ABSTRACT
Integrins are associated with tumour cell survival and progression, and their expression has been shown to be increased in tumours. Thus, four novel conjugates of the tripeptide integrin ligand Arg-Gly-Asp (RGD) and the cytotoxic agent paclitaxel (cRGD-PTX) were prepared to investigate the potential of the multivalent presentation of the RGD moiety in improving the antitumor efficacy of PTX by tumour targeting. PTX was conjugated to two or four integrin recognizing ligands. The influence of multivalent presentation on in vitro αvß3-receptor affinity was confirmed. For all the conjugates compared to the previously synthesized monovalent counterparts, an enhancement of the binding strength was observed; this behaviour was more pronounced when considering the tetravalent presented RGD-conjugate. Cell growth inhibition assays on a panel of human tumour cell lines showed remarkable cytotoxic activity for all conjugates with IC50 values in a nanomolar range. Among the four conjugates, the bivalent derivative 3b was selected for in vivo studies in an ovarian carcinoma cell model xenografted in immunodeficient mice. A marked antitumor activity was observed, similar to that of PTX, but with a much more favourable toxicity profile. Overall, the novel cRGD-PTX conjugates disclosed here represent promising candidates for further advancement in the domain of targeted anti-tumour therapy.
Subject(s)
Drug Delivery Systems , Drug Design , Paclitaxel/chemical synthesis , Paclitaxel/pharmacology , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/pharmacology , Protein Multimerization , Animals , Antineoplastic Agents/pharmacology , Biotinylation , Cell Line, Tumor , Female , Humans , Integrin alphaVbeta3/metabolism , Mice, Nude , Peptides, Cyclic/chemistry , Vitronectin/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Since response to platinum-based therapy in non-small-cell lung cancer (NSCLC) is poor, the present study was designed to rationally identify novel drug combinations in cell models including the A549 cell line and the cisplatin-resistant subline A549/Pt, characterized by reduced sensitivity to cisplatin-induced apoptosis and by upregulation of efflux transporters of the ATP binding cassette (ABC) superfamily. Given the molecular features of these cells, we focused on compounds triggering apoptosis through different mechanisms, such as the mitochondria-targeting drug arsenic trioxide and the phenanthridine analog sanguinarine, which induce apoptosis through the extrinsic pathway. Sanguinarine, not recognized by ABC transporters, could overcome cisplatin resistance and, when used in combination with arsenic trioxide, was synergistic in A549 and A549/Pt cells. The arsenic trioxide/sanguinarine cotreatment upregulated genes implicated in apoptosis activation through the extrinsic pathway. Drug combination experiments indicated that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment improved arsenic trioxide/sanguinarine efficacy, a feature associated with a striking apoptosis induction, particularly in the cisplatin-resistant variant. Thus, a synergistic interaction between sanguinarine and arsenic trioxide could be obtained independent of relative cell sensitivity to arsenic trioxide, and an enhanced apoptosis induction could be achieved in combination with TRAIL through modulation of the extrinsic apoptotic pathway. Antitumor activity studies supported the interest of drug combinations including TRAIL in NSCLC, indicating that drug-resistant NSCLC cells can efficiently be killed by the combination of proapoptotic agents. Our results suggest that the molecular changes occurring in treated cells may be exploited to rationally hit surviving cells.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Arsenicals/pharmacology , Benzophenanthridines/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Isoquinolines/pharmacology , Lung Neoplasms/drug therapy , Oxides/pharmacology , TNF-Related Apoptosis-Inducing Ligand/metabolism , Arsenic Trioxide , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cisplatin/pharmacology , DNA Damage , Drug Resistance, Neoplasm , Drug Synergism , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacologyABSTRACT
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 (72 h exposure), with the IC50 being around 40 microM. The growth-inhibitory effect of Ru(acac)3 ranged over 100 microM, while Cr(III) and Rh(III) complexes were completely devoid of antitumor action in vitro. Ru(tfac)3 exhibited strong potential for apoptosis induction on A2780 cells (up to 40%) and caused cell cycle arrest in the S phase as well as decrease of the percent of G1 and G2 cells. Ru(acac)3-induced apoptosis was slightly higher than 10%, whereas activation of caspase-3 in HeLa cells was moderate. DNA binding study revealed that only Cr(acac)3 was capable of binding DNA, while Cr(III) and Ru(III) compounds possess potential to inhibit DNA/RNA synthesis. In conclusion, only Ru(III) complexes showed potential for antitumor action.
