O câncer de próstata é o tumor maligno mais freqüente nos homens com idade superior a 50 anos, excetuando-se os tumores cutâneos. No Brasil estima-se para o ano de 2012 cerca de 60.180 casos novos deste tipo de neoplasia. Os marcadores tumorais permitem fazer o rastreamento do câncer, o diagnóstico diferencial entre uma neoplasia benigna e maligna, a avaliação de prognóstico e o acompanhamento terapêutico, assim como a detecção da recidiva tumoral. Dentre estes marcadores tumorais, tem-se dado muito atenção para as proteínas que mediam e participam da progressão do ciclo celular. A ciclina D1 é uma proteína nuclear de vida curta que é destruída pela via da ubiquitina ATP dependente, e está envolvida na transição celular da fase do ciclo G1 (repouso) para a fase S (síntese) tanto em células normais como em células neoplásicas. A super expressão de ciclina D1 remove a regulação normal do ciclo celular causando proliferação celular descontrolada, um crescimento anormal dos tecidos e a transformação para um fenótipo neoplásico, atuando como oncogene. No presente trabalho foi estudado a expressão de ciclina D1 em adenocarcinomas de próstata, tendo como objetivo avaliar a relação desta proteína com parâmetros epidemiológicos, clínicos e histopatológicos. Adicionalmente também foi feita comparação de escore de Gleason e lateralidade tumoral entre biópsias prostáticas com agulha e de prostatectomias radicais. No ensaio para ciclina D1 foram analisados 85 casos através de imunoistoquímica (IHQ) de material proveniente de prostatectomias radicais diagnosticados com adenocarcinoma de próstata entre os anos de 2005 e 2010 em nosso serviço. O método de avaliação se utilizou de microscopia ótica comum e contagem semi-quantitativa, comparado-se a expressão com achados clínicos, epidemiológicos e histopatológicos utilizando-se Teste T de Fisher, Qui Quadrado, Mann-Whitney, Curva ROC e correlação de Spearman. Os resultados demonstraram correlação positiva de ciclina D1 com escore de Gleason (p<0,05), com volume prostático (p=0,01) e uma tendência a correlação positiva com invasão perineural (p=0,07). Não houve correlação estatística entre ciclina D1 e o aumento de PSA, assim como outros achados histopatológicos. As biópsias prostáticas com agulha apresentaram subestimação em 40% dos casos para escore de Gleason e de 62,3% dos casos para lateralidade tumoral quando comparadas a prostatectomia radical. Já que as taxas de subestimação de escore de Gleason e lateralidade tumoral são relativamente altas e visto a urgência em se padronizar novos biomarcadores para o câncer prostático, sugerimos que ciclina D1 pode ser utilizada como biomarcador em patologia cirúrgica da próstata auxiliando numa gradação histológica mais precisa em biópsias com agulha colaborando para melhor vigilância e escolha terapêutica. / Prostate cancer is the most common malignant tumor in men older than 50 years, except for skin tumors. In Brazil it is estimated for the year 2012 about 60,180 new cases of this type of neoplasm. Tumor markers allow to cancer screening, differential diagnosis between a benign and malignant, assessment of prognosis and therapeutic monitoring, and detection of tumor recurrence. Among these tumor markers, has been given much attention for proteins that mediate and participate in cell cycle progression. Cyclin D1 is a short-lived nuclear protein that is destroyed by the ATP ubiquitin dependent pathway, and is involved in the transition of cell cycle G1 phase (resting) to the S phase (synthesis) cells both in normal and neoplastic cells. The overexpression of cyclin D1 removes the normal regulation of cell cycle causing uncontrolled cell proliferation, abnormal growth of tissues and transformation to a neoplastic phenotype, acting as an oncogene. In the present work we studied the expression of cyclin D1 in prostate adenocarcinomas, and to evaluate the relationship of this protein with epidemiologic factors, clinical and histopathological features. Additionally comparison was also made of Gleason score and laterality between tumor biopsies and prostate needle radical prostatectomies. In the assay for cyclin D1 were 85 cases analyzed by immunohistochemistry (IHC) of material from radical prostatectomies diagnosed with prostate adenocarcinoma between the years 2005 and 2010 at our institution. The evaluation method utilized were light microscopy and semi-quantitative score, comparing the cyclin D1 expression with clinical, epidemiological and histopathological features using Fisher\'s exact test, chi square test, Mann-Whitney test, ROC curve and Spearman correlation. The results showed a positive correlation of cyclin D1 with Gleason score (p <0.05), prostate volume (p = 0.01) and a trend toward positive correlation with perineural invasion (p = 0.07). There was no statistical correlation between cyclin D1 and increased PSA, as well as other histopathologic features. Prostate needle biopsies showed underestimation in 40% of cases for Gleason score and 62.3% of cases for tumor laterality when compared to radical prostatectomy. Since the rates of underestimation of Gleason score and tumor laterality are relatively high and the urgency to standardize new biomarkers for prostate cancer, we suggest that cyclin D1 may be used as biomarkers in surgical pathology of the prostate assisting more accurate histological grading in needle biopsies and collaborating for better surveillance and therapeutic choice.

