Discovery of Ring-Annulated Analogues of Cannabidiol as Potential Anticancer Agents: Synthesis and Biological Evaluation

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“Cannabidiol (CBD) is a nonpsychoactive cannabinoid derived from Cannabis sativa and its potential therapeutic effects extend beyond its well-known antiepileptic properties. Exploring CBD and its analogues as anticancer agents has gained significant attention in recent years.

In this study, a series of novel ring-annulated analogues of CBD with oxazinyl moiety were synthesized and evaluated for their antiproliferative effect.

The analogues 4d and 4h demonstrate promising activity against breast and colorectal cancer. Furthermore, mechanistic insights revealed that the identified candidates arrest the G1 phase of the cell cycle and induce apoptosis via the mitochondrial pathway in breast cancer cell lines.

Notably, CBD ring-annulated analogues 4d or 4h exhibit enhanced solubility, better metabolic stability, and lowered cytochrome P450 (CYP) inhibition liability compared to CBD.

These multifaceted attributes highlight the potential of cannabinoid-based candidates for further preclinical development.”

https://pubmed.ncbi.nlm.nih.gov/39563806/

https://pubs.acs.org/doi/10.1021/acsmedchemlett.4c00233

Combinatorial effects of cannabinoid receptor 1 and 2 agonists on characteristics and proteomic alteration in MDA-MB-231 breast cancer cells

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“Breast cancer is the most common cancer diagnosed in women worldwide. However, the effective treatment for breast cancer progression is still being sought.

The activation of cannabinoid receptor (CB) has been shown to negatively affect breast cancer cell survival.

Our previous study also reported that breast cancer cells responded to various combinations of CB1 and CB2 agonists differently. Nonetheless, the mechanism underlying this effect and whether this phenomenon can be seen in other cancer characteristics remain unknown. Therefore, this study aims to further elucidate the effects of highly selective CB agonists and their combination on triple-negative breast cancer proliferation, cell cycle progression, invasion, lamellipodia formation as well as proteomic profile of MDA-MB-231 breast cancer cells.

The presence of CB agonists, specifically a 2:1 (ACEA: GW405833) combination, prominently inhibited colony formation and induced the S-phase cell cycle arrest in MDA-MB-231 cells. Furthermore, cell invasion ability and lamellipodia formation of MDA-MB-231 were also attenuated by the exposure of CB agonists and their 2:1 combination ratio. Our proteomic analysis revealed proteomic profile alteration in MDA-MB-231 upon CB exposure that potentially led to breast cancer suppression, such as ZPR1/SHC1/MAPK-mediated cell proliferation and AXL/VAV2/RAC1-mediated cell motility pathways.

Our findings showed that selective CB agonists and their combination suppressed breast cancer characteristics in MDA-MB-231 cells. The exposure of CB agonists also altered the proteomic profile of MDA-MB-231, which could lead to cell proliferation and motility suppression.”

https://pubmed.ncbi.nlm.nih.gov/39527598/

“Our study demonstrated that the presence of CB agonists hindered breast cancer cell growth, cell cycle progression, invasion through extracellular matrices and lamellipodia formation. The exposure of specific combination of CB1 and CB2 agonists also enhanced their breast cancer suppression effects. Moreover, breast cancer survival and motility-related proteins affected by the presence of these agonists suggesting the potential pathways underlying their effects were also depicted in this study.”

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0312851

Improved Therapeutic Efficacy of Doxorubicin Chemotherapy With Cannabidiol in 4T1 Mice Breast Cancer Model

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“Background: High dose chemotherapy is one of the therapeutic strategies for breast cancer and doxorubicin (DOX) as a chemotherapy agent is widely used. DOX indication is limited due to its dose-depended cardiotoxicity. Recently, cannabidiol (CBD) shows antitumoral and cardioprotective effects, so we hypothesized that CBD administration with high-dose DOX chemotherapy can improve anticancer activity and reduce cardiotoxic side effects.

