Publications by authors named "Xianjun Fang"

54 Publications

Momordin Ic induces G0/1 phase arrest and apoptosis in colon cancer cells by suppressing SENP1/c-MYC signaling pathway.

J Pharmacol Sci 2021 Aug 11;146(4):249-258. Epub 2021 May 11.

Department of Pharmacy, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, PR China. Electronic address:

Momordin Ic (MI) is a natural pentacyclic triterpenoid enriched in various Chinese natural medicines such as the fruit of Kochia scoparia (L.) Schrad. Studies have shown that MI presents antitumor properties in liver and prostate cancers. However, the activity and potential mechanisms of MI against colorectal cancer remain elusive. Here, we showed that MI inhibited cell proliferation with G0/1 phase cell cycle arrest in colon cancer cells. Moreover, it was observed that MI increased apoptosis compared to untreated cells. Further investigation showed that the SUMOylation of c-Myc was enhanced by MI and led to the down-regulated protein level of c-Myc, which is involved in regulating cell proliferation and apoptosis. SENP1 has been demonstrated to be critical for the SUMOylation of c-Myc. Meanwhile, knockdown of SENP1 by siRNA abolished the effects of MI on c-Myc level and cell viability in colon cancer cells. Together, these results revealed that MI exerted an anti-tumor activity in colon cancer cells via SENP1/c-Myc signaling pathway. These finding provide an insight into the potential of MI for colon cancer therapy.
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http://dx.doi.org/10.1016/j.jphs.2021.04.007DOI Listing
August 2021

A Fenofibrate Diet Prevents Paclitaxel-Induced Peripheral Neuropathy in Mice.

Cancers (Basel) 2020 Dec 29;13(1). Epub 2020 Dec 29.

Department of Pharmacology and Toxicology and Translational Research Initiative for Pain and Neuropathy, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23284, USA.

Background: Paclitaxel-induced peripheral neuropathy (PIPN) is a major adverse effect of this chemotherapeutic agent that is used in the treatment of a number of solid malignancies. PIPN leads notably to burning pain, cold and mechanical allodynia. PIPN is thought to be a consequence of alterations of mitochondrial function, hyperexcitability of neurons, nerve fiber loss, oxidative stress and neuroinflammation in dorsal root ganglia (DRG) and spinal cord (SC). Therefore, reducing neuroinflammation could potentially attenuate neuropathy symptoms. Peroxisome proliferator-activated receptor-α (PPAR-α) nuclear receptors that modulate inflammatory responses can be targeted by non-selective agonists, such as fenofibrate, which is used in the treatment of dyslipidemia.

Methods: Our studies tested the efficacy of a fenofibrate diet (0.2% and 0.4%) in preventing the development of PIPN. Paclitaxel (8 mg/kg) was administered via 4 intraperitoneal (i.p.) injections in C57BL/6J mice (both male and female). Mechanical and cold hypersensitivity, wheel running activity, sensory nerve action potential (SNAP), sciatic nerve histology, intra-epidermal fibers, as well as the expression of PPAR-α and neuroinflammation were evaluated in DRG and SC.

Results: Fenofibrate in the diet partially prevented the development of mechanical hypersensitivity but completely prevented cold hypersensitivity and the decrease in wheel running activity induced by paclitaxel. The reduction in SNAP amplitude induced by paclitaxel was also prevented by fenofibrate. Our results indicate that suppression of paclitaxel-induced pain by fenofibrate involves the regulation of PPAR-α expression through reduction in neuroinflammation. Finally, co-administration of paclitaxel and the active metabolite of fenofibrate (fenofibric acid) did not interfere with the suppression of tumor cell growth or clonogenicity by paclitaxel in ovarian and breast cancer cell lines.

Conclusions: Taken together, our results show the therapeutic potential of fenofibrate in the prevention of PIPN development.
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http://dx.doi.org/10.3390/cancers13010069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795224PMC
December 2020

Lysophosphatidic acid induces tumor necrosis factor-alpha to regulate a pro-inflammatory cytokine network in ovarian cancer.

FASEB J 2020 10 26;34(10):13935-13948. Epub 2020 Aug 26.

Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA, USA.

Epithelial ovarian carcinoma tissues express high levels of tumor necrosis factor-alpha (TNF-α) and other inflammatory cytokines. The underlying mechanism leading to the abnormal TNF-α expression in ovarian cancer remains poorly understood. In the current study, we demonstrated that lysophosphatidic acid (LPA), a lipid mediator present in ascites of ovarian cancer patients, induced expression of TNF-α mRNA and release of TNF-α protein in ovarian cancer cells. LPA also induced expression of interleukin-1β (IL-1β) mRNA but no significant increase in IL-1β protein was detected. LPA enhanced TNF-α mRNA through NF-κB-mediated transcriptional activation. Inactivation of ADAM17, a disintegrin and metalloproteinase, with a specific inhibitor TMI-1 or by shRNA knockdown prevented ovarian cancer cells from releasing TNF-α protein in response to LPA, indicating that LPA-mediated TNF-α production relies on both transcriptional upregulations of the TNF-α gene and the activity of ADAM17, the membrane-associated TNF-α-converting enzyme. Like many other biological responses to LPA, induction of TNF-α by LPA also depended on the transactivation of the epidermal growth factor receptor (EGFR). Interestingly, our results revealed that ADAM17 was also the shedding protease responsible for the transactivation of EGFR by LPA in ovarian cancer cells. To explore the biological outcomes of LPA-induced TNF-α, we examined the effects of a TNF-α neutralizing antibody and recombinant TNF-α soluble receptor on LPA-stimulated expression of pro-tumorigenic cytokines and chemokines overexpressed in ovarian cancer. Blockade of TNF-α signaling significantly reduced the production of IL-8, IL-6, and CXCL1, suggesting a hierarchy of mechanisms contributing to the robust expression of the inflammatory mediators in response to LPA in ovarian cancer cells. In contrast, TNF-α inhibition did not affect LPA-dependent cell proliferation. Taken together, our results establish that the bioactive lipid LPA drives the expression of TNF-α to regulate an inflammatory network in ovarian cancer.
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http://dx.doi.org/10.1096/fj.202001136RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722118PMC
October 2020

Functional analysis of molecular and pharmacological modulators of mitochondrial fatty acid oxidation.

Sci Rep 2020 01 29;10(1):1450. Epub 2020 Jan 29.

Departments of Biochemistry & Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, Virginia, 23298, USA.

