Inhibitory Effects of a New 1H-Pyrrolo[3,2-c]pyridine Derivative, KIST101029, on Activator Protein-1 Activity and Neoplastic Cell Transformation Induced by Insulin-Like Growth Factor-1

Abstract

Diarylureas and diarylamides derivatives are reported to have antitumor activity. Encouraged by the interesting antiproliferative activity of diarylurea and diarylamide derivatives, we synthesized a new series of diarylureas and diarylamides containing pyrrolo[3,2-c]pyridine scaffold. In this study, we demonstrate that a N-(3-(4-benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)phenyl)-4-morpholino-3-(trifluoromethyl)benzamide, KIST101029, inhibits neoplastic cell transformation induced by insulin-like growth factor 1 (IGF-1) in mouse epidermal JB6 Cl41 cells. The KIST101029 compound inhibited mitogen-activated protein kinase/extracellular signal-regulated kinase kinases (MEK), c-jun N-terminal kinases (JNK), and mechanistic target of rapamycin (mTOR) signaling pathways induced by IGF-1 in JB6 Cl41 cells, resulting in the inhibition of c-fos and c-jun transcriptional activity. In addition, the KIST101029 inhibited the associated activator protein-1 (AP-1) transactivation activity and cell transformation induced by IGF-1 in JB6 Cl41 cells. Consistent with these observations, in vivo chorioallantoic membrane assay also showed that the KIST101029 inhibited IGF-1-induced tumorigenicity of JB6 Cl41 cells. Importantly, KIST101029 suppressed the colony formation of A375 cells in soft agar. Taken together, these results indicate that a KIST101029 might exert chemopreventive effects through the inhibition of phosphorylation of MAPK and mTOR signaling pathway.

Many of the processes involved in tumor growth, progression, and metastasis are mediated by signaling pathways initiated by activated receptor tyrosine kinases (RTKs).¹⁾ RAS activation can be exploited by overexpression variety of RTKs, including those for the epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF-1R), or platelet-derived growth factor receptor (PDGFR), or vascular-endothelial growth factors (VEGFR).^2–5) In this way, the majority of human tumors depend on activation of RAS signal transduction pathways to achieve cellular proliferation and survival.⁶⁾ The three mammalian RAF proteins (A, B and CRAF) can be activated by the human oncogene RAS, downstream from which they exert both kinase-dependent and kinase-independent, tumor-promoting functions.⁷⁾ The kinase-dependent functions are mediated chiefly by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-extracellular signal-regulated kinase (MEK/ERK) pathway, whose activation is associated with proliferation in a broad range of human tumors.⁸⁾ Therefore, the development of a small molecule that can inhibit RAF activity is a promising direction for cancer therapy.

Activator protein (AP)-1 acts as pivotal transcription factors involving neoplastic transformation development of cancer, and is regulated by upstream kinases, including mitogen-activated protein kinases (MAPKs), signaling pathways.^9,10) Among the components of the MAPK pathways, the RAF/MEK/ERK cascade has been the focus of cancer chemotherapy because of tis relevance in carcinogenesis. A variety of tumor promoters including IGF-1 are known to induce neoplastic transformation through activation of RAF/MEK/ERK pathway in various cell lines.¹¹⁾ RAF kinases are serine/threonine protein kinases and initiate a mitogenic kinase cascade that ultimately modulates gene expression via the phosphorylation of transcriptional factors, which can have profound effects on cellular proliferation and tumorigenesis.¹²⁾ Because aberrant activation of ERK has been demonstrated in various types of tumors,¹³⁾ the targeted downregulation of ERK through the inhibition of upstream kinases such as RAF is an effective method for intervening in carcinogenesis.

