Parvin Ataie-Kachoie

Gene of the month: Interleukin 6 (IL-6).

The Interleukin 6 (IL-6) gene encodes the classic proinflammatory cytokine IL-6. It is also known as interferon-β2 (IFN-β2), B cell stimulatory factor-2 and hybridoma/plasmacytoma growth factor. IL-6 is a multifunctional cytokine with a central role in many physiological inflammatory and immunological processes. Due to its major role in initiation as well as resolving inflammation, deregulation of IL-6 is a mainstay of chronic inflammatory and autoimmune diseases. Additionally, IL-6 has been shown to be implicated in pathogenesis of many human malignancies. Thus, a better understanding of IL-6 and its role in various pathological conditions could enable the development of strategies to use it as a therapeutic target. This short review focuses on the structure, regulation and biological activities of IL-6. In addition we discuss the role of IL-6 in diseases with inflammatory background and cancer and also the therapeutic applications of anti-IL-6 agents.

Inhibition of the IL-6 signaling pathway: a strategy to combat chronic inflammatory diseases and cancer.

Interleukin (IL)-6 is a pro-inflammatory cytokine that produces multifunctional effects. Deregulated IL-6 production and signaling are associated with chronic inflammatory diseases, auto-immunity and cancer. On this basis, inhibition of IL-6 production, its receptors or the signaling pathways are strategies currently being widely pursued to develop novel therapies for a wide range of diseases. This survey aims to provide an updated account of why IL-6 inhibitors are shaping up to become an important class of drugs potentially useful in the treatment of ailments and in particular in inflammation and cancer. In addition we discuss the role of different agents in modulating IL-6 and also recent clinical studies targeting IL-6 in inflammation-mediated diseases and cancer.

Minocycline Suppresses Interleukine-6, Its Receptor System and Signaling Pathways and Impairs Migration, Invasion and Adhesion Capacity of Ovarian Cancer Cells: In Vitro and In Vivo Studies

Interleukin (IL)-6 has been shown to be a major contributing factor in growth and progression of ovarian cancer. The cytokine exerts pro-tumorigenic activity through activation of several signaling pathways in particular signal transducer and activator of transcription (STAT3) and extracellular signal-regulated kinase (ERK)1/2. Hence, targeting IL-6 is becoming increasingly attractive as a treatment option in ovarian cancer. Here, we investigated the effects of minocycline on IL-6 and its signaling pathways in ovarian cancer. In vitro, minocycline was found to significantly suppress both constitutive and IL-1β or 4-hydroxyestradiol (4-OH-E2)-stimulated IL-6 expression in human ovarian cancer cells; OVCAR-3, SKOV-3 and CAOV-3. Moreover, minocycline down-regulated two major components of IL-6 receptor system (IL-6Rα and gp130) and blocked the activation of STAT3 and ERK1/2 pathways leading to suppression of the downstream product MCL-1. In female nude mice bearing intraperitoneal OVCAR-3 tumors, acute administration (4 and 24 h) of minocycline (30 mg/kg) led to suppression of IL-6. Even single dose of minocycline was effective at significantly lowering plasma and tumor IL-6 levels. In line with this, tumoral expression of p-STAT3, p-ERK1/2 and MCL-1 were decreased in minocycline-treated mice. Evaluation of the functional implication of minocycline on metastatic activity revealed the capacity of minocycline to inhibit cellular migration, invasion and adhesion associated with down-regulation of matrix metalloproteinases (MMP)-2 and 9. Thus, the data suggest a potential role for minocycline in suppressing IL-6 expression and activity. These effects may prove to be an important attribute to the upcoming clinical trials of minocycline in ovarian cancer.

Minocycline inhibits malignant ascites of ovarian cancer through targeting multiple signaling pathways

