TriplatinNC is a highly positively charged, substitutioninert derivative of the phase II clinical... more TriplatinNC is a highly positively charged, substitutioninert derivative of the phase II clinical anticancer drug, BBR3464. Such substitution-inert complexes form a distinct subset of polynuclear platinum complexes (PPCs) interacting with DNA and other biomolecules through noncovalent interactions. Rapid cellular entry is facilitated via interaction with cell surface glycosoaminoglycans and is a mechanism unique to PPCs. Nanoscale secondary ion mass spectrometry (nanoSIMS) showed rapid distribution within cytoplasmic and nucleolar compartments, but not the nucleus. In this article, the downstream effects of nucleolar localization are described. In human colon carcinoma cells, HCT116, the production rate of 47S rRNA precursor transcripts was dramatically reduced as an early event after drug treatment. Transcriptional inhibition of rRNA was followed by a robust G 1 arrest, and activation of apoptotic proteins caspase-8, -9, and -3 and PARP-1 in a p53-independent manner. Using cell synchronization and flow cytometry, it was determined that cells treated while in G 1 arrest immediately, but cells treated in S or G 2 successfully complete mitosis. Twenty-four hours after treatment, the majority of cells finally arrest in G 1 , but nearly one-third contained highly compacted DNA; a distinct biological feature that cannot be associated with mitosis, senescence, or apoptosis. This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems. The combination of DNA compaction and apoptosis by TriplatinNC treatment conferred striking activity in platinum-resistant and/or p53 mutant or null cell lines. Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation, and novel consequences of high-affinity noncovalent DNA binding, producing a new profile and a further shift in the structure−activity paradigms for antitumor complexes.
The treatment of tumor cells with HDAC inhibitors (HDACi) induces a range of effects including ap... more The treatment of tumor cells with HDAC inhibitors (HDACi) induces a range of effects including apoptosis, cell cycle arrest, differentiation and senescence, modulation of immune response, and altered angiogenesis. The single-agent activities of several HDACi have been tested in preclinical and clinical studies and are currently the subject of ongoing clinical trials. Although HDACi have been shown to be effective as a single agent against a defined subset of hematological tumors, less convincing results have been found in the treatment of solid tumors. Since current clinical trials of single-agent HDACi showed limited efficacy, in this review we focus on drug combinations including HDACi with conventional chemotherapeutic agents and novel targeted agents. Particular emphasis has been devoted to combinations effective in solid tumors and to combinations between HDACi and immunotherapies. An outline of novel combination strategies, including a new generation of more potent and specific HDACi (e.g., compounds with adamantine and noradamantane as scaffolds) as well as chemical hybrid molecules, has been provided. The paradoxical role of HDACs as tumor suppressors in developing tumors and as therapeutic targets in established neoplasms has also been considered.
Drug resistance of tumor cells is recognized as the primary cause of failure of chemotherapeutic ... more Drug resistance of tumor cells is recognized as the primary cause of failure of chemotherapeutic treatment of most human tumors. Although pharmacological factors including inadequate drug concentration at the tumor site can contribute to clinical resistance, cellular factors play a major role in chemoresistance of several tumors. Although manifestations of resistance are conventionally referred to as acquired or intrinsic on the basis of the initial response to the first therapy, a common feature is the development of a phenotype resistant to a variety of structurally and functionally distinct agents. In both manifestations, drug resistance is a multifactorial phenomenon involving multiple interrelated or independent mechanisms. A heterogeneous expression of involved mechanisms may characterize tumors of the same type or cells of the same tumor and may at least in part reflect tumor progression. The relevant mechanisms that can contribute to cellular resistance include: increased expression of defense factors involved in reducing intracellular drug concentration; alterations in drug-target interaction; and changes in cellular response, in particular increased cell ability to repair DNA damage or tolerate stress conditions, and defects in apoptotic pathways. This chapter presents an overview of the drug resistance mechanisms.
We read with great interest the Opinion article by Jamie Fletcher and colleagues, ABC transporter... more We read with great interest the Opinion article by Jamie Fletcher and colleagues, ABC transporters in cancer: more than just drug efflux pumps. Nature Rev. Cancer. 10, 147???156 (2010), recently published in Nature Reviews Cancer 1. The authors underline innovative ...
Poisoning of DNA topoisomerase I is the mechanism by which camptothecins interfere with tumor gro... more Poisoning of DNA topoisomerase I is the mechanism by which camptothecins interfere with tumor growth. Although the clinical use of camptothecins has had a significant impact on cancer therapy, de novo or acquired clinical resistance to these drugs is common. Clinical resistance to camptothecins is still a poorly understood phenomenon, likely involving pharmacological and tumor-related factors. Experimental models including yeast and mammalian cell cultures suggest three general mechanisms of camptothecin resistance: i) reduced cellular accumulation of drugs, ii) alteration in the structure/expression of topoisomerase I, and iii) alterations in the cellular response to camptothecin-DNA-ternary complex formation. Some lines of evidence have also suggested links between cellular camptothecin resistance, the existence of a subset of tumor-initiating cells and miRNA deregulation. In this regard, a better definition of the molecular events clarifying the regulation of tumorigenesis and gene expression might contribute to gain insight into the molecular mechanisms on the basis of camptothecin resistance of tumors and to identify new molecular tools for targeting cancer cells. The relevance of these mechanisms to clinical drug resistance has not yet been completely defined, but their evaluation in clinical specimens should help to define personalized treatments including camptothecins as single agents or in combination with other cytotoxic and target-specific anticancer agents. The present review focuses on the cellular/molecular aspects involved in resistance of tumor cells to camptothecins, including the potential role of cancer stem cells and deregulated miRNAs, and on the approaches proposed for overcoming resistance.
