ß-Caryophyllene Inhibits Endothelial Tube Formation by Modulating the Secretome of Hypoxic Lung Cancer Cells-Possible Role of VEGF Downregulation.

ß-Caryophyllene (BCP), a bicyclic sesquiterpene that is a component of the essential oils of various spice and food plants, has been described as a selective CB2 cannabinoid receptor agonist. In the present study, the effect of BCP on angiogenesis was investigated. It was found that conditioned media (CM) from BCP-treated hypoxic A549 lung cancer cells exhibited a concentration-dependent inhibitory effect on human umbilical vein endothelial cell (HUVEC) tube formation induced by CM from vehicle-treated hypoxic A549 cells. There was an associated concentration-dependent decrease in the proangiogenic factor vascular endothelial growth factor (VEGF) in the CM, with both BCP inhibitory effects (tube formation, VEGF secretion) being CB2 receptor-dependent. A reduction of the transcription factor hypoxia-inducible factor 1α (HIF-1α) was furthermore detected. The antiangiogenic and VEGF-lowering properties of BCP were confirmed when CM from another lung cancer cell line, H358, were tested. When directly exposed to HUVECs, BCP showed no significant effect on tube formation, but at 10 µM, impaired VEGF receptor 2 (VEGFR2) phosphorylation triggered by recombinant VEGF in a CB2 receptor-independent manner. In summary, BCP has a dual antiangiogenic effect on HUVECs, manifested in the inhibition of tube formation through modulation of the tumor cell secretome and additionally in the inhibition of VEGF-induced VEGFR2 activation. Because the CB2 agonist has no psychoactive properties, BCP should continue to be evaluated preclinically for further antitumor effects.

Antiangiogenic Action of JZL184 on Endothelial Cells via Inhibition of VEGF Expression in Hypoxic Lung Cancer Cells.

JZL184, an inhibitor of monoacylglycerol lipase (MAGL) and thus of the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG), mediates various anticancer effects in preclinical studies. However, studies on the effect of this or other MAGL inhibitors under hypoxia, an important factor in tumor biology and response to cancer therapy, have not yet been performed in cancer cells. In the present study, the impact of the conditioned media (CM) of A549 and H358 lung cancer cells incubated with JZL184 under hypoxic conditions on the angiogenic properties of human umbilical vein endothelial cells (HUVECs) was investigated. Treatment of HUVECs with CM derived from cancer cells cultured for 48 h under hypoxic conditions was associated with a substantial increase in migration and tube formation compared with unconditioned medium, which was inhibited when cancer cells were incubated with JZL184. In this process, JZL184 led to a significant increase in 2-AG levels in both cell lines. Analysis of a panel of proangiogenic factors revealed inhibition of hypoxia-induced vascular endothelial growth factor (VEGF) expression by JZL184. Antiangiogenic and VEGF-lowering effects were also demonstrated for the MAGL inhibitor MJN110. Receptor antagonist experiments suggest partial involvement of the cannabinoid receptors CB1 and CB2 in the antiangiogenic and VEGF-lowering effects induced by JZL184. The functional importance of VEGF for angiogenesis in the selected system is supported by observations showing inhibition of VEGF receptor 2 (VEGFR2) phosphorylation in HUVECs by CM from hypoxic cancer cells treated with JZL184 or when hypoxic cancer cell-derived CM was spiked with a neutralizing VEGF antibody. On the other hand, JZL184 did not exert a direct effect on VEGFR2 activation induced by recombinant VEGF, so there seems to be no downstream effect on already released VEGF. In conclusion, these results reveal a novel mechanism of antiangiogenic action of JZL184 under conditions of hypoxic tumor-endothelial communication.

A Thia-Analogous Indirubin N-Glycoside Disrupts Mitochondrial Function and Causes the Death of Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells.

Skin cancer is the most common malignant disease worldwide and, therefore, also poses a challenge from a pharmacotherapeutic perspective. Derivatives of indirubin are an interesting option in this context. In the present study, the effects of 3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(ß-d-glucopyranosyl)-oxindole (KD87), a thia-analogous indirubin N-glycoside, on the viability and mitochondrial properties of melanoma (A375) and squamous cell carcinoma cells (A431) of the skin were investigated. In both cell lines, KD87 caused decreased viability, the activation of caspases-3 and -7, and the inhibition of colony formation. At the mitochondrial level, a concentration-dependent decrease in both the basal and ATP-linked oxygen consumption rate and in the reserve capacity of oxidative respiration were registered in the presence of KD87. These changes were accompanied by morphological alterations in the mitochondria, a release of mitochondrial cytochrome c into the cytosol and significant reductions in succinate dehydrogenase complex subunit B (SDHB, subunit of complex II) in A375 and A431 cells and NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8, subunit of complex I) in A375 cells. The effect of KD87 was accompanied by a significant upregulation of the enzyme heme oxygenase-1, whose inhibition led to a partial but significant reduction in the metabolic-activity-reducing effect of KD87. In summary, our data show a mitochondria-targeting effect of KD87 as part of the cytotoxic effect of this compound on skin cancer cells, which should be considered in future studies with this class of compounds.

