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1.
Mol Cancer Res ; 20(8): 1233-1246, 2022 08 05.
Article in English | MEDLINE | ID: mdl-35533313

ABSTRACT

Coevolution of tumor cells and adjacent stromal elements is a key feature during tumor progression; however, the precise regulatory mechanisms during this process remain unknown. Here, we show stromal p53 loss enhances oncogenic KrasG12D, but not ErbB2, driven tumorigenesis in murine mammary epithelia. Stroma-specific p53 deletion increases both epithelial and fibroblast proliferation in mammary glands bearing the KrasG12D oncogene in epithelia, while concurrently increasing DNA damage and/or DNA replication stress and decreasing apoptosis in the tumor cells proper. Normal epithelia was not affected by stromal p53 deletion. Tumors with p53-null stroma had a significant decrease in total, cytotoxic, and regulatory T cells; however, there was a significant increase in myeloid-derived suppressor cells, total macrophages, and M2-polarized tumor-associated macrophages, with no impact on angiogenesis or connective tissue deposition. Stroma-specific p53 deletion reprogrammed gene expression in both fibroblasts and adjacent epithelium, with p53 targets and chemokine receptors/chemokine signaling pathways in fibroblasts and DNA replication, DNA damage repair, and apoptosis in epithelia being the most significantly impacted biological processes. A gene cluster in p53-deficient mouse fibroblasts was negatively associated with patient survival when compared with two independent datasets. In summary, stroma-specific p53 loss promotes mammary tumorigenesis in an oncogene-specific manner, influences the tumor immune landscape, and ultimately impacts patient survival. IMPLICATIONS: Expression of the p53 tumor suppressor in breast cancer tumor stroma regulates tumorigenesis in an oncogene-specific manner, influences the tumor immune landscape, and ultimately impacts patient survival.


Subject(s)
Breast Neoplasms , Oncogenes , Tumor Suppressor Protein p53 , Animals , Breast Neoplasms/genetics , Breast Neoplasms/immunology , Carcinogenesis , Connective Tissue/metabolism , Mice , Proto-Oncogene Proteins p21(ras) , Stromal Cells/pathology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
2.
PLoS One ; 15(2): e0228252, 2020.
Article in English | MEDLINE | ID: mdl-32027675

ABSTRACT

Herpes simplex virus type-1 (HSV-1) can cause severe ocular infection and blindness. We have previously shown that the HSV-1 VC2 vaccine strain is protective in mice and guinea pigs against genital herpes infection following vaginal challenge with HSV-1 or HSV-2. In this study, we evaluated the efficacy of VC2 intramuscular vaccination in mice against herpetic keratitis following ocular challenge with lethal human clinical strain HSV-1(McKrae). VC2 vaccination in mice produced superior protection and morbidity control in comparison to its parental strain HSV-1(F). Specifically, after HSV-1(McKrae) ocular challenge, all VC2 vaccinated- mice survived, while 30% of the HSV-1(F)- vaccinated and 100% of the mock-vaccinated mice died post challenge. VC2-vaccinated mice did not exhibit any symptoms of ocular infection and completely recovered from initial conjunctivitis. In contrast, HSV-1(F)-vaccinated mice developed time-dependent progressive keratitis characterized by corneal opacification, while mock-vaccinated animals exhibited more severe stromal keratitis characterized by immune cell infiltration and neovascularization in corneal stroma with corneal opacification. Cornea in VC2-immunized mice exhibited significantly increased infiltration of CD3+ T lymphocytes and decreased infiltration of Iba1+ macrophages in comparison to mock- or HSV-1(F)-vaccinated groups. VC2 immunization produced higher virus neutralization titers than HSV-1(F) post challenge. Furthermore, VC-vaccination significantly increased the CD4 T central memory (TCM) subsets and CD8 T effector memory (TEM) subsets in the draining lymph nodes following ocular HSV-1 (McKrae) challenge, then mock- or HSV-1(F)-vaccination. These results indicate that VC2 vaccination produces a protective immune response at the site of challenge to protect against HSV-1-induced ocular pathogenesis.


Subject(s)
Herpes Simplex Virus Vaccines/immunology , Herpes Simplex/prevention & control , Herpesvirus 1, Human/pathogenicity , Animals , Antigens, Viral/immunology , Cornea/pathology , Cornea/virology , Female , Herpes Simplex/pathology , Herpes Simplex/veterinary , Herpesvirus 1, Human/metabolism , Humans , Injections, Intramuscular , Mice , Mice, SCID , Vaccination
3.
Am J Physiol Lung Cell Mol Physiol ; 317(2): L212-L221, 2019 08 01.
Article in English | MEDLINE | ID: mdl-31090436

ABSTRACT

Maternal smoking during pregnancy and exposure of infants to cigarette smoke are strongly associated with adverse health effects in childhood including higher susceptibility to respiratory viral infections. Human respiratory syncytial virus (HRSV) is the most important cause of lower respiratory tract infection among young infants. Exacerbation of respiratory disease, including HRSV bronchiolitis and higher susceptibility to HRSV infection, is well correlated with previous smoke exposure. The mechanisms of recurrence and susceptibility to viral pathogens after passive smoke exposure are multifactorial and include alteration of the structural and immunologic host defenses. In this work, we used a well-established mouse model of in utero smoke exposure to investigate the effect of in utero smoke exposure in HRSV-induced pathogenesis. Sample analysis indicated that in utero exposure led to increased lung inflammation characterized by an increased influx of neutrophils to the airways of the infected mice and a delayed viral clearance. On the other hand, decreased HRSV-specific CD8+ T-cell response was observed. These findings indicate that cigarette smoke exposure during pregnancy alters HRSV-induced disease as well as several aspects of the neonatal immune responses.


