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Cannabidiol Induces Cell Cycle Arrest and Cell Apoptosis in Human Gastric Cancer SGC-7901 Cells

1 Yantai Key Laboratory of Pharmacology of Traditional Chinese Medicine in Tumor Metabolism, School of Integrated Traditional Chinese and Western Medicine, Binzhou Medical University, Yantai 264003, China

Yao Qin

1 Yantai Key Laboratory of Pharmacology of Traditional Chinese Medicine in Tumor Metabolism, School of Integrated Traditional Chinese and Western Medicine, Binzhou Medical University, Yantai 264003, China

Zhaohai Pan

1 Yantai Key Laboratory of Pharmacology of Traditional Chinese Medicine in Tumor Metabolism, School of Integrated Traditional Chinese and Western Medicine, Binzhou Medical University, Yantai 264003, China

Minjing Li

1 Yantai Key Laboratory of Pharmacology of Traditional Chinese Medicine in Tumor Metabolism, School of Integrated Traditional Chinese and Western Medicine, Binzhou Medical University, Yantai 264003, China

Xiaona Liu

1 Yantai Key Laboratory of Pharmacology of Traditional Chinese Medicine in Tumor Metabolism, School of Integrated Traditional Chinese and Western Medicine, Binzhou Medical University, Yantai 264003, China

Xiaoyu Chen

1 Yantai Key Laboratory of Pharmacology of Traditional Chinese Medicine in Tumor Metabolism, School of Integrated Traditional Chinese and Western Medicine, Binzhou Medical University, Yantai 264003, China

Guiwu Qu

2 School of Gerontology, Binzhou Medical University, Yantai 264003, China

Ling Zhou

3 School of Pharmacy, Binzhou Medical University, Yantai 264003, China

Maolei Xu

3 School of Pharmacy, Binzhou Medical University, Yantai 264003, China

Qiusheng Zheng

1 Yantai Key Laboratory of Pharmacology of Traditional Chinese Medicine in Tumor Metabolism, School of Integrated Traditional Chinese and Western Medicine, Binzhou Medical University, Yantai 264003, China

Defang Li

1 Yantai Key Laboratory of Pharmacology of Traditional Chinese Medicine in Tumor Metabolism, School of Integrated Traditional Chinese and Western Medicine, Binzhou Medical University, Yantai 264003, China

1 Yantai Key Laboratory of Pharmacology of Traditional Chinese Medicine in Tumor Metabolism, School of Integrated Traditional Chinese and Western Medicine, Binzhou Medical University, Yantai 264003, China

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Abstract

The main chemical component of cannabis, cannabidiol (CBD), has been shown to have antitumor properties. The present study examined the in vitro effects of CBD on human gastric cancer SGC-7901 cells. We found that CBD significantly inhibited the proliferation and colony formation of SGC-7901 cells. Further investigation showed that CBD significantly upregulated ataxia telangiectasia-mutated gene (ATM) and p53 protein expression and downregulated p21 protein expression in SGC-7901 cells, which subsequently inhibited the levels of CDK2 and cyclin E, thereby resulting in cell cycle arrest at the G0–G1 phase. In addition, CBD significantly increased Bax expression levels, decreased Bcl-2 expression levels and mitochondrial membrane potential, and then upregulated the levels of cleaved caspase-3 and cleaved caspase-9, thereby inducing apoptosis in SGC-7901 cells. Finally, we found that intracellular reactive oxygen species (ROS) increased after CBD treatment. These results indicated that CBD could induce G0–G1 phase cell cycle arrest and apoptosis by increasing ROS production, leading to the inhibition of SGC-7901 cell proliferation, thereby suggesting that CBD may have therapeutic effects on gastric cancer.

1. Introduction

Gastric cancer is a common malignant tumor that originates in the gastric mucosal epithelium [1]. Gastric cancer can be further classified according to the disease site, which includes gastric cardia cancer, gastric cancer, and gastric antrum cancer [2]. Recent epidemiological data have indicated that there are about 951,000 new cases of gastric cancer around the world and about 723,000 deaths due to gastric cancer, thereby ranking gastric cancer as the fifth most common and third most malignant tumor with the highest mortality rate [3]. Countries including Japan, South Korea, and China are at high risk for gastric cancer [4]. Chinese gastric cancer morbidity and mortality account for 42.6% and 45.0% of global gastric cancer morbidity and mortality, respectively [5]. Gastric cancer is the most common malignant tumor in China [6], with regional differences in incidence. The incidences of gastric cancer in the northwest and eastern coastal areas of China are significantly higher than in the southern regions [7]. The high-risk age of the disease is above 50 years old, and the ratio of male to female risk is 2:1 [8]. In the past few decades, due to an increase in pressure at work and in daily life, dietary changes, and Helicobacter pylori infections, gastric cancer has been observed to have an earlier onset [9].

Current treatment schemes for gastric cancer mainly include surgery and chemotherapy: However, their long-term effects are not ideal [10]. For instance, tumor recurrence has been reported in half of the patients who undergo surgery [11]. In addition, chemotherapy, which induces tumor cell cytotoxicity and eventually death [12], has not significantly improved the overall survival of patients with gastric cancer because of poor selectivity and toxicity [2,10,13,14]. Thus, the traditional treatment schemes for gastric cancer do not meet clinical needs, and the development of novel treatment methods is imperative [15]. It is thus important to study the effect of traditional Chinese medicine on malignant tumors, to explore its mechanism of action, and to develop new and effective anti-tumor drugs [16].

Cannabidiol (CBD) is the main chemical component of the medicinal plant cannabis (Cannabis sativa L). This cannabinoid is extracted from female cannabis plants and is nonaddictive [17]. Studies have shown that CBD inhibits tumor cell proliferation, metastasis, or the induction of autophagy or apoptosis [18,19]. CBD and tamoxifen have been cocultured with C6 glioma cells, which showed an inhibitory effect on C6 glioma cells [20]. CBD induces apoptosis in human glioma cells U87 and U373 through mechanisms such as the activation of caspase and the involvement of reactive oxygen species (ROS) [21]. CBD induces mouse lymphoma EL-4 cells and Jurkat leukemia cell apoptosis by regulating NOX4 and p22phox expression, which in turn leads to an increase in reactive oxygen species (ROS) level [22]. CBD plays an antiprostate cancer role by inhibiting prostate cancer cell proliferation or inducing apoptosis [23]. CBD can also inhibit the growth and metastasis of breast cancer cells through the epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) [24] and protein kinase B (AKT)/mTOR/4EBP1 [25] signaling pathways. In addition, CBD exerts a good safety profile while exhibiting significant anticancer effects [19,26]. However, the effects of CBD on protein expression in gastric cancer cells and the underlying mechanism of action are unclear.

To explore the antitumor effects of CBD on gastric cancer, we selected human gastric cancer SGC-7901 cells as a research object. Preliminary experiments have shown that CBD can significantly inhibit the proliferation and induce apoptosis in SGC-7901 cells, suggesting that CBD may be a potential chemotherapeutic drug for gastric cancer. However, its specific mechanism of action is still unclear. In this study, we explored the in vitro effects of CBD on human gastric cancer SGC-7901 cells and its molecular mechanisms.

2. Methods

2.1. Cell Culture

Human gastric cancer SGC-7901 cells were obtained from Cell Bank, Typical Culture Preservation Commission, Chinese Academy of Sciences (Shanghai, China). The cells were cultured with RPMI 1640 (SH30809.01, GE Healthcare Life Sciences Hyclone Laboratories, Logan, UT, USA) containing 10% fetal calf serum (REF10091-48, Gibco, Invitrogen), 0.1 µg penicillin, and 0.1 µg/L streptomycin (P1400, Solarbio, Beijing, China) and were incubated in a 5% CO2 incubator (HF90/HF240, Heal Force, Shanghai, China) at 37 °C.

2.2. Cell Counting Kit-8 (CCK-8) Assay

The effect of CBD on the viability of SGC-7901 cells was determined using a CCK-8 assay. SGC-7901 cells were cultured in RPMI 1640 medium and, upon reaching the logarithmic growth phase, were digested with 0.25% trypsin + 0.02% ethylene diamine tetraacetic acid (EDTA), centrifuged at 600× g for 3 min, and collected. After counting, 100 µL of the cell suspension was seeded in each well of a 96-well plate at a density of 1.2 × 10 5 cells/mL. After 24 h of culture, culture medium containing 5, 10, 20, 30, and 40 µg/mL of CBD was added and further cultured for another 24 h in the incubator. After 24 and 48 h of culture, 10 µL of CCK-8 solution was added to each well and incubated for 2–4 h. Then, the plate was shaken for 1 min in the dark, and the absorbance of each well at a wavelength of 450 nm was detected using a Thermo 3001 multifunction microplate (DMI3000, Leica, Germany) reader. The absorbance of the cells treated with 0.1% DMSO in RPMI 1640 was used as a control (survival rate: 100%). IC50 (half maximal inhibitory concentration) indicates a drug concentration resulting in a 50% reduction in cell survival.