Inhibition of Monoacylglycerol Lipase Decreases Angiogenic Features of Endothelial Cells via Release of Tissue Inhibitor of Metalloproteinase-1 from Lung Cancer Cells.

Despite the well-described anticarcinogenic effects of endocannabinoids, the influence of the endocannabinoid system on tumor angiogenesis is still debated. In the present study, conditioned medium (CM) from A549 and H358 lung cancer cells treated with ascending concentrations of the monoacylglycerol lipase (MAGL) inhibitor JZL184 and 2-arachidonoylglycerol (2-AG), a prominent MAGL substrate, caused a concentration-dependent reduction in human umbilical vein endothelial cell (HUVEC) migration and tube formation compared with CM from vehicle-treated cancer cells. Comparative experiments with MAGL inhibitors JW651 and MJN110 showed the same results. On the other hand, the angiogenic properties of HUVECs were not significantly altered by direct stimulation with JZL184 or 2-AG or by exposure to CM of JZL184- or 2-AG-treated non-cancerous bronchial epithelial cells (BEAS-2B). Inhibition of HUVEC migration and tube formation by CM of JZL184- and 2-AG-treated A549 cells was abolished in the presence of the CB1 antagonist AM-251. Increased release of tissue inhibitor of metalloproteinase-1 (TIMP-1) from JZL184- or 2-AG-stimulated A549 or H358 cells was shown to exert an antiangiogenic effect on HUVECs, as confirmed by siRNA experiments. In addition, JZL184 caused a dose-dependent regression of A549 tumor xenografts in athymic nude mice, which was associated with a decreased number of CD31-positive cells and upregulation of TIMP-1-positive cells in xenograft tissue. In conclusion, our data suggest that elevation of 2-AG by MAGL inhibition leads to increased release of TIMP-1 from lung cancer cells, which mediates an antiangiogenic effect on endothelial cells.

A conserved long-distance telomeric silencing mechanism suppresses mTOR signaling in aging human fibroblasts.

Telomeres are repetitive nucleotide sequences at the ends of each chromosome. It has been hypothesized that telomere attrition evolved as a tumor suppressor mechanism in large long-lived species. Long telomeres can silence genes millions of bases away through a looping mechanism called telomere position effect over long distances (TPE-OLD). The function of this silencing mechanism is unknown. We determined a set of 2322 genes with high positional conservation across replicatively aging species that includes known and candidate TPE-OLD genes that may mitigate potentially harmful effects of replicative aging. Notably, we identified PPP2R2C as a tumor suppressor gene, whose up-regulation by TPE-OLD in aged human fibroblasts leads to dephosphorylation of p70S6 kinase and mammalian target of rapamycin suppression. A mechanistic link between telomeres and a tumor suppressor mechanism supports the hypothesis that replicative aging fulfills a tumor suppressor function and motivates previously unknown antitumor and antiaging strategies.

Impact of Cannabinoid Compounds on Skin Cancer.

Drugs targeting the endocannabinoid system are of interest as potential systemic chemotherapeutic treatments and for palliative care in cancer. In this context, cannabinoid compounds have been successfully tested as a systemic therapeutic option in preclinical models over the past decades. Recent findings have suggested an essential function of the endocannabinoid system in the homeostasis of various skin functions and indicated that cannabinoids could also be considered for the treatment and prophylaxis of tumour diseases of the skin. Cannabinoids have been shown to exert their anticarcinogenic effects at different levels of skin cancer progression, such as inhibition of tumour growth, proliferation, invasion and angiogenesis, as well as inducing apoptosis and autophagy. This review provides an insight into the current literature on cannabinoid compounds as potential pharmaceuticals for the treatment of melanoma and squamous cell carcinoma.

The Endocannabinoid System as a Pharmacological Target for New Cancer Therapies.