Sorafenib (Nexavar®) is a diarylurea derivative that has been extensively used in clinical trials.¹⁴⁾ Sorafenib is an oral multikinase inhibitor that targets 2 classes of kinases, which are known to be involved in both tumor proliferation and angiogenesis.¹⁵⁾ It inhibits RAF kinases (Raf-1 and B-Raf), as well as proangiogenic receptor tyrosine kinases of the PDGFR and VEGFR family.¹⁶⁾ The antiproliferative activity of sorafenib against melanoma is assumed to be due to B-Raf inhibition and induction of apoptosis in a caspase-independent manner.¹⁷⁾ The present study aimed to elucidate the mechanism of the antitumorigenic effects of a new diarylurea and diarylamide derivative, KIST101029, on IGF-induced neoplastic transformation of JB6 Cl41 cells. Here, we demonstrate that KIST101029 is a potent inhibitor of Raf-1. The inhibition of Raf-1 suppressed the signaling cascades of MEKs-ERKs, c-jun N-terminal kinases (JNKs) c-Jun, and mammalian target of rapamycin (mTOR) signaling, resulted in inhibition of c-fos, c-jun, and AP-1 activity, which subsequently inhibited neoplastic transformation. KIST101029 also inhibited IGF-1-induced tumor progression of JB6 Cl41 cells in chick embryo chorioallantoic membrane (CAM).

Materials and Methods

Reagents and Antibodies

Eagle’s minimal essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, gentamicin, insulin-like growth factor-1 (IGF-1), and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, U.S.A). 3-[4,5-Dimethylthiazol-2-thiazoyl]-2,5-diphenyltetrazolium bromide (MTT) was from Sigma-Aldrich (St. Louis, MO, U.S.A.). Cell Proliferation ELISA, Brdu (colorimetric) kit was from Roche Applied Science (Indianapolis, IN, U.S.A.). Polyvinylidene difluoride (PVDF) membrane was from Millipore (Bedford, MA, U.S.A.). Antibodies against phospho-MEK1/2, -ERK1/2, -p90RSK, -JNK, -c-Jun (Ser63), MEK1/2, ERK1/2, and JNK1/2 were purchased from Cell Signaling Tech. Inc. (Beverly, MA, U.S.A.); antibodies against p90RSK, c-Jun, c-Fos, goat anti-mouse immunoglobulin G (IgG) horse-radish peroxidase (HRP), and goat anti-rabbit IgG HRP were from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). The jetPEI cationic polymer transfection reagent was from Polyplus-transfection (New York, NY, U.S.A.). The Dual-luciferase reporter assay kit was purchased from Promega (Madison, WI, U.S.A.).

Cell Culture and Transfection

JB6 Cl41 mouse epidermal cells were cultured in supplemented with 5% FBS, at 37°C in humidified air containing 5% CO₂. A375 human melanoma cells were cultured in DMEM supplemented with 10% FBS, at 37°C. The DNA transfection of the cells was performed using a jetPEI cationic polymer transfection reagent (Polyplus-transfection). Dimethyl-sulfoxide (DMSO) was used as a vehicle to dissolve the compound and a final concentration of 0.1% DMSO (v/v) was used for each treatment.

3-[4,5-Dimethylthiazol-2-thiazolyl]-2,5-diphenyltetrazolium Bromide (MTT) Assay

The MTT assay was performed to check cell viability. In brief, cells (1×10⁵ cells/mL) were seeded in 96-well plates with 100 µL of cell suspension in each well. After culturing for 24 h, cells were treated with KIST101029. The cells were then treated with 5 µg/mL MTT solution (10 µL/well) and incubated for 3 h, after which the purple formazan formed by the live cells was dissolved in 0.04 N HCl in isopropanol (100 µL/well) and the absorbance was measured at 570 nm on a TriStar LB 941 (Berthold Tech. GmbHand Co., KG, Germany).