Subject(s)
Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus, Human/pathogenicity , T-Lymphocytes/immunology , Tobacco Smoke Pollution/adverse effects , Animals , Animals, Newborn , Disease Models, Animal , Female , Humans , Lung/pathology , Lung/virology , Male , Neutrophils/immunology , Neutrophils/virology , Pneumonia/immunology , Pneumonia/virology , Respiratory Tract Infections/immunology , Respiratory Tract Infections/virology , Smoke/adverse effects , T-Lymphocytes/pathology , T-Lymphocytes/virology
4.
Curr Clin Microbiol Rep ; 5(1): 55-65, 2018 Mar.
Article in English | MEDLINE | ID: mdl-30560044

ABSTRACT

PURPOSE: The earliest host-virus interactions occur during virus attachment and entry into cells. These initial steps in the virus lifecycle influence the outcome of infection beyond delivery of the viral genome into the cell. Herpesviruses alter host signaling pathways and processes during attachment and entry to facilitate virus infection and modulate innate immune responses. We suggest in this review that understanding these early signaling events may inform the rational design of therapeutic and prevention strategies for herpesvirus infection, as well as the engineering of viral vectors for immunotherapy purposes. RECENT FINDINGS: Recent reports demonstrate that modulation of Herpes Simplex Virus Type-1 (HSV-1) entry results in unexpected enhancement of antiviral immune responses. SUMMARY: A variety of evidence suggests that herpesviruses promote specific cellular signaling responses that facilitate viral replication after binding to cell surfaces, as well as during virus entry. Of particular interest is the ability of the virus to alter innate immune responses through these cellular signaling events. Uncovering the underlying immune evasion strategies may lead to the design of live-attenuated vaccines that can generate robust and protective anti-viral immune responses against herpesviruses. These adjuvant properties may be extended to a variety of heterologous antigens expressed by herpesviral vectors.

5.
Front Cardiovasc Med ; 5: 114, 2018.
Article in English | MEDLINE | ID: mdl-30211171

ABSTRACT

Myocardial infarction (MI), which occurs often due to acute ischemia followed by reflow, is associated with irreversible loss (death) of cardiomyocytes. If left untreated, MI will lead to progressive loss of viable cardiomyocytes, deterioration of cardiac function, and congestive heart failure. While supplemental oxygen therapy has long been in practice to treat acute MI, there has not been a clear scientific basis for the observed beneficial effects. Further, there is no rationale for the amount or duration of administration of supplemental oxygenation for effective therapy. The goal of the present study was to determine an optimum oxygenation protocol that can be clinically applicable for treating acute MI. Using EPR oximetry, we studied the effect of exposure to supplemental oxygen cycling (OxCy) administered by inhalation of 21-100% oxygen for brief periods (15-90 min), daily for 5 days, using a rat model of acute MI. Myocardial oxygen tension (pO2), cardiac function and pro-survival/apoptotic signaling molecules were used as markers of treatment outcome. OxCy resulted in a significant reduction of infarct size and improvement of cardiac function. An optimal condition of 30-min OxCy with 95% oxygen + 5% CO2 under normobaric conditions was found to be effective for cardioprotection.

6.
Cancer Res ; 78(7): 1739-1750, 2018 04 01.
Article in English | MEDLINE | ID: mdl-29339537

ABSTRACT

The initial molecular events that lead to malignant transformation of the fimbria of the fallopian tube (FT) through high-grade serous ovarian carcinoma (HGSC) remain poorly understood. In this study, we report that increased expression of signal transducer and activator of transcription 3 (pSTAT3 Tyr705) and suppression or loss of protein inhibitor of activated STAT3 (PIAS3) in FT likely drive HGSC. We evaluated human tissues-benign normal FT, tubal-peritoneal junction (TPJ), p53 signature FT tissue, tubal intraepithelial lesion in transition (TILT), serous tubal intraepithelial carcinoma (STIC) without ovarian cancer, and HGSC for expression of STAT3/PIAS3 (compared with their known TP53 signature) and their target proliferation genes. We observed constitutive activation of STAT3 and low levels or loss of PIAS3 in the TPJ, p53 signature, TILT, and STIC through advanced stage IV (HGSC) tissues. Elevated expression of pSTAT3 Tyr705 and decreased levels of PIAS3 appeared as early as TPJ and the trend continued until very advanced stage HGSC (compared with high PIAS3 and low pSTAT3 expression in normal benign FT). Exogenous expression of STAT3 in FT cells mediated translocation of pSTAT3 and c-Myc into the nucleus. In vivo experiments demonstrated that overexpression of STAT3 in FT secretory epithelial cells promoted tumor progression and metastasis, mimicking the clinical disease observed in patients with HGSC. Thus, we conclude that the STAT3 pathway plays a role in the development and progression of HGSC from its earliest premalignant states.Significance: Concomitant gain of pSTAT3 Tyr705 and loss of PIAS3 appear critical for initiation and development of high-grade serous carcinoma. Cancer Res; 78(7); 1739-50. ©2018 AACR.


