Prostaglandin D2-ethanolamide induces skin cancer apoptosis by suppressing the activity of cellular antioxidants.

The combined incidence of melanoma and non-melanoma skin cancer (NMSC) is greater than the incidence of all other malignancies in the US. Previously, we demonstrated that the endocannabinoid, arachidonoyl-ethanolamide (AEA), was a potent inducer of apoptosis in NMSC. The metabolism of AEA to the prostaglandin, PGD2-EA, was a prerequisite for AEA cytotoxicity. However, the mechanism of PGD2-EA cell death has not been clearly defined. In the present study, we report that PGD2-EA causes apoptosis in melanoma and NMSC cells. Mass spectrometry analysis revealed that PGD2-EA was dehydrated to three J-series prostaglandins; PGJ2-EA, Δ12PGJ2-EA, and 15deoxy,Δ12,14 PGJ2-EA. PGD2-EA inhibited the antioxidant activity of glutathione and thioredoxin which then caused oxidative stress. This increase in oxidative stress was accompanied by the activation of endoplasmic reticulum (ER) stress and apoptosis. The effect of PGD2-EA was independent of DP1, DP2, and PPARγ receptors suggesting that PGD2-EA cytotoxicity was mediated by its metabolic product, 15dPGJ2-EA.

Anandamide-induced endoplasmic reticulum stress and apoptosis are mediated by oxidative stress in non-melanoma skin cancer: Receptor-independent endocannabinoid signaling.

Endocannabinoids are neuromodulatory lipids that regulate central and peripheral physiological functions. Endocannabinoids have emerged as effective antitumor drugs due to their ability to induce apoptosis in various cancer studies. The G-protein coupled cannabinoid receptors (CB1 and CB2) and the TRPV1 ion channel were reported to mediate the antiproliferative activity of endocannabinoids. However, receptor-independent effects also account for their activity. Our previous studies showed that the antiproliferative activity of anandamide (AEA) was regulated by cyclooxygenase-2 (COX-2) via induction of endoplasmic reticulum (ER) stress. We also determined that AEA induced oxidative stress. However, the role of oxidative stress, the cannabinoid receptors, and TRPV1 in AEA-induced ER stress-apoptosis was unclear. Therefore, the current study examines the role of oxidative stress in ER stress-apoptosis and investigates whether this effect is modulated by CB1, CB2, or TRPV1. In non-melanoma skin cancer (NMSC) cells, AEA reduced the total intracellular level of glutathione and induced oxidative stress. To evaluate the importance of oxidative stress in AEA-induced cell death, the antioxidants, N-acetylcysteine (NAC) and Trolox, were utilized. Each antioxidant ameliorated the antiproliferative effect of AEA. Furthermore, Trolox inhibited AEA-induced CHOP10 expression and caspase 3 activity, indicating that oxidative stress was required for AEA-induced ER stress-apoptosis. On the other hand, selective blockade of CB1, CB2, and TRPV1 did not inhibit AEA-induced oxidative stress or ER stress-apoptosis. These findings suggest that AEA-induced ER stress-apoptosis in NMSC cells is mediated by oxidative stress through a receptor-independent mechanism. Hence, receptor-independent AEA signaling pathways may be targeted to eliminate NMSC. © 2015 Wiley Periodicals, Inc.

Arachidonoyl-ethanolamide activates endoplasmic reticulum stress-apoptosis in tumorigenic keratinocytes: Role of cyclooxygenase-2 and novel J-series prostamides.

