Cannabigerol Induces Autophagic Cell Death by Inhibiting EGFR-RAS Pathways in Human Pancreatic Ductal Adenocarcinoma Cell Lines.

Pancreatic ductal adenocarcinoma (PDAC) is the most frequent infiltrating type of pancreatic cancer. The poor prognosis associated with this cancer is due to the absence of specific biomarkers, aggressiveness, and treatment resistance. PDAC is a deadly malignancy bearing distinct genetic alterations, the most common being those that result in cancer-causing versions of the KRAS gene. Cannabigerol (CBG) is a non-psychomimetic cannabinoid with anti-inflammatory properties. Regarding the anticancer effect of CBG, up to now, there is only limited evidence in human cancers. To fill this gap, we investigated the effects of CBG on the PDAC cell lines, PANC-1 and MIAPaCa-2. The effect of CBG activity on cell viability, cell death, and EGFR-RAS-associated signaling was investigated. Moreover, the potential synergistic effect of CBG in combination with gemcitabine (GEM) and paclitaxel (PTX) was investigated. MTT was applied to investigate the effect of CBG on PDAC cell line viabilities. Annexin-V and Acridine orange staining, followed by cytofluorimetric analysis and Western blotting, were used to evaluate CBG's effect on cell death. The modulation of EGFR-RAS-associated pathways was determined by Western blot analysis and a Milliplex multiplex assay. Moreover, by employing the MTT data and SynergyFinder Plus software analysis, the effect of the combination of CBG and chemotherapeutic drugs was determined.

Synthesis of Carlina Oxide Analogues and Evaluation of Their Insecticidal Efficacy and Cytotoxicity.

Compounds isolated from botanical sources represent innovative and promising alternatives to conventional insecticides. Carlina oxide is a compound isolated from Carlina acaulis L. (Asteraceae) essential oil (EO) with great potential as bioinsecticide, being effective on various arthropod vectors and agricultural pests, with moderate toxicity on non-target species. Since the production from the wild source is limited, there is the need of exploring new synthetic routes for obtaining this compound and analogues with improved bioactivity and lower toxicity. Herein, the chemical synthesis of carlina oxide analogues was developed. Their insecticidal activity was assessed on the vectors Musca domestica L. and Culex quinquefasciatus Say, and their cytotoxicity was evaluated on a human keratinocyte cell line (HaCaT). The compounds' activity was compared with that of the natural counterparts EO and carlina oxide. In housefly tests, the analogues were comparably effective to purified carlina oxide. In Cx. quinquefasciatus assays, the meta-chloro analogue provided a significantly higher efficacy (LC50 of 0.71 µg mL-1) than the EO and carlina oxide (LC50 1.21 and 1.31 µg mL-1, respectively) and a better safety profile than carlina oxide on keratinocytes. Overall, this study can open the way to an agrochemical production of carlina oxide analogues employable as nature-inspired insecticides.

Encapsulation of Hemp (Cannabis sativa L.) Essential Oils into Nanoemulsions for Potential Therapeutic Applications: Assessment of Cytotoxicological Profiles.

Industrial hemp (Cannabis sativa L.), due to its bioactive compounds (terpenes and cannabinoids), has gained increasing interest in different fields, including for medical purposes. The evaluation of the safety profile of hemp essential oil (EO) and its encapsulated form (nanoemulsion, NE) is a relevant aspect for potential therapeutic applications. This study aimed to evaluate the toxicological effect of hemp EOs and NEs from cultivars Carmagnola CS and Uso 31 on three cell lines selected as models for topical and inhalant administration, by evaluating the cytotoxicity and the cytokine expression profiles. Results show that EOs and their NEs have comparable cytotoxicity, if considering the quantity of EO present in the NE. Moreover, cells treated with EOs and NEs showed, in most of the cases, lower levels of proinflammatory cytokines compared to Etoposide used as a positive control, and the basal level of inflammatory cytokines was not altered, suggesting a safety profile of hemp EOs and their NEs to support their use for medical applications.

