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PDf file - 1273KB, Top nine networks revealed by Ingenuity Pathway Analysis.

PDF file - 40KB, Frequency of clinicopathologic characteristics (A) and multivariate analysis of association between CpG-signature and overall survival (B) of ovarian cancer patients.

PDF file - 372KB, RAD23B down-modulation in IGROV-1 cells significantly reduced cisplatin cytotoxicity.

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PDF file - 1436KB, Plot of p-values obtained by Cox hazard analysis comparing the entire gene set with the gene sets obtained removing each single gene.

PDF file - 94KB, List of published datasets used for in silico analyses.

PDF file - 1759KB, Pearson's correlations between gene expression levels determined by qRT-PCR and oligonucleotide microarray for RAD51C, RAD54B, SIRT1 and RAD23B.

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PDF file - 55KB, List of genes present in Figure 1, Figure 2, Figure 3, Figure 7 and Supplementary Figure 1.

PDF file - 51KB, Frequency of clinicopathologic characteristics (A) and multivariate analysis of association between CpG-signature and overall/relapse-free survival (B) of breast cancer patients.

PDF file - 239KB, Kaplan-Meier plot of percent survivors over time among OVCAR-5 ovarian tumor-bearing athymic mice.

PDF file - 5781KB, Heat-map of the 27 modulated genes in the DNA repair pathway obtained in IGROV-1 ovarian tumors from CpG-ODN-treated athymic mice.

PDF file - 335KB, Ascitic fluid from CpG-ODN-treated mice determines DNA-repair genes modulation.

PDF file - 1571KB, Microarray analysis of DNA repair pathway genes in lung mucosa from mice treated with CpG-ODN intranasally.

Synthetic oligodeoxynucleotides expressing CpG motifs (CpG-ODN) are a Toll-like receptor 9 (TLR9) agonist that can enhance the antitumor activity of DNA-damaging chemotherapy and radiation therapy in preclinical mouse models. We hypothesized that the success of these combinations is related to the ability of CpG-ODN to modulate genes involved in DNA repair. We conducted an in silico analysis of genes implicated in DNA repair in data sets obtained from murine colon carcinoma cells in mice injected intratumorally with CpG-ODN and from splenocytes in mice treated intraperitoneally with CpG-ODN. CpG-ODN treatment caused downregulation of DNA repair genes in tumors. Microarray analyses of human IGROV-1 ovarian carcinoma xenografts in mice treated intraperitoneally with CpG-ODN confirmed in silico findings. When combined with the DNA-damaging drug cisplatin, CpG-ODN significantly increased the life span of mice compared with individual treatments. In contrast, CpG-ODN led to an upregulation of genes involved in DNA repair in immune cells. Cisplatin-treated patients with ovarian carcinoma as well as anthracycline-treated patients with breast cancer who are classified as “CpG-like” for the level of expression of CpG-ODN modulated DNA repair genes have a better outcome than patients classified as “CpG-untreated-like,” indicating the relevance of these genes in the tumor cell response to DNA-damaging drugs. Taken together, the findings provide evidence that the tumor microenvironment can sensitize cancer cells to DNA-damaging chemotherapy, thereby expanding the benefits of CpG-ODN therapy beyond induction of a strong immune response. Cancer Res; 71(20); 6382–90. ©2011 AACR.

Bis-2,3-heteroarylmaleimides and polyheterocondensed imides joined through nitrogen atoms of the N,N′-bis(ethyl)-1,3-propanediamine linker were prepared from substituted maleic anhydrides and symmetrical diamines in good to satisfactory yields and short reaction times using microwave heating. The novel molecules were shown to inhibit proliferation of human tumor cells (NCI-H460 lung carcinoma) and rat aortic smooth muscle cells (SMCs) with variable potencies. Compound 11a, the most potent one of the series, showed IC50 values comparable to those observed for the leading molecule elinafide in both cell lines, but with a higher selectivity toward human tumor cells. Compound 11a affected G1/S phase transition of the cell cycle, showed in vitro DNA intercalating activity and in vivo antitumor activity. A thorough structural analysis of the 11a-DNA complex was also made by mean of NMR and computational techniques.

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 + , epithelial-specific 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.