Metastasis is the main reason for lung cancer related mortality but little is known about specifi... more Metastasis is the main reason for lung cancer related mortality but little is known about specific determinants of successful dissemination from primary tumors and metastasis initiation. Here we show that CD133+/CXCR4+ cancer initiating cells (CICs) directly isolated from patient-derived xenografts (PDX) of non-small cell lung cancer (NSCLC) are endowed with superior ability to seed and initiate metastasis at distant organs. We additionally report that CXCR4 inhibition successfully prevents the increase of cisplatin-resistant CD133+/CXCR4+ cells in residual tumors and their metastatization. Immunophenotypic analysis of lung tumor cells intravenously injected or spontaneously disseminated to murine lungs, demonstrates the survival advantage and increased colonization ability of a specific subset of CD133+/CXCR4+ with reduced expression of epithelial cell adhesion molecule (EpCAM-), that also shows the greatest in vitro invasive potential. We next prove that recovered disseminated cel...
The cellular response to the antitumor drug cisplatin is complex, and resistance is widespread. T... more The cellular response to the antitumor drug cisplatin is complex, and resistance is widespread. To gain insights into the global transcriptional response and mechanisms of resistance, we used microarrays to exam- ine the fission yeast cell response to cisplatin. In two iso- genic strains with differing drug sensitivity, cisplatin ac- tivated a stress response involving glutathione-S-trans- ferase, heat shock, and
Platinum drugs have been widely used for the treatment of several solid tumors. Although DNA has ... more Platinum drugs have been widely used for the treatment of several solid tumors. Although DNA has been recognized as the primary cellular target for these agents, there are unresolved issues concerning their effects and the molecular mechanisms underlying the antitumor efficacy. These cytotoxic agents interact with sub-cellular compartments other than the nucleus. Here, we review how such emerging phenomena contribute to the pharmacologic activity as well as to drug resistance phenotypes. DNA-unrelated effects of platinum drugs involve alterations at the plasma membrane and in endo-lysosomal compartments. A direct interaction with the mitochondria also appears to be implicated in drug-induced cell death. Moreover, the pioneering work of a few groups has shown that platinum drugs can act on the tumor microenvironment as well, and potentiate antitumor activity of the immune system. These poorly understood aspects of platinum drug activity sites may be harnessed to enhance their antitumor efficacy. A complete understanding of DNA-unrelated effects of platinum compounds might reveal new aspects of drug resistance allowing the implementation of the antitumor therapeutic efficacy of platinum compound-based regimens and minimization of their toxic side effects.
Medicinal chemistry (Shāriqah (United Arab Emirates)), 2006
The aim of this study was to investigate cellular response to several ruthenium(III), chromium(II... more The aim of this study was to investigate cellular response to several ruthenium(III), chromium(III) and rhodium(III) compounds carrying bidentate beta-diketonato ligands: [(acac)--acetylacetonate ligand, (tfac)--trifluoroacetylacetonate ligand]. Cell sensitivity studies were performed on several cell lines (A2780, cisplatin-sensitive and -resistant U2-OS and U2-OS/Pt, HeLa, B16) using growth-inhibition assay. Effect of intracellular GSH depletion on cell sensitivity to the agents was analyzed in A2780 cells. Flow cytometry was used to assess apoptosis by Annexin-V-FITC/PI staining, and to analyze induction of caspase-3 activity. Possible DNA binding/damaging affinity was investigated, by inductively coupled mass spectrometry, and by 14C-thymidine / 3H-uridine incorporation assay. Cell sensitivity studies showed that the pattern of sensitivity to Ru(tfac)3 complex of the two cisplatin-sensitive/-resistant osteosarcoma cell lines, U2-OS and U2-OS/Pt, was similar to that of A2780 cells...
The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae have b... more The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae have become valuable tools for the study of basic cellular functions of eukaryotic cells, including DNA repair mechanisms and cell cycle control. Since the major signaling pathways and cellular processes involved in cellular response to cytotoxic agents are conserved between yeasts and mammalian cells, these simple eukaryotic systems could be excellent models for the identification of molecular/cellular mechanisms of sensitivity to antitumor drugs. We describe relevant biological features of yeast cells and potential applications derived by their genetic manipulation. In particular, we have outlined the role of genes involved in repair processes and in checkpoint control, with specific reference to genes regulating radiation-sensitivity. Specific examples are provided concerning the use of both yeasts in understanding the mechanism of action of platinum compounds and topoisomerase inhibitors. ...
Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 2000
The p53 gene plays a critical role in cellular response to DNA damage and has been implicated in ... more The p53 gene plays a critical role in cellular response to DNA damage and has been implicated in the response to platinum compounds in ovarian carcinoma patients. Because taxanes could induce p53-independent apoptosis, we assessed the relevance of p53 gene status to response in ovarian carcinoma patients receiving paclitaxel and platinum-containing chemotherapy. Forty-eight previously untreated patients with advanced disease received standard paclitaxel/platinum-based chemotherapy. In tumor specimens collected at the time of initial surgery, before therapy, p53 gene status and expression were examined by single-strand conformation polymorphism, sequence analysis, and immunohistochemical analysis. Microsatellite instability analysis was performed on available samples from 30 patients. Thirty-four (71%) of the 48 patients had a clinical response. Pathologic complete remission was documented in 13 (27%) of 48 patients. p53 mutations were detected in 29 (60%) of 48 tumors. Among the pat...
