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Ferrocene compounds have attracted significant interest as biologically active derivatives. Introduction of the ferrocene moiety in drug-like molecules improves the physicochemical properties and stability of drug candidates, while the transition metals coordinated to biologically active ligands participate in metal-specific processes which enhance drug delivery and biological activity. As part of our research of ferrocene derivatives and their transition metal complexes, we have prepared dipicolylamine, iminodiacetamide and bis-triazole derivatives connected to ferrocene through an ethylenediamine linker. The conjugates were used as ligands in the synthesis of Zn(II), Ni(II) and Cu(II) metal complexes. The stoichiometry and stereochemistry of the obtained complexes were examined by NMR and UV-Vis spectroscopy. We have obtained two crystal structures of metal complexes with bis-triazole ligands and one with an iminodiacetamide ligand. Investigation of antitumor activity is in progress.

Most epithelial ovarian cancer patients are diagnosed with advanced-stage disease due to the late appearance of symptoms and lack of early diagnostic methods/markers. The major problem for successful therapy is the development of tumour drug resistance during carcinogenesis (20-30%) and upon exposure to chemotherapy. The ovarian cancer cell line OVCAR-3/CBP was established by treatment of the ovarian adenocarcinoma cell line OVCAR-3 with long-term, stepwise selection in carboplatin (CBP) up to 25 μM. The variant is ~1.5-2-fold resistant to CBP, with cross-resistance to paclitaxel (TAX, ~4-fold), and presents with a mesenchymal-like phenotype. The increased expression of NHE-1, ATP7-B and decreased expression of ABCC2 and CTR-1 implied decreased total cell platination as a possible mode of CBP resistance, which was confirmed by flame atomic absorption spectrometry. Despite the increased level of ABCB1 transcripts, OVCAR-3/CBP did not efflux [3H]-docetaxel differently compared to parental cells, and the P-glycoprotein inhibitor PSC-833 did not alter these drug accumulation profiles. This indicates that the TAX resistance in OVCAR-3/CBP is non-MDR, but is associated with elevated TUBB3 (class III beta-tubulin) content along with total α- and β-tubulin relative to parental OVCAR-3 cells. In summary, drug selection with carboplatin in an ovarian cancer cell line resulted in non-MDR cross-resistance to paclitaxel. Experiments investigating the functional significance of altered tubulin content and microtubule dynamicity in response to drug exposure in OVCAR-3/CBP are on-going.

Amino acid bioconjugates of triphenlyphosphanes are successfully used as monodentate ligands in rhodium catalyzed asymmetric hydrogenations. A major feature of those metal complexes is the formation of chiral secondary structure responsible for chiral induction in the catalytic cycle. Apart from their excellent catalytic properties, organometallic ruthenium complexes with nitrogen or phosphane ligands showed promising results in cytotoxicity studies. In addition, the catalytic activity of ruthenium complexes has recently been used as a new approach to anticancer drug design. Herein, we explore the synthesis and characterization of (p-cymene)ruthenium complexes 1 and 2, bearing phosphane ligands with chiral or non-chiral amino acid esters. The spacer between the phosphorus and the amino acid was varied in order to investigate hydrogen bonding propensity of the amino acid substituents. Catalytic and biological properties will be reported.