N6-isopentenyladenosine (i6A), a member of the cytokinin family of plant hormones, has potent in vitro antitumour activity in different types of human epithelial cancer cell lines. Gene expression profile analysis of i6A-treated cells revealed induction of genes (e.g., PPP1R15A, DNAJB9, DDIT3, and HBP1) involved in the negative regulation of cell cycle progression and reportedly up-regulated during cell cycle arrest in stress conditions. Of 6 i6A analogues synthesized, only the 1 with a saturated double bond of the isopentenyl side chain had in vitro antitumour activity, although weaker than that of i6A, suggesting that i6A biological activity is highly linked to its structure. In vivo analysis of i6A and the active analogue revealed no significant inhibition of cancer cell growth in mice by either reagent. Thus, although i6A may inhibit cell proliferation by regulating the cell cycle, further studies are needed to identify active analogues potentially useful in vivo. © 2008 Wiley-Liss, Inc.

In previous studies, we have documented the potential therapeutic advantages of camptothecin analogs modified at the 7-position, i.e., 7-oxyiminomethyl derivatives. The present study was performed to explore the therapeutic potential of novel hydrophilic derivatives of this series. With one exception (ST1976), the tested camptothecins exhibited a reduced antiproliferative activity and all compounds retained ability to stabilize the topoisomerase I-mediated cleavable complex. The two analogs (ST1976 and ST1968) characterized by the presence of a free amino group in the side chain also exhibited the formation of persistent cleavable complexes. The most potent compound, ST1976 (7-(4-aminobenzyl)oxyiminomethylcamptothecin), was selected for evaluation of its preclinical profile of antitumor activity in a large panel of human tumor xenografts. As expected on the basis of the introduction of a hydrophilic substituent, the novel camptothecin was a substrate for BCRP. However, in spite of an apparent recognition by BCRP, ST1976 was effective following oral administration. The antitumor activity was evaluated using various schedules and routes of administration (i.v. and p.o.). ST1976 exhibited a remarkable activity in all tested tumors and was effective in a number of tumors which are resistant to irinotecan. The biological and pharmacological profile of ST1976 supports the therapeutic potential of camptothecins containing hydrophilic substituents at the 7-position. On the basis of its excellent activity in preclinical models, ST1976 is a promising candidate for clinical development.

Hepatocyte growth factor (HGF) and its tyrosine kinase receptor Met play a pivotal role in the tumor metastatic phenotype and represent attractive therapeutic targets. We investigated the biochemical and biological effects of the tyrosine kinase inhibitor RPI-1 on the human lung cancer cell lines H460 and N592, which express constitutively active Met. RPI-1-treated cells showed down-regulation of Met activation and expression, inhibition of HGF/Met-dependent downstream signaling involving AKT, signal transducers and activators of transcription 3 and paxillin, as well as a reduced expression of the proangiogenic factors vascular endothelial growth factor and basic fibroblast growth factor. Cell growth in soft agar of H460 cells was strongly reduced in the presence of the drug. Furthermore, RPI-1 inhibited both spontaneous and HGF-induced motility/invasiveness of both H460 and human endothelial cells. Targeting of Met signaling by alternative methods (Met small interfering RNA and anti-phosphorylated Met antibody intracellular transfer) produced comparable biochemical and biological effects. Using the spontaneously metastasizing lung carcinoma xenograft H460, daily oral treatment with well-tolerated doses of RPI-1 produced a significant reduction of spontaneous lung metastases (−75%; P < 0.001, compared with control mice). In addition, a significant inhibition of angiogenesis in primary s.c. tumors of treated mice was observed, possibly contributing to limit the development of metastases. The results provide preclinical evidence in support of Met targeting pharmacologic approach as a new option for the control of tumor metastatic dissemination. [Mol Cancer Ther 2006;5(9):2388–97]

Recent evidence indicates that the success of molecular targeted therapies may depend on the identification of drug targets which are essential for the survival of subsets of tumors. RET oncogenes that have been implicated in the development of thyroid carcinomas are emerging as potential therapeutic targets. In the present study, we investigated the efficacy and the cellular bases of antitumor activity of the indolinone Ret tyrosine kinase inhibitor RPI-1 against large established s.c. TT tumor xenograft, a human medullary thyroid carcinoma (MTC) harboring oncogenic MEN-2A-type RET mutation. Oral treatment with RPI-1 caused growth arrest or regression in 81% treated tumors. Following treatment suspension, tumor inhibition was maintained (51%, P

Breast cancer–initiating cells have been recently identified in breast carcinoma as CD44+/CD24−/low cells, which exclusively retain tumorigenic activity and display stem cell–like properties. However, at present, direct evidence that breast cancer–initiating cells can be propagated in vitro is still lacking. We report here the isolation and in vitro propagation of breast cancer–initiating cells from three breast cancer lesions and from an established breast carcinoma cell line. Our breast carcinoma–derived cultures encompassed undifferentiated cells capable of self-renewal, extensive proliferation as clonal nonadherent spherical clusters, and differentiation along different mammary epithelial lineages (ductal and myoepithelial). Interestingly, cultured cells were CD44+/CD24− and Cx43−, overexpressed neoangiogenic and cytoprotective factors, expressed the putative stem cell marker Oct-4, and gave rise to new tumors when as few as 103 cells were injected into the mammary fat pad of SCID mice. Long-term cultures of breast tumorigenic cells with stem/progenitor cell properties represent a suitable in vitro model to study breast cancer–initiating cells and to develop therapeutic strategies aimed at eradicating the tumorigenic subpopulation within breast cancer.