Subject(s)
Apoptosis/drug effects , Cell Cycle/drug effects , Chromium/pharmacology , Rhodium/pharmacology , Ruthenium/pharmacology , Caspase 3 , Caspases/metabolism , Cell Line, Tumor , DNA, Neoplasm/biosynthesis , DNA, Neoplasm/drug effects , Flow Cytometry , Humans , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/drug effectsABSTRACT
To evaluate the relevance of fragile histidine triad (FHIT) status in relation to drug treatment, we analyzed the sensitivity of the Fhit-negative non-small cell lung cancer (NSCLC) cell line NCI-H460 to different drugs, after treatment with an adenoviral vector expressing the FHIT transgene. Expression of Fhit resulted in reduced sensitivity to etoposide, doxorubicin, and topotecan. This feature was associated with Fhit-induced downregulation of DNA topoisomerases I and II. In contrast, expression of Fhit did not modulate sensitivity to Taxol, but produced a slight increase in sensitivity to cisplatin, as shown by colony-forming assays. Analysis of apoptosis revealed that, after cisplatin exposure, the number of apoptotic cells was two-fold higher in Fhit-expressing H460 cells. Moreover, it appeared that wildtype p53 was required for sensitization to cisplatin because the effect was marginal in A549 and Calu-1 cells, where the p53 pathway is altered and simultaneous restoration of p53 and Fhit in Calu-1 cells increased cisplatin sensitivity. Fhit could also partially restore sensitivity to cisplatin in Bcl-2- and Bcl-x(L)-overexpressing H460 cells that are normally resistant to this drug. Our results support the possible relevance of FHIT in cisplatin-based chemotherapy as well as in the reversal of drug resistance in NSCLC.
Subject(s)
Acid Anhydride Hydrolases/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Cisplatin/pharmacology , Gene Expression Regulation, Neoplastic , Gene Transfer Techniques , Lung Neoplasms/drug therapy , Neoplasm Proteins/genetics , Antineoplastic Agents/pharmacology , Apoptosis , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Resistance , Humans , In Situ Nick-End Labeling , TransgenesABSTRACT
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 examine the fission yeast cell response to cisplatin. In two isogenic strains with differing drug sensitivity, cisplatin activated a stress response involving glutathione-S-transferase, heat shock, and recombinational repair genes. Genes required for proteasome-mediated protein degradation were up-regulated in the sensitive strain, whereas genes for DNA damage recognition/repair and for mitotic progression were induced in the resistant strain. The response to cisplatin overlaps in part with the responses to cadmium and the DNA-damaging agent methylmethane sulfonate. The different gene groups involved in the cellular response to cisplatin help the cells to tolerate and repair DNA damage and to overcome cell cycle blocks. These findings are discussed with respect to known cisplatin response pathways in human cells.
Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Schizosaccharomyces/drug effects , DNA Damage , Drug Resistance, Fungal , Gene Expression/drug effects , Hydrogen-Ion Concentration , Schizosaccharomyces/genetics , Transcription, Genetic/drug effectsABSTRACT
We used cDNA arrays to monitor modulation of mRNA expression after exposure to a multinuclear platinum complex (BBR3464) in a human cervix squamous cell carcinoma cell line (A431) and in a cisplatin-resistant subline (A431/Pt) exhibiting collateral sensitivity to BBR3464. In parental A431cells, the drug induced at least twofold up-regulation of 15 genes including cell cycle and growth regulators, tumor suppressors and signal transduction genes. In cisplatin-resistant A431/Pt cells, BBR3464increased the expression of 15 genes such as apoptosis regulators and genes involved in the DNA damage response. Interestingly, BBR3464induced up-regulation of anti-metastatic factors together with down-regulation of several pro-metastatic factors. Cell cycle analysis indicated a marked G2arrest in treated A431cells, whereas an apoptotic response was documented in A431/Pt cells. These differential patterns of transcriptional profile in sensitive and resistant cells are consistent with a role for cell cycle regulation in the response to BBR3464.
Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Squamous Cell/drug therapy , Gene Expression Profiling , Gene Expression Regulation , Organoplatinum Compounds/therapeutic use , Uterine Cervical Neoplasms/drug therapy , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/physiology , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Line, Tumor , Drug Resistance, Neoplasm , Female , Humans , Oligonucleotide Array Sequence Analysis , Organoplatinum Compounds/pharmacology , RNA, Messenger/metabolismABSTRACT
Although cisplatin is effective in the treatment of different types of tumors, resistance to treatment is a major limitation. In an attempt of overcoming resistance mechanisms, a large effort has been made to generate compounds with a different geometry. At present, the most clinically relevant compounds include mononuclear (i.e. oxaliplatin) as well as multinuclear platinum complexes (i.e. BBR 3464). The mechanisms of cellular response to platinum complexes have not been completely elucidated. Among the main pathways affecting cell sensitivity of these drugs a role for p53 has been proposed at least for cisplatin and BBR 3464. Our results indicate that, also in the case of oxaliplatin, cytotoxicity is modulated by this pathway. Indeed, the effect of oxaliplatin could be reduced in tumor cells expressing mutant p53. The DNA mismatch repair system also appears to be critical in regulating cellular sensitivity to cisplatin because the loss of DNA mismatch repair results in low level of resistance to cisplatin, but not to oxaliplatin. Thus, platinum compounds are endowed with differential capability to activate pathways of p53-dependent or independent apoptosis, and differential recognition by specific cellular systems is likely to be the critical determinant of the cell fate (death/survival) after drug exposure. Further molecular studies are required to better define the precise contribution of such pathways to the cellular responses of the clinically relevant platinum complexes. A complete understanding of the molecular basis of sensitivity to platinum drugs is expected to provide useful insights for the optimization of tumor treatment.
Subject(s)
Antineoplastic Agents/pharmacology , Base Pair Mismatch/physiology , DNA Repair/physiology , Neoplasms/drug therapy , Neoplasms/metabolism , Organoplatinum Compounds/pharmacology , Tumor Suppressor Protein p53/physiology , Clinical Trials as Topic , DNA Adducts/metabolism , DNA Damage , Drug Resistance, Neoplasm , Humans , Neoplasms/pathologyABSTRACT
Mononuclear and multinuclear platinum complexes are known to induce distinct types of DNA lesions and exhibit different profiles of antitumor activity, in relation to p53 mutational status. In this study, we investigated the cellular effects of exposure to two platinum compounds (cisplatin and the multinuclear platinum complex BBR 3464), in the osteosarcoma cell line, U2-OS, carrying the wild-type p53 gene and capable of undergoing apoptosis or cell cycle arrest in response to diverse genotoxic stresses. In spite of the ability of both compounds to up-regulate p53 at cytotoxic concentrations, exposure to BBR 3464 resulted in cell cycle arrest but only cisplatin was capable of inducing significant levels of apoptosis and phosphorylation at the Ser15 residue of p53. The cisplatin-induced protein phosphorylation, not detectable in cells treated with BBR 3464, was associated with RPA phosphorylation, a specific up-regulation of Bax and down-regulation of p21(WAF1). Cells treated with BBR 3464 displayed a different cellular response with evidence of cytostasis associated with a high induction of p21(WAF1). The regulation of p21(WAF1) after cisplatin or BBR 3464 exposure required a p53 signal, as documented using stable transfectants expressing a dominant-negative form of p53 (175(his)). Taken together, these results indicate that cellular response to different genotoxic lesions (i.e. apoptosis or growth arrest) is associated with a specific recognition of DNA damage and a different p53-mediated signaling pathway. Multinuclear platinum complexes could be regarded as useful tools for investigating the p53-mediated process of cell cycle arrest in response to DNA damage.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , DNA Damage/drug effects , Neoplasms/drug therapy , Platinum Compounds/pharmacology , Tumor Suppressor Protein p53/drug effects , Amino Acid Sequence/drug effects , Amino Acid Sequence/physiology , Apoptosis/physiology , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Division/drug effects , Cell Division/physiology , Cisplatin/pharmacology , Cytotoxins/pharmacology , DNA Damage/physiology , Humans , Neoplasms/metabolism , Neoplasms/physiopathology , Phosphorylation/drug effects , Serine/drug effects , Serine/metabolism , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
Based on the role of p53 in the control of apoptosis following DNA damage, the status of the TP53 gene has been implicated as a major determinant of tumour responsiveness to cytotoxic therapies. In spite of the high frequency of TP53 mutations, small-cell lung cancer (SCLC) is recognised as one of the most chemoresponsive solid tumours. Since the relevance of the TP53 gene status in the modulation of tumour responsiveness is dependent on the molecular/biological context, in the present study, we have examined the relationship between chemosensitivity and susceptibility to apoptosis of a TP53-mutant human SCLC cell line. The cell line, in spite of TP53 mutation, retained an efficient response to genotoxic stress as documented by cells ability to modulate the p53 protein, arrest in the G1 and G2 phases of the cell cycle and its marked susceptibility to apoptosis following treatment with DNA damaging agents. Exposure to DNA-damaging agents caused an increase of c-Myc, a DNA damage-responsive transcription factor. An analysis of damage-induced apoptosis in the presence of an anti-Fas/CD95 inhibitory antibody indicated that Fas/CD95 was not required for the apoptotic response. The results support an implication of c-myc in sensitising cells to apoptosis, since inhibition of c-Myc expression with an antisense oligodeoxynucleotide (AS-ODN) almost abolished the drug-induced apoptotic response. In conclusion, the present results support a role for c-myc in the induction of apoptosis by genotoxic stress in the absence of a functional p53 and provide new insights into the mechanisms that may influence apoptosis in TP53-mutant cells. Elucidation of this pathway and of the possible cooperation with p53-dependent mechanisms may provide a basis for therapeutic intervention.
