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Metastatic melanoma (MM) is the deadliest form of skin cancer in the United States, with one person dying from the disease every hour. Metastasis occurs when cells from the primary tumor enter the systemic circulation and grow at local and distant organ sites. The most common sites of melanoma metastasis are the lymph nodes, bone, brain, and lungs. Melanoma that has metastasized is not effectively treated with cytotoxic therapeutics that are currently available, although immunotherapeutic and targeted agents produce more favorable outcomes. Despite the availability of these effective agents, MM is still associated with high mortality rates. In order to develop new and highly effective agents that robustly eliminate MM, current models of metastatic disease in immunocompetent animals must be improved. Several in vivo models, including those that look at endocannabinoids and their metabolites, focus on injecting tumor cells directly into the circulation via the tail vein to allow lesions to form within the lungs. While these models permit rapid metastasis at relevant sites, they lack primary tumor formation and therefore do not mimic the clinical disease. Other MM models employ subcutaneous tumor inoculation to permit primary tumors to spontaneously metastasize. However, the subcutaneous space is not an orthotopic site for melanoma. Therefore, well-established models of spontaneous metastasis from orthotopic sites must be improved to more accurately recapitulate MM and study the effect of endocannabinoids and their metabolites in this disease. The goal of the current study is to modify an existing syngeneic, orthotopic model of spontaneous MM to more precisely reproduce clinical features of metastasis. In this model, B16F10 melanoma cells that expressed firefly luciferase were inoculated on the dorsal ear of C57BL/6 mice. Spontaneous metastasis was then facilitated by removing the primary lesion to prolong the duration before humane endpoints were reached. Metastatic tumor formation in the draining lymph node and at distant sites was observed by using the IVIS Lumina imaging system and by gross examination of target organs after euthanasia. According to our preliminary data, primary tumors developed after an average of 2-3 weeks and metastatic lesions formed in the draining lymph node approximately 8 weeks after inoculation. Phenotypic characteristics that served as markers of metastasis and determinants of primary tumor removal were also identified. Histopathologic analysis of the primary and metastatic tumors revealed that the morphologic appearance of the lesions was consistent with patient melanoma. Further optimization of this model will allow us to mimic MM more precisely. Animal tumor models with greater translational value will provide a more comprehensive understanding of the metastatic process and, ultimately, the effects of endocannabinoids and their metabolites in MM. Citation Format: Rukiyah T. Van Dross, Ariel Myers, Kathleen Thayne. Defining an orthotopic, syngeneic metastatic melanoma model for characterizing the immunotherapeutic activity of the prostamide, 15dPMJ2 [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B34.

Penelitian ini bertujuan untuk mengetahui pengaruh metode proyek terhadap kemampuan sains pada anak. Penelitian ini menggunakan jenis pre-eksperimen dan desain One Shoot Case Study dengan sampel terdri dari 18 anak. Teknik analisis data menggunakan Uji t. Hasil analisis thitung = 6,62 yang diperoleh lebih besar dari nilai ttabel = 1,74, sehingga terdapat pengaruh mentode proyek terhadap kemampuan sains anak TK B PAUD Terpadu Happy Kids Palembang. Hal ini terbukti dari nilai rata-rata posttest anak sebesar 77,33 dan berada pada kategori Berkembang Sesuai Harapan (BSH) dengan indikator melakukan percobaan pencampuran warna adalah indikator dengan nilai paling menonjol. Sebanyak 55,55 % berada dalam kategori BSB, 33,33% berada pada kategori BSH, 11,11% pada kategori MB dan tidak ada anak termasuk kategori BB. Sebanyak 40% anak berada pada kategori BSB, 35,2% berada pada kategori BSH, 23,52% berada pada kategori MB dan tidak ada anak yang berada pada kategori BB. Kata Kunci: Metode, Metode proyek, kemampuan sains