Purpose: Gimatecan, a novel oral lipophilic camptothecin characterized by favorable features at molecular/cellular level and by a promising profile of preclinical activity, is currently in clinical phase I/II. The aim of the study was to additionally investigate the therapeutic potential of the drug in human tumor xenografts growing in different organs as models representative of tumor growth in the clinical setting. Experimental Design: The models include two orthotopic central nervous system tumors, two melanomas growing intracranially, and an ovarian carcinoma growing i.p. In addition, gimatecan was tested against experimental lung metastases of two tumor types (lung and ovarian carcinomas). Gimatecan was delivered by oral gavage according to various schedules (daily or intermittent). The time (in days) mice required to show evident signs of disease was used as end point for drug efficacy. Results: Gimatecan was highly effective in delaying disease manifestations in all tumor systems investigated. In the intracranially growing tumors, a significant time increase (versus control mice) was achieved by the drug administered according to all of the schedules. In addition, almost all treated mice were alive and tumor-free at the end of the experiment in the metastatic models and in the ascitic ovarian tumor. The daily prolonged treatment schedule was the best one. Conclusions: In all tumor systems investigated, including orthotopic tumor growth models and lung metastases, the oral administration of gimatecan showed a therapeutic benefit in terms of survival increase. The good oral availability allowed a prolonged daily treatment regimen, which seems the most promising to exploit the therapeutic potential of the drug.

Following the realisation that DNA topoisomerase I is a useful therapeutic target to be exploited for the design of potential inhibitors, topoisomerase I inhibitors now represent an established class of effective agents. In spite of intense efforts in the field, only camptothecins have a clinical relevance. Several options in chemical manipulation of natural camptothecin have been explored to overcome the major drawbacks of the drug, which include water insolubility, lactone instability, reversibility of the drug-target interaction and drug resistance. Several analogues are currently in clinical development, including water soluble camptothecins, lipophilic camptothecins and polymer-bound camptothecins. The therapeutic advantages of novel camptothecins over the two analogues (topotecan and irinotecan) approved for clinical use remain to be defined. This article is an overview of the relevant features of the analogues that are undergoing clinical development.

The ability of melanoma cells to evade engagement of apoptosis plays a significant role in their resistance to chemotherapy. In an attempt to lower the apoptotic threshold of melanoma cells as a possible strategy to increase their drug sensitivity, we generated a hammerhead ribozyme to down-regulate the expression of the anti-apoptotic protein survivin. The JR8 human melanoma cell line was stably transfected with the active ribozyme RZsurv (targeting the 3' end of the GUC 294 triplet in the exon 3 of the survivin mRNA) or the catalytically inactive ribozyme mutRZsurv (carrying a mutation in the catalytic core of RZsurv). Two polyclonal cell populations expressing the active (JR8/ RZsurv) or the mutant (JR8/mutRZsurv) ribozyme were selected for the study. JR8/RZsurv cells were characterized by a markedly lower survivin protein level than JR8 parental cells, whereas a negligible reduction in survivin expression was observed in JR8/mutRZsurv cells. JR8/ RZsurv cells showed a significantly increased sensitivity to the topoisomerase-I inhibitor topotecan (as detected by clonogenic cell survival) compared with JR8/mutRZsurv cells. Moreover, the extent of drug-induced apoptosis (in terms of percentage of apoptotic nuclei and level of caspase-9 and caspase-3 catalytic activity) was significantly greater in JR8/RZsurv than in JR8/mutRZsurv cells. Finally, an increased antitumor activity of oral topotecan was observed in JR8/RZsurv cells grown as xenograft tumors in athymic nude mice compared with JR8/mutRZsurv cells. These results demonstrate that attenuation of survivin expression renders human melanoma cells more susceptible to topotecan-induced apoptosis and more responsive to in vivo treatment, and support the concept that survivin is an attractive target for new therapeutic interventions in melanoma.

