Differential Effects of Integrin αv Knockdown and Cilengitide on Sensitization of Triple-Negative Breast Cancer and Melanoma Cells to Microtubule Poisons.

Low survival rates of patients with metastatic triple-negative breast cancer (TNBC) and melanoma, in which current therapies are ineffective, emphasize the need for new therapeutic approaches. Integrin ß1 appears to be a promising target when combined with chemotherapy, but recent data have shown that its inactivation increases metastatic potential owing to the compensatory upregulation of other integrin subunits. Consequently, we analyzed the potential of integrin subunits αv, α3, or α4 as targets for improved therapy in seven TNBC and melanoma cell lines. Experiments performed in an integrin αvß1-negative melanoma cell line, MDA-MB-435S, showed that knockdown of integrin subunit αv increased sensitivity to microtubule poisons vincristine or paclitaxel and decreased migration and invasion. In the MDA-MB-435S cell line, we also identified a phenomenon in which change in the expression of one integrin subunit changes the expression of other integrins, leading to an unpredictable influence on sensitivity to anticancer drugs and cell migration, referred to as the integrin switching effect. In a panel of six TNBCs and melanoma cell lines, the contribution of integrins αv versus integrins αvß3/ß5 was assessed by the combined action of αv-specific small interfering RNA or αvß3/ß5 inhibitor cilengitide with paclitaxel. Our results suggest that, for TNBC, knockdown of integrin αv in combination with paclitaxel presents a better therapeutic option than a combination of cilengitide with paclitaxel; however, in melanoma, neither of these combinations is advisable because a decreased sensitivity to paclitaxel was observed.

Integrin αVß3 silencing sensitizes malignant glioma cells to temozolomide by suppression of homologous recombination repair.

Integrins have been suggested as possible targets in anticancer therapy. Here we show that knockdown of integrins αVß3, αVß5, α3ß1 and α4ß1 and pharmacological inhibition using a cyclo-RGD integrin αVß3/αVß5 antagonist sensitized multiple high-grade glioma cell lines to temozolomide (TMZ)-induced cytotoxicity. The greatest effect was observed in LN229 cells upon integrin ß3 silencing, which led to inhibition of the FAK/Src/Akt/NFκB signaling pathway and increased formation of γH2AX foci. The integrin ß3 knockdown led to the proteasomal degradation of Rad51, reduction of Rad51 foci and reduced repair of TMZ-induced DNA double-strand breaks by impairing homologous recombination efficiency. The down-regulation of ß3 in Rad51 knockdown (LN229-Rad51kd) cells neither further sensitized them to TMZ nor increased the number of γH2AX foci, confirming causality between ß3 silencing and Rad51 reduction. RIP1 was found cleaved and IκBα significantly less degraded in ß3-silenced/TMZ-exposed cells, indicating inactivation of NFκB signaling. The anti-apoptotic proteins Bcl-xL, survivin and XIAP were proteasomally degraded and caspase-3/-2 cleaved. Increased H2AX phosphorylation, caspase-3 cleavage, reduced Rad51 and RIP1 expression, as well as sustained IκBα expression were also observed in mouse glioma xenografts treated with the cyclo-RGD inhibitor and TMZ, confirming the molecular mechanism in vivo. Our data indicates that ß3 silencing in glioma cells represents a promising strategy to sensitize high-grade gliomas to TMZ therapy.

Discovery of 'click' 1,2,3-triazolium salts as potential anticancer drugs.

BACKGROUND: In order to increase the effectiveness of cancer treatment, new compounds with potential anticancer activities are synthesized and screened. Here we present the screening of a new class of compounds, 1-(2-picolyl)-, 4-(2-picolyl)-, 1-(2-pyridyl)-, and 4-(2-pyridyl)-3-methyl-1,2,3-triazolium salts and 'parent' 1,2,3-triazole precursors. METHODS: Cytotoxic activity of new compounds was determined by spectrophotometric MTT assay on several tumour and one normal cell line. Effect of the selected compound to bind double stranded DNA (ds DNA) was examined by testing its influence on thermal stability of calf thymus DNA while its influence on cell cycle was determined by flow cytometric analysis. Generation of reactive oxygen species (ROS) was determined by addition of specific substrate 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA). RESULTS: Parent triazoles were largely inactive, while some of the triazolium salts were highly cytotoxic for HeLa cells. Triazolium salts exhibited high cell-type dependent cytotoxicity against different tumour cells. Selected compound (4-(4-methoxyphenyl)-3-methyl-1-(2-picolyl)-1H-1,2,3-triazolium hexafluorophosphate(V) (2b) was significantly more cytotoxic against tumour cells than to normal cells, with very high therapeutic index 7.69 for large cell lung carcinoma H460 cells. Additionally, this compound was similarly cytotoxic against parent laryngeal carcinoma HEp-2 cells and their drug resistant 7T subline, suggesting the potential of this compound in treatment of drug resistant cancers. Compound 2b arrested cells in the G1 phase of the cell cycle. It did not bind ds DNA, but induced ROS in treated cells, which further triggered cell death. CONCLUSIONS: Our results suggest that the 'click' triazolium salts are worthy of further investigation as anti-cancer agents.