Multinuclear platinum compounds have been designed to circumvent the cellular resistance to conve... more Multinuclear platinum compounds have been designed to circumvent the cellular resistance to conventional platinum-based drugs. In an attempt to examine the cellular basis of the preclinical antitumor efficacy of a novel multinuclear platinum compound (BBR 3464) in the treatment of cisplatin-resistant tumors, we have performed a comparative study of cisplatin and BBR 3464 in a human osteosarcoma cell line (U2-OS) and in an in vitro selected cisplatin-resistant subline (U2-OS/Pt). A marked increase of cytotoxic potency of BBR 3464 in comparison with cisplatin in U2-OS cells and a complete lack of cross-resistance in U2-OS/Pt cells were found. A detailed analysis of the cisplatin-resistant phenotype indicated that it was associated with reduced cisplatin accumulation, reduced interstrand cross-link (ICL) formation and DNA platination, microsatellite instability, and reduced expression of the DNA mismatch repair protein PMS2. Despite BBR 3464 charge and molecular size, in U2-OS and U2-O...
The aim of our research work was to find modulator of ABCC1/MRP1 protein which is related to the ... more The aim of our research work was to find modulator of ABCC1/MRP1 protein which is related to the multidrug resistance of cancer cells. Thus, concerning the commonly known biological activity of oleanolic acid, we decided to test a group of its semisynthetic derivatives. Materials and Methods: We tested four oleanolic acid derivatives which have been chemically modified comparing to the parental compound base structure at C-3, C-11 and C-28 positions: methyl 3,11-dioxoolean-12-en-28-oate, methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate ( , 12abromo-3-hydroxyiminoolean-28->13-olide and methyl 3,12-dioxo-12a-aza-C-homoolean-28-oate. As an experimental model two cell lines were used: HL-60 (acute promyelocytic leukemia cells) and its multidrug resistant subline HL-60/AR overexpressing ABCC1 gene. MTT test was performed to assess the potency of compounds in reduction of leukemic cells viability. The influence of the compounds on the MRP1 function was checked using fluorescent MRP1 substrate − calcein accumulation and retention assay with cytometric detection method and changes in MRP1 protein level were measured using SDS-PAGE electrophoresis and Western Blot technique. Results: The studied oleanolic acid derivatives showed high activity revealing significant reduction of leukemic cells viability (MTT assay). All of tested compounds were more effective than parental compound. Methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate (HIMOXOL) possessing = NOH group in C-3 position showed even higher activity against multidrug resistant cells than against wild type after 72 h of treatment (IC 50 : 3.17±0.48 mM, 4.68±0.28 mM, respectively). Test assessing the ABCC1 function modulating activity (calcein retention assay) gave the information that HIMOXOL used in 10 mM almost completely blocked calcein efflux from HL-60/AR cells within three hours after the loading of the cells with calcein-AM. Calcein accumulation test confirmed effectiveness of the compound. Moreover 24 h treatment of HL-60/AR cells with 5, 10 and 20 mM HIMOXOL yielded dose dependent reduction of MRP1 protein level. Conclusion: Obtained results prove that oleanolic acid derivative − methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate is efficient ABCC1/MRP1 modulator able to decrease level of MRP1 protein and inhibit transport of its substrate out of the cell.
TriplatinNC is a highly positively charged, substitutioninert derivative of the phase II clinical... more TriplatinNC is a highly positively charged, substitutioninert derivative of the phase II clinical anticancer drug, BBR3464. Such substitution-inert complexes form a distinct subset of polynuclear platinum complexes (PPCs) interacting with DNA and other biomolecules through noncovalent interactions. Rapid cellular entry is facilitated via interaction with cell surface glycosoaminoglycans and is a mechanism unique to PPCs. Nanoscale secondary ion mass spectrometry (nanoSIMS) showed rapid distribution within cytoplasmic and nucleolar compartments, but not the nucleus. In this article, the downstream effects of nucleolar localization are described. In human colon carcinoma cells, HCT116, the production rate of 47S rRNA precursor transcripts was dramatically reduced as an early event after drug treatment. Transcriptional inhibition of rRNA was followed by a robust G 1 arrest, and activation of apoptotic proteins caspase-8, -9, and -3 and PARP-1 in a p53-independent manner. Using cell synchronization and flow cytometry, it was determined that cells treated while in G 1 arrest immediately, but cells treated in S or G 2 successfully complete mitosis. Twenty-four hours after treatment, the majority of cells finally arrest in G 1 , but nearly one-third contained highly compacted DNA; a distinct biological feature that cannot be associated with mitosis, senescence, or apoptosis. This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems. The combination of DNA compaction and apoptosis by TriplatinNC treatment conferred striking activity in platinum-resistant and/or p53 mutant or null cell lines. Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation, and novel consequences of high-affinity noncovalent DNA binding, producing a new profile and a further shift in the structure−activity paradigms for antitumor complexes.