ST1481, a lead compound of a novel potent 7-substituted lipophilic camptothecin series, is able to overcome several mechanisms of drug resistance and was selected for clinical development. This study was designed to examine the antitumor activity of ST1481 in the treatment of preclinical models of human p53-defective hormone-refractory prostate carcinoma (DU145, PC3, and JCA-1) and to explore the cellular bases of the efficacy of camptothecins. A cellular pharmacology study (cytotoxicity, apoptosis, cellular drug accumulation, DNA damage, and cell cycle perturbation) was performed in DU145 and PC3 cells, characterized by a different cell cycle checkpoint status. The introduction of wild-type p53 in PC3 cells appreciably decreased the drug sensitivity. The 7-substituted camptothecins exhibited a high cytotoxic potency that paralleled their relative ability to induce DNA damage and a substantially increased cellular accumulation as compared to topotecan. The cytotoxic effect of camptothecins in DU145 cells was associated with arrest in S phase and early activation of apoptosis, whereas PC3 cells responded to drugs by a persistent block in G2 phase with a cytostatic effect and a late apoptosis. The efficiency of S phase checkpoint in DU145 cells was supported by a time-dependent decrease of DNA synthesis following treatment. In spite of an apparent cytostatic response and apoptosis resistance, the PC3 tumor was more responsive to in vivo treatment with camptothecins than the DU145 model. Indeed, the therapeutic outcome did not reflect the cell susceptibility to early activation of apoptosis. We suggest that cell death in PC3 cells is a delayed event consequent to persistent arrest in G2 and insufficient repair of DNA damage. ST1481 was appreciably more effective than topotecan in all tested tumors. In conclusion, the results indicated a relevant efficacy of camptothecins against human prostate carcinoma models, in spite of p53 alterations. Although p53 status could influence DNA damage and cell cycle checkpoints, p53 mutation was not a determinant of resistance. The results support that, in addition to the extent and persistence of topoisomerase I-mediated DNA damage, cell cycle checkpoints and DNA damage signaling pathways are critical determinants of tumor responsiveness to camptothecins. A role of cell cycle checkpoints activated by DNA damage in cell response is supported by the modulation of transcriptional profile.

Breast tumor growth and metastasization are both hormone-sensitive and angiogenesis-dependent. Recent work carried out in our laboratory on a transgenic model of breast cancer displaying many similarities to its human counterpart, has shown that liposome-mediated angiostatin cDNA delivery partially inhibits both local and metastatic growth. However, it is now recognized that anti-angiogenesis strategy alone cannot completely arrest tumor growth and spread, and this led to the suggestion that approaches based on different molecular mechanisms could usefully be combined. In the present work, we investigated whether tamoxifen, a classical antiestrogen agent widely used in human therapy, could improve the results obtained with angiostatin alone. Further reduction of local growth was achieved with the combined regimen with respect to angiostatin or tamoxifen alone, while, as expected, no metastatic growth was detected in either group. We therefore conclude that a combination of angiogenesis inhibitors with antiestrogen drugs might be useful in humans and that other associations between conventional and gene transfer-mediated therapy are worth investigating and will soon become important components of anticancer therapy.

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.

MEN 10755 is a disaccharide anthracycline endowed with a broader spectrum of antitumour activity than doxorubicin (DOX). To investigate the cellular and molecular basis of its action, cytotoxic activity, drug uptake, subcellular localisation, induction of DNA damage, and apoptosis were assessed in the human A2780 ovarian carcinoma cell line. Experiments with radiolabelled anthracyclines indicated that MEN 10755 exhibited reduced cellular accumulation and a different subcellular distribution (higher cytoplasmic/nuclear ratio) than DOX. In spite of the lower nuclear concentration, MEN 10755 was as potent as DOX in eliciting DNA single- and double-strand breaks, G2/M cell arrest, and apoptosis. Sequencing of drug-induced topoisomerase II cleavage sites showed a common DNA cleavage pattern for MEN 10755 and DOX. Cleavage sites were always characterised by the presence of adenine in −1 position. However, the extent of DNA cleavage stimulation induced by MEN 10755 was greater than that produced by DOX. Reversibility studies showed that MEN 10755-stimulated DNA cleavage sites were more persistent than those induced by DOX, thus suggesting a more stable interaction of the drug in the ternary complex. As a whole, the study indicated that the cellular pharmacokinetics of MEN 10755 substantially differs from that of DOX, showing a lower uptake and a different subcellular disposition. In spite of the apparently unfavourable cellular pharmacokinetics, MEN 10755 was still as potent as DOX in inducing topoisomerase-mediated DNA damage. Although the extent and persistence of protein-associated DNA breaks may contribute to the cytotoxic effects, the drug’s efficacy as apoptosis inducer and antitumour agent could not be adequately explained on the basis of DNA damage mediated by the known target (i.e. topoisomerase II), thus supporting additional cellular effects that may be relevant in cellular response.