The effects of cannabidiol via TRPV2 channel in chronic myeloid leukemia cells and its combination with imatinib.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by accumulation of immature cells in bone marrow and peripheral blood. Although successful results were obtained with tyrosine kinase inhibitors, several patients showed resistance. For this reason, the identification of new strategies and therapeutic biomarkers represents an attractive goal. The role of transient receptor potential (TRP) ion channels as possible drug targets has been elucidated in different types of cancer. Among natural compounds known to activate TRPs, cannabidiol (CBD) displays anticancer properties. By using FACS analysis, confocal microscopy, gene silencing, and cell growth assay, we demonstrated that CBD, through TRPV2, inhibits cell proliferation and cell cycle in CML cells. It promoted mitochondria dysfunction and mitophagy as shown by mitochondrial mass reduction and up-regulation of several mitophagy markers. These effects were associated with changes in the expression of octamer-binding transcription factor 4 and PU.1 markers regulated during cellular differentiation. Interestingly, a synergistic effect by combining CBD with the standard drug imatinib was found and imatinib-resistant cells remain susceptible to CBD effects. Therefore, the targeting of TRPV2 by using CBD, through the activation of mitophagy and the reduction in stemness, could be a promising strategy to enhance conventional therapy and improve the prognosis of CML patients.

The Effects of Cannabidiol and Prognostic Role of TRPV2 in Human Endometrial Cancer.

Several studies support, both in vitro and in vivo, the anti-cancer effects of cannabidiol (CBD), a transient receptor potential vanilloid 2 (TRPV2) ligand. TRPV2, often dysregulated in tumors, is associated with altered cell proliferation and aggressiveness. Endometrial cancer (EC) is historically divided in type I endometrioid EC and type II non-endometrioid EC, associated with poor prognosis. Treatment options with chemotherapy and combinations with radiation showed only limited efficacy. Since no data are reported concerning TRPV2 expression as well as CBD potential effects in EC, the aim of this study was to evaluate the expression of TRPV2 in biopsies and cell lines as well as the effects of CBD in in vitro models. Overall survival (OS), progression-free survival (PFS), cell viability, migration, and chemo-resistance have been evaluated. Results show that TRPV2 expression increased with the malignancy of the cancer tissue and correlated with shorter PFS (p = 0.0224). Moreover, in vitro TRPV2 over-expression in Ishikawa cell line increased migratory ability and response to cisplatin. CBD reduced cell viability, activating predominantly apoptosis in type I cells and autophagy in mixed type EC cells. The CBD improved chemotherapeutic drugs cytotoxic effects, enhanced by TRPV2 over-expression. Hence, TRPV2 could be considered as a marker for optimizing the therapy and CBD might be a useful therapeutic option as adjuvant therapy.

Anticancer effect of minor phytocannabinoids in preclinical models of multiple myeloma.

Multiple myeloma (MM) is a blood cancer caused by uncontrolled growth of clonal plasmacells. Bone disease is responsible for the severe complications of MM and is caused by myeloma cells infiltrating the bone marrow and inducing osteoclast activation. To date, no treatment for MM is truly curative since patients relapse and become refractory to all drug classes. Cannabinoids are already used as palliative in cancer patients. Furthermore, their proper anticancer effect was demonstrated in many cancer models in vitro, in vivo, and in clinical trials. Anyway, few information was reported on the effect of cannabinoids on MM and no data has been provided on minor phytocannabinoids such as cannabigerol (CBG), cannabichromene (CBC), cannabinol (CBN), and cannabidivarin (CBDV). Scientific literature also reported cannabinoids beneficial effect against bone disease. Here, we examined the cytotoxic activity of CBG, CBC, CBN, and CBDV in vitro in MM cell lines, their effect in modulating MM cells invasion toward bone cells and the bone resorption. Subsequently, according to the in vitro results, we selected CBN for in vivo study in a MM xenograft mice model. Results showed that the phytocannabinoids inhibited MM cell growth and induced necrotic cell death. Moreover, the phytocannabinoids reduced the invasion of MM cells toward osteoblast cells and bone resorption in vitro. Lastly, CBN reduced in vivo tumor mass. Together, our results suggest that CBG, CBC, CBN, and CBDV can be promising anticancer agents for MM.

Copper-Based Complexes with Adamantane Ring-Conjugated bis(3,5-Dimethyl-pyrazol-1-yl)acetate Ligand as Promising Agents for the Treatment of Glioblastoma.