Non-melanoma skin cancer and other epithelial tumors overexpress cyclooxygenase-2 (COX-2), differentiating them from normal cells. COX-2 metabolizes arachidonic acid to prostaglandins including, the J-series prostaglandins, which induce apoptosis by mechanisms including endoplasmic reticulum (ER) stress. Arachidonoyl-ethanolamide (AEA) is a cannabinoid that causes apoptosis in diverse tumor types. Previous studies from our group demonstrated that AEA was metabolized by COX-2 to J-series prostaglandins. Thus, the current study examines the role of COX-2, J-series prostaglandins, and ER stress in AEA-induced apoptosis. In tumorigenic keratinocytes that overexpress COX-2, AEA activated the PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE1), and activating transcription factor-6 (ATF6) ER stress pathways and the ER stress apoptosis-associated proteins, C/EBP homologous protein-10 (CHOP10), caspase-12, and caspase-3. Using an ER stress inhibitor, it was determined that ER stress was required for AEA-induced apoptosis. To evaluate the role of COX-2 in ER stress-apoptosis, HaCaT keratinocytes with low endogenous COX-2 expression were transfected with COX-2 cDNA or an empty vector and AEA-induced ER stress-apoptosis occurred only in the presence of COX-2. Moreover, LC-MS analysis showed that the novel prostaglandins, 15-deoxyΔ(12,14) PGJ2 -EA and Δ(12) PGJ2 /PGJ2-EA, were synthesized from AEA. These findings suggest that AEA will be selectively toxic in tumor cells that overexpress COX-2 due to the metabolism of AEA by COX-2 to J-series prostaglandin-ethanolamides (prostamides). Hence, AEA may be an ideal topical agent for the elimination of malignancies that overexpress COX-2.

Arachidonoyl ethanolamide (AEA)-induced apoptosis is mediated by J-series prostaglandins and is enhanced by fatty acid amide hydrolase (FAAH) blockade.

The endocannabinoid arachidonoyl ethanolamide (AEA) is a potent inducer of tumor cell apoptosis however its mechanism of cytotoxicity is unclear. A previous report from our laboratory showed that AEA induced cell death in a cyclooxygenase-2 (COX-2)-dependent manner and in this report our data indicate that AEA-induced apoptosis is mediated by COX-2 metabolic products of the J-series. In experiments conducted with JWF2 keratinocytes which over-express COX-2, AEA caused a concentration-regulated increase in J-series prostaglandin production and apoptosis. Similarly, cell treatment with exogenously added J-series prostaglandins (15-deoxy, Δ(12,14) PGJ(2) and PGJ(2)) induced apoptosis. AEA-induced apoptosis was inhibited by the antioxidant, N-acetyl cysteine, indicating that reactive oxygen species generation was required for apoptosis. Using antagonists of cannabinoid receptor 1, cannabinoid receptor 2, or transient receptor potential cation channel, subfamily V, member 1, it was observed that cannabinoid receptor inhibition did not block AEA-mediated cell death. In contrast, an inhibitor of fatty acid amide hydrolase (FAAH) potentiated AEA-induced J-series PG synthesis and apoptosis. These results suggest that the metabolism of AEA to J-series PGs regulates the induction of apoptosis in cells with elevated COX-2 levels. Our data further indicate that the proapoptotic activity of AEA can be enhanced by combining it with an inhibitor of FAAH. As such, AEA may be an effective agent to eliminate tumor cells that over-express COX-2.

Kinin B1R Activation Induces Endoplasmic Reticulum Stress in Primary Hypothalamic Neurons.

The endoplasmic reticulum (ER) is a key organelle involved in homeostatic functions including protein synthesis and transport, and the storage of free calcium. ER stress potentiates neuroinflammation and neurodegeneration and is a key contributor to the pathogenesis of neurogenic hypertension. Recently, we showed that kinin B1 receptor (B1R) activation plays a vital role in modulating neuroinflammation and hypertension. However, whether B1R activation results in the progression and enhancement of ER stress has not yet been studied. In this brief research report, we tested the hypothesis that B1R activation in neurons contributes to unfolded protein response (UPR) and the development of ER stress. To test this hypothesis, we treated primary hypothalamic neuronal cultures with B1R specific agonist Lys-Des-Arg9-Bradykinin (LDABK) and measured the components of UPR and ER stress. Our data show that B1R stimulation via LDABK, induced the upregulation of GRP78, a molecular chaperone of ER stress. B1R stimulation was associated with an increased expression and activation of transmembrane ER stress sensors, ATF6, IRE1α, and PERK, the critical components of UPR. In the presence of overwhelming ER stress, activated ER stress sensors can lead to oxidative stress, autophagy, or apoptosis. To determine whether B1R activation induces apoptosis we measured intracellular Ca2+ and extracellular ATP levels, caspases 3/7 activity, and cell viability. Our data show that LDABK treatment does increase Ca2+ and ATP levels but does not alter caspase activity or cell viability. These findings suggest that B1R activation initiates the UPR and is a key factor in the ER stress pathway.

