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// Jung Nyeo Chun 1, 2, * , Soonbum Park 1, * , Sanghoon Lee 3 , Jae-Kyung Kim 1 , Eun-Jung Park 1 , MinJi Kang 1 , Hye Kyung Kim 4, 5 , Jong Kwan Park 5 , Insuk So 1, 2 and Ju-Hong Jeon 1, 2 1 Department of Physiology and Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, Korea 2 Institute of Human-Environment Interface Biology, Seoul National University, Seoul 03080, Korea 3 Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112-5650, USA 4 College of Pharmacy, Kyungsung University, Busan 48434, Korea 5 Department of Urology, Medical School, and Institute for Medical Sciences, Chonbuk National University, Jeonju 54896, Korea * These authors contributed equally to this work Correspondence to: Ju-Hong Jeon, email: jhjeon2@snu.ac.kr Keywords: schisandra chinensis; schisandrol B; schisandrin B; TGFβ1; NF-κB Received: June 30, 2017 Accepted: November 15, 2017 Published: December 14, 2017 ABSTRACT Aberrant transforming growth factor β1 (TGFβ1) signaling plays a pathogenic role in the development of vascular fibrosis. We have reported that Schisandra chinensis fruit extract (SCE), which has been used as a traditional oriental medicine, suppresses TGFβ1-mediated phenotypes in vascular smooth muscle cells (VSMCs). However, it is still largely unknown about the pharmacologic effects of SCE on various TGFβ1 signaling components. In this study, we found that SCE attenuated TGFβ1-induced NF-κB activation and nuclear translocation in VSMCs. Among the five active ingredients of SCE that were examined, schisandrol B (SolB) and schisandrin B (SchB) most potently suppressed TGFβ1-mediated NF-κB activation. In addition, SolB and SchB effectively inhibited IKKα/β activation and IκBα phosphorylation in TGFβ1-treated VSMCs. The pharmacologic effects of SolB and SchB on NF-κB activation were independent of the Smad-mediated canonical pathway. Therefore, our study demonstrates that SCE and its active constituents SolB and SchB suppress TGFβ1-mediated NF-κB signaling pathway in a Smad-independent mechanism. Our results may help further investigations to develop novel multi-targeted therapeutic strategies that treat or prevent vascular fibrotic diseases.

Geraniol is a dietary monoterpene alcohol that is found in the essential oils of aromatic plants. To date, experimental evidence supports the therapeutic or preventive effects of geraniol on different types of cancer, such as breast, lung, colon, prostate, pancreatic, and hepatic cancer, and has revealed the mechanistic basis for its pharmacological actions. In addition, geraniol sensitizes tumor cells to commonly used chemotherapy agents. Geraniol controls a variety of signaling molecules and pathways that represent tumor hallmarks; these actions of geraniol constrain the ability of tumor cells to acquire adaptive resistance against anticancer drugs. In the present review, we emphasize that geraniol is a promising compound or chemical moiety for the development of a safe and effective multi-targeted anticancer agent. We summarize the current knowledge of the effects of geraniol on target molecules and pathways in cancer cells. Our review provides novel insight into the challenges and perspectives with regard to geraniol research and to its application in future clinical investigation.

Cyclosporin A (CsA) has antitumor effects on various cancers including prostate cancer. However, its antitumor mechanism is poorly understood. In this study, we showed that AMP-activated protein kinase (AMPK) mediates the antitumor effect of CsA on prostate cancer cells. CsA attenuated cell growth by inducing a G1 arrest through the inhibition of mTOR complex 1 (mTORC1) signaling. In this context, Akt was paradoxically activated downstream of the EGF receptor (EGFR)-mediated increase in phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) production. However, CsA also caused a Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ)-dependent activation of AMPK, which inhibits mTORC1 signaling; this led to ineffective Akt signaling. An EGFR or Akt inhibitor increased the growth suppressive activity of CsA, whereas the combination of an AMPK inhibitor and CsA markedly rescued cells from the G1 arrest and increased cell growth. These results provide novel insights into the molecular mechanisms of CsA on cancer signaling pathways.

Angiotensin II (Ang II), a potent precursor of hypertrophy and heart failure, upregulates neuronal nitric oxide synthase (nNOS or NOS1) in the myocardium. Here, we investigate the involvement of type 1 and 2 angiotensin receptors (AT1R and AT2R) and molecular mechanisms mediating Ang II-upregulation of nNOS. Our results showed that pre-treatment of left ventricular (LV) myocytes with antagonists of AT1R or AT2R (losartan, PD123319) and ROS scavengers (apocynin, tiron or PEG-catalase) blocked Ang II-upregulation of nNOS. Surface biotinylation or immunocytochemistry experiments demonstrated that AT1R expression in plasma membrane was progressively decreased (internalization), whereas AT2R was increased (membrane trafficking) by Ang II. Inhibition of AT1R or ROS scavengers prevented Ang II-induced translocation of AT2R to plasma membrane, suggesting an alignment of AT1R-ROS-AT2R. Furthermore, Ang II increased eNOS-Ser(1177) but decreased eNOS-Thr(495), indicating concomitant activation of eNOS. Intriguingly, ROS scavengers but not AT2R antagonist prevented Ang II-activation of eNOS. NOS inhibitor (L-NG-Nitroarginine Methyl Ester, L-NAME) or eNOS gene deletion (eNOS(-/-)) abolished Ang II-induced membrane trafficking of AT2R, nNOS protein expression and activity. Mechanistically, S-nitrosation of AT2R was increased by sodium nitroprusside (SNP), a NO donor. Site-specific mutagenesis analysis reveals that C-terminal cysteine 349 in AT2R is essential in AT2R translocation to plasma membrane. Taken together, we demonstrate, for the first time, that Ang II upregulates nNOS protein expression and activity via AT1R/ROS/eNOS-dependent S-nitrosation and membrane translocation of AT2R. Our results suggest a novel crosstalk between AT1R and AT2R in regulating nNOS via eNOS in the myocardium under pathogenic stimuli.