Badan akreditasi merupakan lembaga yang bekerja berdasarkan dasar hukum yang kuat dalam sistem pendidikan nasional, dan secara kelembagaan yang telah memiliki tujuan, fungsi, prinsip, kewenangan dan tugas yang jelas, walaupun dalam melaksanakan kegiatannya masih menghadapi beberapa kendala. Salah satu tujuan pembangunan Indonesia adalah untuk mencerdaskan kehidupan bangsa oleh sebab itu Undang-Undang dasar 1945 menyebutkan pada pasal 33 bahwa rakyat Indonesia berhak mendapatkan pendidikan sehingga pemerintah berkewajiban mewujudkan sistem pendidikan Indonesia, dan memberi Anggaran pendidikan paling kurang sebanyak 20% dari anggaran pendapatan Negara atau anggaran pendapatan daerah. Agar masyarakat mendapatkan pendidikan yang bermutu, maka fasilitas pemerintah sebagai wujud kewajiban pemerintah, adalah menciptakan mekanisme dan sistem agar pendirian satuan pendidikan dapat memenuhi syarat dan bertanggungjawab atas pelayanan yang diberikan sehingga memberi kualitas pendidikan yang memenuhi harapan masyarakat. Oleh karena itu, sejak berdiri, berkembang dan sepanjang keberadaan satuan pendidikan sudah semestinya mendapatkan penilaian dan evaluasi agar memenuhi standar yang diharapkan, yaitu sesuai dengan Peraturan Pemerintah (PP) No. 19 Tahun 2005 tentang Standar Nasional Pendidikan.Kata Kunci: badan akreditasi, nasional, madrasah, sekolah

Metastatic melanoma (MM) is the deadliest form of skin cancer in the United States with one person dying from the disease every hour. Metastasis occurs when cells from the primary tumor enter the systemic circulation and grow at local and distant organ sites. The most common sites of melanoma metastasis are the lymph nodes, bone, brain, and lungs. Melanoma that has metastasized is not effectively treated with cytotoxic therapeutics that are currently available, although immunotherapeutic and targeted agents produce more favorable outcomes. Despite the availability of these effective agents, MM is still associated with high mortality rates. In order to develop new and highly effective agents that robustly eliminate MM, current models of metastatic disease in immunocompetent animals must be improved. Several in vivo models focus on injecting tumor cells directly into the circulation via the tail vein to allow lesions to form within the lungs. While these models permit rapid metastasis at a relevant sites, they lack primary tumor formation and therefore, do not mimic the clinical disease. Other MM models employ subcutaneous tumor inoculation to permit primary tumors to spontaneously metastasize. However, the subcutaneous space is not an orthotopic site for melanoma. Therefore, well-established models of spontaneous metastasis from orthotopic sites must be improved to more accurately, recapitulate MM. The goal of the current study is to modify an existing syngeneic, orthotopic model of spontaneous MM to more precisely reproduce clinical features of metastasis. In this model, B16F10 melanoma cells that expressed firefly luciferase were inoculated on the dorsal ear of C57BL/6 mice. Spontaneous metastasis was then facilitated by removing the primary lesion to prolong the duration before humane endpoints were reached. Metastatic tumor formation in the draining lymph node and at distant sites were observed by using the IVIS Lumina imaging system and by gross examination of target organs after euthanasia. According to our preliminary data, primary tumors developed after an average of 2.8 weeks. Metastatic lesions formed in the draining lymph node approximately 8.8 weeks after inoculation and distant metastasis were subsequently observed. Phenotypic characteristics that served as markers of metastasis and determinants of primary tumor removal were also identified. Histopathologic analysis of the primary and metastatic tumors revealed that the morphological appearance of the lesions was consistent with patient melanoma. Further optimization of this model will allow us to mimic MM more precisely. Animal tumor models with greater translational value will provide a more comprehensive understanding of the metastatic process as well as the effects of clinical and experimental therapeutics. Citation Format: Ariel L. Myers, Kathleen A. Thayne, Rukiyah Van Dross, Gina Murray. Optimization of a syngeneic animal model for metastatic melanoma: From ear to lymph node and beyond [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4606.