Subject(s)
Apoptosis/genetics , Carcinoma, Small Cell/genetics , DNA Damage/genetics , Genes, p53 , Lung Neoplasms/genetics , Proto-Oncogene Proteins c-myc/metabolism , Apoptosis/drug effects , Apoptosis/radiation effects , Carcinoma, Small Cell/metabolism , Cell Cycle/drug effects , Cell Cycle/radiation effects , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , DNA, Neoplasm/genetics , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Doxorubicin/pharmacology , Gamma Rays , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lung Neoplasms/metabolism , Mutation , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins c-myc/genetics , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolismABSTRACT
Relevant drawbacks of the molecular structure and mechanism of the action of camptothecins are the instability of the E ring lactone and the reversibility of drug-target interaction. Such features are expected to limit the clinical efficacy of conventional camptothecins. In an attempt to overcome these limitations and to improve the pharmacological profile of camptothecins, a novel series of seven modified lipophilic analogues was synthesized based on the hypothesis that lipophilicity could promote a rapid cellular accumulation and stabilization of drug-target interaction. A novel analogue (ST1481) of the series, characterized by a potent antitopoisomerase and cytotoxic activity, was selected for preclinical development. A detailed preclinical study of ST1481 was performed in the H460 non-small cell lung tumor model using oral administration and various treatment schedules. Under all of the conditions, ST1481 exhibited an impressive efficacy in terms of tumor growth inhibition (tumor volume inhibition percentage > 99%), log(10) cell kill, rate of complete responses (including "cures"), and an improvement of the therapeutic index compared with topotecan (used as the reference drug). The cytotoxic potency was also reflected by the in vivo potency, because the drug activity was observed at doses as low as 0.25 mg/kg with the daily schedule. In contrast to topotecan, no cross-resistance to ST1481 was found in ovarian carcinoma cells overexpressing P-glycoprotein (A2780/DX). A similar trend in the improvement of activity was also observed in the same tumor model growing in vivo with a 100% rate of complete tumor regressions. A rapid intestinal absorption and good oral bioavailability were supported by in vivo distribution studies, because the peak values of drug accumulation were found from 1 to 2 h after administration. The relevant liver accumulation may account for a marked effect of ST1481 against liver metastases induced by the ovarian carcinoma IGROV-1. In conclusion, the results support the hypothesis that a potent lipophilic camptothecin with a proper substituent at the position 7 may have therapeutic advantages likely related to a rapid intracellular uptake and tissue distribution, stabilization of the drug-target complex, and good oral bioavailability. Overall, the results support the preclinical interest of ST1481 in terms of efficacy, potency, toxicity profile, and ability to overcome multidrug resistance.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Camptothecin/pharmacology , Administration, Oral , Animals , Antineoplastic Agents, Phytogenic/pharmacokinetics , Camptothecin/analogs & derivatives , Camptothecin/pharmacokinetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Doxorubicin/pharmacology , Drug Administration Schedule , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Female , Humans , Liver Neoplasms, Experimental/prevention & control , Liver Neoplasms, Experimental/secondary , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Tissue Distribution , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVES: Systematic investigation of a novel series of intercalating agents, 9-aza-anthrapyrazoles, has led to the identification of a promising analogue, BBR 3438. This study describes the antitumour efficacy of the novel compound in human prostate carcinoma models and the molecular/cellular basis of its activity. METHODS AND RESULTS: The novel 9-aza-anthrapyrazole BBR 3438 was significantly more effective than doxorubicin and losoxantrone (DuP-941) in two of the three tested prostate carcinoma models. The superior activity was more evident in PC3 tumour, since BBR 3438 produced an appreciable rate of complete tumour regressions. Under these conditions, the drug-induced antiproliferative activity paralleled delayed apoptosis. Tumour response to in vivo drug treatment was associated with an early down-regulation of Bcl-2, which was somewhat more marked for the aza compound. In fact, the 9-aza-anthrapyrazole induced DNA cleavage in vitro with isolated DNA topoisomerase II (isoform alpha) and DNA strand breaks in prostatic carcinoma cells. Although the molecular effects of losoxantrone and the 9-aza analogue on the enzyme target were comparable, the cytotoxic effects of BBR 3438 could be enhanced by long-term exposure as a consequence of favourable cellular accumulation and prominent DNA-binding affinity. In addition, a lower reduction potential of the 9-aza-anthrapyrazole in comparison with classical anthrapyrazoles suggests an increased ability of the drug to induce oxidative stress following free radical production, which may be a contributing factor in determining the long-term response (i.e. delayed cell death) to genotoxic damage. CONCLUSIONS: BBR 3438 exhibited a unique profile of preclinical activity with a superior efficacy against prostatic carcinoma models compared to reference compounds (doxorubicin and losoxantrone). The antitumour efficacy of BBR 3438 against prostatic carcinoma could be the result of a combination of favourable events, including enhanced intracellular accumulation and an increased DNA-binding affinity favouring the accumulation of multiple sublethal or lethal damage. In spite of its enhanced cytotoxic potency, the 9-aza compound was better tolerated in vivo than losoxantrone, thus improving the therapeutic index. The preclinical profile of efficacy against prostatic carcinoma, a tumour resistant to conventional antitumour drugs, makes the novel 9-aza-anthrapyrazole BBR 3438 a promising candidate for clinical evaluation.