Fatty acid oxidation (FAO) is a key bioenergetic pathway often dysregulated in diseases. The current knowledge on FAO regulators in mammalian cells is limited and sometimes controversial. Previous FAO analyses involve nonphysiological culture conditions or lack adequate quantification. We herein described a convenient and quantitative assay to monitor dynamic FAO activities of mammalian cells in physiologically relevant settings. The method enabled us to assess various molecular and pharmacological modulators of the FAO pathway in established cell lines, primary cells and mice. Surprisingly, many previously proposed FAO inhibitors such as ranolazine and trimetazidine lacked FAO-interfering activity. In comparison, etomoxir at low micromolar concentrations was sufficient to saturate its target proteins and to block cellular FAO function. Oxfenicine, on the other hand, acted as a partial inhibitor of FAO. As another class of FAO inhibitors that transcriptionally repress FAO genes, antagonists of peroxisome proliferator-activated receptors (PPARs), particularly that of PPARα, significantly decreased cellular FAO activity. Our assay also had sufficient sensitivity to monitor upregulation of FAO in response to environmental glucose depletion and other energy-demanding cues. Altogether this study provided a reliable FAO assay and a clear picture of biological properties of potential FAO modulators in the mammalian system.
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http://dx.doi.org/10.1038/s41598-020-58334-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989517PMC
January 2020

Berberine suppresses colon cancer cell proliferation by inhibiting the SCAP/SREBP-1 signaling pathway-mediated lipogenesis.

Biochem Pharmacol 2020 04 23;174:113776. Epub 2019 Dec 23.

Department of Pharmacy, Nanjing First Hospital, Nanjing Medical University, Nanjing 210029, PR China; School of Basic Medicine & Clinical Pharmacy, China Pharmaceutical University, Nanjing 211198, PR China. Electronic address:

Lipid metabolism is a significant section of energy homeostasis, and it affects the development of various cancers. Previous studies have revealed that berberine has strong anticancer and blood lipid-lowering effects. Here, we further investigated the effects of berberine on cell proliferation and lipogenesis in colon cancer cells and the relationship between the two effects. We found that berberine inhibited cell proliferation by inducing G0/G1 phase cell cycle arrest in colon cancer cells. Moreover, the expressions of key lipogenic enzymes were down-regulated by berberine and led to the suppressed lipid synthesis, which was linked to cell proliferation via Wnt/β-catenin pathway. Importantly, berberine inhibited sterol regulatory element-binding protein-1 (SREBP-1) activation and SREBP cleavage-activating protein (SCAP) expression, resulting in the downregulation of these lipogenic enzymes. Knockdown of SCAP by shRNA could abolish the effect of berberine on SREBP-1 activation. Besides the inhibitory effects in vitro, berberine suppressed the growth and lipogenesis of colon cancer xenograft in a SCAP-dependent manner as well. Together, our results suggest that berberine may serve as a candidate against tumor growth of colon cancer partially through targeting SCAP/SREBP-1 pathway driving lipogenesis.
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http://dx.doi.org/10.1016/j.bcp.2019.113776DOI Listing
April 2020

Immunometabolism: A new target for improving cancer immunotherapy.

Adv Cancer Res 2019 17;143:195-253. Epub 2019 Apr 17.

Department of Human & Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, VA, United States; VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, VA, United States; VCU Massey Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, VA, United States. Electronic address:

Fundamental metabolic pathways are essential for mammalian cells to provide energy, precursors for biosynthesis of macromolecules, and reducing power for redox regulation. While dysregulated metabolism (e.g., aerobic glycolysis also known as the Warburg effect) has long been recognized as a hallmark of cancer, recent discoveries of metabolic reprogramming in immune cells during their activation and differentiation have led to an emerging concept of "immunometabolism." Considering the recent success of cancer immunotherapy in the treatment of several cancer types, increasing research efforts are being made to elucidate alterations in metabolic profiles of cancer and immune cells during their interplays in the setting of cancer progression and immunotherapy. In this review, we summarize recent advances in studies of metabolic reprogramming in cancer as well as differentiation and functionality of various immune cells. In particular, we will elaborate how distinct metabolic pathways in the tumor microenvironment cause functional impairment of immune cells and contribute to immune evasion by cancer. Lastly, we highlight the potential of metabolically reprogramming the tumor microenvironment to promote effective and long-lasting antitumor immunity for improved immunotherapeutic outcomes.
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http://dx.doi.org/10.1016/bs.acr.2019.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6822174PMC
July 2020

LPA receptor 4 deficiency attenuates experimental atherosclerosis.

J Lipid Res 2019 05 22;60(5):972-980. Epub 2019 Feb 22.

Division of Cardiovascular Medicine, Gill Heart and Vascular Institute, University of Kentucky, Lexington, KY 40536; Veterans Affairs Medical Center, Lexington, KY 40511. Electronic address:

The widely expressed lysophosphatidic acid (LPA) selective receptor 4 (LPAR4) contributes to vascular development in mice and zebrafish. LPAR4 regulates endothelial permeability, lymphocyte migration, and hematopoiesis, which could contribute to atherosclerosis. We investigated the role of LPAR4 in experimental atherosclerosis elicited by adeno-associated virus expressing PCSK9 to lower LDL receptor levels. After 20 weeks on a Western diet, cholesterol levels and lipoprotein distribution were similar in WT male and mice ( = 0.94). The atherosclerotic lesion area in the proximal aorta and arch was ∼25% smaller in mice ( = 0.009), and less atherosclerosis was detected in mice at any given plasma cholesterol. Neutral lipid accumulation in aortic root sections occupied ∼40% less area in mice ( = 0.001), and CD68 expression was ∼25% lower ( = 0.045). No difference in α-smooth muscle actin staining was observed. Bone marrow-derived macrophages isolated from mice displayed significantly increased upregulation of the M2 marker in response to LPA compared with WT cells. In aortic root sections from mice, heightened M2 "repair" macrophage marker expression was detected by CD206 staining ( = 0.03). These results suggest that LPAR4 may regulate the recruitment of specific sets of macrophages or their phenotypic switching in a manner that could influence the development of atherosclerosis.
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http://dx.doi.org/10.1194/jlr.M091066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6495174PMC
May 2019

Ovarian Cancer Relies on Glucose Transporter 1 to Fuel Glycolysis and Growth: Anti-Tumor Activity of BAY-876.

Cancers (Basel) 2018 Dec 31;11(1). Epub 2018 Dec 31.

Department of Biochemistry & Molecular Biology, Virginia Commonwealth University School of Medicine, 1101 East Marshall Street, Richmond, VA 23298, USA.

