Targeting bladder cancer: Potent anti-cancer effects of cannabichromene and delta-9-tetrahydrocannabinol-rich Cannabis sativa strains

Abstract

Objective

This study aimed to explore the anticancer potential of Cannabis sativa (C. sativa) strains, specifically PARIS, Dairy Queen (DQ), and super cannabidiol (sCBD), on bladder cancer cells. Given the increasing interest in cannabinoids like cannabichromene (CBC) and delta-9-tetrahydrocannabinol (THC) for their therapeutic properties, we evaluated their cytotoxic effects on urothelial carcinoma (UC) cell lines and their ability to inhibit cell migration and induce apoptosis in both two-dimensional cell models and three-dimensional ex vivo organ cultures (EVOCs).

Methods

C. sativa strains were screened for their cytotoxicity against UC cell lines (HTB-4 and HTB-9) using XTT assays. Their phytocannabinoid content was analyzed using high-performance liquid chromatography. We employed fluorescence-activated cell-sorting to determine apoptosis and cell cycle, migration assays to determine cell migration, and EVOCs to evaluate the cytotoxic effect on UC. Gene expression was determined by quantitative polymerase chain reaction.

Results

Three commercial C. sativa strains, PARIS, DQ, and sCBD, were found to have the most potent anticancer effects on bladder cancer cells. All extracts contain CBC and THC at different concentrations. In XTT assays on UC cell lines, PARIS had a half-maximal inhibitory concentration (IC₅₀) of 21.58 μg/mL, while DQ and sCBD had similar cytotoxic activity with IC₅₀ values for 48-h treatment of 17.99 μg/mL and 17.88 μg/mL, respectively. DQ and sCBD extracts were found to significantly reduce cell migration and increase the percentage of cells in S phase and G₂/M phase within the cell population. In EVOCs, the extracts initiated cell death with the expression of apoptosis-related genes increased following exposure to treatment.

Conclusion

The findings suggest that C. sativa strains PARIS, DQ, and sCBD, containing CBC and THC, exhibit significant anticancer activity against UC cell lines and ex vivo models. These results underscore the therapeutic potential of CBC- and THC-rich C. sativa extracts in bladder cancer treatment.

1. Introduction

Urothelial carcinoma (UC) represents the sixth most commonly diagnosed cancer in men and the 13th most common cancer within the general population [1]. Most patients are diagnosed in the early stages of the disease, in which tumor resection is the first approach. However, UC is a recurrent disease and even after complete removal of a primary tumor, disease progression and disease recurrence are very common.

The local application of chemotherapy or immunomodulatory drugs is the mainstay of treatment for localized disease after surgical resection, with different efficacy for the prevention of recurrence or progression. However, treatment-refractory UC is common. Therefore, new therapeutic formulations with high efficiency and acceptable side-effect profiles are still needed.

A large retrospective epidemiological study revealed that cannabis use among the general population may be associated with a reduced incidence of bladder cancer [2]. This association remained unexplained. We have previously shown that cannabis-derived compounds have cytotoxic synergistic activity against UC cell lines [3]. Our work demonstrated a consistent inhibitory effect of cannabichromene (CBC) and delta-9-tetrahydrocannabinol (THC) at well-defined concentrations and ratios on UC cell proliferation, migration, cell cycle arrest, and treatment-induced apoptosis of UC cells.

Currently, cannabis use in the population of the oncologic patients is directed towards palliative care and the reduction of chemotherapy-related side effects. However, since it first emerged with preliminary evidence for the antitumor effect [4], there has been mounting evidence for the antitumor effect of cannabis-derived compounds on epithelial cancers. THC produced a cytotoxic effect on breast cancer cells with a more potent activity when combined with other plant-derived compounds [5]. Literature review reveals that THC decreased cell viability and proliferation through the apoptotic pathway in pancreatic cancer cells [6], hepatocellular carcinoma [7], prostate carcinoma cells [8], colon cancer cells [9], and uterine cervix carcinoma cells [10] among others. In addition, cannabinoids were shown to improve the cytotoxicity of chemotherapeutic drugs, induce profound apoptosis and tumor shrinkage [11,12], and enhance the anti-tumor effect of the hormonal therapy for prostate carcinoma cells [13].