Cell Proliferation Assay (Bromodeoxyuridine (BrdU) Incorporation)

JB6 Cl41 cells were seeded (1×10⁴ cells/well) in 96-well plates in 100 µL of 5% FBS-MEM. After 24 h, the cells were either treated or not treated with 50 ng/mL IGF-1 together with KIST101029 for 48 h, labeled with 10 µL/well BrdU labeling solution, and then reincubated for additional 4 h at 37°C in a 5% CO₂ atmosphere. After sucked the media, Fix Denat solution was added in each well, incubated at room temperature (RT) for 30 min, and then removed. Anti-BrdU-peroxidase (POD) working solution was added in each well and incubated for further 90 min at RT. The cells were then washed with washing solution for 3 times and 100 µL of substrate solution was added in each well and incubated for 30 min. Cell proliferations was estimated by measuring the absorbance at 370 nm.

Immunoblot Analysis

The cells were disrupted in lysis buffer [50 mM Tris (pH 7.5), 5 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 1 mM dithiothreitol, 0.01% Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail]. The proteins were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked and hybridized with the appropriate primary antibody overnight at 4°C. The protein bands were visualized using chemiluminescence detection kit (Amersham HRP Chemiluminescent Substrates, Amersham Biosciences, Piscataway, NJ, U.S.A.) after hybridization with the HRP-conjugated secondary antibody from rabbits or mice. The LAS4000-mini (FUJIFILM, Tokyo, Japan) was used for chemiluminescence detection.

Reporter Gene Assays

The reporter gene assay for firefly luciferase activity was performed using lysates from AP-1-, c-jun-, or c-fos-luc-transfected JB6 Cl41 cells. In addition, the reporter gene vector pRL-TK-luciferase plasmid (Promega) was co-transfected into each cell line and the renilla luciferase activity generated by this vector was used to normalize the results for transfection efficiency. Cell lysates were prepared by first washing the transfected JB6 Cl41 cells once with phosphate-buffered saline (PBS) at RT. After removing the PBS completely, passive lysis buffer (PLB, Promega) was added, and then cells were incubated at RT for 1 h with gentle shaking. The supernatant fraction was used to measure firefly and renilla luciferase activities. Cell lysates (50 µL each) were mixed with 50 µL of luciferase assay II reagent (Promega), and firefly luciferase light emission was measured by TriStar LB 941 (Berthold Tech. GmbHand Co., KG, Germany). Subsequently, 50 µL of renilla luciferase substrate was added in order to normalize the firefly luciferase data. The c-fos-luc promoter (pFos-WT GL3) and c-jun-luc promoter (JC6GL3) constructs were kindly provided by Dr. Ron Prywes (Columbia University, New York, NY, U.S.A.). The AP-1 luciferase reporter plasmid (−73/+63 collagenase-luciferase) was kindly provided by Dr. Dong Zigang (Hormel Institute, University of Minnesota, Austin, MN, U.S.A.).

Anchorage-Independent Cell Transformation Assay (Soft Agar Assay)

The effect of KIST101029 in the IGF-1 induced cell transformation was investigated in JB6 Cl41 cells. In brief, 8×10³ cells were exposed to IGF-1 (50 ng/mL) with or without KIST101029 in 1 mL of 0.3% basal medium Eagle (BME) agar containing 10% FBS, 2 mM L-glutamine, and 25 µg/mL gentamicin. The cultures were maintained at 37°C, in a 5% CO₂ incubator for 2 weeks, and cell colonies were scored using an Axiovert 200 M fluorescence microscope and Axio Vision software (Carl Zeiss Inc., Thornwood, NY, U.S.A.).

Chorioallantoic Membrane (CAM) Assay

Briefly, fertilized chicken eggs were transferred to an egg incubator and allowed to grow for 10 d. After this, JB6 Cl41 cells (2×10⁶) together with IGF-1 or KIST101029 were placed on the exposed CAM, the eggs were incubated in a humidified incubator at 37°C for 7 d. After 7 d, images were digitally recorded at ×15 magnifications with an SZ-61 zoom stereomicroscope (Olympus, Center Valley, PA, U.S.A.). Tumor areas were analyzed with Image J digital imaging software (download from the NIH website).

