Development of a Novel Azaspirane That Targets the Janus Kinase-Signal Transducer and Activator of Transcription (STAT) Pathway in Hepatocellular Carcinoma in Vitro and in Vivo*

Background: Constitutive activation of STAT3 is associated with the progression of hepatocellular carcinoma (HCC), and abrogation of STAT3 signaling is a potential target for HCC treatment. Results: A novel azaspirane modulates the JAK-STAT pathway in HCC. Conclusion: The lead compound induces apoptosis by down-regulating STAT3 signaling. Significance: This investigation reports a novel inhibitor of the JAK-STAT pathway with the potential to target various cancers. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates genes involved in cell growth, proliferation, and survival, and given its association with many types of cancers, it has recently emerged as a promising target for therapy. In this work, we present the synthesis of N-substituted azaspirane derivatives and their biological evaluation against hepatocellular carcinoma (HCC) cells (IC50 = 7.3 μm), thereby identifying 2-(1-(4-(2-cyanophenyl)1-benzyl-1H-indol-3-yl)-5-(4-methoxy-phenyl)-1-oxa-3-azaspiro(5,5) undecane (CIMO) as a potent inhibitor of the JAK-STAT pathway with selectivity over normal LO2 cells (IC50 > 100 μm). The lead compound, CIMO, suppresses proliferation of HCC cells and achieves this effect by reducing both constitutive and inducible phosphorylation of JAK1, JAK2, and STAT3. Interestingly, CIMO displayed inhibition of Tyr-705 phosphorylation, which is required for nuclear translocation of STAT3, but it has no effect on Ser-727 phosphorylation. CIMO accumulates cancer cells in the sub-G1 phase and decreases STAT3 in the nucleus and thereby causes down-regulation of genes regulated via STAT3. Suppression of STAT3 phosphorylation by CIMO and knockdown of STAT3 mRNA using siRNA transfection displayed a similar effect on the viability of HCC cells. Furthermore, CIMO significantly decreased the tumor development in an orthotopic HCC mouse model through the modulation of phospho-STAT3, Ki-67, and cleaved caspase-3 in tumor tissues. Thus, CIMO represents a chemically novel and biologically in vitro and in vivo validated compound, which targets the JAK-STAT pathway as a potential cancer treatment.

[1]  Yate-Ching Yuan,et al.  Dual inhibition of Janus and Src family kinases by novel indirubin derivative blocks constitutively‐activated Stat3 signaling associated with apoptosis of human pancreatic cancer cells , 2013, Molecular oncology.

[2]  S. Murase,et al.  Neuronal activity‐dependent STAT3 localization to nucleus is dependent on Tyr‐705 and Ser‐727 phosphorylation in rat hippocampal neurons , 2014, The European journal of neuroscience.

[3]  G. Sethi,et al.  γ‐Tocotrienol is a novel inhibitor of constitutive and inducible STAT3 signalling pathway in human hepatocellular carcinoma: potential role as an antiproliferative, pro‐apoptotic and chemosensitizing agent , 2011, British journal of pharmacology.

[4]  Delong Liu,et al.  Dysregulation of JAK-STAT pathway in hematological malignancies and JAK inhibitors for clinical application , 2013, Biomarker Research.

[5]  R. Jove,et al.  Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein. , 1995, Science.

[6]  Hayato Nakagawa,et al.  Molecular Mechanisms of Liver Injury and Hepatocarcinogenesis: Focusing on the Role of Stress-Activated MAPK , 2012, Pathology research international.

[7]  J. Bromberg.  Stat proteins and oncogenesis. , 2002, The Journal of clinical investigation.

[8]  Andreas Bender,et al.  In Silico Target Predictions: Defining a Benchmarking Data Set and Comparison of Performance of the Multiclass Naïve Bayes and Parzen-Rosenblatt Window , 2013, J. Chem. Inf. Model..

[9]  J. Turkson,et al.  Novel peptidomimetic inhibitors of signal transducer and activator of transcription 3 dimerization and biological activity. , 2004, Molecular cancer therapeutics.

[10]  S. Eschrich,et al.  Persistent Activation of Stat3 Signaling Induces Survivin Gene Expression and Confers Resistance to Apoptosis in Human Breast Cancer Cells , 2006, Clinical Cancer Research.

[11]  J. Montgomery,et al.  Synthesis of potential anticancer agents. Pyrido[4,3-b][1,4]oxazines and pyrido[4,3-b][1,4]thiazines. , 1983, Journal of medicinal chemistry.

[12]  Z. Estrov,et al.  Preclinical characterization of atiprimod, a novel JAK2 AND JAK3 inhibitor , 2011, Investigational New Drugs.

[13]  Taher Abbasi,et al.  Suppression of Signal Transducer and Activator of Transcription 3 Activation by Butein Inhibits Growth of Human Hepatocellular Carcinoma In Vivo , 2010, Clinical Cancer Research.

[14]  A. Levitzki,et al.  Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor , 1996, Nature.

[15]  Xin Chen,et al.  Simplified staurosporine analogs as potent JAK3 inhibitors. , 2007, Bioorganic & medicinal chemistry letters.

[16]  L. Koniaris,et al.  Interleukin-6 mediates G(0)/G(1) growth arrest in hepatocellular carcinoma through a STAT 3-dependent pathway. , 2008, The Journal of surgical research.

[17]  Basappa,et al.  A small oxazine compound as an anti-tumor agent: a novel pyranoside mimetic that binds to VEGF, HB-EGF, and TNF-α. , 2010, Cancer letters.

[18]  J. Darnell,et al.  Mapping of Stat3 serine phosphorylation to a single residue (727) and evidence that serine phosphorylation has no influence on DNA binding of Stat1 and Stat3. , 1997, Nucleic acids research.