The recent progresses in understanding of cancer glycolytic phenotype have offered new strategies to manage ovarian cancer and other malignancies. However, therapeutic targeting of glycolysis to treat cancer remains unsuccessful due to complex mechanisms of tumor glycolysis and the lack of selective, potent and safe glycolytic inhibitors. Recently, BAY-876 was identified as a new-generation inhibitor of glucose transporter 1 (GLUT1), a GLUT isoform commonly overexpressed but functionally poorly defined in ovarian cancer. Notably, BAY-876 has not been evaluated in any cell or preclinical animal models since its discovery. We herein took advantage of BAY-876 and molecular approaches to study GLUT1 regulation, targetability, and functional relevance to cancer glycolysis. The anti-tumor activity of BAY-876 was evaluated with ovarian cancer cell line- and patient-derived xenograft (PDX) models. Our results show that inhibition of GLUT1 is sufficient to block basal and stress-regulated glycolysis, and anchorage-dependent and independent growth of ovarian cancer cells. BAY-876 dramatically inhibits tumorigenicity of both cell line-derived xenografts and PDXs. These studies provide direct evidence that GLUT1 is causally linked to the glycolytic phenotype in ovarian cancer. BAY-876 is a potent blocker of GLUT1 activity, glycolytic metabolism and ovarian cancer growth, holding promise as a novel glycolysis-targeted anti-cancer agent.
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http://dx.doi.org/10.3390/cancers11010033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356953PMC
December 2018

Fatty acid oxidation: An emerging facet of metabolic transformation in cancer.

Cancer Lett 2018 10 10;435:92-100. Epub 2018 Aug 10.

Department of Biochemistry & Molecular Biology, School of Medicine, Virginia Commonwealth University, Richmond, VA, 23298, USA. Electronic address:

Cancer cells undergo metabolic reprogramming such as enhanced aerobic glycolysis, mutations in the tricarboxylic acid cycle enzymes, and upregulation of de novo lipid synthesis and glutaminolysis. These alterations are pivotal to the development and maintenance of the malignant phenotype of cancer cells in unfavorable tumor microenvironment or metastatic sites. Although mitochondrial fatty acid β-oxidation (FAO) is a primary bioenergetic source, it has not been generally recognized as part of the metabolic landscape of cancer. The last few years, however, have seen a dramatic change in the view of cancer relevance of the FAO pathway. Many recent studies have provided significant evidence to support a "lipolytic phenotype" of cancer. FAO, like other well-defined metabolic pathways involved in cancer, is dysregulated in diverse human malignancies. Cancer cells rely on FAO for proliferation, survival, stemness, drug resistance, and metastatic progression. FAO is also reprogrammed in cancer-associated immune and other host cells, which may contribute to immune suppression and tumor-promoting microenvironment. This article reviews and puts into context our current understanding of multi-faceted roles of FAO in oncogenesis as well as anti-cancer therapeutic opportunities posed by the FAO pathway.
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http://dx.doi.org/10.1016/j.canlet.2018.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240910PMC
October 2018

Berberine inhibits macrophage M1 polarization via AKT1/SOCS1/NF-κB signaling pathway to protect against DSS-induced colitis.

Int Immunopharmacol 2018 Apr 24;57:121-131. Epub 2018 Feb 24.

Department of Pharmacy, Nanjing First Hospital, Nanjing Medical University, Nanjing 210029, PR China; School of Basic Medicine & Clinical Pharmacy, China Pharmaceutical University, Nanjing 210009, PR China. Electronic address:

Berberine has been reported to have protective effects in colitis treatment. However, the detailed mechanisms remain unclear. Herein, we demonstrated that berberine could protect against dextran sulfate sodium (DSS)-induced colitis in mice by regulating macrophage polarization. In the colitis mouse model, berberine ameliorated DSS-induced colon shortening and colon tissue injury. Moreover, berberine-treated mice showed significant reduction in the disease activity index (DAI), pro-inflammatory cytokines expression and macrophages infiltration compared with the DSS-treated mice. Notably, berberine significantly reduced the percentage of M1 macrophages. In vitro analysis also confirmed the inhibitory effects of berberine on macrophages M1 polarization in RAW267.4 cells. Further investigation showed that berberine promoted AKT1 expression in mRNA and protein level. Silence of AKT1 abolished the inhibitory effect of berberine on macrophages M1 polarization. The berberine-induced AKT1 expression promoted suppressers of cytokine signaling (SOCS1) activation, which inhibited nuclear factor-kappa B (NF-κB) phosphorylation. In addition, we also found that berberine activated AKT1/SOCS1 signaling pathway but inhibited p65 phosphorylation in macrophages in vivo. Therefore, we concluded that berberine played a regulatory role in macrophages M1 polarization in DSS-induced colitis via AKT1/SOCS1/NF-κB signaling pathway. This unexpected property of berberine may provide a potential explanation for its protective effects in colitis treatment.
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http://dx.doi.org/10.1016/j.intimp.2018.01.049DOI Listing
April 2018

The lignan manassantin is a potent and specific inhibitor of mitochondrial complex I and bioenergetic activity in mammals.

J Biol Chem 2017 12 18;292(51):20989-20997. Epub 2017 Oct 18.

From the Departments of Biochemistry and Molecular Biology,

Dineolignans manassantin A and B from the plant are used in traditional medicine to manage a wide range of ailments such as edema, jaundice, and gonorrhea. Cell-based studies have identified several molecular target candidates of manassantin including NF-κB, MAPK, STAT3, and hypoxia-inducible factor 1α (HIF-1α). It is unclear whether or how these structurally diverse proteins or pathways mediate any of the medical benefits of manassantin Moreover, it has recently been reported that manassantin causes developmental arrest in zebrafish by inhibiting the mitochondrial complex I, but it is unknown whether manassantin inhibits mitochondrial respiration in intact mammalian cells and live animals. Here, we present direct evidence that manassantin potently and specifically inhibits the mitochondrial complex I and bioenergetic activity in mammalian systems. Manassantin had no effect on complex II- or complex IV-mediated respiration. Of note, it decreased NADH-ubiquinone reductase activity but not the activity of NADH-ferricyanide reductase. Treatment with manassantin reduced cellular ATP levels and concomitantly stimulated AMP-activated protein kinase and As an adaptive response to manassantin-induced bioenergetic deficiency, mammalian cells up-regulated aerobic glycolysis, a process mediated by AMP-activated protein kinase (AMPK) independently of HIF-1α. Together these results demonstrate a biologically important activity of manassantin in the control of complex I-mediated respiration and its profound effects on oxygen utilization, energy homeostasis, and glucose metabolism in mammalian cells.
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http://dx.doi.org/10.1074/jbc.M117.812925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743073PMC
December 2017

Nicotine Prevents and Reverses Paclitaxel-Induced Mechanical Allodynia in a Mouse Model of CIPN.

