Robert Orlando

Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis

Mucositis is a debilitating adverse effect of chemotherapy and radiation treatment. It is important to develop a simple and reliable in vitro model, which can routinely be used to screen new drugs for prevention and treatment of mucositis. Furthermore, identifying cell and molecular stresses especially in the initiation phase of mucositis in this model will help towards this end. We evaluated a three-dimensional (3-D) human oral cell culture that consisted of oral keratinocytes and fibroblasts as a model of oral mucositis. The 3-D cell culture model was irradiated with 12 or 2 Gy. Six hours after the irradiation we evaluated microscopic sections of the cell culture for evidence of morphologic changes including apoptosis. We used microarrays to compare the expression of several genes from the irradiated tissue with identical genes from tissue that was not irradiated. We found that irradiation with 12 Gy induced significant histopathologic effects including cellular apoptosis. Irradiation significantly affected the expression of several genes of the NF-kB pathway and several inflammatory cytokines, such as IL-1B, 1L-8, NF-kB1, and FOS compared to tissue that was not irradiated. We identified significant upregulation of several genes that belong to damage-associated molecular patterns (DAMPs) such as HMB1, S100A13, SA10014, and SA10016 in the 3-D tissues that received 12 Gy but not in tissues that received 2 Gy. In conclusion, this model quantifies radiation damage and this is an important first step towards the development 3-D tissue as a screening tool.

DMH1, a Small Molecule Inhibitor of BMP Type I Receptors, Suppresses Growth and Invasion of Lung Cancer

The bone morphogenetic protein (BMP) signaling cascade is aberrantly activated in human non-small cell lung cancer (NSCLC) but not in normal lung epithelial cells, suggesting that blocking BMP signaling may be an effective therapeutic approach for lung cancer. Previous studies demonstrated that some BMP antagonists, which bind to extracellular BMP ligands and prevent their association with BMP receptors, dramatically reduced lung tumor growth. However, clinical application of protein-based BMP antagonists is limited by short half-lives, poor intra-tumor delivery as well as resistance caused by potential gain-of-function mutations in the downstream of the BMP pathway. Small molecule BMP inhibitors which target the intracellular BMP cascades would be ideal for anticancer drug development. In a zebrafish embryo-based structure and activity study, we previously identified a group of highly selective small molecule inhibitors specifically antagonizing the intracellular kinase domain of BMP type I receptors. In the present study, we demonstrated that DMH1, one of such inhibitors, potently reduced lung cell proliferation, promoted cell death, and decreased cell migration and invasion in NSCLC cells by blocking BMP signaling, as indicated by suppression of Smad 1/5/8 phosphorylation and gene expression of Id1, Id2 and Id3. Additionally, DMH1 treatment significantly reduced the tumor growth in human lung cancer xenograft model. In conclusion, our study indicates that small molecule inhibitors of BMP type I receptors may offer a promising novel strategy for lung cancer treatment.

Prevention of skin carcinogenesis by the β-blocker carvedilol.

The stress-related catecholamine hormones and the α- and β-adrenergic receptors (α- and β-AR) may affect carcinogenesis. The β-AR GRK/β-arrestin biased agonist carvedilol can induce β-AR–mediated transactivation of the EGFR. The initial purpose of this study was to determine whether carvedilol, through activation of EGFR, can promote cancer. Carvedilol failed to promote anchorage-independent growth of JB6 P+ cells, a skin cell model used to study tumor promotion. However, at nontoxic concentrations, carvedilol dose dependently inhibited EGF-induced malignant transformation of JB6 P+ cells, suggesting that carvedilol has chemopreventive activity against skin cancer. Such effect was not observed for the β-AR agonist isoproterenol and the β-AR antagonist atenolol. Gene expression, receptor binding, and functional studies indicate that JB6 P+ cells only express β2-ARs. Carvedilol, but not atenolol, inhibited EGF-mediated activator protein-1 (AP-1) activation. A topical 7,12-dimethylbenz(α)anthracene (DMBA)-induced skin hyperplasia model in SENCAR mice was utilized to determine the in vivo cancer preventative activity of carvedilol. Both topical and oral carvedilol treatment inhibited DMBA-induced epidermal hyperplasia (P < 0.05) and reduced H-ras mutations; topical treatment being the most potent. However, in models of established cancer, carvedilol had modest to no inhibitory effect on tumor growth of human lung cancer A549 cells in vitro and in vivo. In conclusion, these results suggest that the cardiovascular drug carvedilol may be repurposed for skin cancer chemoprevention, but may not be an effective treatment of established tumors. More broadly, this study suggests that β-ARs may serve as a novel target for cancer prevention. Cancer Prev Res; 8(1); 27–36. ©2014 AACR.