Introduction: Integrins are cell-surface adhesion molecules that connect cells to components of the extracellular matrix. They play key roles in the regulation of tumor cell adhesion, migration, invasion and survival to antitumor drugs. In the present study we investigate the susceptibility of tongue squamous carcinoma cells Cal27 with de novo expression of integrin αvβ3 to antitumor drugs. Materials and methods: Mock transfected cell clone Cal27-Φ or cell clones Cal27-2B1 and Cal27-2B3 were obtained by stable transfection of Cal27 cells with empty plasmid pcDNA3 or a plasmid containing integrin β3 subunit gene, respectively. The sensitivity of cells to antitumor drugs was determined using MTT assay. Flow cytometry analysis was used to determine the expression of integrins αvβ3 and αvβ5 on the cell surface. Knockdown of integrin subunits αv and β5, Src and ILK was performed by transfection of specific siRNAs. The activity of Src kinase (pSrc(Y418)) was inhibited using dasatinib. The expressions of Src/pSrc(Y418) and ILK were measured by Western blot. Results and discussion: Cell clones Cal27-2B1 and Cal27-2B3 as compared to parental cell line Cal27 and control clone Cal27-Φ demonstrate: (i) de novo expression of integrin αvβ3, (ii) increased expression of integrin αvβ5, (iii) increased adhesion properties to fibronectin and vitronectin ; (iv) resistance to cisplatin, doxorubicin, mitomycin C and 5-fluorouracil ; (v) increased amount of ILK and (vi) decreased amount of Src and pSrc (Y418). ILK knockdown in Cal27-2B1 and Cal27-2B3 had different effects on the cell survival upon treatment with the aforementioned antitumor drugs ruling out its involvement in the resistance to these agents. The Src knockdown in Cal27 cells, as well as concomitant treatment with dasatinib, increased resistance to the four antitumor drugs to the level observed in cell clones Cal27-2B1 and Cal27-2B3. Conclusion: Since dasatinib treatment in Cal27 mimics the effect of a decreased amount of pSrc (Y418) observed in integrin αvβ3-expressing cell clones Cal27-2B1 and Cal27-2B3, we conclude that integrin αvβ3-induced downregulation of pSrc (Y418) is responsible for drug resistance. Given the strong attention that dasatinib receives as a potential drug for cancer therapy in combination with conventional chemotherapy, it is intriguing that decreased Src (Y418) activity confers a resistance to various antitumor drugs.

Modern synthetic chemistry is powered by new synthetic protocols that allow the rapid generation of compound scaffolds. Copper-catalysed azide–alkyne cycloaddition reaction, also referred to as the ‘Click’ reaction, producing 1, 4-disubstituted-1, 2, 3-triazoles (‘Click’ triazoles) is one of that kind. 1-(2-Picolyl)-, 4-(2-picolyl)-, 1-(2-pyridyl)-, and 4-(2-pyridyl)-3-methyl-1, 2, 3-triazolium salts have been synthesized and tested for their anticancer activity on human cervical carcinoma HeLa cells. In general, the analogues that are functionalized at the triazole ring with strongly electron donating substituent were more potent in comparison to those with the electron neutral or electron accepting groups. The most active compound was 4-(4-methoxyphenyl)-3-methyl-1-(2-picolyl)-1H-1, 2, 3-triazolium hexafluorophosphate(V) (AB144), with very high cytotoxicity. Its anticancer activity was cell type dependent, being most cytotoxic against large-cell lung carcinoma H460 cells. It is of outmost importance that AB144 was significantly more cytotoxic against tumor cells than to normal cells, with very low therapeutic index (0.0658) for H460 cells. Additionally, this compound was similarly cytotoxic against parent laryngeal carcinoma HEp-2 cells and their carboplatin and cisplatin resistant 7T subline what is, having in mind the importance of inhibitory effect of drug resistance on the success of cancer treatment, of great importance. On the basis of the structural features of triazolium salts it was anticipated that DNA would be the primary target. The fact that no influence of AB144 on thermal stability of calf thymus DNA could be observed, however, discarded this possibility. Compound AB144 arrests tumor cells in the G1 phase of the cell cycle, and induces apoptosis. While our data suggest that the triazolium salt AB144 is an anticancer molecule which preferentially target malignant cells, it holds a promise as simple to make, low molecular mass, non-metalic potential anti-cancer agents.

