KiSS-1 Modulation by Epigenetic Agents Improves the Cisplatin Sensitivity of Lung Cancer Cells.

Epigenetic alterations my play a role in the aggressive behavior of Non-Small Cell Lung Cancer (NSCLC). Treatment with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA, vorinostat) has been reported to interfere with the proliferative and invasive potential of NSCLC cells. In addition, the DNA methyltransferase inhibitor azacytidine (AZA, vidaza) can modulate the levels of the metastasis suppressor KiSS-1. Thus, since cisplatin is still clinically available for NSCLC therapy, the aim of this study was to evaluate drug combinations between cisplatin and SAHA as well as AZA using cisplatin-sensitive H460 and -resistant H460/Pt NSCLC cells in relation to KiSS-1 modulation. An analysis of drug interaction according to the Combination-Index values indicated a more marked synergistic effect when the exposure to SAHA or AZA preceded cisplatin treatment with respect to a simultaneous schedule. A modulation of proteins involved in apoptosis (p53, Bax) was found in both sensitive and resistant cells, and compared to the treatment with epigenetic agents alone, the combination of cisplatin and SAHA or AZA increased apoptosis induction. The epigenetic treatments, both as single agents and in combination, increased the release of KiSS-1. Finally, the exposure of cisplatin-sensitive and -resistant cells to the kisspeptin KP10 enhanced cisplatin induced cell death. The efficacy of the combination of SAHA and cisplatin was tested in vivo after subcutaneous inoculum of parental and resistant cells in immunodeficient mice. A significant tumor volume inhibition was found when mice bearing advanced tumors were treated with the combination of SAHA and cisplatin according to the best schedule identified in cellular studies. These results, together with the available literature, support that epigenetic drugs are amenable for the combination treatment of NSCLC, including patients bearing cisplatin-resistant tumors.

Synthesis of N-oxyamide analogues of protein kinase B (Akt) targeting anionic glycoglycerolipids and their antiproliferative activity on human ovarian carcinoma cells.

N-Oxyamides of bioactive anionic glycoglycerolipids based on 2-O-ß-D-glucosylglycerol were efficiently prepared. However, the oxidation step of the primary hydroxyl group of the glucose moiety in the presence of the N-oxyamide function appeared to be a difficult task that was nevertheless conveniently achieved for the first time by employing a chemoenzymatic laccase/TEMPO procedure. The obtained N-oxyamides exhibited a higher inhibition of proliferation of ovarian carcinoma IGROV-1 cells in serum-free medium than in complete medium, similarly to the corresponding bioactive esters. Stability and serum binding studies indicated that the observed reduced activity of the compounds in complete medium could be mainly due to a binding effect of serum proteins rather than the hydrolytic degradation of glycoglycerolipid acyl chains. Furthermore, the results of the cellular studies under serum-free conditions suggested that the N-oxyamide group could increase the antiproliferative activity of a glycoglycerolipid independently of the presence of the anionic carboxylic group. Cellular studies in other cell lines besides IGROV-1 also support a certain degree of selectivity of this series of compounds for tumor cells with Akt hyperactivation.

The deubiquitinase USP8 regulates ovarian cancer cell response to cisplatin by suppressing apoptosis.

The identification of therapeutic approaches to improve response to platinum-based therapies is an urgent need for ovarian carcinoma. Deubiquitinases are a large family of ubiquitin proteases implicated in a variety of cellular functions and may contribute to tumor aggressive features through regulation of processes such as proliferation and cell death. Among the subfamily of ubiquitin-specific peptidases, USP8 appears to be involved in modulation of cancer cell survival by still poorly understood mechanisms. Thus, we used ovarian carcinoma cells of different histotypes, including cisplatin-resistant variants with increased survival features to evaluate the efficacy of molecular targeting of USP8 as a strategy to overcome drug resistance/modulate cisplatin response. We performed biochemical analysis of USP8 activity in pairs of cisplatin-sensitive and -resistant cells and found increased USP8 activity in resistant cells. Silencing of USP8 resulted in decreased activation of receptor tyrosine kinases and increased sensitivity to cisplatin in IGROV-1/Pt1 resistant cells as shown by colony forming assay. Increased cisplatin sensitivity was associated with enhanced cisplatin-induced caspase 3/7 activation and apoptosis, a phenotype also observed in cisplatin sensitive cells. Increased apoptosis was linked to FLIPL decrease and cisplatin induction of caspase 3 in IGROV-1/Pt1 cells, cisplatin-induced claspin and survivin down-regulation in IGROV-1 cells, thereby showing a decrease of anti-apoptotic proteins. Immunohistochemical staining on 65 clinical specimens from advanced stage ovarian carcinoma indicated that 40% of tumors were USP8 positive suggesting that USP8 is an independent prognostic factor for adverse outcome when considering progression free survival as a clinical end-point. Taken together, our results support that USP8 may be of diagnostic value and may provide a therapeutic target to improve the efficacy of platinum-based therapy in ovarian carcinoma.

