Department of Pharmacology and Toxicology Works
Permanent URI for this collection
Browse
Recent Submissions
Item Over-expression of asparagine synthetase and matrix metalloproteinase 19 confers cisplatin sensitivity in nasopharyngeal carcinoma cells(American Association for Cancer Research, 2013) Liu, Ran-Yi; Dong, Zizheng; Liu, Jianguo; Zhou, Ling; Huang, Wenlin; Khoo, Sok Kean; Zhang, Zhongfa; Petillo, David; Teh, Bin Tean; Qian, Chao-Nan; Zhang, Jian-Ting; Pharmacology and Toxicology, School of MedicinePlatinum-based concurrent chemoradiotherapy is considered a standard treatment approach for locoregionally advanced nasopharyngeal carcinoma. However, only a minority of patients benefit from this treatment regimen compared with radiotherapy alone. Identification of a set of molecular markers predicting sensitivity of platinum-based chemotherapy may contribute to personalized treatment of patients with nasopharyngeal carcinoma for better clinical outcome with less toxicity. Previously, we generated a cisplatin-sensitive nasopharyngeal carcinoma cell line, S16, by clonal selection from CNE-2 cells and found that eIF3a is upregulated and contributes to cisplatin sensitivity by downregulating the synthesis of nucleotide excision repair proteins. In this study, we conducted a gene expression profiling analysis and found three other genes, asparagine synthetase (ASNS), choriogonadotropin α subunit (CGA), and matrix metalloproteinase 19 (MMP19), that are upregulated in the cisplatin-sensitive S16 cells compared with the CNE-2 cells. However, only ASNS and MMP19, but not CGA, contributes to cisplatin sensitivity by potentiating cisplatin-induced DNA damage and apoptosis. Thus, ASNS and MMP19, along with eIF3a, are the sensitivity factors for cisplatin treatment and may serve as potential candidate molecular markers for predicting cisplatin sensitivity of advanced nasopharyngeal carcinoma.Item Structural Basis for the Modulation of the Neuronal Voltage-Gated Sodium Channel NaV1.6 by Calmodulin(Springer Nature, 2013) Reddy Chichili, Vishnu Priyanka; Xiao, Yucheng; Seetharaman, J.; Cummins, Theodore R.; Sivaraman, J.; Pharmacology and Toxicology, School of MedicineThe neuronal-voltage gated sodium channel (VGSC), Na(V)1.6, plays an important role in propagating action potentials along myelinated axons. Calmodulin (CaM) is known to modulate the inactivation kinetics of Na(V)1.6 by interacting with its IQ motif. Here we report the crystal structure of apo-CaM:Na(V)1.6IQ motif, along with functional studies. The IQ motif of Na(V)1.6 adopts an α-helical conformation in its interaction with the C-lobe of CaM. CaM uses different residues to interact with Na(V)1.6IQ motif depending on the presence or absence of Ca²⁺. Three residues from Na(V)1.6, Arg1902, Tyr1904 and Arg1905 were identified as the key common interacting residues in both the presence and absence of Ca²⁺. Substitution of Arg1902 and Tyr1904 with alanine showed a reduced rate of Na(V)1.6 inactivation in electrophysiological experiments in vivo. Compared with other CaM:Na(V) complexes, our results reveal a different mode of interaction for CaM:Na(V)1.6 and provides structural insight into the isoform-specific modulation of VGSCs.Item Protein arginine methylation of non-histone proteins and its role in diseases(Taylor & Francis, 2014) Wei, Han; Mundade, Rasika; Lange, Kevin C.; Lu, Tao; Pharmacology and Toxicology, School of MedicineProtein arginine methyltransferases (PRMTs) are a family of enzymes that can methylate arginine residues on histones and other proteins. PRMTs play a crucial role in influencing various cellular functions, including cellular development and tumorigenesis. Arginine methylation by PRMTs is found on both nuclear and cytoplasmic proteins. Recently, there is increasing evidence regarding post-translational modifications of non-histone proteins by PRMTs, illustrating the previously unknown importance of PRMTs in the regulation of various cellular functions by post-translational modifications. In this review, we present the recent developments