The rapid evolution of techniques for measuring gene expression makes available substantial data ... more The rapid evolution of techniques for measuring gene expression makes available substantial data which require careful analysis. In particular, relative quantification based on microfluidic cards allows performing of rapid large scale analyses. In the present study, we employed ovarian carcinoma cell lines resistant to cisplatin (IGROV-1/Pt1) or to a camptothecin (IGROV-1CPT/L), both characterized by a complex pattern of resistance to multiple agents, to examine the expression of genes of the superfamily of ATP binding-cassette (ABC) transporters by TaqMan microfluidic cards with the aim of developing an analytical tool to process data in this particular framework. The transcript quantification was based on the comparative threshold cycle method, which compares the expression of a target gene normalized to the expression of one or more reference genes (relative quantification). To process expression of ABC transporters, we applied a statistical procedure based on multivariate approaches and re-sampling techniques. The transporters that were significantly modulated included members of the ABCA, ABCB, ABCC and ABCG subgroups. A consistent up-regulation of ABCC2 as compared with the parental IGROV-1 cell line was observed in the IGROV-1/Pt1 cells, whereas down-regulation of ABCC6 and ABCG1 was found in IGROV-1/CPT-L cells. The use of rigorous analytic tools for gene expression data in preclinical models may lead to the identification of signatures to test in ovarian carcinoma clinical samples. Moreover, the developed procedure may be useful in the analysis of relative quantification data obtained with microfluidic cards in different experimental settings.
Non-Small-Cell Lung Cancer (NSCLC) remains an aggressive and fatal disease with low responsivenes... more Non-Small-Cell Lung Cancer (NSCLC) remains an aggressive and fatal disease with low responsiveness to chemotherapy, frequent drug resistance development and metastatic behavior. Platinum-based therapy is the standard of care for NSCLC with limited benefits. Since epigenetic alterations have been implicated in the aggressive behaviour of lung cancer, the purpose of the present study was to examine the capability of the pan-histone deacetylase inhibitor SAHA and of ST3595, a novel hydroxamate-based compound, to interfere with proliferative and invasive potential of NSCLC cells. We used two NSCLC cell lines (H460 and A549) and the cisplatin-resistant variants (H460/Pt, A549/Pt), to mimic a frequent clinical condition. The resistant models exhibited increased invasive properties as compared to parental cells, features associated with a wide modulation of the level of angiogenesis- and invasion-related factors in the cell conditioned media. The levels of urokinase-type plasminogen activator, IL8, and macrophage migration inhibitory factor were increased in the conditioned media from both H460/Pt and A549/Pt cells. SAHA and ST3595 induced a strong inhibition of cell invasive properties, which was more marked after ST3595 exposure. Both HDAC inhibitors up-regulated the metastasis suppressor KiSS1 at the mRNA level. Forced expression of KiSS1 significantly decreased the invasive capability of drug-resistant cells. ST3595 displayed an anti-metastatic effect in tumors associated with decreased of phosphorylation of Src. Our data indicate that HDAC inhibitors are effective in NSCLC cell systems. The ability of ST3595 to counteract the invasive potential of resistant cells through mechanisms involving KiSS1 is an interesting novel finding.
Ovarian cancer is the main cause of death from gynaecological malignancies. In spite of the effic... more Ovarian cancer is the main cause of death from gynaecological malignancies. In spite of the efficacy of platinum-paclitaxel treatment in patients with primary epithelial ovarian carcinoma, platinum-based chemotherapy is not curative and resistance remains one of the most important causes of treatment failure. Although ABC transporters have been implicated in cellular resistance to multiple drugs, the clinical relevance of these efflux pumps is still poorly understood. Thus, we examined the prognostic role of transporters of the MRP family (i.e., ABCC1/MRP1, ABCC4/MRP4) to gain insights into their clinical impacts. A case material of 127 patients with ovarian carcinoma at different stages and histotypes was used. The expression of MRP1 and MRP4 was examined by immunohistochemistry using tissue microarrays in tumor specimens collected at the time of initial surgery expression. We found an association between MRP1 expression and grading, and we observed that MRP4 displayed an unfavourable impact on disease relapse in multivariate analysis (HR = 2.05, 95% CI: 1.01-4.11; = 0.045). These results suggest that in epithelial ovarian cancer, MRP1 may be a marker for aggressiveness because its expression was associated with tumor grade and support that MRP4 may play an unfavourable role in disease outcome.
Proceedings of the National Academy of Sciences, 2009
The identification of lung tumor-initiating cells and associated markers may be useful for optimi... more The identification of lung tumor-initiating cells and associated markers may be useful for optimization of therapeutic approaches and for predictive and prognostic information in lung cancer patients. CD133, a surface glycoprotein linked to organ-specific stem cells, was described as a marker of cancer-initiating cells in different tumor types. Here, we report that a CD133 ؉ , epithelialspecific antigen-positive (CD133 ؉ ESA ؉ ) population is increased in primary nonsmall cell lung cancer (NSCLC) compared with normal lung tissue and has higher tumorigenic potential in SCID mice and expression of genes involved in stemness, adhesion, motility, and drug efflux than the CD133 ؊ counterpart. Cisplatin treatment of lung cancer cells in vitro resulted in enrichment of CD133 ؉ fraction both after acute cytotoxic exposure and in cells with stable cisplatin-resistant phenotype. Subpopulations of CD133 ؉ ABCG2 ؉ and CD133 ؉ CXCR4 ؉ cells were spared by in vivo cisplatin treatment of lung tumor xenografts established from primary tumors. A tendency toward shorter progression-free survival was observed in CD133 ؉ NSCLC patients treated with platinum-containing regimens. Our results indicate that chemoresistant populations with highly tumorigenic and stem-like features are present in lung tumors. The molecular features of these cells may provide the rationale for more specific therapeutic targeting and the definition of predictive factors in clinical management of this lethal disease.
