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In this study, we demonstrated that bcl-2 overexpression in human melanoma cells consistently enhanced the activity of multiple metastasis-related proteinases, in vitro cell invasion, and in vivo tumor growth. In particular, by using the M14 parental cell line, the MN8 control clone, and two bcl-2 overexpressing derivatives, we found that bcl-2 overexpressing cells exposed to hypoxia, when compared to parental cells, expressed higher level of several metalloproteases (MMPs) such as MMP-2, MMP-7, MT1-MMP, and tissue inhibitors of metalloproteases-1 and -2. Moreover, bcl-2 overexpression in melanoma cells enhanced in vitro invasion on matrigel and, in vivo tumor growth. The more aggressive behavior of bcl-2 transfectants tumors is significantly associated to an increase in MMP-2 expression as well as in a more elevated microvessel density as compared to the parental line. Taken together, our data suggest that bcl-2 plays a pivotal role in the regulation of molecules associated with the migratory and invasive phenotype, contributing, in cooperation to hypoxia, to tumor progression., ((c) 2005 Wiley-Liss, Inc.)

We have previously demonstrated that bcl-2 overexpression in tumor cells exposed to hypoxia increases the expression of vascular endothelial growth factor (VEGF) gene through the hypoxia-inducible factor-1 (HIF-1). In this article, we demonstrate that exposure of bcl-2 overexpressing melanoma cells to hypoxia induced phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2 proteins. On the contrary, no modulation of these pathways by bcl-2 was observed under normoxic conditions. When HIF-1alpha expression was reduced by RNA interference, AKT and ERK1/2 phosphorylation were still induced by bcl-2. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways reduced the induction of VEGF and HIF-1 in response to bcl-2 overexpression in hypoxia. No differences were observed between control and bcl-2-overexpressing cells in normoxia, in terms of VEGF protein secretion and in response to PI3K and MAPK inhibitors. We also demonstrated that RNA interference-mediated down-regulation of bcl-2 expression resulted in a decrease in the ERK1/2 phosphorylation and VEGF secretion only in bcl-2-overexpressing cell exposed to hypoxia but not in control cells. In conclusion, our results indicate, for the first time, that bcl-2 synergizes with hypoxia to promote expression of angiogenesis factors in melanoma cells through both PI3K- and MAPK-dependent pathways.

The purpose of this study was to evaluate the antitumoral activity of different gemcitabine-based combination on an experimental model of human breast cancer, in order to identify the most effective treatment and to provide a rationale for clinical investigations. To this end, CG5 breast cancer cells were treated in vitro with gemcitabine followed by epirubicin, doxorubicin, docetaxel or paclitaxel. The reversed sequence was also investigated. Results, analyzed by multiple drug effect/combination index (CI) isobologram, demonstrated that the combination gemcitabine/paclitaxel was the most active showing synergism with a CI of about 0.5 in the two sequences employed. Moreover, the synergistic interaction of gemcitabine and paclitaxel was correlated to a block of the cells in the G0/G1 compartment of cell cycle and to an increase of apoptotic cells compared to each drug. Based on these evidences, the antitumoral efficacy of gemcitabine/paclitaxel combination has been studied in vivo. Mice bearing CG5 human breast xenografts treated with paclitaxel and gemcitabine in combination showed a significant higher inhibition of tumor growth (approximately 70%) compared to that with either agent alone (25%). In conclusion, this study suggests that paclitaxel is the most promising agent for combination protocols with gemcitabine and supports the use of gemcitabine/paclitaxel combination in the clinical management of advanced breast cancer.

Clusterin (CLU) is a heterodimeric secreted glycoprotein implicated in several physiological and pathological processes including cancer. Although recent data showed that overexpression of CLU is closely associated with disease progression in patients with breast tumor, the functional role of CLU expression in this tumor hystotype remains to be determined. The objectives in this study were to evaluate CLU expression levels after treatment with Trastuzumab, a HER2-targeted monoclonal antibody used in the clinical management of advanced breast cancer patients, and to test the usefulness of combined treatment with OGX-011, the second generation 2'-methoxyethyl gapmer oligonucleotides targeting the CLU gene, and Trastuzumab in this tumor hystotype. By using the HER-2 gene amplified-BT474 human breast cancer cells, we found Trastuzumab decreased HER-2 expression and inhibited cell proliferation without affecting apoptosis. Interestingly, Trastuzumab treatment up-regulated CLU protein expression in a dose-dependent fashion. We therefore hypothesized that the treatment with OGX-011, by blocking Trastuzumab-induced CLU expression, might potentiate the growth-inhibitory effect of Trastuzumab alone. Although OGX-011 had no effect on the behavior of the BT474 cells when used alone, it significantly enhanced the sensitivity of cells to Trastuzumab. A significant increase in the percentage of apoptotic cells, analyzed in terms of annexin V positivity and cleavage of poly(ADP-ribose) polymerase, was observed after combined treatment with OGX-011 plus Trastuzumab but not with either agent alone. Altogether our findings suggest that combined targeting of HER-2 and CLU may represent a novel, rational approach to breast cancer therapy., ((c) 2005 Wiley-Liss, Inc.)

The anti-apoptotic protein Bcl-2 has been implicated in the intrinsic resistance of melanoma to chemotherapy. The aim of this study was to investigate the effects of anti-Bcl-2 oligonucleotide oblimersen on the antitumour activity of gimatecan, a novel lipophilic camptothecin currently undergoing clinical phase II studies. Results showed a reduced sensitivity of melanoma cells to gimatecan following Bcl-2 transfection and inversely, increased cell sensitivity to gimatecan in combination with oblimersen. In in vivo studies performed in two melanoma xenografts expressing different Bcl-2 levels, the antitumour activity of oblimersen itself was modest, but the combination with gimatecan produced a significant therapeutic advantage. The combination therapy inhibited tumour growth and delayed regrowth of the two tumours tested. The enhancement of antitumour activity was observed at doses that were tolerated well. The effects of oblimersen on antitumour activity and toxicity of gimatecan were dose-dependent. The capability of oblimersen to improve the efficacy of gimatecan supports the therapeutic potential of the drug combination in the treatment of human melanoma.

Purpose: bcl-2 and c-myc oncogenes are frequently overexpressed in different human tumors, including melanoma. Here, we evaluate the combined efficacy of two antisense oligonucleotides targeting bcl-2 mRNA (ODN bcl-2) and c-myc mRNA (ODN c-myc) in combination with cis-diammine dichloroplatinum (cisplatin, DDP) on three human melanoma lines (LM, NG, and M20)., Experimental Design: Two different sequences were designed to treat tumor-bearing mice: in the first one, ODN bcl-2 at a dose of 0.2 mg/day x4, followed by DDP given i.p. at a dose of 3.3 mg/kg/day x3 and ODN c-myc i.v. at 0.5 mg/day x7, whereas the other sequence consisted of ODN c-myc given as first agent followed by DDP and ODN bcl-2 at the same doses. Mice received three complete cycles of treatment in 1-week intervals., Results: The treatment sequence with ODN bcl-2/DDP/ODN c-myc combination completely inhibited growth in NG tumor and induced a 35-day delay in LM tumor growth. In contrast, the M20 tumor growth was unaffected by the combination. A discrete amount of c-Myc and bcl-2 protein expression in both LM and NG tumors was detected, whereas no detectable levels of the two proteins were observed in M20 tumors. Compared with the other combination, the sequence (ODN bcl-2/DDP/ODN c-myc) produced the most effective results, producing a significant decrease in bcl-2 and c-Myc protein expression, which in turn significantly increased the survival of NG- and LM-bearing mice, with 4 mice out of 11 and 1 out of 7 mice being cured, respectively. Finally, this combination increased the apoptotic rate and produced an antiangiogenetic effect., Conclusions: These results show that an antisense approach to the treatment of melanoma xenografts overexpressing either bcl-2 or c-myc oncogenes represents a successful strategy to improve the response to chemotherapy in melanoma, with particular attention to the treatment sequence.