Subject(s)
Adenocarcinoma/drug therapy , Antineoplastic Agents/therapeutic use , Ethanolamines/therapeutic use , Intercalating Agents/therapeutic use , Prostatic Neoplasms/drug therapy , Pyrazoles/therapeutic use , Pyrazolones , Animals , Anthraquinones/therapeutic use , Antigens, Neoplasm , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Division/drug effects , DNA Damage , DNA Topoisomerases, Type II/metabolism , DNA, Neoplasm/drug effects , DNA-Binding Proteins , Doxorubicin/therapeutic use , Ethanolamines/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Genes, bcl-2 , Humans , Intercalating Agents/pharmacology , Male , Mice , Mice, Nude , Molecular Structure , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Oxidation-Reduction , Oxidative Stress , Phosphorylation , Protein Processing, Post-Translational/drug effects , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Pyrazoles/pharmacology , Remission Induction , Xenograft Model Antitumor AssaysABSTRACT
In an attempt to synthesize potential anticancer agents acting by inhibition of topoisomerase I (Topo I) a new series of oxyiminomethyl derivatives in position 7 of camptothecin (CPT) was prepared. The synthesis relied on the condensation of 20S-CPT-7-aldehyde or 20S-CPT-7-ketones with alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl O-substituted hydroxylamines. The compounds were tested for their cytotoxic activity in vitro against H460 non-small lung carcinoma cell line, the activity being for 24 out of 37 compounds in the 0.01-0.3 microM range. A QSAR analysis indicated that lipophilicity is the main parameter correlated with cytotoxicity. Investigation of the DNA-Topo I-drug cleavable complex showed a rough parallelism between cytotoxicity and inhibition of Topo I. Persistence of the DNA cleavage after NaCl-mediated disruption of the ternary complex suggests that for the most potent compounds, e.g., 15, the cytotoxicity was at least in part related to stabilization of the complex, as also supported by the persistence of the DNA-enzyme complex in drug-treated cells. The in vivo antitumor efficacy of the most potent analogue (15) was evaluated in direct comparison with topotecan using human lung tumor xenograft models. In the range of optimal doses (2-3 mg/kg), the improved efficacy of 15 was documented in terms of inhibition of tumor growth and rate of complete response.
Subject(s)
Antineoplastic Agents/chemical synthesis , Camptothecin/analogs & derivatives , Camptothecin/chemical synthesis , Imines/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Camptothecin/chemistry , Camptothecin/pharmacology , DNA/chemistry , DNA/metabolism , DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/metabolism , Drug Screening Assays, Antitumor , Humans , Imines/chemistry , Imines/pharmacology , Immunoblotting , Inhibitory Concentration 50 , Mice , Mice, Nude , Quantitative Structure-Activity Relationship , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
We selected a mitoxantrone-resistant HT29 colon carcinoma cell line (HT29/MIT) that exhibited a very high degree of resistance to the selecting agent and marked resistance to topotecan and SN38, but limited resistance to doxorubicin. The development of drug resistance was independent of expression of P-glycoprotein or multidrug resistance-associated protein but was associated with high up-regulation of the breast carcinoma resistance protein (BCRP) as shown by Western blot analysis. BCRP overexpression was associated with a reduced intracellular accumulation of topotecan, a known substrate for BCRP. Conversely, a lipophilic 7-modified camptothecin analogue (ST1481) displayed a complete lack of cross-resistance in HT29/MIT cells, suggesting that the drug was not a substrate for BCRP because no defects in intracellular accumulation were found. This conclusion is consistent with the antitumor efficacy of ST1481 against a BCRP-expressing tumor. These results may have therapeutic implications because the antitumor efficacy of ST1481 is in part related to a good bioavailability after oral administration, and the drug is currently under Phase I clinical evaluation.
Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Camptothecin/analogs & derivatives , Camptothecin/pharmacology , HT29 Cells/drug effects , Mitoxantrone/pharmacology , Neoplasm Proteins , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/biosynthesis , ATP-Binding Cassette Transporters/genetics , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents, Phytogenic/pharmacokinetics , Antineoplastic Agents, Phytogenic/pharmacology , Camptothecin/pharmacokinetics , DNA Damage , Drug Resistance, Multiple/genetics , Drug Resistance, Neoplasm , Female , Gene Expression , HT29 Cells/metabolism , Humans , Mice , Mice, Nude , Mitoxantrone/pharmacokinetics , Multidrug Resistance-Associated Proteins , Topotecan/pharmacokinetics , Topotecan/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
A series of new 7-iminomethyl derivatives of camptothecin were obtained from camptothecin-7-aldehyde and aromatic, alicyclic and aliphatic amines. Their hydrogenation led to the corresponding amines. All the imines and the less polar amines showed a marked increase of the cytotoxic activity against H460 non-small lung carcinoma cell line, with respect to topotecan. The lipophilicity of the substituent in position 7 of camptothecin seems to play an important role for cytotoxic potency. The 7-phenyliminomethyl derivative showed efficacy comparable to topotecan in vivo against NSCLC H460 xenografted in athymic nude mice.
Subject(s)
Antineoplastic Agents, Phytogenic/chemical synthesis , Camptothecin/analogs & derivatives , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Camptothecin/chemical synthesis , Camptothecin/pharmacology , Carcinoma, Non-Small-Cell Lung/pathology , Cell Division/drug effects , Combinatorial Chemistry Techniques , Drug Screening Assays, Antitumor , Humans , Inhibitory Concentration 50 , Lung Neoplasms , Mice , Mice, Nude , Neoplasm Transplantation , Structure-Activity Relationship , Survival Rate , Tumor Cells, Cultured/drug effectsABSTRACT
The natural alkaloid camptothecin is the lead compound of a new class of antitumor agents with a unique mechanism of action (i.e. inhibition of DNA topoisomerase I). The pharmacological interest of these agents has generated a large number of derivatives and analogues endowed with potent cytotoxic activity, two of them being in clinical use as antitumor drugs. We have synthesized a new series of camptothecins substituted in position 7 with an alkyl or alkenyl chain bearing cyano and/or carbethoxy groups. These compounds showed potent cytotoxic activity in vitro against the human non-small-cell lung carcinoma H460 cell line, most of them exhibiting IC(50) values in the 0.05-1 microM range, more active than topotecan used as a reference compound. In particular 7-cyano-20S-camptothecin (5a) showed high in vitro cytotoxicity against a topotecan-resistant H460 cell subline (H460/TPT) and a cisplatin-resistant ovarian carcinoma subline (IGROV-1/Pt 1). In an in vivo evaluation of the antitumor activity, 5a appeared significantly more effective than topotecan in the H460 tumor model and comparable with topotecan in a small-cell lung carcinoma model and a colon carcinoma model. The efficacy and good tolerability of this compound increase interest for further preclinical development.
Subject(s)
Antineoplastic Agents/chemical synthesis , Camptothecin/analogs & derivatives , Camptothecin/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Camptothecin/chemistry , Camptothecin/pharmacology , Drug Screening Assays, Antitumor , Humans , Mice , Mice, Nude , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
The biochemical basis of the anti-proliferative effect of exogenous glutathione was investigated in A2780 ovarian carcinoma cells. Previous observations have implicated gamma-glutamyl transpeptidase-mediated pro-oxidant reactions as a primary mechanism of the extracellular effects of glutathione. In 2 cell lines (A2780 and IGROV-1), glutathione led to H(2)O(2) production, but only A2780 cells, characterized by low expression of detoxification enzymes, were sensitive to the thiol compound. In A2780 cells, glutathione exposure resulted in DNA single-strand break formation, as measured by alkaline elution. Glutathione-induced DNA damage generated significant levels of apoptosis in A2780 cells, but not in IGROV-1 cells. The capability of glutathione to induce apoptosis was associated with cleavage of poly(ADP-ribose)polymerase and with generation of a low-molecular-weight form of the pro-apoptotic protein bax. In A2780 cells, glutathione exposure was followed by p21 and Bax induction and p53 up-regulation, as expected for genotoxic stress. Consistently, analysis of cell-cycle perturbations demonstrated the occurrence of G(2)M accumulation after exposure to glutathione, similar to what was observed for H(2)O(2). Taken together, these results indicate that the cytotoxic effect of extracellular glutathione, related to membrane metabolism, is mediated by production of H(2)O(2) leading to DNA damage and a cellular response involving p53. These findings might also provide insights into the cellular and molecular determinants of chemosensitivity to DNA damaging agents, as oxidative stress is implicated in p53-dependent apoptosis.