Increased serum levels of KiSS1-derived peptides in non-small cell lung cancer patient liquid biopsies and biological relevance.

Background: The secreted products of the metastasis suppressor gene KiSS1 may represent useful biomarkers in non-small cell lung cancer (NSCLC) but their levels in patients have remained poorly investigated. We previously found that forced expression of KiSS1 decreased the invasive capability of NSCLC drug-resistant cells and a pro-apoptotic role for KiSS1 has been proposed in head and neck cancer. Thus, we designed a translational investigation including a pilot study to analyze KiSS1 levels in liquid biopsies, and in vitro experiments to explore the biological relevance of KiSS1 modulation. Methods: KiSS1-derived peptide levels in liquid biopsies from 60 NSCLC patients were assayed by ELISA. Preclinical experiments were carried out using quantitative real time polymerase chain reaction (qRT-PCR), ELISA, annexin V-binding and caspase activation assays. Results: We compared KiSS1 release in 3 different matrices (serum, plasma and urine) and the highest levels were detectable in serum (range, 0-4.5 ng/mL). We observed increased levels of seric KiSS1 in NSCLC patients as compared to healthy donors. KiSS1 serum concentrations, after surgical procedure and/or adjuvant therapy. We observed differences among disease stages in urine samples. In preclinical models, KiSS1 mRNA levels were increased by short term exposure to azacytidine, enhanced KiSS1 release was induced by the combination of azacytidine and cisplatin and KiSS1-derived peptides enhanced cisplatin-induced apoptosis. KiSS1 increase was observed upon exposure neurons-enriched cultures to tumor cell conditioned medium. Conclusions: Our results showing a peculiar modulation of KiSS1 levels in liquid biopsies of NSCLC patients and a regulation of cisplatin-induced apoptosis by KiSS1-derived peptides support an involvement of KiSS1 in cell response to treatment and highlight its promising features as a potential biomarker in NSCLC.

Caffeic acid phenethyl ester targets ubiquitin-specific protease 8 and synergizes with cisplatin in endometrioid ovarian carcinoma cells.

Deubiquitinases (DUBs) mediate the removal of ubiquitin from diverse proteins that participate in the regulation of cell survival, DNA damage repair, apoptosis and drug resistance. Previous studies have shown an association between activation of cell survival pathways and platinum-drug resistance in ovarian carcinoma cell lines. Among the strategies available to inhibit DUBs, curcumin derivatives appear promising, thus we hypothesized their use to enhance the efficacy of cisplatin in ovarian carcinoma preclinical models. The caffeic acid phenethyl ester (CAPE), inhibited ubiquitin-specific protease 8 (USP8), but not proteasomal DUBs in cell-free assays. When CAPE was combined with cisplatin in nine cell lines representative of various histotypes a synergistic effect was observed in TOV112D cells and in the cisplatin-resistant IGROV-1/Pt1 variant, both of endometrioid type and carrying mutant TP53. In the latter cells, persistent G1 accumulation upon combined treatment associated with p27kip1 protein levels was observed. The synergy was not dependent on apoptosis induction, and appeared to occur in cells with higher USP8 levels. In vivo antitumor activity studies supported the advantage of the combination of CAPE and cisplatin in the subcutaneous model of cisplatin-resistant IGROV-1/Pt1 ovarian carcinoma as well as CAPE activity on intraperitoneal disease. This study reveals the therapeutic potential of CAPE in cisplatin-resistant ovarian tumors as well as in tumors expressing USP8.

Synergistic Interaction of Histone Deacetylase 6- and MEK-Inhibitors in Castration-Resistant Prostate Cancer Cells.