[19]  Basappa,et al.  Synthesis, biological evaluation and in silico and in vitro mode-of-action analysis of novel dihydropyrimidones targeting PPAR-γ , 2014 .

[20]  R. Lai,et al.  Loss of SHP1 enhances JAK3/STAT3 signaling and decreases proteosome degradation of JAK3 and NPM-ALK in ALK+ anaplastic large-cell lymphoma. , 2006, Blood.

[21]  G. Rao,et al.  Cytosolic Phospholipase A2 Is an Effector of Jak/STAT Signaling and Is Involved in Platelet-derived Growth Factor BB-induced Growth in Vascular Smooth Muscle Cells* , 2003, The Journal of Biological Chemistry.

[22]  Andreas Bender,et al.  From in silico target prediction to multi-target drug design: current databases, methods and applications. , 2011, Journal of proteomics.

[23]  J. Ihle.  STATs: Signal Transducers and Activators of Transcription , 1996, Cell.

[24]  C. Desbois-Mouthon,et al.  Targeting STAT3 in hepatocellular carcinoma: sorafenib again…. , 2011, Journal of hepatology.

[25]  Xin Chen,et al.  Synthetic staurosporines via a ring closing metathesis strategy as potent JAK3 inhibitors and modulators of allergic responses. , 2009, Bioorganic & medicinal chemistry letters.

[26]  T. Smithgall,et al.  Activation of STAT3 by the Src Family Kinase Hck Requires a Functional SH3 Domain* , 2002, The Journal of Biological Chemistry.

[27]  R. Gish,et al.  Hepatocellular carcinoma: overcoming challenges in disease management. , 2006, Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association.

[28]  G. Sethi,et al.  Emodin Suppresses Migration and Invasion through the Modulation of CXCR4 Expression in an Orthotopic Model of Human Hepatocellular Carcinoma , 2013, PloS one.

[29]  Z. Estrov,et al.  Atiprimod blocks STAT3 phosphorylation and induces apoptosis in multiple myeloma cells , 2005, British Journal of Cancer.

[30]  A. Seligman,et al.  Synthesis of potential anticancer agents. , 1967, Journal of medicinal chemistry.

[31]  J. McMurray.  A new small-molecule Stat3 inhibitor. , 2006, Chemistry & biology.

[32]  M. Mitchell,et al.  Artemin Reduces Sensitivity to Doxorubicin and Paclitaxel in Endometrial Carcinoma Cells through Specific Regulation of CD24. , 2010, Translational oncology.

[33]  D. Frank.  STAT3 as a central mediator of neoplastic cellular transformation. , 2007, Cancer letters.

[34]  Pavel Kovarik,et al.  Serine phosphorylation of STATs , 2000, Oncogene.

[35]  Basappa,et al.  Novel Synthetic Biscoumarins Target Tumor Necrosis Factor-α in Hepatocellular Carcinoma in Vitro and in Vivo* , 2014, The Journal of Biological Chemistry.

[36]  K. Anderson,et al.  Azaspirane (N-N-diethyl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) inhibits human multiple myeloma cell growth in the bone marrow milieu in vitro and in vivo. , 2005, Blood.

[37]  S. Verstovsek,et al.  Investigational Janus kinase inhibitors , 2013, Expert opinion on investigational drugs.

[38]  P. Galle,et al.  Future perspectives in hepatocellular carcinoma. , 2010, Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver.

[39]  G. Sethi,et al.  Garcinol, a Polyisoprenylated Benzophenone Modulates Multiple Proinflammatory Signaling Cascades Leading to the Suppression of Growth and Survival of Head and Neck Carcinoma , 2013, Cancer Prevention Research.

[40]  A. Zauberman,et al.  Stress activated protein kinase p38 is involved in IL-6 induced transcriptional activation of STAT3 , 1999, Oncogene.

[41]  J. Luk,et al.  Celastrol Suppresses Growth and Induces Apoptosis of Human Hepatocellular Carcinoma through the Modulation of STAT3/JAK2 Signaling Cascade In Vitro and In Vivo , 2012, Cancer Prevention Research.

[42]  Basappa,et al.  Simple and an efficient method for the synthesis of 1-[2-dimethylamino-1-(4-methoxy-phenyl)-ethyl]-cyclohexanol hydrochloride: (+/-) venlafaxine racemic mixtures. , 2004, Bioorganic & medicinal chemistry letters.

[43]  M. Karin,et al.  Obesity, inflammation, and liver cancer. , 2012, Journal of hepatology.

[44]  Basappa,et al.  Synthesis and Characterization of Novel 2-Amino-Chromene-Nitriles that Target Bcl-2 in Acute Myeloid Leukemia Cell Lines , 2014, PloS one.

[45]  Xiaomin Chen,et al.  STAT3 is constitutively phosphorylated on serine 727 residues, binds DNA, and activates transcription in CLL cells. , 2010, Blood.

[46]  Suyun Huang.  Regulation of Metastases by Signal Transducer and Activator of Transcription 3 Signaling Pathway: Clinical Implications , 2007, Clinical Cancer Research.

[47]  Mei Su,et al.  Discovery of 1-methyl-1H-imidazole derivatives as potent Jak2 inhibitors. , 2014, Journal of medicinal chemistry.

[48]  B. Aggarwal,et al.  Targeting Inflammatory Pathways for Prevention and Therapy of Cancer: Short-Term Friend, Long-Term Foe , 2009, Clinical Cancer Research.

[49]  J. Whang‐Peng,et al.  Clinical Development and Future Direction for the Treatment of Hepatocellular Carcinoma , 2010 .