Corrigendum: Preclinical and Clinical Assessment of Cannabinoids as Anti-Cancer Agents.

[This corrects the article DOI: 10.3389/fphar.2016.00361.].

Apigenin prevents UVB-induced cyclooxygenase 2 expression: coupled mRNA stabilization and translational inhibition.

Cyclooxygenase 2 (COX-2) is a key enzyme in the conversion of arachidonic acid to prostaglandins, and COX-2 overexpression plays an important role in carcinogenesis. Exposure to UVB strongly increased COX-2 protein expression in mouse 308 keratinocytes, and this induction was inhibited by apigenin, a nonmutagenic bioflavonoid that has been shown to prevent mouse skin carcinogenesis induced by both chemical carcinogens and UV exposure. Our previous study suggested that one pathway by which apigenin inhibits UV-induced and basal COX-2 expression is through modulation of USF transcriptional activity in the 5' upstream region of the COX-2 gene. Here, we found that apigenin treatment also increased COX-2 mRNA stability, and the inhibitory effect of apigenin on UVB-induced luciferase reporter gene activity was dependent on the AU-rich element of the COX-2 3'-untranslated region. Furthermore, we identified two RNA-binding proteins, HuR and the T-cell-restricted intracellular antigen 1-related protein (TIAR), which were associated with endogenous COX-2 mRNA in 308 keratinocytes, and apigenin treatment increased their localization to cell cytoplasm. More importantly, reduction of HuR levels by small interfering RNA inhibited apigenin-mediated stabilization of COX-2 mRNA. Cells expressing reduced TIAR showed marked resistance to apigenin's ability to inhibit UVB-induced COX-2 expression. Taken together, these results indicate that in addition to transcriptional regulation, another mechanism by which apigenin prevents COX-2 expression is through mediating TIAR suppression of translation.

Damage-associated molecular pattern (DAMP) activation in melanoma: investigation of the immunogenic activity of 15-deoxy, Δ12,14 prostamide J2.

Metastatic melanoma is the most deadly skin neoplasm in the United States. Outcomes for this lethal disease have improved dramatically due to the use of both targeted and immunostimulatory drugs. Immunogenic cell death (ICD) has emerged as another approach for initiating antitumor immunity. ICD is triggered by tumor cells that display damage-associated molecular patterns (DAMPs). These DAMP molecules recruit and activate dendritic cells (DCs) that present tumor-specific antigens to T cells which eliminate neoplastic cells. Interestingly, the expression of DAMP molecules occurs in an endoplasmic reticulum (ER) stress-dependent manner. We have previously shown that ER stress was required for the cytotoxic activity of the endocannabinoid metabolite, 15-deoxy, Δ12,14 prostamide J2 (15dPMJ2). As such, the current study investigates whether 15dPMJ2 induces DAMP signaling in melanoma. In B16F10 cells, 15dPMJ2 caused a significant increase in the cell surface expression of calreticulin (CRT), the release of ATP and the secretion of high-mobility group box 1 (HMGB1), three molecules that serve as surrogate markers of ICD. 15dPMJ2 also stimulated the cell surface expression of the DAMP molecules, heat shock protein 70 (Hsp70) and Hsp90. In addition, the display of CRT and ATP was increased by 15dPMJ2 to a greater extent in tumorigenic compared to non-tumorigenic melanocytes. Consistent with this finding, the activation of bone marrow-derived DCs was upregulated in co-cultures with 15dPMJ2-treated tumor compared to non-tumor melanocytes. Moreover, 15dPMJ2-mediated DAMP exposure and DC activation required the electrophilic cyclopentenone double bond within the structure of 15dPMJ2 and the ER stress pathway. These results demonstrate that 15dPMJ2 is a tumor-selective inducer of DAMP signaling in melanoma.