TriplatinNC is a highly positively charged, substitutioninert derivative of the phase II clinical... more TriplatinNC is a highly positively charged, substitutioninert derivative of the phase II clinical anticancer drug, BBR3464. Such substitution-inert complexes form a distinct subset of polynuclear platinum complexes (PPCs) interacting with DNA and other biomolecules through noncovalent interactions. Rapid cellular entry is facilitated via interaction with cell surface glycosoaminoglycans and is a mechanism unique to PPCs. Nanoscale secondary ion mass spectrometry (nanoSIMS) showed rapid distribution within cytoplasmic and nucleolar compartments, but not the nucleus. In this article, the downstream effects of nucleolar localization are described. In human colon carcinoma cells, HCT116, the production rate of 47S rRNA precursor transcripts was dramatically reduced as an early event after drug treatment. Transcriptional inhibition of rRNA was followed by a robust G 1 arrest, and activation of apoptotic proteins caspase-8, -9, and -3 and PARP-1 in a p53-independent manner. Using cell synchronization and flow cytometry, it was determined that cells treated while in G 1 arrest immediately, but cells treated in S or G 2 successfully complete mitosis. Twenty-four hours after treatment, the majority of cells finally arrest in G 1 , but nearly one-third contained highly compacted DNA; a distinct biological feature that cannot be associated with mitosis, senescence, or apoptosis. This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems. The combination of DNA compaction and apoptosis by TriplatinNC treatment conferred striking activity in platinum-resistant and/or p53 mutant or null cell lines. Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation, and novel consequences of high-affinity noncovalent DNA binding, producing a new profile and a further shift in the structure−activity paradigms for antitumor complexes.
The treatment of tumor cells with HDAC inhibitors (HDACi) induces a range of effects including ap... more The treatment of tumor cells with HDAC inhibitors (HDACi) induces a range of effects including apoptosis, cell cycle arrest, differentiation and senescence, modulation of immune response, and altered angiogenesis. The single-agent activities of several HDACi have been tested in preclinical and clinical studies and are currently the subject of ongoing clinical trials. Although HDACi have been shown to be effective as a single agent against a defined subset of hematological tumors, less convincing results have been found in the treatment of solid tumors. Since current clinical trials of single-agent HDACi showed limited efficacy, in this review we focus on drug combinations including HDACi with conventional chemotherapeutic agents and novel targeted agents. Particular emphasis has been devoted to combinations effective in solid tumors and to combinations between HDACi and immunotherapies. An outline of novel combination strategies, including a new generation of more potent and specific HDACi (e.g., compounds with adamantine and noradamantane as scaffolds) as well as chemical hybrid molecules, has been provided. The paradoxical role of HDACs as tumor suppressors in developing tumors and as therapeutic targets in established neoplasms has also been considered.
Drug resistance of tumor cells is recognized as the primary cause of failure of chemotherapeutic ... more Drug resistance of tumor cells is recognized as the primary cause of failure of chemotherapeutic treatment of most human tumors. Although pharmacological factors including inadequate drug concentration at the tumor site can contribute to clinical resistance, cellular factors play a major role in chemoresistance of several tumors. Although manifestations of resistance are conventionally referred to as acquired or intrinsic on the basis of the initial response to the first therapy, a common feature is the development of a phenotype resistant to a variety of structurally and functionally distinct agents. In both manifestations, drug resistance is a multifactorial phenomenon involving multiple interrelated or independent mechanisms. A heterogeneous expression of involved mechanisms may characterize tumors of the same type or cells of the same tumor and may at least in part reflect tumor progression. The relevant mechanisms that can contribute to cellular resistance include: increased expression of defense factors involved in reducing intracellular drug concentration; alterations in drug-target interaction; and changes in cellular response, in particular increased cell ability to repair DNA damage or tolerate stress conditions, and defects in apoptotic pathways. This chapter presents an overview of the drug resistance mechanisms.
We read with great interest the Opinion article by Jamie Fletcher and colleagues, ABC transporter... more We read with great interest the Opinion article by Jamie Fletcher and colleagues, ABC transporters in cancer: more than just drug efflux pumps. Nature Rev. Cancer. 10, 147???156 (2010), recently published in Nature Reviews Cancer 1. The authors underline innovative ...
Poisoning of DNA topoisomerase I is the mechanism by which camptothecins interfere with tumor gro... more Poisoning of DNA topoisomerase I is the mechanism by which camptothecins interfere with tumor growth. Although the clinical use of camptothecins has had a significant impact on cancer therapy, de novo or acquired clinical resistance to these drugs is common. Clinical resistance to camptothecins is still a poorly understood phenomenon, likely involving pharmacological and tumor-related factors. Experimental models including yeast and mammalian cell cultures suggest three general mechanisms of camptothecin resistance: i) reduced cellular accumulation of drugs, ii) alteration in the structure/expression of topoisomerase I, and iii) alterations in the cellular response to camptothecin-DNA-ternary complex formation. Some lines of evidence have also suggested links between cellular camptothecin resistance, the existence of a subset of tumor-initiating cells and miRNA deregulation. In this regard, a better definition of the molecular events clarifying the regulation of tumorigenesis and gene expression might contribute to gain insight into the molecular mechanisms on the basis of camptothecin resistance of tumors and to identify new molecular tools for targeting cancer cells. The relevance of these mechanisms to clinical drug resistance has not yet been completely defined, but their evaluation in clinical specimens should help to define personalized treatments including camptothecins as single agents or in combination with other cytotoxic and target-specific anticancer agents. The present review focuses on the cellular/molecular aspects involved in resistance of tumor cells to camptothecins, including the potential role of cancer stem cells and deregulated miRNAs, and on the approaches proposed for overcoming resistance.