J Pharmacol Exp Ther 2018 01 17;364(1):110-119. Epub 2017 Oct 17.

Departments of Pharmacology and Toxicology (S.L.K., W.T., D.B., J.A.M., L.D.S., A.H.L., M.I.D., D.A.G.), Neurology (Z.-J.C.), Internal Medicine (E.D.F.), Biochemistry and Molecular Biology (X.F.), and Anatomy and Neurobiology (J.W.B.), and Massey Cancer Center (D.A.G.), Virginia Commonwealth University, Richmond, Virginia; and Experimental Animals Breeding and Research Center, Uludag University, Bursa, Turkey (D.B.).

Chemotherapy-induced peripheral neuropathy (CIPN), a consequence of peripheral nerve fiber dysfunction or degeneration, continues to be a dose-limiting and debilitating side effect during and/or after cancer chemotherapy. Paclitaxel, a taxane commonly used to treat breast, lung, and ovarian cancers, causes CIPN in 59-78% of cancer patients. Novel interventions are needed due to the current lack of effective CIPN treatments. Our studies were designed to investigate whether nicotine can prevent and/or reverse paclitaxel-induced peripheral neuropathy in a mouse model of CIPN, while ensuring that nicotine will not stimulate lung tumor cell proliferation or interfere with the antitumor properties of paclitaxel. Male C57BL/6J mice received paclitaxel every other day for a total of four injections (8 mg/kg, i.p.). Acute (0.3-0.9 mg/kg, i.p.) and chronic (24 mg/kg per day, s.c.) administration of nicotine respectively reversed and prevented paclitaxel-induced mechanical allodynia. Blockade of the antinociceptive effect of nicotine with mecamylamine and methyllycaconitine suggests that the reversal of paclitaxel-induced mechanical allodynia is primarily mediated by the 7 nicotinic acetylcholine receptor subtype. Chronic nicotine treatment also prevented paclitaxel-induced intraepidermal nerve fiber loss. Notably, nicotine neither promoted proliferation of A549 and H460 non-small cell lung cancer cells nor interfered with paclitaxel-induced antitumor effects, including apoptosis. Most importantly, chronic nicotine administration did not enhance Lewis lung carcinoma tumor growth in C57BL/6J mice. These data suggest that the nicotinic acetylcholine receptor-mediated pathways may be promising drug targets for the prevention and treatment of CIPN.
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http://dx.doi.org/10.1124/jpet.117.243972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738719PMC
January 2018

CRH promotes human colon cancer cell proliferation via IL-6/JAK2/STAT3 signaling pathway and VEGF-induced tumor angiogenesis.

Mol Carcinog 2017 Nov 13;56(11):2434-2445. Epub 2017 Jul 13.

Department of Pharmacology, Nanjing Medical University, Nanjing, China.

Corticotrophin-releasing hormone (CRH) has been demonstrated to participate in various diseases. Our previous study showed that its receptor CRHR1 mediated the development of colitis-associated cancer in mouse model. However, the detailed mechanisms remain unclear. In this study, we explored the oncogenetic role of CRH/CRHR1 signaling in colon cancer cells. Cell proliferation and colony formation assays revealed that CRH contributed to cell proliferation. Moreover, tube formation assay showed that CRH-treated colon cancer cell supernatant significantly promoted tube formation of human umbilical vein endothelial cells (HUVECs). And these effects could be reversed by the CRHR1 specific antagonist Antalarmin. Further investigation showed that CRH significantly upregulated the expressions of interlukin-6 (IL-6) and vascular endothelial growth factor (VEGF) through activating nuclear factor-kappa B (NF-κB). The CRH-induced IL-6 promoted phosphorylation of janus kinase 2 (JAK2) and signal transducers and activators of transcription 3 (STAT3). STAT3 inhibition by Stattic significantly inhibited the CRH-induced cell proliferation. In addition, silence of VEGF resulted in declined tube formation induced by CRH. Taken together, CRH/CRHR1 signaling promoted human colon cancer cell proliferation via NF-κB/IL-6/JAK2/STAT3 signaling pathway and tumor angiogenesis via NF-κB/VEGF signaling pathway. Our results provide evidence to support a critical role for the CRH/CRHR1 signaling in colon cancer progression and suggest its potential utility as a new therapeutic target for colon cancer.
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http://dx.doi.org/10.1002/mc.22691DOI Listing
November 2017

The role of cPLA2 in Methylglyoxal-induced cell apoptosis of HUVECs.

Toxicol Appl Pharmacol 2017 05 22;323:44-52. Epub 2017 Mar 22.

Jiangsu Provincial Key Lab of Cardiovascular Diseases and Molecular intervention, Department of Pharmacology, Nanjing Medical University, Nanjing 210029, PR China. Electronic address:

Methylglyoxal (MGO), a highly reactive dicarbonyl compound, is mainly formed as a byproduct of glycolysis. Elevated MGO level is known to induce apoptosis of vascular endothelial cells, which is implicated with progression of atherosclerosis and diabetic complications. However, the underlying mechanisms have not been exhaustively investigated yet. Here, we further characterized the mechanisms how MGO induced apoptosis in human umbilical vein endothelial cells (HUVECs). Our data revealed that cytosolic phospholipase A2 (cPLA2) played an important role in MGO-induced cell apoptosis. It was found that MGO could increase both the activity and expression of cPLA2. Inhibition of cPLA2 by Pyrrophenone (PYR) or siRNA significantly attenuated the MGO-induced apoptosis. Additionally, MGO time-dependently decreased the phosphorylation of nuclear factor κB (NF-κB). Pretreatment of the cells with NF-κB inhibitor, BAY11-7082, further increased MGO-induced apoptosis of HUVECs, indicating that NF-κB played a survival role in this MGO-induced apoptosis. Furthermore, in the presence of si-cPLA2 or PYR, MGO no longer decreased NF-κB phosphorylation. Beyond that, the antioxidant N-acetyl cysteine (NAC) could reverse the changes of both cPLA2 and NF-κB caused by MGO. p38, the upstream of cPLA2, was also significantly phosphorylated by MGO. However, p38 inhibitor failed to reverse the apoptosis induced by MGO. This study gives an important insight into the downstream signaling mechanisms of MGO, cPLA2-NF-κB, in endothelial apoptosis.
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http://dx.doi.org/10.1016/j.taap.2017.03.020DOI Listing
May 2017

Carnitine palmitoyltransferase 1A functions to repress FoxO transcription factors to allow cell cycle progression in ovarian cancer.

Oncotarget 2016 Jan;7(4):3832-46

Departments of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA.