Heme-Dependent ER Stress Apoptosis: A Mechanism for the Selective Toxicity of the Dihydroartemisinin, NSC735847, in Colorectal Cancer Cells.

Colorectal cancer (CRC) is a leading cause of cancer death in the United States. Artemisinin derivatives, including the dihydroartemisinin (DHA) monomers, are widely used as clinical agents for the treatment of malaria. Numerous studies demonstrate that these molecules also display antineoplastic activity with minimal toxicity. Of interest, dimeric DHA molecules are more active than their monomeric counterparts. Our previous data showed that the DHA dimer, NSC735847, was a potent inducer of death in different cancer cell types. However, the mechanism of action and activity of NSC735847 in colon cancer cells was not explored. The present study investigated the anticancer activity of NSC735847 and four structurally similar analog in human tumorigenic (HT-29 and HCT-116) and non-tumorigenic (FHC) colon cell lines. NSC735847 was more cytotoxic toward tumorigenic than non-tumorigenic colonocytes. In addition, NSC735847 exhibited greater cytotoxicity and tumor selectivity than the NSC735847 derivatives. To gain insight into mechanisms of NSC735847 activity, the requirement for endoplasmic reticulum (ER) stress and oxidative stress was tested. The data show that ER stress played a key role in the cytotoxicity of NSC735847 while oxidative stress had little impact on cell fate. In addition, it was observed that the cytotoxic activity of NSC735847 required the presence of heme, but not iron. The activity of NSC735847 was then compared to clinically utilized CRC therapeutics. NSC735847 was cytotoxic toward colon tumor cells at lower concentrations than oxaliplatin (OX). In addition, cell death was achieved at lower concentrations in colon cancer cells that were co-treated with folinic acid (Fol), 5-FU (F), and NSC735847 (FolFNSC), than Fol, F, and OX (FolFOX). The selective activity of NSC735847 and its ability to induce cytotoxicity at low concentrations suggest that NSC735847 may be an alternative for oxaliplatin in the FolFOX regimen for patients who are unable to tolerate its adverse effects.

Impaired mitochondrial function of alveolar macrophages in carbon nanotube-induced chronic pulmonary granulomatous disease.

Human exposure to carbon nanotubes (CNT) has been associated with the development of pulmonary sarcoid-like granulomatous disease. Our previous studies demonstrated that multi-walled carbon nanotubes (MWCNT) induced chronic pulmonary granulomatous inflammation in mice. Granuloma formation was accompanied by decreased peroxisome proliferator-activated receptor gamma (PPARγ) and disrupted intracellular lipid homeostasis in alveolar macrophages. Others have shown that PPARγ activation increases mitochondrial fatty acid oxidation (FAO) to reduce free fatty acid accumulation. Hence, we hypothesized that the disrupted lipid metabolism suppresses mitochondrial FAO. To test our hypothesis, C57BL/6 J mice were instilled by an oropharyngeal route with 100 µg MWCNT freshly suspended in 35 % Infasurf. Control sham mice received vehicle alone. Sixty days following instillation, mitochondrial FAO was measured in permeabilized bronchoalveolar lavage (BAL) cells. MWCNT instillation reduced the mitochondrial oxygen consumption rate of BAL cells in the presence of palmitoyl-carnitine as mitochondrial fuel. MWCNT also reduced mRNA expression of mitochondrial genes regulating FAO, carnitine palmitoyl transferase-1 (CPT1), carnitine palmitoyl transferase-2 (CPT2), hydroxyacyl-CoA dehydrogenase subunit beta (HADHB), and PPARγ coactivator 1 alpha (PPARGC1A). Importantly, both oxidative stress and apoptosis in alveolar macrophages and lung tissues of MWCNT-instilled mice were increased. Because macrophage PPARγ expression has been reported to be controlled by miR-27b which is known to induce oxidative stress and apoptosis, we measured the expression of miR-27b. Results indicated elevated levels in alveolar macrophages from MWCNT-instilled mice compared to controls. Given that inhibition of FAO and apoptosis are linked to M1 and M2 macrophage activation, respectively, the expression of both M1 and M2 key indicator genes were measured. Interestingly, results showed that both M1 and M2 phenotypes of alveolar macrophages were activated in MWCNT-instilled mice. In conclusion, alveolar macrophages of MWCNT-instilled mice had increased miR-27b expression, which may reduce the expression of PPARγ resulting in attenuation of FAO. This reduction in FAO may lead to activation of M1 macrophages. The upregulation of miR-27b may also induce apoptosis, which in turn can cause M2 activation of alveolar macrophages. These observations indicate a possible role of miR-27b in impaired mitochondrial function in the chronic activation of alveolar macrophages by MWCNT and the development of chronic pulmonary granulomatous inflammation.