Metastasis is the main reason for lung cancer related mortality but little is known about specifi... more Metastasis is the main reason for lung cancer related mortality but little is known about specific determinants of successful dissemination from primary tumors and metastasis initiation. Here we show that CD133+/CXCR4+ cancer initiating cells (CICs) directly isolated from patient-derived xenografts (PDX) of non-small cell lung cancer (NSCLC) are endowed with superior ability to seed and initiate metastasis at distant organs. We additionally report that CXCR4 inhibition successfully prevents the increase of cisplatin-resistant CD133+/CXCR4+ cells in residual tumors and their metastatization. Immunophenotypic analysis of lung tumor cells intravenously injected or spontaneously disseminated to murine lungs, demonstrates the survival advantage and increased colonization ability of a specific subset of CD133+/CXCR4+ with reduced expression of epithelial cell adhesion molecule (EpCAM-), that also shows the greatest in vitro invasive potential. We next prove that recovered disseminated cel...
The cellular response to the antitumor drug cisplatin is complex, and resistance is widespread. T... more The cellular response to the antitumor drug cisplatin is complex, and resistance is widespread. To gain insights into the global transcriptional response and mechanisms of resistance, we used microarrays to exam- ine the fission yeast cell response to cisplatin. In two iso- genic strains with differing drug sensitivity, cisplatin ac- tivated a stress response involving glutathione-S-trans- ferase, heat shock, and
Platinum drugs have been widely used for the treatment of several solid tumors. Although DNA has ... more Platinum drugs have been widely used for the treatment of several solid tumors. Although DNA has been recognized as the primary cellular target for these agents, there are unresolved issues concerning their effects and the molecular mechanisms underlying the antitumor efficacy. These cytotoxic agents interact with sub-cellular compartments other than the nucleus. Here, we review how such emerging phenomena contribute to the pharmacologic activity as well as to drug resistance phenotypes. DNA-unrelated effects of platinum drugs involve alterations at the plasma membrane and in endo-lysosomal compartments. A direct interaction with the mitochondria also appears to be implicated in drug-induced cell death. Moreover, the pioneering work of a few groups has shown that platinum drugs can act on the tumor microenvironment as well, and potentiate antitumor activity of the immune system. These poorly understood aspects of platinum drug activity sites may be harnessed to enhance their antitumor efficacy. A complete understanding of DNA-unrelated effects of platinum compounds might reveal new aspects of drug resistance allowing the implementation of the antitumor therapeutic efficacy of platinum compound-based regimens and minimization of their toxic side effects.
Medicinal chemistry (Shāriqah (United Arab Emirates)), 2006
The aim of this study was to investigate cellular response to several ruthenium(III), chromium(II... more The aim of this study was to investigate cellular response to several ruthenium(III), chromium(III) and rhodium(III) compounds carrying bidentate beta-diketonato ligands: [(acac)--acetylacetonate ligand, (tfac)--trifluoroacetylacetonate ligand]. Cell sensitivity studies were performed on several cell lines (A2780, cisplatin-sensitive and -resistant U2-OS and U2-OS/Pt, HeLa, B16) using growth-inhibition assay. Effect of intracellular GSH depletion on cell sensitivity to the agents was analyzed in A2780 cells. Flow cytometry was used to assess apoptosis by Annexin-V-FITC/PI staining, and to analyze induction of caspase-3 activity. Possible DNA binding/damaging affinity was investigated, by inductively coupled mass spectrometry, and by 14C-thymidine / 3H-uridine incorporation assay. Cell sensitivity studies showed that the pattern of sensitivity to Ru(tfac)3 complex of the two cisplatin-sensitive/-resistant osteosarcoma cell lines, U2-OS and U2-OS/Pt, was similar to that of A2780 cells...
The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae have b... more The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae have become valuable tools for the study of basic cellular functions of eukaryotic cells, including DNA repair mechanisms and cell cycle control. Since the major signaling pathways and cellular processes involved in cellular response to cytotoxic agents are conserved between yeasts and mammalian cells, these simple eukaryotic systems could be excellent models for the identification of molecular/cellular mechanisms of sensitivity to antitumor drugs. We describe relevant biological features of yeast cells and potential applications derived by their genetic manipulation. In particular, we have outlined the role of genes involved in repair processes and in checkpoint control, with specific reference to genes regulating radiation-sensitivity. Specific examples are provided concerning the use of both yeasts in understanding the mechanism of action of platinum compounds and topoisomerase inhibitors. ...
Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 2000
The p53 gene plays a critical role in cellular response to DNA damage and has been implicated in ... more The p53 gene plays a critical role in cellular response to DNA damage and has been implicated in the response to platinum compounds in ovarian carcinoma patients. Because taxanes could induce p53-independent apoptosis, we assessed the relevance of p53 gene status to response in ovarian carcinoma patients receiving paclitaxel and platinum-containing chemotherapy. Forty-eight previously untreated patients with advanced disease received standard paclitaxel/platinum-based chemotherapy. In tumor specimens collected at the time of initial surgery, before therapy, p53 gene status and expression were examined by single-strand conformation polymorphism, sequence analysis, and immunohistochemical analysis. Microsatellite instability analysis was performed on available samples from 30 patients. Thirty-four (71%) of the 48 patients had a clinical response. Pathologic complete remission was documented in 13 (27%) of 48 patients. p53 mutations were detected in 29 (60%) of 48 tumors. Among the pat...
