Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Anti-Cancer Agents) - Volume 14, Issue 1, 2014

Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter activity. Treatment of MCF-7 cells with 1alpha, 25-dihydroxyvitamin D3 (1,25D) down-regulated endogenous FAK and up-regulated TXNIP protein level, and treatment with 5-FU decreased FAK protein expression and up-regulated TXNIP protein expression in 293 cells. Moreover, silencing of FAK with siRNA increased TXNIP protein expression, while overexpression of FAK inhibited TXNIP protein expression in 293 cells. In addition, treatment of DBTRG glioblastoma cells with FAK inhibitor Y15 increased TXNIP mRNA, decreased cancer cell viability and increased apoptosis. These results for the first time demonstrate FAK-regulated TXNIP expression which is important for apoptotic, survival and oxidative stress signaling pathways in cancer cells.

Focal adhesion is known to be highly expressed and activated in glioma cells. Recently, we demonstrated that FAK autophosphorylation inhibitor, Y15 significantly decreased tumor growth of DBTRG and U87 cells, especially in combination with temozolomide. In the present report, we performed gene expression analysis in these cells to reveal genes affected by Y15, temozolomide and combination of Y15 and temozolomide. We tested the effect of Y15 on gene expression by Illumina Human HT12v4 microarray assay and detected 8087 and 6555 genes, which were significantly either up- or down-regulated by Y15-treatment in DBTRG and U87 cells, respectively (p<0.05). Moreover, DBTRG and U87 cells treated with Y15 changed expression of 1332 and 462 genes more than 1.5 fold, p<0.05, respectively and had 237 common genes affected by Y15. The common genes up-regulated by Y15 included GADD45A, HSPA6 (heat-shock 70); DUSP1, DUSP 5 (dual-phosphatase 5); CDKN1A (p21) and common down-regulated genes included kinesins, such as KIF11, 14, 20A, 20B; topoisomerase II, TOP2A; cyclin F; cell cycle protein: BUB1; PARP1, POLA1. In addition, we detected genes affected by temozolomide and by combination of Y15 and temozolomide treatment in U87 cells. Among genes up-regulated by Y15 and temozolomide more significantly than by each agent alone were: COX7B; interferon, gamma-inducible transcript: IFI16; DDIT4; GADD45G and down-regulated: KIF3A, AKT1; ABL; JAK1, GLI3 and ALDH1A3. Thus, microarray gene expression analysis can be effective in establishing genes affected in response to FAK inhibitor alone and in response to combination of Y15 with temozolomide that is important for glioblastoma therapy.

Focal Adhesion Kinase is a 125 kDa non-receptor kinase and overexpressed in many types of tumors. Recently, short noncoding RNAs, called microRNAs have been discovered as regulators of gene expression mainly through binding to the untranslated region (UTR) of mRNA. In this report we show that MiR-138 and MiR-135 down-regulated FAK expression in cancer cells. MiR-138 and MiR-135 inhibited FAK protein expression in different cancer cell lines. The computer analysis of 3’FAK-untranslated region (FAKUTR) identified one conserved MiR-138 binding site (CACCAGCA) at positions 3514-3521 and one conserved MiR-135 (AAGCCAU) binding site at positions 4278-4284 in the FAK-UTR. By a dual-luciferase assay we demonstrate that MiR-138 and MiR-135 directly bound the FAK untranslated region using FAK-UTR-Target (FAK-UTR) luciferase plasmid and inhibited its luciferase activity. The sitedirected mutagenesis of the MiR-138 and MiR-135 binding sites in the FAK-UTR abrogated MiR-138 and MiR-135-directed inhibition of FAK-UTR. Real-time PCR demonstrated that cells transfected with MiR-138 and MiR-135 expressed decreased FAK mRNA levels. Moreover, stable expression of MiR-138 and MiR-135 in 293 and HeLa cells decreased cell invasion and increased sensitivity to 5- fluorouracil (5-FU), FAK inhibitor, Y15, and doxorubicin. In addition, MiR-138 significantly decreased 293 xenograft tumor growth in vivo. This is the first report on regulation of FAK expression by MiR-135 and MiR138 that affected invasion, drug sensitivity, and tumor growth in cancer cells, which is important to the development of FAK-targeted therapeutics and understanding their novel regulations and functions.