Statistical Analysis

Statistical calculations were carried out with Prism 4 for Macintosh (GraphPad Software Inc., La Jolla, CA, U.S.A.). Results are expressed as the mean±standard error of triplicate measurements of three independent experiments. Student’s t-test was used for statistical analyses; p values <0.05 were considered significant.

Results

KIST101029 Inhibits IGF-1-Induced Cell Proliferation of JB6 Cl41 Cells via Its Direct Interaction with Raf-1

Sorafenib (BAY 43–9006) is a synthetic molecule that can be broadly defined as a bi-aryl urea, which was originally identified through inhibition of Raf-1 kinase biochemical and cellular mechanistic assays.^14,18) Therefore, we designed and synthesized a new sorafenib analog, KIST101029, possessing 1H-pyrrolo[3,2-c]pyridine scaffold (Fig. 1A),¹⁹⁾ and evaluated the effects of its derivative, KIST101029, on the cell viability and cell proliferation of JB6 Cl41 mouse epidermal cells by MTT assay and BrdU incorporation assay, respectively. KIST101029 at lower concentrations from 0.01 µM to 0.5 µM did not affect cell viability at 96 h in JB6 Cl41 cells, whereas significantly decreased the cell viability of JB6 Cl41 cells at higher concentrations from 1 µM to 10 µM, dose-dependently (Fig. 1B). To also examine the effect of KIST101029 on cell proliferation induced by insulin like growth factor-1 (IGF-1), JB6 Cl41 cells were treated with IGF-1 in an absence or presence of KIST101029. The result showed that KIST101029 significantly reduced the IGF-1-induced cell proliferation after treatment of KIST101029, at concentrations from 0.1 µM to 0.5 µM, dose-dependently (Fig. 1C). To determine whether the inhibition of IGF-1-induced cell proliferation induced by KIST101029 was caused by a directed interaction with Raf-1, we next performed pull-down assay. We transfected Myc-tagged Raf-1 in HEK 293 cells. The cells lysates were incubated with KIST101029-biotin immunoprecipitated using the anti-biotin antibody and blotted with the anti-Myc antibody. The results showed that the exogenously expressed Raf-1 was found in the KIST101029-biotin-immunoprecipitated-sepharose beads, but not sepharose beads (Fig. 1D), suggesting that KIST101029 directly bind with Raf-1 in vitro.

Fig. 1. Effects of KIST101029, on Cell Viability and Cell Proliferation of JB6 Cl41 Cells

(A) Chemical structure of N-(3,4-benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)phenyl)-4-morpholino-3-(trifluoromethyl)benzamide, KIST101029. (B) Cells were seeded and cultured for 96 h in 5% FBS/MEM at 37°C in 5% CO₂ atmosphere. Then, the cells were treated with KIST101029 dose-dependently as indicated. Cell viability was measured by MTT assay, as described in Materials and Methods. Data are represented as the means±S.D. as determined from triplicate experiments. (C) Cells were treated with different concentrations of KIST101029 in an absence or presence of 50 ng/mL IGF-1 for 48h, and then cell proliferation was estimated using BrdU incorporation assay. Columns, mean of triplicate measurements of three experiments; bars, S.D. * p<0.05, compared with control cells. (D) KIST101029-biotin specifically binds with Raf-1 in vitro. HEK 293 cells were transfected with pcDNA4-Myc-Raf-1. At 48 h after transfection, cells were harvested, lysed, and used for pull down assay. The Raf-1-KIST101029-biotin binding in vitro was confirmed by immunoblotting using an antibody against Myc-Raf-1. Lane 1, whole-cell lysates from Myc-Raf-1-overexpressing cells (input control); lane 2, pcDNA3-transfected whole-cell lysates precipitated with anti-biotin antibody and KIST101029-biotin; lane 3, Myc-Raf-1-transfected whole-cell lysates precipitated only with anti-biotin antibody; lane 4, Myc-Raf-1-whole-cell lysates precipitated with anti-biotin antibody and KIST101029-biotin. Immunoblotting analysis was also performed for Myc-Raf-1 and β-actin by taking the protein from the total cell lysate.