in the regulation of non-histone proteins by PRMTs.Item Expression of DNA Translesion Synthesis Polymerase η in Head and Neck Squamous Cell Cancer Predicts Resistance to Gemcitabine and Cisplatin-Based Chemotherapy(Public Library of Science, 2013-12-20) Zhou, Wendi; Chen, Yih-wen; Liu, Xiyong; Chu, Peiguo; Loria, Sofia; Wang, Yafan; Yen, Yun; Chou, Kai-Ming; Pharmacology and Toxicology, School of MedicinePurpose: The development of resistance against anticancer drugs has been a persistent clinical problem for the treatment of locally advanced malignancies in the head and neck mucosal derived squamous cell carcinoma (HNSCC). Recent evidence indicates that the DNA translesion synthesis (TLS) polymerase η (Pol η; hRad30a gene) reduces the effectiveness of gemcitabine/cisplatin. The goal of this study is to examine the relationship between the expression level of Pol η and the observed resistance against these chemotherapeutic agents in HNSCC, which is currently unknown. Methods: Sixty-four mucosal derived squamous cell carcinomas of head and neck (HNSCC) from 1989 and 2007 at the City of Hope National Medical Center (Duarte, CA) were retrospectively analyzed. Pretreatment samples were immunostained with anti-Pol η antibody and the correlation between the expression level of Pol η and clinical outcomes were evaluated. Forty-nine cases treated with platinum (n=40) or gemcitabine (n=9) based chemotherapy were further examined for Pol η expression level for comparison with patient response to chemotherapy. Results: The expression of Pol η was elevated in 67% of the head and neck tumor samples. Pol η expression level was significantly higher in grade 1 to grade 2 tumors (well to moderately differentiated). The overall benefit rate (complete response+ partial response) in patients treated with platinum and gemcitabine based chemotherapy was 79.5%, where low Pol η level was significantly associated with high complete response rate (p=0.03), although not associated with overall survival. Furthermore, no significant correlation was observed between Pol η expression level with gender, age, tobacco/alcohol history, tumor stage and metastatic status. Conclusions: Our data suggest that Pol η expression may be a useful prediction marker for the effectiveness of platinum or gemcitabine based therapy for HNSCC.Item Long-Acting Microparticle Formulation of Griseofulvin for Ocular Neovascularization Therapy(Wiley, 2024) Chobisa, Dhawal; Muniyandi, Anbukkarasi; Sishtla, Kamakshi; Corson, Timothy W.; Yeo, Yoon; Pharmacology and Toxicology, School of MedicineNeovascular age-related macular degeneration (nAMD) is a leading cause of vision loss in older adults. nAMD is treated with biologics targeting vascular endothelial growth factor; however, many patients do not respond to the current therapy. Here, a small molecule drug, griseofulvin (GRF), is used due to its inhibitory effect on ferrochelatase, an enzyme important for choroidal neovascularization (CNV). For local and sustained delivery to the eyes, GRF is encapsulated in microparticles based on poly(lactide-co-glycolide) (PLGA), a biodegradable polymer with a track record in long-acting formulations. The GRF-loaded PLGA microparticles (GRF MPs) are designed for intravitreal application, considering constraints in size, drug loading content, and drug release kinetics. Magnesium hydroxide is co-encapsulated to enable sustained GRF release over >30 days in phosphate-buffered saline with Tween 80. Incubated in cell culture medium over 30 days, the GRF MPs and the released drug show antiangiogenic effects in retinal endothelial cells. A single intravitreal injection of MPs containing 0.18 µg GRF releases the drug over 6 weeks in vivo to inhibit the progression of laser-induced CNV in mice with no abnormality in the fundus and retina. Intravitreally administered GRF MPs prove effective in preventing CNV, providing proof-of-concept toward a novel, cost-effective nAMD therapy.Item Prostate cancer ETS rearrangements switch a cell migration gene expression program from RAS/ERK to PI3K/AKT