Cell death induction and intracellular vesicle formation in human colorectal cancer cells treated with Δ9-Tetrahydrocannabinol

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“Background: Δ9-Tetrahydrocannabinol (Δ9-THC) is a principal psychoactive extract of Cannabis sativa and has been traditionally used as palliative medicine for neuropathic pain. Cannabidiol (CBD), an extract of hemp species, has recently attracted increased attention as a cancer treatment, but Δ9-THC is also requiring explored pharmacological application.

Objective: This study evaluated the pharmacological effects of Δ9-THC in two human colorectal cancer cell lines. We investigated whether Δ9-THC treatment induces cell death in human colorectal cancer cells.

Methods: We performed an MTT assay to determine the pharmacological concentration of Δ9-THC. Annxein V and Western blot analysis confirmed that Δ9-THC induced apoptosis in colorectal cancer cells. Metabolic activity was evaluated using MitoTracker staining and ATP determination. We investigated vesicle formation by Δ9-THC treatment using GW9662, known as a PPARγ inhibitor.

Results: The MTT assay showed that treatment with 40 μM Δ9-THC and above inhibited the proliferation of colorectal cancer cells. Multiple intracytoplasmic vesicles were detected upon microscopic observation, and fluorescence-activated cell sorting analysis showed cell death via G1 arrest. Δ9-THC treatment increased the expression of cell death marker proteins, including p53, cleaved PARP-1, RIP1, and RIP3, suggesting that Δ9-THC induced the death of colorectal cancer cells. Δ9-THC treatment also reduced ATP production via changes in Bax and Bcl-2. Δ9-THC regulated intracytoplasmic vesicle formation by modulating the expression of PPARγ and clathrin, adding that antiproliferative activity of Δ9-THC was also affected.

Conclusion: In conclusion, Δ9-THC regulated two functional mechanisms, intracellular vesicle formation and cell death. These findings can help to determine how cannabinoids can be used most effectively to improve the efficacy of cancer treatment.”



Identification of phenolic compounds from inflorescences of non-psychoactive Cannabis sativa L. by UHPLC-HRMS and in vitro assessment of the antiproliferative activity against colorectal cancer

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“Phenolic compounds from Cannabis sativa L. (Cannabaceae family), in particular cannflavins, are known to possess several biological properties. However, their antiproliferative activity, being of great interest from a medicinal chemistry point of view, has not been deeply investigated so far in the literature. In the light of this, the aim of this study was to obtain an enriched fraction of polyphenols (namely PEF) from inflorescences of a non-psychoactive C. sativa (hemp) variety and to evaluate its antiproliferative activity against cancer cells, capitalizing on a new and selective extraction method for hemp polyphenols, followed by preparative flash column chromatography. Untargeted metabolomics, using a new method based on ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS), was applied here for the first time to fully characterize PEF. Then, the main phenolic compounds were quantified by HPLC-UV. The antiproliferative activity of PEF and of the isolated compounds was assessed in vitro for the first time against Caco-2 and SW480 human colon adenocarcinoma cell lines providing promising IC50 values, in comparison with the reference drug used in therapy for this cancer type. Based on these results, PEF can be considered as a new highly potential therapeutic product to be further investigated against colorectal cancer, thanks to the possible synergistic interaction of its compounds.”



Single-cell analyses reveal cannabidiol rewires tumor microenvironment via inhibiting alternative activation of macrophage and synergizes with anti-PD-1 in colon cancer

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“Colorectal tumors often create an immunosuppressive microenvironment that prevents them from responding to immunotherapy.

Cannabidiol (CBD) is a non-psychoactive natural active ingredient from the cannabis plant that has various pharmacological effects, including neuroprotective, antiemetic, anti-inflammatory, and antineoplastic activities.

This study aimed to elucidate the specific anticancer mechanism of CBD by single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) technologies.

Here, we report that CBD inhibits colorectal cancer progression by modulating the suppressive tumor microenvironment (TME).

Our single-cell transcriptome and ATAC sequencing results showed that CBD suppressed M2-like macrophages and promoted M1-like macrophages in tumors both in strength and quantity. Furthermore, CBD significantly enhanced the interaction between M1-like macrophages and tumor cells and restored the intrinsic anti-tumor properties of macrophages, thereby preventing tumor progression. Mechanistically, CBD altered the metabolic pattern of macrophages and related anti-tumor signaling pathways.

We found that CBD inhibited the alternative activation of macrophages and shifted the metabolic process from oxidative phosphorylation and fatty acid oxidation to glycolysis by inhibiting the phosphatidylinositol 3-kinase-protein kinase B signaling pathway and related downstream target genes. Furthermore, CBD-mediated macrophage plasticity enhanced the response to anti-programmed cell death protein-1 (PD-1) immunotherapy in xenografted mice.