Subject(s)
Apoptosis/drug effects , DNA Damage , Glutathione/pharmacology , Hydrogen Peroxide/metabolism , Proto-Oncogene Proteins c-bcl-2 , Cell Cycle/drug effects , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , Cyclins/genetics , DNA, Neoplasm/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Genes, p53 , Growth Inhibitors/pharmacology , Humans , Inactivation, Metabolic , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Oxidative Stress , Poly(ADP-ribose) Polymerases/metabolism , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Tumor Cells, Cultured/drug effects , Tumor Suppressor Protein p53/biosynthesis , bcl-2-Associated X ProteinABSTRACT
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-OS/Pt cells, BBR 3464 accumulation and DNA-bound platinum were much higher than those observed for cisplatin. In contrast, the frequency of ICLs after exposure to BBR 3464 was very low. The time course of ICL formation after drug removal revealed a low persistence of these types of DNA lesions induced by BBR 3464, in contrast to an increase of DNA lesions induced by cisplatin, suggesting that components of the DNA repair pathway handle the two types of DNA lesions differently. The cellular response of HCT116 mismatch repair-deficient cells was consistent with a lack of influence of mismatch repair status on BBR 3464 cytotoxicity. Because BBR 3464 produces high levels of lesions different from ICLs, likely including intra-strand cross-links and monoadducts, the ability of the triplatinum complex to overcome cisplatin resistance appears to be related to a different mechanism of DNA interaction (formation of different types of drug-induced DNA lesions) as compared with conventional mononuclear complexes rather than the ability to overcome specific cellular alterations.
Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Organoplatinum Compounds/pharmacology , Apoptosis , Carboplatin/pharmacology , Cisplatin/pharmacokinetics , DNA Adducts/metabolism , DNA Damage , DNA Ligases/biosynthesis , DNA Ligases/physiology , DNA Repair , Drug Resistance, Neoplasm , Humans , Osteosarcoma/metabolism , Osteosarcoma/pathology , Tumor Cells, CulturedABSTRACT
In an attempt to examine the cellular changes associated with cisplatin resistance, we selected a cisplatin-resistant (A43 1/Pt) human cervix squamous cell carcinoma cell line following continuous in vitro drug exposure. The resistant subline was characterized by a 2.5-fold degree of resistance. In particular, we investigated the expression of cellular defence systems and other cellular factors probably involved in dealing with cisplatin-induced DNA damage. Resistant cells exhibited decreased platinum accumulation and reduced levels of DNA-bound platinum and interstrand cross-link frequency after short-term drug exposure. Analysis of the effect of cisplatin on cell cycle progression revealed a cisplatin-induced G2M arrest in sensitive and resistant cells. Interestingly, a slowdown in S-phase transit was found in A431/Pt cells. A comparison of the ability of sensitive and resistant cells to repair drug-induced DNA damage suggested that resistant cells were able to tolerate higher levels of cisplatin-induced DNA damage than their parental counterparts. Analysis of the expression of proteins involved in DNA mismatch repair showed a decreased level of MSH2 in resistant cells. Since MSH2 seems to be involved in recognition of drug-induced DNA damage, this change may account for the increased tolerance to DNA damage observed in the resistant subline. In conclusion, the involvement of accumulation defects and the increased tolerance to cisplatin-induced DNA damage in these cisplatin-resistant cells support the notion that multiple changes contribute to confer a low level of cisplatin resistance.
Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , DNA Damage , DNA/drug effects , Apoptosis/drug effects , Cell Cycle/drug effects , DNA/biosynthesis , DNA Adducts/drug effects , DNA Repair , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , Glutathione/metabolism , Humans , Platinum/pharmacokinetics , Tumor Cells, CulturedABSTRACT
The role of genes that affect response to radiation in determining sensitivity to platinum-containing compounds was studied using a panel of 23 strains of the yeast Schizosaccharomyces pombe. The radiation-hypersensitive mutants all had the same genetic background and most of them contained mutations that disabled either cell cycle checkpoints or DNA repair. The tested platinum compounds included cisplatin and two complexes containing diaminocyclohexane (oxaliplatin and tetraplatin), two ammine/cyclohexylamine complexes with different orientation of the leaving groups (JM216 and JM335) and a multinuclear platinum complex (BBR 3464). The cytotoxic effect of the selected platinum complexes was evaluated by using a microtiter growth inhibition assay with a 48 hr exposure to drug. The mutants fell into three groups with respect to sensitivity to cisplatin: four mutants (rad2, -7, -11, -15) exhibited minimal change in sensitivity; fifteen mutants (rad4-6, -8-10, -12-14, -16-17, -19-21, and -22) were 5.1-21.7-fold hypersensitive; only rad1 and -3 mutants, defective in checkpoints, and rad18, defective in repair, displayed a marked hypersensitivity. None of the mutants demonstrated appreciable change in sensitivity to JM216 presumably as a consequence of a lack of resistance of the wild-type strain, whereas a moderate increase in sensitivity to JM335 was observed for most of the mutants, and hypersensitivity to BBR3464 was observed only in rad1 and -3. No relevant changes in sensitivity to tetraplatin were observed. Most of the mutants, with the exception of rad2, -7, and -15, were hypersensitive to oxaliplatin. These findings demonstrate that specific mutations have disparate effects on the profile of sensitivity to different members of the same class of cytotoxic agents, which provides genetic evidence that different mechanisms are involved in differential cytotoxicity induced by Pt compounds. The results also demonstrate the utility of such a panel of mutants, constructed on the same genetic background, for detecting specific cellular response; presumably, this reflects the recognition or processing of specific DNA adducts. In conclusion, because the rad1 and rad3 gene products are determinants of cellular response to a large number of platinum-containing compounds, the present results support a critical role of genes involved in cell cycle control in cellular sensitivity to these agents.
Subject(s)
Cisplatin/pharmacology , DNA, Fungal/drug effects , Radiation-Sensitizing Agents/pharmacology , Schizosaccharomyces/drug effects , Cell Cycle/drug effects , DNA Repair/drug effects , Microbial Sensitivity Tests , Mutation , Platinum Compounds/pharmacology , Schizosaccharomyces/geneticsABSTRACT
BACKGROUND: Alteration in apoptosis pathways (in particular mutations of p53 gene) may result in resistance of ovarian carcinoma to cisplatin. However, cisplatin resistance is likely to be multifactorial. An understanding of the molecular alterations associated with the development of resistance may be of considerable relevance in an attempt to optimize the therapeutic approach. STUDY DESIGN: Two cisplatin-resistant sublines (IGROV-1/Pt0.5 and IGROV-1/Pt1), both characterized by mutant p53 (Cancer Res 1996; 56: 556-62), but with different degree of resistance were studied in terms of pattern of cross-resistance, susceptibility to drug-induced apoptosis, expression of gluthathione-dependent system, cellular pharmacokinetics, drug-induced DNA damage. The resistance index (ratio between the IC50 of resistant and sensitive cells) after a 96-hour drug exposure was 10 for IGROV-1/Pt0.5 and 14 for IGROV-1/Pt1 cells. RESULTS: Resistant cells were cross-resistant to DNA-damaging agents and, interestingly, they had a collateral sensitivity to Taxol. The cellular response to Taxol paralleled the drug ability to induce apoptosis. The intracellular glutathione level was significantly increased in IGROV-1/Pt cells compared to the sensitive counterpart. In contrast, glutathione S-transferase level was consistently reduced in both sublines. gamma-Glutamyl transpeptidase activity, which was lower in resistant than in sensitive cells, was not directly correlated with glutathione level, thus suggesting a complex regulation of cellular glutathione content. In the resistant cells with the highest glutathione content, a reduced level of cisplatin-induced cross-link was found. Analysis of DNA platination revealed a slight decrease of DNA-bound platinum only in IGROV-1/Pt1 cells. Again, this reduction is consistent with a protective role for glutathione. The expression of metallothionein IIa was increased in both resistant variants. CONCLUSIONS: Multiple changes are involved in acquired resistance of ovarian carcinoma cells including reduced susceptibility to apoptosis as consequence of inactivation of p53 and expression of defence mechanisms. The relative contribution is related to the degree of drug resistance. In particular, the glutathione-dependent system could have a role only in the development of a high degree of resistance. Finally, the finding that Taxol was very effective in inducing apoptosis in resistant sublines with p53 mutation supports the expression of an intact p53-independent pathway of apoptosis and suggests the pharmacological interest of Taxol in the treatment of p53-mutated tumors.