These findings are a solid basis for further investigation of cannabis-derived compounds with therapeutic potential in clinical applications. Here, we translate these results to therapeutic avenues by showing that commercial cannabis strains, which contain the relevant composition, act to reduce cell and tissue viability and cell migration of UC cell lines (T24 and HTB-9) [[14], [15], [16], [17], [18]] and inhibit tumor survival in tissue cultures from UC tumors.

2. Materials and methods

2.1. Plant growth and extract preparation

Inflorescences of three Cannabis sativa (C. sativa) strains were used for plant extract preparation: PARIS (IM Cannabis Corp., Israel), Super Cannabidiol (sCBD, a high cannabidiol strain; IM Cannabis Corp., Israel) and Dairy Queen (DQ, a high THC strain; IM Cannabis Corp., Israel). The inflorescences were immediately frozen at −20 °C using liquid nitrogen to preserve their chemical integrity. Frozen inflorescences were ground into a fine powder using a mortar and pestle. The ground material was then transferred into 15 mL tubes. Absolute ethanol was added to each inflorescence powder sample at a sample-to-ethanol ratio of 1:4 (solids:solvent ratio). The mixture was thoroughly agitated on a shaker for 30 min to ensure efficient extraction of cannabinoids and other phytochemicals. Following extraction, the mixture was filtered through a 0.2 μm polyvinyl difluoride syringe filter to remove any solid particulate matter. The filtrate was collected into new tubes. The solvent was then evaporated under a stream of nitrogen to obtain a concentrated dry extract. The concentrated dry extract underwent decarboxylation, a process that converts cannabinoid acids (e.g., THC acid and cannabidiolic acid) into their neutral forms (THC and cannabidiol, respectively). This was achieved by heating the extract to 220 °C for 10 min. The decarboxylated extract was weighed and resuspended in absolute methanol (MeOH) to achieve the desired concentration. The resuspended extract was then filtered through a 0.45 μm syringe filter to ensure a clear solution devoid of any remaining particulates.

2.2. Standard material preparation

Mitomycin C (MMC) was dissolved in water at a stock concentration of 800 μg/mL.

2.3. Chemical analysis

High-performance liquid chromatography analysis was performed using an Agilent 1260 Infinity II LC system (Agilent Technologies, Santa Clara, CA, USA). Analysis was carried out using isocratic separation with acetonitrile (20%) and water with 5 mmol/L ammonium formate and 0.1% formic acid (80%) at a constant flow rate of 1.5 mL/min.

2.4. Cell cultures

The UC cell line T24 (ATCC® HTB-4™, Manassas, VA, USA) was cultured in McCoy's 5A growth medium (Biological Industries, Kibbutz Beit Haemek, Israel, Cat. No. 01-075-1A). Similarly, the UC cell line 5637 (ATCC® HTB-9™) was maintained under appropriate culture conditions.

2.5. Cell proliferation assay

The XTT assay (Cell Proliferation Kit II, Biological Industries, Kibbutz Beit Haemek, Israel) was carried out on T24 and HTB-9™ cells. Briefly, 1×10⁴ cells/well were seeded in 96-well plates. MeOH was used as the vehicle control at the same concentration as the treatments, i.e., at concentrations that do not lead to cell death. MMC was used as a positive control. The cell viability was calculated relative to the vehicle control after subtracting the blank.

2.6. Apoptosis assay and cell cycle analysis

T24 cells were treated with cannabis compounds or with MeOH as a vehicle control. Cell cycle was determined at 24 h and apoptosis was determined at 48 h after the treatment; staining and detection followed the manufacturer's instructions. Briefly, for the apoptosis assay, a MEBCYTO® Apoptosis Kit with Annexin V-FITC (Milan, Italy) and propidium iodide (PI, ab14083, Abcam, Cambridge, UK) was used. Cells were seeded in 6-well plates, at a density of 5×10⁵ cells/well. The following day, the media were replaced with fresh media containing treatments and vehicle control. Next, cells were harvested and stained using 10 μL of Annexin V-FITC solution (MEBCYTO, Milan, Italy) and 5 μL of PI working solution (ab14083, Abcam, Cambridge, UK), and flow cytometry was performed using a Fortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Cells were considered to be apoptotic if they were Annexin V⁺/PI⁻ (early apoptotic) or Annexin V⁺/PI⁺ (late apoptotic). Live cells were defined as Annexin V⁻/PI⁻, and cells that were Annexin V⁻/PI⁺ were defined as necrotic.

