Gene Profiling of Cannabis-sativa-mediated Apoptosis in Human Melanoma Cells

Anticancer Research: 43 (3)

“Background/aim: Malignant melanoma is an aggressive skin cancer, accounting for the majority of skin cancer deaths. Prognosis is often poor and finding effective treatment remains a challenge. Tetrahydrocannabinol (THC) and cannabidiol (CBD) are main bioactive components of Cannabis sativa plant extracts that have been shown to exert anti-tumor effects. In this study, we aimed to perform gene expression analysis of human melanoma A375 cells following stimulation with C. sativa extracts.

Materials and methods: Gene expression profiles of A375 human melanoma and Vero (control) cell lines were evaluated by RNA sequencing and quantitative real-time PCR.

Results: Flow cytometry showed that the THC+CBD cannabis fractions induced apoptosis on A375 cells. Induction of apoptosis was accompanied by a notable up-regulation of DNA damage inducible transcript 3 (DDIT), nerve growth factor receptor (NGFR), colony-stimulating factor 2 (CSF2), growth arrest and DNA damage inducible beta (GADD45B), and thymic stromal lymphopoietin (TSLP) genes and down-regulation of aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), cyclin E2 (CCNE2), integrin subunit alpha 9 (ITGA9), proliferating cell nuclear antigen (PCNA) and E2F transcription factor 1 (E2F1) genes. Treatment of A375 cells with the THC+CBD fraction inhibited the phosphorylation of ERK1/2 signaling pathway, which regulates melanoma cell proliferation. We showed that the THC+CBD combination disrupted melanoma cell migration.

Conclusion: Use of C. sativa-derived extracts containing equal amounts of THC and CBD is proposed as a potential treatment of melanoma.”

Rare Phytocannabinoids Exert Anti-Inflammatory Effects on Human Keratinocytes via the Endocannabinoid System and MAPK Signaling Pathway


“Increasing evidence supports the therapeutic potential of rare cannabis-derived phytocannabinoids (pCBs) in skin disorders such as atopic dermatitis, psoriasis, pruritus, and acne. However, the molecular mechanisms of the biological action of these pCBs remain poorly investigated.

In this study, an experimental model of inflamed human keratinocytes (HaCaT cells) was set up by using lipopolysaccharide (LPS) in order to investigate the anti-inflammatory effects of the rare pCBs cannabigerol (CBG), cannabichromene (CBC), Δ9-tetrahydrocannabivarin (THCV) and cannabigerolic acid (CBGA). To this aim, pro-inflammatory interleukins (IL)-1β, IL-8, IL-12, IL-31, tumor necrosis factor (TNF-β) and anti-inflammatory IL-10 levels were measured through ELISA quantification. In addition, IL-12 and IL-31 levels were measured after treatment of HaCaT cells with THCV and CBGA in the presence of selected modulators of endocannabinoid (eCB) signaling. In the latter cells, the activation of 17 distinct proteins along the mitogen-activated protein kinase (MAPK) pathway was also investigated via Human Phosphorylation Array.

Our results demonstrate that rare pCBs significantly blocked inflammation by reducing the release of all pro-inflammatory ILs tested, except for TNF-β. Moreover, the reduction of IL-31 expression by THCV and CBGA was significantly reverted by blocking the eCB-binding TRPV1 receptor and by inhibiting the eCB-hydrolase MAGL. Remarkably, THCV and CBGA modulated the expression of the phosphorylated forms (and hence of the activity) of the MAPK-related proteins GSK3β, MEK1, MKK6 and CREB also by engaging eCB hydrolases MAGL and FAAH.

Taken together, the ability of rare pCBs to exert an anti-inflammatory effect in human keratinocytes through modifications of eCB and MAPK signaling opens new perspectives for the treatment of inflammation-related skin pathologies.”