Neutrophils are an important cell in host defence and migrate rapidly to sites of inflammation when the host is compromised (e.g., in infection or wounding). There, they produce and/or release inflammatory mediators (e.g., LTB4, TNF, IL-8) and ingest and degrade pathogens (e.g., by release of granule proteins and reactive oxygen species). Neutrophils then undergo apoptosis and are cleared by phagocytes such as macrophages, to allow efficient resolution of inflammation. Inducing neutrophil apoptosis by pharmacological means could be a therapeutic strategy to dampen inflammation in diseases where neutrophils are prevalent, e.g., acute respiratory distress syndrome (ARDS) and rheumatoid arthritis (RA). Inhibition of cyclin-dependent kinases (CDKs) using CDK inhibitor (CDKi) compounds induces mammalian neutrophil apoptosis in vitro, and can drive resolution of inflammation in vivo in mouse models. Evidence indicated that this is due to inhibition of CDK9 and CDK7-mediated transcription of the anti-apoptotic protein Mcl-1. The hypothesis of this project was that CDK9, CDK7 and Mcl-1 are pivotal regulators of resolution of inflammation in vivo. The model selected to test this hypothesis was tailfin injury of embryonic zebrafish (Danio rerio). Zebrafish are optically transparent and reporter transgenic lines with neutrophils labelled by enhanced GFP (EGFP - Tg[mpx:EGFP]i114) and macrophages (Tg[MPEG1:mCherry]) have been created, permitting the imaging of the behaviour of these cells in vivo. The model of tailfin transection was chosen to cause an inflammatory response in these animals, with neutrophil and macrophage recruitment to the tailfin. This response was manipulated using CDKi compounds and specific gene knockdowns (using morpholino and CRISPR/cas9 technologies). It was shown that CDKi compounds could reduce neutrophil numbers at 24 h post-injury at the transected tailfin, but did not affect macrophage numbers. The CDKi AT7519 increased neutrophil apoptosis at 12 h post-injury. Specific CDK9 knockdown using morpholinos or CRISPR/cas9 also reduced neutrophilic inflammation at the tailfin 24 h after transection, accompanied by increased apoptosis levels at 8 h in the morpholino-treated group. Inhibition of an endogenous CDK9 inhibitor, LaRP7, had the opposite effect and increased neutrophil numbers; and could oppose the neutrophil- reducing effect of AT7519 and CDK9 morpholino knockdown. Preliminary genetic knockdown studies into the roles of CDK7 and Mcl-1 have been carried out. Taken together, the results demonstrate CDK9 is important in the resolution of neutrophilic inflammation, indicating that manipulation of CDK9 activity could be a good target for therapeutic intervention in inflammatory disease.

