug-resistant lung and breast cancers.

Methods: Several cannabinoids including cannabichromene (CBC), cannabidiolic acid, cannabidivarin, cannabinol, cannabigerol and terpenes; β-caryophyllene (BC), bisabolol, myrcene, linalool, limonene, geraniol, nerolidol, α-pinene, α-terpineol, and γ-terpinene were screened for their anticancer activity against doxorubicin- resistant (DOX-RT) MDA-MB-231 breast cancer, and osimertinib-resistant H1975 (with EGFR, T790M, L858R mutations) and PDX lung cancer cell lines (with EFGR, L858R mutations) using 2D cytotoxicity and 3D spheroids assays. Combination index (CI) values of cannabinoids with BC were evaluated using compusyn software. Other assays such as colony formation, wound healing, apoptotic assay, and cell-cycle analysis by flow cytometry were done to investigate the possible mechanisms underlying the synergism of the combinations investigated. In-vivo studies were conducted to further test the efficacy of combinations. Briefly, 5 million DOX-RT MDA-MB-231 cells were injected subcutaneously into the right flank region of BALB/c nude mice. The animals were treated after the tumor volume reached 180 – 200 mm3. The animals were administered CBC (15mg/kg, i.p.), BC (100mg/kg, i.p.), and CBC+BC thrice a week for two weeks. Western blotting and qPCR was done to understand the molecular mechanisms.

Results: It was observed that CBC, CBDA with BC over a concentration range of 0.3-10μM has shown strong synergism with CI values of 0.1-0.4 whereas other combinations of CBG+BC, CBDV+BC, and CBN+BC have shown synergism at low concentrations but strong antagonism at high concentrations (5, 10μM). CBC+BC was the most potent combination among the in-vitro studies, and hence, was further evaluated in in-vivo tumor xenografts. The tumor volume in the combination group was reduced two-fold relative to individual treatments (p<0.001) and fourfold relative to control (p<0.0001). Western blot analysis of tumor xenografts revealed downregulation of apoptotic markers: PARP (p<0.0001), mTOR (p<0.001), pAMPK (p<0.001), surviving (p<0.0001); autophagy markers, LC3 (p<0.001) and Glypican 5 (p<0.0001); and migration markers, vimentin (p<0.0001) and integrin (p<0.0001). Immune checkpoint proteins PD-L1 (p<0.0001) and PD-1 (p<0.0001) were also downregulated by combination treatment. Few of these markers like PD-L1, PARP, mTOR were further confirmed by qPCR and found to be significantly down regulated as well. CBC was found to induce apoptosis and autophagy considerably more potently than BC; whereas BC inhibited cancer cell migration which explains the synergism of CBC+BC.

Conclusion: The results obtained emphasizes that the combination of CBC and BC can successfully overcome doxorubicin resistance in TNBC.