KIST101029 Suppresses Signaling Cascade of MEKs/ERKs, JNKs/c-Jun, and mTOR/p70S6K Induced by IGF-1 in JB6 Cl41 Cells

IGF-1 exerts both proliferative and anti-apoptotic effects via activating two distinct signaling pathways, mitogen activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K).^20–22) Therefore, we hypothesized that KIST101029 might inhibit these signaling pathway leading to anti-proliferative effects on JB6 Cl41 cells. To examine the effect of KIST101029 on phosphorylation of MAPKs induced by IGF-1, JB6 Cl41 cells were treated with IGF-1 in the absence or presence of various concentrations of KIST101029 and immunoblotted with anti-phospho antibodies against MEK1/2, ERK1/2, and p90RSK. The results showed that KIST101029 suppressed IGF-1-induced phosphorylation of MEK1/2, ERK1/2, and p90RSK in a dose dependent manner (Fig. 2A). Constitutively active ERK signaling pathway upregulates JNK and activates c-Jun oncogene and its downstream targets including RACK1 and cyclin D1.²³⁾ We next examined whether KIST101029 downregulates JNKs pathways induced by IGF-1. The results showed that KIST101029 dose dependently inhibited IGF-1-induced phosphorylation of JNK1/2 as well as c-Jun (Fig. 2B). We next investigated whether KIST101029 inhibits AKT/mTOR/p70S6K signaling. As shown in Fig. 3C, KIST101029 did not inhibit the phosphorylation of AKT induced by IGF-1 (Fig. 2C). However, KIST101029 inhibited IGF-1-induced phosphorylation of p70S6K via activation of Raptor (Fig. 2D). These overall data suggest that KIST101029 has inhibitory effects on the signaling pathway of MAPK as well as mTOR.

Fig. 2. Effects of KIST101029 on IGF-1-Induced Phosphorylation of MEK, ERK1/2, p90RSK, JNK and c-Jun in JB6 Cl41 Cells

(A–D) Cells were starved for 24 h, pretreated with KIST101029 at the indicated concentrations (0.05, 0.1, and 0.2 µM) for 24 h, then stimulated with 50 ng/mL IGF-1 for 15 min, and harvested. The levels of phosphorylated and total proteins related with MEK-ERK (A), JNK-c-Jun (B), and AKT-p70S6K (C and D) signaling cascades in whole cell lysates were determined by immunoblotting analysis using specific antibodies against the corresponding proteins, respectively.

KIST101029 Inhibits IGF-1-Induced c-fos, c-jun, and AP-1 Activation

c-Jun and c-Fos are nuclear proto-oncoproteins whose expression is stimulated by a variety of growth-promoting agents and activated oncogenes.²⁴⁾ AP-1 is mainly composed with Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2) and induced by several external stimuli, such as EGF, which increase MAPK activity.²⁴⁾ To investigate whether KIST101029 suppress the IGF-1-induced c-fos or c-jun transcriptional activity, we took advantage of the availability of the reporter plasmid carrying the luc gene under the control of the murine c-fos or c-jun promoter. Twenty-four hours after transfection with these reporters in JB6 Cl41 cells, cells were starved for another 12 h by incubating in serum-deprived MEM at 37°C in a 5% CO₂ atmosphere. At 12 h of starvation, cells were pretreated or not treated with KIST101029 for 12 h, and then treated or not treated with IGF-1 (50 ng/mL) for additional 12 h. The results showed that IGF-1 promoted c-fos or c-jun transcriptional activity was significantly suppressed by KIST101029 (Figs. 3A, 3B). To determine whether the antitumorigenetic effect of KIST101029 is responsible for the inhibition of AP-1 activation response to IGF-1, we next cotransfected the AP-1 luciferase reporter plasmid and pRL-TK-luciferase plasmid into cells. The IGF-1-induced AP-1 activity was significantly inhibited by KIST101029 similar to that observed for c-jun or c-fos activity (Fig. 3C). These data indicated that the suppression of the c-jun, c-fos, and AP-1 promoters by IGF-1 is one of the mechanisms explaining the anti-proliferative effect of KIST101029.