BACKGROUND: Nitric oxide-releasing nonsteroidal antiinflammatory drugs (NO-NSAIDs) are reported to be safer than NSAIDs because of their lower gastric toxicity. We compared the effect of a novel NO-releasing derivate, NCX 4040, with that of aspirin and its denitrated analog, NCX 4042, in in vitro and in vivo human colon cancer models and investigated the mechanisms of action underlying its antitumor activity. METHODS: In vitro cytotoxicity was evaluated on a panel of colon cancer lines (LoVo, LoVo Dx, WiDr and LRWZ) by sulforhodamine B assay. Cell cycle perturbations and apoptosis were evaluated by flow cytometry. Protein expression was detected by Western blot. In the in vivo experiments, tumor-bearing mice were treated with NCX 4040, five times a week, for six consecutive weeks. RESULTS: In the in vitro studies, aspirin and NCX 4042 did not induce an effect on any of the cell lines, whereas NCX 4040 produced a marked cytostatic dose-related effect, indicating a pivotal role of the -NO2 group. Furthermore, in LoVo and LRWZ cell lines, we observed caspase-9 and -3-mediated apoptosis, whereas no apoptotic effect was observed after drug exposure in WiDr or LoVo Dx cell lines. In in vivo studies, both NCX 4040 and its parental compound were administered per os. NCX 4040 induced a 40% reduction in tumor weight. Conversely, aspirin did not influence tumor growth at all. CONCLUSIONS: NCX 4040, but not its parental compound, aspirin, showed an in vitro and in vivo antiproliferative activity, indicating its potential usefulness to treat colon cancer.

Purpose: In addition to their original function as cell cycle regulators, retinoblastoma (Rb) family members were recently reported to modulate the sensitivity of cancer cells to chemotherapeutic agents. The purpose of this study is to investigate the possible role of pRb2/p130 in the sensitivity of ovarian cancer to camptothecin, doxorubicin, and taxol., Experimental Design: pRb2/p130 was overexpressed in the CAOV-3 ovarian cancer cell line, and the effect of pRb2/p130 overexpression on sensitivity to apoptosis trigged by IC(50) doses of different drugs was evaluated by various methods, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and Western blot analyses., Results: The results reported in this study support the conclusion that overexpression of pRb2/p130 in the CAOV-3 ovarian cancer cell line lacking wild-type p53 is able to inhibit apoptosis triggered by camptothecin and doxorubicin through the c-Jun NH(2)-terminal kinase signaling transduction pathway. Conversely, taxol-induced cell death is not influenced by the pRb2/p130 protein level., Conclusions: A careful analysis of pRb2/p130 expression in tumor specimens could help to identify the best clinical protocol to be used for each patient, improving efficacy and tolerance and therefore offering additional progress in the treatment of advanced ovarian cancer.

Purpose: To investigate the possible existence of an antiapoptotic cross-talk between HER-2 and antiapoptotic Bcl-2 family members., Experimental Design: Bcl-2 and Bcl-XL expression and apoptosis induction were analyzed in HER-2 gene-amplified (BT474) and nonamplified (ZR 75-1) breast cancer cell lines exposed to trastuzumab, alone or in combination with either Bcl-2/Bcl-XL bispecific antisense oligonucleotides (AS-4625) or the small-molecule Bcl-2 antagonist HA14-1., Results: In addition to HER-2 and epidermal growth factor receptor, trastuzumab down-regulated Bcl-2, but not Bcl-XL, protein, and mRNA expression in BT474 cells. Interestingly, trastuzumab-induced down-regulation of HER-2 and Bcl-2 was also observed in three of five and two of three breast cancer patients undergoing trastuzumab treatment, respectively. Despite Bcl-2 down-regulation, however, trastuzumab only marginally increased the rate of apoptosis (7.3 +/- 3.5%). We therefore investigated whether a combination of AS-4625 and trastuzumab might increase proapoptotic efficiency. AS-4625 treatment of BT474 cells decreased both Bcl-2 and Bcl-XL expression, resulting in a 21 +/- 7% net apoptosis induction; the combination of AS-4625 followed by trastuzumab resulted in a significantly stronger induction of apoptosis (37 +/- 6%, P <0.01) that was not observed with the reverse treatment sequence (trastuzumab followed by AS-4625). Similar results were obtained with the Bcl-2 antagonist HA14-1; indeed, exposure of BT474 cells to HA14-1 followed by trastuzumab resulted in a striking proapoptotic synergism (combination index=0.58 +/- 0.18), as assessed by isobologram analysis., Conclusions: Altogether our findings suggest that combined targeting of HER-2 and Bcl-2 may represent a novel, rational approach to more effective breast cancer therapy.

This study had two goals: 1) to evaluate the biological effect of the novel pentacyclic acridine 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) on human melanoma lines possessing long telomeres, and 2) to elucidate the relationship between G-quadruplex-based telomerase inhibitor-induced cellular effects and telomere length/dysfunction. The cellular pharmacological effects of RHPS4 have been evaluated by treating melanoma lines with increasing concentrations of RHPS4. A dose-dependent inhibition of cell proliferation was observed in all the lines during short-term treatment. Flow cytometric analysis demonstrated that RHPS4 induced a dose-dependent accumulation of cells in the S-G(2)/M phase of cell cycle. The RHPS4-induced cell cycle alteration was irreversible even at low doses, and the cells died from apoptosis. At high RHPS4 concentration, apoptosis was accompanied by the induction of a senescence phenotype: large cell size, vacuolated cytoplasm, and beta-galactosidase activity. The short-term biological activity of RHPS4 was not caused by telomere shortening, but it was associated with telomere dysfunction, in terms of presence of telomeric fusions, polynucleated cells, and typical images of telophase bridge. In conclusion, our results demonstrate that the G-quadruplex ligand RHPS4 can function in a telomere length-independent manner through its ability to cause telomere-capping alteration.

The aim of this study was to assess whether lonidamine (LND) interferes with some steps in angiogenesis progression. We report here, for the first time, that LND inhibited angiogenic-related endothelial cell functions in a dose-dependent manner (1-50 microg/ml). In particular, LND decreased proliferation, migration, invasion, and morphogenesis on matrigel of different endothelial cell lines. Zymographic and Western blot analysis assays showed that LND treatment produced a reduction in the secretion of matrix metalloproteinase-2 and metalloproteinase-9 by endothelial cells. Vessel formation in a matrigel plug was also reduced by LND. The viability, migration, invasion, and matrix metalloproteinase production of different tumor cell lines were not affected by low doses of LND (1-10 microg/ml), whereas 50 microg/ml LND, which corresponds to the dose used in clinical management of tumors, triggered apoptosis both in endothelial and tumor cells. Together, these data demonstrate that LND is a compound that interferes with endothelial cell functions, both at low and high doses. Thus, the effect of LND on endothelial cell functions, previously undescribed, may be a significant contributor to the antitumor effect of LND observed for clinical management of solid tumors.

Resistance to chemotherapy is a common feature of malignant gliomas. This resistance is mediated by receptor tyrosine kinase (RTK)-regulated signaling. p21-Ras protein is pivotal in the propagation of the signal originated from many RTKs. Our aim was to investigate whether inhibition of Ras pathway affects the response to cisplatin in malignant gliomas. We found an enhanced sensitivity to cisplatin of two glioblastoma cell lines expressing dominant negative Ras. Moreover, DN-Ras expressing cells, implanted in nude mice, resulted in being extremely sensitive to cisplatin. The growth of all the tumors was significantly inhibited by combining DN-Ras adenovirus infection with cisplatin treatment. The majority of glioma cells expressing DN-Ras underwent apoptosis in response to cisplatin. In vivo, DN-Ras alone did not influence the growth of tumors, suggesting that the effects of Ras-inhibition observed in vitro could not be extrapolated in vivo. The survival signal pathway transduced by Ras was essentially mediated by inhibition of caspase-9 cleavage via PI3K/Akt.