Melanoma is the most aggressive and deadly form of cutaneous cancer in the United States, representing a major clinical challenge. Our lab previously demonstrated that the endocannabinoid metabolite, 15-deoxy, Δ12,14 prostamide J2 (15d-PMJ2), was an effective and selective inducer of apoptotic cell death in B16F10 murine melanoma cells. Furthermore, we found that 15d-PMJ2 mediated apoptosis was regulated by endoplasmic reticulum (ER) stress. Numerous studies have determined that ER stress causes Ca+2 flux, and that this action initiates cellular apoptosis. As such, we investigated Ca+2 mobilization in the context of 15d-PMJ2-mediated ER stress. In B16F10 melanoma cells, 15d-PMJ2 significantly increased cytosolic Ca+2 levels. Blockade of Ca+2 channels using ruthenium red (a pan Ca+2 channel inhibitor) or 2-APB (an IP3 selective inhibitor) decreased the cytotoxicity of 15d-PMJ2. To investigate the role of ER stress in Ca+2 mobilization, cells were pretreated with the ER stress inhibitors, phenylbutyric acid (PBA) and GSK2602 (a PERK-selective inhibitor). Both inhibitors significantly lowered cytosolic Ca+2 levels suggesting Ca+2 flux is mediated by ER stress. Moreover, BAP31, an important regulator of endoplasmic Ca+2 release and ER stress was activated following exposure to 15d-PMJ2. To understand the impact of elevated Ca+2 levels, we investigated cysteine proteases (calpains) and mitochondrial Ca+2, both of which induce apoptosis in a Ca+2-dependent manner. Our results show that mitochondrial Ca+2 levels were significantly increased by 15d-PMJ2 and that this effect was prevented by blocking ER stress. In contrast, 15d-PMJ2 increased calpain-2 activity however, the inhibition of calpain had no effect on the cytotoxicity of 15d-PMJ2. These findings indicate that mitochondrial Ca2+ flux, but not calpains regulate ER stress apoptosis induced by 15d-PMJ2. It has been reported that the activity of J-series prostaglandins is conferred by an electrophilic double bond contained within its cyclopentenone ring. To evaluate the significance of this moiety in 15d-PMJ2, a neutral analog (neutral-15d-PMJ2) lacking the double bond was synthesized. We determined that that the accumulation of intracellular and mitochondrial Ca2+ was dependent on the electrophilic double bond, suggesting it is a critical structural component required for activity. Taken together, these data indicate that the mobilization of calcium to the cytoplasm and mitochondria may critically regulate ER stress apoptosis that is mediated by 15d-PMJ2. Citation Format: Daniel Alexander Ladin, Estefani Cota, Rukiyah Van Dross. Regulation of ER stress-mediated cell death by calcium mobilization: A potential mitochondrial pathway for 15-deoxy-Δ12,14prostamide J2cytotoxicity in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2663.