Cordycepin, also known as 3-deoxyadenosine, is an analogue of adenosine extracted from the traditional Chinese medicine “Dong Chong Xia Cao”. Cordycepin is an active small molecular weight compound and is implicated in modulating multiple physiological functions including immune activation, anti-aging and anti-tumor effects. Several studies have indicated that cordycepin suppresses tumor progression. However, the signaling pathways involved in cordycepin regulating cancer cell motility, invasiveness and epithelial-mesenchymal transition (EMT) remain unclear. In this study, we found that cordycepin inhibits hepatocellular carcinoma (HCC) cell proliferation and migration/invasion. Treatment of cordycepin results in the increasing expression of epithelial marker, Ecadherin while no significant effect was found on N-cadherin α-catenin and β-catenin. Furthermore, although the expression of focal adhesion kinase (FAK) was slightly reduced, the level of phosphorylated FAK was significantly reduced by the treatment of cordycepin. In addition, cordycepin significantly suppresses the expression of integrin α3, integrin α6 and integrin β1 which are crucial interacting partners of FAK in regulating the focal adhesion complex. These results suggest cordycepin may contribute to EMT, antimigration/ invasion and growth inhibitory effects of HCC by suppressing E-cadherin and integrin/FAK signaling. Thus, cordycepin is a potential therapeutic or supplementary agent for preventing HCC tumor progression.

S100 inflammatory proteins have been previously shown to modulate breast cancer processes. More specifically, genome-wide transcriptome studies associate S100A8 and S100A9 members to breast cancer progression and malignancy. Findings have shown that S100A8 and S100A9 can signal and regulate cancer cell behavior through both extracellular and intracellular-initiated cascades. However, functional studies exploring the effects of S100 proteins are often contradictory leaving ambiguity and a paucity of data relating to the specific function of S100A8 and S100A9 in breast cancer progression. In this study we sought to better define the functions of intracellular expressed S100A8 and S100A9 on key signaling and cellular processes driving breast cancer malignancy. We observed that extracellular treatments of the MCF7 breast cancer cell line with S100A8 and S100A9 proteins induces cell proliferation. In contrast, intracellular recombinant expression of S100A8 and S100A9 led to growth suppression. Furthermore our analysis revealed that intracellular-expressed S100A8 and S100A9 promote an epithelial-like phenotype through the induction of key markers, such as Ecadherin, integrin alpha-5 and Zona Occludens 1 (ZO-1). Concomitantly, S100A8 and S100A9 negatively regulate the activity of the promalignant Focal Adhesion Kinase-1 (FAK) signaling cascade leading to changes in cell adhesion and invasion properties. Our results uncover important differences in intracellular versus extracellular initiated S100A8 and S100A9 signaling cascades and their effects on mammary epithelial growth. Importantly, S100A8 and S100A9 appear to suppress breast cancer malignancy through an increase in mesenchymal to epithelial transitioning. Our findings shed insight into S100 protein involvement in breast cancer invasiveness and metastasis and clarify some of the controversies relating to these proteins in breast cancer processes.

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 the individual protein tyrosine kinase, focal adhesion kinase (FAK) and its interaction with the transcription factors, MYCN, p53, and Mdm2, and how their interactions modulate the growth and malignancy of neuroblastomas.

Estrogens are known to be important in breast cancer growth in both pre- and post-menopausal women. Although circulating estrogen concentrations are very low after menopause, peripheral tissues generate sufficient concentrations to stimulate tumor growth. As aromatase is the rate-limiting enzyme in estrogen biosynthesis, inhibitors of this enzyme represent effective targeted therapy for breast cancer. Three compounds are now FDA approved and have become the first-choice endocrine drugs for postmenopausal breast cancer patients, since they are associated with superior activity and better general tolerability when compared with the estrogen receptor modulator tamoxifen. Nevertheless, some questions concerning the use of aromatase inhibitors for the treatment of breast cancer still need to be addressed, mainly related to their side-effects and the development of resistance, making research in this field still appealing. Many research groups, including our own, are still dealing with the search of new compounds that possess aromatase inhibitory properties. In this review an update of the latest achievements in the field of nonsteroidal aromatase inhibitors will be given.

Most prostate and breast cancers are hormone dependent. The inhibition of steroid 17α-hydroxylase/17,20- lyase (CYP17), which is a crucial enzyme for steroid hormone biosynthesis, is widely used to treat androgen-dependent prostate cancer (PC). CYP17 has dual enzymatic activity: 17alpha-hydroxylase activity (utilizing delta4- C21 steroids as substrates) and the 17,20-lyase activity (using delta5- C21 steroids as substrates). The steroid biosynthetic pathway is directed to either the production of corticosteroids or sex hormones depending on the activity of CYP17. In this review, the current information on the genetics, molecular structure, substrate specificity and inhibitors of CYP17 is analyzed and discussed.