Multinuclear platinum compounds have been designed to circumvent the cellular resistance to conve... more Multinuclear platinum compounds have been designed to circumvent the cellular resistance to conventional platinum-based drugs. In an attempt to examine the cellular basis of the preclinical antitumor efficacy of a novel multinuclear platinum compound (BBR 3464) in the treatment of cisplatin-resistant tumors, we have performed a comparative study of cisplatin and BBR 3464 in a human osteosarcoma cell line (U2-OS) and in an in vitro selected cisplatin-resistant subline (U2-OS/Pt). A marked increase of cytotoxic potency of BBR 3464 in comparison with cisplatin in U2-OS cells and a complete lack of cross-resistance in U2-OS/Pt cells were found. A detailed analysis of the cisplatin-resistant phenotype indicated that it was associated with reduced cisplatin accumulation, reduced interstrand cross-link (ICL) formation and DNA platination, microsatellite instability, and reduced expression of the DNA mismatch repair protein PMS2. Despite BBR 3464 charge and molecular size, in U2-OS and U2-O...
The aim of our research work was to find modulator of ABCC1/MRP1 protein which is related to the ... more The aim of our research work was to find modulator of ABCC1/MRP1 protein which is related to the multidrug resistance of cancer cells. Thus, concerning the commonly known biological activity of oleanolic acid, we decided to test a group of its semisynthetic derivatives. Materials and Methods: We tested four oleanolic acid derivatives which have been chemically modified comparing to the parental compound base structure at C-3, C-11 and C-28 positions: methyl 3,11-dioxoolean-12-en-28-oate, methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate ( , 12abromo-3-hydroxyiminoolean-28->13-olide and methyl 3,12-dioxo-12a-aza-C-homoolean-28-oate. As an experimental model two cell lines were used: HL-60 (acute promyelocytic leukemia cells) and its multidrug resistant subline HL-60/AR overexpressing ABCC1 gene. MTT test was performed to assess the potency of compounds in reduction of leukemic cells viability. The influence of the compounds on the MRP1 function was checked using fluorescent MRP1 substrate − calcein accumulation and retention assay with cytometric detection method and changes in MRP1 protein level were measured using SDS-PAGE electrophoresis and Western Blot technique. Results: The studied oleanolic acid derivatives showed high activity revealing significant reduction of leukemic cells viability (MTT assay). All of tested compounds were more effective than parental compound. Methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate (HIMOXOL) possessing = NOH group in C-3 position showed even higher activity against multidrug resistant cells than against wild type after 72 h of treatment (IC 50 : 3.17±0.48 mM, 4.68±0.28 mM, respectively). Test assessing the ABCC1 function modulating activity (calcein retention assay) gave the information that HIMOXOL used in 10 mM almost completely blocked calcein efflux from HL-60/AR cells within three hours after the loading of the cells with calcein-AM. Calcein accumulation test confirmed effectiveness of the compound. Moreover 24 h treatment of HL-60/AR cells with 5, 10 and 20 mM HIMOXOL yielded dose dependent reduction of MRP1 protein level. Conclusion: Obtained results prove that oleanolic acid derivative − methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate is efficient ABCC1/MRP1 modulator able to decrease level of MRP1 protein and inhibit transport of its substrate out of the cell.
TriplatinNC is a highly positively charged, substitutioninert derivative of the phase II clinical... more TriplatinNC is a highly positively charged, substitutioninert derivative of the phase II clinical anticancer drug, BBR3464. Such substitution-inert complexes form a distinct subset of polynuclear platinum complexes (PPCs) interacting with DNA and other biomolecules through noncovalent interactions. Rapid cellular entry is facilitated via interaction with cell surface glycosoaminoglycans and is a mechanism unique to PPCs. Nanoscale secondary ion mass spectrometry (nanoSIMS) showed rapid distribution within cytoplasmic and nucleolar compartments, but not the nucleus. In this article, the downstream effects of nucleolar localization are described. In human colon carcinoma cells, HCT116, the production rate of 47S rRNA precursor transcripts was dramatically reduced as an early event after drug treatment. Transcriptional inhibition of rRNA was followed by a robust G 1 arrest, and activation of apoptotic proteins caspase-8, -9, and -3 and PARP-1 in a p53-independent manner. Using cell synchronization and flow cytometry, it was determined that cells treated while in G 1 arrest immediately, but cells treated in S or G 2 successfully complete mitosis. Twenty-four hours after treatment, the majority of cells finally arrest in G 1 , but nearly one-third contained highly compacted DNA; a distinct biological feature that cannot be associated with mitosis, senescence, or apoptosis. This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems. The combination of DNA compaction and apoptosis by TriplatinNC treatment conferred striking activity in platinum-resistant and/or p53 mutant or null cell lines. Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation, and novel consequences of high-affinity noncovalent DNA binding, producing a new profile and a further shift in the structure−activity paradigms for antitumor complexes.