“Overall, this proof of concept, which shows that in inflamed human keratinocytes, rare pCBs can indeed interact with specific eCB system elements, opens new perspectives for possible treatments of inflammation-related skin diseases. Incidentally, such interactions between pCBs and eCB system seems to hold therapeutic potential well beyond the skin, such as possible treatments reported for autism spectrum disorders and cancer”

“Effects of Rare Phytocannabinoids on the Endocannabinoid System of Human Keratinocytes”

Cannabidiol mediates epidermal terminal differentiation and redox homeostasis through aryl hydrocarbon receptor (AhR)-dependent signaling

Home Page: Journal of Dermatological Science

“Background: Cannabidiol, a non-psychoactive phytocannabinoid, has antioxidant and anti-inflammatory activity in keratinocytes. However, the signaling pathway through which cannabidiol exerts its effect on keratinocytes or whether it can modulate keratinocyte differentiation has not been fully elucidated yet.

Objective: We investigated whether cannabidiol modulates epidermal differentiation and scavenges reactive oxygen species through the aryl hydrocarbon receptor (AhR) in keratinocytes and epidermal equivalents.

Methods: We investigated the cannabidiol-induced activation of AhR using AhR luciferase reporter assay, qRT-PCR, western blot, and immunofluorescence assays. We also analyzed whether keratinocyte differentiation and antioxidant activity are regulated by cannabidiol-induced AhR activation.

Results: In both keratinocytes and epidermal equivalents, cannabidiol increased both the mRNA and protein expression of filaggrin, involucrin, NRF2, and NQO1 and the mRNA expression of the AhR target genes, including CYP1A1 and aryl hydrocarbon receptor repressor. Additionally, cannabidiol showed antioxidant activity that was attenuated by AhR knockdown or co-administration with an AhR antagonist. Moreover, cannabidiol increased the ratio of OVOL1/OVOL2 mRNA expression, which is a downstream regulator of AhR that mediates epidermal differentiation. In addition to increased expression of barrier-related proteins, cannabidiol-treated epidermal equivalent showed a more prominent granular layer than the control epidermis. The increased granular layer by cannabidiol was suppressed by the AhR antagonist.

Conclusion: Cannabidiol can be a modulator of the AhR-OVOL1-filaggrin axis and AhR-NRF2-NQO1 signaling, thus indicating a potential use of cannabidiol in skin barrier enhancement and reducing oxidative stress.”

Phytochemical Constituents and Derivatives of Cannabis sativa; Bridging the Gap in Melanoma Treatment


“Melanoma is deadly, physically impairing, and has ongoing treatment deficiencies. Current treatment regimens include surgery, targeted kinase inhibitors, immunotherapy, and combined approaches. Each of these treatments face pitfalls, with diminutive five-year survival in patients with advanced metastatic invasion of lymph and secondary organ tissues. Polyphenolic compounds, including cannabinoids, terpenoids, and flavonoids; both natural and synthetic, have emerging evidence of nutraceutical, cosmetic and pharmacological potential, including specific anti-cancer, anti-inflammatory, and palliative utility. Cannabis sativa is a wellspring of medicinal compounds whose direct and adjunctive application may offer considerable relief for melanoma suffers worldwide. This review aims to address the diverse applications of C. sativa‘s biocompounds in the scope of melanoma and suggest it as a strong candidate for ongoing pharmacological evaluation.”

“In conclusion, there is a complex array of effects that polyphenolic and cannabinoid compounds elicit in relation to melanoma. Multiple biochemical and genetic cascades are regulated through the presence of these natural substances. Polyphenolic compounds emergingly demonstrate a significant capacity to mediate many of the impacts of cancer, including pain, inflammation and invasiveness. Combined administration of polyphenol compounds has shown existing promise for improvement of potency and bioactivity of these substances. To combat the complexity of cancer, new pharmacological perspectives are necessary. Accordingly, plant polyphenols, particularly those of cannabis provide a deep well of structural potential for the emergence of novel drugs with multi-applicability to the total sphere of cancer treatment. This is merely the budding tip of biocompounds available for exploration in plant-based medicine and is a substantive base for future research.”

Formulation and development of novel lipid-based combinatorial advanced nanoformulation for effective treatment of non-melanoma skin cancer

International Journal of Pharmaceutics

“Non-melanoma skin cancer is one of the most common malignancies reported with high number of morbidities, demanding an advanced treatment option with superior chemotherapeutic effects. Due to high degree of drug resistance, conventional therapy fails to meet the desired therapeutic efficacy. To break the bottleneck, nanoparticles have been used as next generation vehicles that facilitate the efficient interaction with the cancer cells.