Taken together, we provide new insights into the anti-tumor effects of CBD.”


“CBD shapes the TME to prevent tumor progression.”


Cannabidiol-induced crosstalk of apoptosis and macroautophagy in colorectal cancer cells involves p53 and Hsp70

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“Although it has been established that cannabidiol (CBD), the major non-psychoactive constituent of cannabis, exerts antitumoral activities, the exact mechanism(s) via which tumor cells are killed by CBD are not well understood.

This study provides new insights into the potential mechanisms of CBD-induced mutual antagonism of apoptosis and macroautophagy using wild type (HCT116 p53wt, LS174T p53wt), knockout (HCT116 p53-/-) and mutant (SW480 p53mut) human colorectal cancer cells (CRC).

CBD causes a more pronounced loss in the viability of p53wt cells than p53-/- and p53mut cells, and a 5-week treatment with CBD reduced the volume of HCT116 p53wt xenografts in mice, but had no effect on the volume of HCT116 p53-/- tumors.

Mechanistically, we demonstrate that CBD only significantly elevates ROS production in cells harboring wild-type p53 (HCT116, LS174T) and that this is associated with an accumulation of PARP1. CBD-induced elevated ROS levels trigger G0/G1 cell cycle arrest, a reduction in CDK2, a p53-dependent caspase-8/9/3 activation and macroautophagy in p53wt cells. The ROS-induced macroautophagy which promotes the activation of keap1/Nrf2 pathway might be positively regulated by p53wt, since inhibition of p53 by pifithrin-α further attenuates autophagy after CBD treatment.

Interestingly, an inhibition of heat shock protein 70 (Hsp70) expression significantly enhances caspase-3 mediated programmed cell death in p53wt cells, whereas autophagy-which is associated with a nuclear translocation of Nrf2-was blocked.

Taken together, our results demonstrate an intricate interplay between apoptosis and macroautophagy in CBD-treated colorectal cancer cells, which is regulated by the complex interactions of p53wt and Hsp70.”



Antitumor Effects of Cannabis sativa Bioactive Compounds on Colorectal Carcinogenesis

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“Cannabis sativa is a multipurpose plant that has been used in medicine for centuries. Recently, considerable research has focused on the bioactive compounds of this plant, particularly cannabinoids and terpenes. Among other properties, these compounds exhibit antitumor effects in several cancer types, including colorectal cancer (CRC). Cannabinoids show positive effects in the treatment of CRC by inducing apoptosis, proliferation, metastasis, inflammation, angiogenesis, oxidative stress, and autophagy. Terpenes, such as β-caryophyllene, limonene, and myrcene, have also been reported to have potential antitumor effects on CRC through the induction of apoptosis, the inhibition of cell proliferation, and angiogenesis. In addition, synergy effects between cannabinoids and terpenes are believed to be important factors in the treatment of CRC. This review focuses on the current knowledge about the potential of cannabinoids and terpenoids from C. sativa to serve as bioactive agents for the treatment of CRC while evidencing the need for further research to fully elucidate the mechanisms of action and the safety of these compounds.”


“Data suggest that cannabinoids exert advantages in the treatment of CRC, mostly by inducing apoptosis, although some evidence also points out that they may target other key therapeutic events, such as proliferation, metastasis, inflammation, angiogenesis, oxidative stress, and autophagy. The currently available data on this subject refer mostly to the C. sativa major cannabinoids, i.e., CBD, THC, and CBG, but several pieces of evidence suggest that minor cannabinoids and other bioactive compounds such as terpenes also may hold potential as therapeutic agents for CRC. Data also suggest that certain combinations of cannabinoids and terpenes in C. sativa extracts can lead to a synergistic action known as the “entourage effect,” which has been linked to certain pharmacological benefits. The potential therapeutic benefits of the cannabinoids and terpenes from this plant make them key candidates for further drug development.”


Cannabinoid compounds in combination with curcumin and piperine display an anti-tumorigenic effect against colon cancer cells

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“Currently, use of cannabinoids is limited to improve adverse effects of chemotherapy and their palliative administration during treatment is curiously concomitant with improved prognosis and regressed progression in patients with different tumor types. Although, non-psychoactive cannabidiol (CBD) and cannabigerol (CBG) display antineoplastic effects by repressing tumor growth and angiogenesis both in cell line and animal models, their use as chemotherapeutic agents is awaiting further investigation. Both clinical and epidemiological evidence supported by experimental findings suggest that micronutrients such as curcumin and piperine may present a safer strategy in preventing tumorigenesis and its recurrence. Recent studies demonstrated that piperine potentiates curcumin’s inhibitory effect on tumor progression via enhancing its delivery and therapeutic activity. In this study, we investigated a plausible therapeutic synergism of a triple combination of CBD/CBG, curcumin, and piperine in the colon adenocarcinoma using HCT116 and HT29 cell lines. Potential synergistic effects of various combinations including these compounds were tested by measuring cancer cell proliferation and apoptosis. Our findings revealed that different genetic backgrounds of HCT116 and HT29 cell lines resulted in divergent responses to the combination treatments. Triple treatment showed synergism in terms of exhibiting anti-tumorigenic effects by activating the Hippo YAP signaling pathway in the HCT116 cell line.”