For determination of the cell cycle phases, cells were seeded in 6-well plates at a concentration of 5×10⁵ cells/well. After 24 h of incubation, the media were replaced with new media containing treatments and vehicle control. Cells from each well were then harvested and fixed with 70% cold ethanol at 4 °C for at least 1 h. The fixed cells were then pelleted and washed twice with 1 mL of PBS. The cell pellet was then stained by resuspending in 250 μL of PI solution (50 μg/mL) containing RNase A (100 μg/mL) for 30 min in darkness. Then 200 μL of PBS was added to each tube and the cells were analyzed using a Fortessa flow cytometer.

2.7. Cell migration and cell invasion assays

For the cell migration assay, T24 cells were seeded into a 96-well tissue culture plate, 2×10⁴ cells/well. After 24 h, the cell monolayer was scratched perpendicularly across the center of the well with a 200 μL pipette tip. Immediately after scratching, the culture medium was aspirated and 100 μL of treatment solution was added. Images were acquired at 0, 10 h, 13 h, and 15 h following scratching, and the gap area was measured using ImageJ (National Institutes of Health, Bethesda, MD, USA) (n=12). At each time point, we measured the unoccupied area, subtracted it from the initial unoccupied area (time 0), divided that by the initial unoccupied area, and multiplied that by 100 to get the percentage of scratch closure.

2.8. Quantitative real-time polymerase chain reaction

T24 cells were treated with cannabis compounds or MeOH as a vehicle control for 6 h. Cells were then harvested and total RNA was isolated. The RNA was reverse transcribed. The expression of each target gene was normalized to the expression of hypoxanthine phosphoribosyltransferase (HPRT; gene ID 3251) mRNA using the 2^−ΔΔCt method to calculate the difference (Δ) in threshold cycle (Ct) between the target gene and HPRT gene (ΔCt=Ct of target gene–Ct of HPRT). Experiments were repeated four times (four biological repeats), with two more technical replicates. Primers targeting caspase 3 and caspase 7 were as follows: for caspase 3, forward 5’-GAGGCCGACTTCTTGTATGC-3’ (SEQ ID NO: 5) and reverse 5’-CGGTTAACCCGGGTAAGAAT-3’ (SEQ ID NO: 6); for caspase 7, forward 5’-GAAGAGGCTCCTGGTTTGTG-3’ (SEQ ID NO: 7) and reverse 5’-TCTCATGGAAGTGTGGGTCA-3’ (SEQ ID NO: 8).

2.9. Biopsy harvest and treatments

Tissues from UC patients were obtained during transurethral tumor resection, at the Chaim Sheba Medical Center, Ramat-Gan, Israel. All patients involved in this study gave written informed consent according to the institutional review board requirements.

2.10. Ex vivo organ cultures (EVOCs)

EVOCs were performed to evaluate the effect of the same extracts and cannabinoids on UC tumors using tumor samples taken from patients at our institute. The effect of cannabis-derived compounds was tested using continuous exposure and repeated exposure 2 h daily, at a concentration of 100 μg/mL. The survival of EVOC tumor cells was evaluated by a pathologist, who was blinded to treatment groups. Drug-related cell death was assessed using hematoxylin and eosin stain.

The study was conducted in accordance with the principles outlined in the Declaration of Helsinki and was approved by the Institutional Review Board of Chaim Sheba Medical Center (6960-20-SMC). Prior to their involvement, all patients provided informed consent.

