(M1130-05-29) Synergistic Anticancer Activity of Cannabinoids and Terpenes in Xenograft Mouse Model of Triple Negative Breast Cancer

Purpose: Combination therapy, a treatment modality that studies the effect of two or more drugs is the most common strategy used to overcome drug resistance in cancer. Cannabinoids like CBD have shown potential as anticancer agents. Terpenes have also demonstrated to exhibit anticancer activity. The purpose of this study was to evaluate the effect of combinations of various cannabinoids and terpenes against overcoming breast cancer resistance.

Methods: Several cannabinoids such as cannabichromene (CBC), cannabidiolic acid (CBDA), cannabidivarin (CBDV), cannabinol (CBN), cannabigerol (CBG) were screened for their anticancer activity against MDA MB 231 DOX RT, H1975 OSM RT, PDX Lung cancer cell lines using 2D cytotoxicity and 3D spheroids assay. Terpenes such as β-caryophyllene, bisabolol, myrcene, linalool, limonene, geraniol, neralidol, α-pinene, α-terpineol, γ-terpinene were evaluated individually against MDA MB 231 DOX RT, H1975 OSM RT, PDX Lung cancer cell lines for their anticancer potential in 2D cytotoxicity and 3D spheroid assay. The potential cannabinoids (all) and terpenes (β-caryophyllene) obtained from the individual screening were further investigated in combinations against resistance cancer cell lines. The mode of action of combinations, either synergistic, additive or antagonistic was determined from combination index (CI) values using compusyn software. Other assays such as colony formation assay, wound healing assay, Apoptosis assay, cell cycle analysis were also performed with the potential combinations. Compiling all the in-vitro results one combination (CBC+BC) was taken further for in-vivo studies. The animal model was developed by injecting 5 million MDA MB 231 DOX RT cells by S.C route in the right flank region of athymic nude mice. When once after the tumor volume reached 180-200mm³, the animals were administered CBC (15mg/kg, i.p.), BC (100mg/kg, i.p.) and CBC+BC thrice a week for two weeks. The tumor volumes were measured using vernier caliper and the tumor volume was calculated using the formula Tumor volume = 1/2 xy², where ‘x’ and ‘y’ represent the length and width of the tumors. The animals were euthanized, tumors were collected and processed for further studies.

Results: The IC50 values of cannabinoids were found to be 9.36±0.22µM against all the three resistant cancer cell lines in a 2D cytotoxicity assay. The IC50 values of β-caryophyllene and bisabolol were found to be 32.47±0.84µM and 80.50±0.94µM respectively in 2D cytotoxicity assay. Other terpenes screened had IC50 values more than 150µM and hence were not studied further. The IC50 values of combinations of cannabinoids and β-caryophyllene decreased by 10 folds and were 1.34± 0.05µM for all the combinations. Same pattern was replicated in 3D spheroid assay i.e., the IC50 values dropped from 45.83±0.52µM to 7.24±0.74µM. The combination index values were found to be < 1 at all concentrations for the selected combinations suggesting synergism. The colony formation ability of cells post treatment with combinations decreased significantly (p < 0.01) which was confirmed with staining as well. CBC+BC was found to be the most potential combination from all the in-vitro screening studies and was taken further for in vivo studies. CBC+BC combination controlled tumor progression significantly in comparison to individual treatment groups. The rate of tumor growth was maintained at 1700mm³ till 14 days post-treatment for the combination group whereas in the individual treatments the rate of tumor growth reached 2000mm³ after 7 days of treatment. Also, the animals of the combination group maintained their weight and remained healthy for 2 weeks relative to control and individual treatments.

Conclusion: From the 2D cytotoxicity studies, it was observed that CBC along with BC was the most potent combination with combination index value of 0.1 for all the concentrations evaluated. It was further validated by 3D spheroids assay. From all the other In-vitro studies such as wound healing, colony formation, apoptosis, cell cycle analysis and in vivo xenograft studies, it can be concluded that CBC+BC is the most potent combination against resistant TNBC.

Figure 1 In-vitro studies showing antitumor activity of combinations of cannabinoids and terpenes in comparison to individual treatments A) 2D In-vitro cytotoxicity assay of i) Cannabinoids ii) Terpenes iii) Combinations of cannabinoids with β-caryophyllene demonstrating that combinations are effective by 10 folds than individual treatments B) In-vitro cytotoxicity studies with 3D spheroids with MDA-MB231 Dox resistant cells C) Cell cycle analysis by flow cytometry using Propidium Iodide demonstrating that the 90% of cells remain in G1 phase post treatment with combinations compared to individual treatments which had 80% cells in G1 phase D) Apoptosis assay using Acridine Orange and Ethidium Bromide demonstrating relatively 60% of cells undergoing apoptosis in combination treatments whereas 40% in individual treatments E) Cell migration assay suggesting a significant decrease in migration ability of cells treated with combinations compared to individual treatments F) Colony formation assay showing that very few/no colonies in combination groups as compared to individual treatments.

Figure 2 In-vivo studies on Xenograft mouse model of Triple Negative Breast Cancer demonstrating significant control on tumor progression in combination group relative to individual groups.