Cannabis sativa a Potential Anticancer Treatment in Melanoma Cancer Cells

Abstract

Introduction

Melanoma cancer is the most aggressive skin cancer type with a poor prognosis. Chemotherapy has been used in the past in treating melanoma cancer, however, the method usually results in toxicity. Therefore, a call has been made to develop cancer treatments using medicinal plants to reduce drug toxicity. The aim of this study was to establish the role of RBBP6 in melanoma cancer development and progression. The hypothesis of this study was that RBBBP6 is highly expressed in melanoma cancer and serves as an early biomarker. Results obtained supported the hypothesis since high expression of RBBP6 in melanoma cancer was observed in untreated control melanoma cells.

Methodology

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Cytosmart fluorescence, and quantitative Polymerase Chain Reaction (qPCR) were used.

Results

The IC₅₀ values of Cannabis sativa EtOH and MeOH extracts were obtained at 51.31315 and 57.34135 µg/mL, respectively, for A375 cells. Normal cells were used to check if there are any serious cytotoxic effects, IC₅₀ values for Cannabis sativa EtOH and MeOH extracts were obtained at 79.577850 and 59.5754 µg/mL, respectively. RMG-1 cells showed IC₅₀ at 84.748 and 34.66475 µg/mL for Cannabis sativa EtOH and MeOH extracts, respectively. CYTOSMART LUX3 bright field microscope was used for morphological analysis of both A375 and RMG-1 cells and changes were observed after treating with Cannabis sativa EtOH and MeOH extracts. Apoptosis analysis to determine the mode of cell death was done using a CYTOSMART fluorescence microscope. Early apoptotic effects were observed in both Cannabis sativa EtOH and MeOH extracts on A375 cells. Early apoptotic effects were also observed after treating RMG-1 cells with EtOH extract. Caspase 3/7 activity was done on both A375 and RMG-1 cells, and caspase 3/7 activity were increased after treating with Cannabis sativa EtOH and MeOH extracts. Real Time Polymerase Chain Reaction (RT-PCR) showed that RBBP6 and BCl₂ were down-regulated after treating with Cannabis sativa EtOH and MeOH extracts.

Conclusion

siRNA co-treatment with EtOH and MeOH extracts resulted in low expression of both RBBP6 and BCl₂. P53 was upregulated after treatment with the extracts and co-treatment with the extracts and siRNA.

Introduction

Melanoma is the fifth and sixth most common cancer in men and women, respectively, with a lifetime cumulative risk of developing melanoma cancer of 1.72 for men and 1.22 for women. ¹ Gender differences have been shown to play a role in melanoma incidences.

Despite many attempts to develop melanoma therapies, patients with highly advanced diseases continue to have a poor prognosis. ² Fortunately, over decades there has been an improvement in the survival rates (five years survival rate) of invasive melanoma from 82% in 1979% to 90% in 2002. ¹ This improved survival rate is suspected to be a result of early detection since the primary treatment of melanoma remains to be surgical excision. ³ However, late diagnosis is related to later stages of the disease, and patients with regional lymphatic or metastatic disease respond badly to conventional radiation and chemotherapy, with 10% to 50% survival within 5 years. ² Early detection and therapy are promising to also cut the costs of treating melanoma, about US$1.6 billion is expected to be spent in the United States in 2030 in treating melanoma cancer. ⁴ Tsao et al ² found that the cost of treatment of melanoma increases with the stage of the disease, less than 20% of patients with stage III and stage IV disease were responsible for 90% of the total annual cost for treating melanoma in 1997, which was approximately US$563 million. Thus, accurate staging, early detection, and improved therapeutic strategies are associated with positive outcomes and each is important for successful management of melanoma cancer in patients. ⁵ However, given the low incidence and prevalence of melanoma cancer, it becomes too expensive to screen the whole general population and this will result in a low yield of screening. ⁶ Therefore, screening should be done on individuals with a high risk of developing melanoma cancer to enhance the screening population. ⁷ In this study, we hypothesize that because of its antioxidant properties, cannabis sativa induces cell death in skin cancer. The significance of the study is to test the apoptotic properties of Cannabis sativa and its metabolites.

Materials and Methods

Cell Culture

Before the experiments could begin, the melanoma cancer cell line, A375, ovarian cancer cell line, RMG-1, and normal fibroblast cell line, human embryonic kidney 293 (HEK293) were used as the experimental cell line to study the effects of Cannabis sativa on melanoma cancer were authenticated using multiplex polymerase chain reaction (PCR) assays that target short tandem repeat (STR) markers. The cells were purchased from American Type Culture Collection (ATCC).

Plant Extract

Cannabis sativa leaves were washed and frozen in liquid nitrogen, the plant components were then mixed by blending and weighed. The leaves were then extracted using absolute EtOH and MeOH for 24 h at room temperature. The extract suspension extract was then filtered using Whatman® qualitative filter paper overnight. The filtrate was then dried at 45 °C using rotavapor at low pressure. Once they were dried, the extracts were then weighed and dissolved in 100% dimethyl sulfoxide (DMSO) to the required concentration and stored at 4 °C as stock. The plant was supplied by Prof LR Motadi and taken to the botany department of Northwest University for voucher and verification as Cannabis sativa.