Here we evaluated the antitumor efficacy of vincristine (VCR) encapsulated in sphingomyelin/cholesterol liposomes (SM/Chol) on drug-resistant human solid tumors. We firstly used the M14 human melanoma line and the counterpart resistant derivative, M14/R. The M14/R, selected after doxorubicin exposure, was cross resistant to VCR: the in vitro treatment with free VCR reduced the survival of M14, while M14/R line was completely resistant to VCR. Encapsulation in liposomes improved the efficacy of VCR in M14 cells and sensitized the M14/R line to the drug. Experiments in vivo confirmed these results. The treatment of M14 bearing mice with VCR resulted in marked reduction of tumor growth, while no antitumoral effect was observed in M14/R tumors. The administration of VCR encapsulated in liposomes was able to sensitize M14/R tumors to the drug, the antitumoral effect being comparable to that observed in M14 tumors after the same treatment. By injecting animals with the same dose of liposomal VCR fractionated into 3 daily injections and administering repeated cycles of treatment, to a marked improvement of the antitumor activity of liposomal VCR was observed. TUNEL assay in tumor sections indicated that the improved efficacy of liposomal VCR was related to the induction of massive necrosis and apoptosis. To confirm the efficacy of liposomal VCR on drug-resistant tumors, MCF7 breast and LoVo colon carcinomas, sensitive and resistant to VCR treatment, were also employed. The results showed that the treatment with liposomal VCR of mice bearing breast or colon resistant tumors reduced the tumor mass and delayed the tumor regrowth to the same extent observed in the sensitive counterpart. Together, these results demonstrate the ability of VCR encapsulated in liposomes in sensitizing drug resistant tumors of different histotypes., (Copyright 2004 Wiley-Liss, Inc.)

We have targeted the urokinase-type plasminogen activator receptor (uPAR) with phosphorothioate antisense oligonucleotides (aODN) in vitro to evaluate the anti-invasive and anti-proliferative effects of uPAR down-regulation, as well as in vivo to evaluate anti-tumor and anti-metastatic activity. aODN-dependent uPAR downregulation in vitro was induced in cells of human melanoma, mammary carcinoma, ovarian carcinoma and SV-40-transformed embryonic lung fibroblasts. uPAR was determined by an antibody-based assay and by semiquantitative polymerase chain reaction. Cell invasion was evaluated by Matrigel invasion assay and cell proliferation by direct cell counting. aODN reduced uPAR, invasion and proliferation in all the treated cell lines. Following aODN treatment, human melanoma cells exhibited a strong decrease of uPAR-dependent ERK1/2 activation and were used in vivo to control metastasis in CD-1 male nude (nu/nu) mice by uPAR aODN injection. 60 mice were injected in the hind leg muscles with a suspension of 10(6) melanoma cells. After 4 days, when a tumor mass of about 350 mg was evident in all the mice injected, 20 mice were treated i.v. with aODN and 20 with dODN at 0.5 mg/day for 5 consecutive days. Twenty control mice were not treated. A second and third cycle of treatment was administered at 2-day intervals. Treatment with aODN resulted into a 78% reduction of lung metastases and 45% reduction of the primary tumor mass with no loss of body weight. Our results suggest to evaluate the utility of uPAR aODN in controlling the metastatic spreading of human melanoma., (Copyright 2004 Wiley-Liss, Inc.)

Here we investigate the mechanism(s) involved in the c-Myc-dependent drug response of melanoma cells. By using three M14-derived c-Myc low-expressing clones, we demonstrate that alkylating agents, cisplatin and melphalan, trigger apoptosis in the c-Myc antisense transfectants, but not in the parental line. On the contrary, topoisomerase inhibitors, adriamycin and camptothecin, induce apoptosis to the same extent regardless of c-Myc expression. Because we previously demonstrated that c-Myc downregulation decreases glutathione (GSH) content, we evaluated the role of GSH in the apoptosis induced by the different drugs. In control cells treated with one of the alkylating agents or the others, GSH depletion achieved by L-buthionine-sulfoximine preincubation opens the apoptotic pathway. The apoptosis proceeded through early Bax relocalization, cytochrome c release, and concomitant caspase-9 activation, whereas reactive oxygen species production and alteration of mitochondria membrane potential were late events. That GSH was determining in the c-Myc-dependent drug-induced apoptosis was demonstrated by altering the intracellular GSH content of the c-Myc low-expressing cells up to the level of controls. Indeed, GSH ethyl ester-mediated increase of GSH abrogated apoptosis induced by cisplatin and melphalan by inhibition of Bax/cytochrome c redistribution. The relationship among c-Myc, GSH content, and the response to alkylating agent has been also evaluated in the M14 Myc overexpressing clones as well as in the melanoma JR8 c-Myc antisense transfectants. All together, these results demonstrate that GSH plays a key role in governing c-Myc-dependent drug-induced apoptosis.

We have previously demonstrated that Bcl-2 overexpression in human breast carcinoma and melanoma cells synergizes with hypoxia to increase angiogenesis through up-regulation of vascular endothelial growth factor. In this work we demonstrated, for the first time, that Bcl-2 overexpression in cancer cells exposed to hypoxia modulates urokinase plasminogen activator receptor (uPAR) expression through Sp1 transcription factor and that the extracellular signal-regulated kinase (ERK) pathway plays a role in Sp1 transcriptional activity. In particular, an increase in uPAR protein and mRNA expression was found in melanoma bcl-2 transfectants grown under hypoxia when compared with control cells, and a decrease of uPAR protein expression was induced by treatment of cells with specific bcl-2 antisense oligonucleotides. Up-regulation of uPAR expression was accompanied by increased Sp1 protein expression, stability, serine phosphorylation, and DNA binding activity. Treatment of cells with mitramycin A, an inhibitor of Sp1 activity, confirmed the role of Sp1 transcriptional activity in uPAR induction by Bcl-2. The contribution of the ERK pathway in Sp1-increased transcriptional activity was demonstrated by the use of chemical inhibition. In fact, ERK kinase activation was induced in Bcl-2-overexpressing cells exposed to hypoxia, and the ERK kinase inhibitor UO126 was able to down-regulate Sp1 phosphorylation and DNA binding activity. Using a human breast carcinoma line, we obtained data supporting our findings with melanoma cells and identified a link between the induction of Sp1 and uPAR expression as a common bcl-2-controlled phenomenon in human tumors. In conclusion, our results strongly indicate that up-regulation of uPAR expression by Bcl-2 in hypoxia is modulated by Sp1 DNA binding activity through the ERK signaling pathway.

We have recently reported that bcl-2 overexpression and hypoxia synergistically interact to modulate vascular endothelial growth factor (VEGF) and in vivo angiogenesis in tumour cells through VEGF mRNA stabilization and hypoxia-inducible factor 1-mediated transcriptional activity. Bcl-2 antisense treatment has shown promising clinical results in patients with malignant melanoma. In the present study, we demonstrated that the bcl-2/bcl-xL bispecific antisense oligonucleotide 4625 inhibits bcl-2 expression and angiogenesis in two bcl-2 overexpressing clones derived from the M14 human melanoma cell line. The antiangiogenic effect was determined in in vitro and in vivo angiogenesis assays. In particular, a reduction of hypoxia-induced VEGF secretion was observed after 4625 treatment, and the conditioned medium (CM) of bcl-2 overexpressing clones treated with 4625 and exposed to hypoxic conditions resulted in decreased endothelial cell proliferation when compared to CM of untreated control cells. In addition, we found that CM of 4625 antisense-treated bcl-2 transfectants inhibited in vivo vessel formation in matrigel plugs implanted subcutaneously in C57/B16 mice. Our findings confirm that bcl-2 plays a crucial role in melanoma angiogenesis and demonstrate for the first time that downregulation of bcl-2 by antisense treatment has potential to inhibit angiogenesis independent of its effect on cell survival. The use of 4625 in cancer therapy is suggested as an approach to facilitate simultaneously tumour cell apoptosis and inhibit tumour angiogenesis.