Subject(s)
Cystadenocarcinoma, Serous/genetics , Fallopian Tube Neoplasms/genetics , Molecular Chaperones/genetics , Ovarian Neoplasms/genetics , Precancerous Conditions/genetics , Protein Inhibitors of Activated STAT/genetics , STAT3 Transcription Factor/genetics , Animals , Cell Line, Tumor , Cell Movement , Cell Transformation, Neoplastic/genetics , Cystadenocarcinoma, Serous/pathology , Fallopian Tube Neoplasms/pathology , Fallopian Tubes/pathology , Female , Humans , Mice , Ovarian Neoplasms/pathology , Precancerous Conditions/pathology , Proto-Oncogene Proteins c-myc/metabolism , STAT3 Transcription Factor/metabolism , Tumor Suppressor Protein p53/genetics
7.
J Virol ; 91(12)2017 06 15.
Article in English | MEDLINE | ID: mdl-28404844

ABSTRACT

Vaccination remains the best option to combat equine herpesvirus 1 (EHV-1) infection, and several different strategies of vaccination have been investigated and developed over the past few decades. Herein, we report that the live-attenuated herpes simplex virus 1 (HSV-1) VC2 vaccine strain, which has been shown to be unable to enter into neurons and establish latency in mice, can be utilized as a vector for the heterologous expression of EHV-1 glycoprotein D (gD) and that the intramuscular immunization of mice results in strong antiviral humoral and cellular immune responses. The VC2-EHV-1-gD recombinant virus was constructed by inserting an EHV-1 gD expression cassette under the control of the cytomegalovirus immediate early promoter into the VC2 vector in place of the HSV-1 thymidine kinase (UL23) gene. The vaccines were introduced into mice through intramuscular injection. Vaccination with both the VC2-EHV-1-gD vaccine and the commercially available vaccine Vetera EHVXP 1/4 (Vetera; Boehringer Ingelheim Vetmedica) resulted in the production of neutralizing antibodies, the levels of which were significantly higher in comparison to those in VC2- and mock-vaccinated animals (P < 0.01 or P < 0.001). Analysis of EHV-1-reactive IgG subtypes demonstrated that vaccination with the VC2-EHV-1-gD vaccine stimulated robust IgG1 and IgG2a antibodies after three vaccinations (P < 0.001). Interestingly, Vetera-vaccinated mice produced significantly higher levels of IgM than mice in the other groups before and after challenge (P < 0.01 or P < 0.05). Vaccination with VC2-EHV-1-gD stimulated strong cellular immune responses, characterized by the upregulation of both interferon- and tumor necrosis factor-positive CD4+ T cells and CD8+ T cells. Overall, the data suggest that the HSV-1 VC2 vaccine strain may be used as a viral vector for the vaccination of horses as well as, potentially, for the vaccination of other economically important animals.IMPORTANCE A novel virus-vectored VC2-EHV-1-gD vaccine was constructed using the live-attenuated HSV-1 VC2 vaccine strain. This vaccine stimulated strong humoral and cellular immune responses in mice, suggesting that it could protect horses against EHV-1 infection.


Subject(s)
Herpesviridae Infections/veterinary , Herpesvirus 1, Equid/chemistry , Herpesvirus 1, Equid/immunology , Herpesvirus Vaccines/immunology , Horse Diseases/prevention & control , Viral Envelope Proteins/genetics , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , Disease Models, Animal , Herpesviridae Infections/immunology , Herpesviridae Infections/prevention & control , Herpesvirus 1, Equid/genetics , Herpesvirus Vaccines/administration & dosage , Horse Diseases/virology , Horses , Immunity, Cellular , Immunity, Humoral , Immunization , Injections, Intramuscular , Mice , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunology , Viral Envelope Proteins/immunology , Viral Vaccines/immunology
8.
J Leukoc Biol ; 101(6): 1383-1392, 2017 06.
Article in English | MEDLINE | ID: mdl-28336678

ABSTRACT

Neutrophils are the most abundant leukocytes in human circulation. They are the first immune cell population recruited to the sites of infection. However, the role of neutrophils to regulate host immune responses during respiratory viral infections is largely unknown. To elucidate the role of neutrophils in respiratory antiviral defense, we used an experimental mouse model of human metapneumovirus (HMPV) infection. HMPV, a member of the Paramyxoviridae family, is a leading respiratory pathogen causing severe symptoms, such as bronchiolitis and pneumonia, in young, elderly, and immunocompromised patients. We demonstrate that neutrophils are the predominant population of immune cells recruited into the lungs after HMPV infection. This led us to hypothesize that neutrophils represent a key player of the immune response during HMPV infection, thereby regulating HMPV-induced lung pathogenesis. Specific depletion of neutrophils in vivo using a mAb and simultaneous infection with HMPV exhibited higher levels of inflammatory cytokines, pulmonary inflammation, and severe clinical disease compared with HMPV-infected, competent mice. Interestingly, the lack of neutrophils altered γδ T cell accumulation in the lung. The absence of γδ T cells during HMPV infection led to reduced pulmonary inflammation. These novel findings demonstrate that neutrophils play a critical role in controlling HMPV-induced inflammatory responses by regulating γδ T cell infiltration to the site of infection.