regulation(Springer Nature, 2014-03-19) Selvaraj, Nagarathinam; Budka, Justin A.; Ferris, Mary W.; Jerde, Travis J.; Hollenhorst, Peter C.; Pharmacology and Toxicology, School of MedicineBackground: The RAS/ERK and PI3K/AKT pathways induce oncogenic gene expression programs and are commonly activated together in cancer cells. Often, RAS/ERK signaling is activated by mutation of the RAS or RAF oncogenes, and PI3K/AKT is activated by loss of the tumor suppressor PTEN. In prostate cancer, PTEN deletions are common, but, unlike other carcinomas, RAS and RAF mutations are rare. We have previously shown that over-expression of "oncogenic" ETS transcription factors, which occurs in about one-half of prostate tumors due to chromosome rearrangement, can bypass the need for RAS/ERK signaling in the activation of a cell migration gene expression program. In this study we test the role of RAS/ERK and PI3K/AKT signaling in the function of oncogenic ETS proteins. Results: We find that oncogenic ETS expression negatively correlates with RAS and RAF mutations in prostate tumors. Furthermore, the oncogenic ETS transcription factors only increased cell migration in the absence of RAS/ERK activation. In contrast to RAS/ERK, it has been reported that oncogenic ETS expression positively correlates with PI3K/AKT activation. We identified a mechanistic explanation for this finding by showing that oncogenic ETS proteins required AKT signaling to activate a cell migration gene expression program through ETS/AP-1 binding sequences. Levels of pAKT correlated with the ability of oncogenic ETS proteins to increase cell migration, but this process did not require mTORC1. Conclusions: Our findings indicate that oncogenic ETS rearrangements cause a cell migration gene expression program to switch from RAS/ERK control to PI3K/AKT control and provide a possible explanation for the high frequency of PTEN, but not RAS/RAF mutations in prostate cancer.Item Nerve Growth Factor Mediates a Switch in Intracellular Signaling for PGE2-Induced Sensitization of Sensory Neurons from Protein Kinase A to Epac(Public Library of Science, 2014-08-15) Vasko, Michael R.; Habashy Malty, Ramy; Guo, Chunlu; Duarte, Djane B.; Zhang, Yihong; Nicol, Grant D.; Pharmacology and Toxicology, School of MedicineWe examined whether nerve growth factor (NGF), an inflammatory mediator that contributes to chronic hypersensitivity, alters the intracellular signaling that mediates the sensitizing actions of PGE2 from activation of protein kinase A (PKA) to exchange proteins directly activated by cAMP (Epacs). When isolated sensory neurons are grown in the absence of added NGF, but not in cultures grown with 30 ng/ml NGF, inhibiting protein kinase A (PKA) activity blocks the ability of PGE2 to augment capsaicin-evoked release of the neuropeptide CGRP and to increase the number of action potentials (APs) evoked by a ramp of current. Growing sensory neurons in culture in the presence of increasing concentrations of NGF increases the expression of Epac2, but not Epac1. An intradermal injection of complete Freund's adjuvant into the rat hindpaw also increases the expression of Epac2, but not Epac1 in the dorsal root ganglia and spinal cord: an effect blocked by intraplantar administration of NGF antibodies. Treating cultures grown in the presence of 30 ng/ml NGF with Epac1siRNA significantly reduced the expression of Epac1, but not Epac2, and did not block the ability of PGE2 to augment capsaicin-evoked release of CGRP from sensory neurons. Exposing neuronal cultures grown in NGF to Epac2siRNAreduced the expression of Epac2, but not Epac1 and prevented the PGE2-induced augmentation of capsaicin and potassium-evoked CGRP release in sensory neurons and the PGE2-induced increase in the number of APs generated by a ramp of current. In neurons grown with no added NGF, Epac siRNAs did not attenuate PGE2-induced sensitization. These results demonstrate that NGF, through increasing Epac2 expression, alters the signaling cascade that mediates PGE2-induced sensitization of sensory neurons, thus