Cell and Tissue Culture

In this study, both cancerous and noncancerous cell lines were used. Tumorigenic cell lines used are A375 and RMG-1 for comparison and nontumorigenic cell lines, HEK293, were used to test the treatment. Cell lines were maintained in DMEM (Thermo Fisher Scientific) or DMEM/F12 (Thermo Fisher Scientific) supplemented with 10% of fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% penicillin–streptomycin antibiotic solution (Thermo Fisher Scientific). All three cell lines were maintained in flasks (25 cm³) at 37 °C, in a humidified 5% carbon dioxide (CO₂) incubator (HERACELL 150i CO₂ Incubator, Thermo Fisher Scientific), and 95% relative humidity. Cultured cells were grown as a monolayer and allowed to reach a confluency of about 75% to 80% and were subcultured after every 2 days into bigger flasks (75 cm³). When splitting the cells, the media was disposed of, and cells were washed with a sterile Dulbecco's Phosphate Buffered Saline (DPBS) (Sigma Aldrich). The cells were trypsinized with 500 µL of trypsin (Thermo Fisher Scientific) and incubated at 37 °C until the cells were detached. 5 mL of complete DMEM was added to stop trypsinization. The cells were then centrifuged at 2000/min for 4 min and the supernatant was disposed of, leaving the pellet behind. 1 mL of DMEM was used to resuspend the pellet and 10 µL of the cells was mixed with trypan blue dye (10 µL) for cell counting. Approximately 10 µL of the mixture of cells and dye was added onto a slide and counted using a TC20™ Automated Cell Counter (BIO-RAD).

AlamarBlue

A375, RMG-1, and HEK 293 cell lines were cultured in 96 well tissue culture plates. A cell density of 2.5  ×  10⁴ cells in 90 µL of media per well was added to the plates and incubated overnight before treatment with a range of concentrations of Cannabis sativa extracts (MeOH and EtOH extracts) (500, 250, 125, 62.5, 31.25, 15.625, 7.815, and 3.906 µg/mL). The controls of the experiment were DMEM only (blank), cells treated with DMSO (0.1%), untreated control cells in DMEM, and 100 µM cisplatin (Sigma Aldrich) (positive control). Cells were treated for 24 h. 10 µL of AlamarBlue (Thermo Fisher Scientific) was added to each well at 22 h after treatment and incubated for 2 h at 37 °C in the dark since AlamarBlue is light sensitive. Cell viability was measured using AlamarBlue and readings were obtained in terms of fluorescent values using a microplate reader (BioTek Synergy HT). Samples were exposed to an excitation wavelength of 530 nm and at an emission wavelength of 590 nm. Cell viability percentage was calculated using the formula below:

$$\begin{array}{rl}& \mathrm{Cell}\mathrm{viability}\\ & ={\displaystyle \frac{\mathrm{Absorbance}\mathrm{of}\mathrm{treated}\mathrm{cells}-\mathrm{Absorbance}\mathrm{of}\mathrm{blank}}{\mathrm{Absorbance}\mathrm{of}\mathrm{untreated}\mathrm{control}\mathrm{cells}-\mathrm{Absorbance}\mathrm{of}\mathrm{blank}}}\\ & \times 100\end{array}$$

• IC₅₀ determination

The half-maximal inhibitory concentration (IC₅₀) was obtained using the dose-response graph produced by Compusyn software.

Morphology Analysis

To study the morphology of cancerous cell lines after treatment, A375 and RMG-1 cell lines were seeded at a density of 2.5  ×  10⁴ cells in a 6-well plate and maintained overnight at 37 °C. The following day, cells were washed with 500 µL of DPBS the following day and treated with IC₅₀ of the drugs and treatment for 24 h. The cells were then viewed using a cytoSMART Lux3 FL live-cell imaging microscope (Brightfield channel) and images were captured.

Apoptosis Detection Assay

The apoptotic effect of Cannabis sativa EtOH and MeOH extracts was investigated using their IC_50s on both A375 and RMG-1 cell lines. The cell density of 1  ×  10⁵ cells per well (100 µL) was seeded in a 96-well plate and incubated overnight. The cells were then treated with IC_50s of the extracts, cisplatin, and DMSO. The following day the cells were then transferred into Eppendorf tubes after detaching with 50 µL trypsin and were centrifuged at 1500/min for 2 min. The supernatant was discarded, followed by the addition of 50 µL DPBS and centrifugation for 2 min at the same speed. The supernatant was discarded again and Annexin V-FITC/PI solution (5 µL Annexin V and 2 µL PI) (Thermo Fisher Scientific) was added into each Eppendorf tube and incubated for 15 min in the dark. The cells were analyzed with a CYTOSMART Lux3 FL live-cell imaging microscope (fluorescence channel) and images were captured.