Fig. 3. Effects of KIST101029 on IGF-1-Induced c-fos, c-jun, and AP-1 Promoter Activity in JB6 Cl41 Cells

(A–C) Cells were transfected with a plasmid mixture containing c-fos-luc (A), c-jun-luc (B), or AP-1-luc promoter gene (C) with the pRL-TK vector. At 24 h after transfection, cells were serum-starved for 12 h, and then pretreated with the indicated concentration of KIST101029 for 12 h and then either exposed or not exposed with 50 ng/mL IGF-1 for 12 h. In all of the promoter assays, the firefly luciferase activity was determined in cell lysates and normalized against renilla luciferase activity, and these luciferase activities are expressed relative to control cells, respectively. Columns, mean of triplicate measurements of two experiments; bars, S.D. * p<0.05, compared with control cells.

KIST101029 Suppressed Cell Transformation of JB6 Cl41 Cells Induced by IGF-1

AP-1 is major transcription factor involved in neoplastic cell transformation of JB6 Cl41 cells induced by various tumor promoters.²⁵⁾ We next examined the effect of KIST101029 on IGF-1-induced cell transformation. JB6 Cl41 cells were treated separately with 50 ng/mL IGF-1 in the absence or presence of various concentrations of KIST101029 in a soft agar matrix and incubated at 37°C in a 5% CO₂ incubator for 14 d. Our results showed that KIST101029 significantly inhibited the IGF-1-induced cell transformation of JB6 cells in a dose-dependent manner (Figs. 4A, 4B). These data strongly indicated that KIST101029 plays an inhibitory role of neoplastic cell transformation in epidermal mouse skin cells stimulated with IGF-1.

Fig. 4. Inhibitory Effects of KIST101029 on IGF-1-Induced Neoplastic Cell Transformation in JB6 Mouse Epidermal Cells

(A and B) Cells were exposed to 50 ng/mL IGF-1 with/without treatment with KIST101029 as indicated in soft agar. The representative colonies from three separate experiments were photographed (A). The colonies were counted under a microscope with the aid of the Image-Pro Plus software program (B). Columns, mean of triplicate samples; bars, S.D. * p<0.05, compared with only IGF-1-treated cells.

KIST101029 Inhibited the IGF-1-Induced Tumor Progression of JB6 Cl41 Cells in Vivo

The in vivo CAM assay with JB6 Cl41 cells was subsequently performed in the absence or presence of IGF-1 with/without KIST101029. The representative images showed that IGF-1 induced the tumor formation in CAM as evident from tumor area. Interestingly, we observed that treatment of KIST101029 significantly inhibited the tumor formation induced by IGF-1 (Fig. 5A). For statistical evaluation, images of sections were digitally recorded and tumor areas were analyzed (Fig. 5B). Similar to ours in vitro soft agar assay, these results indicated that the KIST101029 treatment significantly inhibited IGF-1-induced tumor progression of JB6 Cl41 cells in CAM of chicken embryos compared with control group.

Fig. 5. In Vivo Effects of KIST101029 on the IGF-1-Induced Tumorigenesis

(A and B) Fertilized chicken eggs were implanted with JB6 Cl41 cells with/without IGF-1 and KIST101029 for 15 d. Representative pictures of the CAM (A) and measured tumor area (B) are shown. Error bars indicate the means±standard deviation of six samples per group from two independent experiments. * p<0.05, compared with control groups or only IGF-1-treated group, respectively.