The sugar moiety is an essential component of anthracycline antibiotics for their topoisomerase poisoning activity and antitumor efficacy. Since the sugar interacts with the minor groove, modifications in this moiety could enhance the recognition potential of the drug at the target level. Based on this hypothesis, novel anthracyclines, disaccharides lacking the amino group in the first (aglycone-linked) sugar, were designed. The 3'-amino group in the first sugar was replaced by an hydroxyl group, and the second sugar residue was bound to the first sugar via an alpha (1-4) linkage. The cytotoxic and antitumor activities of disaccharide analogs of idarubicin were critically dependent on the optimal (axial) orientation of the second sugar residue. Although configurational requirements of the sugar moiety for optimal drug activity support a critical role of the external (non-intercalating) drug domains in the interaction of anthracyclines with the DNA-topoisomerase (ternary complex), the antitumor efficacy of disaccharide analogs is not fully explained by effects mediated by the nuclear enzyme target. The development of this novel disaccharide series may provide insights for a rational synthesis of anthracycline analogs with improved pharmacological profile.

To approach the question of hyaluronan catabolism in tumours, we have selected the cancer cell line H460M, a highly metastatic cell line in the nude mouse. H460M cells release hyaluronidase in culture media at a high rate of 57 pU/cell/h, without producing hyaluronan. Hyaluronidase was measured in the H460M cell culture medium at the optimum pH 3.8, and was not found above pH 4.5, with the enzyme-linked sorbent assay technique and zymography. Tritiated hyaluronan was digested at pH 3.8 by cells or cell membranes as shown by gel permeation chromatography, but no activity was recorded at pH 7 with this technique. Hyaluronan was digested in culture medium by tumour slices, prepared from tumours developed in nude mice grafted with H460M cells, showing that hyaluronan could be digested in complex tissue at physiological pH. Culture of tumour slices with tritiated acetate resulted in the accumulation within 2 days of radioactive macromolecules in the culture medium. The radioactive macromolecular material was mostly digested by Streptomyces hyaluronidase, showing that hyaluronan was its main component and that hyaluronan synthesis occurred together with its digestion. These results demonstrate that the membrane-associated hyaluronidase of H460M cells can act in vivo, and that hyaluronan, which is synthesised by the tumour stroma, can be made soluble and reduced to a smaller size by tumour cells before being internalised and further digested.

BACKGROUND RORα is a transcription factor which belongs to the family of orphan nuclear receptors. The regulatory functions of this receptor are still poorly understood. However, response elements for RORα are present on the promoter of cell cycle-related genes suggesting that it might be involved in the control of cell proliferation. In this study, we investigated the expression and the possible function of RORα in a human androgen-independent prostate cancer cell line (DU 145). The thiazolidinedione-derivative CGP 52608 has been utilized as the specific ligand and activator of RORα. METHODS The effects of CGP 52608 on DU 145 cell proliferation and cell cycle distribution were analyzed by hemocytometer and by FACS analysis, respectively. The expression of RORα as well as the effects of RORα activation on the expression of cell cycle-related genes were evaluated by RT-PCR. To clarify whether RORα activation might affect the proliferation of prostate cancer cells also in vivo, nude mice bearing DU 145 tumor xenografts were treated with CGP 52608 at different doses and the growth of the tumors was followed by caliper measurement. RESULTS RORα is expressed in DU 145 cells and the treatment of the cells with the thiazolidinedione-derivative CGP 52608 brought about a dose-dependent and significant decrease of cell proliferation. Ligand-induced activation of RORα affected cell cycle distribution, inducing an accumulation in the G0/G1 phase and a decrease in the S phase. This effect was accompanied by an increased expression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and a decreased expression of cyclin A. The growth of DU 145 tumors in nude mice was significantly reduced by treatment with CGP 52608. CONCLUSIONS These data indicate that, in androgen-independent DU 145 prostate cancer cells, activation of the orphan nuclear receptor RORα inhibits cell growth, both in vitro and in vivo. RORα also induces cell cycle arrest, possibly through the modulation of the expression of cell cycle-related genes. Prostate 46:327–335, 2001. © 2001 Wiley-Liss, Inc.