Integrin αvß3 expression in tongue squamous carcinoma cells Cal27 confers anticancer drug resistance through loss of pSrc(Y418).

Integrins play key roles in the regulation of tumor cell adhesion, migration, invasion and sensitivity to anticancer drugs. In the present study we investigate the mechanism of resistance of tongue squamous carcinoma cells Cal27 with de novo integrin αvß3 expression to anticancer drugs. Cal27-derived cell clones, obtained by transfection of plasmid containing integrin subunit ß3 cDNA, as compared to control cells demonstrate: expression of integrin αvß3; increased expression of integrin αvß5; increased adhesion to fibronectin and vitronectin; resistance to cisplatin, mitomycin C, doxorubicin and 5-fluorouracil; increased migration and invasion, increased amount of integrin-linked kinase (ILK) and decreased amounts of non-receptor tyrosine kinase (Src) and pSrc(Y418). Knockdown of ILK and integrin ß5 in cells expressing integrin αvß3 ruled out their involvement in drug resistance. Opposite, Src knockdown in Cal27 cells which led to a reduction in pSrc(Y418), as well as treatment with the pSrc(Y418) inhibitors dasatinib and PP2, conferred resistance to all four anticancer drugs, indicating that the loss of pSrc(Y418) is responsible for the observed effect. We identified differential integrin signaling between Cal27 and integrin αvß3-expressing cells. In Cal27 cells integrin αv heterodimers signal through pSrc(Y418) while this is not the case in integrin αvß3-expressing cells. Finally, we show that dasatinib counteracts the effect of cisplatin in two additional head and neck squamous cell carcinoma (HNSCC) cell lines Cal33 and Detroit562. Our results suggest that pSrc(Y418) inhibitors, potential drugs for cancer therapy, may reduce therapeutic efficacy if combined with chemotherapeutics, and might not be recommended for HNSCC treatment.

Antitumour action on human glioblastoma A1235 cells through cooperation of bee venom and cisplatin.

Cisplatin (cDDP) is one of the most widely used anticancer-drugs in both therapy and research. However, cDDP-resistance is the greatest obstacle for the successful treatment of cancer patients. In the present study, the possible joint anticancer effect of bee venom (BV), as a natural toxin, and cDDP towards human glioblastoma A1235 cells was evaluated. Treatment with BV alone in concentrations of 2.5-30 µg/ml displayed dose-dependent cytotoxicity towards A1235 cells, as evaluated with different cytotoxicity assays (MTT, Cristal violet and Trypan blue exclusion assay), with an IC50 value of 22.57 µg/ml based on the MTT results. Furthermore, BV treatment induced necrosis, which was confirmed by typical morphological features and fast staining with ethidium-bromide dye. Pre-treatment with BV induced cell sensitization to cDDP, indicating that BV could improve the killing effect of selected cells when combined with cDDP. The isobologram method used to determine the extent of synergism in combining two agents to examine their possible therapeutic effect showed that combined treatment induced an additive and/or synergistic effect towards selected cells depending on the concentration of both. Hence, a greater anticancer effect could be triggered if BV was used in the course of chemotherapy. The obtained results indicate that joint treatment with BV could be useful from the point of minimizing the cDDP concentration during chemotherapy, thus reducing and/or postponing the development of drug resistance. Our data, in accordance with previously reported results, suggests that BV could be used in the development of a new strategy for cancer treatment.

The 1,3-diaryltriazenido(p-cymene)ruthenium(II) complexes with a high in vitro anticancer activity.