Cancer cells rely on hyperactive de novo lipid synthesis for maintaining malignancy. Recent studies suggest involvement in cancer of fatty acid oxidation, a process functionally opposite to lipogenesis. A mechanistic link from lipid catabolism to oncogenic processes is yet to be established. Carnitine palmitoyltransferase 1 (CPT1) is a rate-limiting enzyme of fatty acid β-oxidation (FAO) that catalyzes the transfer of long-chain acyl group of the acyl-CoA ester to carnitine, thereby shuttling fatty acids into the mitochondrial matrix for β-oxidation. In the present study, we demonstrated that CPT1A was highly expressed in most ovarian cancer cell lines and primary ovarian serous carcinomas. Overexpression of CPT1A correlated with a poor overall survival of ovarian cancer patients. Inactivation of CPT1A decreased cellular ATP levels and induced cell cycle arrest at G0/G1, suggesting that ovarian cancer cells depend on or are addicted to CPT1A-mediated FAO for cell cycle progression. CPT1A deficiency also suppressed anchorage-independent growth and formation of xenografts from ovarian cancer cell lines. The cyclin-dependent kinase inhibitor p21WAF1 (p21) was identified as most consistently and robustly induced cell cycle regulator upon inactivation of CPT1A. Furthermore, p21 was transcriptionally upregulated by the FoxO transcription factors, which were in turn phosphorylated and activated by AMP-activated protein kinase and the mitogen-activated protein kinases JNK and p38. Our results established the oncogenic relevance of CPT1A and a mechanistic link from lipid catabolism to cell cycle regulation, suggesting that CPT1A could be a prognostic biomarker and rational target for therapeutic intervention of cancer.
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http://dx.doi.org/10.18632/oncotarget.6757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4826173PMC
January 2016

Sperm Associated Antigen 6 (SPAG6) Regulates Fibroblast Cell Growth, Morphology, Migration and Ciliogenesis.

Sci Rep 2015 11 20;5:16506. Epub 2015 Nov 20.

Department of Obstetrics &Gynecology, Virginia Commonwealth University, Richmond, VA, 23298.

Mammalian Spag6 is the orthologue of Chlamydomonas PF16, which encodes a protein localized in the axoneme central apparatus, and regulates flagella/cilia motility. Most Spag6-deficient mice are smaller in size than their littermates. Because SPAG6 decorates microtubules, we hypothesized that SPAG6 has other roles related to microtubule function besides regulating flagellar/cilia motility. Mouse embryonic fibroblasts (MEFs) were isolated from Spag6-deficient and wild-type embryos for these studies. Both primary and immortalized Spag6-deficient MEFs proliferated at a much slower rate than the wild-type MEFs, and they had a larger surface area. Re-expression of SPAG6 in the Spag6-deficient MEFs rescued the abnormal cell morphology. Spag6-deficient MEFs were less motile than wild-type MEFs, as shown by both chemotactic analysis and wound-healing assays. Spag6-deficient MEFs also showed reduced adhesion associated with a non-polarized F-actin distribution. Multiple centrosomes were observed in the Spag6-deficient MEF cultures. The percentage of cells with primary cilia was significantly reduced compared to the wild-type MEFs, and some Spag6-deficient MEFs developed multiple cilia. Furthermore, SPAG6 selectively increased expression of acetylated tubulin, a microtubule stability marker. The Spag6-deficient MEFs were more sensitive to paclitaxel, a microtubule stabilizer. Our studies reveal new roles for SPAG6 in modulation of cell morphology, proliferation, migration, and ciliogenesis.
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http://dx.doi.org/10.1038/srep16506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4653743PMC
November 2015

Lysophosphatidic Acid Up-Regulates Hexokinase II and Glycolysis to Promote Proliferation of Ovarian Cancer Cells.

Neoplasia 2015 Sep;17(9):723-734

Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298. Electronic address:

Lysophosphatidic acid (LPA), a blood-borne lipid mediator, is present in elevated concentrations in ascites of ovarian cancer patients and other malignant effusions. LPA is a potent mitogen in cancer cells. The mechanism linking LPA signal to cancer cell proliferation is not well understood. Little is known about whether LPA affects glucose metabolism to accommodate rapid proliferation of cancer cells. Here we describe that in ovarian cancer cells, LPA enhances glycolytic rate and lactate efflux. A real time PCR-based miniarray showed that hexokinase II (HK2) was the most dramatically induced glycolytic gene to promote glycolysis in LPA-treated cells. Analysis of the human HK2 gene promoter identified the sterol regulatory element-binding protein as the primary mediator of LPA-induced HK2 transcription. The effects of LPA on HK2 and glycolysis rely on LPA2, an LPA receptor subtype overexpressed in ovarian cancer and many other malignancies. We further examined the general role of growth factor-induced glycolysis in cell proliferation. Like LPA, epidermal growth factor (EGF) elicited robust glycolytic and proliferative responses in ovarian cancer cells. Insulin-like growth factor 1 (IGF-1) and insulin, however, potently stimulated cell proliferation but only modestly induced glycolysis. Consistent with their differential effects on glycolysis, LPA and EGF-dependent cell proliferation was highly sensitive to glycolytic inhibition while the growth-promoting effect of IGF-1 or insulin was more resistant. These results indicate that LPA- and EGF-induced cell proliferation selectively involves up-regulation of HK2 and glycolytic metabolism. The work is the first to implicate LPA signaling in promotion of glucose metabolism in cancer cells.
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http://dx.doi.org/10.1016/j.neo.2015.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4611075PMC
September 2015

In-depth LC-MS/MS analysis of the chicken ovarian cancer proteome reveals conserved and novel differentially regulated proteins in humans.

Anal Bioanal Chem 2015 Sep 10;407(22):6851-63. Epub 2015 Jul 10.

W.M. Keck FTMS Laboratory for Human Health Research, Department of Chemistry, North Carolina State University, 2620 Yarbrough Dr., Box 8204, Raleigh, NC, 27695, USA.

