Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Anti-Cancer Agents) - Volume 13, Issue 4, 2013

Focal Adhesion Kinase (FAK) is overexpressed in many types of tumors and plays an important role in survival. We developed a novel approach, targeting FAK-protein interactions by computer modeling and screening of NCI small molecule drug database. In this report we targeted FAK and Mdm-2 protein interaction to decrease tumor growth. By macromolecular modeling we found a model of FAK and Mdm-2 interaction and performed screening of >200,000 small molecule compounds from NCI database with drug-like characteristics, targeting the FAK-Mdm-2 interaction. We identified 5'-O-Tritylthymidine, called M13 compound that significantly decreased viability in different cancer cells. M13 was docked into the pocket of FAK and Mdm-2 interaction and was directly bound to the FAK-N terminal domain by ForteBio Octet assay. In addition, M13 compound affected FAK and Mdm-2 levels and decreased complex of FAK and Mdm-2 proteins in breast and colon cancer cells. M13 re-activated p53 activity inhibited by FAK with Mdm-2 promoter. M13 decreased viability, clonogenicity, increased detachment and apoptosis in a dose-dependent manner in BT474 breast and in HCT116 colon cancer cells in vitro. M13 decreased FAK, activated p53 and caspase-8 in both cell lines. In addition, M13 decreased breast and colon tumor growth in vivo. M13 activated p53 and decreased FAK in tumor samples consistent with decreased tumor growth. The data demonstrate a novel approach for targeting FAK and Mdm-2 protein interaction, provide a model of FAK and Mdm-2 interaction, identify M13 compound targeting this interaction and decreasing tumor growth that is critical for future targeted therapeutics.

Focal Adhesion Kinase (FAK) is a non-receptor kinase that is overexpressed in many types of tumors and plays a key role in cell adhesion, spreading, motility, proliferation, invasion, angiogenesis, and survival. Recently, FAK has been proposed as a target for cancer therapy, and we performed computer modeling and screening of the National Cancer Institute (NCI) small molecule compounds database to target the ATP-binding site of FAK, K454. More than 140,000 small molecule compounds were docked into the crystal structure of the kinase domain of FAK in 100 different orientations using DOCK5.1 that identified small molecule compounds, targeting the K454 site, called A-compounds. To find the therapeutic efficacy of these compounds, we examined the effect of twenty small molecule compounds on cell viability by MTT assays in different cancer cell lines. One compound, A18 (1,4-bis(diethylamino)-5,8- dihydroxy anthraquinon) was a mitoxantrone derivative and significantly decreased viability in most of the cells comparable to the to the level of FAK kinase inhibitors TAE-226 (Novartis, Inc) and PF-573,228 (Pfizer). The A18 compound specifically blocked autophosphorylation of FAK like TAE-226 and PF-228. ForteBio Octet Binding assay demonstrated that mitoxantrone (1,4-dihydroxy- 5,8-bis[2-(2-hydroxyethylamino) ethylamino] anthracene-9,10-dione directly binds the FAK-kinase domain. In addition, mitoxantrone significantly decreased the viability of breast cancer cells in a dose-dependent manner and inhibited the kinase activity of FAK and Y56/577 FAK phosphorylation at 10-20 μM. Mitoxantrone did not affect phosphorylation of EGFR, but decreased Pyk-2, c-Src, and IGF-1R kinase activities. The data demonstrate that mitoxantrone decreases cancer viability, binds FAK-Kinase domain, inhibits its kinase activity, and also inhibits in vitro kinase activities of Pyk-2 and IGF-1R. Thus, this novel function of the mitoxantrone drug can be critical for future development of anti-cancer agents and FAK-targeted therapy research.

Focal adhesion kinase (FAK) is implicated in cancer cell survival, proliferation and migration. Expression of FAK expression is elevated and associated with tumor progression and metastasis in various tumors, including hepatocellular carcinoma (HCC). Increased 14-3-3ε expression is shown to be a potential prognostic factor to predict higher risk of distant metastasis and worse overall survival in HCC. The aim of this study is to investigate whether FAK is associated or regulated by 14-3-3ε to modulate tumor progression in HCC. In this study, 114 primary HCC tumors including 34 matched metastatic tumors were subjected to immunohistochemistry analysis of FAK and 14-3-3ε expression. Overexpression of FAK was significantly associated with increased risk of extrahepatic metastasis (p=0.027) and reduced 5-year overall survival rate (p=0.017). A significant correlation of FAK and 14-3-3ε expression was observed in primary tumor (p<0.001) and also metastatic tumors. Furthermore, overexpression of 14-3-3ε induced FAK expression and promoter activity which were determined by Western blotting analysis and luciferase-reporter assay. Moreover, 14-3-3ε enhanced NFκB activation and increased nuclear translocation of NFκB. Results from chromatin immunoprecipitation assay revealed that 14-3-3ε induced NFκB binding on FAK promoter region. These findings suggest that FAK expression is correlated with and upregulated by 14-3-3ε via activation of NFκB. Target to suppress or inactivate FAK alone, or combine with 14-3-3ε is thus considered as the potential therapeutic strategy for preventing HCC tumor progression.