1,3-Diaryltriazenes (1) were let to react with [RuCl2(p-cymene)]2 in the presence of trimethylamine to give neutral 1,3-diaryltriazenido(p-cymene)ruthenium(II) complexes, [RuCl(p-cymene)(ArNNNAr)] (2). The molecular composition of the products 2 was confirmed by NMR spectroscopy and mass spectrometry. The structures of the selected complexes were confirmed by a single crystal X-ray analysis. All triazenido-ruthenium complexes were highly cytotoxic against human cervical carcinoma HeLa cells with IC50 below 6µM, as determined by a spectrophotometric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) method. The most active was [RuCl(p-cymene)(ArNNNAr)] (Ar=4-Cl-3-(CF3)-C6H3) (2g) with IC50 of 0.103±0.006µM. In comparison with the data for the non-coordinated triazenes 1, the triazenido-ruthenium complexes 2 exhibited up to 560-times higher activity. Three selected complexes were highly cytotoxic also against several tumor cell lines: laryngeal carcinoma HEp-2 cells and their drug-resistant HEp-2 subline (7T), colorectal carcinoma HCT-116 cells, lung adenocarcinoma H460 cells, and mammary carcinoma MDA-MB-435 cells. The compounds 2g and [RuCl(p-cymene)(ArNNNAr)] (Ar=4-I-C6H4) (2j) were similarly cytotoxic against parental and drug-resistant cells. Time and dose dependent accumulation of the cells in the S phase of the cell cycle was induced by the compound 2g, triggering apoptosis. Our preliminary results indicate triazenido-ruthenium complexes as promising anticancer drug candidates.

An axial chirality and disorder of positional isomers in a crystal of highly cytotoxic 3-acetyl-1,3-bis(2-chloro-4-nitrophenyl)-1E-triazene.

Biologically active 4-nitro-substituted 1,3-diaryltriazene, a chemical analogue of 1,3-bis(4'-amidinophenyl)-triazene-berenil®, belongs to the novel, chemically modified class of potent antitumor agents. Its structural characterization by X-ray analysis and 1H NMR spectroscopy is performed to determine molecular overall conformation in view of its possible interaction to DNA.

Platinum-induced kidney damage: Unraveling the DNA damage response (DDR) of renal tubular epithelial and glomerular endothelial cells following platinum injury.

BACKGROUND: Platinum compounds are potent anticancer drugs but also evoke considerable normal tissue damage. Here, we elucidate the molecular mechanisms contributing to the nephrotoxic effects of cisplatin. METHODS: We comparatively investigated the stress responses of rat kidney tubular (NRK-52E) and glomerular cells (RGE) following treatment with cisplatin (CisPt), oxaliplatin (OxaliPt) and carboplatin (CarboPt). To this end, cell viability, apoptosis, cell cycle progression, DNA damage response (DDR) and repair of DNA adducts were investigated. RESULTS: CisPt reduced the viability of tubular NRK-52E and glomerular RGE cells most efficiently. Cytotoxicity evoked by CarboPt occurred with a delay, which might be related to a retarded formation of Pt-(GpG) intrastrand crosslinks. RGE cells were more sensitive towards all platinum compounds than NRK-52E cells. Platinum drugs efficiently induced caspase-mediated apoptosis in tubular cells, while RGE cells favored G2/M arrest when treated with equitoxic platinum doses. Mitotic index of NKR-52E and RGE cells was worst affected by OxaliPt. Activation of the DDR was strikingly agent- and cell type-specific. Most comprehensive and substantial stimulation of DDR mechanisms was provoked by CisPt. Repair of Pt-(GpG) intrastrand crosslinks was best in RGE, which was reflected by high mRNA expression of nucleotide excision repair (NER) factors. CONCLUSIONS: There are substantial differences regarding the cause of sensitivity and mechanisms of DDR between tubular and glomerular cells following platinum injury. CisPt is the most potent stimulator of the DDR. We hypothesize that specific DNA adducts and thereby forcefully activated pro-toxic DDR mechanisms contribute to the exceptionally high acute nephrotoxicity of CisPt.

Combined antitumor effects of bee venom and cisplatin on human cervical and laryngeal carcinoma cells and their drug resistant sublines.