2.11. Cleaved caspase 3 immunostaining

Slides following fixation were stained with cleaved caspase 3 antibody (1:500, #D175, Cell Signaling Technology, Danvers, MA, USA) with the BOND automated immunohistochemistry system (Leica Biosystems, Wetzlar, Germany) using ethylenediaminetetraacetic acid antigen retrieval (20 min) and the Bond Polymer Refine Detection Kit (#DS9800, Leica, Wetzlar, Germany). The stained tissue sections were examined under a microscope, and representative images were captured using QuPath, an open-source imaging system (Edinburgh, UK).

2.12. Statistical analysis

All statistical analyses were performed using SPSS version 25.0 (IBM Corp., Armonk, NY, USA). Data are presented as mean with standard deviation (SD). The half-maximal inhibitory concentration (IC₅₀) values were calculated using non-linear regression analysis with GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, USA). The comparisons between treatment groups and controls were performed using one-way analysis of variance followed by Tukey's post hoc test for multiple comparisons. The non-parametric Kruskal-Wallis H test was used to compare drug-related responses in EVOCs, with Dunn's post hoc test for pairwise comparisons. Spearman's rank correlation coefficient was employed to assess the relationship between cannabinoid concentrations and cytotoxic effects. The statistical significance was set at p<0.05 for all tests.

3. Results

3.1. Identification of the cytotoxicity of C. sativa strains on UC cells

C. sativa strains were screened to evaluate their cytotoxic activity against bladder cancer cell lines, namely T24 and HTB-9. MeOH was used as a vehicle for extracting several C. sativa strains. The extracts were screened and IC₅₀ values were calculated (Supplementary Table 1). The concentrations and contents of phytocannabinoids in the extracts were determined using high-performance liquid chromatography. Among the screened strains, a particular strain from the Israeli gene bank previously demonstrated the highest cytotoxic effect on UC cells, with a specific composition within the extract responsible for initiating the inhibitory effect [3]. Additionally, three commercial strains, namely “PARIS” (IM Cannabis Corp., Israel), “DQ”, and “sCBD” exhibited potent anticancer effects (Supplementary Table 1). The total phytocannabinoid concentration was the highest in the DQ extract (82.75%) and the lowest in the sCBD extract (66.44%) in the current extraction. CBC accounted for 1.51%, 4.65%, and 2.68% of the cannabinoid content in the DQ, sCBD, and PARIS extracts, respectively (Supplementary Table 1). THC levels were measured to be 93.35%, 1.78%, and 35.10% in the DQ, sCBD, and PARIS extracts, respectively. The cytotoxic activity of PARIS, DQ, and sCBD whole extracts against UC cell lines was evaluated (Fig. 1). The PARIS strain exhibited an IC₅₀ of 21.58 μg/mL. DQ and sCBD had similar cytotoxic activity on T24 cells (with IC₅₀ values for 48-h treatment of 17.99 μg/mL and 17.88 μg/mL, respectively). The IC₅₀ values of DQ and sCBD were further examined in a second cell line (HTB-9) with the values of 15.35 μg/mL and 16.28 μg/mL, respectively. Furthermore, once cytotoxic activity and effective doses were evaluated for a short treatment duration of 2 h, IC₅₀ values of 20.95 μg/mL and 25.83 μg/mL for DQ and sCBD on T24 cells, respectively, were recorded (Fig. 1). The same 2-h treatment with DQ and sCBD was evaluated on HTB-9 cells, with IC₅₀ values of 23.18 μg/mL and 20.71 μg/mL, respectively. In the comparative analysis of all tested extracts, we observed a positive correlation between the concentration of THC and the combined THC and CBC concentration with the IC₅₀ that was calculated for the whole extract (Spearman's r=0.74 for both parameters, p=0.05). This indicates that a higher concentration of THC or total THC and CBC correspond to a lower cytotoxic effect (Supplementary Table 2). It should be noted that the calculation of combined CBC and THC is heavily influenced by the THC concentrations due to the low CBC concentration present in each extract. However, we did not find any correlation between the total cannabinoid concentration, the ratio of CBC to THC, total CBC, total CBD, and the IC₅₀ values.

Figure 1.