Neuroblastoma is the most common extracranial solid tumor of childhood and is responsible for over 15% of pediatric cancer deaths. Neuroblastoma tumorigenesis and malignant transformation is driven by overexpression and dominance of cell survival pathways and a lack of normal cellular senescence or apoptosis. Therefore, manipulation of cell survival pathways may decrease the malignant potential of these tumors and provide avenues for the development of novel therapeutics. This review focuses on several facets of cell survival pathways including protein kinases (PI3K, AKT, ALK, and FAK), transcription factors (NF-κB, MYCN and p53), and growth factors (IGF, EGF, PDGF, and VEGF). Modulation of each of these factors decreases the growth or otherwise hinders the malignant potential of neuroblastoma, and many therapeutics targeting these pathways are already in the clinical trial phase of development. Continued research and discovery of effective modulators of these pathways will revolutionize the treatment of neuroblastoma.

This review is focused on the role of Focal Adhesion Kinase (FAK) signaling in cancer stem cells. The recent data demonstrate the important role of FAK in cancer stem cell proliferation, differentiation, motility, and invasion. We showed recently that the transcription factor Nanog binds the FAK promoter and up-regulates FAK expression, and that FAK binds Nanog and phosphorylates it. This review discusses the interaction of FAK, Nanog, Oct-3/4, and Sox-2 signaling pathways that are critical for the regulation of cancer stem cells. The cross-linked signaling of FAK with p53 and Nanog signaling in cancer stem cell and function and targeted therapeutics approaches are discussed.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase implicated in carcinogenesis through its pleitropic effects on cell proliferation, survival and metastasis. This article provides a summary of the existing data implicating FAK in lung cancer.

Focal adhesion kinase (FAK), hyaluronan (HA), and hyaluronan synthase-3 (HAS3) have been implicated in cancer growth and progression. FAK inhibition with the small molecule inhibitor Y15 decreases colon cancer cell growth in vitro and in vivo. HAS3 inhibition in colon cancer cells decreases FAK expression and activation, and exogenous HA increases FAK activation. We sought to determine the genes affected by HAS and FAK inhibition and hypothesized that dual inhibition would synergistically inhibit viability. Y15 (FAK inhibitor) and the HAS inhibitor 4-methylumbelliferone (4-MU) decreased viability in a dose dependent manner; viability was further inhibited by treatment with Y15 and 4-MU in colon cancer cells. HAS inhibited cells treated with 2μM of Y15 showed significantly decreased viability compared to HAS scrambled cells treated with the same dose (p<0.05) demonstrating synergistic inhibition of viability with dual FAK/HAS inhibition. Microarray analysis showed more than 2-fold up- or down-regulation of 121 genes by HAS inhibition, and 696 genes by FAK inhibition (p<0.05) and revealed 29 common genes affected by both signaling. Among the genes affected by FAK or HAS3 inhibition were genes, playing role in apoptosis, cell cycle regulation, adhesion, transcription, heatshock and WNT pathways. Thus, FAK or HAS inhibition decreases SW620 viability and affects several similar genes, which are involved in the regulation of tumor survival. Dual inhibition of FAK and HAS3 decreases viability to a greater degree than with either agent alone, and suggests that synergistic inhibition of colon cancer cell growth can result from affecting similar genetic pathways.

Pancreatic cancer is one of the most lethal diseases with no effective treatment. Previously, we have shown that FAK is overexpressed in pancreatic cancer and plays a key role in cancer cell survival and proliferation. FAK has been shown to interact with growth factor receptors including cMET and IGF-1R. As a novel therapeutic approach, we targeted the protein interaction of FAK with growth factor receptors to block tumor growth, alter signaling pathways and sensitize cells to chemotherapy. We have selected a small molecule compound (INT2-31) that decreases phosphorylation of AKT via disrupting interaction of FAK with cMET and IGF-1R. Our results demonstrate that interaction of a small molecule compound with FAK decreases phosphorylation of FAK Y397 while increasing FAK Y407 phosphorylation, without inhibiting the kinase activity of FAK and dramatically reduces downstream signaling to AKT. Our lead compound, INT2-31, demonstrates significant inhibition of tumor cell growth in two orthotopic models of pancreatic cancer. In addition, INT2-31increases sensitivity to gemcitabine chemotherapy in a direct fresh biopsy xenograft model of pancreatic cancer growth.