Purpose: Temozolomide (TMZ) is a DNA methylating agent that has shown promising antitumor activity in recent clinical trials against high grade gliomas, metastatic melanoma, and brain lymphoma. In this study, we tested whether systemic administration of GPI 15427, a novel poly(ADP-ribose) polymerase (PARP-1) inhibitor capable of crossing the blood-brain barrier, could enhance the efficacy of TMZ against metastatic melanoma, glioblastoma multiforme, and lymphoma growing in the brain., Experimental Design: Murine B16 melanoma or L5178Y lymphoma cells were injected intracranially in syngeneic mice. An orthotopic xenograft of the human SJGBM2 glioblastoma multiforme was implanted in nude mice. Animals were treated with TMZ + GPI 15427 using a schedule of 40 mg/kg/i.v. GPI 15427 + 100 mg/kg/i.p. TMZ for 3 days. The efficacy of drug treatment was assessed by: (a) the increase of mouse survival and life span; and (b) the suppression of melanoma metastases to lung after i.v. injection of B16 cells., Results: In all models, systemic administration of GPI 15427 shortly before TMZ significantly increased life span of tumor-bearing mice with respect to untreated controls or to groups treated with either GPI 15427 or TMZ only. Moreover, GPI 15427 increased the antimetastatic effect of TMZ., Conclusions: These data indicate that systemic administration of the poly(ADP-ribose) polymerase-1 inhibitor GPI 15427 significantly enhances TMZ antitumor efficacy against solid or hematological neoplasias even when located at the central nervous system site.

The current direction in cancer research is rational drug design, which is based on the evidence that transformed cells are characterized by alterations of genes devoted to the regulation of both cell proliferation and apoptosis. A variety of approaches have been carried out to develop new agents selective for cancer cells. Among these, antisense oligonucleotides (ASOs) are one of such class of new agents able to inhibit specifically the synthesis of a particular cancer-associated protein by binding to protein-encoding RNA, thereby preventing RNA function. In the past decade, several ASOs have been developed and tested in preclinical and clinical studies. Many have shown convincing in vitro reduction in target gene expression and promising activity against a wide variety of tumors. However, because of the multigenic alterations of tumors, the use of ASOs as single agents does not seem to be effective in the treatment of malignancies. Antisense therapy that interferes with signaling pathways involved in cell proliferation and apoptosis are particularly promising in combination with conventional anticancer treatment. An overview of the progress of ASOs used in combination therapy is provided.

Here, we show that inhibition of c-Myc causes a proliferative arrest of M14 melanoma cells through cellular crisis, evident by the increase in size, multiple nuclei, vacuolated cytoplasm, induction of senescence-associated beta-galactosidase activity and massive apoptosis. The c-Myc-induced crisis is associated with decreased human telomerase reverse transcriptase expression, telomerase activity, progressive telomere shortening, glutathione (GSH), depletion and, increased production of reactive oxygen species. Treatment of control cells with L-buthionine sulfoximine decreases GSH to levels of c-Myc low expressing cells, but it does not modify the growth kinetic of the cells. Surprisingly, when GSH is increased in the c-Myc low expressing cells by treatment with N-acetyl-L-cysteine, cells escape crisis. To test the hypothesis that both oxidative stress and telomerase dysfunction are involved in the c-Myc-dependent crisis, we directly inhibited telomerase function and glutathione levels. Inactivation of telomerase, by expression of a catalytically inactive, dominant negative form of reverse transcriptase, reduces cellular lifespan by inducing telomere shortening. Treatment of cells with L-buthionine sulfoximine decreases GSH content and accelerates cell crisis. Analysis of telomere status demonstrated that oxidative stress affects c-Myc-induced crisis by increasing telomere dysfunction. Our results demonstrate that inhibition of c-Myc oncoprotein induces cellular crisis through cooperation between telomerase dysfunction and oxidative stress.

Our aim was 2-fold: to investigate the role of alpha-tocopherol supplementation on the antitumor activity of DDP and to evaluate the effect of alpha-tocopherol on the survival and neurotoxicity of DDP-treated mice. Experiments performed on the M14 human melanoma line demonstrated that alpha-tocopherol supplementation did not influence the efficacy of DDP; the inhibition of cell survival and of the in vivo tumor growth after treatment with alpha-tocopherol and DDP combination was similar to that observed after DDP alone. Conversely, alpha-tocopherol was also able to increase survival of mice treated with a high dose of DDP. While DDP alone produced death in about 70% of mice, the combination reduced deaths to about 30%. Analysis of oxidative stress markers and peroxidative damage in organs indicated that the protective effect of alpha-tocopherol was mainly related to its antioxidant activity. A significant increase in the concentration of TBARS and decreased PUFAs and catalase activity were observed after DDP treatment, while with alpha-tocopherol the levels of these markers were comparable to those observed in untreated mice. Histologic analysis performed on peripheral nerve revealed that alpha-tocopherol also protected mice from severe neurologic damage induced by DDP treatment. In conclusion, our results demonstrate that alpha-tocopherol protects against the systemic toxicity and neurotoxicity induced by DDP without interfering with its antitumor activity and suggest that this combination is a promising strategy to improve the therapeutic index of DDP-based chemotherapy., (Copyright 2003 Wiley-Liss, Inc.)

The aim of this study was to investigate the role of telomerase function on the chemosensitivity of melanoma cells. To this end, ecteinascidin-743 (ET-743) and cisplatin [cis-diamminedichloroplatinum(II) (CDDP)], two DNA-interacting drugs that invariably cause an arrest in the G(2)/M phase, and 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid (LND), a mitochondria-targeting drug inducing a G(1) block, were used. As experimental model, human melanoma clones showing reduced human telomerase reverse transcriptase (hTERT) expression and telomerase activity and characterized by telomere dysfunction were used. Reconstitution of telomerase activity by exogenous hTERT expression improved telomere function and reduced the sensitivity to CDDP and ET-743 without affecting LND susceptibility. The decreased sensitivity to CDDP and ET-743 was mainly caused by the ability of cells to recover from drug-induced damage, evaluated in terms of both chromosomal lesions and cell survival. The ability of hTERT-reconstituted cells to recover from drug-induced damage was attributable to the restoration of cell cycle progression. In fact, the cells without hTERT restoration remained for a prolonged time in the G(2)/M phase, and this cell cycle alteration made irreversible the drug-induced S-G(2)/M block and led to the activation of apoptotic program. On the contrary, the hTERT-reconstituted cells progressed quickly through the cell cycle, thus acquiring the capacity to recover from drug-induced block and to protect themselves from the G(2)/M phase-specific drug-triggered apoptosis.

The objective of this article is to dissect the mechanisms by which the down-regulation of c-Myc induces programmed cell death in melanoma cells. In stable and doxycycline-inducible M14 melanoma cells, down-regulation of c-Myc induced apoptosis subsequent to a decrease in the intracellular reduced glutathione content and a concomitant accumulation of its oxidized form. This redox alteration was associated with a decrease of the enzyme activities of gamma-glutamyl-cysteine synthetase and NADPH-dependent GSSG reductase, as well as a consequent glutathione release in the extracellular medium. Cytochrome c was released into the cytosol at very early stages of apoptosis induction, long before detectable production of reactive oxygen species and activation of caspase-9 and -3. Macroarray analysis revealed that down-regulation of c-Myc produced striking changes in gene expression in the section related to metabolism, where the expression of gamma-glutamyl-cysteine synthetase and GSSG reductase was found to be significantly reduced. The addition of N-acetyl-l-cysteine or glutathione ethyl ester inhibited the apoptotic process, thus confirming the key role of glutathione in programmed cell death induced by c-Myc.