2.3. Hoechst 33258 Staining

Approximately 2 mL of the SGC-7901 cell suspension was seeded into six-well plates at a density of 2.5 × 10 5 cells/mL. After 24 h of culture, the CBD culture medium containing 10, 20, and 40 µg/mL was added, and the plate was incubated for 24 h. After incubation, the culture solution was removed by aspiration, and 0.5 mL of fixative was added to each well and incubated for at least 10 min (can be overnight at 4 °C). Then, the fixative was removed, and the cells were washed twice with phosphate buffered solution (PBS) for 3 min each time, with shaking. Approximately 0.5 mL of 2-µg/mL Hoechst 33258 was then added to stain the cells at room temperature for 5 min with shaking. To remove the staining solution, the cells were rinsed twice with PBS for 3 min each time, with shaking. A drop of a fluorescent antiquenching agent was placed on the slide, covered with a coverslip, and the cells were allowed to come into contact with the sealing solution, avoiding air bubbles. Fluorescence microscopy (DMI3000, Leica, Germany) could detect blue nuclei.

2.4. Colony Formation Assay

The SGC-7901 cells at the logarithmic growth phase were digested with 0.25% trypsin + 0.02% EDTA and centrifuged at 800× g for 3 min to collect the cells. After counting, the cell concentration was adjusted, and 2 mL of the cell suspension containing 300 cells was seeded into each well of a six-well plate. After 48 h of culture, the CBD culture medium containing 10, 20, and 40 µg/mL was added, and after culturing for another 24 h, the fresh medium was replaced, and cell growth was monitored each day. Cells were cultured for 14 days and then dyed. The medium was discarded, and the cells were washed twice with PBS. The cells were fixed with a 4% cell fixing solution for 15 min, and then the cell fixing solution was discarded. The cells were then stained with gentian violet for 10 min and then slowly rinsed with running water. The plates were then observed for the formation of colonies. Images were captured using a light microscope (XDS-3, OPTIKA, Ponteranica, BG, Italy). Clones containing more than 50 cells were counted and used in calculating the rate of colony formation.

2.5. Cell Cycle Analysis

The cell cycle distribution of CBD-treated SGC-7901 cells was determined by flow cytometry. The SGC-7901 cells were treated with different concentrations of CBD or treated with 24 h of CBD and subsequently incubated with fresh culture media for 24 h. Then the cells (approximately 1 × 10 6 cells per well) were harvested and fixed overnight in 70% ethanol at 4 °C. After fixation, the cells were centrifuged at 3000× g for 5 min to remove the ethanol. Then, the cells were washed with PBS, treated with 100 µL of RNase A, resuspended, and incubated at 37 °C for 30 min in the dark. Fluorescence detection of propidium iodide (PI)-DNA complexes was determined by Epics XL flow cytometry (Beckman Coulter, Inc, Brea, CA, USA). Cell distribution in different stages of the cell cycle was analyzed using WinMDI 2.8 software (Scripps Research, La Jolla, CA, USA), and cell cycle distribution was calculated [27,28].

2.6. Annexin V-FITC/Propidium Iodide (PI) Double Staining Assay

The Annexin V-FITC/PI double staining assay was performed according to the manufacturer’s instructions. Briefly, SGC-7901 cells in a logarithmic growth phase were digested with 0.25% trypsin + 0.02% EDTA and centrifuged at 600× g for 3 min to collect cells. The cell density of the suspension was adjusted to 2.5 × 10 5 cells/mL, and 2 mL of the suspension was loaded into each well. After 24 h of culture, CBD culture medium containing 10, 20, and 40 µg/mL was added, and the cultures were incubated for 24 h in the incubator. The cells were collected and centrifuged at 2000× g for 3 min at room temperature. The cells were resuspended in precooled 1× PBS and centrifuged at 2000× g for 3 min, and the cells were washed. The cells were resuspended by adding 500 µL of 1× binding buffer. Then, 5 µL of Annexin V-FITC was added to the suspension, mixed well, and incubated for 15 min at room temperature. The cells were then stained with 5 µL of a PI staining solution before loading into a flow cytometer. Fluorescence intensity was measured using a FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA), and the apoptotic rates of CBD-treated cells were analyzed using FACSDiva software (version 6.1.3; Becton Dickinson, Franklin Lakes, NJ, USA).

2.7. Mitochondrial Membrane Potential Assay

The JC-1 method was performed according to the manufacturer′s instructions. Briefly, SGC-7901 cells in a logarithmic growth phase were digested with 0.25% trypsin + 0.02% EDTA and centrifuged at 600× g for 3 min to collect cells. The cell density of the suspension was adjusted to 2.5 × 10 5 cells/mL, and 2 mL of the suspension was added to each well of a six-well plate. After 24 h of culture, CBD medium containing 10, 20, and 40 μg/mL was added, and the culture was cultured in an incubator for 24 h. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (10 mM) was added to the cell culture medium at 1:1000 and diluted to 10 μM, and the positive control cells were treated for 20 min. The medium in the six-well plate was aspirated, the cells were washed once with PBS, 1 mL of the cell culture solution was added, 1 mL of JC-1 (CBIC2(3)) staining working solution was added, and the mixture was thoroughly mixed and incubated at 37 °C for 20 min. The supernatant was aspirated and washed twice with JC-1 staining buffer (1×), and 2 mL of the cell culture medium was added and observed under a fluorescence microscope.

On the other hand, the mitochondrial membrane potential of CBD-treated SGC-7901 cells was determined by flow cytometer. Briefly, SGC-7901 cells in a logarithmic growth phase were digested with 0.25% trypsin + 0.02% EDTA and centrifuged at 600× g for 3 min to collect cells. The cell density of the suspension was adjusted to 2.5 × 10 5 cells/mL, and 2 mL of the suspension was added to each well of a six-well plate. After 24 h of culture, CBD medium containing 10, 20, and 40 μg/mL was added, and the culture was cultured in an incubator for 24 h. CCCP (10 mM) was added to the cell culture medium at 1:1000 and diluted to 10 μM, and the positive control cells were treated for 20 min. The medium was discarded and the cells were washed once with PBS. Then 1 mL of the cell culture media and 1 mL of JC-1 staining working solution was added and mixed well, and subsequently incubated at 37 °C for 20 min. After incubation, the cells were collected and centrifuged at 2000× g for 3 min at room temperature. The cells were resuspended in prechilled 1× JC-1 staining buffer, centrifuged at 2000× g for 3 min, and washed twice. The cells were resuspended by the addition of 500 μL of 1x JC-1 staining buffer and then loaded onto a flow cytometer. Fluorescence intensity was measured using a FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

2.8. Reactive Oxygen Species Assay

The ROS method was measured according to the manufacturer′s instructions. Briefly, SGC-7901 cells in a logarithmic growth phase were digested with 0.25% trypsin + 0.02% EDTA and centrifuged at 600× g for 3 min to collect cells. The cell density of the suspension was adjusted to 2.5 × 10 5 cells/mL, and 2 mL of the suspension was added to each well of a six-well plate. After 24 h of culture, CBD medium containing 10, 20, and 40 μg/mL was added, and the culture was cultured in an incubator for 24 h. Rosup (50 mg/mL) was added to the cell culture medium at 1:1000, and the positive control cells were treated for 20 min. The medium was aspirated in a six-well plate, the cells were washed once with PBS, 1 mL of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was added, and everything was mixed well and incubated at 37 °C for 20 min. The supernatant was aspirated and washed three times with serum-free medium, and 2 mL of serum-free medium was added and observed under a fluorescence microscope.