OBJECTIVES To evaluate the effect of minocycline on the expression of cytokines and growth factors responsible for malignant ascite formation. METHODS In vitro, cells obtained from malignant ascites were pre-treated with minocycline (0-100 μmol/L) and exposed briefly to hypoxia. In vivo, female nude mice bearing OVCAR-3 tumors were treated orally in drinking water with minocycline for 4 weeks. Plasma, ascites, and tumors were analyzed. RESULTS Minocycline blocked hypoxia-induced surge in interleukin-6 (IL-6), its soluble receptor (sIL-6R) and vascular endothelial growth factor (VEGF) levels in concentration-dependent manner. In mice, orally administered minocycline led to dramatic reduction in tumor weight and malignant ascite volume. IL-6, sIL6R and in particular VEGF levels were highly suppressed in plasma, ascite fluid and tumor tissue by minocycline. In addition, tumors from minocycline treated mice expressed profoundly lower levels of phosphorylated extracellular regulated kinases (p-Erk1/2) and p-Akt. Minocycline was also effective at suppressing transforming growth factor beta (TGF-β1) and increasing vascular endothelial cadherin (VE-cadherin) expression thereby providing molecular confirmation for its effects on malignant ascite formation. CONCLUSION Orally administered minocycline is highly effective in suppressing ovarian cancer-induced malignant ascites by targeting cytokines and growth factors essential for tumor growth and malignant ascite formation.

Minocycline Targets the NF-κB Nexus through Suppression of TGF-β1-TAK1-IκB Signaling in Ovarian Cancer

Substantial evidence supports the critical role of NF-κB in ovarian cancer. Minocycline, a tetracycline, has been shown to exhibit beneficial effects in this malignancy through regulation of a cohort of genes that overlap significantly with the NF-κB transcriptome. Here, it was examined whether or not the molecular mechanism could be attributed to modulation of NF-κB signaling using a combination of in vitro and in vivo models. Minocycline suppressed constitutive NF-κB activation in OVCAR-3 and SKOV-3 ovarian carcinoma cells and was correlated with attenuation of IκBα kinase (IKK) activation, IκBα phosphorylation and degradation, and p65 phosphorylation and nuclear translocation. The inhibition of IKK was found to be associated with suppression of TGF-β-activated-kinase-1 (TAK1) activation and its dissociation from TAK1-binding-protein-1 (TAB1), an indispensable functional mediator between TGF-β and TAK1. Further studies demonstrated that minocycline downregulated TGF-β1 expression. Enforced TGF-β1 expression induced NF-κB activity, and minocycline rescued this effect. Consistent with this finding, TGF-β1 knockdown suppressed NF-κB activation and abrogated the inhibitory effect of minocycline on this transcription factor. These results suggest that the minocycline-induced suppression of NF-κB activity is mediated, in part, through inhibition of TGF-β1. Furthermore, the influence of minocycline on NF-κB pathway activation was examined in female nude mice harboring intraperitoneal OVCAR-3 tumors. Both acute and chronic administration of minocycline led to suppression of p65 phosphorylation and nuclear translocation accompanied by downregulation of NF-κB activity and endogenous protein levels of its target gene products. These data reveal the therapeutic potential of minocycline as an agent targeting the pro-oncogenic TGF-β—NF-κB axis in ovarian cancer. Implications: This preclinical study lends support to the notion that ovarian cancer management would benefit from administration of minocycline. Mol Cancer Res; 11(10); 1279–91. ©2013 AACR.

p70 Ribosomal protein S6 kinase (Rps6kb1): an update.

The Rps6kb1 gene encodes the 70 kDa ribosomal protein S6 kinase (p70S6K), which is a serine/threonine kinase regulated by phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. p70S6K plays a crucial role in controlling cell cycle, growth and survival. The PI3K/mTOR signalling pathway is one of the major mechanisms for controlling cell survival, proliferation and metabolism and is the central regulator of translation of some components of protein synthesis system. Upon activation, this kinase phosphorylates S6 protein of ribosomal subunit 40S resulting in selective translation of unique family of mRNAs that contain oligopyrimidine tract on 5’ transcriptional site (5′TOP). 5′TOP mRNAs are coding the components of translational apparatus including ribosomal proteins and elongation factors. Due to the role of p70S6K in protein synthesis and also its involvement in a variety of human diseases ranging from diabetes and obesity to cancer, p70S6K is now being considered as a new therapeutic target for drug development. Furthermore, p70S6K acts as a biomarker for response to immunosuppressant as well as anticancer effects of inhibitors of the mTOR. Because of the narrow therapeutic index of mTOR inhibitors, drug monitoring is essential, and this is usually done by measuring blood drug levels, therapeutic response and drug-induced adverse effects. Recent studies have suggested that plasma p70S6K is a reliable index for the monitoring of patient response to mTOR inhibitors. Therefore, a better understanding of p70S6K and its role in various pathological conditions could enable the development of strategies to aid diagnosis, prognosis and treatment schedules.

