Identification of Synergistic Interaction Between Cannabis-Derived Compounds for Cytotoxic Activity in Colorectal Cancer Cell Lines and Colon Polyps That Induces Apoptosis-Related Cell Death and Distinct Gene Expression

Abstract

Introduction: Colorectal cancer remains the third most common cancer diagnosis and fourth leading cause of cancer-related mortality worldwide. Purified cannabinoids have been reported to prevent proliferation, metastasis, and induce apoptosis in a variety of cancer cell types. However, the active compounds from Cannabis sativa flowers and their interactions remain elusive. Research Aim: This study was aimed to specify the cytotoxic effect of C. sativa-derived extracts on colon cancer cells and adenomatous polyps by identification of active compound(s) and characterization of their interaction. Materials and Methods: Ethanol extracts of C. sativa were analyzed by high-performance liquid chromatography and gas chromatograph/mass spectrometry and their cytotoxic activity was determined using alamarBlue-based assay (Resazurin) and tetrazolium dye-based assay (XTT) on cancer and normal colon cell lines and on dysplastic adenomatous polyp cells. Annexin V Assay and fluorescence-activated cell sorting (FACS) were used to determine apoptosis and cell cycle, and RNA sequencing was used to determine gene expression. Results: The unheated cannabis extracts (C2F), fraction 7 (F7), and fraction 3 (F3) had cytotoxic activity on colon cancer cells, but reduced activity on normal colon cell lines. Moreover, synergistic interaction was found between F7 and F3 and the latter contains mainly cannabigerolic acid. The F7 and F7+F3 cytotoxic activity involved cell apoptosis and cell cycle arrest in S or G0/G1 phases, respectively. RNA profiling identified 2283 differentially expressed genes in F7+F3 treatment, among them genes related to the Wnt signaling pathway and apoptosis-related genes. Moreover, F7, F3, and F7+F3 treatments induced cell death of polyp cells. Conclusions:C. sativa compounds interact synergistically for cytotoxic activity against colon cancer cells and induce cell cycle arrest, apoptotic cell death, and distinct gene expression. F3, F7, and F7+F3 are also active on adenomatous polyps, suggesting possible future therapeutic value.

Keywords: Cannabis; apoptosis; cell cycle arrest; colorectal cancer; cytotoxicity; synergism.

FIG. 1.

Dose–effect curves of Cannabis sativa ethanol extracts on the viability of HCT 116 colon cancer and CCD-18Co colon healthy cells. Dose–effect curves of C. sativa ethanol extracts of fresh inflorescences (C2F) (A; IC50=83.9±0.9 μg/mL, 95% confidence interval=83.2–84.7), heated inflorescences (C2B) (B; IC50=84.1±1.3 μg/mL, 95% confidence interval=83.1–85.2) on the viability of HCT 116 colon cancer cells, and C2F (C; IC50=144.2±1.1 μg/mL, 95% confidence interval=142.9–145.5), C2B (D; IC50=54.63±2.03 μg/mL, 95% confidence interval=53.09–56.17) on the viability of CCD-18Co colon healthy cells. Cell viability determined by alamarBlue fluorescence (Resazurin assay). HCT 116 and CCD-18Co cells were seeded (10,000 per well) in triplicate in 100 μL of growing media and incubated for 24 h at 37°C in a humidified 5% CO₂-95% air atmosphere. Cells were treated with C2F, C2B at different dilutions along with 50 ng/mL TNF-α for 16 h. The cells were next incubated with alamarBlue for 4 h. Relative fluorescence at the excitation/emission of 544/590 nm was measured. Values were calculated as the percentage of live cells relative to the nontreated control (cells without TNF-α and treatments) after reducing the autofluorescence of alamarBlue without cells (n=3). For dose–response assays, data points were connected by nonlinear regression lines of the sigmoidal dose–response relation. GraphPad Prism was used to produce dose–response curve and IC50 doses for C2F and C2B. TNF, tumor necrosis factor.

FIG. 2.