In spite of new knowledge on prostate cancer molecular landscape, this has been only partially translated to the therapeutic setting. The activation of Ras/Mitogen-activated protein kinase (MAPK) signaling plays an important role in progression of prostate cancer in which deregulation of histone deacetylases (HDAC) is frequent. Based on the notion that HDAC inhibitors may reactivate the expression of genes favoring cell response to drugs, the aim of this study was to investigate the interaction between the HDAC6-specific inhibitor ricolinostat (ACY1215) and the MEK-inhibitor selumetinib (AZD6244) to identify effective combinations in prostate cancer models. Using cell lines exhibiting differential activation of survival pathways (PC3, DU145, 22Rv1) and following different treatment schedules, a synergistic interaction was observed in all cell models, the drug combination being particularly effective in 22Rv1 cells. Marginal levels of apoptosis were observed in PC3 cells after combined treatment, whereas higher levels were achieved in DU145 and 22Rv1 cells. RNAi-mediated knockdown of HDAC6 in selumetinib-treated 22Rv1 cells resulted in increased apoptosis. Combined treatment suppressed the constitutively deregulated survival pathways in all cell lines. A decrease of androgen receptor (AR)-dependent gene (KLK2, DUSP1) mRNA levels was observed in 22Rv1 treated cells, associated with increased AR cytoplasmatic expression, suggesting AR signaling down-regulation, not involving Hsp90 acetylation. When a taxane was used in combination with AZD6244 and ACY1215 by a simultaneous schedule, a synergistic cytotoxic effect together with increased apoptosis was evidenced in all cell models. These results support a rational use of targeted agents to improve prostate cancer cell apoptotic response.

AXL Downstream Targeting Unravels Synergistic Drug Combinations in Ovarian Carcinoma Cells.

BACKGROUND: Platinum-based therapy represents the main pharmacological treatment for ovarian carcinoma. Since molecular targeting of receptor tyrosine kinases (RTK) affects factors that may modulate drug response, the aim of this study was to examine whether downstream targets of AXL RTK could be exploited to improve cell response to cisplatin. MATERIALS AND METHODS: Inhibitors of p38 (SB203580) and of signal transducer and activator of transcription 3 (stattic) were employed in combination with cisplatin in ovarian carcinoma cell lines. Apoptosis assay and western blot analysis were performed to evaluate cell response after treatment. RESULTS: SB203580 produced a synergistic effect in combination with cisplatin in cisplatin-resistant IGROV-1/Pt1 cells. In addition, a favorable drug interaction was observed in A2780 cells when pre-incubated with cisplatin prior to stattic. The analysis of cell response after combined treatment showed down-regulation of the pro-apoptotic protein BCL2-associated agonist of cell death (BAD). CONCLUSION: Our results support the notion that downstream targets of AXL in ovarian carcinoma cells can be exploited to increase cisplatin activity in ovarian carcinoma models.

FoxO-1 contributes to the efficacy of the combination of the XPO1 inhibitor selinexor and cisplatin in ovarian carcinoma preclinical models.

The XPO1/CRM1 inhibitor selinexor (KPT-330), is currently being evaluated in multiple clinical trials as an anticancer agent. XPO1 participates in the nuclear export of FoxO-1, which we previously found to be decreased in platinum-resistant ovarian carcinoma. The aim of this study was to determine whether enriching FoxO-1 nuclear localization using selinexor would increase ovarian cancer cell sensitivity to cisplatin. Selinexor, as a single agent, displayed a striking antiproliferative effect in different ovarian carcinoma cell lines. A schedule-dependent synergistic effect of selinexor in combination with cisplatin was found in cisplatin-sensitive IGROV-1, the combination efficacy being more evident in sensitive than in the resistant cells. In IGROV-1 cells, the combination was more effective when selinexor followed cisplatin exposure. A modulation of proteins involved in apoptosis (p53, Bax) and in cell cycle progression (p21WAF1) was found by Western blotting. Selinexor-treated cells exhibited enriched FoxO-1 nuclear staining. Knock-down experiments with RNA interference indicated that FOXO1-silenced cells displayed a reduced sensitivity to selinexor. FOXO1 silencing also tended to reduce the efficacy of the drug combination at selected cisplatin concentrations. Selinexor significantly inhibited tumor growth, induced FoxO-1 nuclear localization and improved the efficacy of cisplatin in IGROV-1 xenografts. Taken together, our results support FoxO-1 as one of the key factors promoting sensitivity towards selinexor and the synergistic interaction between cisplatin and selinexor in ovarian carcinoma cells with selected molecular backgrounds, highlighting the need for treatment regimens tailored to the molecular tumor features.