In the present study, we investigated the possible combined anticancer ability of bee venom (BV) and cisplatin towards two pairs of tumour cell lines: parental cervical carcinoma HeLa cells and their cisplatin-resistant HeLa CK subline,as well as laryngeal carcinoma HEp-2 cells and their cisplatin-resistant CK2 subline. Additionally, we identified several peptides of BV in the BV sample used in the course of the study and determined the exact concentration of MEL. BV applied alone in concentrations of 30 to 60 µg ml(­1) displayed dose-dependent cytotoxicity against all cell lines tested. Cisplatin-resistant cervical carcinoma cells were more sensitive to BV than their parental cell lines (IC(50) values were 52.50 µg ml(­1) for HeLa vs.47.64 µg ml(­1) for HeLa CK cells), whereas opposite results were obtained for cisplatin-resistant laryngeal carcinoma cells (IC(50) values were 51.98 µg ml(­1) for HEp-2 vs. > 60.00 µg ml(­1) for CK2 cells). Treatment with BV alone induced a necrotic type of cell death, as shown by characteristic morphological features, fast staining with ethidium-bromide and a lack of cleavage of apoptotic marker poly (ADP-ribose) polymerase (PARP) on Western blot. Combined treatment of BV and cisplatin induced an additive and/or weak synergistic effect towards tested cell lines, suggesting that BV could enhance the killing effect of selected cells when combined with cisplatin. Therefore, a greater anticancer effect could be triggered if BV was used in the course of chemotherapy. Our results suggest that combined treatment with BV could be useful from the point of minimizing the cisplatin concentration during chemotherapy, consequently reducing and/or postponing the development of cisplatin resistance.

Combining [arene-Ru] with azocarboxamide to generate a complex with cytotoxic properties.

Azocarboxamide (azcH) has been combined for the first time with [Ru-Cym] to generate metal complexes with N,N- and N,O-coordination mode, [(Cym)Ru(azc)Cl] and [(Cym)Ru(azcH)Cl](+) [PF6 ](-). Geometric and electronic structures of the complexes are reported along with their in vitro activities against different tumour cell lines and preliminary results on solution chemistry. Compound [(Cym)Ru(azc)Cl] exhibited remarkable cytotoxic properties. It was cell-type specific and had comparable IC50 values towards both cancer cells and their drug-resistant subline. A tenfold increase in the sensitivity towards [(Cym)Ru(azc)Cl] was noted for the tumour cells with depleted intracellular glutathione (GSH) level, suggesting the essential role of GSH in cell response to this compound.

FTIR spectroscopy reveals lipid droplets in drug resistant laryngeal carcinoma cells through detection of increased ester vibrational bands intensity.

The major obstacle to successful chemotherapy of cancer patients is drug resistance. Previously we explored the molecular mechanisms of curcumin cross-resistance in carboplatin resistant human laryngeal carcinoma 7T cells. Following curcumin treatment we found a reduction in curcumin accumulation, and reduced induction of reactive oxygen species (ROS) and their downstream effects, compared to parental HEp-2 cells. In order to shed more light on mechanisms involved in drug resistance of 7T cells, in the present study we applied Fourier transform infrared (FTIR) spectroscopy, a technique that provides information about the nature and quantities of all molecules present in the cell. By comparing the spectra from parental HEp-2 cells and their 7T subline, we found an increase in the intensity of ester vibrational bands in 7T cells. This implied an increase in the amount of cholesteryl esters in resistant cells, which we confirmed by an enzymatic assay. Since cholesteryl esters are localized in lipid droplets, we confirmed their higher quantity and serum dependency in 7T cells compared to HEp-2 cells. Moreover, treatment with oleic acid induced more lipid droplets in 7T when compared to HEp-2 cells, as shown by flow cytometry. We can conclude that along with previously determined molecular mechanisms of curcumin resistance in 7T cells, these cells exhibit an increased content of cholesteryl esters and lipid droplets, suggesting an alteration in cellular lipid metabolism as a possible additional mechanism of drug resistance. Furthermore, our results suggest the use of FTIR spectroscopy as a promising technique in drug resistance research.

Increased adenovirus Type 5 mediated transgene expression due to RhoB down-regulation.

Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking.

3-Acetyl-bis(2-chloro-4-nitrophenyl)triazene is a potent antitumor agent that induces oxidative stress and independently activates the stress-activated protein kinase/c-Jun NH2-terminal kinase pathway.