Molecular Signatures in the Prevention of Radiation Damage by the Synergistic Effect of N-Acetyl Cysteine and Qingre Liyan Decoction, a Traditional Chinese Medicine, Using a 3-Dimensional Cell Culture Model of Oral Mucositis

Qingre Liyan decoction (QYD), a Traditional Chinese medicine, and N-acetyl cysteine (NAC) have been used to prevent radiation induced mucositis. This work evaluates the protective mechanisms of QYD, NAC, and their combination (NAC-QYD) at the cellular and transcriptional level. A validated organotypic model of oral mucosal consisting of a three-dimensional (3D) cell tissue-culture of primary human keratinocytes exposed to X-ray irradiation was used. Six hours after the irradiation, the tissues were evaluated by hematoxylin and eosin (H and E) and a TUNEL assay to assess histopathology and apoptosis, respectively. Total RNA was extracted and used for microarray gene expression profiling. The tissue-cultures treated with NAC-QYD preserved their integrity and showed no apoptosis. Microarray results revealed that the NAC-QYD caused the upregulation of genes encoding metallothioneins, HMOX1, and other components of the Nrf2 pathway, which protects against oxidative stress. DNA repair genes (XCP, GADD45G, RAD9, and XRCC1), protective genes (EGFR and PPARD), and genes of the NFκB pathway were upregulated. Finally, tissue-cultures treated prophylactically with NAC-QYD showed significant downregulation of apoptosis, cytokines and chemokines genes, and constrained damage-associated molecular patterns (DAMPs). NAC-QYD treatment involves the protective effect of Nrf2, NFκB, and DNA repair factors.

Inhibition of Neoplastic Transformation and Chemically-Induced Skin Hyperplasia in Mice by Traditional Chinese Medicinal Formula Si-Wu-Tang

Exploring traditional medicines may lead to the development of low-cost and non-toxic cancer preventive agents. Si-Wu-Tang (SWT), comprising the combination of four herbs, Rehmanniae, Angelica, Chuanxiong, and Paeoniae, is one of the most popular traditional Chinese medicines for women’s diseases. In our previous studies, the antioxidant Nrf2 pathways were strongly induced by SWT in vitro and in vivo. Since Nrf2 activation has been associated with anticarcinogenic effects, the purpose of this study is to evaluate SWT’s activity of cancer prevention. In the Ames test, SWT demonstrated an antimutagenic activity against mutagenicity induced by the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). In JB6 P+ cells, a non-cancerous murine epidermal model for studying tumor promotion, SWT inhibited epidermal growth factor (EGF)-induced neoplastic transformation. The luciferase reporter gene assays demonstrated that SWT suppressed EGF-induced AP-1 and TNF-α-induced NF-κB activation, which are essential factors involved in skin carcinogenesis. In a DMBA-induced skin hyperplasia assay in ‘Sensitivity to Carcinogenesis’ (SENCAR) mice, both topical and oral SWT inhibited DMBA-induced epidermal hyperplasia, expression of the proliferation marker Proliferating cell nuclear antigen (PCNA), and H-ras mutations. These findings demonstrate, for the first time, that SWT prevents tumor promoter and chemical-induced carcinogenesis in vitro and in vivo, partly by inhibiting DNA damage and blocking the activation of AP-1 and NF-κB.

Topically Applied Carvedilol Attenuates Solar Ultraviolet Radiation Induced Skin Carcinogenesis