O câncer de próstata é o tumor maligno mais freqüente nos homens com idade superior a 50 anos, excetuando-se os tumores cutâneos. No Brasil estima-se para o ano de 2012 cerca de 60.180 casos novos deste tipo de neoplasia. Os marcadores tumorais permitem fazer o rastreamento do câncer, o diagnóstico diferencial entre uma neoplasia benigna e maligna, a avaliação de prognóstico e o acompanhamento terapêutico, assim como a detecção da recidiva tumoral. Dentre estes marcadores tumorais, tem-se dado muito atenção para as proteínas que mediam e participam da progressão do ciclo celular. A ciclina D1 é uma proteína nuclear de vida curta que é destruída pela via da ubiquitina ATP dependente, e está envolvida na transição celular da fase do ciclo G1 (repouso) para a fase S (síntese) tanto em células normais como em células neoplásicas. A super expressão de ciclina D1 remove a regulação normal do ciclo celular causando proliferação celular descontrolada, um crescimento anormal dos tecidos e a transformação para um fenótipo neoplásico, atuando como oncogene. No presente trabalho foi estudado a expressão de ciclina D1 em adenocarcinomas de próstata, tendo como objetivo avaliar a relação desta proteína com parâmetros epidemiológicos, clínicos e histopatológicos. Adicionalmente também foi feita comparação de escore de Gleason e lateralidade tumoral entre biópsias prostáticas com agulha e de prostatectomias radicais. No ensaio para ciclina D1 foram analisados 85 casos através de imunoistoquímica (IHQ) de material proveniente de prostatectomias radicais diagnosticados com adenocarcinoma de próstata entre os anos de 2005 e 2010 em nosso serviço. O método de avaliação se utilizou de microscopia ótica comum e contagem semi-quantitativa, comparado-se a expressão com achados clínicos, epidemiológicos e histopatológicos utilizando-se Teste T de Fisher, Qui Quadrado, Mann-Whitney, Curva ROC e correlação de Spearman. Os resultados demonstraram correlação positiva de ciclina D1 com escore de Gleason (p<0,05), com volume prostático (p=0,01) e uma tendência a correlação positiva com invasão perineural (p=0,07). Não houve correlação estatística entre ciclina D1 e o aumento de PSA, assim como outros achados histopatológicos. As biópsias prostáticas com agulha apresentaram subestimação em 40% dos casos para escore de Gleason e de 62,3% dos casos para lateralidade tumoral quando comparadas a prostatectomia radical. Já que as taxas de subestimação de escore de Gleason e lateralidade tumoral são relativamente altas e visto a urgência em se padronizar novos biomarcadores para o câncer prostático, sugerimos que ciclina D1 pode ser utilizada como biomarcador em patologia cirúrgica da próstata auxiliando numa gradação histológica mais precisa em biópsias com agulha colaborando para melhor vigilância e escolha terapêutica. / Prostate cancer is the most common malignant tumor in men older than 50 years, except for skin tumors. In Brazil it is estimated for the year 2012 about 60,180 new cases of this type of neoplasm. Tumor markers allow to cancer screening, differential diagnosis between a benign and malignant, assessment of prognosis and therapeutic monitoring, and detection of tumor recurrence. Among these tumor markers, has been given much attention for proteins that mediate and participate in cell cycle progression. Cyclin D1 is a short-lived nuclear protein that is destroyed by the ATP ubiquitin dependent pathway, and is involved in the transition of cell cycle G1 phase (resting) to the S phase (synthesis) cells both in normal and neoplastic cells. The overexpression of cyclin D1 removes the normal regulation of cell cycle causing uncontrolled cell proliferation, abnormal growth of tissues and transformation to a neoplastic phenotype, acting as an oncogene. In the present work we studied the expression of cyclin D1 in prostate adenocarcinomas, and to evaluate the relationship of this protein with epidemiologic factors, clinical and histopathological features. Additionally comparison was also made of Gleason score and laterality between tumor biopsies and prostate needle radical prostatectomies. In the assay for cyclin D1 were 85 cases analyzed by immunohistochemistry (IHC) of material from radical prostatectomies diagnosed with prostate adenocarcinoma between the years 2005 and 2010 at our institution. The evaluation method utilized were light microscopy and semi-quantitative score, comparing the cyclin D1 expression with clinical, epidemiological and histopathological features using Fisher\'s exact test, chi square test, Mann-Whitney test, ROC curve and Spearman correlation. The results showed a positive correlation of cyclin D1 with Gleason score (p <0.05), prostate volume (p = 0.01) and a trend toward positive correlation with perineural invasion (p = 0.07). There was no statistical correlation between cyclin D1 and increased PSA, as well as other histopathologic features. Prostate needle biopsies showed underestimation in 40% of cases for Gleason score and 62.3% of cases for tumor laterality when compared to radical prostatectomy. Since the rates of underestimation of Gleason score and tumor laterality are relatively high and the urgency to standardize new biomarkers for prostate cancer, we suggest that cyclin D1 may be used as biomarkers in surgical pathology of the prostate assisting more accurate histological grading in needle biopsies and collaborating for better surveillance and therapeutic choice.