Caspase 3/7 Assay

A cell density of 1  ×  10⁵ cells (2 mL) was plated into five cell culture dishes for both A375 and RMG-1 cell lines. Four dishes were treated with 2 mL of the treatments (DMSO, cisplatin, EtOH extract, and MeOH extract) and only culture medium was added to the untreated control dish. All cells were washed in 500 µL of DPBS before treating. The cells were then harvested into 2 mL Eppendorf tubes using 500 µL trypsin. The tubes were then centrifuged for 2 min at a speed of 2000/min. The supernatant was then discarded and 500 µL of DPBS was added into the same tubes followed by centrifugation at the same speed. The supernatant was then discarded and 50 µL of media was added to the pellet and cells resuspended. 25 µL of the cell suspension was transferred into the 96-well black polystyrene plate. 25 µL volume of caspase-Glo 3/7 reagent (Promega) was added to the Promega Glomax® 96 microplate luminometer wells and the plate was then covered with a foil. All steps were conducted in a laminar flow. The foiled plate was incubated in a shaker for 15 min at maximum speed. Caspase-Glo 3/7 development and absorbance of the colored product were then recorded on a microplate reader at a wavelength of 405 nm after 15 min. The experiment was done in duplicate and caspase 3/7 was represented as the mean of Relative Light Units (RLU). Caspase 3/7 activity was calculated using the following formula:

$$\mathrm{RLU}=\mathrm{Luminescence}(\mathrm{treatment})\text{–}\mathrm{Luminescence}(\mathrm{blank})$$

Gene Silencing by siRNA Transfection

siRNAs synthesized by Ambion Inc, Huntingdon, UK were designed by Prof Motadi to target mRNA regions of RBBP6. Transfection was performed following the manufacturer's protocol. About 1  ×  10⁵ A375 cells were cultured in cell culture dishes and were transfected by 40 nM Silencer Select siRNA (Ambion). The cells were returned to their normal culture conditions for 6 h and then co-treated with 100 µM cisplatin, IC_50S of Cannabis sativa EtOH, and MeOH extracts.

Real-Time PCR

RNA was isolated with Trizol® reagent (Invitrogen), including a DNase digestion step, with the Real Star Kit (Durviz, Valencia, Spain). cDNA was reverse transcribed from RNA using the First Strand cDNA Synthesis Kit (Promega). TaqMan probes were obtained from Applied Biosystems (Foster City, CA). Amplifications were run in a 7900 Real-time PCR System (Applied Biosystems). Each value was adjusted by using 18S RNA levels as a reference.

Statistical Analysis

All data were reported as mean  ±  standard deviation, and the significance levels were calculated using the student's t-test and one-way analysis of variance (ANOVA) analysis. Statistical significance was defined as a P-value of .05 or .01 or less. Software called SPSS/Win11.0 was used for the statistical analysis.

Results

Cytotoxicity Effects of Cannabis sativa Extract on A375

In A375 cells were treated with various concentrations of EtOH extracts (Figure 1A), whereby the cell viability is seen to be decreasing with increasing concentrations. Higher concentrations such as 500 µg/mL were highly cytotoxic to A375 cells, and this can be seen by a decrease in cell viability (2.73%), whereas lower concentrations were not as effective since 3.90 and 7.81 µg/mL resulted in 102.8% and 96.64% cell viability, respectively. The effects of high concentrations (500 µg/mL) were highly significant compared to untreated control cells. DMSO was used as a negative control, and it gave the expected results of 99.64% cell viability which was nonsignificant. The same was seen in the positive control where cisplatin treatment resulted in cell viability of 35.05% and its effects were highly significant. When melanoma cell lines (A375) were treated with MeOH extracts (Figure 1B), a highly significant difference was seen at higher concentrations compared to lower concentrations. The highest concentration used (500 µg/mL) resulted in 1.216% cell viability and lower concentrations (3.90 and 7.81 µg/mL) had a nonsignificant difference (103.7% and 102.1% cell viability, respectively) when compared to untreated control. However, cisplatin showed expected cell viability (46.60%) which was highly significant and DMSO did not affect the proliferation of A375 cell lines. Compusyn determined that 50% of the cells died with EtOH extract at 51.31 and 57.34 µg/mL of MeOH extract.

Figure 1.

Cell viability assay of A375 cells after being treated with several concentrations of Cannabis sativa EtOH and MeOH extracts for 24 h. DMSO and cisplatin were used as controls. Data was expressed as mean  ±  SD, whereby ns represents P > .05, * represents P ≤ .05, ** represents P ≤ .01, *** represents P ≤ .001, and **** represents P ≤ .0001 (A). A dose–effect graph was obtained from Compusyn software by using duplicates to deduce the IC₅₀ value of the treatment (B).