The aim of this paper was to study the molecular mechanisms by which bcl-2 increases hypoxia-induced vascular endothelial growth factor (VEGF) expression. We demonstrated that bcl-2 overexpression in M14 human melanoma cell line enhances hypoxia-induced VEGF mRNA stability and promoter activation. In particular, the half-life of the message was longer in bcl-2 transfectants (approximately 330 min) than in control cells (approximately 180 min). In addition, bcl-2 overexpression increased VEGF promoter activity through the hypoxia-inducible factor-1 (HIF-1) transcription factor. Increased HIF-1a protein expression and DNA binding activity were detected in bcl-2 overexpressing cells compared with control cells. An enhanced functional activity of secreted VEGF was found both in in vitro and in vivo angiogenic assays, and the use of VEGF specific antibodies validated the role of VEGF on bcl-2-induced angiogenesis. Taken together our results indicate that bcl-2 plays an important role in melanoma angiogenesis, and that VEGF mRNA stabilization and HIF-1-mediated transcriptional activity are two important control points in bcl-2/hypoxia-induced VEGF expression.

c-Myc is involved in the control of telomerase activity through its ability to induce the expression of the catalytic subunit of the enzyme, the human telomerase reverse transcriptase (hTERT). Our aim was to study whether telomerase plays a critical role in c-Myc-dependent tumorigenicity of melanoma cells. By using M14-derived clones, expressing low levels of c-Myc, we demonstrated that the down-regulation of c-Myc reduced cell proliferation rate, cloning efficiency and tumorigenicity and increased the apoptotic rate. Decreased tumorigenic potential correlated with reduced hTERT gene expression, telomerase activity and telomere shortening. Introduction of wild-type hTERT into these cells increased their proliferation rate and partially re-established their tumorigenic potential, at early passages, even though the apoptotic rate of the population remained unaltered. After several in vitro passages, hTERT-mediated cell proliferation made the tumorigenic potential of the c-Myc low-expressing clones comparable to that of the M14 parental line. Over-expression of the mutant biologically inactive hTERT did not drive cells to proliferate. In conclusion, our results demonstrate that the reconstitution of high levels of telomerase activity reverses the low tumorigenicity due to low c-Myc expression.

Constitutive bcl-2 overexpression increases the tumorigenic and metastatic potential of doxorubicin-resistant, estrogen-independent, MCF-7 ADR human breast cancer cells. We evaluated the sensitivity to taxanes (paclitaxel, docetaxel and IDN 5109) of 2 bcl-2-overexpressing MCF-7 ADR clones and control neomycin-transfected MCF-7 ADR neo cells. The 2 bcl-2-overexpressing MCF-7 ADR clones were relatively resistant to all 3 taxanes, whereas the MCF-7 ADR neo cells were relatively resistant to paclitaxel and docetaxel, but sensitive to IDN 5109. We found that both MCF-7 ADR neo and bcl-2-overexpressing MCF-7 ADR clones express high levels of the epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor-alpha (TGF-alpha). Therefore, we tested the growth inhibitory effect of ZD1839 (Iressa, AstraZeneca, Macclesfield, UK), an orally active, selective EGFR tyrosine kinase inhibitor (EGFR-TKI) that is in clinical development. ZD1839 inhibited the growth in soft agar of all 3 clones in a dose-dependent manner (IC(50) of approximately 0.1 microm). This effect was accompanied by a dose-dependent inhibition of EGFR tyrosine autophosphorylation and of the production of TGF-alpha, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). To determine whether the blockade of EGFR signaling might affect the sensitivity of bcl-2-overexpressing MCF-7 ADR cells to taxanes, cells were treated with ZD1839 in combination with paclitaxel, docetaxel or IDN 5109, and dose-dependent cooperative growth inhibition as well as apoptosis potentiation were observed. Combined treatment with IDN 5109 and ZD1839 also resulted in a significant inhibition of bcl-2 expression in bcl-2-overexpressing MCF-7 ADR cells. These results demonstrate the ability of ZD1839 to overcome taxane resistance in a model of hormone-independent, multidrug-resistant, human breast cancer., (Copyright 2002 Wiley-Liss, Inc.)

Temozolomide (TZM) is a DNA-methylating agent that has recently been introduced into various clinical trials for treatment of solid or hematologic neoplasias, including brain lymphomas. In the current study, we have investigated whether the antitumor activity of TZM could be selectively enhanced at the central nervous system (CNS) site by intracerebral injection of a poly(ADP-ribose) polymerase (PARP) inhibitor. Mice were injected intracranially with lymphoma cells. The PARP inhibitor NU1025 (1 mg/animal) was delivered intracerebrally, whereas TZM was given as a single or a fractionated dose of 200 mg/kg by intraperitoneal administration. Results indicated that this drug combination significantly enhanced the survival of tumor-bearing mice and that this fractionated modality of treatment was the most effective schedule. Increased survival time was related to a marked reduction of tumor growth, as evidenced by histologic studies. Treatment with TZM alone was ineffective. This is the first report exploring in vivo the combination of TZM with PARP inhibitor for intracerebral neoplasias.

The aim of this study was to evaluate the role of bcl-2 in 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) sensitivity of the ADFS human glioblastoma cell line in vitro and in vivo. To this end, the ADFS line expressing a low level of the bcl-2 protein was transfected with a bcl-2 expression vector. We found that bcl-2 overexpressing clones were less sensitive to in vitro BCNU treatment than the control clone. Cell cycle analysis demonstrated that while BCNU induced a consistent block in S/G2-M phases of the cell cycle in the control clone, it did not affect the cell cycle phase distribution of the two bcl-2 transfectants. The different sensitivity to BCNU was unrelated to the ability of bcl-2 to inhibit apoptosis, while bcl-2 appeared to protect bcl-2 transfectants from BCNU toxicity through an increase of catalase activity. The ability of the catalase inhibitor, sodium azide, to increase the BCNU sensitivity of the bcl-2 transfectants to levels of the BCNU-treated control clone substantiated the role of the catalase activity. The effect of bcl-2 in reducing sensitivity to BCNU was also confirmed by in vivo experiments. Xenografts of bcl-2 overexpressing tumors were less sensitive to BCNU treatment than xenografts originating from control cells., (Copyright 2001 Wiley-Liss, Inc.)

Our aim in this work was to define the role of c-Myc in the susceptibility to cisplatin [cis-diamminedichloroplatinum(II) (CDDP)] in human melanoma cells. Two M14 melanoma cell clones obtained by transfection and expressing six to ten times lower c-Myc protein levels than the parental cells and the control clone were employed. Analysis of survival curves demonstrates an increase in CDDP sensitivity in c-Myc low-expressing clones if compared with the control clone and the parental line. The enhanced sensitivity is unrelated to the impairment in enzymatic DNA repair activity. Cell cycle analysis demonstrates that although the control clone is able to completely recover from the CDDP-induced S-G(2)/M block, this arrest is prolonged in c-Myc low-expressing clones and a fraction of cells undergoes apoptosis. Although no changes in P53, Bax, Bcl-2, and Bcl-x(L/S) protein levels are observed, apoptosis is associated with the formation of reactive oxygen species (ROS), activation of caspase-1, caspase-3 and cleavage of the specific caspase substrate poly-ADP-ribose polymerase. The use of the antioxidant N-acetyl cysteine and caspase inhibitors prevents CDDP-induced apoptosis in c-Myc low-expressing clones, demonstrating that ROS, caspase-1, and caspase-3 are required for apoptotic cell death. Moreover, ROS generation depends on caspase-1-like activation because the Ac-YVAD-cho inhibitor abrogates CDDP-induced ROS in the c-Myc low-expressing clones.