providing a novel mechanism for maintaining PGE2-induced hypersensitivity during inflammation.Item A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in calcium-induced Toxoplasma gondii egress(Public Library of Science, 2022-10-20) Nofal, Stephanie D.; Dominicus, Caia; Broncel, Malgorzata; Katris, Nicholas J.; Flynn, Helen R.; Arrizabalaga, Gustavo; Botté, Cyrille Y.; Invergo, Brandon M.; Treeck, Moritz; Pharmacology and Toxicology, School of MedicineFundamental processes that govern the lytic cycle of the intracellular parasite Toxoplasma gondii are regulated by several signalling pathways. However, how these pathways are connected remains largely unknown. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels. We show that both egress inducers trigger indistinguishable signalling responses and provide evidence for a positive feedback loop linking calcium and cyclic nucleotide signalling. Using WT and conditional knockout parasites of the non-essential calcium-dependent protein kinase 3 (CDPK3), which display a delay in calcium inonophore-mediated egress, we explore changes in phosphorylation and lipid signalling in sub-minute timecourses after inducing Ca2+ release. These studies indicate that cAMP and lipid metabolism are central to the feedback loop, which is partly dependent on CDPK3 and allows the parasite to respond faster to inducers of egress. Biochemical analysis of 4 phosphodiesterases (PDEs) identified in our phosphoproteomes establishes PDE2 as a cAMP-specific PDE which regulates Ca2+ induced egress in a CDPK3-independent manner. The other PDEs display dual hydrolytic activity and play no role in Ca2+ induced egress. In summary, we uncover a positive feedback loop that enhances signalling during egress, thereby linking several signalling pathways.Item The agony and the efficacy: central mechanisms of GLP-1 induced adverse events and their mitigation by GIP(Frontiers Media, 2025-02-03) Douros, Jonathan D.; Flak, Jonathan N.; Knerr, Patrick J.; Pharmacology and Toxicology, School of MedicineItem Molecular and Synaptic Signatures in Mouse Models of Late‐Onset Alzheimer’s Disease Independent of Amyloid and Tau Pathology(Wiley, 2025-01-03) Oblak, Adrian L.; Pharmacology and Toxicology, School of MedicineBackground: MODEL‐AD (Model Organism Development and Evaluation for Late‐onset AD) is developing, characterizing, and distributing novel mouse models expressing humanized, clinically relevant genetic risk factors. Models expressing human‐relevant risk genetic risk factors are expected to better phenocopy LOAD than widely used transgenic models. Method: Here, two genetic risk factors APOE4 and Trem2*R47H, were incorporated into C57BL/6J (B6) mice along with humanized amyloid‐beta to produce the LOAD2 model. LOAD2 and control mice were aged up to 24 months with some being provided in the absence or presence of normal chow or a high fat/high sugar diet (LOAD2 HFD) from two months of age. A phenotyping pipeline was employed to evaluate disease outcomes observed in human patients, including in vivo imaging, brain and blood biomarker and cytokine analyses, multi‐omics (transcriptomics and proteomics), neuropathology and behavior. Result: By 18 months, unlike control mice (e.g., LOAD2 mice fed a control diet, CD), LOAD2 HFD mice presented subtle but significant loss of neurons in the cortex, elevated levels of insoluble AΒ42 in the brain, and increased plasma neurofilament light chain (NfL). Transcriptomics and proteomics showed changes in gene/proteins relating to a variety of disease‐relevant processes including lipid metabolism and synaptic function. In vivo imaging revealed an age‐dependent reduction in brain region volume (MRI) and neurovascular uncoupling (PET/CT). LOAD2 HFD mice also showed a learning deficit based on a Touchscreen cognitive assay. Conclusion: Despite the absence of hallmark amyloid and Tau pathologies, collectively these data support the use of LOAD2 HFD mice reveal this model as important for preclinical studies that target other features of LOAD independent of amyloid and tau.