Cytotoxicity Effects of Cannabis sativa Extract on HEK293

When a normal cell line (HEK293) was treated with ethanol extract (Figure 2A), higher concentrations resulted in lower cell viability, and this can be seen by 250 µg/mL resulting in 13.37% viable cells and these effects were highly significant when compared to untreated control cells. However, lower concentrations resulted in higher cell viability, for example, 3.90 µg/mL yielded 111.8% viable cells and their effects were nonsignificant. Negative control (DMSO) did not have any significant difference in the cell viability of HEK293 cells (103.1%) when compared to the untreated control cell line. On the other hand, cisplatin showed expected cell viability (49.16 µg/mL) which was highly significant. IC₅₀ (Figure 2B) was determined at a concentration of 79.577850 µg/mL. Normal epithelial cells (HEK293) were treated with MeOH extract (Figure 2A) and high concentrations induced very high cell death with 250 µg/mL resulting in 2.43% cell viability whereas low concentrations did not show many antiproliferative effects (3.90 µg/mL caused 106.4% cell viability). The effects of high concentration (250 µg/mL) were highly significant whereas the low concentration effect was nonsignificant. DMSO did not have antiproliferative effects on HEK293 cell lines resulting in 94.08% cell viability which was nonsignificant when compared to untreated control cells. Cisplatin treatment resulted in 46.06% cell death which was highly significant. The concentration at which 50% of the cells died was determined as 59.57 µg/mL (Figure 2B).

Figure 2.

Cell viability assay HEK293 cells after being treated with several concentrations of EtOH extract for 24 h. DMSO and cisplatin were used as controls. Data was expressed as mean  ±  SD, whereby ns represents P > .05, * represents P ≤ .05, ** represents P ≤ .01, *** represents P ≤ .001 and **** represents P ≤ .0001 (A). A dose–effect graph was obtained from Compusyn software by using duplicates to deduce the IC₅₀ value of the treatment (B).

Cytotoxicity Effects of cannabis sativa Extract on RMG-1 for Comparison

The antiproliferative effects of EtOH extract were observed on ovarian cell lines (RMG-1) (Figure 3A), whereby high concentrations such as 500 µg/mL resulted in 2.309% cell viability and 250 µg/mL yielded 7.76% cell viability and it was highly significant. However, low concentrations did not have much effect on the proliferation of RMG-1 cells, and 3.906 µg/mL caused 117.2% cell viability. Cisplatin treatment gave results of 40.15% cell viability and DMSO as negative control did not have any antiproliferative effects on the cells resulting in 113.6% cell viability. Cisplatin cytotoxicity was highly significant when compared to untreated control cells. However, DMSO had nonsignificant effects. The concentration at which 50% cell death occurred was determined by Compusyn software (Figure 3B) at 84.74 µg/mL. Antiproliferative effects were observed at all concentrations of MeOH extract (Figure 3B) when treating ovarian cells. However, the effect was proportional to the concentration, that is, the higher the concentration of the drug, the higher the antiproliferative effects. A low concentration (3.90 µg/mL) caused 81.94% cell viability and a higher concentration (500 µg/mL) resulted in 5.41% cell viability. At High concentrations such as 500 µg/mL, cytotoxicity was highly significant when compared to untreated control cell lines whereas low concentration (3.90 µg/mL) was nonsignificant. Negative control (DMSO) gave expected results of 107.4% cell viability which was nonsignificant when compared to untreated control cells. Positive control (cisplatin) gave expected cell viability of 33.47% and it was highly significant. IC₅₀ value was determined as 34.66475 µg/mL (Figure 3B).

Figure 3.

Cell viability assay of RMG-1 cells after being treated with several concentrations of EtOH extract for 24 h. DMSO and cisplatin were used as controls. Data was expressed as mean  ±  SD, whereby ns represents P > .05, * represents P ≤ .05, ** represents P ≤ .01, *** represents P ≤ .001 and **** represents P ≤ .0001 (A). A dose–effect graph was obtained from Compusyn software by using duplicates to deduce the IC₅₀ value of the treatment (B).

Morphological Analysis Using Brightfield Microscope

Morphological Effects of Cannabis sativa EtOH and MeOH Extracts on A375 and RMG-1 (Comparison Analysis Cell Line)

A375 and RMG-1 (Figures 4 and 5) cell lines were treated with Cannabis sativa EtOH and MeOH extracts and 24 h posttreatment; cell morphology was changed. The morphological analysis for both A375 and RMG-1 cells showed that untreated control cells were not floating, and the morphology was still the same after 24 h. The cells treated with DMSO (negative control) did not have any change in morphology compared to untreated control cells. A decrease in cell count of cells attached on the surface of the well in all other treatments was observed with cisplatin (positive control) which showed cell rounding which demonstrates apoptosis. However, with EtOH and MeOH extracts cell rounding and cell detachment were observed, demonstrating apoptosis.

Figure 4.

Microscopic images of A375 cell morphological changes after 24 h treatments: (A) untreated control, (B) DMSO, (C) cisplatin, (D) EtOH extract, and (E) MeOH extract.

Figure 5.

Microscopic images of RMG-1 cell morphological changes after 24 h treatments: (A) untreated control, (B) DMSO, (C) cisplatin, (D) EtOH extract, and (E) MeOH extract.