The new ligand L2Ad, obtained by conjugating the bifunctional species bis(3,5-dimethylpyrazol-1-yl)-acetate and the drug amantadine, was used as a chelator for the synthesis of new Cu complexes 1-5. Their structures were investigated by synchrotron radiation-induced X-ray photoelectron spectroscopy (SR-XPS), near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, and by combining X-ray absorption fine structure (XAFS) spectroscopy techniques and DFT modeling. The structure of complex 3 was determined by single-crystal X-ray diffraction analysis. Tested on U87, T98, and U251 glioma cells, Cu(II) complex 3 and Cu(I) complex 5 decreased cell viability with IC50 values significantly lower than cisplatin, affecting cell growth, proliferation, and death. Their effects were prevented by treatment with the Cu chelator tetrathiomolybdate, suggesting the involvement of copper in their cytotoxic activity. Both complexes were able to increase ROS production, leading to DNA damage and death. Interestingly, nontoxic doses of 3 or 5 enhanced the chemosensitivity to Temozolomide.

Calcium influx, oxidative stress, and apoptosis induced by TRPV1 in chronic myeloid leukemia cells: Synergistic effects with imatinib.

Introduction: Calcium flux is the master second messenger that influences the proliferation-apoptosis balance. The ability of calcium flux alterations to reduce cell growth makes ion channels interesting targets for therapy. Among all, we focused on transient receptor potential vanilloid 1, a ligand-gated cation channel with selectivity for calcium. Its involvement in hematological malignancies is poorly investigated, especially in the field of chronic myeloid leukemia, a malignancy characterized by the accumulation of immature cells. Methods: FACS analysis, Western blot analysis, gene silencing, and cell viability assay were performed to investigate the activation of transient receptor potential vanilloid 1, by N-oleoyl-dopamine, in chronic myeloid leukemia cell lines. Results: We demonstrated that the triggering of transient receptor potential vanilloid 1 inhibits cell growth and promotes apoptosis of chronic myeloid leukemia cells. Its activation induced calcium influx, oxidative stress, ER stress, mitochondria dysfunction, and caspase activation. Interestingly, a synergistic effect exerted by N-oleoyl-dopamine and the standard drug imatinib was found. Conclusion: Overall, our results support that transient receptor potential vanilloid 1 activation could be a promising strategy to enhance conventional therapy and improve the management of chronic myeloid leukemia.

Acaricidal Activity of Bufadienolides Isolated from Drimia pancration against Tetranychus urticae, and Structural Elucidation of Arenobufagin-3-O-α-L-rhamnopyranoside.

Chemical characterization of the bulbs of Drimia pancration was conducted to isolate four steroidal saponins (1-4). Earlier, we focused on the structural elucidation of compounds 1-3. Herein, by means of 1H-NMR, 13C-NMR, Nuclear Overhauser Effects (NOE), and 2D-NMR spectra, the full stereochemical structure of 4 is reported, and all the 1H and 13C signals are assigned. Compounds 1-4 were tested for their acaricidal properties against the two-spotted spider mite Tetranychus urticae. Our results showed excellent activity of compound 1, with an LD50 (µg/cm2) of 0.29 and a LD90 (µg/cm2) of 0.96, whereas compounds 2, 3, and 4 showed moderate activity. Furthermore, the acaricidal and cytotoxic properties of the crude extract were also investigated. Of note, after 96 h of exposure, the acaricidal activity of compound 1 was higher than that of the positive control, hexythiazox. Indeed, for compound 1, LD50 and LD90 were 0.29 and 0.96 µg/cm2, respectively, while hexythiazox LD50(90) was 18.7 (132.5) µg/cm2. Additionally, D. pancration extract, after 72 h, induced a high cytotoxic effect in HaCaT and THP-1 cell lines, with an IC50 of 7.37 ± 0.5 µg/mL and 3.50 ± 0.15 µg/mL, respectively. Overall, D. pancration can be considered as a green source of novel acaricides effective against mites of agricultural importance, such as T. urticae, pending proper field validation and the assessment of non-target effects on other invertebrate species.

Unveiling the Molecular Mechanisms Driving the Capsaicin-Induced Immunomodulatory Effects on PD-L1 Expression in Bladder and Renal Cancer Cell Lines.