In addition, the ROS levels of CBD-treated SGC-7901 cells were determined by flow cytometer. Briefly, SGC-7901 cells in a logarithmic growth phase were digested with 0.25% trypsin + 0.02% EDTA and centrifuged at 600× g for 3 min to collect cells. The cell density of the suspension was adjusted to 2.5 × 10 5 cells/mL, and 2 mL of the suspension was added to each well of a six-well plate. After 24 h of culture, CBD medium containing 10, 20, and 40 μg/mL was added, and the culture was cultured in an incubator for 24 h. Rosup (50 mg/mL) was added to the cell culture medium at 1:1000, and the positive control cells were treated for 20 min. The medium was aspirated in a six-well plate, the cells were washed once with PBS, 1 mL of DCFH-DA was added, and everything was mixed well and incubated at 37 °C for 20 min. The supernatant was aspirated, and the cells were collected and centrifuged at 2000× g for 3 min at room temperature and washed three times with serum-free medium. The cells were resuspended in serum-free medium and centrifuged at 2000× g for 3 min, and the cells were washed 3 times. The cells were resuspended by the addition of 500 μL of serum-free medium and then loaded onto a flow cytometer. Fluorescence intensity was measured using a FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

2.9. Western Blotting Analysis

The SGC-7901 cells (density: 1 × 10 6 /well) were seeded into 100-mm culture dishes and treated with different concentrations of CBD for 24 h. After treatment, the cells were harvested and mixed with a cell lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 0.1 mM phenylmethanesulfonylfluoride, 0.1 mM sodium orthovanadate, 0.5 mM dithiothreitol, 10× protease inhibitor; pH 6.8) on ice for 30 min. The suspension was then centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was collected and stored at −20 °C. Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Solarbio, Beijing, China). A cell lysate having a protein content of 40 mg and an equal volume of sodium dodecyl sulfate (SDS) loading dye (2% SDS, 10% sucrose, 0.002% bromophenol blue, 5% 2-mercaptoethanol, 625 mM Tris; pH 6.8) was subsequently separated on ta 12.5% SDS-PAGE with an iridescent protein molecular marker (Solarbio, Beijing, China). After two hours of operation at 110 V, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, REF 88520) using a Trans-Blot ® SD semidry transfer cell (Bio-Rad). The membrane was blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 120 min, and then incubated overnight at 4 °C with primary antibodies (some were purchased from Abcam, Cambridge, UK; the others were purchased from cell signaling technology, Boston, United States) as follow: p21 (1:1000, ab109199), Bcl-xl/Bcl-2-associated death prompter (BAD, 1:2000, ab32445), cyclin-dependent kinase 2 (CDK2, 1:1000, ab32147), B-cell lymphoma-2 (Bcl-2, 1:500, ab196495), ataxia telangiectasia-mutated gene (ATM, 1:2000, ab199726), Cyclin E (1:5000, ab194070), Caspase-3 (1:1000, #9662), p53 (1:1000, #9284), apoptotic protease activating factor-1 (Apaf-1, 1:1000, #5088), cytochrome C (1:1000, #4280), Caspase-9 (1:1000, #9508), Bcl-2-associated X (BAX, 1:1000, #2772), active Caspase-3 (Cleaved Caspase-3, 1:1000, #9664), and active Caspase-9 (Cleaved Caspase-9, 1:1000, #52873)) containing 5% BSA. Subsequently, the membrane was washed with TBS-T buffer for 45 min and then hybridized with the appropriate horseradish-conjugated secondary antibody for 40 min. An electro-chemi-luminescence (ECL) chromogenic solution was added for color development, and the target gene was detected using an immunoblot imaging system. Immunoreactive bands were visualized using a Novex™ ECL chemiluminescent substrate reagent kit (WP20005; Thermo Fisher Scientific, Waltham, MA, USA) using a film processor (BioSpectrum Imaging System, Upland, CA, USA). Image-Pro Plus 6.0 (IPP6) software was used to calculate the gray-scale values of each band [29].

2.10. Statistical Analysis

The experiments were conducted at least three times. The data were presented as the mean ± standard deviation. Statistical analyses were performed using the SPSS 21.0 software package (version 21.0, SPSS Inc., Chicago, IL, USA). Student’s t-test or one-way ANOVA was used to calculate the statistical difference. Differences with p < 0.05 were considered statistically significant.

3. Results

3.1. In Vitro Antigastric Cancer Effects of CBD

To study the effect of CBD on gastric cancer in vitro, we treated the gastric cancer SGC-7901 cells with different concentrations of CBD for 24 and 48 h. The activity of SGC-7901 cells after CBD treatment was detected by the CCK-8 assay. The results showed that CBD (5–40 μg/mL) significantly inhibited SGC-7901 cell proliferation in a concentration-dependent manner, with an IC50 value of 23.4 μg/mL after 24 h of treatment ( Figure 1 a). The inhibition rate was significantly higher in SGC-7901 cells after 48 h of treatment with CBD than after 24 h of treatment ( Figure 1 a). In addition, the cell colony formation assay demonstrated that CBD effectively inhibited colony formation in SGC-7901 cells at concentrations of 10, 20, and 40 μg/mL ( Figure 1 b,c).

Inhibition of proliferation of SGC-7901 cells by cannabidiol (CBD). (a) SGC-7901 cells were seeded into 96-well plates, and cells were treated with different concentrations of CBD for 24 and 48 h. The survival rate of cells treated with CBD was measured by the CCK-8 method. (b) Statistical analysis of cell colony formation rate in CBD-treated SGC-7901 cells. (c) Representative images of cell colonies in CBD-treated SGC-7901 cells. All data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 compared to the control (24 h). ## p < 0.01 compared to the control (48 h). & p < 0.05 compared to the 24-h CBD-treated cells.

3.2. CBD Induces Cell Cycle Arrest at the G0–G1 Phase in SGC-7901 Cells

To further investigate the inhibitory effect of CBD on the proliferation of SGC-7901 cells, we examined the cell cycle distribution of SGC-7901 cells after 24 h of treatment with CBD. Flow cytometry analysis showed that the proportion of SGC-7901 cells that were at the G0–G1 phase significantly increased after treatment with CBD compared to the control group ( Figure 2 a,b). It should be noted that the CBD-induced G0–G1 phase arrest was obviously released in SGC 7901 cells after 24 h of incubation with fresh culture media, as evidenced by no significant difference in the proportion of SGC-7901 cells at the G0–G1 phase being found between the 20-μg/mL CBD treatment group and the control group ( Figure 2 c,d). When the G1/S cycle is retarded, the content of the CDK2/cyclin E complex is correspondingly reduced [30]. Therefore, we examined the expression levels of CDK2 and cyclin E in cells after CBD treatment, which were significantly lower than the control group ( Figure 2 e,f). These results indicated that CBD could effectively induce cell cycle arrest at the G0–G1 phase by inhibiting CDK2 and cyclin E expression.

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CBD induced G0–G1 cell cycle arrest of SGC-7901 cells. (a) Flow cytometry was used to determine the cell cycle distribution of CBD-treated SGC-7901 cells. (b) Statistical analysis of the cell cycle distribution of SGC-7901 cells treated by CBD. (c) SGC-7901 cells were treated with CBD for 24 h, followed by 24 h of incubation with fresh culture media (FM), and then the cycle distribution was examined by flow cytometry. (d) Statistical analysis of the cell cycle distribution of SGC-7901 cells after 24 h of CBD treatment and 24 h of incubation with fresh culture media (FM). (e) The expression levels of CDK2 and cyclin E protein in CBD-treated SGC-7901 cells were detected by western blotting. (f) Statistical analysis of the levels of CDK2 and cyclin E in CBD-treated SGC-7901 cells. The ratio of protein levels was normalized according to the values of the control. All data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 compared to the control.

3.3. Effects of CBD on the ATM/p53/p21 Signaling Pathway

ATM is activated during DNA damage, which in turn can upregulate the expression level of P21, downregulate the expression level of P53 [31], and subsequently inhibit the formation of CDK2/cyclin E complexes [32]. Previous experiments have shown that CBD can induce cell cycle arrest and decrease the expression of CDK2/cyclin E in SGC-7901 cells. Therefore, we examined the protein expression levels of ATM, p53, and p21 in SGC-7901 cells after CBD treatment and found that both ATM and p21 expression were elevated, whereas that of the p53 protein decreased ( Figure 3 a,b).

Effect of CBD on ATM/P53/P21 signaling protein levels. (a) Western blotting was used to detect the protein expression levels of ATM, p53, and p21 in SGC-7901 cells after 24 h of treatment with CBD. (b) Statistical analysis of the levels of ATM, p53, and p21 in SGC-7901 cells treated with CBD. The ratio of protein levels was normalized according to the values of the control. All data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 compared to the control.

3.4. CBD Promotes Apoptosis in SGC-7901 Cells

To explore whether the observed inhibition of the proliferation effect of CBD is related to cell apoptosis, Hoechst 33258 staining of nuclei was performed. Fluorescence microscopy revealed that there were more abnormal nuclei in the CBD administration group compared to the control. These nuclei had distinct apoptotic features such as nuclear contraction, irregular condensation of chromatin, and apoptotic bodies ( Figure 4 a). We also analyzed the apoptotic rate of CBD-treated SGC-7901 cells using an Annexin V FITC/PI double staining kit and flow cytometry. We found that as the concentration of CBD increased, the percentage of apoptotic cells in the SGC-7901 population increased ( Figure 4 b,c). These results indicated that CBD effectively induced apoptosis in SGC-7901 cells.