Effect of Cannabis sativa C2F and HPLC fractions (F1–F9) in different combinations on HCT 116 cell viability. (A) Determination of HCT 116 cell viability using XTT assay as a function of live cell number. Cells were seeded and treated with C. sativa ethanol extracts (C2F) F1–F9, excluding F7 (HPLC fractions of C2F) and F1–F9, including F7 (HPLC fractions of C2F) at the IC50 dose of C2F crude (58 μg/mL), and F1–F9 diluted as C2F crude along with 50 ng/mL of TNF-α for 48 h. The cells were then incubated with XTT reagent for 2 h. Absorbance was recorded at 490 nm with 650 nm of reference wavelength. Values were calculated as the percentage of live cells relative to the nontreated (cells without TNF-α and treatments) control after reducing the absorbance without cells. (B) Determination of synergism of C2F Fraction 7 (F7) in combination with Fraction 2 (F2) and Fraction 3 (F3) on HCT 116 cell viability using XTT assay as a function of live cell number. Cells were seeded and treated with IC50 doses of F2 (7 μg/mL), F3 (36 μg/mL), F7 (20 μg/mL), the combinations of F2+F3, F7+F2, and F7+F3, along with 50 ng/mL of TNF-α for 48 h. Subsequently, the cells were incubated with XTT reagent for 2 h. Absorbance was recorded at 490 nm with 650 nm of reference wavelength. Values were calculated as the percentage of live cells relative to the nontreated control (cells without TNF-α and treatments) after reducing the absorbance without cells. Error bars indicate ±SE (n=3). Levels with different letters are significantly different from all combinations of pairs by Tukey–Kramer HSD. HPLC, high-performance liquid chromatography; HSD, honest significant difference.

FIG. 3.

Determination of apoptosis or necrosis as cytotoxic effect of F7, F3, or F7+F3 on HCT 116 cells. HCT 116 cells were treated with F7 (20 μg/mL), F3 (36 μg/mL), the combination of F7 with F3 and solvent control (methanol) along with TNF-α (50 ng/mL) for 24 h (A) or 48 h (B). The treated cells were harvested and analyzed in FACS following Annexin V-FITC and PI staining. Shown are the percentages of live, necrotic, early, and late apoptosis cells, analyzed from 10,000 events per treatment. FACS, fluorescence-activated cell sorting; PI, propidium iodide.

FIG. 4.

Determination of stages of cell cycle arrest induced by F7, F3, or F7+F3 in HCT 116 cells. Starved HCT 116 cells were treated with F7 (20 μg/mL), F3 (36 μg/mL), the combination of F7 with F3 and solvent control (methanol) along with TNF-α (50 ng/mL) for 24 h. The treated cells were harvested, fixed, and analyzed in FACS following PI staining. The percentage of cells in Sub-G0, G0/G1, S, and G2/M phase were analyzed from 10,000 events per treatment.

FIG. 5.

Hierarchical clustering and Venn diagram of genes significantly differentially expressed in HCT 116 cells treated with F7, F3, or F7+F3. (A) Hierarchical clustering and Pearson correlations among the four conditions based on the gene expression (counts-per-million) followed by a log2 transform. Pearson correlations were calculated with the R software. (B) Venn diagrams illustrating the relationships between significantly differentially expressed genes in the three treatments against the control.

FIG. 6.

Genetic pathways of genes differentially expressed in HCT 116 cells treated with F7+F3 versus control for Wnt signaling pathways. Pathways determined according to KEGG (www.genome.jp/kegg/). Green boxes—significantly upregulated genes; red boxes—significantly downregulated genes (edgeR; more than twofold and padj <0.05). Light green boxes—nonsignificant upregulated genes; pink boxes—nonsignificant downregulated genes. Yellow boxes denote genes with multiple gene annotations that encompass both significantly upregulated and downregulated genes; light yellow boxes denote genes with multiple gene annotations that encompass both nonsignificant upregulated and nonsignificant downregulated genes.

FIG. 7.

Genetic pathways of genes differentially expressed in HCT 116 cells treated with F7+F3 versus control for apoptotic signaling pathways. Pathways determined according to KEGG (www.genome.jp/kegg/). Green boxes—significantly upregulated genes; red boxes—significantly downregulated genes (edgeR; more than twofold and padj <0.05). Light green boxes—nonsignificant upregulated genes; pink boxes—nonsignificant downregulated genes. Yellow boxes denote genes with multiple gene annotations that encompass both significantly upregulated and downregulated genes; light yellow boxes denote genes with multiple gene annotations that encompass both nonsignificant upregulated and nonsignificant downregulated genes.