The blockade of the PD-L1/PD-1 immune checkpoint has promising efficacy in cancer treatment. However, few patients with bladder cancer (BC) or renal cell carcinoma (RCC) respond to this approach. Thus, it is important to implement a strategy to stimulate the immune anti-tumor response. In this scenario, our study evaluated the effects of a low capsaicin (CPS) dose in BC and RCC cell lines. Western blot, qRT-PCR and confocal microscopy were used to assess PD-L1 mRNA and protein expression. Alterations to the cellular oxidative status and changes to the antioxidant NME4 levels, mRNA modulation of cytokines, growth factors, transcriptional factors and oncogene, and the activation of Stat1/Stat3 pathways were examined using Western blot, cytofluorimetry and qRT-PCR profiling assays. In BC, CPS triggers an altered stress oxidative-mediated DNA double-strand break response and increases the PD-L1 expression. On the contrary, in RCC, CPS, by stimulating an efficient DNA damage repair response, thus triggering protein carbonylation, reduces the PD-L1 expression. Overall, our results show that CPS mediates a multi-faceted approach. In modulating PD-L1 expression, there is a rationale for CPS exploitation as a stimulus that increases BC cells' response to immunotherapy or as an immune adjuvant to improve the efficacy of the conventional therapy in RCC patients.

Evening Primrose Oil Improves Chemotherapeutic Effects in Human Pancreatic Ductal Adenocarcinoma Cell Lines-A Preclinical Study.

Evening Primrose oil (EPO), obtained from the seeds of Evening Primrose (Oenothera L.), is largely used as a dietary supplement, especially after cancer diagnosis. Human pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease correlated with poor clinical prognosis and a very low response rate to common chemotherapy. The aim of this work was to study the potential ability of EPO to improve the effects of chemotherapeutic drugs in PANC-1 and MIAPaCa-2 cell lines. Cytotoxicity, cell death, reactive oxygen species (ROS) production and EPO anticancer activity associated with the main chemotherapeutic drugs commonly used in therapy were investigated. Results showed that EPO reduced PDAC cell viability and increased paclitaxel efficacy. This evidence suggests that EPO may be used as a potential supplement to increase chemotherapeutic efficacy in PDAC therapy.

Transient Receptor Potential (TRP) Channels in Haematological Malignancies: An Update.

Transient receptor potential (TRP) channels are improving their importance in different cancers, becoming suitable as promising candidates for precision medicine. Their important contribution in calcium trafficking inside and outside cells is coming to light from many papers published so far. Encouraging results on the correlation between TRP and overall survival (OS) and progression-free survival (PFS) in cancer patients are available, and there are as many promising data from in vitro studies. For what concerns haematological malignancy, the role of TRPs is still not elucidated, and data regarding TRP channel expression have demonstrated great variability throughout blood cancer so far. Thus, the aim of this review is to highlight the most recent findings on TRP channels in leukaemia and lymphoma, demonstrating their important contribution in the perspective of personalised therapies.

Evaluation of anti-inflammatory and immunoregulatory activities of Stimunex® and Stimunex D3® in human monocytes/macrophages stimulated with LPS or IL-4/IL-13.

Macrophages exert an important role in maintaining and/or ameliorating the inflammatory response. They are involved in the activation of an immune response to pathogens, with a balance between the immunomodulatory role and tissue integrity maintenance, however, excessive macrophage activity promotes tissue injury and chronic disease pathogenesis. There is a high interest in evaluating the anti-inflammatory properties of new botanical preparations. Stimunex® and Stimunex D3® are two food supplements formulated as syrups, containing the extract of elderflower (Sambucus nigra, Caprifoliaceae), standardized in polyphenol (6%) and anthocyanins (4%), associated with wellmune WGP® ß-glucan, with the addiction of vitamin D3 (in Stimunex D3® formulation). The aim of the work was the evaluation of Stimunex® and Stimunex D3® activity in human polarized-macrophages, in order to support their use as supplement for preventing and reducing the inflammatory processes. In primary human stimulated macrophages, both syrups were able to revert LPS- and IL-4/IL-13-mediated response, reducing the release of several pro-inflammatory cytokines. Results support that these standardized botanical preparations fortified with ß-glucan, may have a potential use in the prevention and coadjuvant management of inflammatory process as respiratory recurrent infections and other similar conditions. Moreover, the addition of vitamin D3 revealed to be an advantage in Stimunex D3® for its important role in maintaining and enhancing the innate immune response.

Cannabidiol and Oxygen-Ozone Combination Induce Cytotoxicity in Human Pancreatic Ductal Adenocarcinoma Cell Lines.