A variety of cytotoxic agents effective as antitumor drugs are known to kill tumor cells through induction of apoptosis as the most relevant modality of cell death. A specific role for the protein Bcl-2 in the cell death pathway induced by antimicrotubule agents has been proposed, because Bcl-2 phosphorylation occurs in response to microtubule damage. In this study, we compared efficacy, apoptosis, and Bcl-2 phosphorylation in the Bcl-2-overexpressing MX-1 human breast carcinoma xenograft after treatment with cytotoxic agents characterized by different mechanisms of action. We demonstrated that, in addition to antimicrotubule agents, effective DNA-damaging agents were also able to induce Bcl-2 phosphorylation irrespective of the type of genotoxic lesion. A comparison of effects of drugs belonging to the same class but endowed with a different antitumor activity (i.e. cisplatin versus a novel multinuclear platinum complex and doxorubicin versus a disaccharide analogue) showed a correlation between drug efficacy, apoptotic response, and Bcl-2 phosphorylation. In conclusion, overexpression of Bcl-2 did not counteract the apoptotic effects of a number of cytotoxic agents and could not be regarded as a mechanism of cellular resistance. Since Bcl-2 phosphorylation is a common event in response to different types of cytotoxic damage and is not only related to microtubule dysfunction, we suggest that many cell death pathways converge on Bcl-2 and protein phosphorylation is a step of the signaling cascade activated by diverse stimuli and likely related to the onset of drug-induced apoptosis.

Experiments have been performed to clarify whether LHRH agonists might decrease growth of hormone-unresponsive prostate cancer in vivo. Male nude mice were injected s.c. with the human androgen-independent prostate tumor DU 145 cells; osmotic minipumps releasing the LHRH agonist Zoladex (LHRH-A) for 14 days were simultaneously implanted under the skin. Treatment with LHRH-A induced a significant decrease in tumor growth up to the end of the treatment. In subsequent experiment, minipumps releasing LHRH-A were implanted in nude mice either 7 or 14 days after cell inoculation. When the treatment was started 7 days after inoculation of the cells, tumor growth was significantly decreased up to 28 days; thereafter, tumor volume remained lower than in controls, although not significantly. When LHRH-A was administered beginning 14 days after cell inoculation, tumor growth was not significantly affected at any time interval considered. LHRH-A did not appear to induce apoptosis in DU 145 cells, at least on the basis of the apoptotic index and immunohistochemical staining of the p53 protein. On the other hand, treatment with LHRH-A was accompanied by a significant decrease of the concentration of epidermal growth factor receptors in DU 145 prostate cancer specimens. Our results show that the LHRH agonist used significantly inhibits the growth of DU 145 androgen-independent prostate tumor xenografts in nude mice.

Background: Although doxorubicin re mains one of the most effective agent for the treatment of solid tumors, then is an intensive effort to synthesize doxo rubicin analogues (compounds with similar chemical structures) that may have improved antitumor properties We have synthesized a novel doxorubi cin disaccharide analogue (MEN 10755) and have characterized some of its relevant biochemical, biologic, an pharmacologic properties. Methods. The antitumor activity of this com-pound (MEN 10755) was studied in a panel of human tumor xenografts, including xenografts of A2780 ovarian tumor cells, MX-1 breast carcinoma cells, and POVD small-cell lung cancer cells. MEN 10755 was compared with doxorubicin according to the optimal dose and schedule for each drug. The drug's cytotoxic effects, induction of DNA damage, and intracellular accumulation were studied in A2780 cells. DNA cleavage mediated by the enzyme topoisomerase II was investigated in vitro by incubating fragments of simian virus 40 DNA with the purified enzyme at various drug concentrations and analyzing the DNA cleavage-intensity patterns. Drug-induced apoptosis (programmed cell death) in tumors was determined with the use of MX-1 and POVD tumor-bearing athymic Swiss nude mice. Results: MEN 10755 was more effective than doxorubicin as a topoisomerase II poison and stimulated DNA fragmentation at lower intracellular concentrations. In addition, MEN 10755 exhibited striking antitumor activity in the treatment of human tumor xenografts, including those of the doxo-rubicin-resistant breast carcinoma cell line MX-1. Conclusions: The high antitumor activity of MEN 10755 in human tumor xenografts, including doxorubi-cin-resistant xenografts, and its unique pharmacologic and biologic properties make this disaccharide analogue a promising candidate for clinical evaluation.