Abstract not available

"September, 2002." / Bibliography: leaves 93-106. / 106 leaves : ill. (some col.), plates ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / In this thesis the biochemical and genetic characterization of Fkh2p identifies it as a major component of SFF. It has been shown to bind DNA in an Mcm1p dependent manner and the Fkh2p DNA binding domain is essential for this interaction. The protein interaction domain of Mcm1p has been demonstrated to be essential for ternary complex formation. Fkh2p, along with a functionally redundant protein Fkh1p, has been show to control the periodic expression of the CLB2 cluster genes. The functional characterisation of the Fkh2p domains reveals an important role for both the Forkhead associated domain and the C-terminus. Ndd1p. another protein important for mitotic progression, is shown to be important for CLB2 cluster regulation by de-repressing Fkh2p and activating gene expression. The role of cdk activity is shown to act through the CLB2 cluster upstream activating sequences, possibly through Ndd1p. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2003

Deregulated cell proliferation, resulting from disruption of cell cycle control, is characteristic of many cancers. In normal cells, cell cycle progression is mediated by a family of cyclin dependent kinases (Cdks) that are positively regulated by associated cyclins. The activities of these cyclin-Cdk complexes are regulated by two protein families: the inhibitors of Cdk4 (INK4) and the kinase inhibitor proteins (KIP). p27 is a KIP family member that can inhibit cyclin E-Cdk2 activity. It also plays a role in the assembly and nuclear import of cyclin D-Cdk4 in early G1. p27 has been shown to be deregulated in human cancers by accelerated proteolysis, sequestration in cyclin D-Cdk complexes, and mislocalization to the cytoplasm. The causes of these alterations are not fully understood, but result, at least in part, from changes in signal transduction pathways that alter p27 phosphorylation and function. Activation of both the Ras/Raf/ mitogen activated protein kinase (MAPK) and the phospho-inositol 3' kinase (PI3K) pathways have been shown to alter p27 function and to activate p27 degradation in different cell types. In this thesis, I have investigated the roles played by two kinases downstream of PI3K, protein kinase B (PKB) and p90 ribosomal S6 kinase (RSK1), in regulation of p27 function. I observed that PKB-mediated phosphorylation of p27 promotes p27-cyclin D1-Cdk4 assembly. p27 phosphorylation by RSK1 alters the interaction of p27 with cytoskeleton proteins to promote cell motility. I observed that PKB activation and the appearance of p27pT157 and p27pT198 in early G1 precede p27-cyclin D1-Cdk4 assembly. PI3K/PKB inhibition dissociates cellular p27-cyclin D1-Cdk4 and p27T157A, p27T198A and p27T157A/T198A bind cellular cyclin D1 and Cdk4 poorly. Cellular p27pT157 and p27pT198 co-precipitate with Cdk4 but not Cdk2. p27 phosphorylation by PKB increases the ability of p27 to assemble cyclin D1-Cdk4 in vitro, but yields inactive Cdk4. While Src does not affect p27's ability to assemble cyclin D1-Cdk4, Src treatment yields catalytically active p27-cyclin D1-Cdk4. Thus, while PKB dependent p27 phosphorylation promotes p27-cyclin D1-Cdk4 assembly, tyrosine phosphorylation of p27 is required for activation of p27-cyclin D1-Cdk4 complexes. Constitutive activation of PKB and Abl or Src family kinases in cancers would drive p27 phosphorylation, increase cyclin D1-Cdk4 assembly and activation, and reduce the cyclin E-Cdk2 inhibitory function of p27. Combined therapy with both Src and PI3K/PKB inhibitors may reverse this process. While RSK1 has been shown to phosphorylate p27, the key phosphorylation sites and the consequence of this phosphorylation event were not fully elucidated. I have shown that RSK1 activation in early G1 precedes p27 phosphorylation at T157 and T198 in synchronized cell populations. Overexpression of RSK1 causes resistance to G1 arrest by TGF-â. Moreover, cells overexpressing RSK1 show an increase in p27 phosphorylation at T198, increased p27 stability, and an increase in p27 binding to Cdk4. In addition, RSK1-transfectants have increased cytoplasmic p27, associated with increased cell motility and inhibition of RhoA. p27 phosphorylation by recombinant RSK1 increases p27 binding to RhoA, while p27T157A/T198A shows reduced association with RhoA in cells. Thus, phosphorylation of p27 at T198 by RSK1 promotes its binding to RhoA and loss of actin stress fiber stability. Oncogenic RSK1 activation may promote increased cancer cell migration and cancer metastasis. Taken together our results indicate that oncogenic activation of the PI3K pathway can contribute to loss of cyclin E-Cdk2 inhibitory action of p27 by at least two mechanisms. Activation of PKB and RSK1 signaling would promote cytoplasmic mislocalization of p27, p27-RhoA binding and inhibition of the RhoA pathway to augment cell motility. In addition, these phosphorylation events on p27 would increase the assembly of p27-cyclin D1-Cdk4 as a first step in a chain of events that would promote that nuclear import and activation of D-type cyclin Cdk complexes, shifting the equilibrium away from the Cdk2 inhibitory action of p27.