“This study demonstrates that combination of curcumin, piperine and cannabinoid variants inhibit cell proliferation and induce apoptosis drastically in distinct models of colorectal cancer. Intriguingly, our findings point out that the compounds of interest, each of which are already known for their anti-tumorigenic and preventive role in colon cancer as single agents, displayed an augmented therapeutic effect in the cell lines tested. In the HT29 cell line, CBG significantly reduced cell proliferation and induced apoptosis as a monotherapy agent, whereas these anti-tumorigenic effects were overridden in the presence of curcumin/piperine. Therefore, findings from this study suggest a benefit in using cannabinoid compounds as single anti-cancer agents in the treatment of those colon carcinoma tumors that carry a genetic profile similar to that of the HT29 cell line. One major limitation of the current study was to reconcile these findings with the cannabinoid receptor 1 (CB1 receptor) and cannabinoid receptor 2 (CB2 receptor) expression profile of the cell lines used. Therefore, in future studies the link between the anti-tumorigenic effects of single cannabinoid compounds or their cocktails and the cannabinoid receptor expression should be interrogated to shed light on the differences in the responses of these cells to distinct cannabinoid-based regimens. In addition to the cannabinoid receptor status, role of other mutations in driver genes should be subject to more rigorous mechanistic studies to fully understand their role in determining the drug mechanism of action and the response to distinct treatment schemes involving cannabinoids as single agents their various combinations.”


Therapeutic targeting of the tumor microenvironments with cannabinoids and their analogs: Update on clinical trials

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“Cancer is a major global public health concern that affects both industrialized and developing nations. Current cancer chemotherapeutic options are limited by side effects, but plant-derived alternatives and their derivatives offer the possibilities of enhanced treatment response and reduced side effects.

A plethora of recently published articles have focused on treatments based on cannabinoids and cannabinoid analogs and reported that they positively affect healthy cell growth and reverse cancer-related abnormalities by targeting aberrant tumor microenvironments (TMEs), lowering tumorigenesis, preventing metastasis, and/or boosting the effectiveness of chemotherapy and radiotherapy.

Furthermore, TME modulating systems are receiving much interest in the cancer immunotherapy field because it has been shown that TMEs have significant impacts on tumor progression, angiogenesis, invasion, migration, epithelial to mesenchymal transition, metastasis and development of drug resistance.

Here, we have reviewed the effective role of cannabinoids, their analogs and cannabinoid nano formulations on the cellular components of TME (endothelial cells, pericytes, fibroblast and immune cells) and how efficiently it retards the progression of carcinogenesis is discussed. The article summarizes the existing research on the molecular mechanisms of cannabinoids regulation of the TME and finally highlights the human studies on cannabinoids’ active interventional clinical trials.

The conclusion outlines the need for future research involving clinical trials of cannabinoids to demonstrate their efficacy and activity as a treatment/prevention for various types of human malignancies.”



The protective effect of cannabinoids against colorectal cancer cachexia through modulation of inflammation and immune responses

Biomedicine & Pharmacotherapy

“Cancer cachexia is a multifactorial disorder characterized by weight loss and muscle wasting, and there are currently no FDA-approved medications. In the present study, upregulation of six cytokines was observed in serum samples from patients with colorectal cancer (CRC) and in mouse models. A negative correlation between the levels of the six cytokines and body mass index in CRC patients was seen. Gene Ontology analysis revealed that these cytokines were involved in regulating T cell proliferation. The infiltration of CD8+ T cells was found to be associated with muscle atrophy in mice with CRC. Adoptive transfer of CD8+ T cells isolated from CRC mice resulted in muscle wasting in recipients.

The Genotype-Tissue Expression database showed that negative correlations between the expression of cachexia markers and cannabinoid receptor 2 (CB2) in human skeletal muscle tissues. Pharmacological treatment with Δ9-tetrahydrocannabinol (Δ9-THC), a selective CB2 agonist or overexpression of CB2 attenuated CRC-associated muscle atrophy. In contrast, knockout of CB2 with a CRISPR/Cas9-based strategy or depletion of CD8+ T cells in CRC mice abolished the Δ9-THC-mediated effects.