Metabolism of anandamide by COX-2 is necessary for endocannabinoid-induced cell death in tumorigenic keratinocytes.

Nonmelanoma skin cancer is the most prevalent cancer in the United States with approximately 1.25 million new cases diagnosed each year. Cyclooxygenase-2 (COX-2) expression is commonly elevated in these and other epithelial tumors. Cyclooxygenases metabolize arachidonic acid to prostaglandins, which promote growth and survival of tumor cells. COX-2 also metabolizes endocannabinoids forming prostaglandin-ethanolamides (PG-EA); however, the role of these lipid molecules in tumor cell survival is unclear. The goal of this research is to determine if the metabolic products of COX-2 contribute to endocannabinoid-induced cell death. Anandamide [also known as arachidonyl ethanolamide (AEA)] induced cell death in the COX-2 overexpressing squamous carcinoma cell line JWF2. In contrast, AEA did not initiate cell death in HaCaT keratinocytes, which express low basal levels of COX-2. Resistance to AEA-mediated cell death in HaCaT cells was reversed by overexpressing COX-2 in these cells. Next, ELISA assays were carried out to identify prostaglandins involved in AEA-mediated cell death. D-type prostaglandins were predominantly formed in AEA-exposed JWF2 cells although significant increases in E- and F-type prostaglandins were also seen. Cells were then treated with various prostaglandins or PG-EA to determine the contribution of each to AEA-induced cell death. PGD(2) and PGD(2)-EA were found to be cytotoxic to JWF2 keratinocytes and the PGD(2) dehydration products, PGJ(2) and 15-deoxy Delta(12,14) PGJ(2), were also potent inducers of cell death. These results suggest that AEA selectively induces cell death in tumorigenic keratinocytes due to COX-2 overexpression and the resulting metabolism of AEA to cytotoxic prostaglandins.

Cannabinoids Modulate Neuronal Activity and Cancer by CB1 and CB2 Receptor-Independent Mechanisms.

Cannabinoids include the active constituents of Cannabis or are molecules that mimic the structure and/or function of these Cannabis-derived molecules. Cannabinoids produce many of their cellular and organ system effects by interacting with the well-characterized CB1 and CB2 receptors. However, it has become clear that not all effects of cannabinoid drugs are attributable to their interaction with CB1 and CB2 receptors. Evidence now demonstrates that cannabinoid agents produce effects by modulating activity of the entire array of cellular macromolecules targeted by other drug classes, including: other receptor types; ion channels; transporters; enzymes, and protein- and non-protein cellular structures. This review summarizes evidence for these interactions in the CNS and in cancer, and is organized according to the cellular targets involved. The CNS represents a well-studied area and cancer is emerging in terms of understanding mechanisms by which cannabinoids modulate their activity. Considering the CNS and cancer together allow identification of non-cannabinoid receptor targets that are shared and divergent in both systems. This comparative approach allows the identified targets to be compared and contrasted, suggesting potential new areas of investigation. It also provides insight into the diverse sources of efficacy employed by this interesting class of drugs. Obtaining a comprehensive understanding of the diverse mechanisms of cannabinoid action may lead to the design and development of therapeutic agents with greater efficacy and specificity for their cellular targets.