Previously, we described the synthesis and biological activity of a new class of anticancer molecules that preferentially target malignant cells and may serve as potential antitumor agents. Among several synthesized agents, we selected 3-acetyl-1,3-bis(2-chloro-4-nitrophenyl)-1-triazene (8b) as a representative of the group of 4-nitro-substituted 1,3-diaryltriazenes. The aim of this study was to further investigate the mechanism of cell response to the 8b compound. The HeLa human cervical carcinoma cell line was used as an experimental model to further investigate the mechanism of cell response to the 8b compound. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to address cell survival, and western blot (immunoblotting) was used for the expression of relevant proteins after 8b drug exposure. The pretreatment of HeLa cells with salubrinal, a specific inhibitor of endoplasmic reticulum (ER) stress, confirmed the importance of ER stress in apoptosis induced by 8b. We also demonstrate that 8b triggers the activation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) in a time-dependent and dose-dependent manner. Moreover, the inhibition of SAPK/JNK activity by JNK II before 8b treatment increased the survival rate of HeLa cells relative to survival in the presence of 8b alone, indicating the importance of this kinase in cell death. The simultaneous inhibition of ER stress induction and SAPK/JNK activation increased the survival of HeLa cells upon 8b treatment more than inhibition of both pathways independently, suggesting the separate triggering of both signaling pathways. Our data indicate that cytotoxic activity of the novel compound 8b is based on its ability to induce ER stress and SAPK/JNK signaling pathways independently, driving cells to cell death.

Synthesis and biological activity of new diazenedicarboxamides as potential anticancer agents.

To increase the effectiveness of cancer treatment, more effective anti-cancer drugs, as well as the new improved strategies of cancer treatment, are urgently needed. Our previous results have shown that various diazenes are cytotoxic to different tumor cells and can even revert the resistance to cisplatin and vincristine. We also demonstrated that unsymmetrical diazenedicarboxamides 1 and 2 exhibited promising cytotoxicity. The aim of the present study was to synthesize new diazenedicarboxamides with acceptable solubility and good cytotoxicity. Here we report the synthesis and biological evaluation of new N,N'-disubstituted diazenedicarboxamides. We found that a modification of either 1 or 2 led to the more active compounds. The most effective among them was diazenedicarboxamide 11, which can be considered as a new potential anticancer agent for the tumors of different origin, as well as for the drug resistant tumors.

Endoplasmic reticulum stress is involved in the response of human laryngeal carcinoma cells to Carboplatin but is absent in Carboplatin-resistant cells.

The major obstacle of successful tumor treatment with carboplatin (CBP) is the development of drug resistance. In the present study, we found that following treatment with CBP the amount of platinum which enters the human laryngeal carcinoma (HEp2)-derived CBP-resistant (7T) cells is reduced relative to the parental HEp2. As a consequence, the formation of reactive oxidative species (ROS) is reduced, the induction of endoplasmic reticulum (ER) stress is diminished, the amount of inter- and intrastrand cross-links is lower, and the induction of apoptosis is depressed. In HEp2 cells, ROS scavenger tempol, inhibitor of ER stress salubrinal, as well as gene silencing of ER stress marker CCAAT/enhancer-binding protein (CHOP) increases their survival and renders them as resistant to CBP as 7T cell subline but did not influence the survival of 7T cells. Our results suggest that in HEp2 cells CBP-induced ROS is a stimulus for ER stress. To the contrary, despite the ability of CBP to induce formation of ROS and activate ER stress in 7T cells, the cell death mechanism in 7T cells is independent of ROS induction and activation of ER stress. The novel signaling pathway of CBP-driven toxicity that was found in the HEp2 cell line, i.e. increased ROS formation and induction of ER stress, may be predictive for therapeutic response of epithelial cancer cells to CBP-based therapy.

Design and evaluation of biological activity of diazenecarboxamide-extended cisplatin and carboplatin analogues.

Construction of a library of structurally diverse diazenecarboxamide-extended cis-[Pt(2-picolyl-1,2,3-triazole)Cl2,] and cis-[Pt(propan-1,3-diamine)CBDCA] (CBDCA = 1,1 -cyclobutanedicarboxylate) complexes 1-4 is described. These compounds retain oxidative properties of parent diazenecarboxamides against glutathione as demonstrated by NMR spectroscopy and high resolution mass spectrometry experiments. Cytotoxic activity of 1-4 was investigated against human cervical carcinoma HeLa cells. Four library members were found to possess moderate cytotoxic activity. Some model compounds were also examined, returning [PtCl2L2] (L = 1-(2-picolyl)-4-phenyl-1H-1,2,3-triazole) as the most potent under this investigation with IC50 of 19.05 microM, comparable to that of cisplatin (IC50 = 16.3 microM).

Accurate models for P-gp drug recognition induced from a cancer cell line cytotoxicity screen.