Apoptosis Detection Assay

Apoptosis Analysis of Cannabis sativa EtOH and MeOH Extracts on A375 and RMG-1 Cell Lines

Since morphological changes were observed in Figures 4 and 5, apoptosis was then analyzed using CytoSMART Lux 3 fluorescence microscopy. Both cell lines were stained with Annexin V/PI dye (Figures 6 and 7). Annexin V detects cellular apoptosis (green), and PI (red) detects necrotic and late apoptotic cells and untreated control cells remained unstained. In the A375 cell line (Figure 6), untreated control cells (A) gave expected results of very low early apoptotic cell count (1.5 cell count) and DMSO (B) had 2.5 early apoptotic cell count which was nonsignificant when compared to untreated control cells. However, the positive control (cisplatin) had a low early apoptotic cell count of 13 cells count compared to EtOH (50 cell count) and MeOH extracts (109.5 cell count). Cell counts for cisplatin (C) and EtOH extracts (D) were nonsignificant (F) compared to untreated control cells, whereas MeOH (E) was slightly significant. Late apoptotic/necrotic cell count for untreated control cells was 0.5 cell count whereas negative control DMSO showed only 1 cell count which was nonsignificant. Cisplatin had a low late apoptotic/necrotic cell count of 1 cell count compared to 1.5 cell count of EtOH and 3.5 cell count of MeOH extracts. All the late/necrotic cell counts for the treatments were nonsignificant compared to the untreated control cell line.

Figure 6.

Fluorescence microscopy images showing apoptosis of A375 cells after 24 h treatment: (A) untreated control, (B) DMSO, (C) cisplatin, (D) EtOH extract, and (E) MeOH extract. Graphical representation showing the cell count of early apoptotic cells (F) and late apoptotic or necrotic cells (G) is also represented. The color yellow/green indicates the apoptosis positivity. Data was expressed as the mean  ±  SD, whereby ns represents P > .05, * represents P ≤ .05, ** represents P ≤ .01, *** represents P ≤ .001 and **** represents P ≤ .0001.

Figure 7.

Fluorescence microscopy images showing apoptosis of RMG-1 cells after 24 h treatment: (A) untreated control, (B) DMSO, (C) cisplatin, (D) EtOH extract, and (E) MeOH extract. Graphical representation showing the cell count of early apoptotic cells (F) and late apoptotic or necrotic cells (G) is also represented. The yellow/green indicates the positive cells for apoptosis. Data was expressed as the mean  ±  SD, whereby ns represents P > .05, * represents P ≤ .05, ** represents P ≤ .01, *** represents P ≤ .001, and **** represents P ≤ .0001.

In the RMG-1 cell line (Figure 7), untreated control cells (A) and DMSO-treated cells (B) gave the same cell count of 1.5 cell count for early apoptotic cells. On the other hand, positive control (cisplatin) had a high early apoptotic cell count of 29.50 compared to MeOH extract (E) cell count which was 8 cell counts. EtOH extract (D) showed an early apoptotic cell count of 39.50 which was slightly significant when compared to untreated control cells (F). A late apoptotic/necrotic cell count of untreated control cells was found to be 1 cell count whereas DMSO-treated cells showed a cell count of 3.50 cell count which was nonsignificant when compared to untreated control cells. Cisplatin (C) had a high late apoptotic/necrotic cell count of 20.50 compared to EtOH (18.50 cell counts) and MeOH extracts (8 cell counts). Cisplatin and EtOH (D) had a slightly significant cell count when compared to untreated control cells (F).

Caspase Assay

Effects of Cannabis sativa EtOH and MeOH Extracts on A375 and RMG-1 (Comparison Analysis Cell Line) Caspase 3/7 Activity

A caspase 3/7 assay to detect caspase 3/7 activity was carried out on A375 and RMG-1 cells following 24-hour treatment with Cannabis sativa EtOH and MeOH extracts. This assay was performed to determine if apoptosis/cell death observed from cytotoxicity and apoptosis analysis was due to caspase 3/7 activity. As shown in Figure 8A and B, the caspase 3/7 activity of both EtOH and MeOH extracts in both cell lines was detected. In the A375 cell line (A), the caspase 3/7 activity of EtOH extract (8836 RLU) was found to be higher than that of MeOH extract (3301 RLU), whereas the untreated control cell lines expressed caspase 3/7 activity of 4560 RLU which was higher than MeOH extract. However, DMSO (negative control) gave expected caspase 3/7 activity (7036 RLU) and positive control (55 596 RLU). Cisplatin caspase 3/7 activity was significant when compared to the untreated control cell line. On the other hand, in the RMG-1 cell line (B), caspase 3/7 activity of both EtOH extract (11 767 RLU) and MeOH extract (3465 RLU) was higher than that of the untreated control cell line (386 RLU). Negative control (DMSO) maintained the expected trend of slightly higher caspase 3/7 activity (665 RLU) than the untreated control. The positive control gave expected results of the highest caspase 3/7 activity than other treatments at 86 313 RLU and it was significant when compared to untreated control cells.

Figure 8.

Caspase 3/7 activity after treatment of A375 (A) and RMG-1 (B) with IC₅₀ of Cannabis sativa EtOH and MeOH extracts. DMSO (0.1%) and cisplatin (100 µM) were used as controls. Data was expressed as the mean  ±  SD, whereby ns represents P > .05, * represents P ≤ .05, ** represents P ≤ .01, *** represents P ≤ .001 and **** represents P ≤ .0001.