1,25-(OH)2 vitamin D3 inhibits cell proliferation of a variety of cancers including prostate. In the human prostate cancer cell line LNCaP, 1,25-(OH)2 vitamin D3-mediated growth inhibition is attributed to cell cycle G1 accumulation which correlates with a robust decrease of cyclin-dependent kinase 2 (CDK2) activity and pronounced relocalization of CDK2 into the cytoplasm. Nuclear targeting CDK2 blocks the 1,25-(OH)2 vitamin D3-mediated growth inhibition and cell cycle G1 accumulation. Further, the nuclear targeted CDK2 blocks 1,25-(OH)2 vitamin D3-mediated inhibition of CDK2 activity and nuclear exclusion in LNCaP cells. Therefore, CDK2 cytoplasmic relocalization is the key mechanism for 1,25-(OH)2 vitamin D3 effects. Since cyclin E is important for CDK2 nuclear localization and activation, 1,25-(OH)2 vitamin D3 may exert its effects through regulation of cyclin E. Cyclin E but not a cyclin E mutant deficient in CDK2 binding reverses 1,25-(OH)2 vitamin D3-mediated antiproliferation which suggests the involvement of cyclin E as a mechanism. However, the studies showed no effects of 1,25-(OH)2 vitamin D3 on cyclin E levels, intracellular localization or binding to CDK2. In order to develop a model for studying 1,25-(OH)2 vitamin D3-mediated antiproliferative effects, LNCaP vitD.R cell line, a vitamin D resistant LNCaP derivative, was generated by continuously culturing of LNCaP cells in medium supplemented with 10 nM 1,25-(OH)2 vitamin D3 for over 9 months. The initial characterization of this cell line showed complete resistance to 1,25-(OH)2 vitamin D3-mediated effects. Analysis of vitamin D regulation of VDR target gene expression revealed that vitamin D resistance in LNCaP vitD.R cells was not due to deregulation of VDR signaling. HDAC inhibitor Trichostatin A (TSA) did not confer sensitivity of LNCaP vitD.R cells to vitamin D treatment suggested the resistance to 1,25(OH)2 vitamin D3 effect of LNCaP vitD.R cells is not due to histone deacetylase remodeling of the chromatin structure which leads to inhibition of gene transcription. While the partial sensitization of LNCaP vitD.R cells to 1,25(OH)2 vitamin D3 effect by demethylation reagent 5-Aza-2¡¯-deoxycytidine treatment suggested a set of genes involved in 1,25(OH)2 vitamin D3-mediated antiproliferative effects is silenced via hypermethylation in LNCaP vitD.R cells. These results suggested LNCaP vitD. R cell line is a useful tool and further studies to elucidate the genes involved in this effect will help uncover the mechanisms of 1,25(OH)2 vitamin D3-mediated antiproliferative effects.