Method: Mice breast cancer model established by injecting 4T1 cell lines. One group was not injected by 4T1 cells as a not cancerous group and received normal saline (NS, 0.1 mL). In cancerous groups, first group was considered as cancerous control and received NS (0.1 mL); the second group received CBD (5 mg/kg, IP) on Days 1,7, and 14; in the third group DOX (5 mg/kg, IV) as CBD schedule was administrated; the fourth group treated with CBD 1 day before DOX injection as pretreatment, and the last group was treated with CBD and DOX at same time with previous doses and schedules. On Day 21, all mice were sacrificed, heart and lungs tissues were obtained and histological sections were isolated. SOD2, iNOS, MMP2, MMP9 were evaluated through western blot and TUNEL test preformed for breast tumor.

Results: Tumor size and weight significantly decreased in DOX, pretreatment CBD + DOX and CBD + DOX groups. Administration of CBD with DOX could not prevent weight loss. TUNEL test demonstrated the highest tumor cell apoptosis in pretreatment CBD + DOX and CBD + DOX. In lungs belonged to CBD + DOX, there was not any sign of metastasis. Cardiac histopathological examination of pretreatment CBD + DOX and CBD + DOX did not show any sign of congestion or inflammation. In CBD + DOX SOD2 increased, also iNOS, MMP2, and MMP9 decreased compared to DOX.

Conclusions: This study demonstrated that simultaneous administration of CBD and DOX can increase antitumoral effect and reduce DOX cardiotoxicity. Nevertheless, CBD can induce cardiotoxicity as administrated alone.”

https://pubmed.ncbi.nlm.nih.gov/39503169/

“This study demonstrated the potent efficacy of cannabidiol in mouse breast cancer model with high-dose chemotherapy on the antitumor, anti-metastasis and cardioprotective roles against doxorubicin. Simultaneous administration of cannabidiol with high-dose doxorubicin not only improved the antitumor and anti-metastasis efficacy, but also could reduce cardiotoxicity by decreasing MMP2 and MMP9 and improving cardiac function by decreasing iNOS. Furthermore, cannabidiol could improve antioxidant system by increasing SOD2. Eventually, these findings demonstrated cannabidiol as a potential effective agent in coadministration with doxorubicin at the same time in improving anticancer effects and reducing cardiotoxicity.”

https://onlinelibrary.wiley.com/doi/10.1002/cam4.70395

The molecular anti-metastatic potential of CBD and THC from Lebanese Cannabis via apoptosis induction and alterations in autophagy

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“The medicinal plant Cannabis sativa L. (C. sativa) is currently being extensively studied to determine the full extent of its therapeutic pharmacological potential. Δ9-tetrahydocannabinol (THC) and cannabidiol (CBD) are the most thoroughly investigated compounds.

We aimed to explore the anticancer activity of cannabinoids mixture isolated from the Lebanese C. sativa plant in ratios comparable to the local medicinal plant, to elucidate its mechanism of action in breast cancer cells in vitro.

Cells were subjected to cytotoxicity assay, cell cycle analysis, Annexin V/PI dual staining, cell death ELISA, immunofluorescence, in addition to western blot analysis of apoptotic and autophagy markers. We further evaluated the anti-metastatic effect of cannabinoids on MDA-MB-231 using the scratch wound-healing, trans-well migration and invasion assays.

Our results revealed the promising therapeutic benefits of CBD/THC on inhibiting the growth of breast cancer cells by promoting cellular fragmentation, phosphatidylserine translocation to the outer membrane leaflet and DNA fragmentation in both cell lines while inhibiting the motility of the triple negative breast cancer cells.

In our study, CBD/THC mixture was found to exhibit a pro-apoptotic activity via the activation of the mitochondrial apoptotic pathway, independent from ROS production while also suggesting the activation of a caspase-dependent apoptotic pathway. Even though autophagy was altered upon exposure to the cannabinoid mixture, our data suggested that it is not the mechanism responsible of inducing cell death.