Axl molecular targeting counteracts aggressiveness but not platinum-resistance of ovarian carcinoma cells.

Ovarian carcinoma, the most common gynaecological cancer, is characterized by high lethality mainly due to late diagnosis and treatment failure. The efficacy of platinum drug-based therapy in the disease is limited by the occurrence of drug resistance, a phenomenon often associated with increased metastatic potential. Because the Tyr-kinase receptor Axl can be deregulated in ovarian carcinoma and plays a pro-metastatic/anti-apoptotic role, the aim of this study was to examine if Axl inhibition modulates drug resistance and aggressive features of ovarian carcinoma cells, using various pairs of cisplatin-sensitive and -resistant cell lines. We found that mRNA and protein levels of Axl were increased in the platinum-resistant IGROV-1/Pt1 and IGROV-1/OHP cell lines compared to the parental IGROV-1 cells. IGROV-1/Pt1 cells displayed increased migratory and invasive capabilities. When Axl was silenced, these cells exhibited reduced growth and invasive/migratory capabilities compared to control siRNA-transfected cells, associated with decreased p38 and STAT3 phosphorylation. In keeping with this evidence, pharmacological inhibition of p38 and STAT3 decreased IGROV-1/Pt1 invasive capability. Molecular inhibition of Axl did not sensitize IGROV-1/Pt1 cells to cisplatin, but enhanced ErbB3 activation in IGROV-1/Pt1 cells and suppressed the clonogenic capability of various ovarian carcinoma cell lines. The combination of cisplatin and AZD8931, a small molecule which inhibits ErbB3, produced a synergistic effect in IGROV-1/Pt1 cells. Thus, Axl targeting per se reduces invasive capability of drug-resistant cells, but sensitization to cisplatin requires the concomitant inhibition of additional survival pathways.

PKC-alpha modulation by miR-483-3p in platinum-resistant ovarian carcinoma cells.

The occurrence of drug resistance limits the efficacy of platinum compounds in the cure of ovarian carcinoma. Since microRNAs (miRNAs) may contribute to this phenomenon by regulating different aspects of tumor cell response, the aim of this study was to exploit the analysis of expression of miRNAs in platinum sensitive/resistant cells in an attempt to identify potential regulators of drug response. MiR-483-3p, which may participate in apoptosis and cell proliferation regulation, was found up-regulated in 4 platinum resistant variants, particularly in the IGROV-1/Pt1 subline, versus parental cells. Transfection of a synthetic precursor of miR-483-3p in IGROV-1 parental cells elicited a marked up-regulation of the miRNA levels. Growth-inhibition and colony-forming assays indicated that miR-483-3p over-expression reduced cell growth and conferred mild levels of cisplatin resistance in IGROV-1 cells, by interference with their proliferative potential. Predicted targets of miR-483-3p included PRKCA (encoding PKC-alpha), previously reported to be associated to platinum-resistance in ovarian carcinoma. We found that miR-483-3p directly targeted PRKCA in IGROV-1 cells. In keeping with this finding, cisplatin sensitivity of IGROV-1 cells decreased upon molecular/pharmacological inhibition of PKC-alpha. Overall, our results suggest that overexpression of miR-483-3p by ovarian carcinoma platinum-resistant cells may interfere with their proliferation, thus protecting them from DNA damage induced by platinum compounds and ultimately representing a drug-resistance mechanism. The impairment of cell growth may account for low levels of drug resistance that could be relevant in the clinical setting.

Targeting peptidyl-prolyl isomerase pin1 to inhibit tumor cell aggressiveness.