Inhibitory concentrations derived from dose-effect curves of the crude extract of F7 (85% cannabichromene and 15% delta-9-tetrahydrocannabinol), PARIS, DQ, and sCBD extracts on the viability of UC cell lines. The cell viability was determined by an XTT assay. Values represent means with standard deviations of cell viability assays and their calculated half-maximal inhibitory concentrations, compared to vehicle control (n=2). IC₅₀, half-maximal inhibitory concentration; F7, standard mix 7; DQ, Dairy Queen; sCBD, super cannabidiol; UC, urothelial carcinoma.

3.2. Determination of the effect of treatments with DQ and sCBD extracts on cell motility

As previously demonstrated, treatments based on CBD, CBC, and THC inhibited cell migration [3]. Herein, the effects on cell migration of whole cannabis extracts of the two most active strains, DQ and sCBD, containing CBC and THC were examined using the scratch assay. Treatments consisted of DQ (17.99 μg/mL), sCBD (17.88 μg/mL), MMC (4.00 μg/mL; positive control), or MeOH (vehicle control) (Fig. 2). A marked reduction in the ability of the cells to migrate into the scratch was noted for cells treated with DQ or sCBD crude extracts in comparison to the control: mean scratch reduction (closure) for sCBD and DQ was lower than the control at 15 h following the treatment (39.3% and 66.4%, respectively, both p<0.05). DQ whole extract was more effective than sCBD crude extract in reducing cell migration (mean scratch reduction of 33.5% [SD 4.2%] vs. 60.6% [SD 4.1%], p<0.05) (Fig. 2).

Figure 2.

The scratch assay showing T24 cell migration over time (0, 10 h, 13 h, and 15 h) following the treatment with sCBD (17.88 μg/mL), DQ (17.99 μg/mL), MMC (4.00 μg/mL; positive control), or MeOH (1.8% v/v; vehicle control). (A) Graphical representation. Groups labeled with different letters (e.g., a and b) are significantly different from each other based on the Tukey-Kramer honest significant difference test (p<0.05). (B) Representative images. The closure of the scratch area was monitored to assess the effects of each treatment on cell migration. MMC, mitomycin C; sCBD, super cannabidiol; DQ, Dairy Queen; MeOH, methanol.

3.3. Determination of the effect of treatments with DQ and sCBD extracts on the cell cycle and apoptosis in T24 cells

The treatment with DQ and sCBD extracts led to a cell cycle arrest seen as an increase in the percentage of T24 cells found in S phase (52.3% and 47.2% for DQ and sCBD, respectively) compared to the control (19.6%), whereas MMC led to 34.4% of cells in S phase (Fig. 3A). The cell population treated with DQ or sCBD extracts also showed an increase in G₂/M phase cells (15.6% and 15.8%, respectively; Fig. 3A). MMC led to 28.1% of cells in G₂/M phase, whereas nearly no cells were in G₂/M phase in the control (Fig. 3A).

Figure 3.

The cell cycle analysis and apoptosis assessment following treatment with DQ, sCBD, MMC (positive control), and MeOH (vehicle control; 3% v/v) in T24 cells. (A) Determination of the stages of the cell cycle after 24 h; (B) Proportion of viable, apoptotic, or necrotic cells after 48 h. MMC, mitomycin C; sCBD, super cannabidiol; DQ, Dairy Queen; MeOH, methanol. Note: error bars indicate standard error (n=4). Groups labeled with different letters (e.g., a and b) are significantly different from each other based on the Tukey-Kramer honest significant difference test (p<0.05). Groups sharing at least one letter (e.g., a or ab) are not significantly different.

The treatment with DQ or sCBD for 48 h led to 64.1% or 84.0% of cells in apoptosis, respectively. Only 16.5% of cells were undergoing apoptosis in the vehicle control (Fig. 3B). The treatment with MMC led to 55.9% apoptotic cells (Fig. 3B). Only low levels of necrosis were recorded with the sCBD treatment, not significantly different from the vehicle control (14.3% and 1.7%, respectively; Fig. 3B). However, both DQ and MMC led to relatively high levels of cell necrosis (35.3% and 25.1%, respectively).