In conclusion, our study demonstrates the promising therapeutic benefits of CBD and THC isolated from the Lebanese C. sativa plant on breast cancer cells in vitro.”

https://pubmed.ncbi.nlm.nih.gov/39463375/

“Our study showed that CBD and THC isolated from the Lebanese cannabis strains, in ratios comparable to the medicinal plants, exhibit promising effect on breast cancer cell lines. The anticancer activity of this mixture was revealed by its ability to promote cellular fragmentation, phosphatidylserine translocation and DNA fragmentation while inhibiting the motility of aggressive breast cancer cells. Our results showed a pro-apoptotic activity on MDA-MB-231 and MCF-7 cells via the activation of the intrinsic apoptotic pathway. Moreover, we found that even if autophagy was altered in breast cancer cell lines, it is not the major mechanism leading to cellular death. Also, we demonstrated that this mixture was effective in halting the progression of breast cancer cells via the suppression of cancer cell migration and invasion.”

https://www.nature.com/articles/s41598-024-76340-x

Molecular Targets of Minor Cannabinoids in Breast Cancer: In Silico and In Vitro Studies

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“Background: Breast cancer therapy has been facing remarkable changes. Classic treatments are now combined with other therapies to improve efficacy and surpass resistance. Indeed, the emergence of resistance demands the development of novel therapeutic approaches. Due to key estrogen signaling, estrogen receptor-positive (ER+) breast cancer treatment has always been focused on aromatase inhibition and ER modulation. Lately, the effects of phytocannabinoids, mainly Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), have been evaluated in different cancers, including breast. However, Cannabis sativa contains more than 120 phytocannabinoids less researched and understood.

Methods: Here, we evaluated, both in silico and in vitro, the ability of 129 phytocannabinoids to modulate important molecular targets in ER+ breast cancer: aromatase, ER, and androgen receptor (AR).

Results: In silico results suggested that some cannabinoids may inhibit aromatase and act as ERα antagonists. Nine selected cannabinoids showed, in vitro, potential to act either as ER antagonists with inverse agonist properties, or as ER agonists. Moreover, these cannabinoids were considered as weak aromatase inhibitors and AR antagonists with inverse agonist action.

Conclusions: Overall, we present, for the first time, a comprehensive analysis of the actions of the phytocannabinoids in targets of ER+ breast tumors, pointing out their therapeutic potential in cancer and in other diseases.”

https://pubmed.ncbi.nlm.nih.gov/39338407/

“From the best of our knowledge, this is the first study exploring the molecular targets of minor cannabinoids and, together with previous studies, it reinforces the importance and therapeutic potential of cannabinoids in breast cancer, paving the way for novel and alternative therapeutic approaches and highlighting the medicinal potential of Cannabis.”

https://www.mdpi.com/1424-8247/17/9/1245

Cannabidiol enhances Atezolizumab efficacy by upregulating PD-L1 expression via the cGAS-STING pathway in triple-negative breast cancer cells

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“The treatment of patients with triple negative breast cancer (TNBC) relies on cytotoxic therapy. Currently, atezolizumab and chemotherapy can be combined in patients with TNBC. However, this approach is not effective for all patients with low reactivity to atezolizumab. As there is a lack of alternative treatment options, new anti-cancer drugs are urgently needed to enhance atezolizumab reactivity against TNBC. Recent strategies have focused on regulating the expression of programmed death-ligand 1 (PD-L1) or enhancing immune response activation by combining anti-cancer drugs with immune checkpoint inhibitors (ICIs).

Cannabidiol (CBD), a cannabinoid component derived from the cannabis plant, has been reported to have anti-cancer therapeutic potential because of its capacity to induce apoptotic cell death in tumor cells while avoiding cytotoxicity in normal cells.

Previous studies have demonstrated the effects of CBD on apoptosis in various cancer cell types. However, the potential role of CBD as an immune modulator in the regulation of PD-L1 expression and anti-cancer immune responses remains to be explored.