CBD could effectively induce apoptosis in SGC-7901 cells. (a) After 24 h of treatment with CBD, SGC-7901 cells showed typical morphological changes after staining with Hoechst 33258. (b) After 24 h of treatment with CBD, the apoptosis rate of SGC-7901 cells was detected by Annexin V-FITC/PI double staining. (c) Statistical analysis of the apoptotic rate of the SGC-7901 cell population after CBD treatment. All data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 compared to the control.

3.5. Effect of CBD on Apoptosis-Related Proteins

In the above experiments, we observed that CBD could effectively induce apoptosis in SGC-7901 cells. To explore the intrinsic mechanism of this effect, we performed western blot analysis to identify the core proteins associated with apoptosis. The results showed that CBD significantly reduced the expression of caspase-3 and caspase-9 at the same time and markedly increased the content of cleaved caspase-3 and cleaved caspase-9 ( Figure 5 a,b). We also investigated the proteins that activate the caspase family of proteins, such as cytochrome C and Apaf-1, by western blotting. The results showed that CBD remarkably increased cytoplasm cytochrome C and Apaf-1 protein expression levels in SGC-7901 cells ( Figure 5 c,d). These results implied that CBD induced SGC-7901 cell apoptosis through a mitochondrial-dependent apoptosis pathway.

Effect of CBD on the expression of apoptosis-related proteins. (a) After 24 h of treatment with CBD, the protein expression levels of caspase-9, cleaved caspase-9, caspase-3, and cleaved caspase-3 in SGC-7901 cells were determined by western blotting. (b) Statistical analysis of protein relative levels of caspase-9, cleaved caspase-9, caspase-3, and cleaved caspase-3 in SGC-7901 cells treated with CBD. The ratio of protein levels was normalized according to the values of the control. (c) After 24 h of treatment with CBD, the levels of cytoplasm cytochrome C and Apaf-1 in SGC-7901 cells were determined by western blotting. (d) Statistical analysis of the levels of cytoplasm cytochrome C and Apaf-1 in CBD-treated SGC-7901 cells. The ratio of protein levels was normalized according to the values of the control. All data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 compared to the control.

3.6. The Effect of CBD on the Mitochondrial Apoptosis Signaling Pathway

Next, the levels of mitochondrial apoptosis-related proteins in CBD-treated SGC-7901 cells were examined by western blotting. We found that CBD significantly increased the protein expression levels of Bad and Bax and decreased that of Bcl-2 relative to the control group ( Figure 6 a,b). We also further examined the mitochondrial membrane potential of CBD-treated SGC-7901 cells using JC-1 staining. Flow cytometry analysis showed that the ratio of red to green fluorescence significantly decreased in CBD-treated SGC-7901 cell groups compared to the control group ( Figure 6 c,d). Fluorescence microscopy revealed a significant increase in green fluorescence intensity and a decrease in red fluorescence intensity in CBD-treated SGC-7901 cells ( Figure 6 e). The results confirmed that CBD induced SGC-7901 cell apoptosis via the mitochondrial-dependent apoptosis pathway.

Effect of CBD on the expression of signaling molecules in the mitochondrial apoptotic pathway. (a) After 24 h of treatment with CBD, the protein levels of Bad, Bax, and Bcl-2 in CBD-treated SGC-7901 cells were determined by western blotting. (b) Statistical analysis of relative protein expression levels of Bad, Bax, and Bcl-2 in CBD-treated SGC-7901 cells. The ratio of the proteins was normalized according to the values of the control. (c) After 24 h of treatment with CBD, the changes in the mitochondrial membrane potential in SGC-7901 cells were detected using a JC-1 (CBIC2(3)) mitochondrial membrane potential assay kit. (d) Quantitative analysis of the mitochondrial membrane potential of SGC-7901 cells. (e) After 24 h of treatment with CBD, SGC-7901 cells were stained with JC-1 dye, and the fluorescence distribution was observed under a fluorescence microscope. All data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 compared to the control.

3.7. The Effect of CBD on the ROS Levels of SGC-7901 Cells

Based on the antioxidant activity of CBD, we investigated the changes in ROS levels in CBD-treated SGC-7901 cells using a DCFH-DA fluorescence probe. The DCFH-DA probe is nonfluorescent in its initial form, but it can be easily oxidized by intracellular ROS, leading to the formation of fluorescent product dichlorofluorescein (DCF) [33]. After 2′,7′-dichlorofluorescein diacetate (DCFH-DA) staining, flow cytometry analysis showed that the intensity of green fluorescence (DCF) was markedly enhanced in CBD-induced SGC-7901 cells compared to the control ( Figure 7 a,b).

Effect of CBD on reactive oxygen species (ROS) levels in SGC-7901 cells. (a) After 24 h of treatment with CBD, the changes in reactive oxygen species (ROS) content in SGC-7901 cells were determined using an ROS assay kit. (b) After staining with an ROS species assay kit, the fluorescence distribution in CBD-treated SGC-7901 cells was observed under a fluorescence microscope.

4. Discussion

CBD is the main chemical in and a nonaddictive component of the medical plant cannabis [34]. Studies have shown that CBD can inhibit tumor cell proliferation and metastasis or induce autophagy or apoptosis [35] in various cancers such as glioma [36], leukemia [37], prostate cancer [38], and breast cancer [39]. In this study, we found that CBD could arrest SGC-7901 cells at the G0–G1 phase and induce apoptosis of SGC-7901 cells by activating the mitochondrial apoptosis pathway.

Checkpoints are important regulatory nodes of the cell cycle. Cells can only enter the next cell cycle after passing these checkpoints [40]. The function of the G0–G1 phase detection point is to integrate and transmit complex intracellular and extracellular signals, such as various growth factors, mitogens, and DNA damage, as well as to determine whether cells are undergoing division and apoptosis [41]. When the cells begin to synthesize DNA during G1–S conversion, CDK2 will combine with its regulatory subunit cyclin E to form a Cdk2/Cyclin E complex, leading to Rb phosphorylation, and then E2F factor is released and cells are accelerated into the S phase [42]. During G0–G1 cycle arrest, the content of the CDK2/cyclin E complex decreases accordingly [43]. Our data showed that the protein expression levels of CDK2 and cyclin E in the SGC-7901 cells decreased after CBD treatment, which in turn reduced the formation of CDK2/cyclin E complexes, ultimately arresting SGC-7901 cells at the G0–G1 phase.

The cell cycle refers to the entire process of continuous cell division. When cell cycle arrest occurs during cell division, it is often due to damage or errors that are difficult to repair during cell division [44]. This theory suggests that CBD-induced G1 arrest may be due to DNA damage that is difficult to repair in cells. At the same time, intracellular DNA is damaged, ATM and ATR (ATM and Rad 3-related) are the centers of the stress response, and ATM/ATR signaling is activated [45]. The ATM/ATR signaling pathway can repair damaged DNA by modulating the activity of various proteins. At present, it is generally believed that the ATM/ATR signaling pathway mediates G0–G1 arrest by regulating the expression of p53. ATM can directly regulate p53, which increases the p53 protein level, which in turn enhances p21 transcription [46]. Activation of ATM during DNA damage can upregulate the expression of the p21 protein and downregulate p53 protein expression. Finally, the formation of the CDK2/cyclin E complexes is inhibited, and the cell cycle is arrested at the G0–G1 phase. The results of this study are consistent with the above theory. After treatment with CBD, ATM protein expression levels increased, and the ATM protein was activated in SGC-7901 cells. Meanwhile, CBD increased the expression of p21 and downregulated the expression of p53 in SGC-7901 cells, which in turn led to cell cycle arrest at the G0–G1 phase.

Apoptosis is a programmed cell death of the body’s cells [47]. A recent study showed that CBD can induce apoptosis in breast cancer cells [48]. This study found that the levels of cleaved caspase-3 and -9 were upregulated in SGC-7901 cells after 24 h of treatment with CBD, subsequently inducing cell apoptosis. Considering that the mitochondria-mediated caspase-dependent pathway is a major apoptotic pathway, we then examined the levels of anti- and pro-apoptotic Bcl-2 family proteins in the mitochondria-dependent apoptotic pathway. We found that CBD upregulated Bax and downregulated Bcl-2 in CBD-treated SGC-7901 cells, leading to a decrease in the ratio of Bcl-2/Bax.

Mitochondrial membrane permeability increases and mitochondrial transmembrane potential decreases when the ratio of Bcl-2/Bax decreases [49]. Large amounts of the apoptotic initiator mitochondrial cytochrome C then flow into the cytoplasm. Cytochrome C in the cytoplasm activates Apaf-1 and caspase-9 and -3, which cleaves DNA and produces apoptotic bodies that ultimately lead to apoptosis [50]. In our study, we further examined the expression levels of the aforementioned proteins in the mitochondrial apoptotic signaling pathway. Our data showed that CBD significantly decreased the mitochondrial transmembrane potential, released mitochondrial cytochrome C into the cytoplasm, and activated Apaf-1 and caspase-9 and -3, which ultimately resulted in SGC-7901 cell apoptosis.