Tumor cell growth, even in advanced stages of ovarian cancer, is nearly always restricted to the peritoneal cavity; therefore, repeated intraperitoneal injections of oligodeoxynucleotides containing dinucleotides with unmethylated CpG motifs (CpG-ODN) recruiting and activating innate effector cells throughout the abdominal cavity to the tumor site might control tumor cell growth and ascites formation. After a single CpG-ODN treatment, in IGROV-1 ovarian tumor ascites-bearing athymic mice, the number of tumor cells declined rapidly and markedly, and ascites volumes declined shortly after treatment (5 h), increasing thereafter at a slower rate than in controls. When administered every 7 days for 4 weeks, CpG-ODN had only a marginal effect on survival time, whereas administration 5 days/wk for 3 or 4 weeks led to a significantly increased survival time as compared with controls (P

Melanospheres, the melanoma cells that grow as nonadherent colonies and that show in vitro self-renewing capacity and multipotency, were selected from melanoma specimens or from melanoma cell lines. Melanospheres were highly tumorigenic, and intradermal injections in severe combined immunodeficient (SCID) mice of as few as 100 cells generated tumors that maintained tumorigenic potential into subsequent recipients. Primary and serially transplanted xenografts recapitulated the phenotypic features of the original melanoma of the patient. Melanoma cells cultured in the presence of fetal calf serum (FCS) were also tumorigenic in SCID mice, although with lower efficiency; these xenografts showed a homogeneous phenotype for the expression of melanoma-associated markers, Melan-A/Mart-1, HMB45, and MITF, and contained cells with features of fully differentiated cells. Melanospheres were heterogeneous for the expression of stem cell markers and showed a significantly enhanced expression of the Nanog and Oct3/4 transcription factors when compared with adherent melanoma cells. No direct and unique correlation between any of the examined stem cell markers and in vivo tumorigenicity was found. Taken together, our data provide further evidence on the heterogeneous nature of human melanomas and show that melanospheres and their corresponding tumors, which are generated in vivo in immunocompromised mice, represent a model to investigate melanoma biology. © 2010 The Society for Investigative Dermatology.

There is indication that tumor growth is sustained by subpopulation of cells with stem-like features but little is known on their genomic characterization and their genetic stability. We report a detailed molecular cytogenetic characterization using Spectral Karyotyping and fluorescent in situ hybridization of parental serum-cultured adherent cells and their sphere-growing stem-like counterpart before and after differentiation from six cell lines established from solid tumors. Our findings indicate increased cytogenetic complexity in sphere-growing stem-like and their differentiated adherent cells compared to parental adherent component suggesting the existence within cell lines of heterogeneous and genetically unstable subpopulations of cells endowed with stem-like features.

Purpose: Oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN) are potent activators of innate and adaptive immunity. Recognition of CpG-ODN is mediated by Toll-like receptor 9 expressed by immune cells, endothelial and epithelial cells, and fibroblasts. We examined the antitumor effect of CpG-ODN and the role of administration route on human ovarian cancers growing in the peritoneal cavity of nude mice.Experimental Design: Mice implanted i.p. with human ovarian carcinoma cells were treated i.p., s.c., or i.v. and assessed for survival and tumor-free incidence. Peritoneal washings were analyzed for keratinocyte chemokine production and for functional and phenotypic profiles as indicators of the cell types involved in mediating the antitumor effects.Results: IGROV-1-bearing mice treated i.p. survived significantly longer than those treated i.v. or s.c. (P = 0.0005), and nearly half of them (8 of 17) were tumor-free by the end of the experiment, a rate never achieved using a variety of chemotherapeutic drugs. High rates of tumor-free mice were observed in three other ovarian tumor xenografts treated i.p. Compared with peritoneal washings of mice treated s.c. or i.v., those from mice treated i.p. showed the highest level of serum and tissue keratinocyte chemokine, the highest number of natural killer cells and neutrophils, and the highest antiproliferative activity in vitro.Conclusions: The superior antitumor effect obtained by locoregional administration of CpG-ODN in i.p. tumor-bearing mice with a limited adaptive immune response points to the importance of innate effector cells amplification at the site of tumor growth and suggests the promise of i.p. CpG-ODN in clinical trials for ovarian cancer.

ST1968 is a novel hydrophilic camptothecin (CPT) derivative of the 7-oxyiminomethyl series. Because ST1968 retained ability to form remarkably stable cleavable complexes, this study was done to investigate its preclinical profile of antitumor activity in a large panel of human tumor models, including irinotecan-resistant tumors. Although less potent than SN38 in vitro, i.v. administered ST1968 caused a marked tumor inhibition, superior to that of irinotecan, in most tested models. ST1968 exhibited an impressive activity against several tumors including models of ovarian and colon carcinoma in which a high rate of cures was observed. In the most responsive tumors, complete and persistent tumor regressions were achieved even with low suboptimal doses. Even tumors derived from intrinsically resistant cells exhibited a significant responsiveness. Histologic analysis of treated tumors supports a contribution of both proapoptotic and antiangiogenic effects to ST1968 antitumor efficacy. A study done in yeast cells transformed with CPT-resistant mutant forms of topoisomerase I documented that, in contrast to other tested CPT, ST1968 was active against yeasts expressing the mutant K720E enzyme. Based on its outstanding efficacy superior to that of irinotecan and of its good therapeutic index, ST1968 has been selected for clinical development. [Mol Cancer Ther 2008;7(7):2051–9]