Abstract LB-005: Monepantel a new first in class potent inhibitor of P70S6K potentiates the anti-tumor effects of gemcitabine and doxorubicin: In vitro and in vivo studies

Aims: Monepantel (MPL) is a new nematode-specific anthelmintic agent. We have recently communicated the preliminary results showing the anti-tumor activity of this agent in ovarian cancer through attenuation of mTOR/P70S6K pathway. As emerging data indicate that mTOR inhibitors are most effective when combined with other target agents, we evaluated whether MPL could favorably be combined with the clinically approved chemotherapeutic agents to improve therapeutic efficacy. Methods: The effects of MPL and/or chemotherapeutic agents gemcitabine (Gem)/ doxorubicin (Dox) on the growth of a panel of cancer cell lines were determined using SRB assay. In vitro drug synergy was determined using combination index (CI) methods derived from Chou-Talalay equations using CalcuSyn software. For in vivo studies, we evaluated the effect of MPL, alone and in combination with Gem/ Dox on the growth of established human ovarian (OVCAR-3) tumors implanted subcutaneously in BALB/C nude mice. Toxicity was evaluated measuring animal weight. In vivo combination effects was determined using Fractional Tumor Volume (FTV) method. Results: In vitro, MPL in combination with Gem or Dox synergistically reduced survival rates of a panel of malignant cells from different origins while having no additive effect upon non-malignant cell (HOSE) survival rates. In vivo, antitumor activity was observed in all treatment groups compared to the mock-treated animals after 4 weeks of treatment. However combination therapy with MPL and chemotherapeutics significantly attenuated tumor growth, compared to monotherapy without showing any toxicity. Combining MPL (50 mg/kg) increased the tumor inhibitory effect of low dose Gem (2 mg/kg), high dose Gem (5 mg/kg), low dose Dox (2 mg/kg) and high dose Dox (5 mg/kg) by 32.29, 35.1, 15.2 and 24.38% respectively. Moreover, MPL (25 mg/kg) enhanced the tumor inhibitory rate of Gem (5 mg/kg) by 36.26%. These resulted in complete tumor growth inhibition in Gem 5 mg/kg + MPL 25 or 50 mg/kg and Dox 2 or 5 mg/kg+ MPL 50 mg/kg treatment groups. Assessment of therapeutic synergy with FTV method revealed strong synergy in Gem 5 mg/kg + MPL 25 or 50 mg/kg, Gem 2 mg/kg + MPL 50 mg/kg and Dox 2 or 5 mg/kg+ MPL 50 mg/kg treatment groups with odds ratio of 2.48, 2.59, 1.48, 1.63 and 2.97 (>1 indicates synergy) respectively. Conclusion: These findings provide a rational to further investigate MPL in combination with standard chemotherapeutics as novel combination regimens which could hopefully provide strong anticancer synergy. Citation Format: Parvin A. Ataie-Kachoie, Javed Akhter, David L. Morris. Monepantel a new first in class potent inhibitor of P70S6K potentiates the anti-tumor effects of gemcitabine and doxorubicin: In vitro and in vivo studies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-005. doi:10.1158/1538-7445.AM2015-LB-005

Abstract 3689: Minocycline regulates hypoxia-inducible factor-1α expression through the induction of its degradation in ovarian cancer: in vitro and in vivo studies

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Experimental data suggest therapeutic advantage from selective disruption of the hypoxia response in cancer. We recently found that minocycline decreases tumor VEGF expression in ovarian tumor xenografts in mice and herein report a companion laboratory study to test if this effect was the result of hypoxia-inducible factor (HIF) inhibition. Human ovarian carcinoma cell lines (OVCAR-3, SKOV-3 and A2780) were treated with minocycline in normoxic and hypoxic conditions and also in the presence of hypoxia-mimetic agent deferoxamine in vitro. Two different in vivo experiments investigated miocycline effects after single dose (30 mg/kg, 4 or 24 h) or longer exposure in nude mice bearing OVCAR-3 xenografts. Treatment with minocycline inhibited the accumulation of HIF-1alpha in a concentration-dependent fashion in vitro. A similar effect of minocycline was seen in vivo. Furthermore, the inhibtory effect of minocycline on HIF-1alpha was found to be associated with a significant decrease in HIF-1alpha half-life showing that minocycline enhances degradtion of HIF-1alpha. These data warrents further evaluation of minocycline in the treatment of ovarian cancer. Citation Format: Parvin Ataie-Kachoie, David L. Morris, Mohammad H. Pourgholami. Minocycline regulates hypoxia-inducible factor-1α expression through the induction of its degradation in ovarian cancer: in vitro and in vivo studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3689. doi:10.1158/1538-7445.AM2014-3689

Abstract A7: Minocycline regulates Hypoxia-Inducible Factor-1α expression through the induction of its degradation in ovarian cancer: in vitro and in vivo studies.