Marine ecosystems constitute a huge reservoir of biologically active secondary metabolites. Consequently during the last past few decades, several marine-derived molecules have been approved for anticancer treatment or are under clinical trials. This review reports the present state of the art of the sixteen molecules approved or currently on the clinical pipeline for anticancer chemotherapy. The molecules are classified according to their current status in the phase (approved / phase IV / phase III / phase II / phase I) and data are updated to April 2012.

A series of naphthalene diimide (ND)-based mono-, bis-, and tris-intercalators are synthesized and evaluated for their anticancer activities. All compounds show anticancer activities in the micromolar range. Among them the bis-intercalator is the most promising. Experimental results indicate that (i) target compounds intercalate DNA and (ii) the bis-intercalator with the optimal linker shows considerably more affinity to DNA than corresponding mono-and tris-intercalators. Spectroscopic measurements indicate that the ND groups bind to the double-stranded DNA (ds-DNA) in a classical threading intercalation mode, while the cationic linker reinforces the intercalation via electrostatic interaction with ds-DNA. In vitro cytotoxicity of the bis-intercalator towards a number of cancer cells, such as C6, HeLa, and MDA-435S, is tested and compared to that of normal cells. Attractive anticancer activity is observed with the bisintercalator, which provides a new lead in the anticancer drug design strategy.

The tetrapyrrolic macrocycle and the functional groups at its periphery allow for a variety of modifications aimed at multifunctional therapeutic compounds. In particular, conjugation of boron polyhedra yields dual efficacy antitumor photo/ radiosensitizers. Structural optimization of these agents presumes the identification of macromolecules that bind and transport boronated tetrapyrroles. Using spectroscopic methods we demonstrated that methylpheophorbide a forms complexes with serum albumin and low density lipoproteins (LDL) whereas two diboronated derivatives, 13(2),17(3)-[di(o-carboran-1-yl)methoxycarbonyl]pheophorbide a and 13(2),17(3)-[di(1-carba-closo-dodecaboran-1-yl)methoxycarbonyl]pheophorbide a, were capable of binding to LDL but not to albumin. Molecular modeling showed a mode of interaction of methylpheophorbide a with the amino acid residues in the albumin’s hemin binding site. In contrast, for diboronated derivatives such interactions are sterically hindered by boron polyhedra, in line with experimentally determined lack of complex formation with albumin. These data strongly suggest that LDL might be the preferred carrier for polycarborane containing methylpheophorbide a derivatives.

The initiation stage of liver cancer is closely related to abnormal cell proliferation as observed for other types of carcinogenesis. Recently, we isolated a glycoprotein from Styrax japonica Siebold et al Zuccarini (SJSZ glycoprotein), which consists of a carbohydrate moiety (52.64%) and a protein moiety (47.36%). In this study, the antitumoric mechanism of SJSZ glycoprotein during the initiation stage in N-Methyl-N`-nitro-N-nitrosoguanidine (MNNG; 40 mg/kg, BW)-induced ICR was investigated. First, we evaluated the activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), thiobarbituric acid-reactive substances (TBARS), and activities of antioxidative enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)] in mouse liver tissue and serum. The alpha-fetoprotein (AFP), cell cycle-related factors [cyclin D1/ cyclin dependent kinase (CDK) 4], cell cycle inhibitors (CKIs; p53, p21, and p27), and proliferating cell nuclear antigen (PCNA) were then assessed using Western Blot analysis. The results of this analysis showed that the SJSZ glycoprotein (10 mg/kg, BW) decreased the levels of LDH, ALT, TBARS, and the expression of AFP but it increased the activity of hepatic anti-oxidant enzymes (SOD, GPx and CAT). In addition, the SJSZ glycoprotein (10 mg/kg, BW)was shown to decrease the expression of cyclin D1/CDK4 and PCNA and increase the expression of CKIs (p53, p21, and p27). The results in this study indicate that the SJSZ glycoprotein displays anti-oxidative stress and anti-cell proliferation activity in MNNGinduced ICR.