P-glycoprotein (P-gp, MDR1) is a promiscuous drug efflux pump of substantial pharmacological importance. Taking advantage of large-scale cytotoxicity screening data involving 60 cancer cell lines, we correlated the differential biological activities of ∼13,000 compounds against cellular P-gp levels. We created a large set of 934 high-confidence P-gp substrates or nonsubstrates by enforcing agreement with an orthogonal criterion involving P-gp overexpressing ADR-RES cells. A support vector machine (SVM) was 86.7% accurate in discriminating P-gp substrates on independent test data, exceeding previous models. Two molecular features had an overarching influence: nearly all P-gp substrates were large (>35 atoms including H) and dense (specific volume of <7.3 Å(3)/atom) molecules. Seven other descriptors and 24 molecular fragments ("effluxophores") were found enriched in the (non)substrates and incorporated into interpretable rule-based models. Biological experiments on an independent P-gp overexpressing cell line, the vincristine-resistant VK2, allowed us to reclassify six compounds previously annotated as substrates, validating our method's predictive ability. Models are freely available at http://pgp.biozyne.com .

Carboplatin resistant human laryngeal carcinoma cells are cross resistant to curcumin due to reduced curcumin accumulation.

Curcumin is a natural compound that exhibits a wide range of beneficial effects, among them the anti-tumor activity. Recently it was shown that curcumin may be efficient against drug resistant tumor cells. The goal of our investigation was to examine if human laryngeal carcinoma cells resistant to carboplatin display sensitivity to curcumin, as compared to parental cells, and if this sensitivity is altered, to determine the molecular mechanisms that are responsible for it. We found that carboplatin resistant 7T cells were also cross resistant to curcumin. After the treatment with equimolar concentration of curcumin, 7T cells exhibited lower intracellular accumulation of curcumin which coincided with reduced formation of reactive oxygen species (ROS), diminished lipid and DNA damage followed by reduced induction of apoptosis and expression of heat shock protein 70 (Hsp70), as compared to parental HEp-2 cells. However, after the treatment with equitoxic concentration of curcumin, intracellular accumulation and all the explored downstream effects were similar in both cell lines suggesting that resistance of 7T cells to curcumin was based on its reduced intracellular accumulation. Since curcumin accumulates mostly in the membranes, we explored the fatty acid composition of both cell lines, but we did not find any difference between them.

Statins and cancer: current and future prospects.

Statins are inhibitors of 3-hydroxy-methylglutaryl (HMG) CoA reductase. They exhibit effects beyond cholesterol reduction, including anticancer activity. This review presents the effects of statins in vitro and their possible molecular anticancer mechanisms and critically discusses the data regarding the role of statins in cancer prevention. Finally, this review focuses on the use of statins combined with other chemotherapeutics to increase the effectiveness of cancer treatments. Despite rare and inconclusive clinical data, the preclinical results strongly suggest that such combined treatment could be a promising new strategy for the treatment of certain tumor types.

Increased expression of the coxsackie and adenovirus receptor downregulates αvß3 and αvß5 integrin expression and reduces cell adhesion and migration.

AIMS: Coxsackie and adenovirus receptor (CAR) is a tumor suppressor and a primary receptor for adenovirus type 5 (Ad5). Our study aims to examine the influence of forced expression of CAR in rhabdomyosarcoma cells (RD) on expression levels of integrins implicated in Ad5 entry, and the effect of CAR on cell-extracellular matrix adhesion and migration. MAIN METHODS: CAR expressing clones were established from RD cells by stable transfection. Flow cytometry was used to evaluate the expression of CAR and integrins. Adhesion was measured in plates previously coated with vitronectin or fibronectin. Boyden chambers were used to investigate migration. Transfection of cells with siRNA was used to achieve integrin silencing. Ad5-mediated transgene expression was measured by ß-gal staining. KEY FINDINGS: Increased expression of CAR in RD cells reduces the expression of αvß3 and αvß5 integrins. Cells overexpressing CAR exhibit significantly reduced adhesion to vitronectin and fibronectin, and reduced cell migration. Specifically silencing αvß3 integrin in RD cells reduced cell migration indicating that reduced migration could be the consequence of αvß3 integrin downregulation. This study also demonstrates the negative effect of reduced levels of αvß3 and αvß5 integrins on Ad5-mediated transgene expression with Ad5 retargeted to αv integrins. SIGNIFICANCE: The pharmacological upregulation of CAR aimed to increase Ad5-mediated transgene expression may actually downregulate αvß3 and αvß5 integrins and thus alter Ad5-mediated gene transfer. The mechanism of decreased cell migration, a prerequisite for metastasis and invasion, due to increased CAR expression may be explained by reduced αvß3 integrin expression.