The endoplasmic reticulum (ER) is a cellular organelle that is primarily responsible for oxidative protein folding. When the protein folding load in cells exceeds the protein folding capacity, ER stress occurs. The ER stress pathway is a prominent regulator of cancer cell death and as a result, ER stress inducers are being exploited pharmacologically. Cytotoxic ER stress is typically regulated by the transcription factor, C/EBP homologous protein 10 (CHOP10). Transcriptional products of CHOP10 include pro-apoptotic genes, such as tribbles-related protein 3 (TRB3), death receptor-5 (DR5), and ER oxidoreductase 1α (ERO1α). Our previous data showed that apoptosis mediated by 15deoxy, Δ12,14 prostamide J2 (15dPMJ2) occurred in an ER stress-dependent manner. However, the signaling pathway that regulates 15dPMJ2 mediated ER stress apoptosis has not been identified. Therefore, the goal of this study is to determine the role of CHOP10 and its transcriptional targets in ER stress apoptosis that is induced by 15dPMJ2 in colon cancer cells. According to our data, 15dPMJ2 was 3-fold more effective in inducing apoptosis in the human colon cancer cell line, HCT116, compared to the non-tumorigenic colon cell line, FHC. The induction of apoptosis in HCT116 cells occurred coincident with a significant increase in CHOP10 protein expression. In addition, blockade of the ER stress pathway with 4-phenylbuterate (PBA) or salubrinal prevented apoptosis indicating that cell death is reliant upon ER stress. To determine the role of CHOP10 in this process, CRISPR/Cas9 CHOP10 knockout HCT116 cells (CHOP10-KO-HCT116) were generated. As anticipated, 15dPMJ2 increased CHOP10 expression in wt-HCT116 cells but not in CHOP10-KO-HCT116 cells. Furthermore, 15dPMJ2 increased the expression of ERO1α, DR5, TRB3, and it induced apoptosis in wt-HCT116 cells, but not in CHOP10-KO cells. Interestingly, the induction of TRB3, but not DR5 or ERO1α, was completely inhibited in cells devoid of CHOP10 expression. Therefore, the activity of protein kinase B (PKB)/Akt, a known TRB3 target, was investigated. CHOP10 stimulation by 15dPMJ2 inactivated Akt in wt-HCT116 cells, but not in CHOP10-KO cells. These findings suggest the importance of CHOP10, TRB3, and Akt in the control of the ER stress-mediated apoptosis in response to 15dPMJ2 treatment. Thus, 15dPMJ2 is a potential chemotherapeutic agent that inhibits PKB/Akt survival signaling by upregulating the CHOP10/TRB3 pathway to prevent colon cancer growth. Citation Format: Hussam Albassam, Daniel A. Ladin, Rukiyah Van Dross. CHOP10/TRB3/Akt signaling regulates ER stress apoptosis in colon cancer cells treated with prostamide-J [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3873.

Melanoma is the most lethal form of skin cancer in the United States. Current trends show that melanoma incidence has been increasing for the last 30 years. Chemotherapeutic agents for advanced stage metastatic melanoma have limited efficacy. Although the use of immune-modulating agents such as checkpoint inhibitors has significantly improved therapeutic outcomes, these agents can cause life-threatening autoimmune side effects. Therefore, immunotherapeutic agents that can preferentially eliminate melanoma cells are needed. Agents that induce the release of damage-associated molecular patterns (DAMPs) can stimulate antitumor immunity. DAMPs are produced by damaged or dying cancer cells and include signals such as expression of cell surface calreticulin (ecto-CRT) and the secretion of ATP. DAMPs can recruit and activate immune cells such as dendritic cells which in turn stimulate tumor-specific cytotoxic T cells to kill cancer cells in a process referred to as immunogenic cell death. The elicitation of DAMPs depends on endoplasmic reticulum stress (ER) and oxidative stress. Our group found that our patented agent, 15-deoxy, Δ12,14-prostaglandin J2-ethanolamide (15dPMJ2), inhibits melanoma tumor growth. In addition, 15dPMJ2 induces DAMPs in melanoma cells via an ER stress dependent mechanism. The purpose of this study was to investigate the selectivity of DAMP induction in melanoma by 15dPMJ2, the effect of 15dPMJ2-treated cells on the activation of bone marrow-derived dendritic cells (BMDCs) and the role of oxidative stress in this process. To determine if 15dPMJ2 causes tumor-selective activation of BMDCs, we first examined ecto-CRT and the release of ATP in B16F10 melanoma cells and Melan-A non-tumorigenic melanocytes. Our results showed that 15dPMJ2 significantly increased DAMP expression in tumorigenic compared to non-tumorigenic melanocytes. Importantly, we found that 15dPMJ2-treated B16F10 cells induced maturation of BMDCs as manifested by an increase in the expression of MHCII, CD80 and CD86. However, 15dPMJ2-treated Melan-A cells did not activate BMDCs. In this study, we also tested the role of oxidative stress in 15dPMJ2-induced DAMPs. Our results showed that the antioxidant Trolox significantly decreased 15dPMJ2 induced ecto-CRT and ATP release in B16F10 cells. However, Trolox did not inhibit the activation of BMDCs by 15dPMJ2-treated B16F10 cells. These results suggest that oxidative stress plays an important role in 15dPMJ2-induced DAMPs, but it is not required for the BMDC activation. In conclusion, 15dPMJ2can selectively induce DAMPs in melanoma which results in the activation of BMDCs. Hence, 15dPMJ2 is a potential small molecule immunotherapeutic for melanoma. Citation Format: Ahmed Elhassanny, Rene Escobedo, Daniel Ladin, Rukiyah Van Dross. Damage-associated molecular pattern (DAMP) induction by 15-deoxy, Δ12,14-prostaglandin J2-ethanolamide: Examination of tumor selectivity, dendritic cell activation and oxidative stress in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3276.