In this study, we found that CBD stimulated PD-L1 expression in TNBC cells, which significantly induced the CBD-mediated cGAS-STING pathway activation. Taken together, we demonstrated that the combination of CBD and anti-PD-L1 antibody enhances the anti-cancer immune response in vitro and in vivo experiments.

Our findings identified the mechanism of PD-L1 regulation by CBD in TNBC cells and suggested that CBD could be a potential candidate for the development of new combinatorial strategies with ICIs in TNBC patients.”

https://pubmed.ncbi.nlm.nih.gov/39226389/

https://aacrjournals.org/cancerimmunolres/article/doi/10.1158/2326-6066.CIR-23-0902/747763/Cannabidiol-enhances-Atezolizumab-efficacy-by

Cannabinoids and triple-negative breast cancer treatment

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“Triple-negative breast cancer (TNBC) accounts for about 10-20% of all breast cancer cases and is associated with an unfavorable prognosis. Until recently, treatment options for TNBC were limited to chemotherapy. A new successful systemic treatment is immunotherapy with immune checkpoint inhibitors, but new tumor-specific biomarkers are needed to improve patient outcomes.

Cannabinoids show antitumor activity in most preclinical studies in TNBC models and do not appear to have adverse effects on chemotherapy.

Clinical data are needed to evaluate efficacy and safety in humans. Importantly, the endocannabinoid system is linked to the immune system and immunosuppression. Therefore, cannabinoid receptors could be a potential biomarker for immune checkpoint inhibitor therapy or a novel mechanism to reverse resistance to immunotherapy. In this article, we provide an overview of the currently available information on how cannabinoids may influence standard therapy in TNBC.”

https://pubmed.ncbi.nlm.nih.gov/39176080/

“Selective CB2R agonists and antagonists are needed to develop potential anti-cancer drugs that target the endocannabinoid system,”

https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1386548/full

Identification of Genes Hub Associated with Triple-Negative Breast Cancer and Cannabidiol Analogs Potential Inhibitory Agents: An In-silico Study

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“Objective: Triple-negative breast cancer presents a significant challenge in oncology due to its complex treatment and aggressive nature. This subtype lacks common cancer cell receptors like estrogen, progesterone, and human epidermal growth factor receptor 2 receptors. This study aimed to identify, through bioinformatic analysis, the key genes associated with triple-negative breast cancer. In addition, CBD analogs with potential inhibitory effects on these genes were evaluated through docking and molecular dynamics.

Methods: Gene expression profiles from the GSE178748 dataset were analyzed, focusing on MDA-MB-231 breast cancer cell lines. Differentially expressed genes were determined through protein-protein interaction networks and subsequently validated. Additionally, the inhibitory effects of cannabidiol analogs on these hub genes were assessed using molecular docking and dynamics.

Results: Analysis of the hub highlighted RPL7A, NHP2L1, and PSMD11 as significant players in TNBC regulation. Ligand 44409296 showed the best affinity energy with RPL7A, while 166505341 exhibited the highest affinity with NHP2L1 and PSMD11, surpassing CBD. Analyses of RMSD, RMSF, SASA, and Gyration Radius indicated structural stability and interactions of the proteins with ligands over time. MMGBSA calculations showed favorable binding energies for the ligands with the target proteins.

Conclusion: In conclusion, this study identified key genes, namely RPL7A, NHP2L1, and PSMD11, associated with triple-negative breast cancer and demonstrated promising interactions with cannabidiol analogs, particularly 44409296 and 166505341. These findings suggest potential therapeutic targets and highlight the relevance of further clinical investigations. Additionally, the ligands exhibited favorable ADME properties and low toxicity, underscoring their potential in future drug development for TNBC treatment.”

https://pubmed.ncbi.nlm.nih.gov/38809637/

https://journal.waocp.org/article_91159.html

Cannabis and cancer: unveiling the potential of a green ally in breast, colorectal, and prostate cancer

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“Cancer comes in second place on the list of causes of death worldwide. In 2018, the 5-year prevalence of breast cancer (BC), prostate cancer (PC), and colorectal cancer (CRC) were 30%, 12.3%, and 10.9%, respectively.

