Cannabidiol rather than Cannabis sativa extracts inhibit cell growth and induce apoptosis in cervical cancer cells

Abstract

Background: Cervical cancer remains a global health related issue among females of Sub-Saharan Africa, with over half a million new cases reported each year. Different therapeutic regimens have been suggested in various regions of Africa, however, over a quarter of a million women die of cervical cancer, annually. This makes it the most lethal cancer amongst black women and calls for urgent therapeutic strategies. In this study we compare the anti-proliferative effects of crude extract of Cannabis sativa and its main compound cannabidiol on different cervical cancer cell lines.

Methods: To achieve our aim, phytochemical screening, MTT assay, cell growth analysis, flow cytometry, morphology analysis, Western blot, caspase 3/7 assay, and ATP measurement assay were conducted.

Results: Results obtained indicate that both cannabidiol and Cannabis sativa extracts were able to halt cell proliferation in all cell lines at varying concentrations. They further revealed that apoptosis was induced by cannabidiol as shown by increased subG0/G1 and apoptosis through annexin V. Apoptosis was confirmed by overexpression of p53, caspase 3 and bax. Apoptosis induction was further confirmed by morphological changes, an increase in Caspase 3/7 and a decrease in the ATP levels.

Conclusions: In conclusion, these data suggest that cannabidiol rather than Cannabis sativa crude extracts prevent cell growth and induce cell death in cervical cancer cell lines.

Keywords: Apoptosis; Cannabidiol; Cannabis sativa; Cervical cancer.

Fig. 1

Representative cell viability bar graphs of cervical cancer cell lines. MTT assay was conducted to determine IC₅₀ following incubation of SiHa, HeLa, and ME-180 cells with different doses of butanol extract (a, b, c), hexane extract (d, e, f), and cannabidiol extract (g, h, i) for a period of 24 h. Data was expressed as mean value ± standard deviation (SD). The level of significance was determined using Students t-Test with ^nsrepresenting p > 0.05, ***represents p < 0.001, **represents p < 0.01, and *represents p < 0.05

Fig. 2

xCELLigence analysis of the cell growth pattern after treatment of cervical cancer cells with Cannabis sativa extracts and cannabidiol. SiHa (a, d, g), HeLa (b, e, h), and ME-180 (c, f, i) cells were seeded for a period of 22–24 h, followed by treatment with IC₅₀ concentration of butanol (a, b, c), hexane (d, e, f), and cannabidiol (g, h, i)

Fig. 3

Apoptosis assessment following treatment of cervical cancer cells with IC₅₀ concentrations of Cannabis sativa extract and cannabidiol. These bar graphs are a representative of apoptosis induction in SiHa (a and d), HeLa (b and e), and ME-180 (c and f) cells. Cells were treated with IC₅₀ of Cannabis sativa extracts and cannabidiol for a period of 24 h and further stained with Annexin-V/PI. Data represented as mean ± standard deviation with ***p < 0.001, **p < 0.01 and ^ns p > 0.05 representing the level of significance in comparison to the untreated

Fig. 4

Morphological analysis and assessment of apoptosis in SiHa cells stained with DAPI and Annexin V dye. Cells were incubated with IC₅₀ of Cannabis sativa extracts for a period of 24 h. Cells were stained with Annexin V and counterstained with DAPI. BX63-fluorescence confocal microscopy was used to visualize the cells

Fig. 5

Morphological analysis and assessment of apoptosis in HeLa cells stained with DAPI and Annexin V dye. Cells were incubated with IC₅₀ of Cannabis sativa extracts for a period of 24 h. Cells were stained with Annexin V and counterstained with DAPI. BX63-fluorescence confocal microscopy was used to visualize the cells

Fig. 6

Caspase 3/7 activity after treatment of SiHa, HeLa, and ME-180 cells with IC₅₀ of Cannabis sativa extract and cannabidiol. Cells were treated with IC₅₀ of Cannabis sativa and cannabidiol extracts for a period of 24 h. Caspase 3/7 reagent was added to the treated cells for 1 h. Luminescence–was measured using GLOMAX instrument in RLU. Data represented as mean ± standard deviation with ***p < 0.001, **p < 0.01, and *p < 0.05 representing the level of significance in comparison to the untreated

Fig. 7

Bar graphs representing changes in the ATP levels following treatment of cervical cancer cells with Cannabis sativa and cannabidiol. Cells were treated with IC₅₀ of both Cannabis sativa extracts and cannabidiol for a period of 2–24 h. Untreated and camptothecin were included as controls for comparative purposes. The level of significance was determined using Students t-Test with ***p < 0.001, **p < 0.01, *p < 0.05, and ^ns p > 0.05 in comparison to the untreated

Fig. 8

Representative bar graph of the cervical cancer cell cycle before and after treatment with Cannabis sativa extracts and cannabidiol. Cells were harvested and treated with camptothecin and the IC₅₀ concentrations of Cannabis sativa extracts and cannabidiol. Bar graph a and d represents SiHa cells, b and e represents HeLa cells, c and f represents ME-180 cells. Data represented as mean ± standard deviation with ***p < 0.001, **p < 0.01, *p < 0.05, and ^ns p > 0.05 representing the level of significance in comparison to the untreated

Fig. 9

Western blot analysis of the protein expression before and after 24 h treatment with IC₅₀ of Cannabis sativa extracts and cannabidiol. SiHa (a and d), HeLa (b and e), and ME-180 (c and f) cells were treated for a period of 24 h and protein lysates were separated using SDS-PAGE gel. Untreated protein was used as a control. Antibodies against pro-apoptotic proteins (p53 and Bax) and anti-apoptotic proteins (Bcl-2 and RBBP6), Initiator caspase-9 and effecter caspase-3 were included to elucidate apoptosis induction

Fig. 10

A densitometry analysis SiHa protein was performed using ImageJ quantification software to measure the relative band intensity. CPT represents camptothecin. Data represented as mean ± standard deviation with ***p < 0.001, **p < 0.01 and ^ns p > 0.05 representing the level of significance in comparison to the untreated represent the western blot analysis of SiHa and HeLa cells. The genes analyzed are p53 and RBBP6 including caspases. Equal amount of protein (conc) was loaded in each well. Note that the darker the bands increased expression of the gene

Fig. 11

A densitometry analysis HeLa protein was performed using ImageJ quantification software to measure the relative band intensity. CPT represents camptothecin. Data represented as mean ± standard deviation with ***p < 0.001, **p < 0.01 and ^ns p > 0.05 representing the level of significance in comparison to the untreated

Fig. 12

A densitometry analysis ME-180 protein was performed using ImageJ quantification software to measure the relative band intensity. CPT represents camptothecin. Data represented as mean ± standard deviation with ***p < 0.001, **p < 0.01 and ^ns p > 0.05 representing the level of significance in comparison to the untreated