15-deoxy, Δ

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.

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.

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/m2), then treated with apigenin (0–50 μmol/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. [Cancer Res 2008;68(8):3057–65]

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 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. © 2006 Wiley-Liss, Inc.

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 apigenin9s 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.

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. © 2015 Wiley Periodicals, Inc.

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.

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.

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.

The Pneumocystis carinii topoisomerase I-encoding gene has been cloned and sequenced, and the expressed enzyme interactions with several classes of topoisomerase I poisons have been characterized. The P. carinii topoisomerase I protein contains 763 amino acids and has a molecular mass of ca. 90 kDa. The expressed enzyme relaxes supercoiled DNA to completion and has no Mg 2+ requirement. Cleavage assays reveal that both the human and P. carinii enzymes form covalent complexes in the presence of camptothecin, Hoechst 33342, and the terbenzimidazole QS-II-48. As with the human enzyme, no cleavage is stimulated in the presence of 4′,6′-diamidino-2-phenylindole (DAPI) or berenil. A yeast cytotoxicity assay shows that P. carinii topoisomerase I is also a cytotoxic target for the mixed intercalative plus minor-groove binding drug nogalamycin. In contrast to the human enzyme, P. carinii topoisomerase I is resistant to both nitidine and potent protoberberine human topoisomerase I poisons. The differences in the sensitivities of P. carinii and human topoisomerase I to various topoisomerase I poisons support the use of the fungal enzyme as a molecular target for drug development. Additionally, we have characterized the interaction of pentamidine with P. carinii topoisomerase I. We show, by catalytic inhibition, cleavage, and yeast cytotoxicity assays, that pentamidine does not target topoisomerase I.