Transglutaminase2 (TG2) is a multi-functional protein involved in various cellular processes, including apoptosis, differentiation, wound healing, and angiogenesis. The malfunction of TG2 causes many human disease including inflammatory disease, celiac disease, neurodegenerative diseases, tissue fibrosis, and cancers. Protein cross-linking activity, which is representative of TG2, is activated by calcium ions and suppressed by GTP. Here, we elucidated the structure of TG2 in complex with its endogenous inhibitor, GTP. Our structure showed why GTP is the optimal nucleotide for interacting with and inhibiting TG2. In addition, sequence comparison provided information describing the evolutionary scenario of GTP usage for controlling the activity of TG2.

Aberrant expression of cell cycle regulators have been implicated in prostate cancer development and progression. Therefore, understanding transcriptional networks controlling the cell cycle remain a challenge in the development of prostate cancer treatment. In this study, we found that icilin, a super-cooling agent, down-regulated the expression of cell cycle signature genes and caused G1 arrest in PC-3 prostate cancer cells. With reverse-engineering and an unbiased interrogation of a prostate cancer-specific regulatory network, master regulator analysis discovered that icilin affected cell cycle-related transcriptional modules and identified E2F1 transcription factor as a target master regulator of icilin. Experimental analyses confirmed that icilin reduced the activity and expression levels of E2F1. These results demonstrated that icilin inactivates a small regulatory module controlling the cell cycle in prostate cancer cells. Our study might provide insight into the development of cell cycle-targeted cancer therapeutics.

Menthol, a naturally occurring monoterpene, is used in foods, cosmetic products, and topical therapeutic preparations. It also exerts cytotoxic activity against several cancer cell types, including prostate cancer cells. However, little is known about the mechanism of menthol action against prostate cancer cells. In this study, we investigated the effect of menthol on the gene expression profile of PC-3 prostate cancer cells using DNA microarray analyses. Gene set enrichment analysis revealed that menthol primarily affects the expression of cell cycle-related genes. Subsequent experimental analyses validated that menthol induces G2/M arrest. Particularly, menthol markedly down-regulated polo-like kinase 1 (PLK1), a key regulator of G2/M phase progression and inhibited its downstream signaling. Our computational analyses and experimental validation provide a basis for future investigations that are aimed at elucidating the action of menthol on cell cycle control in prostate cancer cells.

Cytoplasmic presence of Hsp60, which is principally a nuclear gene-encoded mitochondrial chaperonin, has frequently been stated, but its role in intracellular signaling is largely unknown. In this study, we demonstrate that the cytosolic Hsp60 promotes the TNF-alpha-mediated activation of the IKK/NF-kappaB survival pathway via direct interaction with IKKalpha/beta in the cytoplasm. Selective loss or blockade of cytosolic Hsp60 by specific antisense oligonucleotide or neutralizing antibody diminished the IKK/NF-kappaB activation and the expression of NF-kappaB target genes, such as Bfl-1/A1 and MnSOD, which thus augmented intracellular ROS production and ASK1-dependent cell death, in response to TNF-alpha. Conversely, the ectopic expression of cytosol-targeted Hsp60 enhanced IKK/NF-kappaB activation. Mechanistically, the cytosolic Hsp60 enhanced IKK activation via upregulating the activation-dependent serine phosphorylation in a chaperone-independent manner. Furthermore, transgenic mouse study showed that the cytosolic Hsp60 suppressed hepatic cell death induced by diethylnitrosamine in vivo. The cytosolic Hsp60 is likely to be a regulatory component of IKK complex and it implicates the first mitochondrial factor that regulates cell survival via NF-kappaB pathway.

IkappaB kinase (IKK), the pivotal kinase in signal-dependent activation of nuclear factor-kappaB (NF-kappaB), is composed of multiple protein components, including IKK alpha/beta/gamma core subunits. To investigate the regulation of the IKK complex, we immunoaffinity purified the IKK complex, and by MALDI-TOF mass spectrometry identified a splice variant of zinc finger protein 268 (ZNF268) as a novel IKK-interacting protein. Both the full-length and the spliced form of the ZNF268 protein were detected in a variety of mammalian tissues and cell lines. The genes were cloned and expressed by in vitro transcription/translation. Several deletion derivatives, such as KRAB domain (KRAB) on its own, the KRAB/spacer/4-zinc fingers (zF4), and the spacer/ 4-zinc fingers (zS4), were ectopically expressed in mammalian cells and exhibited had different subcellular locations. The KRAB-containing mutants were restricted to the nucleus, while zS4 was localized in the cytosol. TNF-alpha-induced NF-kappaB activation was examined using these mutants and only zS4 was found to stimulate activation. Collectively, the results indicate that a spliced form of ZNF268 lacking the KRAB domain is located in the cytosol, where it seems to play a role in TNF-alpha-induced NF-kappaB activation by interacting with the IKK complex.