3.4. Determination of the effects of treatments with DQ and sCBD extracts on caspase gene expression in T24 cell lines

Caspase gene expression was measured in T24 cells treated for 2 h with DQ or sCBD. The expression of caspase 3 mRNA was upregulated at an early stage (2 h after treatment washout), and caspase 7 mRNA expression was elevated at 6 h after the treatment washout (Fig. 4). Caspase 8, caspase 9, or Fas-associated death domain did not exhibit a consistent change (not shown).

Figure 4.

Quantitative RT-PCR-based determination of the mRNA levels in the T24 cell line. (A and B) Casp 3 expression; (C and D) Casp 7 expression. Cells were harvested 2 h (A and C) or 6 h (B and D) following the treatment with MMC, sCBD, or DQ. Transcript values were determined as the ratio of the target gene to the reference gene (HPRT). Values were calculated relative to the mean expression of target genes in treated versus control groups using the 2^−ΔΔCt method; the MeOH (1.5% v/v) treatment served as solvent (vehicle) control; error bars indicate standard error (n=3). Casp, caspase; MMC, mitomycin C; sCBD, super cannabidiol; DQ, Dairy Queen; MeOH, methanol; RT-PCR, reverse transcription polymerase chain reaction; HPRT, hypoxanthine-guanine phosphoribosyltransferase. Note: groups labeled with different letters (e.g., a and b) are significantly different from each other based on the Tukey-Kramer honest significant difference test (p<0.05). Groups sharing at least one letter (e.g., a and ab) are not significantly different.

3.5. Investigating the effects of PARIS, DQ, and sCBD extracts on cell viability in UC tumors obtained during transurethral resection of bladder tumors

Tissues from six patients were included in the drug-related response assessment. EVOCs were extracted from tissues of all six patients and were treated with C. sativa extracts PARIS, DQ, and sCBD at a concentration of 100 μg/mL. Of them, three EVOCs were treated with PARIS, six with sCBD, and five with DQ. Continuous drug exposure and repeated 2-h drug exposure protocols were evaluated and compared for each extract. MMC was used as a positive control (800 μg/mL), while MeOH was used as a vehicle control (10% v/v MeOH). All tissues showed drug-related tumor cell death (Figure 5, Figure 6), with prominent cleaved caspase 3 staining (Fig. 5B), indicating a significant induction of apoptosis in the treated samples. Continuous drug exposure to each cannabis extract showed a strong histological drug-related response with tumor cell death. The mean drug-related response for sCBD was 100% (SD 0%), for DQ 100% (SD 0%), and for PARIS 92% (SD 12%) compared to MeOH and MMC with 18% (SD 6.1%) and 48% (SD 45%), respectively. EVOCs in the 2-h repeated protocol exhibited mean drug-related responses of 93% (SD 14%) for sCBD and 65% (SD 14%) for DQ, compared to MeOH and MMC with 18% (SD 6.1%) and 48% (SD 45%), respectively. The PARIS extract did not show a marked effect on UC EVOC viability when the 2-h protocol was conducted, nor a statistically significant difference compared to MeOH. However, it is noted that the sample size for the PARIS EVOC group was too small.

Figure 5.

Cytotoxic effects of cannabis extracts on high-grade urothelial carcinoma EVOCs. (A) After tissue harvest, EVOCs were treated in a continuous manner. Hematoxylin & eosin staining shows marked loss of viable tumor cells. (B) The immunohistochemical staining using the cleaved caspase 3 antibody. The treatment with whole cannabis extracts demonstrates prominent apoptotic staining, indicating a significant induction of apoptosis in the treated samples. MMC, mitomycin C; sCBD, super cannabidiol; DQ, Dairy Queen; MeOH, methanol; EVOCs, ex vivo organ cultures; GZ, gemcitabine. Scale bars: 50 μm (main images) and 10 μm (insets) for Fig. A; 100 μm (main images) and 20 μm (insets) for Fig. B.

Figure 6.