Non-melanoma skin cancer (NMSC) is the most common cancer in the United States. The absence of selective toxicity is a major problem that limits the utility of chemotherapeutic and radiation therapy for NMSCs. Our previous data showed that the endocannabinoid, anandamide, selectively induced apoptosis in non-melanoma skin cancer (NMSC) cells which overexpress cyclooxygenase-2. The cytotoxic effect of anandamide was mediated by the production of ethanolamide-conjugated D- and J-series prostaglandins (PGs), also known as prostamide D2 (PMD2) and prostamide J2 (PMJ2), respectively. The aim of the current study was to determine if tumorigenic keratinocytes are more sensitive to PMD2 than non-tumorigenic cells and to examine whether the cytotoxicity of PMD2 is mediated by its downstream metabolite PMJ2. To determine if PMD2 demonstrates preferential toxicity, tumorigenic (JWF2) and non-tumorigenic (HaCaT) keratinocytes were utilized. A significant reduction in cell viability was observed in JWF2 but not in HaCaT cells treated with PMD2. In tumorigenic keratinocytes, PMD2 induced apoptotic cell death, oxidative stress and increased the expression of apoptotic ER stress protein, C/EBP homologous protein-10 (CHOP10). In addition, use of the antioxidant, trolox, and ER stress inhibitor, salubrinal, inhibited the cytotoxic effect of PMD2. Furthermore, the use of prostaglandin D receptor (DP1 and DP2) antagonists did not inhibit PMD2-induced apoptosis indicating that the activity was mediated by a receptor-independent pathway. Similar effects were observed in keratinocytes treated with the structurally-related arachidonic acid metabolite, prostaglandin D2 (PGD2). Interestingly, PMD2 increased the production of J-series prostaglandins in both tumorigenic and non-tumorigenic keratinocytes. LC-ESI-MS/MS analysis detected ethanolamide- conjugated J series PG (15-deoxy, Δ12,14 prostamide J2, 15d-PMJ2) in PMD2-treated cell culture media. Since selective inhibitors of the J-series prostaglandins are not available, 15d-PMJ2 was synthesized to examine its direct activity. Exogenous 15d-PMJ2 mimicked the activity of PMD2 demonstrating preferential cytotoxicity towards tumorigenic compared to non-tumorigenic keratinocytes. In addition, 15d-PMJ2 induced oxidative stress, ER stress and apoptosis in tumorigenic keratinocytes. These findings suggest that the cytotoxicity of PMD2 is mediated by 15dPMJ2. Since PMD2 and its metabolite 15d-PMJ2 are preferentially toxic towards tumorigenic cells, PMD2 or 15d-PMJ2 may be an ideal topical treatment for NMSC that will elicit minimal toxicity in healthy surrounding skin cells. Citation Format: Eman Soliman, Daniel Ladin, Hussam Albassam, Ahmed E. Elhassanny, Andrew Morris, Allison Danell, Rukiyah Van Dross. A novel J-series prostamide mediates D-series prostamide-induced apoptosis in skin cancer: receptor-independent signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 123. doi:10.1158/1538-7445.AM2017-123

Many epithelial cancers have been shown to overexpress the enzyme cyclooxygenase-2 (COX-2), an enzyme responsible for metabolizing anandamide (AEA) to prostamides. AEA has demonstrated cytotoxicity in COX-2 overexpressing cancers via its metabolism to novel J-series prostamides, namely 15d-PMJ2. Fatty acid amide hydrolase (FAAH) degrades AEA into arachidonic acid and ethanolamine (EA), limiting the cytotoxic capability of AEA. Cell lines with high FAAH expression have demonstrated resistance to AEA. By understanding the metabolic characteristics of FAAH, we can design AEA analogs which circumvent FAAH breakdown. To examine the effects of altering polarity, steric bulk, and functional groups on AEA-mediated cytotoxicity, we investigated known AEA derivatives which possess these properties. Arvanil, Arachidonoyl Diethanolamine (ADA), Arachidonoyl Serinol (AS), and R1-methanandamide (m-AEA) add steric bulk to the molecule via aromatic rings, extra EA arms and additional alcohol/methyl functional groups respectively. Arachidonoyl glycine (NAGly) substitutes the terminal EA alcohol with a carboxylic acid increasing polarity. Arachidonoyl-2’-chloroethylamine (AC) substitutes the terminal EA alcohol with a highly soluble chlorine. Furthermore, it is known that Arvanil, ADA, AS, and m-AEA resist degradation of the molecule by FAAH and NAGly is a known substrate of COX-2. Therefore our goal was to determine which structural modifications improve AEA-mediated cytotoxicity. To determine this, JWF2 tumorigenic keratinocytes were exposed to differing concentrations of the AEA analogs for 24 hours and cell viability was measured by conducting MTS assays. Arvanil demonstrated a 90% reduction in cell viability, NAGly demonstrated a 70% reduction in cell viability, and m-AEA demonstrated a 100% reduction in cell viability at 20 µM, the optimal dosage of AEA. Due to the fact that ADA, AS, and AC did not show a significant reduction in cell viability these molecules were not further examined. MTS assays were conducted in other epithelial cancer cell lines with varying expressions of COX-2 and FAAH. Each cell line was exposed to varying concentrations of either NAGly, Arvanil, or both for 24 hours. NAGly demonstrated a 40% reduction in cell viability in HT-29 colon cancer cells (low COX-2, high FAAH). Arvanil demonstrated a 60% reduction in A431 tumorigenic keratinocytes. NAGly demonstrated a 60% reduction in cell viability and Arvanil demonstrated a 100% reduction in cell viability in patient-derived primary melanoma. These findings suggest that modulation and substitute to the core structure of AEA will result in decreased susceptibility to FAAH degradation and enhanced antineoplastic activity. Citation Format: Andrew Morris, Eman Soliman, Rukiyah Van Dross, Colin Burns. Structural modification of the chemotherapeutic anandamide: Designing anti-cancer agents and investigating their COX-2 metabolic products [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2193. doi:10.1158/1538-7445.AM2017-2193