Previous studies have demonstrated that the continuous increase of intracellular ROS leads to DNA damage [51,52]. After the “sensing” or “detection” of DNA damage, the ATM/ATR signal is activated, which subsequently upregulates p21 and downregulates p53, leading to cell cycle arrest at the G0–G1 phase by decreasing the level of the CDK2/cyclin E complexes [53]. Moreover, the continuous increase in intracellular ROS levels leads to the continuous opening of mitochondrial permeability transition pore (mPTP), which reduces the mitochondrial transmembrane potential [54], and the release of cytochrome C into the cytoplasm, which results in a decrease in mitochondrial membrane potential and induces cell apoptosis through the mitochondrial-dependent pathway [55]. Considering the increase in the ROS levels in Jurkat leukemia EL-4 cells induced by CBD [22], we examined the ROS levels in CBD-treated SGC-7901 cells and found that the intracellular ROS levels in SGC-7901 cells significantly increased after CBD treatment. Taken together, these results indicated that CDB-induced cell cycle arrest and cell apoptosis of SGC-7901 cells were associated with the increasing intracellular ROS levels.

However, some limitations to the antitumor effects of CBD in this study should be noted. First, the antigastric cancer effect of CBD was only examined in one human gastric cancer cell line, SGC-7901, and thus other gastric cancer cells (e.g., human gastric cancer BGC-823 cells and mouse gastric cancer MFC cells) should be employed to further explore the therapeutic effects of CBD. Second, the change in ROS levels was examined in CBD-treated SGC-7901 cells, so some antioxidants could be employed to evaluate the role of ROS in CBD-induced apoptosis of SGC 7901 cells. In addition, the cytotoxicity of CBD in normal cells was not determined. However, we searched for reports of the cytotoxicity of CBD in normal cells. It has been reported that no significant effects on physiological parameters (heart rate, blood pressure, and body temperature) and psychological functions are found after CBD treatment, and high doses up to 1500 mg/day of CBD are tolerated well in humans [26], implying that CBD has no obvious cytotoxicity in normal tissues/cells.

In conclusion, our results indicated that CBD upregulated CDK2 and cyclin E in SGC-7901 cells by increasing ATM expression, which in turn induced cell cycle arrest at the G0–G1 phase. CBD also upregulated Bax and Bad and downregulated Bcl-2 in SGC-7901 cells, which in turn activated the mitochondrial-dependent apoptotic pathway and ultimately induced apoptosis. These findings may be utilized in the development of CBD as a potential drug for the treatment of gastric cancer.

Author Contributions

Data curation, Z.P., M.L., X.L. and X.C.; Formal analysis, G.Q., L.Z. and M.X.; Writing—original draft, X.Z. and Y.Q.; Writing—review & editing, Q.Z. and D.L.

Funding

This study was supported by the National Natural Science Foundation of China (81872162 and 81602556 to Defang Li, 31870338 to Qiusheng Zheng), the Shandong Provincial Natural Science Foundation, China (ZR2017JL030 to Defang Li, and ZR2016HL55 to Guiwu Qu), the Key Research and Development Program of Shandong Province of China (GG201809270118 to Qiusheng Zheng), Taishan Scholars Construction Engineering of Shandong Province (to Defang Li), the Yantai High-End Talent Introduction Plan “Double Hundred” (to Defang Li), the Scientific Research Foundation of Binzhou Medical University (Grant no. BY2016KYQD01 to Defang Li), and the Dominant Disciplines’ Talent Team Development Scheme of Higher Education of Shandong Province (to Defang Li).

Conflicts of Interest

The authors declare no conflict of interest.

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Cannabis, cannabinoids and cancer – the evidence so far

The current consensus is that, right now, there isn’t a large enough body of evidence to prove that cannabis (or any of its active compounds or derivatives) can reliably treat any form of cancer but the medical use of cannabis to treat cancer-related chronic pain is approved in the UK.

Cancer Research UK does not have an organisational policy on the legal status of cannabis, its use as a recreational drug, or its medical use diseases other than cancer. But we are supportive of properly conducted scientific research into cannabis and its derivatives that could benefit cancer patients and we will continue to monitor developments in the fields and evidence as it emerges.

For the last couple of decades, one of the most talked about discussions online is whether or not cannabis can treat cancer.

Claims that there is solid “proof” that cannabis or cannabinoids can cure cancer are highly misleading. Unfortunately, there are many unreliable sources of information about cannabis, particularly online.

This post contains up-to-date, evidence-based information on cannabis and cancer.

The basics

What is cannabis?

Cannabis is a plant grown and cultivated commercially across the globe. It is known by many names depending on its preparation and quality, including marijuana, trees, pot, dank, grass, green, kush, weed, hemp, hash, loud, and herb. These usually refer to the dried form or resin of the flowers or leaves of the plant.

There are multiple species of cannabis plant, including Cannabis sativa, Cannabis indica and Cannabis ruderalis.

For thousands of years, it has been used recreationally, religiously, and medically. Records from Ancient Egypt, India, and China show that physicians would use the plant as part of treating ailments such as haemorrhoids, insomnia, and for other pain relief.

In the Western world, cannabis emerged as a mainstream medicine in the 1840s and was noted for its sedative, anti-inflammatory, pain relief, and anticonvulsant effects.

Scientists have identified multiple active compounds within cannabis (known as cannabinoids) that play a role in cannabis’ effects, including the psychoactive delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD).

Cannabinoids – what are they?

Cannabinoids are compounds that can interact with a system inside the body known as the endocannabinoid system.

Most commonly, the term “cannabinoid” is used to refer to the compounds found in cannabis (and other plants). As the body naturally produces cannabinoids itself (known as endocannabinoids), a more accurate term for these is phytocannabinoids (meaning “cannabinoids from plants”).

Researchers have found that cannabis contains over 450 different chemical compounds, many of which are cannabinoids.

The two main cannabinoids of interest to researchers are:

  • Delta-9-tetrahydrocannabinol (THC) – a psychoactive substance that can affect how the brain works, creating a ‘high’ feeling.
  • Cannabidiol (CBD) – may relieve pain, lower inflammation and decrease anxiety without any psychoactive effects.

Is cannabis legal in the UK?

In the UK, medical use of cannabis was legalised in November 2018 and the UK is one of the world’s largest exporters of legal cannabis. However, cannabis is still classified as a class B drug in the UK, meaning that it is illegal to possess or supply it for personal recreational use.

  • Products without THC – legal to buy in the UK as supplements (such as CBD oil or hemp oil).
  • Products containing THC – illegal in the UK for recreational purposes (such as cannabis flower, cannabis oil, edibles, etc).
  • Medicines derived from cannabis – legal in the UK for certain healthcare professionals to prescribe (such as Sativex and Nabilone).

Medical cannabis is only legal when prescribed by a specialist consultant and GPs are not allowed to prescribe cannabis-derived medicines. NHS guidance states that medical cannabis should only be prescribed when there is clear published evidence of its benefit and other treatment options have been exhausted.

How do cannabinoids work inside the body?

Our bodies naturally produce our own cannabinoids (known as endocannabinoids).

These interact with molecules found on the surface of cells (cannabinoid receptors). One type of is densely packed inside the brain and second type is found in our immune tissues.

These compounds and receptors form the endocannabinoid system, a network that is involved in the control and regulation of multiple functions within the body – including memory, sleep, learning, eating, pain control, inflammation, and immune system.

As THC, CBD and other cannabinoids look similar to the endocannabinoids inside the body, they are able to interact with these receptors and affect how the system functions.

This is why some researchers think that cannabinoids have the potential to control some of the most common and debilitating symptoms of cancer and its treatments, including nausea and vomiting, loss of appetite, and pain.

Is all cannabis is the same?

Like how beer, wine, and vodka all have differing levels of alcohol and other ingredients, different strains/types of cannabis have varying levels of THC, CBD, and other compounds. This means that different strains of cannabis can have different effects on the body.

Additionally, its effects also depend on how cannabis is taken, most commonly by inhaling (smoking or vaping) or ingesting (edibles).

When it is inhaled, THC enters the lungs where it passes directly into your bloodstream and then your brain quickly. The effects of inhaled cannabis fade faster than cannabis taken by mouth.

When ingested (such as when it’s used in oils/drinks/baked goods/sweets), edible cannabis travels first to your stomach then to your liver before getting into your bloodstream and brain. The liver converts THC into a stronger compound and this (combined with the THC from the original product) adds to the intensity of the high.

Are there cannabis-based medicines?

Some cannabis-based products are available on prescription. The following medicines are sometimes prescribed to help relieve symptoms.