Experimental data suggest therapeutic advantage from selective disruption of the hypoxia response in cancer. We recently found that minocycline decreases tumor VEGF expression in ovarian tumor xenografts in mice and herein report a companion laboratory study to test if this effect was the result of hypoxia-inducible factor (HIF) inhibition. Human ovarian carcinoma cell lines (OVCAR-3, SKOV-3 and A2780) were treated with minocycline in normoxic and hypoxic conditions and also in the presence of hypoxia-mimetic agent deferoxamine in vitro. Two different in vivo experiments investigated miocycline effects after single dose (30 mg/kg, 4 or 24 h) or longer exposure in nude mice bearing OVCAR-3 xenografts. Treatment with minocycline inhibited the accumulation of HIF-1alpha in a concentration-dependent fashion in vitro. A similar effect of minocycline was seen in vivo. Furthermore, the inhibtory effect of minocycline on HIF-1alpha was found to be associated with a significant decrease in HIF-1alpha half-life showing that minocycline enhances degradtion of HIF-1alpha. These data warrents further evaluation of minocycline in the treatment of ovarian cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A7. Citation Format: Parvin Ataie-Kachoie, Mohammad H. Pourgholami, David L. Morris. Minocycline regulates Hypoxia-Inducible Factor-1α expression through the induction of its degradation in ovarian cancer: in vitro and in vivo studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A7.

Abstract 5603: Minocycline abrogates IL-6 signaling and inhibits migration, invasion and adhesion capacity of ovarian cancer cells.

Aims: Minocycline (7-dimethylamino-6-desoxytertracycline) has been recognized as a drug with interesting pleiotropic properties including suppression of pro-inflammatory cytokine levels. The beneficial effects of minocycline arisen from suppression of proinflammatory cytokines and in particular IL-6 have been elucidated in central nervous system. However, this drug has never been examined in cancers with aberrant IL-6 expression. Considering the key role of IL-6 and its signaling pathways in the growth and progression of ovarian cancer, we evaluated whether minocycline modulates IL-6, its receptor system and signaling pathways in ovarian cancer. Methods: Expression of IL-6 in control and minocycline-treated ovarian cancer cell lines (OVCAR-3, SKOV-3 and CAOV-3) was determined by immunocytochemistry. ELISA was used to quantify the effects of minocycline on both constitutive and IL-1β or 4-OH-estradiol stimulated IL-6 expression in these cell lines. The effects of minocycline on IL-6R expression was assessed by immunocytochemistry and western blot. Activation of STAT3 and ERK1/2, two major pathways down-stream of IL-6 were evaluated by western blotting. To further characterize the influence of minocycline against IL-6 bioactivity, the effect of the drug on the functional status of SKOV-3 cells was examined by adhesion assay and transwell migration and invasion assay. Finally, to determine the effect of minocycline on IL-6 expression in vivo, female nude mice bearing intraperitoneal OVCAR-3 tumors were treated with a single 30 mg/kg dose of minocycline administered intraperitoneally for 4 and 24 h. The IL-6 expression in plasma and tumor samples excised from these animals was evaluated by ELISA and western blot. Results: We found that minocycline was potent in suppressing both constitutive and stimulated IL-6 expression in vitro. Analysis of plasma and tumors also revealed substantial down-regulation of IL-6 in minocycline-treated mice in vivo. Moreover, minocycline blocked the expression of IL-6R and phosphorylation of STAT3 and ERK1/2 time-dependently. It was also found that minocycline-treated cells exhibit dose-dependent attenuation of cell adhesion, migration and invasion capacity. Conclusion: These results demonstrated that minocycline effectively blocks IL-6 signaling in ovarian cancer. Blockage of IL-6 signaling may provide benefits for the treatment of this deadly cancer. Citation Format: Parvin Ataie-Kachoie, David L. Morris, Mohammad H. Pourgholami. Minocycline abrogates IL-6 signaling and inhibits migration, invasion and adhesion capacity of ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5603. doi:10.1158/1538-7445.AM2013-5603