Several miRNA-200 family members (miR-200a, 200b, 200c, 429, and 141) have been associated with the regulation of epithelial-mesenchymal transition (EMT). EMT is involved in cancer cell invasion and metastasis, and has been implicated in cellular resistance to taxane drugs. We have generated a series of taxane resistant models of ovarian cancer cells that demonstrate a strong EMT phenotype associated with the downregulation of miRNA-200 family members. OVCAR-3/TP variants are five-fold resistant to paclitaxel compared to parental OVCAR-3 cells, and manifest marked decreases in miR-200c and 141 content, modest decreases in miR-200a, 200b, and 429, upregulation of the mesenchymal markers vimentin and fibronectin, and downregulation of E-cadherin. In order to examine the role of miRNA-200 family members in paclitaxel resistance, OVCAR-3/TP cells were infected with retroviral particles carrying either an empty mCherry-retroviral construct, the miR-200ab429 (OVCAR-3/TPab429) cluster or the miR-200c141 (OVCAR-3/TPc141) cluster. The introduction of miRNA-200 family members to mesenchymal OVCAR-3/TP cells resulted in downregulation of the EMT transcriptional regulators, ZEB1 and ZEB2, and partial reversion of vimentin, fibronectin and E-cadherin to the levels in epithelial OVCAR-3 parental cells. Despite the increased miR-200ab429 and miR-200c141 expression and the reversal of the EMT phenotype in the OVCAR-3/TP cells, paclitaxel resistance was not altered. In contrast, the miRNA-200 expressing cell lines are two to four-fold resistant to carboplatin under identical experimental conditions. This finding is observed more in OVCAR-3/TPc141 compared to OVCAR-3/TPab429, which implies a different role of these two miRNA clusters in determining cellular responses to carboplatin. These data were confirmed by transfecting OVCAR-3/TP cells with 50 and 100 nM mirVana miRNA mimics (Life Technologies, Grand Island, NY) specific for miR-200c and 141, resulting in no change in paclitaxel sensitivity but three-fold resistance to carboplatin. Similar data were obtained for two additional ovarian cancer cell lines (MES-OV and Hey). We conclude that increased expression of miR-200c and 141 and their mimics in paclitaxel resistant OVCAR-3/TP cells does not affect cellular sensitivity to paclitaxel, but it does result in resistance to carboplatin.

Background: Ovarian cancer is strongly associated with a pro-inflammatory leukocyte infiltrate, and very high levels of chemokines are found in ascites, including CCL2. CNTO 888, a neutralizing anti-CCL2 antibody, could inhibit the pro-tumor inflammatory infiltrate and tumor growth, thus providing clinical benefit to ovarian cancer patients. It was reported in a study of prostate cancer angiogenesis that CCL2 stimulated tumor cells to produce pro-angiogenic factors instead of directly stimulating bone marrow endothelial cells (Zhang J et al. 2009). The anti-CCL2 antibody CNTO 888 also had no effect as a monotherapy on capillary tube formation ; however, CCL2 increased VEGF-A mRNA expression levels in PC-3 cells after 4-6 h of treatment. The induction of VEGF-A mRNA expression in PC-3 cells was blocked by pretreatment with a neutralizing antibody, indicating this induction was mediated by CCL2. CCL2 has been shown to be overexpressed in tumor cell lines resistant to taxanes. CCL2 may be induced by chemotherapy and mediate chemoresistance to taxanes. Moreover, this upregulation of CCL2 by taxane-based chemotherapy has been shown to occur via the JNK pathway. Study design: Our overall hypothesis is that CCL2 neutralization can inhibit tumor growth of taxane resistant metastatic ovarian cancer, and that stroma plays an important role in promoting tumor growth. This project is focused on three pairs of taxane resistant variants developed in the lab: ES-2/TP80, MES-OV/TP40 and OVCAR-3/TP20. These three cell models exhibit alterations in tubulin expression and dynamics along with other non-MDR1/P-glycoprotein mechanisms of resistance to taxanes. Using quantitative PCR, we observed elevated CCL2 expression (7x, 45x and 9x, respectively) in these cell models relative to parental controls, and confirmed these findings at the protein level by ELISA. CCR2 expression has been determined as well, by qPCR and by FACS analysis. In order to assess tumor growth by bioluminescent imaging, our taxane variants were transduced with the pHR2-gfp/luc lentiviral vector and were sorted in order to obtain homogenous gfp/luc positive population. These cells have been implanted intraperitoneally (i.p.) and subcutaneously (s.c.) in athymic nude, adult female mice. A pilot study has been done to determine the growth of our three cell lines pairs injected subcutaneously or intraperitoneally into mice. Then, Mice have been treated with antibodies to neutralize both the human tumor-derived CCL2 (CNTO 888) and the mouse orthologue of human CCL2, MCP-1 (C1142), with and without chemotherapy (paclitaxel or carboplatin). Bioluminescence images have been acquired in order to evaluate tumor growth during treatment. We have observed a significant difference in efficacy of paclitaxel and carboplatin when the CCL2 blockade is added, compared to the chemotherapy alone.