Nabilone

Nabilone is a drug developed from cannabis. It is licensed for treating severe sickness from chemotherapy that is not controlled by other anti-sickness drugs.

It works very well for some people, but can cause drowsiness or dizziness in others. These side effects can last for a couple of days after you’ve stopped taking it.

Sativex

Sativex (or nabixmols) is a liquid cannabis-based medicine that you spray into your mouth.

Researchers are looking into Sativex as a treatment for cancer related symptoms and for certain types of cancer.

The research

Is Cancer Research UK investigating cannabinoids?

In the past, Cancer Research UK has funded research into cannabinoids, notably the work of Professor Chris Paraskeva in Bristol investigating the properties of cannabinoids as part of his research into the prevention and treatment of bowel cancer. He has published a number of papers detailing lab experiments looking at endocannabinoids as well as THC, and written a review looking at the potential of cannabinoids for treating bowel cancer.

We have also supported the work of Dr Laureano de la Vega , a Cancer Research UK Fellow at the University of Dundee, who in 2019 started to explore if CBD can limit cancer’s ability to spread, using lung and triple negative breast cancer cells grown in the lab.

We’re also involved in the only 2 UK clinical trials of cannabinoids for treating cancer, mentioned above, through our national network of Experimental Cancer Medicine Centres .

Our funding committees have previously received other applications from researchers who want to investigate cannabinoids but these failed to reach our high standards for funding.

If we receive future proposals that meet these stringent requirements, then there is no reason that they wouldn’t be funded, assuming we have the money available.

Scam warning

Unfortunately, some scammers are using the email address [email protected] and claiming to be based at our head office, tricking cancer patients and their families into handing over money for “cannabis oil”, after which they receive nothing in return. This is a scam and has nothing to do with Cancer Research UK or our employees, as we wrote about in 2015. If you believe you have been a victim of this fraud, please contact the police.

How do researchers study cannabis?

Around the world, many researchers are actively investigating cannabis and cannabinoids, and Cancer Research UK is supporting some of this work.

Generally, the cannabis that researchers study isn’t the same as the one as you might see on the street or oils sold in shops.

When researchers conduct rigorous scientific studies, they often use purified forms of the compounds that they are investigating . This gives us more reliable evidence on the effect of different cannabinoids.

Through many detailed experiments – summarised in this review article from the British Journal of Cancer – scientists have discovered that both natural and synthetic cannabinoids have a wide range of effects on cells, which is why there’s interest in finding out whether it can be a part of treating diseases like cancer, as well as help relieving side effects.

Can cannabinoids treat cancer?

As of 2022, several hundreds of scientific papers looking at cannabinoids, the endocannabinoid system, and the relation to cancer have been published. So far these studies simply haven’t found enough robust scientific evidence to prove that these can safely and effectively treat cancer.

This is because the majority of the scientific research investigating whether cannabinoids can treat cancer has been done using cancer cells grown in the lab or animals. While these studies are a vital part of research, providing early indications of the benefits of particular treatments, they don’t necessarily hold true for people.

Much of the research into cannabinoids and cancer so far has been done in the lab

So far, the best results from lab studies have come from using a combination of highly purified THC and CBD . But researchers have also found positive results using man-made cannabinoids, such as a molecule called JWH-133.

There have been intriguing results from lab experiments looking at a number of different cancers, including glioblastoma brain tumours, prostate, breast, lung, and pancreatic cancers. But the take-home message is that different cannabinoids seem to have different effects on various cancer types, so they are far from being a ‘universal’ treatment.

There’s also evidence that cannabinoids have unwanted effects. Although high doses of THC can kill cancer cells, they also harm crucial blood vessel cells. And under some circumstances, cannabinoids can encourage cancer cells to grow, or have different effects depending on the dose used and levels of cannabinoid receptors present on the cancer cells.

Cannabis in clinical trials

To robustly test the potential benefits of cannabinoids in cancer, clinical trials in large numbers of people with control groups of patients – who aren’t given the treatment in question – would be needed.

A few small clinical trials have been set up to test the benefits of cannabinoids for people with glioblastoma multiforme. Results published from a pilot clinical trial where 9 people with advanced, incurable glioblastoma multiforme – the most aggressive brain tumour – were given highly purified THC through a tube directly into their brain showed that THC given in this way is safe and doesn’t seem to cause significant side effects. But as this was an early stage trial without a control group, it couldn’t show whether THC helped to extend patients’ lives.

And a second clinical trial, supported through our Experimental Cancer Medicine Centre (ECMC) Network, tested whether Sativex (nabiximols), a highly purified pharmaceutical-grade extract of cannabis containing THC, CBD, and other cannabinoids could treat people with glioblastoma multiforme brain tumours that have come back after treatment.

In 2021, scientists reported the final results of this phase 1 study to treat people with recurrent glioblastoma with Sativex in combination with the chemotherapy drug, temozolomide. Researchers found that adding Sativex (patients were allowed to choose the amount they took) had acceptable levels of side effects, which included vomiting, dizziness, fatigue, nausea and headache. They also observed that more patients were alive after one year using Sativex (83%) compared to those taking the placebo (44%). However, this phase 1 study only involved 27 patients, which was too small to confirm any potential benefits of Sativex, and was intended to find out if it was safe to take by patients.

This trial is being extended into phase 2 (known as ARISTOCRAT) to explore if this treatment is effective and which patients are most likely to respond to this treatment. It is set to launch at 15 NHS hospitals in 2022, with over 230 patients to be recruited (and making use of the Cancer Research UK Clinical Trials Unit). To find out more about this work, you can listen to our podcast – That Cancer Conversation – where we hear from Professor Susan Short, one of the researchers leading this study.

We’ve also supported a trial that’s testing the benefits of a man-made cannabinoid called dexanabinol in patients with different types of advanced cancer. The trial finished recruiting in 2015 and researchers established a safe dose of the drug, but further development of the drug was stopped due to a lack of evidence around the drug’s effectiveness. Full trials results are yet to be published.

Groups exploring cannabinoids and cancer

    is researching cannabis and cannabinoids for treating cancer to build up the evidence. He is happy to collect individual stories from UK patients and can be contacted by email. is the lead on the ARISTOCRAT trial that is evaluating the combined use of Sativex and the chemotherapy drug temozolomide treat people with recurrent glioblastoma.
  • The Medical Cannabis Research Group at Imperial College London are exploring cannabinoid use as it relates to potential therapies for inflammation and pain linked to cancer.
  • The charity DrugScience are running Project Twenty21, an observational medical cannabis study in the UK. It is gathering data on the efficacy of cannabis-based medicines for a wide range of conditions (including cancer-related pain, nausea, and anxiety).

Can cannabis prevent cancer?

There is no reliable evidence that cannabis can prevent cancer.

There has been some research suggesting that the body’s endocannabinoids (mentioned earlier) can suppress tumour growth.

When it comes to cannabis, experiments where mice were given very high doses of purified THC showed that they seemed to have a lower risk of developing cancer. But this is not enough solid scientific evidence to suggest that cannabinoids or cannabis can cut people’s cancer risk.

Does cannabis cause cancer?

The evidence is a lot less clear when it comes to whether cannabis itself can cause cancer.

This is because most people who use cannabis smoke it mixed with tobacco, a substance that we know causes cancer. Data from 2016 has shown that 77% of UK people surveyed (who smoke weed) reported normally mixing it with tobacco.

This makes it hard for researchers to disentangle the potential impact of cannabis on cancer risk from the impact of the tobacco. As of 2022, we can’t be sure whether the increased risk is due to tobacco or whether cannabis itself also has an independent effect.

We do know from decades of evidence that there is no safe way to use tobacco – it’s addictive and harmful for your health. People who smoke weed mixed with tobacco increase their risk of cancer and other conditions. Tobacco also contains the very addictive substance nicotine. This means people who regularly smoke weed mixed with tobacco may find it harder to stop.

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In addition to this, there have not been published studies looking at cannabis ingestion (such as edibles) and cancer risk, nor vaporised cannabis and cancer risk.

> Read about the free support and quitting tools available to help you to stop smoking for good on our website.

Can cannabis relieve cancer symptoms like pain or sickness?

There’s good evidence that cannabinoids may be beneficial in managing cancer pain and side effects from treatment.

As far back as the 1980s, cannabinoid-based drugs including dronabinol (synthetic THC) and nabilone were used to help reduce nausea and vomiting caused by chemotherapy. But there are now safer and more effective alternatives and cannabinoids tend to only be used where other approaches fail.

In some parts of the world, medical marijuana has been legalised for relieving pain and symptoms (palliative use), including cancer pain. But one of the problems with using herbal cannabis is managing the dose. Smoking cannabis or taking it in the form of tea often provides an inconsistent dose, which may make it difficult for patients to monitor their intake. So, researchers are turning to alternative dosing methods, such as mouth sprays, which deliver a reliable and regulated dose.