Skin is the largest body organ forming a metabolically active barrier between external and internal environments. The metabolic barrier is composed of cytochromes P450 (CYPs) that regulate its homeostasis through activation or inactivation of biologically relevant molecules. In this review we focus our attention on local steroidogenic and secosteroidogenic systems in relation to skin cancer, e.g., prevention, attenuation of tumor progression and therapy. The local steroidogenic system is composed of locally expressed CYPs involved in local production of androgens, estrogens, gluco- and mineralo-corticosteroids from cholesterol (initiated by CYP11A1) or from steroid precursors delivered to the skin, and of their metabolism and/or inactivation. Cutaneous 7-hydroxylases (CYP7A1, CYP7B1 and CYP39) potentially can produce 7-hydroxy/oxy-steroids/sterols with modifying effects on local tumorigenesis. CYP11A1 also transforms 7-dehydrocholesterol (7DHC)→22(OH)7DHC→20,22(OH)2-7DHC→7-dehydropregnenolone, which can be further metabolized to other 5,7- steroidal dienes. These 5,7-dienal intermediates are converted by ultraviolet radiation B (UVB) into secosteroids which show pro-differentiation and anti-cancer properties. Finally, the skin is the site of activation of vitamin D3 through two alternative pathways. The classical one involves sequential hydroxylation at positions 25 and 1 to produce active 1,25(OH)2D3, which is further inactivated through hydroxylation at C24. The novel pathway is initiated by CYP11A1 with predominant production of 20(OH)D3 which is further metabolized to biologically active but non-calcemic D3-hydroxyderivatives. Classical and non-classical (novel) vitamin D analogs show pro-differentiation, anti-proliferative and anticancer properties. In addition, melatonin is metabolized by local CYPs. In conclusion cutaneously expressed CYPs have significant effects on skin physiology and pathology trough regulation of its chemical milieu.

Increasing evidence has accumulated to suggest that vitamin D may reduce the risk of cancer through its biologically active metabolite, 1α,25(OH)2D3, which inhibits proliferation and angiogenesis, induces differentiation and apoptosis, and regulates many other cellular functions. Thus, it is plausible to assume that rapid clearance of 1α,25(OH)2D3 by highly expressed CYP24A1 could interrupt the normal physiology of cells and might be one cause of cancer initiation and progression. In fact, enhancement of CYP24A1 expression has been reported in literature for many cancers. Based on these findings, CYP24A1-specific inhibitors and vitamin D analogs which are resistant to CYP24A1-dependent catabolism might be useful for cancer treatment. CYP24A1-specific inhibitor VID400, which is an azole compound, markedly enhanced and prolonged the antiproliferative activity of 1α,25(OH)2D3 in the human keratinocytes. Likewise, CYP24A1-resistant analogs such as 2α-(3-hydroxypropoxy)-1α,25(OH)2D3 (O2C3) and its C2-epimer ED-71 (Eldecalcitol), and 19nor- 2α-(3-hydroxypropyl)-1α,25(OH)2D3 (MART-10) showed potent biological effects. Our in vivo studies using rats revealed that MART-10 had a low calcemic effect, which is a suitable property as an anticancer drug. Much lower affinity of MART-10 for vitamin D binding protein (DBP) as compared with 1α,25(OH)2D3 may be related to its more potent cellular activities. Based on these results, we conclude that (1) high affinity for VDR, (2) resistance to CYP24A1-dependent catabolism, (3) low affinity for DBP, and (4) low calcemic effect may be required for designing potent vitamin D analogs for cancer treatment.

The expression of CYP27B1 or vitamin D 1α-hydroxylase (1α-OHase) and CYP24A1 in specific tissues may act as the central part between 25-hydroxyvitamin D [25(OH)D] serum levels and the anticancer effects of1α,25-dihydroxyvitamin D [1α,25(OH)2D3],alternative splicing of these enzymes may affect their biological functions. Here, we describe the expression of CYP24A1 and its splicing variants detected in breast cells and tissues. Manifestation of CYP24A1 mRNA was measured by RT-PCR followed by western blot analysis for protein expression. In MCF-7 cells, the expression of CYP24A1 protein was reduced by about 57% compared to MCF-10F cells. Western blot analysis revealed a signal band at 56 kDa, with additional bands detected at 42 and 44 kDa. The expression of CYP24A1 mRNA was reduced by about 58% in breast cancer tissues. We found only one signal in the benign tissues at 56 kDa in western blot, whereas in malignant tissue, an additional band was detected at 40kDa. Our results suggest that alternative splicing of CYP24A1 may lead to a catalytically dysfunctional enzyme.