The rapid evolution of techniques for measuring gene expression makes available substantial data ... more The rapid evolution of techniques for measuring gene expression makes available substantial data which require careful analysis. In particular, relative quantification based on microfluidic cards allows performing of rapid large scale analyses. In the present study, we employed ovarian carcinoma cell lines resistant to cisplatin (IGROV-1/Pt1) or to a camptothecin (IGROV-1CPT/L), both characterized by a complex pattern of resistance to multiple agents, to examine the expression of genes of the superfamily of ATP binding-cassette (ABC) transporters by TaqMan microfluidic cards with the aim of developing an analytical tool to process data in this particular framework. The transcript quantification was based on the comparative threshold cycle method, which compares the expression of a target gene normalized to the expression of one or more reference genes (relative quantification). To process expression of ABC transporters, we applied a statistical procedure based on multivariate approaches and re-sampling techniques. The transporters that were significantly modulated included members of the ABCA, ABCB, ABCC and ABCG subgroups. A consistent up-regulation of ABCC2 as compared with the parental IGROV-1 cell line was observed in the IGROV-1/Pt1 cells, whereas down-regulation of ABCC6 and ABCG1 was found in IGROV-1/CPT-L cells. The use of rigorous analytic tools for gene expression data in preclinical models may lead to the identification of signatures to test in ovarian carcinoma clinical samples. Moreover, the developed procedure may be useful in the analysis of relative quantification data obtained with microfluidic cards in different experimental settings.
Non-Small-Cell Lung Cancer (NSCLC) remains an aggressive and fatal disease with low responsivenes... more Non-Small-Cell Lung Cancer (NSCLC) remains an aggressive and fatal disease with low responsiveness to chemotherapy, frequent drug resistance development and metastatic behavior. Platinum-based therapy is the standard of care for NSCLC with limited benefits. Since epigenetic alterations have been implicated in the aggressive behaviour of lung cancer, the purpose of the present study was to examine the capability of the pan-histone deacetylase inhibitor SAHA and of ST3595, a novel hydroxamate-based compound, to interfere with proliferative and invasive potential of NSCLC cells. We used two NSCLC cell lines (H460 and A549) and the cisplatin-resistant variants (H460/Pt, A549/Pt), to mimic a frequent clinical condition. The resistant models exhibited increased invasive properties as compared to parental cells, features associated with a wide modulation of the level of angiogenesis- and invasion-related factors in the cell conditioned media. The levels of urokinase-type plasminogen activator, IL8, and macrophage migration inhibitory factor were increased in the conditioned media from both H460/Pt and A549/Pt cells. SAHA and ST3595 induced a strong inhibition of cell invasive properties, which was more marked after ST3595 exposure. Both HDAC inhibitors up-regulated the metastasis suppressor KiSS1 at the mRNA level. Forced expression of KiSS1 significantly decreased the invasive capability of drug-resistant cells. ST3595 displayed an anti-metastatic effect in tumors associated with decreased of phosphorylation of Src. Our data indicate that HDAC inhibitors are effective in NSCLC cell systems. The ability of ST3595 to counteract the invasive potential of resistant cells through mechanisms involving KiSS1 is an interesting novel finding.
Ovarian cancer is the main cause of death from gynaecological malignancies. In spite of the effic... more Ovarian cancer is the main cause of death from gynaecological malignancies. In spite of the efficacy of platinum-paclitaxel treatment in patients with primary epithelial ovarian carcinoma, platinum-based chemotherapy is not curative and resistance remains one of the most important causes of treatment failure. Although ABC transporters have been implicated in cellular resistance to multiple drugs, the clinical relevance of these efflux pumps is still poorly understood. Thus, we examined the prognostic role of transporters of the MRP family (i.e., ABCC1/MRP1, ABCC4/MRP4) to gain insights into their clinical impacts. A case material of 127 patients with ovarian carcinoma at different stages and histotypes was used. The expression of MRP1 and MRP4 was examined by immunohistochemistry using tissue microarrays in tumor specimens collected at the time of initial surgery expression. We found an association between MRP1 expression and grading, and we observed that MRP4 displayed an unfavourable impact on disease relapse in multivariate analysis (HR = 2.05, 95% CI: 1.01-4.11; = 0.045). These results suggest that in epithelial ovarian cancer, MRP1 may be a marker for aggressiveness because its expression was associated with tumor grade and support that MRP4 may play an unfavourable role in disease outcome.
Proceedings of the National Academy of Sciences, 2009
The identification of lung tumor-initiating cells and associated markers may be useful for optimi... more The identification of lung tumor-initiating cells and associated markers may be useful for optimization of therapeutic approaches and for predictive and prognostic information in lung cancer patients. CD133, a surface glycoprotein linked to organ-specific stem cells, was described as a marker of cancer-initiating cells in different tumor types. Here, we report that a CD133 ؉ , epithelialspecific antigen-positive (CD133 ؉ ESA ؉ ) population is increased in primary nonsmall cell lung cancer (NSCLC) compared with normal lung tissue and has higher tumorigenic potential in SCID mice and expression of genes involved in stemness, adhesion, motility, and drug efflux than the CD133 ؊ counterpart. Cisplatin treatment of lung cancer cells in vitro resulted in enrichment of CD133 ؉ fraction both after acute cytotoxic exposure and in cells with stable cisplatin-resistant phenotype. Subpopulations of CD133 ؉ ABCG2 ؉ and CD133 ؉ CXCR4 ؉ cells were spared by in vivo cisplatin treatment of lung tumor xenografts established from primary tumors. A tendency toward shorter progression-free survival was observed in CD133 ؉ NSCLC patients treated with platinum-containing regimens. Our results indicate that chemoresistant populations with highly tumorigenic and stem-like features are present in lung tumors. The molecular features of these cells may provide the rationale for more specific therapeutic targeting and the definition of predictive factors in clinical management of this lethal disease.
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Papers by Laura Gatti