Melanoma is the most aggressive and deadly form of cutaneous neoplasm in the United States, representing a major clinical challenge. Our lab previously demonstrated that the endocannabinoid, arachidonoyl ethanolamide (AEA), induced cell death in non-melanoma skin cancer (NMSC) cells through the cyclooxygenase-2 (COX-2) mediated formation of novel J-series prostamides (PMJs). We were the first to chemically synthesize the primary metabolite, 15-deoxy-Δ12,14 prostamide J2 (15d-PMJ2), which displayed potent and selective cytotoxicity in NMSC cells. As such, we hypothesize that the selective cytotoxicity of 15d-PMJ2 would be observed in other forms of skin cancer, including melanoma. B16F10 murine melanoma cells and nontumorigenic Melan-A cells were treated with different concentrations of 15d-PMJ2 for 24 hours and cell viability was measured using MTS assays. At 5µM, 15d-PMJ2 decreased viability by 63% in B16F10 cells, while Melan-A viability was not affected. To verify that cell death was due to apoptosis, the cleavage of apoptotic markers caspase-3 and PARP was examined by conducting Western blot analysis. 15d-PMJ2 markedly increased caspase-3 and PARP cleavage only in B16F10 melanoma cells. Previous studies in NMSC indicated that 15d-PMJ2 induced ER-stress and apoptosis. To investigate the mechanism of 15d-PMJ2-mediated death in melanoma, we examined ER-stress responses. Melan-A and B16F10 melanoma cells were treated with 5µM 15d-PMJ2 and evaluated for CHOP10 and p-PERK expression by Western blot analysis. B16F10, but not Melan-A cells exhibited a notable increase in CHOP10 and p-PERK expression when treated with 15d-PMJ2. To further examine the role of ER-stress on 15d-PMJ2 mediated apoptosis, B16F10 cells were pretreated with the ER-stress inhibitors salubrinal and 4-phenylbutyric acid (PBA). Both salubrinal and PBA decreased activation of caspase-3/7, suggesting that ER-stress plays an important role in 15d-PMJ2 mediated tumor cell death. To determine the anti-melanoma activity of 15d-PMJ2 in vivo, B16F10 allograft tumors grown in C57BL/6 mice were dosed subcutaneously with 0.5 or 5.0 mg/kg 15d-PMJ2 for 5 days. Tumors treated with 15d-PMJ2 exhibited significantly reduced growth and mean weights compared to vehicle and untreated animals. TUNEL analysis of tumor tissues indicated a large presence of necrotic and apoptotic cells in 15d-PMJ2-treated tumors compared to vehicle and untreated tumors. To determine whether 15d-PMJ2 induced ER-stress in vivo, tumors were assayed for p-PERK and CHOP10 levels by immunohistochemistry (IHC). These markers were significantly elevated in 15d-PMJ2-treated tumors. Similarly, the viability of primary patient-derived melanoma cells was significantly decreased by 15d-PMJ2. These findings suggest that the novel prostamide, 15d-PMJ2, possesses potent and selective anti-melanoma activity in vitro and in vivo. Citation Format: Daniel A. Ladin, Li V. Yang, Timothy L. Fitzgerald, Rukiyah Van Dross. Novel prostamide, 15-deoxy-delta12,14 prostamide J2, displays activity against melanoma in vitro and in vivo: potential role of endoplasmic reticulum stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3217. doi:10.1158/1538-7445.AM2017-3217