Synthesis and Evaluation of the Novel Prostamide, 15-Deoxy, Δ12,14-Prostamide J2, as a Selective Antitumor Therapeutic.

15-deoxy, Δ12,14-prostaglandin J2-ethanolamide, also known as 15-deoxy, Δ12,14-prostamide J2 (15d-PMJ2) is a novel product of the metabolism of arachidonoyl ethanolamide (AEA) by COX-2. 15d-PMJ2 preferentially induced cell death and apoptosis in tumorigenic A431 keratinocytes and B16F10 melanoma cells compared with nontumorigenic HaCaT keratinocytes and Melan-A melanocytes. Activation of the ER stress execution proteins, PERK and CHOP10, was evaluated to determine whether this process was involved in 15d-PMJ2 cell death. 15d-PMJ2 increased the phosphorylation of PERK and expression of CHOP10 in tumorigenic but not nontumorigenic cells. The known ER stress inhibitors, salubrinal and 4-phenylbutaric acid, significantly inhibited 15d-PMJ2-mediated apoptosis, suggesting ER stress as a primary apoptotic mediator. Furthermore, the reactive double bond present within the cyclopentenone structure of 15d-PMJ2 was identified as a required moiety for the induction of ER stress apoptosis. The effect of 15d-PMJ2 on B16F10 melanoma growth was also evaluated by dosing C57BL/6 mice with 0.5 mg/kg 15d-PMJ2 Tumors of animals treated with 15d-PMJ2 exhibited significantly reduced growth and mean weights compared with vehicle and untreated animals. TUNEL and IHC analysis of tumor tissues showed significant cell death and ER stress in tumors of 15d-PMJ2-treated compared with control group animals. Taken together, these findings suggest that the novel prostamide, 15d-PMJ2, possesses potent antitumor activity in vitro and in vivoMol Cancer Ther; 16(5); 838-49. ©2017 AACR.

Apigenin inhibits COX-2, PGE2, and EP1 and also initiates terminal differentiation in the epidermis of tumor bearing mice.

Non-melanoma skin cancer (NMSC) is the most prevalent cancer in the United States. NMSC overexpresses cyclooxygenase-2 (COX-2). COX-2 synthesizes prostaglandins such as PGE2 which promote proliferation and tumorigenesis by engaging G-protein-coupled prostaglandin E receptors (EP). Apigenin is a bioflavonoid that blocks mouse skin tumorigenesis induced by the chemical carcinogens, 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the effect of apigenin on the COX-2 pathway has not been examined in the DMBA/TPA skin tumor model. In the present study, apigenin decreased tumor multiplicity and incidence in DMBA/TPA-treated SKH-1 mice. Analysis of the non-tumor epidermis revealed that apigenin reduced COX-2, PGE2, EP1, and EP2 synthesis and also increased terminal differentiation. In contrast, apigenin did not inhibit the COX-2 pathway or promote terminal differentiation in the tumors. Since fewer tumors developed in apigenin-treated animals which contained reduced epidermal COX-2 levels, our data suggest that apigenin may avert skin tumor development by blocking COX-2.

Preclinical and Clinical Assessment of Cannabinoids as Anti-Cancer Agents.