Cannabinoids are chemicals derived from the Cannabis sativa plant; the most investigated cannabinoids are cannabinol, delta 9-tetrahydrocannabinol (Δ9-THC), and cannabidiol. In humans, the endogenous endocannabinoid system consists of endocannabinoids, cannabinoids receptors (CBs), and enzymes that degrade the endocannabinoids.

In this review, we will review the most recent literature for evidence that discusses the role of cannabis in the treatment of the three types of neoplasms mentioned.

Studies have proved that BC cells express CB receptors; many in-vivo studies showed that cannabinoids cause apoptosis and inhibit proliferation and migration. Also, researchers found that treating BC mice with THC and JWH-133 (CB2 receptor agonist) slowed the tumor growth.

Regarding CRC, cannabidiol was found to decrease the viability of chemotherapy-resistant CRC cells and inhibit metastasis by antagonizing the G-protein-coupled receptor 55 (GPR55; a novel cannabinoid receptor) necessary for metastasis. Moreover, cannabidiol had anti-angiogenetic effects by reducing the expression of vascular endothelial growth factor (VEGF) in addition to anti-inflammatory effects.

Finally, studies demonstrated that PC cells highly express CB1 and CB2 receptors and that cannabinoids are capable of inhibiting the release of exosomes and microvesicles related to cancer progression. Cannabinoids also have antiproliferative, anti-invasive, anti-fibroblastic, cell cycle arrest, and proapoptotic effects on PC cells.”

https://pubmed.ncbi.nlm.nih.gov/38755733/

“There is growing evidence supporting the role of Cannabinoids in numerous pathological conditions, including their role in several cancer types such as breast, colorectal, and prostate cancer. Accordingly, cannabinoids could have a promising potential as adjunctive therapy for the treatment of these types of cancers.”

https://jcannabisresearch.biomedcentral.com/articles/10.1186/s42238-024-00233-z

Mitochondrial calcium overload contributes to cannabinoid-induced paraptosis in hormone-responsive breast cancer cells

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“Studies have shown that natural products can induce paraptosis in tumour cell lines. Paraptosis is characterized by cytoplasmic vacuolation arising from the endoplasmic reticulum (ER) and mitochondria. The mechanism of paraptosis is unclear; however, dysregulation of Ca2+ homeostasis is believed to affect paraptosis induction.

This study investigated the mechanism of cell death induced by a phytocannabinoid ratio in the MCF7 breast cancer cell line.

The crystal violet assay was used to detect changes in viability and morphology changes were investigated using light and transmission electron microscopy. Various inhibitors, fluorescent staining with high-content screening, and Western blot analysis were used to investigate different cell death mechanisms.

The phytocannabinoid ratio induced significant cell death and cytoplasmic vacuolation in MCF7 cells; however, no apoptosis, necrosis, autophagy, or ferroptosis was detected. Vacuolation induced by phytocannabinoid treatment was inhibited by cycloheximide, suggesting paraptosis induction. The mechanism of paraptosis induction was investigated, and it was found that treatment (1) induced ER dilation and mitochondrial swelling, (2) induced significant ER stress and mitochondrial Ca2+ overload and dysfunction, which appeared to be mediated by the voltage-dependent anion channel, and (3) significantly impaired all mitochondrial metabolic pathways.

The data demonstrated that paraptosis induced by the cannabinoid ratio was mediated by Ca2+ flux from the ER to the mitochondria.

These findings highlight a novel mechanism of cannabinoid-induced cell death and emphasize the anti-cancer potential of cannabinoid ratios, which exhibited enhanced effects compared to individual cannabinoids.”

https://pubmed.ncbi.nlm.nih.gov/38721827/

” In the context of cancer, cannabinoids have primarily been used for their palliative effects to treat the side effects of chemotherapy; however, their tumour-suppressive properties have been known since the first observation of their antitumor effects in 1975.”

https://onlinelibrary.wiley.com/doi/10.1111/cpr.13650