Phosphorothioate c-myc antisense oligodeoxynucleotides [S]ODNs (free INX-6295) were encapsulated in a new liposome formulation and the antitumor activity was compared to the unencapsulated antisense in a human melanoma xenograft. The systemic administration of INX-6295 encapsulated in stabilized antisense lipid particles (SALP INX-6295) improved plasma AUC (area under the plasma concentration-time curve) and initial half-life of free INX-6295, resulting in a significant enhancement in tumor accumulation and improvement in tumor distribution of antisense oligodeoxynucleotides. Animals treated with SALP INX-6295 exhibited a prolonged reduction of c-myc expression, reduced tumor growth and increased mice survival. When administered in combination with cisplatin (DDP), SALP INX-6295 produced a complete tumor regression in approximately 30% of treated mice, which persisted for at least 60 days following the first cycle of treatment. Finally, the median survival of mice treated with DDP/SALP INX-6295 increased by 105% compared to 84% for animals treated with the combination DDP/free INX-6295. These data indicate that the biological activity and the therapeutic efficacy of c-myc antisense therapy may be improved when these agents are administered in lipid-based delivery systems.

The aim of this study was twofold: to assess the relationship between c-Myb and Bcl-x expression and to evaluate the prognostic significance of their expression in colorectal carcinoma (CRC) patients. Analysis of tumors from 91 CRC patients for expression of c-Myb and Bcl-x revealed a significant relationship between these two proteins. Kaplan-Meier's analysis showed an increased risk of relapse and death in patients whose tumor specimens displayed high c-Myb levels and Bcl-x positivity. Similar results were also observed excluding Dukes' D patients. Molecular analysis using three c-Myb-overexpressing LoVo clones indicated that c-Myb overexpression was accompanied by up-regulation of Bcl-x(L) protein and mRNA. Tumors originating from these clones injected in nude mice were significantly larger than those formed in mice injected with parental or vector-transfected LoVo cells. Moreover, tumors derived from parental and control vector-transfected but not from c-Myb-overexpressing LoVo cells showed high frequency of apoptotic cells. These results provide direct evidence of an association between c-Myb and Bcl-x expression and suggest that expression of both molecules might be a useful prognostic marker in CRC.

We have previously demonstrated that bcl-2 overexpression enhances the metastatic potential of the MCF7 ADR human breast cancer cell line resistant to adriamycin by inducing metastasis-associated properties. To further elucidate the relationship between bcl-2 expression and the metastatic potential of the MCF7 ADR line, we evaluated whether bcl-2 could be also involved in the modulation of the angiogenic phenotype. Four bcl-2-overexpressing clones, a control transfectant clone, and the MCF7 ADR parental line were used for in vitro and in vivo experiments. Bcl-2 overexpression enhanced the synthesis of the hypoxia-stimulated VEGF protein and mRNA. Northern blot analysis demonstrated an increased VEGF mRNA expression in bcl-2-overexpressing clones, and reverse transcription-polymerase chain reaction showed higher levels of the VEGF(121) and VEGF(165) mRNA isoforms, which are the most active in eliciting angiogenesis. When incorporated into matrigel, supernatants of bcl-2-transfected cells cultured under hypoxic conditions induced an increased angiogenic response in C57BL/6 mice compared with that of control clone. Tumors from bcl-2 transfectants demonstrated increased VEGF expression and neovascularization as compared to the parental line, whereas the apoptosis in in vivo xenografts was similar in control and bcl-2 transfectants. The effect of bcl-2 on angiogenesis was not mediated by p53 protein. These results demonstrate that bcl-2 and hypoxia can act synergistically to modulate VEGF expression and the in vivo angiogenic response in the MCF7 ADR line.

TGFbeta can modulate neuroblastoma (NB) cell proliferation and differentiation in vitro. In this study we used a NB cell line (LAN-5) which has been shown to partially respond to TGFbeta and to present high levels of TGFbeta receptor type I and low levels of receptor type II (TbetaRII) on the cell surface. To evaluate the role of TbetaRII in mediating TGFbeta effects, LAN-5 cells were transfected with an expression vector containing the human full-length TbetaRII cDNA or with the empty vector pcDNA3. Compared to control CLV3 cells (transfected with empty plasmid) and parental LAN-5 cells, isolated neomycin-resistant clones (CL1 and CL3) expressed higher levels of TbetaRII, had reduced cell growth rate in vitro, and were unable to form tumors in vivo. Furthermore, isolated clones modified their morphology, assuming a terminally differentiated neuronal phenotype. Immunocytochemical staining demonstrated a basal increased expression of neural-specific markers, such as axonal growth-associated protein (GAP43) and neurofilaments (NF200). TGFbeta treatment further increased the synthesis of NF200 and GAP43 in the transfected clones as revealed by Western blot analysis. These data indicate that TbetaRII overexpression potentiates the TGFbeta signal transduction pathway, reverting NB cell neoplastic phenotype with the reduction of proliferation rate and the induction of terminal maturation., (Copyright 2000 Academic Press.)

About 40% of patients with colorectal carcinoma will develop local or distant tumour recurrences. Integrated analyses of bio-pathological markers, predictive of tumour aggressiveness, may offer a more rational approach to planning adjuvant therapy. To this end, we analysed the correlation between p53 accumulation, Bcl-2 expression, DNA ploidy, cell proliferation and conventional clinico-pathological parameters by testing the prognostic significance of these variables in a series of 171 colorectal carcinoma patients with long-term follow-up. The relationships among the various bio-pathological parameters, analysed by multiple correspondence analysis, showed 2 different clinico-biological profiles. The first, characterised by p53 negativity, Bcl-2 positivity, diploidy, low percentage of cells in S-phase (%S-phase), a low Ki-67 score, is associated with Dukes' A-B stage, well differentiated tumours and lack of relapse. The second, defined by p53 positivity, Bcl-2 negativity, aneuploidy, high %S-phase and elevated Ki-67 score, correlates with Dukes' C-D stage, poorly differentiated tumours and presence of relapse. When these parameters were examined according to Kaplan-Meier's method, significantly shorter disease-free (DFS) and overall survival (OS) were also observed in patients bearing p53 positive and Bcl-2 negative tumours, in Dukes' B stage. In multivariate analysis, p53 accumulation and Bcl-2 expression emerged as independent predictors of a worse and better clinical outcome, respectively. Our results indicate that, in colorectal adenocarcinomas, a biological profile, based on the combined evaluation of p53 and Bcl-2, may be useful for identifying high risk patients to be enrolled in an adjuvant setting, mainly in an early stage of the disease. Int. J. Cancer (Pred. Oncol.) 84:545-552, 1999., (Copyright 1999 Wiley-Liss, Inc.)

A phosphorothioate c-myc antisense oligonucleotide was complexed with zinc and encapsulated into injectable biodegradable microspheres. The efficacy of this novel formulation was compared with intravenous administration of the unencapsulated drug in human melanoma and leukemia xenografts in immunocompromised mice. The microencapsulated formulation was more effective as shown by reduced tumor growth, a decreased number of metastases, reduced c-myc expression, and increased survival in the melanoma model, and decreased metastatic potential and increased survival in the leukemia model. These results show that, as has been demonstrated previously with protein and peptide drugs, greater therapeutic efficacy can be obtained when antisense oligonucleotides are delivered from sustained-release formulations.