Ovarian cancer (OVC) remains the most lethal gynecological malignancy in the world due to the combined lack of early-stage diagnostics and effective therapeutic strategies. The development and application of advanced proteomics technology and new experimental models has created unique opportunities for translational studies. In this study, we investigated the ovarian cancer proteome of the chicken, an emerging experimental model of OVC that develops ovarian tumors spontaneously. Matched plasma, ovary, and oviduct tissue biospecimens derived from healthy, early-stage OVC, and late-stage OVC birds were quantitatively characterized by label-free proteomics. Over 2600 proteins were identified in this study, 348 of which were differentially expressed by more than twofold (p ≤ 0.05) in early- and late-stage ovarian tumor tissue specimens relative to healthy ovarian tissues. Several of the 348 proteins are known to be differentially regulated in human cancers including B2M, CLDN3, EPCAM, PIGR, S100A6, S100A9, S100A11, and TPD52. Of particular interest was ovostatin 2 (OVOS2), a novel 165-kDa protease inhibitor found to be strongly upregulated in chicken ovarian tumors (p = 0.0005) and matched plasma (p = 0.003). Indeed, RT-quantitative PCR and Western blot analysis demonstrated that OVOS2 mRNA and protein were also upregulated in multiple human OVC cell lines compared to normal ovarian epithelia (NOE) cells and immunohistochemical staining confirmed overexpression of OVOS2 in primary human ovarian cancers relative to non-cancerous tissues. Collectively, these data provide the first evidence for involvement of OVOS2 in the pathogenesis of both chicken and human ovarian cancer.
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http://dx.doi.org/10.1007/s00216-015-8862-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5266611PMC
September 2015

The role of corticotropin-releasing hormone receptor 1 in the development of colitis-associated cancer in mouse model.

Endocr Relat Cancer 2014 Aug;21(4):639-51

Key Laboratory of Cardiovascular and Molecular InterventionDepartment of Pharmacology, Nanjing Medical University, Nanjing 210029, People's Republic of China

Patients with ulcerative colitis are at a very high risk of developing colorectal cancer. Corticotrophin-releasing hormone (CRH) family peptides and their receptors (CRHRs) are found to modulate inflammation and tumor cell growth. However, the role of CRH family peptides and their receptors in the inflammation-related colon cancer is still unknown. The aim of this study was to investigate the functions of CRHR1 signaling on the development of colitis-associated cancer (CAC). Crhr1-deficient (Crhr1(-/-)) mice were used to explore the role of CRHR1 in the development of azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced CAC. WT (Crhr1(+/+)) littermates were set as control. We found that the expression of CRHR1 and its endogenous ligands: urocortin and CRH were enhanced in the colon of Crhr1(+/+) mice during treatment with AOM and DSS. Tumorigenesis was significantly reduced in Crhr1(-/-) mice, determined by analysis of survival rate (increased by 20%), weight loss (decreased by 10%), tumor formation (decreased by 60% in tumor number), histological scores (decreased by 58%), and cytokine production. During early CAC tumorigenesis, Crhr1(-/-) mice exhibited much less tumorigenesis, accompanied by lower inflammatory response, including decreased IL1β, IL6 and TNFα expression and macrophage infiltration and increased IL10 expression. Moreover, Crhr1(-/-) mice displayed a reduced activation of NFκB and STAT3 phosphorylation with decreased proliferating and enhanced apoptotic cells in the colon. In conclusion, CRHR1 has a proinflammatory and therefore a protumorigenesis effect in terms of CAC, which may be helpful to develop new therapeutic approaches for inflammation and cancer prevention and treatment.
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http://dx.doi.org/10.1530/ERC-14-0239DOI Listing
August 2014

Corticotropin-releasing hormone receptors mediate apoptosis via cytosolic calcium-dependent phospholipase A₂ and migration in prostate cancer cell RM-1.

J Mol Endocrinol 2014 Jun 28;52(3):255-67. Epub 2014 Apr 28.

Jiangsu Provincial Key Lab of Cardiovascular Diseases and Molecular Intervention, Department of Pharmacology, Nanjing Medical University, Nanjing 210029, China.

Peripheral corticotropin-releasing hormone receptors (CRHRs) are G protein-coupled receptors that play different roles depending on tissue types. Previously, we discovered the mechanism of CRHR-mediated apoptosis of mouse prostate cancer cell line (RM-1) to be a change of Bcl-2:Bax ratio, and CRH was found to inhibit transforming growth factor β migration of breast cancer cells via CRHRs. In the present study, we investigated cytosolic calcium-dependent phospholipase A2 (cPLA2) bridging CRHR activations and Bcl-2:Bax ratio and the effect of CRHR activation on cell migration. Silencing of cPLA2 attenuated a CRHR1 agonist, CRH-induced apoptosis, and the decrease of the Bcl-2:Bax ratio, whereas silencing of cPLA2 aggravated CRHR2 agonist, Urocortin 2 (Ucn2)-inhibited apoptosis, and the increase of the Bcl-2:Bax ratio. CRH in a time- and concentration-dependent manner increased cPLA2 expression mainly through interleukin 1β (IL1β) upregulation. Ucn2 decreased cPLA2 expression through neither tumor necrosis factor α nor IL1β. CRH-suppressed decay of cPLA2 mRNA and Ucn2 merely suppressed its production. Overexpression of CRHR1 or CRHR2 in HEK293 cells correspondingly upregulated or downregulated cPLA2 expression after CRH or Ucn2 stimulation respectively. In addition, both CRH and Ucn2 induced migration of RM-1 cells. Our observation not only established a relationship between CRHRs and cell migration but also for the first time, to our knowledge, demonstrated that cPLA2 participates in CRHR1-induced apoptosis and CRHR2-inhibited apoptosis.
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http://dx.doi.org/10.1530/JME-13-0270DOI Listing
June 2014

Apigenin sensitizes colon cancer cells to antitumor activity of ABT-263.

Mol Cancer Ther 2013 Dec 14;12(12):2640-50. Epub 2013 Oct 14.

Corresponding Authors: Huanjie Shao, Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, 1101 East Marshall Street, Richmond, VA 23298.

Apigenin is an edible plant-derived flavonoid that shows modest antitumor activities in vitro and in vivo. Apigenin treatment resulted in cell growth arrest and apoptosis in various types of tumors by modulating several signaling pathways. In the present study, we evaluated interactions between apigenin and ABT-263 in colon cancer cells. We observed a synergistic effect between apigenin and ABT-263 on apoptosis of colon cancer cells. ABT-263 alone induced limited cell death while upregulating expression of Mcl-1, a potential mechanism for the acquired resistance to ABT-263. The presence of apigenin antagonized ABT-263-induced Mcl-1 upregulation and dramatically enhanced ABT-263-induced cell death. Meanwhile, apigenin suppressed AKT and ERK activation. Inactivation of either AKT or ERK by lentivirus-transduced shRNA or treatment with specific small-molecule inhibitors of these pathways enhanced ABT-263-induced cell death, mirroring the effect of apigenin. Moreover, the combination response was associated with upregulation of Bim and activation of Bax. Downregulation of Bax eliminated the synergistic effect of apigenin and ABT-263 on cell death. Xenograft studies in SCID mice showed that the combined treatment with apigenin and ABT-263 inhibited tumor growth by up to 70% without obvious adverse effects, while either agent only inhibited around 30%. Our results demonstrate a novel strategy to enhance ABT-263-induced antitumor activity in human colon cancer cells by apigenin via inhibition of the Mcl-1, AKT, and ERK prosurvival regulators.
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http://dx.doi.org/10.1158/1535-7163.MCT-13-0066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3871201PMC
December 2013

The potential role of anibamine, a natural product CCR5 antagonist, and its analogues as leads toward development of anti-ovarian cancer agents.