Large-scale clinical trials in the UK have been testing whether a mouth spray formulation of Sativex (nabiximols) can help to control severe cancer pain that doesn’t respond to other drugs. Results from these didn’t find any difference in self-reported pain scores between the treatment and the placebo.

Cannabinoids may also have potential in combating the loss of appetite and wasting (cachexia) experienced by some people with cancer, although so far clinical evidence is lacking. One clinical trial comparing appetite in groups of cancer patients given cannabis extract, THC and a placebo didn’t find a difference between the treatments, while another didn’t show any benefit and was closed early.

Questions that still need to be answered

There are still many unanswered questions around the potential for using cannabinoids to treat cancer. It’s not clear:

  • which type of cannabinoid – either natural or synthetic – might be most effective
  • what kind of doses might be needed
  • which types of cancer might respond best to cannabinoids
  • how to avoid the psychoactive effects of THC
  • how best to get cannabinoids, which don’t dissolve easily in water, into cancer cells
  • whether cannabinoids will help to boost or counteract the effects of chemotherapy

These questions must be answered for cannabinoids to be used as safe and effective treatments for cancer patients. It’s the same situation for the many hundreds of other potential cancer drugs being developed and tested in university, charity and industry labs all over the world.

Without doing rigorous scientific research, we will never sift the ‘hits’ from the ‘misses’. If cannabinoids are to get into the clinic, these hurdles first need to be overcome and their benefits proven over existing cancer treatments.

Frequently asked questions (FAQs)

“What’s Cancer Research UK’s view on cannabis and cancer?”

As of 2022, Cannabis is still classified as a class B drug in the UK, meaning that it is illegal to possess or supply it for personal recreational use.

Cancer Research UK does not have an organisational policy on the legal status of cannabis, its use as a recreational drug, or its medical use in any other diseases.

But we are supportive of properly conducted scientific research into cannabis and its derivatives that could benefit cancer patients and we will continue to monitor developments in the fields and evidence as it emerges.

“It’s natural so it must be better, right?”

There’s no doubt that the natural world is a treasure trove of biologically useful compounds, and there are countless examples where these have been harnessed as effective treatments.

Numerous potent cancer drugs have also been developed in this way – purifying a natural compound, improving it and testing it to create a beneficial drug – including taxol, vincristine, vinblastine, camptothecin, colchicine, and etoposide.

But although these purified drugs in controlled high doses can treat cancer, it doesn’t mean that the original plant (or a simple extract) will have the same effect.

So, although cannabis contains certain cannabinoids, it doesn’t automatically follow that cannabis itself can treat cancer.

“But it worked for this patient…”

Doctors sometimes publish case reports about extraordinary or important observations they have seen in their clinic.

For example, there was a case report published in the British Medical Journal describing a woman in her 80s with lung cancer whose tumour shrank after taking CBD oil over several months.

This might seem like a solid bit of proof, but very little reliable information can be taken from a single patient treated with what’s an unknown mix of cannabinoids outside of a controlled clinical setting

The authors state that even though this case appears to demonstrate a possible benefit of CBD oil intake, it’s not possible to confirm that the tumour regression was due to the patient taking CBD oil (as she was also taking drugs for other conditions).

There are also many videos and anecdotes online claiming that people have been completely cured of cancer with cannabis, hemp/cannabis oil or other cannabis derivatives.

Despite what these sources may claim, it’s impossible to tell whether these patients have been ‘cured’ by cannabis or not. There is usually no information about their medical diagnosis, stage of disease, what other cancer treatments they had, or the chemical make-up of their treatment. These sources also only publish the “success stories”, and don’t share how many people who used cannabis or its derivatives had no benefit, or worse, were potentially harmed.

Robust scientific studies describe the detail of experiments and share the results – positive or negative. This is vital for working out whether a potential cancer treatment is truly safe and effective, or not. And publishing this data allows doctors around the world to judge the information for themselves and use it for the benefit and safety of their patients.

This is the standard to which all cancer treatments are held, and it’s one that cannabis and cannabinoids should be held to, too.

“What’s the harm? There’s nothing to lose.”

If someone chooses to complete reject conventional cancer treatment in favour of unproven alternatives, they may miss out on treatment that could save or significantly lengthen their life. They may also miss out on effective symptom relief to control pain or other problems.

Many unproven therapies are also expensive, and aren’t covered by the NHS or medical insurance. In the worst cases, an alternative therapy may even hasten death.

Although centuries of human experimentation tell us that naturally-occurring cannabinoids are broadly safe, they are not without risks. They can increase heart rate, which may cause problems for patients with pre-existing or undiagnosed heart conditions. They can also interact with other drugs in the body, including antidepressants and antihistamines. And they may also affect how the body processes certain chemotherapy drugs, which could cause serious side effects.

As cannabis is illegal for recreational use in the UK, there are further risks associated with using home-made preparations, particularly cannabis oil, such as toxic chemicals left from the solvents used in the preparation process.

Synthetic cannabinoids (sometimes known as spice) are compounds that have been designed to act like the chemicals found in cannabis but with far stronger effects and have harmful side-effects associated.

There are also many internet scams by people offering to sell cannabis preparations. As well as the risk of getting something with completely unknown chemical or medicinal properties and unknown effectiveness, scammers are tricking cancer patients and their families into handing over money for “cannabis oil” which they then never receive.

We understand the desire to try every possible avenue when conventional cancer treatment fails. But there is little chance that an unproven alternative treatment bought online will help, and it may well harm. We recommend that cancer patients talk to their doctor about clinical trials that they may be able to join, giving them access to new drugs in a safe and monitored environment.

“Are cancer charities hiding cannabis as a cure?”

The idea that a cure already exists is one of the many myths that surrounds cancer that we have written about.

This myth is unjust to the thousands of scientists, doctors and nurses working as hard as they can to beat cancer, and to the many thousands of people in the UK and beyond who give up their time and money to fund our work.

History shows that the best way to beat cancer is through rigorous scientific research. This approach has helped to change the face of cancer prevention, diagnosis, treatment, leading to increased survival in the last few decades .

As a research-based organisation, we want to see reliable scientific evidence to support claims made about any cancer treatment, be it conventional or alternative. This is vital because lives are at stake. Some people may think that a cancer patient has nothing to lose by trying an alternative treatment, but there are big risks.

“Big Pharma can’t patent it so they’re not interested.”

Some people argue that the potential of cannabinoids is being ignored by pharmaceutical companies, because they can’t patent the chemicals naturally occurring in cannabis plants. But there are many ways that these compounds can be patented – for example, by developing more effective lab-made versions or better ways to deliver them.

Other people argue that patients should be treated with homegrown cannabis preparations, and that the research being done by companies is solely to make money and prevent patients accessing “the cure”

The best chance of ensuring that the potential benefits of cannabinoids – whether natural or man-made – can be brought to patients is through research using quality-controlled, safe, legal, pharmaceutical grade preparations containing known amounts of the drugs.

This requires time, effort and money, which may come from companies or independent organisations such as charities or governments. And, ultimately, this investment needs to be paid back by sales of a safe, effective new drug.

It’s true that there are issues around drug pricing and availability and we’re pushing for companies to make new treatments available at a fair price. We would hope that if cannabinoids were to be shown to be safe and effective enough to make it to the clinic, they would be made available at a fair price for all patients who might benefit from them.

In summary

Right now, there simply isn’t enough evidence to prove that cannabinoids – whether natural or synthetic – can effectively treat cancer in patients, although research is ongoing. And there’s certainly no evidence that cannabis bought on the street can treat cancer.

We’re supportive of properly conducted scientific research into cannabis and its derivatives that could benefit cancer patients. Many researchers are actively exploring this approach, and Cancer Research UK is supporting, and will continue to support, scientifically robust research into cannabis and cannabinoids that reaches the high-quality standards set by our funding committees.