Gene Expression by RT-qPCR

To evaluate the effects of Cannabis sativa extracts and gene silencing on the expression of different genes, qPCR was performed. For real-time PCR data analysis, the method by Pfaffl was used. RBBP6 gene expression levels were determined after normalization with housekeeping gene GADPH (Figure 9A) whereby on untreated control cells RBBP6 levels were upregulated at a mean factor of 0.85. However, after treating with cisplatin a significant decrease in expression levels of RBBP6 was observed at a mean factor of 0.02. A significant decreases at mean a factor of 0.101 was identified after co-treating with siRNA and cisplatin. A nonsignificance decrease at a mean factor of 0.7 was observed after treating with Cannabis Sativa EtOH extracts. However, after co-treating with siRNA and EtOH extracts, the expression levels of RBPP6 decreased significantly at a mean factor of 0.3. A slightly significant decrease in the expression levels of RBBP6 after treating with Cannabis Sativa MeOH extracts was observed at a mean factor of 0.35 and similar effects were also observed after co-treating with MeOH extract and siRNA. Transfection with siRNA only resulted in a significant decrease in the RBBP6 expressional levels at a mean factor of 0.25. With p53, the expressional levels were low at a mean factor of 0.55 when co-treating with MeOH extract and siRNA as compared to EtOH extract only but there was a nonsignificance increase as compared to untreated control cells. Very low expression levels at a mean ratio of 0.03 were observed after treating the cells with Cannabis Sativa MeOH extracts however P53 expressional levels increased nonsignificantly after co-treating with MeOH extract and siRNA at a mean factor of 0.3. Transfecting the cells with siRNA only resulted in a nonsignificance increase in P53 expressional levels at a mean factor of 0.65. Relative gene expression of anti-apoptotic gene Bcl₂ after normalization by GADPH was also determined whereby untreated control cells showed an upregulation of Bcl₂ at a mean factor of 3.245. However, after treating the cells with cisplatin a highly significant downregulation of Bcl₂ expression levels was observed at a mean factor of 0.115. The same observations were made whereby a highly significant decrease in Bcl₂ expression level was at 0.05 after co-treating with cisplatin and siRNA. A significant decrease was observed at expressional levels of 0.32 after treating with Cannabis Sativa EtOH extract. The same results were seen whereby there was a significant decrease in expression levels of Bcl₂ at a mean factor of 0.02 after co-treating with EtOH extract and siRNA. Treatment with Cannabis Sativa MeOH extracts showed a significant decrease in the expressional levels of Bcl₂ at a mean factor of 0.15. Similar observations of significant downregulation of Bcl₂ were observed at a mean factor of 1.7 after co-treating with MeOH extract and siRNA. siRNA transfection resulted in a reduction of Bcl₂ expression levels at a mean factor of 0.2.

Figure 9.

A diagram representation showing relative RBBP6, p53, and BCl2 gene expression levels after normalization with GADPH. The data was obtained from two biological repeats, and it was expressed as the mean  ±  SD, whereby ns represents P > .05, * represents P ≤ .05, ** represents P ≤ .01, *** represents P ≤ .001 and **** represents P ≤ .0001.

Discussion

Cannabis sativa was selected because of its traditional medical use as a treatment for different diseases. Although cytotoxic effects of Cannabis sativa have been evaluated on other cell lines such as breast, cervical, and prostate cancer cell lines, there is limited to no data on melanoma cancer cell lines. In this study, a cytotoxicity assay was performed on A375 cells to study the antiproliferative effects of Cannabis sativa extracts (EtOH and MeOH). Cisplatin was used as the positive control to compare the cytotoxic effects, type of cell death, and gene expressions, with those of the extracts. DMSO was also used as the negative control to determine its role in the antiproliferative effects caused by the extracts since it was used as the solvent to dissolve the extracts. To be able to make a constructive conclusion on the effects of the extracts on our experimental cell line (A375), HEK293 cells were used to determine if the extracts will not be harmful to neighboring normal cells during treatment. Also, to determine if the extracts will have similar antiproliferative effects in other cancer types/cell lines, RMG-1 cells (ovarian cancer cell line) were used as a comparative cell line.

Different concentrations of Cannabis sativa extracts were used (from 500 to 3.906 µg/mL) to understand the effects of the extracts at a wide range of concentrations, that is, from higher to lower. From the results, as shown in Figures 1 to 3, higher concentrations (eg, 500 and 250 µg/mL) resulted in very high antiproliferative effects and this shows that these concentrations are not ideal to use at high concentrations in all three cell lines studied. The same trend was seen by Zarei and Yaghoobi ⁸ in their study where the cell viability of LCL-PI 11 and MCF-7 cell lines decreased with increasing concentration of the Rosa Beggeriana Schrenk extracts. Although cytotoxic effects were seen in normal cells (HEK293), they were lower than that in the A375 cell line. At lower concentrations (eg, 3.906 and 7.8125 µg/mL), the extracts were seen to promote cell proliferation in all the cell lines except for RMG-1 with MeOH extract. However, all these are expected qualities of plant extracts since different components of plants are known to be either carcinogenic or anticarcinogenic and they are known to act antagonistically towards one another. ⁹ All the results showed that there was no significant difference in the cell viability of DMSO and untreated control cells meaning that all effects observed were a result of extracts only.