PURPOSE: Because the peptidyl-prolyl isomerase PIN1 interacts with multiple protein kinases and phosphoproteins into a network orchestrating the cellular response to various stimuli, there is an increasing interest in exploiting its potential as therapeutic target. In the present study, the effect of targeting PIN1 was investigated in 2 human cancer cell lines characterized by increased aggressive potential, high expression of erbB receptor family members, and defective p53. METHODS: PIN1 silencing was carried out in skin squamous cell carcinoma A431 cells displaying elevated EGFR/HER1 levels and in ovarian adenocarcinoma SKOV-3 cells displaying high levels of erbB2 (HER2). Nonoverlapping siRNA duplexes targeting different regions of PIN1 mRNA were transfected in tumor cells, which were analyzed using Western blotting for the expression of selected proteins. In vivo tumorigenicity studies were carried out in athymic nude mice. RESULTS: A431 and SKOV-3 cell systems were found to be a source of cells with increased aggressive potential, i.e., cancer stem cell-like cells, as defined by the capability to grow as spheres. A marked decrease of PIN1 levels and of sphere-forming capability was observed in PIN1-silenced cells. The expression of phospho-p38 decreased following PIN1 silencing in A431 and SKOV-3 cells, as well as phospho-EGFR levels in A431 - silenced cells. PIN1 inhibition prolonged latency and reduced tumor take and growth of SKOV-3 cells in nude mice. CONCLUSIONS: Our results support that PIN1 may be a valuable target to hit in cancer cells characterized by increased aggressive potential, overexpression of erbB receptor family members, and defective p53.

Histone deacetylase inhibitor-temozolomide co-treatment inhibits melanoma growth through suppression of Chemokine (C-C motif) ligand 2-driven signals.

Target-specific agents used in melanoma are not curative, and chemokines are being implicated in drug-resistance to target-specific agents. Thus, the use of conventional agents in rationale combinations may result in optimization of therapy. Because histone deacetylases participate in tumor development and progression, the combination of the pan-inhibitor SAHA and temozolomide might provide a therapeutic advantage. Here, we show synergism between the two drugs in mutant BRAF cell lines, in association with decreased phosphorylation of cell survival proteins (e.g., C-Jun-N-terminal-kinase, JNK). In the spontaneous ret transgenic mouse melanoma model, combination therapy produced a significant disease onset delay and down-regulation of Chemokine (C-C motif) ligand 2 (CCL2), JNK, and of Myeloid-derived suppressor cell recruitment. Co-incubation with a CCL2-blocking-antibody enhanced in vitro cell sensitivity to temozolomide. Conversely, recombinant CCL2 activated JNK in human tumor melanoma cells. In keeping with these results, the combination of a JNK-inhibitor with temozolomide was synergistic. By showing that down-regulation of CCL2-driven signals by SAHA and temozolomide via JNK contributes to reduce melanoma growth, we provide a rationale for the therapeutic advantage of the drug combination. This combination strategy may be effective because of interference both with tumor cell and tumor microenvironment.

Differential outcome of MEK1/2 inhibitor-platinum combinations in platinum-sensitive and -resistant ovarian carcinoma cells.

Deregulated pro-survival signalling plays a role in ovarian carcinoma drug resistance. Here, we show that cisplatin or oxaliplatin in combination with the MEK1/2 inhibitor CI-1040 resulted in a synergistic effect associated with enhanced apoptotic response in platinum-sensitive cells. The drug combinations were additive in platinum-resistant cells exhibiting increased phospho-ERK1/2, down-regulation of apoptosis-related factors (BAX, PUMA, FOXO1) and of phosphatases inhibiting ERK1/2 (DUSP5, DUSP6). Consistently, FOXO1 knockdown in sensitive cells reduced the efficacy of the combination treatment. Pharmacological targeting of ERK1/2 pathway increases cell sensitivity to platinum compounds by interfering with multiple events, ultimately favouring apoptosis induction in selected molecular backgrounds.

7-Azaindole-1-carboxamides as a new class of PARP-1 inhibitors.

7-Azaindole-1-carboxamides were designed as a new class of PARP-1 inhibitors. The compounds displayed a variable pattern of target inhibition profile that, in part, paralleled the antiproliferative activity in cell lines characterized by homologous recombination defects. A selected compound (1l; ST7710AA1) showed significant in vitro target inhibition and capability to substantially bypass the multidrug resistance mediated by Pgp. In antitumor activity studies against the MX1 human breast carcinoma growth in nude mice, the compound exhibited an effect similar to that of Olaparib in terms of tumor volume inhibition when used at a lower dose than the reference compound. Treatment was well tolerated, as no deaths or significant weight losses were observed among the treated animals.