Subject(s)
Disease Models, Animal , Metapneumovirus/immunology , Neutrophils/immunology , Paramyxoviridae Infections/immunology , Pneumonia/immunology , T-Lymphocytes/immunology , Animals , Cytokines/metabolism , Humans , Mice , Mice, Inbred BALB C , Neutrophils/virology , Paramyxoviridae Infections/virology , Pneumonia/virology , T-Lymphocytes/virology , Virus Replication
9.
Oncogene ; 36(2): 168-181, 2017 01 12.
Article in English | MEDLINE | ID: mdl-27292260

ABSTRACT

Although activation of the STAT3 pathway has been associated with tumor progression in a wide variety of cancer types (including ovarian cancer), the precise mechanism of invasion and metastasis due to STAT3 are not fully delineated in ovarian cancer. We found that pSTAT3 Tyr705 is constitutively activated in patient ascites and ascites-derived ovarian cancer cells (ADOCCs), and the range of STAT3 expression could be very high to low. In vivo transplantation of ADOCCs with high pSTAT3 expression into the ovarian bursa of mice resulted in a large primary tumor and widespread peritoneal metastases. In contrast, ADOCCs with low STAT3 expression or ADOCCs with STAT3 expression knockdown, led to reduced tumor growth and an absence of metastases in vivo. Cytokines derived from the ADOCC culture medium activate the interleukin (IL)-6/STAT pathway in the STAT3 knockout (KO) cells, compensating for the absence of inherent STAT3 in the cells. Treatment with HO-3867 (a novel STAT3 inhibitor at 100 p.p.m. in an orthotopic murine model) significantly suppressed ovarian tumor growth, angiogenesis and metastasis by targeting STAT3 and its downstream proteins. HO-3867 was found to have cytotoxic effects in ex vivo cultures of freshly collected human ovarian cancers, including those resistant to platinum-based chemotherapy. Our results show that STAT3 is necessary for ovarian tumor progression/metastasis and highlight the potential for targeting STAT3 by HO-3867 as a therapeutic strategy for ovarian cancer.


Subject(s)
Ascites/pathology , Ovarian Neoplasms/pathology , STAT3 Transcription Factor/metabolism , Up-Regulation , Animals , Ascites/metabolism , Cell Movement , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockout Techniques , Humans , Mice , Neoplasm Invasiveness , Neoplasm Metastasis , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Piperidones/administration & dosage , Piperidones/pharmacology , STAT3 Transcription Factor/genetics , Signal Transduction/drug effects , Up-Regulation/drug effects , Xenograft Model Antitumor Assays
10.
Cancer Biol Ther ; 17(10): 1107-1115, 2016 10 02.
Article in English | MEDLINE | ID: mdl-27415751

ABSTRACT

We have previously developed a novel class of bi-functional compounds based on a diarylidenyl-piperidone (DAP) backbone conjugated to an N-hydroxypyrroline (-NOH; a nitroxide precursor) group capable of selectively inhibiting STAT3 activation, translocation, and DNA binding activity. HO-4200 and H-4318 are 2 such derivatives capable of inducing apoptosis in ovarian cancer cells through this mechanism and demonstrated efficacy in platinum resistant primary ovarian cancer cell populations and tumor tissues. The improved absorption and cellular uptake of HO-4200 by cancer cells was determined using optical and electron paramagnetic resonance spectrometry. Treatment of ovarian cancer cells with HO-4200 and H-4318 resulted in cleavage of caspase proteins 3, 7, and 9, as well as PARP and inhibition of the pro-survival protein, Bcl-xL, resulting in significantly decreased cell survival and increased apoptosis. HO-4200 and H-4318 significantly inhibit fatty acid synthase (FAS) and pSTAT3 and decreased the expression of STAT3 target proteins: Survivin, c-myc, Bcl-xl, Bcl-2, cyclin D1/D2, and VEGF were suppressed as analyzed using quantitative real time PCR. In addition, HO-4200 and H-4318 significantly inhibited migration/invasion, in primary ovarian cancer cell populations isolated from primary and recurrent ovarian cancer patients. Treatment of freshly collected human ovarian tumor sections with HO-4200 demonstrated significant suppression of pSTAT3 Tyr 705, angiogenesis (VEFG), and markers of proliferation (Ki-67) in ex vivo models. We have shown, for the first time, that the DAP compounds, HO-4200 and H-4318, inhibit cell migration/invasion and induce apoptosis by targeting FAS/STAT3 in human ovarian cancer cells, including primary ovarian cancer cell populations and tumor tissues. Therefore, our results highlight the clinical anti-cancer potential of HO-4200 and H-4318.


Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Ovarian Neoplasms/drug therapy , Piperidones/pharmacology , Drug Resistance, Neoplasm , Female , Humans , STAT3 Transcription Factor/metabolism , Signal Transduction
11.
Gynecol Oncol ; 135(1): 133-41, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25038288

ABSTRACT

OBJECTIVE: Constitutive activation of STAT3 is a hallmark of various human cancers, however an increased pSTAT3 expression in high grade human endometrial cancer has not been reported. In the present study, we examine the expression of STAT family of proteins in endometrial cancer cell lines and the efficacy of HO-3867, a novel STAT3 inhibitor designed in our lab. METHODS: Expression of STAT family proteins was evaluated via Western blot. The cell viability, post-treatment with HO-3867, was assessed using MTT, cell-cycle profile and Annexin assay. In vivo efficacy of HO-3867 was evaluated using xenograft mice. RESULTS: Expression of activated STATs was inconsistent among the cell lines and 18 human endometrial cancer specimens tested. While pSTAT3 Tyr705 was not expressed in any of the cell lines, pSTAT3 Ser727 was highly expressed in endometrial cancer cell lines and tumor specimens. HO-3867 decreased the expression of pSTAT3 Ser727 while total STAT3 remained constant; cell viability decreased by 50-80% and induced G2/M arrest in 55% of Ishikawa cells at the G2/M cell cycle checkpoint. There was an increase in p53, a decrease in Bcl2 and Bcl-xL, and cleavage of caspase-3, caspase-7 and PARP. HO-3867 mediated a dosage-dependent inhibition of the growth of xenografted endometrial tumors. CONCLUSIONS: HO-3867 treatment decreases the high levels of pSTAT3 Ser727 in endometrial cancer cells by inducing cell cycle arrest and apoptosis. This suggests a specific role of serine-phosphorylated STAT3, independent of tyrosine phosphorylation in the oncogenesis of endometrial cancer. HO-3867 could potentially serve as an adjunctive targeted therapy.


Subject(s)
Endometrial Neoplasms/drug therapy , Endometrial Neoplasms/metabolism , Piperidones/therapeutic use , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/biosynthesis , Animals , Cell Line, Tumor , Female , Humans , Mice
12.
Adv Exp Med Biol ; 812: 81-86, 2014.
Article in English | MEDLINE | ID: mdl-24729218

ABSTRACT

The use of electron paramagnetic resonance (EPR) oximetry for oxygen measurements in deep tissues (>1 cm) is challenging due to the limited penetration depth of the microwave energy. To overcome this limitation, implantable resonators, having a small (0.5 mm diameter) sensory loop containing the oxygen-sensing paramagnetic material connected by a pair of twisted copper wire to a coupling loop (8-10 mm diameter), have been developed, which enable repeated measurements of deep-tissue oxygen levels (pO2, partial pressure of oxygen) in the brain and tumors of rodents. In this study, we have demonstrated the feasibility of measuring dynamic changes in pO2 in the heart and lung of rats using deep-tissue implantable oxygen sensors. The sensory loop of the resonator contained lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) crystals embedded in polydimethylsiloxane (PDMS) polymer and was implanted in the myocardial tissue or lung pleura. The external coupling loop was secured subcutaneously above chest. The rats were exposed to different breathing gas mixtures while undergoing EPR measurements. The results demonstrated that implantable oxygen sensors provide reliable measurements of pO2 in deep tissues such as heart and lung under adverse conditions of cardiac and respiratory motions.


Subject(s)
Electron Spin Resonance Spectroscopy/methods , Lung/metabolism , Myocardium/metabolism , Oximetry/methods , Oxygen/metabolism , Animals , Dimethylpolysiloxanes , Rats , Rats, Sprague-Dawley
13.
Cancer Res ; 74(8): 2316-27, 2014 Apr 15.
Article in English | MEDLINE | ID: mdl-24590057

ABSTRACT

STAT3 is well corroborated preclinically as a cancer therapeutic target, but tractable translational strategies for its blockade by small molecule inhibitors have remained elusive. In this study, we report the development of a novel class of bifunctional STAT3 inhibitors, based on conjugation of a diarylidenyl-piperidone (DAP) backbone to an N-hydroxypyrroline (-NOH) group, which exhibits minimal toxicity against normal cells and good oral bioavailability. Molecular modeling studies of this class suggested direct interaction with the STAT3 DNA binding domain. In particular, the DAP compound HO-3867 selectively inhibited STAT3 phosphorylation, transcription, and DNA binding without affecting the expression of other active STATs. HO-3867 exhibited minimal toxicity toward noncancerous cells and tissues but induced apoptosis in ovarian cancer cells. Pharmacologic analysis revealed greater bioabsorption and bioavailability of the active (cytotoxic) metabolites in cancer cells compared with normal cells. The selective cytotoxicity of HO-3867 seemed to be multifaceted, eliciting differential activation of the Akt pathway in normal versus cancer cells. RNAi attenuation experiments confirmed the requirement of STAT3 for HO-3867-mediated apoptosis in ovarian cancer cells. In vivo testing showed that HO-3867 could block xenograft tumor growth without toxic side effects. Furthermore, in primary human ovarian cancer cells isolated from patient ascites, HO-3867 inhibited cell migration/invasion and survival. Our results offer preclinical proof-of-concept for HO-3867 as a selective STAT3 inhibitor to treat ovarian cancer and other solid tumors where STAT3 is widely upregulated.