Cancer is the second leading cause of death in the United States with 1.7 million new cases estimated to be diagnosed in 2016. This disease remains a formidable clinical challenge and represents a substantial financial burden to the US health care system. Therefore, research and development of novel therapeutics for the treatment of cancer is of high priority. Cannabinoids and their derivatives have been utilized for their medicinal and therapeutic properties throughout history. Cannabinoid activity is regulated by the endocannabinoid system (ECS), which is comprised of cannabinoid receptors, transporters, and enzymes involved in cannabinoid synthesis and breakdown. More recently, cannabinoids have gained special attention for their role in cancer cell proliferation and death. However, many studies investigated these effects using in vitro models which may not adequately mimic tumor growth and metastasis. As such, this article aims to review study results which evaluated effects of cannabinoids from plant, synthetic and endogenous origins on cancer development in preclinical animal models and to examine the current standing of cannabinoids that are being tested in human cancer patients.

Receptor-dependent and receptor-independent endocannabinoid signaling: a therapeutic target for regulation of cancer growth.

The endocannabinoid system comprises the G-protein coupled CB1 cannabinoid receptor (CB1R) and CB2 cannabinoid receptor (CB2R), their endogenous ligands (endocannabinoids), and the enzymes responsible for their synthesis and catabolism. Recent works have revealed several important interactions between the endocannabinoid system and cancer. Moreover, it is now well established that synthetic small molecule cannabinoid receptor agonist acting on either CB1R or CB2R or both exerts anti-cancer effects on a variety of tumor cells. Recent results from many laboratories reported that the expression of CB1R and CB2R in prostate cancer, breast cancer, and many other cancer cells is higher than that in corresponding non-malignant tissues. The mechanisms by which cannabinoids acting on CB1R or CB2R exert their effects on cancer cells are quite diverse and complex. Further, several studies demonstrated that some of the anti-proliferative and apoptotic effects of cannabinoids are mediated by receptor-independent mechanisms. In this minireview we provide an overview of the major findings on the effects of endogenous and/or synthetic cannabinoids on breast and prostate cancers. We also provide insight into receptor independent mechanisms of the anti-cancer effects of cannabinoids under in vitro and in vivo conditions.

Enhancement of UVB-induced apoptosis by apigenin in human keratinocytes and organotypic keratinocyte cultures.

Topical application of the bioflavonoid 4',5,7-trihydroxyflavone (apigenin) to mouse skin effectively reduces the incidence and size of skin tumors caused by UVB exposure. The ability to act as a chemopreventive compound indicates that apigenin treatment alters the molecular events initiated by UVB exposure; however, the effects of apigenin treatment on UVB-irradiated keratinocytes are not fully understood. In the present study, we have used three models of human keratinocytes to study the effect of apigenin treatment on UVB-induced apoptosis: HaCaT human keratinocyte cells, primary keratinocyte cultures isolated from human neonatal foreskin, and human organotypic keratinocyte cultures. Each keratinocyte model was exposed to a moderate dose of UVB (300-1,000 J/m(2)), then treated with apigenin (0-50 micromol/L), and harvested to assess apoptosis by Western blot analysis for poly(ADP)ribose polymerase cleavage, annexin-V staining by flow cytometry, and/or the presence of sunburn cells. Apigenin treatment enhanced UVB-induced apoptosis >2-fold in each of the models tested. When keratinocytes were exposed to UVB, apigenin treatment stimulated changes in Bax localization and increased the release of cytochrome c from the mitochondria compared with UVB exposure alone. Overexpression of the antiapoptotic protein Bcl-2 and expression of a dominant-negative form of Fas-associated death domain led to a reduction in the ability of apigenin to enhance UVB-induced apoptosis. These results suggest that enhancement of UVB-induced apoptosis by apigenin treatment involves both the intrinsic and extrinsic apoptotic pathways. The ability of apigenin to enhance UVB-induced apoptosis may explain, in part, the photochemopreventive effects of apigenin.

Modulation of UVB-induced and basal cyclooxygenase-2 (COX-2) expression by apigenin in mouse keratinocytes: role of USF transcription factors.