Cytotoxic effects of cannabis extracts on high-grade urothelial carcinoma using EVOCs harvested from a patient with muscle-invasive bladder cancer. (A) After tissue harvest, EVOCs were treated continuously or for a duration of 2 h with three types of cannabis extracts: PARIS, sCBD, and DQ; (B) The continuous drug exposure and repeated 2 h drug exposure protocol. For this specific patient, the drug-related response for the continuous treatment protocol with PARIS, sCBD, and DQ was 100%, whereas the 2-h protocol regime resulted in responses of 8%, 73%, and 10% for PARIS, sCBD, and DQ, respectively. SD, standard deviation; MMC, mitomycin C; sCBD, super cannabidiol; DQ, Dairy Queen; MeOH, methanol; EVOCs, ex vivo organ cultures. Scale bars for Fig. A: 50 μm for all main images; 20 μm for the inset (PARIS continuous) and 10 μm for all other insets.

In a non-parametric comparison analysis between groups, the Kruskal-Wallis H test indicated that there is a significant difference in the dependent variable between certain groups. Post-hoc analysis showed a significant difference in the mean drug-related response between sCBD and MeOH control (in both the continuous treatment and 2-h protocol, p=0.01 and p=0.02, respectively). Due to a small sample size in the continuous treatment with DQ extract, the difference in mean cannot be evaluated. The mean drug-related response for the 2-h protocol using DQ extract, did not reach statistical significance (p=0.2).

Tumor tissues exposed to the cannabis-derived treatment did not show necrosis of the stromal or muscle layer tissue in both treatment duration protocols.

4. Discussion

The aim of this study was to investigate the cytotoxic effect of commercially available cannabis extracts on UC tumors. The research employed both a classic continuous exposure to the investigated drug and a commonly practiced regimen of 2-h intravesical local treatment [19] along with in vitro and ex vivo investigational assays.

The results showed that certain cannabis extracts can inhibit UC tumor properties by inducing programmed cell death and the expression of apoptosis-related genes, thereby initiating apoptosis and inhibiting cell migration. Furthermore, for certain strains, the short treatment duration demonstrated a comparable effect on cell viability and a longer exposure demonstrated minimal changes in IC₅₀. This suggests that the compounds primarily exert their effect through an initial short duration of exposure, which persists even after the removal of the treatment and the restoration to normal conditions.

These strains were found to initiate apoptosis in three-dimensional tissue cultures of UC tumors. In this assay, sCBD, a high CBD strain containing a relatively high amount of CBC (3.09 μg/mL), demonstrated a statistically significant effect on human UC EVOC viability, with both the continuous and 2-h protocols. In this model, the treatment effect was shown to impact tumor cells, with minimal effect on stromal and muscle tissue in the samples. Interestingly, sCBD was as cytotoxic as DQ, despite having the lowest total cannabinoid-specific content in the investigated extract in comparison to other C. sativa strain extracts.

These findings emphasize the significance of cannabinoid-specific content rather than total cannabinoid concentrations and can provide guidance when selecting cannabis strains for the treatment and support of cancer patients. However, determining the specific composition or content that leads to the highest cytotoxic effect remains inconclusive. It is possible that the study was underpowered to provide a definitive answer, or that a plant-derived whole extract may elicit unexpected reactions. This could be due to the presence of additional compounds in the extract, which may interact synergistically or antagonistically, contributing to the overall effect observed.

C. sativa contains numerous active compounds, including cannabinoids, flavonoids, and terpenes. The concept of personalized medicine based on specific cannabis strain compositions is still a distant goal. However, commercially available cannabis-derived compounds, such as certain cannabinoids, offer the potential for more precise therapeutic interventions. Previous research has demonstrated the pronounced cytotoxic effect of a combination of CBC and THC on UC cell lines [3]. Moreover, the cytotoxic effect of CBC was further investigated in a study by Tomko et al. [20], which explored other compounds found in C. sativa strains, such as cannflavin A, and revealed a synergistic activity with CBC. In addition, CBD, the predominant cannabinoid in the sCBD strain, has been proven to convey pro-apoptotic, anti-proliferative, and cell migration inhibitory effects [3].