Bioorg Med Chem Lett 2012 Aug 16;22(15):5093-7. Epub 2012 Jun 16.

Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298, USA.

Chemokines and their receptors play important roles in the development of primary tumors and their metastases. Particularly CC chemokine receptor 5 (CCR5) and its ligand CC chemokine ligand 5 (CCL5/RANTES) seem to be critical in proliferation and invasion of ovarian cancer, the leading cause of death from gynecological malignancies in the United States. Anibamine, the first natural product CCR5 antagonist, and its analogues were examined for their effects on proliferation of the OVCAR-3 ovarian cancer cells in order to validate their candidacy as leads to develop novel anti-ovarian cancer agents. Acting as CCR5 antagonists, anibamine and its analogues significantly suppressed CCL5-induced intracellular Ca(2+) flux. The compounds also inhibited the proliferation of OVCAR-3 at micromolar to submicromolar range. Moreover, anibamine and several analogues did not show significant cytotoxicity in NIH 3T3 cells at concentrations up to 20μM. Based on these results, anibamine and one of its synthetic analogues were defined as potential leads to develop novel agents against ovarian cancer.
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http://dx.doi.org/10.1016/j.bmcl.2012.05.127DOI Listing
August 2012

Lysophosphatidic acid activates lipogenic pathways and de novo lipid synthesis in ovarian cancer cells.

J Biol Chem 2012 Jul 3;287(30):24990-5000. Epub 2012 Jun 3.

Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, School of Medicine, Richmond, Virginia 23298, USA.

One of the most common molecular changes in cancer is the increased endogenous lipid synthesis, mediated primarily by overexpression and/or hyperactivity of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC). The changes in these key lipogenic enzymes are critical for the development and maintenance of the malignant phenotype. Previous efforts to control oncogenic lipogenesis have been focused on pharmacological inhibitors of FAS and ACC. Although they show anti-tumor effects in culture and in mouse models, these inhibitors are nonselective blockers of lipid synthesis in both normal and cancer cells. To target lipid anabolism in tumor cells specifically, it is important to identify the mechanism governing hyperactive lipogenesis in malignant cells. In this study, we demonstrate that lysophosphatidic acid (LPA), a growth factor-like mediator present at high levels in ascites of ovarian cancer patients, regulates the sterol regulatory element binding protein-FAS and AMP-activated protein kinase-ACC pathways in ovarian cancer cells but not in normal or immortalized ovarian epithelial cells. Activation of these lipogenic pathways is linked to increased de novo lipid synthesis. The pro-lipogenic action of LPA is mediated through LPA(2), an LPA receptor subtype overexpressed in ovarian cancer and other malignancies. Downstream of LPA(2), the G(12/13) and G(q) signaling cascades mediate LPA-dependent sterol regulatory element-binding protein activation and AMP-activated protein kinase inhibition, respectively. Moreover, inhibition of de novo lipid synthesis dramatically attenuated LPA-induced cell proliferation. These results demonstrate that LPA signaling is causally linked to the hyperactive lipogenesis in ovarian cancer cells, which can be exploited for development of new anti-cancer therapies.
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http://dx.doi.org/10.1074/jbc.M112.340083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408203PMC
July 2012

Innate immune agonist, dsRNA, induces apoptosis in ovarian cancer cells and enhances the potency of cytotoxic chemotherapeutics.

FASEB J 2012 Aug 24;26(8):3188-98. Epub 2012 Apr 24.

Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia, USA.

Ovarian cancer is the most lethal gynecological cancer. Here we show that innate immune agonist, dsRNA, directly induces ovarian cancer cell death and identify biomarkers associated with responsiveness to this targeted treatment. Nuclear staining and MTT assays following dsRNA stimulation revealed two subpopulations, sensitive (OVCAR-3, CAOV-3; patient samples malignant 1 and 2) and resistant (DOV-13, SKOV-3). Microarray analysis identified 75 genes with differential expression that further delineated these two subpopulations. qPCR and immunoblot analyses showed increased dsRNA receptor expression after stimulation as compared to resistant and immortalized ovarian surface epithelial cells (e.g., 70-fold with malignant 2, 43-fold with OVCAR-3). Using agonists, antagonists, and shRNA-mediated knockdown of dsRNA receptors, we show that TLR3, RIG-I, and mda5 coordinated a caspase 8/9- and interferon-dependent cell death. In resistant cells, dsRNA receptor overexpression restored dsRNA sensitivity. When dsRNA was combined with carboplatin or paclitaxel, cell viability significantly decreased over individual treatments (1.5- to 7.5-fold). Isobologram analyses showed synergism in dsRNA combinations (CI=0.4-0.82) vs. an additive effect in carboplatin/paclitaxel treatment (CI=1.5-2). Our data identify a predictive marker, dsRNA receptor expression, to target dsRNA responsive populations and show that, in dsRNA-sensitive cells, dsRNA induces apoptosis and enhances the potency of cytotoxic chemotherapeutics.
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http://dx.doi.org/10.1096/fj.11-202333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3405273PMC
August 2012

Characterization of lysophosphatidic acid subspecies produced by autotaxin using a modified HPLC ESI-MS/MS method.

Anal Methods 2011 Nov;3(12):2822-2828

Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, 1101 East Marshall Street, P.O. Box 980614, Richmond, Virginia, 23298-0614 ; Research and Development, Hunter Holmes McGuire Veterans Administration Medical Center, Richmond, Virginia, 23249 ; The Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia, 23298.

Lysophosphatidic acid (LPA) is a bioactive lipid with a plethora of biological functions including roles in cell survival, proliferation, and migration. Although high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC ESI-MS/MS) technology has been used to measure the levels of LPA in human blood, serum and plasma, current methods cannot readily detect the minute levels of LPA from cell culture. In this study, a modified HPLC ESI-MS/MS method with enhanced sensitivity was developed, which allows accurate measurements of LPA levels with a limit of quantitation at approximately 10 femtomoles. The method was validated by quantitation of LPA levels in the media of previously characterized cell lines ectopically expressing autotaxin. Specifically, autotaxin overexpression induced an increase in the 16:0, 18:2, 18:1, 18:0, and 20:4 subspecies of LPA, but not the 22:6 LPA subspecies. Lastly, this HPLC ESI-MS/MS method was cross-validated biological assays previously utilized to assay LPA levels. Hence, this HPLC ESI-MS/MS method will allow researchers to measure LPA levels and also distinguish between specific LPA subspecies for the delineation of individual biological mechanisms.
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http://dx.doi.org/10.1039/C1AY05459GDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956132PMC
November 2011

Lysophosphatidic acid-induced p21Waf1 expression mediates the cytostatic response of breast and ovarian cancer cells to TGFβ.