KIST101029 Suppressed Tumorigenicity of A375 Human Melanoma Cells

Increased signaling through the RAF/MEK/ERK pathway, as a result of autocrine stimulation by basic fibroblast growth factor and hepatocyte growth factor, is implicated in melanocytic tumorigenesis. ²⁶⁾ To confirm that inhibition of the Raf-1/MEK/ERK signaling pathway by KIST101029 leads to the suppression of tumorigenesis, we first examined whether KIST101029 as well as sorafenib, a RAF inhibitor, could inhibit cell viability of A375 human melanoma cells. Results indicate that KIST101029 and sorafenib inhibited cell growth with an IC(50) of 0.96 µM and 5.12 µM in A375 cells, respectively (Fig. 6A). These findings further supported the evidence showing that KIST101029 and sorafenib inhibited the phosphorylation of ERK1/2 as well as MEK1/2 (Fig. 6B). Next, we further examined the inhibitory effect of KIST101029 and sorafenib on the colony formation of A375 cells using soft agar assay. Representative images demonstrate a profound reduction of the colony formation mediated by the treatment of KIST101029 and sorafenib compared with untreated control group (Fig. 6C). For statistical evaluation, the numbers of colonies were digitally recorded and counted. The results showed that KIST101029 and, to a lesser extent, sorafenib significantly inhibited colony formation of A375 cells (Fig. 6D).

Fig. 6. Effects of KIST101029 on Tumorigenicity of A375 Cells

(A) Cells were seeded and cultured for 96 h in 10% FBS/DMEM at 37°C in 5% CO₂ atmosphere. Then, the cells were treated with sorafenib and KIST101029 dose-dependently as indicated. Cell viability was measured by MTT assay, as described in Materials and Methods. Data are represented as the means±S.D. as determined from triplicate experiments. (B) Cells were starved for 24 h, pretreated with sorafenib and KIST101029 at the indicated concentrations for 24 h, and harvested. The levels of phosphorylated and total proteins related with MEK1/2 and ERK1/2 in whole cell lysates were determined by immunoblotting analysis using specific antibodies against the corresponding proteins, respectively. (C and D) A375 cells were treated or not treated with sorafenib and KIST101029, as indicated concentration, in soft agar. The representative colonies from three separate experiments were photographed (C). The colonies were counted under a microscope with the aid of the Image-Pro Plus software program (D). Columns, mean of triplicate samples; bars, S.D. * p<0.05, compared with only DMSO treated cells.

Discussion

There is a significant unmet medical need for the development of effective therapies that can stabilize or slow the progression of solid tumors. The bi-aryl urea sorafenib is an oral multikinase inhibitor that inhibits cell surface tyrosine kinase receptors, such as vascular endothelial growth factor receptors (VEGFR) and platelet-derived growth factor receptor (PDGF), and downstream intracellular serine/threonine kinases, such as Raf-1, wild-type B-Raf and mutant B-Raf, which are involved in tumor cell proliferation and tumor angiogenesis.¹⁵⁾ Currently, sorafenib is the only effective drug for advanced hepatocellular carcinoma (HCC).²⁷⁾ However, accumulating clinical evidence has suggested that this therapeutic agent rarely causes tumor shrinkage.²⁸⁾ The reason for the reduced effect of sorafenib in reducing the size of the tumors is unknown. Basic research studies have suggested that the blockade of Raf signaling might result in unexpected molecular events, such as the reciprocal activation of the Akt oncoprotein.²⁹⁾ In the present study, we identified KIST101029, a new diarylurea and diarylamide derivative, as novel RAF kinase inhibitor that targets tumor cell proliferation and tumorigenesis. KIST101029 exhibited a strong inhibitory effect on the signaling pathway of MAPK as well as mTOR induced by IGF-1. Also, KIST101029 blocked IGF-1-induced c-fos, c-jun, and AP-1 activation, and thereby effectively suppressed neoplastic transformation.