In previous studies, the β-blocker carvedilol inhibited EGF-induced epidermal cell transformation and chemical carcinogen-induced mouse skin hyperplasia. As exposure to ultraviolet (UV) radiation leads to skin cancer, the present study examined whether carvedilol can prevent UV-induced carcinogenesis. Carvedilol absorbs UV like a sunscreen; thus, to separate pharmacological from sunscreen effects, 4-hydroxycarbazole (4-OHC), which absorbs UV to the same degree as carvedilol, served as control. JB6 P+ cells, an established epidermal model for studying tumor promotion, were used for evaluating the effect of carvedilol on UV-induced neoplastic transformation. Both carvedilol and 4-OHC (1 μmol/L) blocked transformation induced by chronic UV (15 mJ/cm2) exposure for 8 weeks. However, EGF-mediated transformation was inhibited by only carvedilol but not by 4-OHC. Carvedilol (1 and 5 μmol/L), but not 4-OHC, attenuated UV-induced AP-1 and NF-κB luciferase reporter activity, suggesting a potential anti-inflammatory activity. In a single-dose UV (200 mJ/cm2)-induced skin inflammation mouse model, carvedilol (10 μmol/L), applied topically after UV exposure, reduced skin hyperplasia and the levels of cyclobutane pyrimidine dimers, IL1β, IL6, and COX-2 in skin. In SKH-1 mice exposed to gradually increasing levels of UV (50–150 mJ/cm2) three times a week for 25 weeks, topical administration of carvedilol (10 μmol/L) after UV exposure increased tumor latency compared with control (week 18 vs. 15), decreased incidence and multiplicity of squamous cell carcinomas, while 4-OHC had no effect. These data suggest that carvedilol has a novel chemopreventive activity and topical carvedilol following UV exposure may be repurposed for preventing skin inflammation and cancer. Cancer Prev Res; 10(10); 598–606. ©2017 AACR.

Abstract 5252: Skin cancer prevention by traditional Chinese medicinal formula Si-Wu-Tang and its constituents

Si-Wu-Tang (SWT), comprising the combination of four herbs, Rehmanniae, Angelica, Chuanxiong and Paeoniae, is one of the most popular Chinese medicines for women9s diseases. Previously we showed that SWT was able to upregulate genes in the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, suggesting a potential application for cancer chemoprevention. The present study examined the chemopreventive activity of SWT using models of skin carcinogenesis. In JB6 P+ cells, a non-cancerous murine epidermal cell line for studying skin tumor promotion, SWT inhibited epidermal growth factor (EGF)-induced neoplastic transformation. In a 7,12-dimethylbenz(a)anthracene (DMBA)-induced murine skin tumorigenesis model, both topical and oral treatment of SWT inhibited epidermal hyperplasia, proliferating cell nuclear antigen expression, and H-ras mutations induced by DMBA treatment. In addition, SWT exhibited a significant antimutagenic activity against DMBA-induced mutagenicity, determined by the Ames Test using Salmonella typhimurium TA100 in the presence of metabolic activator S9 system. To identify active components in SWT, among nine compounds previously reported in commercial SWT products, in silico molecular docking analysis predicted some as potential Nrf2 activators due to an ability of interfering the forming of Nrf2-Keap1 complex. Three of these compounds, gallic acid, Z-liguistilide and senkyunolide A, were confirmed with highest potency of increasing the antioxidant response element luciferase reporter activity, inducing Nrf2-regulated genes Hmox1, Slc7A11 and Nqo1, and inhibiting EGF-induced JB6 P+ transformation. Further mechanistic studies showed that SWT and the three compounds suppressed EGF-induced activation of the activator protein 1 (AP-1), an essential transcription factor involved in skin carcinogenesis. The antimutagenic activity for the three compounds was also confirmed with the Ames Test. In conclusion, these results provide evidence that SWT and its constituents are able to prevent skin cancer, at least partly, by activating the Nrf2 pathway and blocking the activation of AP-1. Thus, this widely used Chinese medicinal formula may provide a promising option toward preventing skin cancer or may be other types of cancer. Citation Format: Kevin Huang, Mandy Liu, Suhui Zhang, Steven Yeung, Andy Chang, Li Qian, Payal Chatterjee, Rui Li, Su Zhou, Nan Mei, Zhijun Wang, Cyrus Parsa, Robert Orlando, Yun Luo, Ying Huang. Skin cancer prevention by traditional Chinese medicinal formula Si-Wu-Tang and its constituents. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5252.