Indole-3-carbinol (I3C) and its oligomeric derivatives have been widely studied for their chemopreventive and chemotherapeutic properties. We have previously shown that the I3C cyclic tetrameric derivative CTet inhibits breast cancer cell proliferation in vitro and in xenotrasplanted tumor. Here we report the antitumoral activity of a mixture of tri- and tetrameric cyclic I3C derivatives (CTr/CTet) both in vitro (MCF-7 and MDA-MB-231 breast cancer cell lines) and in vivo in a tumor xenograft model. CTr/CTet mixture avoids the low solubility drawbacks of CTet, thus favouring its solubilization, and reducing purification process, time and costs. CTr/CTet mixture has been shown to inhibit breast cancer cell proliferation (IC50 = 1.3 and 1.6 μg/ml in MCF-7 and MDAMB- 231, respectively) inducing the G0/1 cell cycle phase accumulation. The main molecular events related to CTr/CTet activity are the overexpression of p21, p27 and GADD45A, nuclear translocation of FOXO3a, inhibition of Akt activity and downregulation of estrogen receptor. In vivo, the growth of xenotransplanted tumor has been inhibited and the pro-tumoral low molecular weight cyclin E downregulation has been detected. Our data indicate that CTr/CTet is a potential anticancer combination agent for both hormoneresponsive and triple-negative breast tumors.

Previous studies have shown that recombinant snake venom cystatin (sv-cystatin) inhibits the invasion and metastasis of tumor cells in vitro and in vivo. The purpose of this study was to investigate the ability of recombinant sv-cystatin to inhibit tumor angiogenesis in vitro and in vivo, and the mechanisms underlying this effect. Recombinant sv-cystatin inhibited proliferation of human umbilical vein endothelial cells (HUVECs) at 100 and 200 μg/mL after 72, 96 and 120 h. Recombinant sv-cystatin also inhibited tumor–endothelial cell adhesion at 25, 50, 100 and 200 μg/mL. Recombinant sv-cystatin inhibited capillary-like tube formation by HUVECs at 10, 25, 50, 100 and 200 μg/mL following 12, 24 and 36 h incubation. Furthermore, recombinant sv-cystatin significantly suppressed microvessel density (MVD) of lung tumor colonies in C57BL/6 mice inoculated in the lateral tail vein with B16F10 melanoma cells. Administration of recombinant sv-cystatin significantly decreased MVD of primary tumor tissues in nude mice implanted subcutaneously with human hepatocellular carcinoma cells (MHCC97H). Exposure of B16F10 and MHCC97H cells to increasing doses of recombinant sv-cystatin suppressed secretion of vascular endothelial growth factor (VEGF)-A165 and basic fibroblast growth factor (bFGF) into the surrounding medium (P<0.05). The expression of fms-related tyrosine kinase 1 (Flt-1) protein in HUVECs was decreased by 25, 50, 100 and 200 μg/mL recombinant sv-cystatin (P<0.05). This study demonstrates that recombinant sv-cystatin inhibits tumor angiogenesis associated with downregulation of VEGF-A165, Flt-1 and bFGF. This suggests that recombinant sv-cystatin may have potential pharmaceutical applications as an antiangiogenic and antimetastatic therapeutic agent.

Cytokinins are phytohormones critically involved in the regulation of plant growth and development. They also affect the proliferation and differentiation of animal cells, thus representing new tools to treat diseases that involve dysfunctional cell growth and/or differentiation. Recently, by performing structure-function studies on human cells, we found that only N6-isopentenyladenosine and its benzyl analogue N6-benzyladenosine suppress the clonogenic activity and the growth of different neoplastic cells. We here broaden our studies on bladder carcinoma T24 cells, because, due to the high recurrence rate of bladder cancer, new active molecules are sought to contrast the growth of this tumor. Early events induced by N6-isopentenyladenosine and N6-benzyladenosine are the alteration of T24 cell morphology and the disorganization of the actin cytoskeleton. After 24 h N6-isopentenyladenosine and N6-benzyladenosine inhibit growth by arresting the cells in the G0/G1 phase of the cell cycle. We also show that the two compounds induce apoptosis, an event linked to the activation of caspase 3. Since DNA damage is a prime factor resulting in cell cycle arrest and apoptosis, it is noteworthy that we do not detect any genotoxic effect upon treatment of T24 cells with N6-isopentenyladenosine and N6- benzyladenosine. Because the disruption of actin filaments leads to G1 arrest and is also implicated in apoptosis, we hypothesize that cytoskeletal rearrangement might be responsible for triggering the antiproliferative and proapotpotic effects of N6-isopentenyladenosine and N6- benzyladenosine in T24 cells.