Pancreatic cancer (PC) is related to lifestyle risks, chronic inflammation, and germline mutations in BRCA1/2, ATM, MLH1, TP53, or CDKN2A. Surgical resection and adjuvant chemotherapy are the main therapeutic strategies but are less effective in patients with high-grade tumors. Oxygen-ozone (O2/O3) therapy is an emerging alternative tool for the treatment of several clinical disorders. O2/O3 therapy has been found to ameliorate mechanisms promoting chronic pain and inflammation, including hypoxia, inflammatory mediators, and infection. The advantages of using cannabinoids have been evaluated in vitro and in vivo models of several human cancers. Regarding PDAC, activation of cannabinoid receptors was found to induce pancreatic cancer cell apoptosis without affecting the normal pancreas cells. In a murine model of PDAC, a combination of cannabidiol (CBD) and gemcitabine increased survival length by nearly three times. Herein, we evaluate the anticancer effect of CBD and O2/O3, alone or in combination, on two human PDAC cell lines, PANC-1 and MiaPaCa-2, examining expression profiles of 92 pancreatic adenocarcinoma associated genes, cytotoxicity, migration properties, and cell death. Finally, we assess the combination effects with gemcitabine and paclitaxel. Summarizing, for the first time the antitumoral effect of combined therapy with CBD and oxygen-ozone therapy in PDAC is evidenced.

Biological Function of PD-L2 and Correlation With Overall Survival in Type II Endometrial Cancer.

In cancer, upregulation of coinhibitory B7 ligands has been associated with immune evasion. So far, anti-programmed death-1 (PD-1) and anti-PD-ligand 1 (PD-L1) antibodies have been used in immuno-oncology, with promising outcomes; however, it is still needed to identify other markers, especially for endometrial cancer (EC). EC is a gynecological malignancy historically classified into two types: type I, with mostly estrogen-dependent endometrioid diseases, and the most aggressive type II, including mainly estrogen-independent and non-endometrioid tumors. PD ligand-2 (PD-L2) is known as the second ligand of the PD-1 receptor and, upon its binding, contributes to T-cell exhaustion. Up to now, very few information are available about PD-L2 in cancers, and no data have been reported for EC. The aim of this work was to characterize the PD-L1 and PD-L2 ligand expression profile in EC cell lines, focusing the attention on the biological role of PD-L2 and its prognostic impact in human type II EC biopsies. Using in silico analysis of TCGA data, we performed a molecular profiling in a cohort of 506 patients, both types I and II, and PD-1 ligands expression was also analyzed in different primary human EC cell lines. Moreover, PD-L2 staining was evaluated in a cohort of human type II EC samples and correlated with the overall survival (OS), progression-free survival (PFS), and additional clinicopathological data. From the in silico analysis, PD-L2 was more expressed than PD-L1 in EC cell lines. PD-L2 was found highly expressed in 64.44% of tumor specimens, predominantly in the serous subtype, in both stromal and epithelial components, while in peritumoral and normal tissues it was predominantly moderate or low. In vitro, we investigated the cell autonomous role of PD-L2 in controlling cell survival, migration, and chemoresistance.

Ascaridole-rich essential oil from marsh rosemary (Ledum palustre) growing in Poland exerts insecticidal activity on mosquitoes, moths and flies without serious effects on non-target organisms and human cells.

Marsh rosemary (Ledum palustre, Ericaceae) has been widely used in the traditional medicine of various regions worldwide, and as insect repellent. Little is known on its essential oil insecticidal potential. This study explored the insecticidal effects of the essential oil obtained from L. palustre growing in Poland on selected insect pests and vectors. GC-MS analysis evidenced an uncommon chemotype characterized by ascaridole (35.3% as sum of cis-ascaridole and isoascaridole) and p-cymene (25.5%). The essential oil was effective against Culex quinquefasciatus, Spodoptera littoralis and Musca domestica, showing LC50/LD50 of 66.6 mg L-1, 117.2 µg larva-1 and 61.4 µg adult-1, respectively. It was not toxic to non-target Eisenia fetida earthworms and moderately toxic to Daphnia magna microcrustaceans, over the positive control α-cypermethrin. The essential oil cytotoxicity on human keratinocytes and fibroblasts showed high IC50 values (71.3 and 84.4 µg mL-1, respectively). Comet assay data highlighted no DNA damages. Based on our findings, this essential oil, characterized by the ascaridole/p-cymene chemotype, could be a candidate for the formulation of botanical insecticides; large-scale production of green insecticides by this rare species may be assured by ex situ cultivation and biotechnological techniques.