The monoclonal antibody trastuzumab binds to the extracellular domain of HER-2/neu and induces clinical responses in breast tumors with HER-2 gene amplification and/or protein overexpression. Its role in other tumor types remains to be investigated. We evaluated the antitumor efficacy of trastuzumab in vitro and in nude mice implanted orthotopically with cells of 3 human pancreatic tumor lines expressing only low levels of HER-2/neu, as determined by flow cytometry. Although none of the 3 cell lines showed growth inhibition when cultured directly with trastuzumab, 2 of them, GER and PaCa3, were sensitive to lysis in antibody-dependent cellular cytotoxicity assay. This pattern of response was recapitulated in tumor-bearing mice repeatedly treated with trastuzumab, in which survival was significantly prolonged as compared with controls (P=0.03 for GER and 0.0008 for PaCa3). Incidence of metastases was also reduced, especially in liver. These preclinical results indicate that trastuzumab can exert an antitumor effect against orthotopic human pancreatic cancer xenografts with low-level HER-2/neu expression and that this effect correlates with the in vitro antibody-dependent cellular cytotoxicity susceptibility, suggesting a different role for HER-2/neu in the therapy of tumor types other than breast cancer.

Gimatecan, a potent lipophilic camptothecin, is active by oral route, which allows a comparison of different treatment schedules. The present study was designed based on previous evidence of improved antitumor efficacy of the combination of topotecan with immunostimulatory CpG-ODN. Two regimens of gimatecan, i.e., a low-dose/protracted treatment (metronomic) and a high-dose/intermittent treatment, were compared in the GER orthotopic human pancreatic tumor model in nude mice. Metronomic gimatecan (0.25 mg/kg per os, daily) significantly increased mice survival time over control mice (154 T/C%, p0.05), in contrast to intermittent treatment-gimatecan (1.5 mg/kg per os, q4d) or intravenous gemcitabine (125 and 128 T/C%, respectively, p0.1). Metronomic gimatecan combined to CpG-ODN (20 microg/mouse intraperitoneal, weekly) was well tolerated and achieved a strong antitumor effect, with a T/C% of 188 on survival time (P0.001 versus control mice), significantly superior to those of the single agent-treated mice (p0.05 versus gimatecan- or CpG-ODN-treated mice). Cellular studies indicated that TRAIL could increase the apoptotic response to gimatecan in GER tumor cells, and TRAIL was released in the peritoneal washings of CpG-ODN-treated mice. In conclusion, the combination of metronomic gimatecan with CpG-ODN was effective and tolerated, and might represent a preclinical basis for the design of clinical studies, even considering the ability of CpG-ODN to stimulate TRAIL release and the high level of TRAIL-receptors expressed in pancreatic tumor cells.

A series of 2,3-heteroarylmaleimides 9 and polyheterocondensed imides 12 were prepared in good yields and short reaction time using a very efficient procedure consisting in the condensation of the corresponding anhydrides and N,N-diethylethylenediamine and microwave heating. The antiproliferative activity of the novel molecules was tested against human tumor cells (NCI-H460 lung carcinoma) and rat aortic smooth muscle cells (SMCs). The IC50 values for the novel molecules ranged from 0.08 to 13.9 μM in SMCs, and from 0.84 to 9 μM in the tumor cell line. The activity profile for compounds 9 and 12 is comparable to that obtained for amonafide in NCI-H460, except for fused imides 12b,i which proved to be about 10-fold more potent. Whereas, in rat SMCs, only the compound 12b was shown to be 10-fold more potent than amonafide. Instead 12c is equipotent to amonafide. These results suggest that the extended π-system and the kind of heteroatom are essential in the binding with the molecular target.

Synthesis of 10 pyrroloiminoquinone derivatives is presented. The strategy is based around the elaboration of a common intermediate by reaction with primary amines. All the compounds obtained have been subjected to antiproliferative activity with three different cell lines (NCI-H460, HeLa, and HL-60). The capacity of 4 selected compounds to affect the enzymatic activity of the nuclear enzyme DNA topoisomerase II and to form the typical DNA fragmentation which occurs in the apoptotic process is discussed here.