The mechanism by which prostate cancer progresses from androgen dependence (AD) to androgen independence/castration resistance (AI/CR) is currently a major focus of prostate cancer-related research. Prostate cancers that progress to a state of AI/CR are typically resistant to most standard types of treatments. Due to its primary role in driving normal prostate cell growth and proliferation, the androgen receptor (AR) is believed to play a key role in progression. Coregulators, or any proteins which may either enhance or abrogate AR activity, are considered to be one of the potential mechanisms by which AR function may become impaired. Cyclin G-associated kinase (GAK) was initially identified as a potential coregulator of AR in a Tup 1 repressed transactivation system. A LNCaP cDNA library was screened for proteins which interacted with the NH2-terminus of AR. GAK was isolated from three independent library clones using two different AR baits (AR 1-549 and AR 1-646). This interaction was confirmed via GST pulldown and coimmunoprecipitation experiments, and preliminary luciferase assays suggested that GAK activates AR in a hormone dependent manner. In this study, my objectives were to validate GAK’s role as a coregulator of AR and to determine if overexpressing GAK affects progression to AI. In vitro luciferase assays whereby GAK was either overexpressed or knocked down in both LNCaP and PC3 cells did not significantly affect AR activity. Xenograft experiments utilizing a doxycycline (DOX) inducible lentiviral LNCaP-GAK overexpressing stable cell line demonstrated that while GAK may not play a significant role in modulating AR activity, it may adopt a more subtle role enhancing tumour take and tumour volume growth rate in vivo. While these results could not confirm GAK to be a direct coregulator of AR, it is entirely possible that GAK may influence prostate cancer progression, albeit indirectly. Recent publications report a growing amount of evidence suggesting GAK’s involvement in the critical cellular process of clathrin coated vesicle endocytosis, the dysregulation of which could potentially indirectly affect AR regulated genes.

<p>Cancer is a disease state that arises as a result of multiple alterations in signaling pathways that are critical for making key cell fate decisions in normal cells. Understanding how these pathways operate under normal circumstances, therefore, is crucial for comprehensive understanding of tumorigenic process. With Myc and E2F pathways being central components for controlling cell proliferation, an important property that defines a cancer cell, as well as expanding roles for microRNAs(miRNA) in control of gene expression, we asked if we may better understand the underlying regulatory (transcription factor, microRNA) structure that contribute to Myc and E2F pathway activities. Through integrative analysis of mRNA and miRNA expression profile, we observe a distinct regulatory pattern in which, in the case of Myc pathway, Myc-induced miRNAs were contributing to the repression of negative regulators of cell cycle, including PTEN, while in case of E2F pathway, E2F-induced miRs were forming an incoherent Feed-Forward Loop(iFFL) with a number of E2F-induced genes including cyclin E. We further demonstrate through functional studies, as well as through single cell imaging of gene expression dynamics that miRNAs, depending on the context of either Myc or E2F pathway, play distinct roles in ensuring that cell fate decisions relevant to these pathways are properly executed.</p> / Dissertation

Spinal cord injury, acute pain, and morphine tolerance are important issues in the clinical practice. A primary injury to the spinal cord causes both morphological and biochemical changes with initiation of the devastating secondary pathophysiological pathways that ultimately destroy CNS cells and cause degeneration of nerve fibers. Tissue injury is associated with sensitization of nociceptors and subsequent changes in the excitability of central neurons, known as central sensitization. Nociceptor sensitization and central sensitization are believed to underlie the development of primary and secondary hyperalgesia, respectively. The most efficacious drugs used to relieve pain are the opioid analgesics. Chronic administration leads to the development of tolerance. Tolerance is manifested as a decreased potency of the drug, so that progressively larger doses must be administered to achieve a given level of analgesia. The processes underlying opioid tolerance still need to be elucidated. Recently, it is found calpain-Cdk5 (cyclin-dependent kinase-5)-p35 pathway modulation implicated in neuroprotection, acute nociceptive response, and morphine analgesia. In this thesis, we evaluate calpain inhibitor-MDL28170 and Cdk5 inhibitor-roscovitine against rat spinal cord hemisection, formalin-induced acute nociceptive responses, and chronic morphine tolerance. We found calpain-Cdk5-p35 pathway inhibition could protect spinal cord hemisection and subsequent neurodegeneration, inhibit formalin-induced flinch response involving DARPP-32 (dopamine and c-AMP regulated phosphoprotein, MW=32 kDa) phosphorylation, and reverse right shifted morphine dose-response curve with upregulated ED50 (50% of effective dose) reduction. Taken together, calpain-Cdk5-p35 pathway inhibition is useful in the management of spinal cord injury, acute inflammatory pain, and attenuate morphine tolerance development with further clinical application.