Cancer cell heterogeneity is a common feature - both between patients diagnosed with the same cancer and within an individual patient’s tumor - and leads to widely different response rates to cancer therapies and the potential for the emergence of drug resistance. Diverse therapeutic approaches have been developed to combat the complexity of cancer, including individual treatment modalities designed to target tumor heterogeneity. This review discusses adenoviral vectors and how they can be modified to replicate in a cancer-specific manner and deliver therapeutic genes under multi-tiered regulation to target tumor heterogeneity, including heterogeneity associated with cancer stem cell-like subpopulations. Strategies that allow for combination of prodrug-activation gene therapy with a novel replication-conditional, heterogeneous tumor-targeting adenovirus are discussed, as are the benefits of using adenoviral vectors as tumor-targeting oncolytic vectors. While the anticancer activity of many adenoviral vectors has been well established in preclinical studies, only limited successes have been achieved in the clinic, indicating a need for further improvements in activity, specificity, tumor cell delivery and avoidance of immunogenicity.

Heterocyclic compounds, analogs and derivatives have attracted attention due to their diverse biological and pharmacological properties. Benzoheterocycles such as benzothiazoles, benzimidazoles and benzoxazoles are constituents of many bioactive heterocyclic compounds, having wider range of applications. They have been extensively studied for their biological activities, and can serve as unique and versatile scaffolds for drug design. The benzothiazole, in the family of heterocyclic compounds has assumed special significance in synthetic chemistry, pharmaceutical chemistry as well as in clinical applications because of its anti-tumor properties. This review is organized in the following ways. It begins with brief introduction on the chemical diversity of synthetic analogs of benzothiazole. After this, drug design strategy and mechanisms of action through its diverse biological targets in which benzothiazole and its derivatives display their anticancer activity are discussed. It ends with the metabolism pattern of benzothiazole and its analogs. Analysis of the structure-activity relationships (SAR), quantitative structure-activity relationships (QSAR) as well as on docking studies of this family of compounds highlights the potential that may lead to the development of novel anticancer agents. Such relationships will definitely create lot of interest among the researchers to synthesize optimized variety of benzothiazole derivatives and to screen them for their anticancer activity.

In the search for new metal-based anticancer agents as effective candidates for cisplatin, a lot of strategies such as synthesis of cisplatin analogs, trans-platinum compounds and non-platinum metal complexes have been put forward in the last forty years. The concept of multinuclearity for improving the chemotherapeutic activity has been proven in multinuclear platinum complexes such as BBR3464, recently, the effective approach has been successfully transferred to ruthenium complexes. In this review, we highlighted the recent progress in multinuclear platinum complexes and ruthenium complexes as anticancer agents, and their novel DNA binding properties such as phosphate clamps, long range DNA cross links, bisintercalation, interduplex cross links and DNA-protein cross-links were summarized to shed light on the rational design of polynuclear complexes as anticancer agents.

Plumbagin [5-hydroxy- 2-methyl-1, 4-naphthaquinone] is a well-known plant derived anticancer lead compound. Several efforts have been made to synthesize its analogs and derivatives in order to increase its anticancer potential. In the present study, plumbagin and its five derivatives have been evaluated for their antiproliferative potential in one normal and four human cancer cell lines. Treatment with derivatives resulted in dose- and time-dependent inhibition of growth of various cancer cell lines. Prescreening of compounds led us to focus our further investigations on acetyl plumbagin, which showed remarkably low toxicity towards normal BJ cells and HepG2 cells. The mechanisms of apoptosis induction were determined by APOPercentage staining, caspase-3/7 activation, reactive oxygen species production and cell cycle analysis. The modulation of apoptotic genes (p53, Mdm2, NF-kB, Bad, Bax, Bcl-2 and Casp-7) was also measured using real time PCR. The positive staining using APOPercentage dye, increased caspase-3/7 activity, increased ROS production and enhanced mRNA expression of proapoptotic genes suggested that acetyl plumbagin exhibits anticancer effects on MCF-7 cells through its apoptosis-inducing property. A key highlighting point of the study is low toxicity of acetyl plumbagin towards normal BJ cells and negligible hepatotoxicity (data based on HepG2 cell line). Overall results showed that acetyl plumbagin with reduced toxicity might have the potential to be a new lead molecule for testing against estrogen positive breast cancer.