Our laboratory has developed several models of paclitaxel-resistant ovarian cancer cell lines characterized with non-MDR1 mechanisms of resistance by selection of wild-type OVCAR-3, ES-2 and MES-OV cells with increasing doses of paclitaxel and the inhibitor of P-glycoprotein, PSC-833. The resulting variants (OVCAR-3/TP, ES- 2/TP and MES-OV/TP) are showing up-regulation of epithelial-mesenchymal transition (EMT) genes ; vimentin, fibronectin, MMP2 and MMP9. Cell migration was markedly increased in resistant variants, but the growth rates of the variants in vitro were slower than parental cells. In contrast, the growth rate of the EMT variant OVCAR-3/TP in athymic mice was markedly higher than tumors from parental cells in both subcutaneous and intraperitoneal xenografts. All three resistant cell variants are characterized with down-regulation of microRNA-200 family members (200a, 200b, 200c, 141, 429). The goal of this project is to investigate the role of the microRNA-200 family in the regulation of EMT in OVCAR-3 cells and their variants, and its possible contribution to drug resistance and enhanced tumorigenesis in vitro and in vivo. The role of the miRNA-200 family will be investigated using two approaches: (1) up-regulation of miRNA-200s by infecting the paclitaxel-resistant OVCAR-3/TP cell line with lentiviral particles carrying the specific genomic sequences of each family member and a GFP marker gene ; and (2) the paclitaxel- sensitive variant OVCAR-3 will be infected with lentiviral particles carrying anti-miRNA constructs. Stable cell clones with up- or down- regulation of miRNA-200s will be sorted by GFP fluorescence. Downstream targets of miRNA-200s will be investigated as well as sensitivity to paclitaxel and cisplatin.

Chronic exposure of tumor cells to carboplatin (CBP) results very often in development of acquired CBP resistance reducing the efficacy of tumor therapy. The analysis of parental human laryngeal carcinoma (HEp2) cell line and its CBP-resistant subline (7T) identified CBP-induced apoptosis as the cause of resistance. Upon CBP treatment of HEp2 and 7T cell lines we found similar pattern of Bcl-2, survivin and p-Bad expression. Bcl-2 silencing did not influence the sensitivity of both cell lines to CBP treatment therefore excluding a main role of the mitochondrial pathway in apoptosis induction. The constitutive level of FasL was higher in sensitive HEp2 as compared to resistant 7T cells. CBP more efficiently increased the transcription of fasL mRNA in HEp2 than in 7T cell line, indicating the role of death receptor pathway in apoptosis induction. After analyzing several signaling pathways which could be involved in decreased apoptosis of 7T cells, we present here the most promising model. Total cell and DNA platination upon CBP treatment were lower in resistant 7T as compared to sensitive HEp2 cells. The decreased platination delayed activation of SAPK/JNK and p38 in 7T cells. Reduced platination of 7T is very likely caused by an increased constitutive expression of ATP7A and NHE1 efflux pumps found in those cells as compared to HEp2 cells. Our results reveal transcriptional alterations in CBP resistant cells that could be involved in acquired drug resistance. Studies on the mechanisms of anticancer drug resistance provide important information about how to improve cancer chemotherapy.