Apigenin is a bioflavonoid with chemopreventive activity against UV- or chemically-induced mouse skin tumors. To further explore the mechanism of apigenin's chemopreventive activity, we determined whether apigenin inhibited UVB-mediated induction of cyclooxygenase-2 (COX-2) expression in mouse and human keratinocytes. Apigenin suppressed the UVB-induced increase in COX-2 protein and mRNA in mouse and human keratinocyte cell lines. UVB radiation of keratinocytes transfected with a mouse COX-2 promoter/luciferase reporter plasmid resulted in a threefold increase in transcription from the promoter, and apigenin inhibited the UV-induced promoter activity at doses of 5-50 microM. Transient transfections with COX-2 promoter deletion constructs and COX-2 promoter constructs containing mutations in specific enhancer elements indicated that the effects of UVB required intact Ebox and ATF/CRE response elements. Electrophoretic mobility shift assays with supershifting antibodies were used to identify USF-1, USF-2, and CREB as proteins binding to the ATF/CRE-Ebox responsive element of the COX-2 promoter. Keratinocytes co-transfected with the COX-2 luciferase reporter and a USF-2 expression vector, alone or in combination with a USF-1 expression vector, exhibited enhanced promoter activity in both UVB-irradiated and nonirradiated cultures. However, COX-2 promoter activity was inhibited in keratinocytes co-transfected with USF-1 alone. Finally, we present data showing that the suppressive effect of apigenin on COX-2 expression could be reversed by co-expression of USF-1 and USF-2. These results suggest that one pathway by which apigenin inhibits COX-2 expression is through modulation of USF transcriptional activity.

Do truncated cyclins contribute to aberrant cyclin expression in cancer?

Cyclin overexpression is found in several types of cancer. Genetic events that place cyclin genes under the control of active promoters or that increase cyclin gene copy number account for most instances of cyclin overexpression. New paradigms for aberrant cyclin expression have been suggested by studies showing that truncated cyclins are expressed in specific subsets of cancer. The altered cyclins lack regulatory sequences (compared to the wild-type protein) that modulate their stability, subcellular localization or cdk-associated kinase activity. In this communication, we review the current literature and assess the role of truncated cyclins D, E, A, B, C and virus encoded-cyclin D (K-cyclin) in the development of cancer. We also report the molecular characteristics, expression patterns and if available, prognostic significance of these proteins.

Inhibition of TPA-induced cyclooxygenase-2 (COX-2) expression by apigenin through downregulation of Akt signal transduction in human keratinocytes.

Apigenin is a nonmutagenic bioflavonoid that has been shown to be an inhibitor of mouse skin carcinogenesis induced by the two-stage regimen of initiation and promotion with dimethylbenzanthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). These DMBA/TPA-induced squamous cell carcinomas overexpress cyclooxygenase-2 (COX-2). Cyclooxygenases are key enzymes required for prostaglandin (PG) synthesis, converting the arachidonic acid (AA) released by phospholipase A2 into prostaglandins. A large body of evidence indicates that the inducible form of cyclooxygenase, COX-2, is involved in tumor promotion and carcinogenesis in a wide variety of tissue types, including colon, breast, lung, and skin. In the present study, we have determined that apigenin inhibited the TPA-induced increase in COX-2 protein and mRNA in the human keratinocyte cell line; HaCaT. The induction of COX-2 elicited by TPA correlated with increased activation of Akt kinase and cell treatment with the PI3 kinase inhibitor, LY294002, blocked TPA induction of COX-2. In cells treated with TPA and apigenin, the inhibition of COX-2 expression correlated with inhibition of Akt kinase activation. Apigenin-mediated inhibition of TPA-induced COX-2 expression was reversed by transient transfection with constitutively active Akt (CA-Akt). Chemical inhibitors of MEK (PD98059), p38 (SB202190), but not JNK (SP600125) blocked TPA induction of COX-2 although apigenin did not inhibit TPA-mediated COX-2 expression through these pathways. The TPA-induced release of AA from HaCaT cells was also inhibited by cell treatment with apigenin. These data show that apigenin inhibits TPA-mediated COX-2 expression by blocking signal transduction of Akt and that apigenin also blocks AA release, which may contribute to its chemopreventive activity.