Despite the long history of cannabinoid use for medicinal and ritual purposes, an endogenous system of cannabinoid-controlled receptors, as well as their ligands and the enzymes that synthesise and degrade them, was only discovered in the 1990s. Since then, the endocannabinoid system has attracted widespread scientific interest regarding new pharmacological targets in cancer treatment among other reasons. Meanwhile, extensive preclinical studies have shown that cannabinoids have an inhibitory effect on tumour cell proliferation, tumour invasion, metastasis, angiogenesis, chemoresistance and epithelial-mesenchymal transition (EMT) and induce tumour cell apoptosis and autophagy as well as immune response. Appropriate cannabinoid compounds could moreover be useful for cancer patients as potential combination partners with other chemotherapeutic agents to increase their efficacy while reducing unwanted side effects. In addition to the direct activation of cannabinoid receptors through the exogenous application of corresponding agonists, another strategy is to activate these receptors by increasing the endocannabinoid levels at the corresponding pathological hotspots. Indeed, a number of studies accordingly showed an inhibitory effect of blockers of the endocannabinoid-degrading enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) on tumour development and spread. This review summarises the relevant preclinical studies with FAAH and MAGL inhibitors compared to studies with cannabinoids and provides an overview of the regulation of the endocannabinoid system in cancer.

Interleukin-1ß Induces Tissue Factor Expression in A549 Cells via EGFR-Dependent and -Independent Mechanisms.

Tissue factor (TF) plays an important role in the progression and angiogenesis of tumor cells. The present study investigated the mechanism of interleukin-1ß (IL-1ß)-induced TF expression in A549 lung cancer cells. Based on mRNA and protein analyses, including appropriate inhibitor experiments, IL-1ß was shown to induce TF expression in a time-dependent manner, mediated by IL-1 receptor-dependent phosphorylation of the mitogen-activated protein kinases (MAPK) p38, p42/44 and c-jun N-terminal kinase (JNK), as well as the Src kinase and the epidermal growth factor receptor (EGFR). Thereby, inhibition of EGFR transactivation by the Src inhibitor PP1 or direct EGFR inhibition by the EGFR tyrosine kinase inhibitor (TKI) erlotinib led to a reduction of IL-1ß-induced TF expression and to a suppression of p42/44 MAPK and EGFR activation, while IL-1ß-induced p38 MAPK and JNK activation remained unchanged. A knockdown of EGFR by siRNA was associated with decreased IL-1ß-mediated p42/44 MAPK activation, which was no longer inhibitable by erlotinib. Concentration-dependent inhibition of IL-1ß-induced TF expression was also observed in the presence of gefitinib and afatinib, two other EGFR TKIs. In summary, our results suggest that IL-1ß leads to increased TF formation in lung cancer cells via both Src/EGFR/p42/44 MAPK-dependent and EGFR-independent signaling pathways, with the latter mediated via p38 MAPK and JNK.

The Monoacylglycerol Lipase Inhibitor JZL184 Inhibits Lung Cancer Cell Invasion and Metastasis via the CB1 Cannabinoid Receptor.

A targeted modulation of the endocannabinoid system is currently discussed as a promising strategy for cancer treatment. An important enzyme for the endocannabinoid metabolism is the monoacylglycerol lipase (MAGL), which catalyzes the degradation of 2-arachidonoylglycerol (2-AG) to glycerol and free fatty acids. In this study, we investigated the influence of MAGL inhibition on lung cancer cell invasion and metastasis. Using LC-MS, significantly increased 2-AG levels were detected in A549 cells treated with the MAGL inhibitor JZL184. In athymic nude mice, JZL184 suppressed metastasis of A549 cells in a dose-dependent manner, whereby the antimetastatic effect was cancelled by the CB1 receptor antagonist AM-251. In vitro, JZL184 induced a time- and concentration-dependent reduction of A549 cell invasion through Matrigel-coated membranes, which was likewise reversed by AM-251. An MAGL inhibition-associated reduction of free fatty acids as a cause of the anti-invasive effect could be excluded by add-back experiments with palmitic acid. Both JZL184 and the MAGL substrate 2-AG led to an increased formation of the tissue inhibitor of metalloproteinase-1 (TIMP-1), whereby a TIMP-1 knockdown using siRNA significantly attenuated the anti-invasive effects of both substances. Decreased invasion and TIMP-1 upregulation was also caused by the MAGL inhibitors JW651 and MJN110 or transfection with MAGL siRNA. A CB1- and TIMP-1-dependent anti-invasive effect was further confirmed for JZL184 in H358 lung cancer cells. In conclusion, MAGL inhibition led to a CB1-dependent decrease in human lung cancer cell invasion and metastasis via inhibition of 2-AG degradation, with TIMP-1 identified as a mediator of the anti-invasive effect.