Rukiyah Van Dross

Apigenin Prevents UVB-Induced Cyclooxygenase 2 Expression: Coupled mRNA Stabilization and Translational Inhibition

ABSTRACT 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 apigenins 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.

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.

The Chemopreventive Bioflavonoid Apigenin Modulates Signal Transduction Pathways in Keratinocyte and Colon Carcinoma Cell Lines

Apigenin is a nonmutagenic chemopreventive agent found in fruits and green vegetables. In this study, we used two different epithelial cell lines (308 mouse keratinocytes and HCT116 colon carcinoma cells) to determine the effect of apigenin on the mitogen-activated protein kinase (MAPK) cascade. Apigenin induced a dose-dependent phosphorylation of both extracellular signal-regulated protein kinase (ERK) and p38 kinase but had little effect on the phosphorylation of c-jun amino terminal kinase (JNK). We used immunoprecipitation-coupled kinase assays to show that apigenin increased the kinase activity of ERK and p38 but not JNK. Consistent with these results, we found that apigenin induced a 7.4-fold induction in the phosphorylation of Elk, the downstream phosphorylation target of ERK kinase. Similarly, apigenin induced a 3.2-fold induction in the phosphorylation of activating transcription factor-2, the downstream phosphorylation target of p38 kinase. Little change was observed in the phosphorylation of c-jun, the phosphorylation target of JNK. These data suggest that part of the chemopreventive activity of apigenin may be mediated by its ability to modulate the MAPK cascade.

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 apigenins 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 µM. 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.

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 ∼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. PGD2 and PGD2‐EA were found to be cytotoxic to JWF2 keratinocytes and the PGD2 dehydration products, PGJ2 and 15‐deoxy Δ12,14 PGJ2, 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.

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, Δ12,14 PGJ2 and PGJ2) 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. Mol. Carcinog.

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.

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‐deoxyΔ12,14PGJ2‐EA and Δ12PGJ2/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.

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.

Do Truncated Cyclins Contribute to Aberrant Cyclin Expression in Cancer

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