Synthesis and biological evaluation (in vitro and in vivo) of cyclic arginine-glycine-aspartate (RGD) peptidomimetic-paclitaxel conjugates targeting integrin αVß3.

A small library of integrin ligand-paclitaxel conjugates 10-13 was synthesized with the aim of using the tumor-homing cyclo[DKP-RGD] peptidomimetics for site-directed delivery of the cytotoxic drug. All the paclitaxel-RGD constructs 10-13 inhibited biotinylated vitronectin binding to the purified αVß3 integrin receptor at low nanomolar concentration and showed in vitro cytotoxic activity against a panel of human tumor cell lines similar to that of paclitaxel. Among the cell lines, the cisplatin-resistant IGROV-1/Pt1 cells expressed high levels of integrin αVß3, making them attractive to be tested in in vivo models. cyclo[DKP-f3-RGD]-PTX 11 displayed sufficient stability in physiological solution and in both human and murine plasma to be a good candidate for in vivo testing. In tumor-targeting experiments against the IGROV-1/Pt1 human ovarian carcinoma xenotransplanted in nude mice, compound 11 exhibited a superior activity compared with paclitaxel, despite the lower (about half) molar dosage used.

Design, synthesis, and biological evaluation of novel cRGD-paclitaxel conjugates for integrin-assisted drug delivery.

The efficacy of taxane-based antitumor therapy is limited by several drawbacks which result in a poor therapeutic index. Thus, the development of approaches that favor selective delivery of taxane drugs (e.g., paclitaxel, PTX) to the disease area represents a truly challenging goal. On the basis of the strategic role of integrins in tumor cell survival and tumor progression, as well as on integrin expression in tumors, novel molecular conjugates were prepared where PTX is covalently attached to either cyclic AbaRGD (Azabicycloalkane-RGD) or AmproRGD (Aminoproline-RGD) integrin-recognizing matrices via structurally diverse connections. Receptor-binding assays indicated satisfactory-to-excellent α(V)ß(3) binding capabilities for most conjugates, while in vitro growth inhibition assays on a panel of human tumor cell lines revealed outstanding cell sensitivity values. Among the nine conjugate ensemble, derivative 21, bearing a robust triazole ring connected to ethylene glycol units by an amide function and showing excellent cell sensitivity properties, was selected for in vivo studies in an ovarian carcinoma model xenografted in immunodeficient mice. Remarkable antitumor activity was attained, superior to that of PTX itself, which was associated with a marked induction of aberrant mitoses, consistent with the mechanism of action of spindle poisons. Overall, the novel cRGD-PTX conjugates disclosed here represent promising candidates for further advancement in the domain of targeted antitumor therapy.

Role of tyrosyl-DNA phosphodiesterase 1 and inter-players in regulation of tumor cell sensitivity to topoisomerase I inhibition.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) plays a unique function as it catalyzes the repair of topoisomerase I-mediated DNA damage. Thus, ovarian carcinoma cell lines exhibiting increased TDP1 levels and resistance to the topoisomerase I poisons campthotecins were used to clarify the role of this enzyme. The camptothecin gimatecan was employed as a tool to inhibit topoisomerase I because it produces a persistent damage. The resistant sublines displayed an increased capability to repair drug-induced single-strand breaks and a reduced amount of drug-induced double-strand breaks, which was enhanced following TDP1 silencing. In loss of function studies using U2-OS cells, we found that TDP1 knockdown did not produce a change in sensitivity to camptothecin, whereas co-silencing of other pathways cooperating with TDP1 in cell response to topoisomerase I poisons indicated that XRCC1 and BRCA1 were major regulators of sensitivity. No change in cellular sensitivity was observed when TDP1 was silenced concomitantly to RAD17, which participates in the stabilization of collapsed replication forks. The expression of dominant-negative PARP1 in cells with reduced expression of TDP1 due to a constitutively expressed TDP1 targeting microRNA did not modulate cell sensitivity to camptothecin. Mild resistance to gimatecan was observed in cells over-expressing TDP1, a feature associated with decreased levels of drug-induced single-strand breaks. In conclusion, since TDP1 alone can account for mild levels of camptothecin resistance, repair of topoisomerase I-mediated DNA damage likely occurs through redundant pathways mainly implicating BRCA1 and XRCC1, but not RAD17 and PARP1. These findings may be relevant to define novel therapeutic strategies.