Mol Cancer Res 2011 Nov 2;9(11):1562-70. Epub 2011 Sep 2.

Virginia Commonwealth University School of Medicine, Department of Biochemistry and Molecular Biology, Richmond, VA 23298, USA.

Lysophosphatidic acid (LPA) is a multifunctional intercellular phospholipid mediator present in blood and other biological fluids. In cancer cells, LPA stimulates expression or activity of inflammatory cytokines, angiogenic factors, matrix metalloproteinases, and other oncogenic proteins. In this study, we showed that LPA upregulated expression of the cyclin-dependent kinase inhibitor p21(Waf1) in TGFβ-sensitive breast and ovarian cancer cells, but not in TGFβ-resistant ones. We examined the possibility that LPA-induced p21 might contribute to the cytostatic response to TGFβ. In serum-free conditions, TGFβ alone induced p21 expression weakly in TGFβ-sensitive cells. Serum or serum-borne LPA cooperated with TGFβ to elicit the maximal p21 induction. LPA stimulated p21 via LPA(1) and LPA(2) receptors and Erk-dependent activation of the CCAAT/enhancer binding protein beta transcription factor independent of p53. Loss or gain of p21 expression led to a shift between TGFβ-sensitive and -resistant phenotypes in breast and ovarian cancer cells, indicating that p21 is a key determinant of the growth inhibitory activity of TGFβ. Our results reveal a novel cross-talk between LPA and TGFβ that underlies TGFβ-sensitive and -resistant phenotypes of breast and ovarian cancer cells.
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http://dx.doi.org/10.1158/1541-7786.MCR-11-0340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3388908PMC
November 2011

Lysophosphatidic acid stimulates gastric cancer cell proliferation via ERK1-dependent upregulation of sphingosine kinase 1 transcription.

FEBS Lett 2010 Sep 7;584(18):4077-82. Epub 2010 Sep 7.

Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298-0614, USA.

In MKN1 gastric cancer cells, lysophosphatidic acid (LPA) upregulates expression of sphingosine kinase 1 (SphK1) and its downregulation or inhibition suppresses LPA mediated proliferation. Although LPA activates numerous signaling pathways downstream of its receptors, including extracellular-signal-regulated kinase 1/2, p38, JNK, and Akt, and the transactivation of the epidermal growth factor receptor, pharmacological and molecular approaches demonstrated that only activation of ERK1, in addition to the CCAAT/enhancer-binding protein β transcription factor, is involved in transcriptional upregulation of SphK1 by LPA. Our data implicate ERK1 as an important mediator of LPA signaling leading to upregulation of SphK1 and point to SphK1 and sphingosine-1-phosphate production as potential therapeutic targets in gastric cancer.
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http://dx.doi.org/10.1016/j.febslet.2010.08.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2947956PMC
September 2010

Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells.

BMB Rep 2010 Aug;43(8):541-6

Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA.

We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29-915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated from conditioned medium of a stable clone by affinity chromatography with Protein A sepharose followed by cleavage with thrombin. The untagged ATX protein was further purified to essential homogeneity by gel filtration chromatography with a yield of approximately 5 mg/liter medium. The purified ATX protein was enzymatically active and biologically functional, offering a useful tool for further biological and structural studies of this important enzyme.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872511PMC
http://dx.doi.org/10.5483/bmbrep.2010.43.8.541DOI Listing
August 2010

Differential requirement of the epidermal growth factor receptor for G protein-mediated activation of transcription factors by lysophosphatidic acid.

Mol Cancer 2010 Jan 14;9. Epub 2010 Jan 14.

Department of Biochemistry, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA.

Background: The role of the epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases (RTKs) in provoking biological actions of G protein-coupled receptors (GPCRs) has been one of the most disputed subjects in the field of GPCR signal transduction. The purpose of the current study is to identify EGFR-mediated mechanisms involved in activation of G protein cascades and the downstream transcription factors by lysophosphatidic acid (LPA).

Results: In ovarian cancer cells highly responsive to LPA, activation of AP-1 by LPA was suppressed by inhibition of EGFR, an effect that could be reversed by co-stimulation of another receptor tyrosine kinase c-Met with hepatocyte growth factor, indicating that LPA-mediated activation of AP-1 requires activity of a RTK, not necessarily EGFR. Induction of AP-1 components by LPA lied downstream of Gi, G12/13, and Gq. Activation of the effectors of Gi, but not Gq or G12/13 was sensitive to inhibition of EGFR. In contrast, LPA stimulated another prominent transcription factor NF-kappaB via the Gq-PKC pathway in an EGFR-independent manner. Consistent with the importance of Gi-elicited signals in a plethora of biological processes, LPA-induced cytokine production, cell proliferation, migration and invasion require intact EGFR.

Conclusions: An RTK activity is required for activation of the AP-1 transcription factor and other Gi-dependent cellular responses to LPA. In contrast, activation of G12/13, Gq and Gq-elicited NF-kappaB by LPA is independent of such an input. These results provide a novel insight into the role of RTK in GPCR signal transduction and biological functions.
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http://dx.doi.org/10.1186/1476-4598-9-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817642PMC
January 2010

Expression of autotaxin and lysophosphatidic acid receptors increases mammary tumorigenesis, invasion, and metastases.

Cancer Cell 2009 Jun;15(6):539-50

Department of Systems Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.

Lysophosphatidic acid (LPA) acts through high-affinity G protein-coupled receptors to mediate a plethora of physiological and pathological activities associated with tumorigenesis. LPA receptors and autotaxin (ATX/LysoPLD), the primary enzyme producing LPA, are aberrantly expressed in multiple cancer lineages. However, the role of ATX and LPA receptors in the initiation and progression of breast cancer has not been evaluated. We demonstrate that expression of ATX or each edg family LPA receptor in mammary epithelium of transgenic mice is sufficient to induce a high frequency of late-onset, estrogen receptor (ER)-positive, invasive, and metastatic mammary cancer. Thus, ATX and LPA receptors can contribute to the initiation and progression of breast cancer.
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http://dx.doi.org/10.1016/j.ccr.2009.03.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157573PMC
June 2009
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