Subject(s)
Ovarian Neoplasms/drug therapy , Piperidones/pharmacology , STAT3 Transcription Factor/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , CHO Cells , Cell Growth Processes/drug effects , Cell Line, Tumor , Cricetulus , Cytotoxicity, Immunologic , Female , Humans , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , STAT3 Transcription Factor/metabolism , Signal Transduction , Transcriptional Activation , Transfection , Xenograft Model Antitumor Assays
14.
Oncoscience ; 1(3): 216-28, 2014.
Article in English | MEDLINE | ID: mdl-25594014

ABSTRACT

Tumor hypoxia, a feature of many solid tumors including ovarian cancer, is associated with resistance to therapies. We previously demonstrated that hypoxic exposure results in increased expression of phosphorylated signal transducer and activator of transcription 3 (pSTAT3). We hypothesized the activation of STAT3 could lead to chemotherapeutic resistance in ovarian cancer cells in hypoxic conditions. In this study, we demonstrate the level of pSTAT3 Tyr705 is increased in the hypoxic regions of human epithelial ovarian cancer (EOC) specimens, as determined by HIF-1α and CD-31 staining. In vitro mutagenesis studies proved that pSTAT3 Tyr705 is necessary for cell survival and proliferation under hypoxic conditions. In addition, we show that S1PR1, a regulator of STAT3 transcription via the JAK/STAT pathway, is highly expressed in hypoxic ovarian cancer cells (HOCCs). Knock down of S1PR1 in HOCCs reduced pSTAT3 Tyr705 levels and was associated with decreased cell survival. Treatment of HOCCs with the STAT3 inhibitor HO-3867 resulted in a rapid and dramatic decrease in pSTAT3 Tyr705 levels as a result of ubiquitin proteasome degradation. STAT3-target proteins Bcl-xL, cyclin D2 and VEGF showed similar decreases in HO-3867 treated cells. Taken together, these findings suggest that activation of STAT3 Tyr705 promotes cell survival and proliferation in HOCCs, and that S1PR1 is involved in the initiation of STAT3 activation. Targeting hypoxia-mediated STAT3 activation represents a therapeutic option for ovarian cancer and other solid tumors.

15.
PLoS One ; 9(12): e116281, 2014.
Article in English | MEDLINE | ID: mdl-25551230

ABSTRACT

INTRODUCTION: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have recently been shown to express key cardiac proteins and improve in vivo cardiac function when administered following myocardial infarction. However, the efficacy of hiPSC-derived cell therapies, in direct comparison to current, well-established stem cell-based therapies, is yet to be elucidated. The goal of the current study was to compare the therapeutic efficacy of human mesenchymal stem cells (hMSCs) with hiPSC-CMs in mitigating myocardial infarction (MI). METHODS: Male athymic nude hyrats were subjected to permanent ligation of the left-anterior-descending (LAD) coronary artery to induce acute MI. Four experimental groups were studied: 1) control (non-MI), 2) MI, 3) hMSCs (MI+MSC), and 4) hiPSC-CMs (MI+hiPSC-derived cardiomyocytes). The hiPSC-CMs and hMSCs were labeled with superparamagnetic iron oxide (SPIO) in vitro to track the transplanted cells in the ischemic heart by high-field cardiac MRI. These cells were injected into the ischemic heart 30-min after LAD ligation. Four-weeks after MI, cardiac MRI was performed to track the transplanted cells in the infarct heart. Additionally, echocardiography (M-mode) was performed to evaluate the cardiac function. Immunohistological and western blot studies were performed to assess the cell tracking, engraftment and cardiac fibrosis in the infarct heart tissues. RESULTS: Echocardiography data showed a significantly improved cardiac function in the hiPSC-CMs and hMSCs groups, when compared to MI. Immunohistological studies showed expression of connexin-43, α-actinin and myosin heavy chain in engrafted hiPSC-CMs. Cardiac fibrosis was significantly decreased in hiPSC-CMs group when compared to hMSCs or MI groups. Overall, this study demonstrated improved cardiac function with decreased fibrosis with both hiPSC-CMs and hMSCs groups when compared with MI group.


Subject(s)
Induced Pluripotent Stem Cells/physiology , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/physiology , Myocardial Infarction/pathology , Myocytes, Cardiac/physiology , Animals , Biomarkers/metabolism , Cells, Cultured , Disease Models, Animal , Echocardiography , Humans , Magnetic Resonance Imaging , Male , Myocardial Infarction/therapy , Rats, Nude
16.
Hypertension ; 61(3): 593-601, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23339168

ABSTRACT

Pulmonary hypertension (PH) that occurs after left-heart failure (LHF), classified as Group 2 PH, involves progressive pulmonary vascular remodeling induced by smooth muscle cell (SMC) proliferation. However, mechanisms involved in the activation of SMCs remain unknown. The objective of this study was to determine the involvement of peroxynitrite and phosphatase-and-tensin homolog on chromosome 10 (PTEN) in vascular SMC proliferation and remodeling in the LHF-induced PH (LHF-PH). LHF was induced by permanent ligation of left anterior descending coronary artery in rats for 4 weeks. MRI, ultrasound, and hemodynamic measurements were performed to confirm LHF and PH. Histopathology, Western blot, and real-time polymerase chain reaction analyses were used to identify key molecular signatures. Therapeutic intervention was demonstrated using an antiproliferative compound, HO-3867. LHF-PH was confirmed by significant elevation of pulmonary artery pressure (mean pulmonary artery pressure/mm Hg: 35.9±1.8 versus 14.8±2.0, control; P<0.001) and vascular remodeling. HO-3867 treatment decreased mean pulmonary artery pressure to 22.6±0.8 mm Hg (P<0.001). Substantially higher levels of peroxynitrite and significant loss of PTEN expression were observed in the lungs of LHF rats when compared with control. In vitro studies using human pulmonary artery SMCs implicated peroxynitrite-mediated downregulation of PTEN expression as a key mechanism of SMC proliferation. The results further established that HO-3867 attenuated LHF-PH by decreasing oxidative stress and increasing PTEN expression in the lung. In conclusion, peroxynitrite and peroxynitrite-mediated PTEN inactivation seem to be key mediators of lung microvascular remodeling associated with PH secondary to LHF.


Subject(s)
Down-Regulation/physiology , Heart Failure/complications , Hypertension, Pulmonary/etiology , PTEN Phosphohydrolase/biosynthesis , Peroxynitrous Acid/metabolism , Animals , Cell Line , Heart Failure/diagnostic imaging , Heart Failure/drug therapy , Heart Failure/metabolism , Humans , Hypertension, Pulmonary/diagnostic imaging , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/pathology , Lung/blood supply , Lung/drug effects , Lung/metabolism , Lung/pathology , Male , Microvessels/drug effects , Microvessels/metabolism , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Oxidative Stress/drug effects , Peroxynitrous Acid/analysis , Piperidones/therapeutic use , Pulmonary Artery/drug effects , Pulmonary Artery/metabolism , Rats , Rats, Sprague-Dawley , Ultrasonography
17.
Cell Biochem Biophys ; 67(2): 363-72, 2013 Nov.
Article in English | MEDLINE | ID: mdl-22205501

ABSTRACT

Pulmonary hypertension (PH) is a disorder of lung vasculature characterized by arterial narrowing. Phosphatase-and-tensin homolog on chromosome 10 (PTEN), associated in the progression of multiple cancers, is implicated in arterial remodeling. However, the involvement of PTEN in PH remains unclear. The objective of the present study was to determine the role of PTEN in pulmonary vascular remodeling using established models of PH. The study used rat models of PH, induced by monocrotaline (MCT) administration (60 mg/kg) or continuous hypoxic exposure (10% oxygen) for 3 weeks. Pulmonary artery smooth muscle cells (SMCs) were used for in vitro confirmation. Development of PH was verified by hemodynamic, morphological and histopathology analyses. PTEN and key downstream proteins in pulmonary and cardiac tissues were analyzed by western blotting and RT-PCR. PTEN was significantly decreased (MCT, 53%; Hypoxia, 40%), pAkt was significantly increased (MCT, 42%; Hypoxia, 55%) in tissues of rats with PH. Similar results were observed in SMCs exposed to hypoxia (1% oxygen) for 48 h. Ubiquitination assay showed that PTEN degradation occurs via proteasomal degradation pathway. Western blotting demonstrated a significant downregulation of cell-cycle regulatory proteins p53 and p27, and upregulation of cyclin-D1 in the lungs of both models. The results showed that PTEN-mediated modulation of PI3K pathway was independent of the focal adhesion kinase and fatty acid synthase. The study, for the first time, established that PTEN plays a key role in the progression of pulmonary hypertension. The findings may have potential for the treatment of pulmonary hypertension using PTEN as a target.


Subject(s)
Heart/physiopathology , Hypertension, Pulmonary/pathology , Hypertension, Pulmonary/physiopathology , Lung/pathology , Lung/physiopathology , Myocardium/pathology , PTEN Phosphohydrolase/metabolism , Animals , Cell Cycle Proteins/metabolism , Cell Proliferation/drug effects , Gene Expression Regulation/drug effects , Humans , Hypertension, Pulmonary/genetics , Hypertension, Pulmonary/metabolism , Hypoxia/complications , Monocrotaline/pharmacology , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , PTEN Phosphohydrolase/genetics , Proteasome Endopeptidase Complex/metabolism , Proteolysis/drug effects , Rats
18.
J Pharmacol Exp Ther ; 339(2): 350-7, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21799049

ABSTRACT

Doxorubicin (DOX) is a drug commonly used for the treatment of cancer. The development of resistance to DOX is common, and high cumulative doses cause potentially lethal cardiac side effects. HO-3867 (3,5-bis(4-fluorobenzylidene)-1-[(2,2,5,5-tetramethyl-2,5-dihydro-1-hydroxy-pyrrol-3-yl)methyl]piperidin-4-one), a synthetic curcumin analog, has been shown to exhibit both anticancer and cardioprotective effects. However, its cardioprotection in the setting of a conventional cancer therapy has not been established. This work investigated the use of HO-3867 and DOX to achieve a complementary outcome, i.e., increased toxicity toward cancer cells, and reduced cardiac toxicity. Combination treatment was investigated using DOX-resistant MCF-7 breast cancer cells [MCF-7 multidrug-resistant (MDR)] and BALB/c mice. Lower doses of HO-3867 and DOX (5 and 2.5 µM, respectively) reduced viability of MCF-7 MDR cells to an extent significantly greater than that when either drug was used alone, an effect equivalent to that induced by exposure to 50 µM DOX. In normal cardiac cells, the loss of viability from combination treatment was significantly lower than that induced by 50 µM DOX. Increases in apoptotic markers, e.g., cleaved caspase-3, and decreases in fatty acid synthase and pAkt expressions were observed by Western blotting. Mice treated with both HO-3867 and DOX showed significant improvement in cardiac functional parameters compared with mice treated with DOX alone. Reduced expression of Bcl-2 and pAkt was observed in mice treated with DOX alone, whereas mice given combination treatment showed levels similar to control. The study indicates that combination treatment of HO-3867 and DOX is a viable option for treatment of cancer with reduced cardiotoxic side effects.


Subject(s)
Antineoplastic Agents/toxicity , Cardiotonic Agents/pharmacology , Doxorubicin/toxicity , Piperidones/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Antioxidants/administration & dosage , Antioxidants/pharmacology , Antioxidants/therapeutic use , Aorta/cytology , Aorta/drug effects , Breast Neoplasms/drug therapy , Cardiotonic Agents/administration & dosage , Cardiotonic Agents/therapeutic use , Caspases/biosynthesis , Cell Line, Tumor , Cyclins/antagonists & inhibitors , Cyclins/biosynthesis , Doxorubicin/administration & dosage , Doxorubicin/therapeutic use , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Echocardiography , Female , Heart/drug effects , Heart Diseases/drug therapy , Humans , Male , Mice , Mice, Inbred BALB C , Piperidones/administration & dosage , Piperidones/therapeutic use , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/biosynthesis , fas Receptor/antagonists & inhibitors , fas Receptor/biosynthesis
19.
Proc Natl Acad Sci U S A ; 107(11): 5142-7, 2010 Mar 16.
Article in English | MEDLINE | ID: mdl-20194734

ABSTRACT

Germline mutations in the tumor suppressor gene PTEN (phosphatase and tensin homology deleted on chromosome 10) cause Cowden and Bannayan-Riley-Ruvalcaba (BRR) syndromes, two dominantly inherited disorders characterized by mental retardation, multiple hamartomas, and variable cancer risk. Here, we modeled three sentinel mutant alleles of PTEN identified in patients with Cowden syndrome and show that the nonsense Pten(4-5) and missense Pten(C124R) and Pten(G129E) alleles lacking lipid phosphatase activity cause similar developmental abnormalities but distinct tumor spectra with varying severity and age of onset. Allele-specific differences may be accounted for by loss of function for Pten(4-5), hypomorphic function for Pten(C124R), and gain of function for Pten(G129E). These data demonstrate that the variable tumor phenotypes observed in patients with Cowden and BRR syndromes can be attributed to specific mutations in PTEN that alter protein function through distinct mechanisms.


Subject(s)
Alleles , Gene Knock-In Techniques , Neoplasms/enzymology , Neoplasms/genetics , PTEN Phosphohydrolase/genetics , Animals , Base Sequence , Cell Proliferation , DNA Mutational Analysis , Disease Progression , Embryo Loss/pathology , Embryonic Development , Gene Silencing , Gene Targeting , Genetic Predisposition to Disease , Mice , Molecular Sequence Data , Mutant Proteins/metabolism , Neoplasms/pathology , Organ Specificity , Point Mutation/genetics , Precancerous Conditions/pathology , Protein Stability , Proto-Oncogene Proteins c-akt/metabolism , Tumor Suppressor Proteins/metabolism
20.
Nature ; 461(7267): 1084-91, 2009 Oct 22.
Article in English | MEDLINE | ID: mdl-19847259

ABSTRACT

The tumour stroma is believed to contribute to some of the most malignant characteristics of epithelial tumours. However, signalling between stromal and tumour cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumours. This was associated with the massive remodelling of the extracellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumours ameliorated disruption of the tumour microenvironment and was sufficient to decrease tumour growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumour stroma of patients with breast cancer. These findings identify the Pten-Ets2 axis as a critical stroma-specific signalling pathway that suppresses mammary epithelial tumours.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Fibroblasts/metabolism , Neoplasms, Glandular and Epithelial/metabolism , Neoplasms, Glandular and Epithelial/pathology , PTEN Phosphohydrolase/metabolism , Stromal Cells/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Cell Transformation, Neoplastic , Extracellular Matrix/metabolism , Gene Deletion , Gene Expression Regulation, Neoplastic , Humans , Immunity, Innate , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Transgenic , PTEN Phosphohydrolase/deficiency , PTEN Phosphohydrolase/genetics , Proto-Oncogene Protein c-ets-2/deficiency , Proto-Oncogene Protein c-ets-2/metabolism
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