Ubiquitin-proteasome genes as targets for modulation of cisplatin sensitivity in fission yeast.

BACKGROUND: The ubiquitin(Ub)-proteasome pathway is implicated in the regulation of a variety of cellular functions and plays a major role in stress response in eukaryotic cells, by targeting misfolded and damaged proteins for degradation. In addition, in the presence of DNA damage, the Ub-proteasome system regulates proteins involved in sensing, repairing, and/or tolerating the damage. Antitumor agents such as cisplatin can activate the pathway, but the role of specific pathway components in cell sensitivity/response to the drug is not known. Since platinum compounds represent clinically relevant antitumor agents and a major limitation to their use is the development of drug resistance, there is an urgent need for identifying targets for improving their efficacy. RESULTS: In the present study, we performed a genome-wide screening for sensitivity to cisplatin using non-essential haploid deletion mutants of the fission yeast Schizosaccharomyces pombe, belonging to a collection of haploid strains constructed through homologous recombination. Using this approach, we identified three Ub-proteasome mutants exhibiting hypersensitivity to cisplatin (ubp16, ubc13 and pmt3) and ten mutants (including ufd2, beta7 20S, rpt6/let1) resistant to the drug. In addition, the importance of lub1 gene emerged from the comparison between the present screening and gene expression profile data previously obtained in fission yeast. CONCLUSIONS: The factors identified in the present study allowed us to highlight most finely the close relationship between the Ub-proteasome system and DNA damage response mechanisms, thus establishing a comprehensive framework of regulators likely relevant also in higher eukaryotes. Our results provide the proof of principle of the involvement of specific genes modulated by cisplatin treatment in cell response to the drug, suggesting their potential role as targets for modulating cisplatin sensitivity. In this regard, the prospective identification of novel targets for modulation of cisplatin sensitivity in an eukaryotic model organism appears particularly intriguing towards the discovery of strategies to overcome cisplatin resistance in human tumors.

Proteomic analysis of cellular response to novel proapoptotic agents related to atypical retinoids in human IGROV-1 ovarian carcinoma cells.

Novel agents characterized by the scaffold of the atypical retinoid ST1926, but containing different chemical functions (carboxylic or hydroxamic acid), exhibit potent proapoptotic activity. In the present paper, we show that the treatment of the IGROV-1 ovarian cancer cell line with compounds sharing structural features with ST1926 (ST1898, ST3595, ST3056) determines a strong inhibition of proliferation mainly due to apoptotic cell death. In an effort to understand the mechanism of action of these compounds, we performed a proteomics analysis of IGROV-1 total lysates and nuclear extracts. Using this approach, we found that deregulation of calcium homeostasis, oxidative stress, cytoskeleton reorganization, and deregulation of proteasome function may represent important pathways involved in response of IGROV-1 cells to the studied compounds. The most prominent effect was down-regulation of factors involved in protein degradation, an event more marked in cells treated with ST3595. In addition, we identified proteins specifically modulated by each treatment, including prohibitin and cochaperone P23 (ST1898), pre-mRNA splicing factor SF2p32 and clathrin light chain (ST3595), as well as Far upstream element (FUSE) binding protein 1 and DNA-binding protein B (ST3056). By identifying proteins modulated by novel proapoptotic agents, this study provides insights into critical aspects of their mechanism of action.

Design, synthesis and anticancer activities of stilbene-coumarin hybrid compounds: Identification of novel proapoptotic agents.

The naturally occurring coumarins and resveratrol, attract great attention due to their wide range of biological properties, including anticancer, antileukemic, antibacterial and anti-inflammatory activities; moreover, their cancer chemopreventive property have been recently emphasized. A novel class of hybrid compounds, obtained by introducing a substituted trans-vinylbenzene moiety on a coumarin backbone, was synthesized and evaluated for the antitumor profile. A number of derivatives showed a good antiproliferative activity, in some cases higher to that of the reference compound resveratrol. The most promising compounds in this series were 14 and 17, endowed with excellent antiproliferative and proapoptotic activities. The present study suggests that the 7-methoxycoumarin nucleus, together with the 3,5-disubstitution pattern of the trans-vinylbenzene moiety, are likely promising structural features to obtain excellent antitumor compounds endowed with a apoptosis-inducing capability.