A study done by Larsson et al ¹⁰ on MCF-7, HCC38, MCF-10A, and MDA-MB-436 cell lines treated with cisplatin gave an average IC₅₀ value of 52 µM (with a range from 10 to 122 µM), therefore in this study, 100 µM was used as the IC₅₀ of the positive control. To confirm that each cell line has its IC₅₀ value, the cell viabilities of the three cell lines treated with 100 µM of cisplatin were all different but within the range of 33% to 50%. On the other hand, with the extracts, different IC₅₀ values were obtained with normal cell lines having an IC₅₀ value of 79.577850 and 59.5754 µg/mL for both EtOH and MeOH extracts, respectively. Since a good drug should not be toxic toward normal cells, a lower IC₅₀ for the cancerous cells was expected if these extracts are good. This is supported by the findings of Kuete et al, ¹¹ whereby they mention the importance of chemotherapy treatment being able to kill only cancerous cells with as little damage as possible to normal cells. However, this was seen in A375 cells whereby the IC₅₀ values were 51.31315 and 57.34135 µg/mL for EtOH and MeOH extracts, respectively. However, the comparative cell line, RMG-1, had a very high IC₅₀ value (84.748 µg/mL) for EtOH extract compared to normal cells ruling out that EtOH extract can be used as a treatment for ovarian cancer since it will kill normal cells. On the other hand, the IC₅₀ of MeOH extract on RMG-1 was very low (34.66475 µg/mL) compared to normal cells making it to be the ideal treatment for ovarian cancer. However, MeOH extract on A375 is seen to have an IC₅₀ very close to that of normal making it not be the best treatment candidate for melanoma cancer and EtOH extract is shown to be the best/ideal treatment for melanoma. All these results show that cytotoxicity is cell-dependent.

Since Cannabis Sativa EtOH and MeOH extracts showed cytotoxic effects in both the experimental cell line (A375) and comparison cell lines (RMG-1), morphological changes after treatment were studied using CYTOSMART Lux3 bright field microscope. The results obtained in Figures 4 and 5 gave an indication that the extracts do induce cytotoxic effects on the cell lines since the cell morphology changed and the cells failed to attach to the surface of the culture dishes which are features of apoptosis. The same effects were seen by Ibiyeye et al, ¹² whereby drug-loaded alginate-chitosan nanoparticles (ACNP) induced morphological changes in the breast cancer cell line MDA-MB-231, which resulted in detachment from the surface of the cell culture dish. Since features of apoptosis were observed, apoptosis analysis using a CYTOSMART fluorescence microscope was carried out which involved counterstaining using Annexin V/PI. In the A375 cell line (Figure 6), there was no significant difference in the cell count of late or necrotic cells in all the treatments (cisplatin, EtOH, and MeOH extracts) as compared to the untreated control cells. However, these cell counts were low compared to their early apoptotic cell counts which showed that the treatments were highly early apoptotic in the A375 cell line. Although it can be said they are apoptotic, the early apoptotic effects of cisplatin and EtOH were considered insignificant as compared to the untreated control cell lines and this could suggest that there should be a change in the experimental setup which could include extending the treatment time over 24 h to see if the treatments won’t have significance apoptotic effects over increased time. MeOH extract early apoptotic effects were slightly significant when compared to cisplatin and EtOH extracts and this could support the cytotoxic results where MeOH extract was seen to be highly cytotoxic on the A375 cell line. In our comparative ovarian cancer cell line (RMG-1) (Figure 7), early and late apoptotic/necrotic cell counts of MeOH extracts were nonsignificant when compared to untreated control cells which showed the same trend as the one observed in EtOH-treated A375 cells, which could suggest that there should be an increase in the experimental time frame to investigate the change in the apoptotic effects over a longer treatment time. This is because in a study done by Hosami et al, ¹³ it was shown that Cannabis sativa extracts induce early apoptosis in a time-dependent manner on A549 cells. However, cisplatin and EtOH extracts showed a slightly significant early apoptotic cell count and late apoptotic/necrotic cell counts. These findings support those of Ferrer et al ¹⁴ findings whereby they found out that cisplatin induces both cell modes (necrotic and apoptotic cell death) in FA (Fanconi anemia) cells in a concentration-dependent manner. Although cell death in both these treatments was significant, early apoptotic cell counts, were higher compared to late apoptotic/necrotic cell counts which suggest that they are highly early apoptotic. The results also supported the findings from cytotoxicity results, whereby EtOH extract was found to be highly cytotoxic in RMG-1 cells compared to EtOH therefore we expect the apoptotic effects of EtOH to be higher.