Here, we developed combined therapy of 5-fluorouracil (5-FU) and cannabidiol (CBD)-loaded nanostructured lipid carrier gel (FU-CBD-NLCs gel). The NLCs were optimized using central composite design that showed an average particle size of 206 nm and a zeta potential of -34 mV. In addition, in vitro and ex vivo drug permeations studies demonstrated the effective delivery of both drugs in the skin layers via lipid structured nanocarriers.

Also, the prepared FU-CBD-NLCs showed promising effect in-vitro cell studies including MTT assays, wound healing and cell cycle as compared to the conventional formulation. Moreover, dermatokinetic studies shows there was superior deposition of drugs at epidermal and the dermal layer when treated with FU-CBD-NLCs.

In the end, overall study offered a novel combinatorial chemotherapy that could be an option for the treatment of non-melanoma skin cancer.”

Cannabidiol and Its Combinations with Nonsteroidal Anti-Inflammatory Drugs Induce Apoptosis and Inhibit Activation of NF-κB Signaling in Vulvar Squamous Cell Carcinoma


“Vulvar squamous cell carcinoma (VSCC) is a rare malignancy with a relatively good prognosis. However, the prognosis remains poor for elderly patients and those with a significant depth of tumor invasion; thus, novel treatment modalities are needed.

The aim of this study was to analyze the impact of cannabidiol (CBD) and its combination with NSAIDs, diclofenac (DIC) and ibuprofen (IBU) on VSCC cells. In this regard, the MTT test was applied for cytotoxicity analysis. Moreover, the influence of CBD, DIC and IBU, as well as their combinations, on apoptosis and cell cycle distribution were analyzed by flow cytometry. The mechanisms of action of the analyzed compounds, including their impact on NF-κB signaling, p53 and COX-2 expression were evaluated using Western blot.

This study shows that CBD and its combinations with NSAIDs are cytotoxic to A431 cells, but they also reduce, in a dose-dependent manner, the viability of immortalized keratinocyte HaCaT cells, and human umbilical vein cell line, EA.hy926. Moreover, the compounds and their combinations induced apoptosis, diminished the NF-κB signaling activation and reduced COX-2 expression.

We conclude that CBD and its combination with DIC or IBU are promising candidates for the adjuvant treatment of high-risk VSCC patients.”

“The results of our study regarding the use of a CDB and NSAIDs, as well as the combi-treatment of CBD together with NSAIDs, provide the foundation for a new approach to therapy of VSCC.”

Photodynamic Therapy Efficacy of Novel Zinc Phthalocyanine Tetra Sodium 2-Mercaptoacetate Combined with Cannabidiol on Metastatic Melanoma


“This work reports for the first time on the synthesis, characterization, and photodynamic therapy effect of a novel water-soluble zinc (II) 2(3), 9(10), 16(17), 23(24)-tetrakis-(sodium 2-mercaptoacetate) phthalocyanine (ZnPcTS41), on metastatic melanoma cells (A375) combined with cannabidiol (CBD). The ZnPcTS41 structure was confirmed using FTIR, NMR, MS, and elemental analysis while the electronic absorption spectrum was studied using UV-VIS. The study reports further on the dose-dependent effects of ZnPcTS41 (1-8 µM) and CBD alone (0.3-1.1 µM) at 636 nm with 10 J/cm2 on cellular morphology and viability. The IC50 concentrations of ZnPcTS41 and CBD were found to be 5.3 µM and 0.63 µM, respectively. The cytotoxicity effects of the ZnPcTS41 enhanced with CBD on A375 cells were assessed using MTT cell viability assay, ATP cellular proliferation and inverted light microscopy. Cell death induction was also determined via Annexin V-FITC-PI. The combination of CBD- and ZnPcTS41-mediated PDT resulted in a significant reduction in cell viability (15%***) and an increase in the late apoptotic cell population (25%*). These findings suggest that enhancing PDT with anticancer agents such as CBD could possibly obliterate cancer cells and inhibit tumor recurrence.”