References and further reading:

  • Cancer Research UK – Cannabis, CBD oil and cancer
  • NHS – Medical cannabis (and cannabis oils)
  • National Cancer Institute (US) – Information about cannabis and cannabinoids for cancer patients
  • National Cancer Institute (US) – Information about cannabis and cannabinoids for health professionals
  • Velasco, G., Sánchez, C. & Guzmán, M. (2012). Towards the use of cannabinoids as antitumour agents, Nature Reviews Cancer, 12 (6) 444. DOI: 10.1038/nrc3247
  • Sarfaraz, S. et al (2008). Cannabinoids for Cancer Treatment: Progress and Promise, Cancer Research, 68 (2) 342. DOI: 10.1158/0008-5472.CAN-07-2785
  • Guindon, J. & Hohmann, A.G. (2011). The endocannabinoid system and cancer: therapeutic implication, British Journal of Pharmacology, 163 (7) 1463. DOI: 10.1111/j.1476-5381.2011.01327.x
  • Engels, F.K. et al (2007). Medicinal cannabis in oncology, European Journal of Cancer, 43 (18) 2644. DOI: 10.1016/j.ejca.2007.09.010
  • Twelves, C., Sabel, M., Checketts, D. et al (2021). A phase 1b randomised, placebo-controlled trial of nabiximols cannabinoid oromucosal spray with temozolomide in patients with recurrent glioblastoma. British Journal of Cancer 124, 1379–1387. DOI: 10.1038/s41416-021-01259-3
  • Cannabinoids in the treatment of chemotherapy-induced nausea and vomiting – Todaro (2012) Journal of the National Comprehensive Cancer Network
  • Bowles, D.W. et al (2012). The intersection between cannabis and cancer in the United States, Critical Reviews in Oncology/Hematology, 83 (1) 10. DOI: 10.1016/j.critrevonc.2011.09.008
  • Hall, W., Christie, M. & Currow, D. (2005). Cannabinoids and cancer: causation, remediation, and palliation, The Lancet Oncology, 6 (1) 42. DOI: 10.1016/S1470-2045(04)01711-5 . , Wai Liu, The Conversation – University of Birmingham
  • Nutt D, Bazire S, Phillips LD, et al (2020) So near yet so far: why won’t the UK prescribe medical cannabis? BMJ Open 10:e038687. DOI: 10.1136/bmjopen-2020-038687
  • Mangal, N., Erridge, S., Habib, N., Sadanandam, A., Reebye, V., & Sodergren, M. H. (2021). Cannabinoids in the landscape of cancer. Journal of cancer research and clinical oncology, 147(9), 2507–2534. DOI: 10.1007/s00432-021-03710-7

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Comments

As a terminal liver cancer patient at the age of 31 I would try anything at this point as I have nothing else to loose. I don’t know why a significant amount of research hasn’t taken place as of yet when it should be done, even if it can help some patients and not all. It’ should be tested and given as a choice. The fact that we can not easily access it either is terrible yes it may not be a cure but it may help in some way and that’s the most important for cancer patients suffering

Thank you for most informative update. It’s a subject I’m most interested in and feel sure that the natural plant can produce some amazing results.

Until you’re a terminal cancer patient you just wont understand the desperation to live as long as possible, even if it were mere days extra time. I would try anything for extra time with loved ones.
I can’t believe there isn’t more research into cannabis and cancer. And for those that say “well it doesn’t work for everybody” guess what conventional cancer treatment doesn’t either.
Stage 4 cancer = no cure, terminal in most cases.

Why is it that charities raking in millions every year can find the evidence of cannabis for not treating cancer but cant find the overwhelming evidence that it can and does treat cancer ?

Great reading I have lung cancer I’m being treated with chemo now and would be interested in a trail it’s small cell lung cancer

Without full spectrum cannabis oil my life around a year into breast cancer I doubt I would be here now

It has enabled me to come off opioids and live a semi normal life

It sickens me to think drs happily give out meds that are killing people but won’t give out a herb that has O deaths yes Zero

I have even contacted professor Mike barns pleading with him to help me find a trial but guess what not one in the uk

The fact cannabis is illegal in this county is all political and NOTHING to do with our health

It’s about time charity’s like yours start campaigning for us, most of us medical cannabis users are spending far to much on it in order to feel well I echo what another commenter said that all stage 4 should be at least offered cannabis as an alternative

Also why can’t the hospital doctors give medical cannabis too relieve sickness and pain of cancer it’s cruel

I think that medical cannabis should be given too all stage 4 cancer patients that are told it’s aggressive and treatment wont help under medical care it could be done safely then with trial an error they will know if it works legalise cannabis for the sick wake up Boris

Thank you for sharing this amazing blog. It is easy to learn and understand. It’s a truly useful blog.

“Why don’t you campaign for cannabis to be legalised?” Your answer was ridiculous that’s all you said was that it’s illegal to possess or buy or what ever I think the question was why won’t you campaign to have it legal so then it can be tested more . Don’t beat around the bush ( No pun intended) just say it’s not worth the effort for the money you would have to spend .

this blog post is very perfect and has a lot of very vital info, thanks so much for this work

We’ve recently seen stories in the press claiming that the US government has “admitted that cannabis kills cancer” (for example, this one in the Metro), based on the observation that pages on the US National Cancer Institute information website carry details of the current scientific evidence around the effects of cannabis and cannabinoids on cancer cells in the lab and animal models.

The first thing to point out is that the NCI’s cancer information website is an independent resource for doctors and the patients, and is not a statement of NIH, NCI or US government policy.

Furthermore, the information on these pages isn’t new, nor is it an ‘admission’ of any kind: the scientific evidence about cannabis, cannabinoids and cancer, which these media stories are referring to, has been openly published on the NCI’s website for several years – for example, see this page from the same section of the NIH website on cannabis and cannabinoids from 2011, accessed via the internet archive.

We often see websites with long lists of scientific papers claiming that cannabis is a “cure” for various cancers. However, when we look at the detail of the data and the experimental detail of the research, it becomes clear that although they may be interesting and build evidence to show that cannabinoids may one day bring benefits for cancer patients, they are far from being a cure.

The main point to realise is that virtually all these studies have been done in cancer cells grown in the lab or in animals. These are quite artificial systems and are much less complex than a real cancer growing in a patient.

For example, most experiments with cells grown in the lab use cancer cells that were originally taken from a tumour many years ago, but have been grown for a long time in the lab – known as cell lines. One problem with such cells is that they are all very similar on a genetic and molecular level, but we know that in real cancers, the cells can be very different from each other and respond in different ways to treatments. Also the usual way of testing cannabinoids in animals has been done by transplanting cancer cells (either mouse or human) into mice. Usually only a small number (5-20) will be used for each experiment.

There’s growing evidence that these particular kinds of models (known as xenografts) aren’t as good at suggesting a treatment could work, compared to more sophisticated genetically engineered animals, as they don’t accurately represent the situation in real tumours. So although these kinds of experiments can point towards useful approaches, as well as revealing the underlying molecular ‘nuts and bolts’ of what’s going on, they can’t tell us if something will definitely treat cancer effectively and safely in human patients. They do not “prove that cannabis cures cancer”, as the headlines would have us believe.

Put simply, Petri dishes are not people. Most chemicals that show promise in lab or animal experiments turn out not to work as well as hoped when tested in patients. These kinds of human studies, known as clinical trials, are the only way we can really know if a cancer treatment is effective. There’s more about clinical trials on our website: http://www.cancerresearchuk.org/cancer-help/trials/types-of-trials/

It’s also important to think about what’s being claimed when people use the word “cure”. To most people, including us, this means that a cancer is completely treated and does not come back. When we look at the data in the papers listed below, none of them come close to showing these kinds of results. For the experiments involving cells grown in the lab, a proportion of the cells are killed or stop growing, but some of them carry on. Similarly in animal experiments, there is no data that shows a 100 per cent success rate for cannabinoids. For example, most mice treated with cannabinoids will still have tumours, although the cancers may be growing more slowly and spread less in some of them.

This isn’t just true for cannabinoids – it’s true for virtually all cancer drugs used today. Cancer is a very complex biological problem – there are hundreds of different types of cancer, each with important molecular and genetic differences. There’s good evidence to show that every individual’s cancer is as unique as they are, and that tumours can evolve and change within the body to become resistant to treatments.

We know that cancer drugs don’t work for everyone all the time – that’s why there’s so much effort going on to find more effective treatments – but it’s vital that doctors have a solid body of evidence showing how well the treatments they’re using are likely to work. If you or someone you loved were going to take any kind of drug, would you be happy if it had only been tested in very high doses on cancer cell lines grown in the lab, or in mice injected with cancer cells? Or would you want to know that it had been trialled in large numbers of people, and there was good data on how effective it is, whether it’s safe in the dose given, what the side effects are, and the proportion of people that can be expected to get better?

This kind of evidence can only come from a combination of lab studies leading to clinical trials. At the moment, while there are hundreds of interesting lab studies of cannabinoids (just some of which are included in the list below) there is only one clinical trial that has been published. So for now, cannabinoids, whether natural or synthetic, are a very long way from being what we would describe as a “cure” for any type of cancer.

We’ve looked at each of the papers in one of the commonly-seen lists (for example, here), and noted down the kinds of experiments they are. Many of them are available as open access papers, so it’s possible to look at the data for yourself. Hopefully this is a useful explanation of the kind of scientific research that is currently ongoing into cannabinoids and cancer, and the process of gathering evidence to show whether a potential cancer therapy works.