Notably, Chen et al. [21] proposed that CBD's cytotoxic effect on T24 cells works via inhibition of the phosphoinositide 3-kinase/Protein Kinase B/mechanistic target of rapamycin (PI3K/Akt/mTOR) pathway, a crucial intracellular signaling pathway that regulates the cell cycle. At the protein level, CBD was found to upregulate apoptotic proteins in T24 cells, such as Bax, cytochrome-c, and caspase 7. Furthermore, Chen et al. [21] investigated the potential mechanism underlying CBD's impact on cell migration and invasion properties, suggesting that CBD suppresses the matrix metalloproteinase-9 activity by inactivating the PI3K/Akt/mTOR and ERK1/2 signaling.

Moreover, cannabis-derived whole extracts have been shown to inhibit cell migration, which may play a major role in inhibiting tumor cell metastasis, as previously demonstrated for CBC, THC, or CBD [3].

Overall, these findings underscore the potential of cannabis-derived compounds as therapeutic agents in cancer treatment and warrant further investigation.

Further research is needed in the field to understand whether high CBC cannabis strains should be prescribed for systemic cannabis treatment in metastatic bladder cancer patients who experience nausea, pain, or other medical conditions. High CBC strains are usually CBD-enriched [22,23]. Caution must be exercised when considering the immunomodulatory effect of CBD. UC cure is assisted by an intact immune function, and the patient's immune system is commonly used for preventive and therapeutic goals. Therefore, the effect of CBD-enriched strains on the relationship between urothelial tumors and the immune system must be investigated.

Two studies have addressed the possible impact of cannabis on patients' response to immune-modulatory agents. Bar-Sela et al. [24] prospectively investigated cannabis users treated with immune checkpoint inhibitors, compared with non-cannabis users. The authors concluded that cannabis users (34 patients) have shorter time to progression (3.4 months vs. 13.1 months, p=0.0025) and shorter median overall survival (6.4 months vs. 28.5 months, p=0.00094), compared to non-cannabis users (68 patients). It should be noted that although both groups were comparable in terms of epidemiological and disease characteristics, there was a trend towards a more severe disease in the cannabis user group (higher number of patients given immune therapy as second line, higher rates of lymphopenic patients, higher alkaline phosphatase levels, etc.). More research is needed to elaborate on the relationship between systemic cannabis use and the tumor-immune system.

Notably, the use of human tumor tissues serves as a proof of concept for the efficacy of these compounds on actual UC tumors and their potential application in local treatments. As mentioned earlier, the disease is often managed through localized interventions like intravesical chemotherapy or immune-mediated therapy (bacillus Calmette-Guérin). However, the local application of whole extracts poses various regulatory and pharmaceutical challenges. Firstly, the cannabinoid composition of C. sativa can be directed but not precisely controlled. Secondly, the whole extract contains hundreds of constituents [25], some of which are biologically active and may have clinical effects that are not always desirable, such as immune modulation. In our perspective, further research should focus on isolating and purifying specific compositions from the whole cannabis extract that can selectively target cancer cells while minimizing impact on surrounding tissues.

5. Conclusion

This study highlights the potential of commercially available cannabis extracts in inhibiting UC tumors through programmed cell death, the expression of apoptosis-related genes, and cell migration inhibition. The findings emphasize the significance of cannabinoid-specific content over total cannabinoid concentrations in determining their cytotoxic effects. While personalized medicine based on specific strain compositions remains a distant goal, certain cannabinoids like CBC, THC, and CBD show promise in exerting cytotoxic effects. Further research is needed to understand their mechanisms of action and their application in cancer treatment.

Author contributions

Study concept and design: Omer Anis, Gil Raviv, Menachem Laufer, Alon Lazarovich, Tomer Drori, Jacob Ramon, Zohar Dotan, Hinanit Koltai.

Data acquisition: Omer Anis, Seegehali M. Anil, Vered Bar, Adi Zundelevich.

Data analysis: Omer Anis, Seegehali M. Anil, Vered Bar, Adi Zundelevich.

Drafting of manuscript: Omer Anis.

Critical revision of the manuscript: Omer Anis, Yaron Shav-Tal, Amos Toren, Dan Dominissini, Hinanit Koltai.

Conflicts of interest

The authors declare no conflict of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: