David J. Tweardy

Requirement of Stat3 but not Stat1 activation for epidermal growth factor receptor- mediated cell growth In vitro.

Stimulation of epidermal growth factor receptor (EGFR) by ligand(s) leads to activation of signaling molecules including Stat1 and Stat3, two members of the signal transducers and activators of transcription (STAT) protein family. Activation of Stat1 and Stat3 was constitutive in transformed squamous epithelial cells, which produce elevated levels of TGF-alpha, and was enhanced by the addition of exogenous TGF-alpha. Targeting of Stat3 using antisense oligonucleotides directed against the translation initiation site, resulted in significant growth inhibition. In addition, cells stably transfected with dominant negative mutant Stat3 constructs failed to proliferate in vitro. In contrast, targeting of Stat1 using either antisense or dominant-negative strategies had no effect on cell growth. Thus, TGF-alpha/EGFR-mediated autocrine growth of transformed epithelial cells is dependent on activation of Stat3 but not Stat1.

Interleukin-6 induces prostate cancer cell growth accompanied by activation of Stat3 signaling pathway

Interleukin‐6 (IL‐6) is a pleiotropic cytokine that regulates growth and differentiation of various types of malignant tumors, including prostate carcinomas. The levels of IL‐6 are elevated in sera of patients with metastatic prostate cancer. In this study, we evaluate the role of IL‐6 in the growth regulation of prostate cancer cells.

Intra-Abdominal Activation of a Local Inflammatory Response Within the Human Muscularis Externa During Laparotomy

ObjectiveTo investigate the initiation of a complex inflammatory response within the human intestinal muscularis intraoperatively so as to determine the clinical applicability of the inflammatory hypothesis of postoperative ileus. Summary Background DataMild intestinal manipulation in rodents initiates the activation of transcription factors, upregulates proinflammatory cytokines, and increases the release of kinetically active mediators (nitric oxide and prostaglandins), all of which results in the recruitment of leukocytes and a suppression in motility (i.e., postoperative ileus). MethodsHuman small bowel specimens were harvested during abdominal procedures at various times after laparotomy. Histochemical and immunohistochemical techniques were applied to intestinal muscularis whole-mounts. Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed for interleukin (IL)-6, IL-1&bgr;, tumor necrosis factor (TNF)-&agr;, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Signal transducers and activators of transcription (STAT) protein phosphorylation was determined by electromobility shift assay. Organ bath experiments were performed on jejunal circular smooth muscle strips. GW274150C and DFU were used in vitro as iNOS and COX-2 inhibitors. ResultsNormal human muscularis externa contained numerous macrophages that expressed increased lymphocyte function associated antigen-1 (LFA-1) immunoreactivity as a function of intraoperative time. RT-PCR demonstrated a time-dependent induction of IL-6, IL-1&bgr;, TNF-&agr;, iNOS, and COX-2 mRNAs within muscularis extracts after incision. Mediators were localized to macrophages with STAT protein activation in protein extracts demonstrating local IL-6 functional activity. DFU alone or in combination with GW274150C increased circular muscle contractility. Specimens harvested after reoperation developed leukocytic infiltrates and displayed diminished in vitro muscle contractility. ConclusionsThese human data demonstrate that surgical trauma is followed by resident muscularis macrophage activation and the upregulation, release, and functional activity of proinflammatory cytokines and kinetically active mediators.

Inhibition of epidermal growth factor receptor gene expression and function decreases proliferation of head and neck squamous carcinoma but not normal mucosal epithelial cells.

Previous reports have shown that fresh tissues and cell lines from patients with squamous cell carcinoma of the head and neck (SCCHN) overexpress transforming growth factor alpha (TGF-α) and its receptor, the epidermal growth factor receptor (EGFR) at both the mRNA and protein levels. Protein localization studies confirm that TGF-α and EGFR are produced by the same epithelial cells in tissues from head and neck cancer patients further supporting an autocrine growth pathway. Using three strategies, we examined the hypothesis that downmodulation of EGFR would reduce the proliferation of SCCHN cells. We targeted EGFR mRNA using antisense oligonucleotides and the mature EGFR protein at two sites, the ligand-binding domain and the kinase domain, and determined the effects of this targeting on SCCHN proliferation. Treatment of several SCCHN cell lines with a pair of antisense oligodeoxynucleotides directed against the translation start site and first intron-exon splice junction of the human EGFR gene resulted in decreased EGFR protein production and inhibited growth by 86% compared to a 13% reduction in cells treated with sense oligonucleotides (P=0.03). Growth inhibition was specific for carcinoma cells since the same EGFR antisense oligonucleotides had no effect on the proliferation of normal mucosa cells harvested from non-cancer patients. Two monoclonal antibodies which block ligand binding to EGFR (MAbs 425 and 528) inhibited the growth of several SCCHN cell lines by up to 97% which suggests that EGFR is participating in an autocrine pathway in SCCHN that is, at least in part, external. An EGFR-specific tyrosine kinase inhibitor (PD 153035) was found to inhibit EGFR phosphorylation in SCCHN cell lines and to reduce growth by 68% although it had no effect on the growth rate of normal mucosal epithelial cells. These experiments indicate that EGFR gene expression and function is critical for SCCHN cell growth but not for growth of normal mucosa cells and therefore may serve as a tumor-specific target for preventive and therapeutic strategies in head and neck cancer.

G-Quartet Oligonucleotides A New Class of Signal Transducer and Activator of Transcription 3 Inhibitors That Suppresses Growth of Prostate and Breast Tumors through Induction of Apoptosis

Stat3 is a signaling molecular and oncogene activated frequently in many human malignancies including the majority of prostate, breast, and head and neck cancers; yet, no current chemotherapeutic approach has been implemented clinically that specifically targets Stat3. We recently developed G-rich oligodeoxynucleotides, which form intramolecular G-quartet structures (GQ-ODN), as a new class of Stat3 inhibitor. GQ-ODN targeted Stat3 protein directly inhibiting its ability to bind DNA. When delivered into cells using polyethyleneimine as vehicle, GQ-ODN blocked ligand-induced Stat3 activation and Stat3-mediated transcription of antiapoptotic genes. To establish the effectiveness of GQ-ODN as a potential new chemotherapeutic agent, we systemically administered GQ-ODN (T40214 or T40231) plus polyethyleneimine or polyethyleneimine alone (placebo) by tail-vein injection into nude mice with prostate and breast tumor xenografts. Whereas the mean volume of breast tumor xenografts in placebo-treated mice increased >7-fold over 18 days, xenografts in the GQ-ODN-treated mice remained unchanged. Similarly, whereas the mean volume of prostate tumor xenografts in placebo-treated mice increased 9-fold over 10 days, xenografts in GQ-ODN-treated mice increased by only 2-fold. Biochemical examination of tumors from GQ-ODN-treated mice demonstrated a significant reduction in Stat3 activation, levels of the antiapoptotic proteins Bcl-2 and Bcl-xL, and an 8-fold increase in the number of apoptotic cells compared with the tumors of placebo-treated mice. Thus, GQ-ODN targeting Stat3 induces tumor cell apoptosis when delivered into tumor xenografts and represents a novel class of chemotherapeutic agents that holds promise for the systemic treatment of many forms of metastatic cancer.

Molecular and functional contractile sequelae of rat intestinal ischemia/reperfusion injury.

BACKGROUND Pathophysiological states that produce intestinal ischemia/reperfusion injury (I/R) initiate an inflammatory cascade and cause ileus. The aims of this study were to investigate the local cellular responses and molecular mechanisms, which contribute to intestinal dysmotility after selective intestinal I/R injury. METHODS ACI rats were subjected to 75 min SMA clamp-induced ischemia followed by reperfusion and were killed at 0 min, 30 min, and 24 hr. Whole mounts of the jejunum were used to immunohistochemically quantify alterations in leukocytes, and circular muscle strips were used to assess organ bath muscle function. Muscularis and mucosa extracts were isolated from the intestine and used for reverse transcription assisted polymerase chain reaction mRNA measurements of granulocyte-colony stimulating factor and interleukin-6, and for determination of nuclear factor kappa B and Stat3 activation. RESULTS Intestinal I/R injury resulted in the significant recruitment of neutrophils and monocytes into the intestinal muscularis and a functional suppression in jejunal circular muscle contractions. These I/R injury induced cellular responses were preceded by the molecular activation of nuclear factor kappa B, up-regulation of granulocyte colony-stimulating factor and interleukin-6 mRNA and phosphorylation of the downstream signaling and transcription factor Stat3. CONCLUSIONS I/R injury evokes a molecular and cellular inflammatory response within the intestinal muscularis that is associated with a subsequent decrease in intestinal motility.

Stat3 signaling in acute myeloid leukemia: ligand-dependent and -independent activation and induction of apoptosis by a novel small-molecule Stat3 inhibitor

Acute myeloid leukemia (AML) is an aggressive malignancy with a relapse rate approaching 50%, despite aggressive chemotherapy. New therapies for AML are targeted at signal transduction pathways known to support blast survival, such as the Stat3 pathway. Aberrant activation of Stat3 has been demonstrated in many different malignancies, including AML, and this finding is frequently associated with more aggressive disease. The objectives of this study were: (1) to characterize Stat3 signaling patterns in AML cells lines and primary pediatric samples; and (2) to test the efficacy and potency of a novel Stat3 inhibitor in inducing apoptosis in AML cells. We found that Stat3 was constitutively activated in 6 of 7 AML cell lines and 6 of 18 primary pediatric AML samples. Moreover, constitutively phosphorylated Stat3 was frequent in samples with normal karyotype but uncommon in samples with t(8;21). Most cell lines and primary samples responded to G-CSF stimulation, although the sensitivity and magnitude of the response varied dramatically. Our novel small-molecule Stat3 inhibitor, C188-9, inhibited G-CSF-induced Stat3 phosphorylation, induced apoptosis in AML cell lines and primary samples, and inhibited AML blast colony formation with potencies in the low micromolar range. Therefore, Stat3 inhibition may be a valuable strategy for targeted therapies for AML.

Downmodulation of TGF-α protein expression with antisense oligonucleotides inhibits proliferation of head and neck squamous carcinoma but not normal mucosal epithelial cells

Interruption of an autocrine growth pathway involving TGF‐α and EGFR may inhibit tumor growth and improve survival in head and neck cancer patients. We previously demonstrated that biopsy specimens and established cell lines from patients with squamous cell carcinoma of the head and neck (SCCHN) overexpress TGF‐α and its receptor, epidermal growth factor receptor (EGFR) at both the mRNA and protein levels. Protein localization studies showed that TGF‐α and EGFR are produced by the same epithelial cells in tissues from head and neck cancer patients further supporting a role for this ligand‐receptor pair in an autocrine growth pathway. To confirm that TGF‐α contributes to autocrine growth, we examined the effect of down regulation of TGF‐α protein on SCCHN cell proliferation. Treatment of 6 SCCHN cell lines with antisense oligodeoxynucleotides targeting the translation start site of human TGF‐α mRNA decreased TGF‐α protein production by up to 93% and reduced cell proliferation by a mean of 76.2% compared to a 9.7% reduction with sense oligonucleotide (range P<0R > = 0.036–0.0001). TGF‐α antisense oligonucleotide exposure also decreased TGF‐α protein levels in normal oropharyngeal mucosal epithelial cells, however their growth rate was not affected. These findings indicate that TGF‐α is participating in an autocrine signaling pathway in transformed, but not in normal mucosal epithelial cells, that promotes proliferation. J. Cell. Biochem. 69:55–62, 1998.

Interleukin-6 production in hemorrhagic shock is accompanied by neutrophil recruitment and lung injury

Hemorrhagic shock (HS) initiates an inflammatory cascade that includes the production of cytokines and recruitment of neutrophils (PMN) and may progress to organ failure, inducing acute respiratory distress syndrome (ARDS). To examine the hypothesis that interleukin-6 (IL-6) contributes to PMN infiltration and lung damage in HS, we examined the lungs of rats subjected to unresuscitated and resuscitated HS for the production of IL-6 and activation of Stat3. Using semiquantitative RT-PCR, we found a striking increase in IL-6 mRNA levels only in resuscitated HS, with peak levels observed 1 h after initiation of resuscitation. Increased IL-6 protein expression was localized to bronchial and alveolar cells. Electrophoretic mobility shift assay of protein extracts from shock lungs exhibited an increase in Stat3 activation with kinetics similar to IL-6 mRNA. In situ DNA binding assay determined Stat3 activation predominantly within alveoli. Intratracheal instillation of IL-6 alone into normal rats resulted in PMN infiltration into lung interstitium and alveoli, marked elevation of bronchoalveolar lavage cellularity, and increased wet-to-dry ratio. These findings indicate that IL-6 production and Stat3 activation occur early in HS and may contribute to PMN-mediated lung injury, including ARDS after HS.Hemorrhagic shock (HS) initiates an inflammatory cascade that includes the production of cytokines and recruitment of neutrophils (PMN) and may progress to organ failure, inducing acute respiratory distress syndrome (ARDS). To examine the hypothesis that interleukin-6 (IL-6) contributes to PMN infiltration and lung damage in HS, we examined the lungs of rats subjected to unresuscitated and resuscitated HS for the production of IL-6 and activation of Stat3. Using semiquantitative RT-PCR, we found a striking increase in IL-6 mRNA levels only in resuscitated HS, with peak levels observed 1 h after initiation of resuscitation. Increased IL-6 protein expression was localized to bronchial and alveolar cells. Electrophoretic mobility shift assay of protein extracts from shock lungs exhibited an increase in Stat3 activation with kinetics similar to IL-6 mRNA. In situ DNA binding assay determined Stat3 activation predominantly within alveoli. Intratracheal instillation of IL-6 alone into normal rats resulted in PMN infiltration into lung interstitium and alveoli, marked elevation of bronchoalveolar lavage cellularity, and increased wet-to-dry ratio. These findings indicate that IL-6 production and Stat3 activation occur early in HS and may contribute to PMN-mediated lung injury, including ARDS after HS.

Chemical probes that competitively and selectively inhibit Stat3 activation.

Signal transducer and activator of transcription (Stat) 3 is an oncogene constitutively activated in many cancer systems where it contributes to carcinogenesis. To develop chemical probes that selectively target Stat3, we virtually screened 920,000 small drug-like compounds by docking each into the peptide-binding pocket of the Stat3 SH2 domain, which consists of three sites—the pY-residue binding site, the +3 residue-binding site and a hydrophobic binding site, which served as a selectivity filter. Three compounds satisfied criteria of interaction analysis, competitively inhibited recombinant Stat3 binding to its immobilized pY-peptide ligand and inhibited IL-6-mediated tyrosine phosphorylation of Stat3. These compounds were used in a similarity screen of 2.47 million compounds, which identified 3 more compounds with similar activities. Examination of the 6 active compounds for the ability to inhibit IFN-γ-mediated Stat1 phosphorylation revealed that 5 of 6 were selective for Stat3. Molecular modeling of the SH2 domains of Stat3 and Stat1 bound to compound revealed that compound interaction with the hydrophobic binding site was the basis for selectivity. All 5 selective compounds inhibited nuclear-to-cytoplasmic translocation of Stat3, while 3 of 5 compounds induced apoptosis preferentially of breast cancer cell lines with constitutive Stat3 activation. Thus, virtual ligand screening of compound libraries that targeted the Stat3 pY-peptide binding pocket identified for the first time 3 lead compounds that competitively inhibited Stat3 binding to its pY-peptide ligand; these compounds were selective for Stat3 vs. Stat1 and induced apoptosis preferentially of breast cancer cells lines with constitutively activated Stat3.