Since apoptosis was seen to have occurred in both A375 and RMG-1 cell lines, caspase 3/7 activity was investigated to determine if the apoptosis was caspase-dependent or not. This is because caspases are known to be mediators of apoptosis. As shown in Figure 8, it was deduced that there was a significant increase in caspase 3/7 activity from cisplatin treatment in both A375 and RMG-1 cell lines. These results were highly expected in the positive control since there is a study done by Garcia-Berrocal et al ¹⁵ which showed a significant increase in the levels of caspase-3/7 activity in the rat cochlea. However, in the A375 cell line, EtOH extract showed an increase in caspase 3/7 activity as compared to MeOH extract, but it was statistically nonsignificant when compared to untreated control cells. Although it was nonsignificant, these results show that apoptosis that occurred was partially caspase-dependent. On the other hand, in RMG-1 cells, an increase in caspase 3/7 activity was observed following EtOH extract treatment as compared to MeOH extract and these results show us that MeOH extract is slightly caspase-dependent and MeOH extract cell death is more likely to be necrotic or autophagic hence very low caspase 3/7 activity. Although caspase 3/7 activity is seen to be increased in both cell lines, the experimental period can be increased to do a time-point study to determine if after maybe 48 h caspase 3/7 activity won’t increase significantly. This is because different extracts have different effects on different cell lines; Lukhele and Motadi ¹⁶ discovered that Cannabis sativa butanol and hexane extracts result in a significant increase of caspase 3/7 activity on cervical cancer cell lines after 24 h.

Since the extracts showed the potential of having anticancer and apoptotic effects, analysis to study the molecular mechanism of three genes known as RBBP6, P53, and Bcl₂ was done. These genes are known to play major roles in cancer with RBBP6 being found to be overexpressed in various cancer types such as colon cancer. ¹⁷ P53 is a tumor suppressor gene and investigations were done on our extracts to check if the apoptosis that occurred was P53-dependent. Bcl₂ is an anti-apoptotic gene that is known to be an important multidrug resistance molecule. ¹⁸ A study has shown overexpression of Bcl₂ to be associated with prevention of cell death. ¹⁹ From the results obtained (Figure 8A), treating melanoma cancer with Cannabis sativa EtOH extracts resulted in the reduction of RBBP6 expressional levels, however, co-treating with EtOH extract and siRNA resulted in a greater increase in reduction of RBBP6 expression levels. This gives an indication that both treatments can be used in the treatment of melanoma. On the other hand, the hypothesis was accepted since overexpression of RBBP6 was observed on untreated control melanoma cells. This can also be seen by the intensity of the RBBP6 band on the gel in Figure 9. Treating with MeOH extract only or co-treating with MeOH and siRNA resulted in similar effects. The differences in the effects that the extracts have on RBBP6 may be due to differences in the chemical composition of the extracts therefore they should be studied further and explored. Although the effects are different, the extracts both reduce RBBP6 levels. The downregulation of RBBP6 after treatment with Cannabis Sativa butanol extracts was also observed by Lukhele and Motadi. ¹⁶ From Figure 9 the results showed that the P53 tumor suppressor gene was downregulated in untreated control melanoma cancer cells. However, after treating with Cannabis sativa EtOH and MeOH extracts, an increase in the P53 expression was observed which showed that the treatments follow a P53-dependent pathway. Treating with MeOH extract and co-treating with siRNA and EtOH extract after GADPH normalization showed low expression of P53. This could suggest that the treatments follow a P53-independent pathway since expressional levels of P53 are too low. Since more than 50% of cancers have mutated P53 gene that often results in chemotherapeutic resistance toward drugs that follow the P53-dependent pathway, compounds that follow the P53-independent pathway will be useful in treating such cancers. siRNA also showed the potential of being a treatment for melanoma cancer since it can also result in P53 upregulation. From Figure 9, Bcl₂ which is an antiapoptotic gene was found to be upregulated on untreated control melanoma cancer cells however, treating with both EtOH and MeOH extracts resulted in its downregulation. Similar results were observed from the co-treatment with siRNA and both extracts. Overexpression of Bcl₂ in different cancer types such as squamous carcinoma, ²⁰ breast, ²¹ lung, ²² melanoma, ²³ and colorectal cancer cells increases the invasive potentials of these cells. A study by Koehler et al ²⁴ also showed that a reduction in Bcl₂ expression results in a reduction of cellular invasiveness meaning that this pro-survival protein promotes cell migration and invasion.

Conclusion

The experimental findings showed that Cannabis sativa EtOH and MeOH extracts induce cell death in cancer cells, however, the extracts are cell dependent. Therefore, the extracts could be promising anticancer agents and their mechanism of action should be explored. Our findings also showed that siRNA transfection and treatment with both extracts resulted in the downregulation of RBBP6 and Bcl₂. Co-treatment of both EtOH and MeOH extracts with siRNA also resulted in the downregulation of RBBP6 and Bcl₂. Since RBBP6 is upregulated in various cancers and some cancers are resistant to chemotherapy, a combination of chemotherapy with Cannabis sativa EtOH and MeOH extracts or siRNA co-treatment with extracts could be a possible treatment for melanoma cancer.