Abstract 1256: The chemopreventive effects of carvedilol on skin carcinogenesis

Skin cancer, the majority of which is non-melanoma skin cancer (NMSC), is the most common type of cancer in the US. The rate of NMSC is increasing mainly due to depletion of the ozone layer and increased exposure to ultraviolet radiation. To reduce the risk of skin cancer, it is recommended to all individuals to limit sun exposure and use sunscreens. Despite these efforts, the incidence of NMSC continues to rise. Novel chemopreventive targets and nontoxic agents are needed. Epidemiological studies identified an association of psychosocial factors such as chronic stress or depression with cancer onset and progression. These factors are partly mediated by activation of the sympathetic nervous system which results in release of norepinephrine and epinephrine, the effects of which are mediated through the alpha- and beta-adrenergic receptors (alpha- and beta-AR). Further, the use of beta-AR antagonists (beta-blockers) has been associated with reduced cancer incidence. However, whether beta-blockers have chemopreventive activity or whether beta-ARs contribute to carcinogenesis is unknown. To determine whether agonizing or antagonizing beta-ARs affect skin cell transformation, we tested the beta-AR agonist isoproterenol (Iso) and antagonist carvedilol on epidermal growth factor (EGF)-mediated transformation of JB6 P+ cells (a skin cell model to study tumor promotion). The cells were treated with EGF (10 ng/mL) plus 0.1 µM or 1.0 µM Iso, yet no effect was observed. However, treatment with carvedilol dose-dependently inhibited the formation of EGF-induced soft agar colonies. Such effect was not caused by growth inhibition, because cytotoxicity and inhibition of cell proliferation were not observed for tested concentrations. Next, the expression of beta1-, 2- and 3-AR was determined in JB6 cells using RT-PCR. The results showed that only beta2-AR was detectable. We also determined whether beta-ARs are expressed in other types of cancer. In tissues derived from a rat model of 7, 12-dimethylbenz[α]anthracene (DMBA)-induced mammary cancer, expression of beta1- and 2-AR was up-regulated in tumors in comparison with normal tissues. The human cancer cell lines A549 and MDA-MB-231 also express beta2-AR. To determine the in vivo chemopreventive activity of carvedilol, we used a skin hyperplasia model in SENCAR mice induced by topical treatment with 100 nM DMBA twice a week for four weeks. Carvedilol was tested topically (5 and 10 µM) or orally (5 and 20 mg/kg), beginning at two weeks before the first dose of DMBA, three times a week, for six weeks. DMBA alone increased the epidermal thickness from the average of 77.8 + 13.6 nm in normal skin to 294 + 49.7 nm in DMBA-treated skin. Both topical and oral carvedilol treatment inhibited DMBA-induced hyperplasia (P Citation Format: Andy Chang, Mandy Liu, Steven Yeung, Jijun Hao, Cyrus Parsa, Robert Orlando, Bradley Andresen, Ying Huang. The chemopreventive effects of carvedilol on skin carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1256. doi:10.1158/1538-7445.AM2014-1256

Abstract 2717: DMH1, a small molecule inhibitor of BMP type I receptors, suppresses growth and invasion of lung cancer

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and their biological activity is mediated by the formation of heterodimeric complexes of BMP type I and type II receptors of serine/threonine kinases. BMPs play an important role in regulating cell proliferation, differentiation and migration, processes that are involved in carcinogenesis of many types of cancer. BMP-2 has been shown overexpressed in human non-small cell lung cancer (NSCLC) and enhances lung tumorigenesis, suggesting that blocking the BMP signaling may be an effective therapeutic approach for NSCLC. Although previous work demonstrated that known protein-based BMP antagonists such as Noggin may reduce lung tumor growth, their clinical application could be limited by short half-lives and poor intra-tumor delivery. In a previous zebrafish embryo-based structure and activity study, we identified DMH1, a highly selective small molecule inhibitor which specifically antagonize the intracellular kinase domains of BMP type I receptors, the critical mediators of BMP signaling. In the present study, we found that DMH1 potently reduced lung cell proliferation, promoted cell death, and decreased cell migration and invasion in vitro by blocking the BMP signaling, as indicated by the reduction of Smad 1/5/8 phosphorylation and gene expression of Id1, Id2 and Id3 in A549 and H460 cells. In an in vivo study, treatment by intraperitoneal injection of DMH1 (5 mg/kg) for four weeks significantly reduced the growth of A549-derived xenograft in SCID mice. In conclusion, our study indicates that small molecule inhibitors of BMP type I receptors may offer a promising novel therapeutic strategy for lung cancer. Citation Format: Ying Huang, Rachel Lee, Andy Chang, Jeffery Fan, Chantelle Labib, Cyrus Parsa, Robert Orlando, Bradley Andresen, Jijun Hao. DMH1, a small molecule inhibitor of BMP type I receptors, suppresses growth and invasion of lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2717. doi:10.1158/1538-7445.AM2014-2717