Ras activation is the first step in activation of the MAPK cascade. Following oncogenic Ras activation, Raf kinase family composed of three members, A-RAF, B-RAF, and Raf-1 (also termed c-Raf), is recruited to the cell membrane through binding to Ras to cause tumorigenic transformation.³⁰⁾ Raf proteins directly activate MEK1/2 via phosphorylation of multiple serine residues. MEK1 and MEK2 are themselves tyrosine and threonine/serine dual-specificity kinases that subsequently phosphorylate threonine and tyrosine residues in ERK1/2 resulting in activation.³¹⁾ A significant role for the MAPK in cancer biology has been well established. The Ras proteins were initially identified as the transforming component of oncogenic viruses for K-Ras and H-Ras, whereas N-Ras was identical as the transforming component of a neuroblastoma.³²⁾ Additional support for the importance of the MAPK pathway in oncogenesis comes form the prevalence of activating mutations among family members in multiple cancer types. Ras mutations are found in up to 30% of all cancers and are particularly common in pancreatic cancers and colon cancers.³³⁾ B-RAF mutations have a more narrow distribution, but are prevalent in a few specific malignancies including melanoma, papillary thyroid cancers, and low-grade ovarian cancers.^34–36) Based on the well-defined role for the Ras-Raf-MEK-ERK pathway in human cancers, therapeutic targeting has been an area of intense investigation.

AP-1 is dimeric transcription factors composed of Jun, Fos or activating transcription fator (ATF) subunits that bind to a common DNA site, the AP-1 binding site.³⁷⁾ AP-1 is regulated mainly by MAPKs.³⁷⁾ Previous studies have demonstrated that importance of the involvement of ERK1/2 in neoplastic cell transformation of JB6 P+ (promotion-sensitive) cells, whereas JB6 P− (promotion-resistance) cells do not respond to EGF as well as TPA due to low level of phosphorylated ERK.¹⁰⁾ Constitutively active mitogen-activated protein (MAP) kinases, including Raf, MEK and ERK, are sufficient to promote cell transformation through activated AP-1-regulated transcription.³⁸⁾ In this study, KIST101029 inhibited IGF-1-induced phosphorylation of MEK and ERK in JB6 Cl41 cells, and this inhibition of the MEK-ERK pathway led to the suppression of neoplastic transformation through the inhibition of AP-1. In addition, JNK involved AP-1 action and neoplastic transformation in JB6 P+ cells.³⁹⁾ Other studies have found that JNK2-deficinent mice failed to induce skin tumorigenesis in response to TPA, which also supports the important role of JNK in skin tumorigenesis.⁴⁰⁾ This prompted us to examine the effect of KIST101029 on IGF-1-induced JNK phosphorylation in JB6 Cl41 cells. KIST101029 inhibited IGF-1-induced JNK as well as c-Jun phosphorylation. These results indicated that the inhibition of ERK as well as JNK by KIST101029 might be responsible for KIST101029’s strong inhibition of neoplastic transformation in soft agar and CAM. Further, in vitro pull-down assays revealed that KIST101029 bound with Raf-1, which may be contribute to the observed reduced kinase activities of MAPKs pathway.

In summary, KIST101029 inhibited IGF-1-induced cell proliferation and neoplastic transformation of JB6 Cl41 cells via its binding with Raf-1. This inhibition was medicated mainly through the blocking of the MEK-ERK, JNK-c-Jun, and mTOR-p70S6K pathway, and subsequent suppression of AP-1 activity. KIST101029 binds with Raf-1. In addition, KIST101029 significantly inhibited the tumor formation induced by IGF-1 in CAM. Together these results suggested that Raf-1 is the most potent molecular target of KIST101029 for suppressing neoplastic transformation.

Acknowledgment

This work was supported by research fund from Chosun University (2010).

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