In this study, we evaluated the role of the c-myc oncogene in response to cisplatin (DDP) treatment using two melanoma lines derived from the primary tumor (LP) and metastatic lymph node (LM) of the same patient. These cell lines, which retain the phenotypic profile of the original tumors, were studied for growth behavior, expression of c-Myc oncoprotein, and HLA-I antigen. The LM line shows a higher tumorigenic ability, an increased expression of c-Myc protein, and a lack of HLA-I antigen, compared with the LP line. In addition, LP tumor was relatively sensitive to DDP administration, whereas LM tumor was resistant to DDP treatment. To verify whether the increased c-Myc expression observed in the LM line might be responsible for DDP resistance, a c-myc antisense phosphorothioate oligodeoxynucleotide ([S]ODN) was used to down-regulate c-Myc expression. The administration of DDP plus c-myc antisense [S]ODNs produced a decrease in c-Myc protein levels of approximately 50%, accompanied by a tumor weight inhibition of 65%, similar to that obtained when the sensitive line was treated with DDP alone (tumor weight inhibition = 70%). Analysis of apoptosis demonstrated that the sensitivity to DDP of the LP line was related to the ability of tumor cells to undergo apoptosis. Conversely, DDP treatment was not able to induce apoptosis in the LM line, whereas apoptosis was evident both after treatment with c-myc antisense [S]ODNs alone and, more extensively, in combination with DDP. Taken together, these results clearly indicate an important role of c-myc oncogene in the resistance of melanoma to DDP and demonstrate that treatment with c-myc antisense [S]ODN sensitizes a human melanoma line to DDP treatment.

Lonidamine (LND), a selective inhibitor of the energy metabolism of tumor cells, induces apoptosis, independently of the p53 gene, in the adriamycin(ADR)-resistant MCF7 breast-cancer cell line (MCF7 ADR). On the contrary, LND fails to activate the apoptotic program in the parental MCF7-sensitive cell line (MCF7 WT). The extent of bcl-2 expression might account for the different effect of LND on these cell lines. In fact, the MCF7 ADR line shows a low level of bcl-2 protein, whereas MCF7 WT expresses a high level of bcl-2. We therefore investigated the relationship between the amount of bcl-2 and the ability of LND to induce apoptosis, using 4 clones over-expressing bcl-2. The effect of bcl-2 on the energy metabolism was also evaluated. We demonstrated that over-expression of bcl-2 inhibited LND-induced apoptosis, while reducing 14CO2 production, oxygen uptake and ATP content, whereas aerobic lactate production was essentially unaffected. In addition, LND decreased the oxidative metabolism of the MCF7 ADR cells to a greater extent than it did in the bcl-2 transfectants.

In this article, we investigated the effect induced by the reintroduction of wild-type p53 (wt-p53) protein on BCNU sensitivity in the ADF glioblastoma line. Using a wt-p53 recombinant adenovirus (Ad-p53), we demonstrated that exogenous wt-p53 expression was able to increase the sensitivity to BCNU in ADF cells. Interestingly, this effect was more evident when Ad-p53 infection was performed after BCNU treatment compared with the opposite sequence. To understand the biological basis of these different behaviors, we analyzed the cell cycle of the differently treated cells. We found that Ad-p53 infection induced a persistent accumulation of cells in the G0/G1 phase while, as expected, BCNU induced a block in the G2-M phase. Ad-p53-->BCNU sequence did not significantly modify the cell cycle profile in respect of Ad-p53 infected cells. In contrast, BCNU-->Ad-p53 sequence provoked G2-M arrest similar to that observed after treatment with BCNU alone, but prevented the later recovery of the cells through the cell cycle, by driving the cells to apoptotic death. These results demonstrate that the administration sequence is important to increase drug sensitivity. To generalize the phenomenon observed on ADF line, the antiproliferative effect of the two different schedules was analyzed on other glioblastoma lines (A172, CRS-A2, U373MG) with different BCNU sensitivity and p53 status. The data obtained confirm that the wt-p53 gene transfer enhances BCNU sensitivity in glioblastoma cells depending on the administration sequence.

Expression of type II receptor of transforming growth factor beta (TbetaRII) is necessary for this factor to inhibit the growth of thyroid epithelial cells. In rat thyroid transformed cells, the resistance to transforming growth factor beta (TGFbeta) is associated with a decreased expression of TbetaRII mRNA and protein. Reduced TbetaRII expression has also been found in human thyroid differentiated and undifferentiated carcinomas. To investigate the role of TbetaRII in modulating the tumorigenic potential of k-ras-transformed thyroid cells, we transfected these cells with an expression vector carrying the human TbetaRII gene, regulated by an inducible promoter. Isolated clones, overexpressing TbetaRII, showed a reduction in the anchorage-dependent and -independent cell growth, compared with control k-ras-transformed cells. When transplanted in athymic nude mice, the transfected clones presented a decrease in tumorigenicity with respect to the highly malignant parental cells. Moreover, the diminished tumorigenic ability of the clones studied was accompanied by a statistically significant reduction in spontaneous and lung artificial metastases. Taken together, our data demonstrate that TbetaRII acts as a potent tumor suppressor gene when overexpressed in malignant thyroid cells.

Objective: To analyze the prognostic value of DNA multiploidy in a prospective study on frozen surgical tissue samples from primary colorectal cancer., Summary Background Data: Survival data from eleven prospective studies collectively comprising about thirteen hundred patients showed that aneuploidy correlated with a 5-year disease-free survival (DFS) significantly poorer than diploidy, and showed the limited prognostic value of results from retrospective studies employing paraffin-embedded material., Methods: Multiple tumor samples of fresh/frozen surgical tissues from 120 colorectal cancer patients who had undergone radical surgery were taken for flow cytometric analysis of DNA content, and proliferative activity, shown as percentage of cells in S-phase (%S). The minimum follow-up of this series was 30 months. Univariate and multivariate analyses determined the independent significance of both clinical and biological variable on DFS., Results: Values of %S equal to or higher than 17.3 correlated with a 5-year DFS poorer than values lower than 17.3 (44.5% vs 85.2% respectively; p = .03), even if only in patients younger than 64. The subgroup with multiploid tumors showed a significantly poorer 5-year DFS (44.5% vs. 62.6% in the non multiploid patients; p = .02). Subgrouping the Dukes'B stage alone by multiploidy, the difference in DFS was much more evident (31.2% vs. 68% respectively; p = .0004) and multivariate analysis showed multiploidy as the only significant variable. Above all, adjuvant therapy did not absolutely modify the unfavorable outcome of the multiploid Dukes'B patients., Conclusions: The prospective evaluation of ploidy allowed us to identify a very high-risk subgroup of patients with multiploid tumors. This biological characterization was easy to demonstrate and, above all in node-negative patients, reliable and very effective in terms of prognosis. The presence of multiploidy should result in a more aggressive therapeutic approach in the adjuvant setting.

To evaluate the role of hyperthermia combined with chemotherapy in the loco-regional treatment of tumors, a retrospective analysis was done with 228 limb melanoma patients treated with hyperthermic antiblastic perfusion (HAP). A series of treatment- and tumor-related prognostic factors was analyzed to establish their influence on tumor response, loco-regional control, and survival. Concerning tumor response, the logistic model showed that the number of lesions and the minimal tumor temperature (min T) maintained their individual predictive values (P < 0.000001 and P = 0.04, respectively). For loco-regional control, only the number of lesions had a significant predictive value. No direct correlation was found between the treatment-related variables and loco-regional control. However, the 5-year survival rate was significantly higher for patients who achieved a complete response (CR) (51.5%, P = 0.0033) as compared to those who did not (33.3%), providing indirect evidence of the role of the treatment. Multivariate analysis showed that both disease-free and overall survival are strongly influenced by numerous clinical variables and the min T always maintained its significance. When analyzing the subgroup of 119 patients evaluable for tumor response, the Cox model selected the tumor response as the dominant factor for both disease-free and overall survival. These data seem to demonstrate that the optimization of treatment parameters is crucial in determining the CR rate, which, in turn, positively affects the disease outcome. HAP is the treatment of choice for recurrent limb melanoma, and hyperthermia plays an important role in exploiting the efficacy of this technique.