Colon cancer is the third most common cancer and the third leading cause of cancer-related death in the United States. The endoplasmic reticulum (ER) is a cellular organelle responsible for protein synthesis and oxidative folding. ER stress occurs when the protein folding load exceeds the protein folding capacity. Low levels of ER stress promote survival while excessive ER stress causes cell death. An important regulator of the cytotoxic ER stress pathway is the transcription factor, C/EBP homologous protein 10 (CHOP10). It has been shown that CHOP10 regulates the transcription of ER oxidoreductase 1α (ERO1α). ERO1α promotes ER luminal oxidation and under conditions of excessive ER stress releases H2O2 into the cytoplasm, resulting in oxidative stress-mediated apoptosis. Previous studies from our laboratory showed that 15deoxy, Δ12,14 prostamide J2 (15d PMJ2) induced ER stress-mediated apoptosis, leading to a reduction in cell viability in tumorigenic keratinocytes and melanocytes. In addition, 15d PMJ2-induced ER stress-apoptosis was decreased in the presence of ER stress inhibitors, 4-phenylbuterate (PBA) and salubrinal. However, the specific pathway involved in 15d PMJ2-induced ER stress-apoptosis have not been identified. In this study, we hypothesize that 15d PMJ2 causes ER stress-mediated apoptosis in colon tumor cells by activating CHOP10 and its downstream transcriptional target, ERO1α. To examine the anti-proliferative effect of 15d PMJ2, tumorigenic colon cells (HCT116) and non-tumorigenic colon cells (FHC) were treated with different concentrations of 15d PMJ2 or 10µM thapsigargin (TG) for 24 hours and cytotoxicity was measured by lactate dehydrogenase (LDH) assay. A significant increase in cell death was observed in HCT116 cells treated with 5µM 15d PMJ2 and this cytotoxic effect was 3-fold greater in HCT116 compared to FHC cells. Apoptotic measurements showed a significant increase in caspase 3/7 activity and annexin-V positivity following 15d PMJ2 treatment. The expression of CHOP10 was significantly enhanced in the 15d PMJ2-treated group. In addition, 15d PMJ2-induced apoptosis was decreased in the presence of ER stress inhibitors, PBA and salubrinal, suggesting that ER stress is essential for 15d PMJ2-induced apoptosis. Western blot analysis revealed an increase in ERO1α protein expression following 15d PMJ2 treatment. Apoptosis measurements showed a significant inhibition in 15d PMJ2-mediated apoptosis in the presence of ERO1α inhibitor, EN460, suggesting that ERO1α is important for 15d PMJ2-induced apoptosis. These findings suggest that 15d PMJ2-induced apoptosis is mediated via the ER stress pathway. CHOP10 and its transcriptional target, ERO1α, play a potential role in 15d PMJ2-induced ER stress-apoptosis, suggesting that 15d PMJ2 could be a potential anti-neoplastic agent with a unique mechanism for colon cancer. Citation Format: Hussam M. Albassam, Daniel A. Ladin, Rukiyah Van Dross. A novel J-series prostamide induces ER stress-mediated apoptosis and upregulates ER oxidoreductase 1 alpha (ERO1α) in human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2097. doi:10.1158/1538-7445.AM2017-2097