This study demonstrates that cannabinoids ameliorate CD8+ T cell infiltration in CRC-associated skeletal muscle atrophy via a CB2-mediated pathway. Serum levels of the six-cytokine signature might serve as a potential biomarker to detect the therapeutic effects of cannabinoids in CRC-associated cachexia.”


“In recent years, researchers have gradually found that marijuana, in addition to recreational use, has potential applications as a supportive therapy or palliative medicine.

In conclusion, our findings indicate that the infiltration of CD8+ T cells in skeletal muscle plays a vital role in CRC-associated muscle atrophy. Treatment with Δ9-THC or CB65 can ameliorate CRC-associated cachexia and muscle atrophy by activating CB2 in CD8+ T cells. Targeting the CB2 receptor in CD8+ T cells should be evaluated as a therapeutic option for CRC patients who develop cachexia, and the six-cytokine signature in serum might serve as a potential biomarker for the therapeutic effects of cannabinoids in CRC-associated cachexia.”


The Anti-Tumorigenic Role of Cannabinoid Receptor 2 in Colon Cancer: A Study in Mice and Humans


“The endocannabinoid system, particularly cannabinoid receptor 2 (CB2 in mice and CNR2 in humans), has controversial pathophysiological implications in colon cancer.

Here, we investigate the role of CB2 in potentiating the immune response in colon cancer in mice and determine the influence of CNR2 variants in humans. Comparing wild-type (WT) mice to CB2 knockout (CB2-/-) mice, we performed a spontaneous cancer study in aging mice and subsequently used the AOM/DSS model of colitis-associated colorectal cancer and a model for hereditary colon cancer (ApcMin/+). Additionally, we analyzed genomic data in a large human population to determine the relationship between CNR2 variants and colon cancer incidence.

Aging CB2-/- mice exhibited a higher incidence of spontaneous precancerous lesions in the colon compared to WT controls. The AOM/DSS-treated CB2-/- and ApcMin/+CB2-/- mice experienced aggravated tumorigenesis and enhanced splenic populations of immunosuppressive myeloid-derived suppressor cells along with abated anti-tumor CD8+ T cells. Importantly, corroborative genomic data reveal a significant association between non-synonymous variants of CNR2 and the incidence of colon cancer in humans.

Taken together, the results suggest that endogenous CB2 activation suppresses colon tumorigenesis by shifting the balance towards anti-tumor immune cells in mice and thus portray the prognostic value of CNR2 variants for colon cancer patients.”



A zebrafish HCT116 xenograft model to predict anandamide outcomes on colorectal cancer

Cell Death & Disease

“Colon cancer is one of the leading causes of death worldwide. In recent years, cannabinoids have been extensively studied for their potential anticancer effects and symptom management. Several in vitro studies reported anandamide’s (AEA) ability to block cancer cell proliferation and migration, but evidence from in vivo studies is still lacking. Thus, in this study, the effects of AEA exposure in zebrafish embryos transplanted with HCT116 cells were evaluated.

Totally, 48 hpf xenografts were exposed to 10 nM AEA, 10 nM AM251, one of the cannabinoid 1 receptor (CB1) antagonist/inverse agonists, and to AEA + AM251, to verify the specific effect of AEA treatment. AEA efficacy was evaluated by confocal microscopy, which demonstrated that these xenografts presented a smaller tumor size, reduced tumor angiogenesis, and lacked micrometastasis formation.

To gain deeper evidence into AEA action, microscopic observations were completed by molecular analyses. RNA seq performed on zebrafish transcriptome reported the downregulation of genes involved in cell proliferation, angiogenesis, and the immune system. Conversely, HCT116 cell transcripts resulted not affected by AEA treatment. In vitro HCT116 culture, in fact, confirmed that AEA exposure did not affect cell proliferation and viability, thus suggesting that the reduced tumor size mainly depends on direct effects on the fish rather than on the transplanted cancer cells.

AEA reduced cell proliferation and tumor angiogenesis, as suggested by socs3 and pcnp mRNAs and Vegfc protein levels, and exerted anti-inflammatory activity, as indicated by the reduction of il-11a, mhc1uba, and csf3b mRNA. Of note, are the results obtained in groups exposed to AM251, which presence nullifies AEA’s beneficial effects.

In conclusion, this study promotes the efficacy of AEA in personalized cancer therapy, as suggested by its ability to drive tumor growth and metastasis, and strongly supports the use of zebrafish xenograft as an emerging model platform for cancer studies.”


“Collectively, our data suggest a pivotal role of AEA in the anti-angiogenic, anti-proliferative, and anti-inflammatory process in intercellular tumor-endothelial cell communication resulting in the containment of tumor and evidenced that zebrafish larvae xenografts constitute a promising fast assay for precision medicine, bridging the gap between genotype and phenotype in an in vivo setting.”