The Synthetic Cannabinoid URB447 Exerts Antitumor and Antimetastatic Effect in Melanoma and Colon Cancer


“The endocannabinoid system is widespread through the body and carries out a wide variety of functions. However, its involvement in other pathologies, such as cancer, still needs further attention. We aim to investigate the role of CB2 receptor during melanoma and colorectal cancer (CRC) aggressiveness and metastatic growth in the liver. We used the synthetic cannabinoid URB447, a known CB2 agonist and CB1 antagonist drug, and studied prometastatic ability of mouse B16 melanoma and MCA38 CRC cells, by means of proliferation, apoptosis, cell cycle, migration and matrix degradation in vitro upon URB447 treatment. We reported a dose-dependent viability decrease in both tumor types. This result is partly mediated by apoptotic cell death and cell cycle arrest in G1/G0 phase, as observed through flow cytometry. Melanoma and CRC cell migration was affected in a dose-dependent fashion as observed through scratch assay, whereas the secretion of matrix degrading proteins metalloprotease 2 (MMP2) and 9 (MMP9) in tumor cells did not significantly change. Moreover, daily treatment of tumor bearing mice with URB447 decreased the development of liver metastasis in a melanoma model in vivo. This proof of concept study points out to the synthetic cannabinoid URB447 as a potential candidate for deeper studies to confirm its potential as antitumor therapy and liver metastasis treatment for CRC and melanoma.”

Photoprotective Effects of Cannabidiol against Ultraviolet-B-Induced DNA Damage and Autophagy in Human Keratinocyte Cells and Mouse Skin Tissue


“Cannabidiol (CBD) has emerged as a phytocannabinoid with various beneficial effects for the skin, including anti-photoaging effects, but its mechanisms of action are not fully elucidated. The study assessed CBD’s photoprotective effects against acute ultraviolet B (UVB)-induced damage in HaCaT human keratinocyte cells and murine skin tissue. CBD (8 μM) alleviated UVB-induced cytotoxicity, apoptosis, and G2/M cell cycle arrest in HaCaT cells. The contents of γH2AX and cyclobutane pyrimidine dimers were decreased after CBD treatment. CBD reduced the production of reactive oxygen species and modulated the expression of antioxidant-related proteins such as nuclear factor erythroid 2-related factor 2 in UVB-stimulated HaCaT cells. Furthermore, CBD mitigated the UVB-induced cytotoxicity by activating autophagy. In addition, a cream containing 5% CBD showed effectiveness against UVB-induced photodamage in a murine model. The CBD cream improved the skin’s condition by lowering the photodamage scores, reducing abnormal skin proliferation, and decreasing expression of the inflammation-related protein cyclooxygenase-2 in UVB-irradiated skin tissue. These findings indicate that CBD might be beneficial in alleviating UVB-induced skin damage in humans. The photoprotective effects of CBD might be attributed to its modulatory effects on redox homeostasis and autophagy.”

“These findings, along with evidence reported from other studies, suggest that CBD is a phytocannabinoid with promising beneficial effects for the skin against UV-induced photodamage.”

Cannabidiol Interacts Antagonistically with Cisplatin and Additively with Mitoxantrone in Various Melanoma Cell Lines-An Isobolographic Analysis


“The medical application of cannabidiol (CBD) has been gathering increasing attention in recent years. This non-psychotropic cannabis-derived compound possesses antiepileptic, antipsychotic, anti-inflammatory and anxiolytic properties. Recent studies report that it also exerts antineoplastic effects in multiple types of cancers, including melanoma.

In this in vitro study we tried to reveal the anticancer properties of CBD in malignant melanoma cell lines (SK-MEL 28, A375, FM55P and FM55M2) administered alone, as well as in combination with mitoxantrone (MTX) or cisplatin (CDDP).

The effects of CBD on the viability of melanoma cells were measured by the MTT assay; cytotoxicity was determined in the LDH test and proliferation in the BrdU test. Moreover, the safety of CBD was tested in human keratinocytes (HaCaT) in LDH and MTT tests.

Results indicate that CBD reduces the viability and proliferation of melanoma-malignant cells and exerts additive interactions with MTX. Unfortunately, CBD produced antagonistic interaction when combined with CDDP. CBD does not cause significant cytotoxicity in HaCaT cell line.

In conclusion, CBD may be considered as a part of melanoma multi-drug therapy when combined with MTX. A special attention should be paid to the combination of CBD with CDDP due to the antagonistic interaction observed in the studied malignant melanoma cell lines.”