This study was designed to assess the efficacy of a new antimelanoma therapeutic strategy that relies on the use of a c-myc antisense 15-mer phosphorothioate oligodeoxynucleotide ([S]ODN), in combination with cisplatin (cis-diamminedichloroplatinum; DDP), which is currently used in the clinical management of melanoma patients. Proliferation and colony formation of melanoma cells were both inhibited by the DDP/c-myc antisense [S]ODN combination to a greater extent than that observed with either agent alone. Inhibition was most effective when DDP was followed by c-myc antisense [S]ODNs. Cell cycle flow cytometric analysis of cells exposed to the two agents either alone or in combination demonstrated that (a) c-myc antisense [S]ODNs induced an accumulation of cells in S phase and apoptosis in a fraction of the cells, detectable at day 5 after the beginning of treatment; (b) DDP induced a block in G2-M phase detectable at day 1, which was partially recovered, and apoptosis similar in extent to that induced by c-myc antisense [S]ODNs; and (c) DDP and c-myc antisense [S]ODNs together induced arrest in G2-M phase, which was maximum at day 3, i.e., delayed as compared to the block induced by DDP. The combination induced a higher percentage of apoptosis, evident at day 3 from the start of treatment, that correlated with a marked reduction in Bcl-2 expression. Mice bearing human melanoma xenografts and treated sequentially with DDP and c-myc antisense [S]ODNs showed a higher inhibition of tumor growth, reduction in the number of lung metastases, and increase in life span compared with those treated with either agent alone. Together, these data lend support to the development of anticancer therapies involving oncogene-targeted antisense ODNs and conventional antineoplastic drugs.

We have studied whether N-methylformamide can affect the expression pattern of adhesion molecules and the attachment behaviour of M14 human melanoma cells. The role of N-methylformamide on experimental and spontaneous pulmonary metastases from M14 cells in nude mice was also investigated. We demonstrate that N-methylformamide in vitro pretreatment of M14 cells, although inducing a significant increase in the expression of alpha2beta1, alpha6beta1 and alpha(v)beta3 integrin receptors, slightly modifies alpha5beta1 heterodimer and beta1 subunit expression. After this modulation, enhancement of cell adhesion to laminin, collagen I, vitronectin and fibrinogen, which is blocked by specific anti-integrin antibodies, also occurs. No changes in binding to fibronectin are observed. In vitro N-methylformamide pretreatment also results in an increased number of experimental nodules and in a decrease in spontaneous metastases. Moreover, in vivo treatment with N-methylformamide significantly reduces the number of spontaneous metastases. Collectively, these data show that N-methylformamide modulates the expression of some adhesion receptors, cell adhesion to laminin, collagen I, vitronectin and fibrinogen as well as the metastatic behaviour of M14 cells. Our data also suggest that the effect of N-methylformamide might be evaluated in combination with antineoplastic agents for the treatment of human melanoma.

Poly(ADP-ribose) polymerase (PARP) is a DNA-binding protein involved in cellular response to various genotoxic agents. To understand the role of PARP in the mechanisms which lead from specific DNA damage to cell death, we studied the effects of PARP inhibition in human lymphoblasts damaged with bleomycin (BLM) and VP16. These agents can induce DNA breakage but through different mechanisms, enabling the study of the different effects of PARP in inducing apoptosis in damaged cells. We demonstrate that in lymphoblasts VP16 treatment induces apoptosis to a greater extent than BLM treatment, and that PARP inhibition reduces VP16-induced apoptosis whereas it has no effect on BLM-induced apoptosis. After VP16 treatment with PARP inhibition, a reduction in the depletion of the proliferative compartment and a G2/M phase arrest are observed. Therefore, the increase in cell viability and the reduction in chromosome damage may both be the result of a prolonged DNA repair time. Hence, PARP appears to play a significant role in VP16-induced apoptosis and not in BLM-induced apoptosis. Since apoptosis is important in tumor treatment these findings might be useful when considering the combined employment of PARP inhibition with antineoplastic drugs.

Bcl-2 protein has been shown to contribute to oncogenesis because it can transform and immortalize cells in cooperation with c-myc, ras, or viral genes. However, in vivo studies have not yet established whether bcl-2 can play a role in metastasis. Here we investigate the potential metastatic role of bcl-2. We introduced the human bcl-2 gene into a low bcl-2 expressing human breast cancer cell line MCF7 ADR. We demonstrate that two bcl-2 overexpressing clones injected intravenously or intramuscularly into nude mice induce a significantly higher number of experimental and spontaneous lung metastases compared to the control transfectant clone. We demonstrate that bcl-2 overexpressing clones are more invasive and migratory in response to chemotactic stimuli than the control transfectant clone. Furthermore, zymographic analysis shows that secretion of 72 and 92 kDa gelatinases increases in the two bcl-2 overexpressing transfectants. Tumors originating from bcl-2 overexpressing clones also show a decrease in the latency period of tumor appearance. In conclusion, our data show that bcl-2 overexpression enhances both tumorigenicity and metastatic potential of MCF7 ADR cells by inducing metastasis-associated properties.

Background/aims: The purpose of this study was to define the prognostic role of DNA ploidy, proliferative index and EGF-R status in resected gastric cancer., Materials and Methods: Ten clinico-pathological parameters and three biological factors obtained from flow cytometry and immunohisto-chemistry were evaluated in a series of 130 gastric cancer patients who received surgical treatment, including 28 stage IV cases (21.6%), using paraffin-embedded and fresh specimens in 77.7% and 22.3% of the cases, respectively. These variables were first analyzed and tested for correlation within the whole series and then weighted against survival in 117 applicable cases through univariate and multivariate analyses., Results: Aneuploidy was significantly related to higher proliferative activity, EGF-R expression and deeper stomach wall infiltration. Higher proliferative activity was significantly related to deeper stomach wall infiltration and larger tumor diameter. The latter showed a significant relationship to EGF-R expression. Univariate analysis showed the significant variables for survival to be DNA ploidy, pT, pN, M, stage, histological type according to Lauren and tumor diameter. Multivariate analysis calculated on these significant variables using the Cox multiple stepwise regression model detected three factors which independently influence survival: pathological stage (p < 0.00001), histological type according to Lauren (p < 0.002) and DNA ploidy (p < 0.03)., Conclusions: DNA ploidy was shown to be a significant prognostic parameter in resected gastric cancer after pathological stage and histological type according to Lauren. The prognostic roles of proliferative activity and EGF-R status require further investigation.

We studied epidermal growth factor receptor (EGF-R) expression in relation to steroid receptor status, flow cytometric DNA content and S-phase fraction (%S) in a selected case series of 129 ductal primary operable breast cancer to determine the possible role of EGF-R in prognosis assessment. EGF-R expression was positively related with proliferation activity, suggesting that EGF-R could be involved in the regulation of breast cancer cell growth. We found about 80% of highly proliferating DNA aneuploid tumors in the EGF-R positive category, while the EGF-R negative tumors showed a lower frequency of highly proliferating DNA aneuploid tumors (57%), confirming the important role of EGF-R in breast cancer aggressiveness and progression. No relationship between EGF-R expression and steroid receptor status was observed. To better understand how EGF-R and estrogen receptor (ER) operate together to stimulate breast cancer cell growth, we analyzed the %S in the two groups of ER negative (ER-) and ER positive (ER+) tumors, stratifying the patients on the basis of EGF-R tumor positivity. Here breast tumor proliferation activity seems mainly to be induced by the stimulus of EGF-R, the %S values of the EGF-R negative tumors in the ER- and ER+ groups being 6.1 and 6.9%, respectively. Instead, the median %S of EGF-R positive tumors was 10% in the ER- class and 14% in the ER+ group. The analysis of the percentages of 5-year patient disease free survival were 84% for patients with EGF-R negative tumors and 61% for patients with EGF-R positive lesions, respectively. The data reported here further show the crucial role of EGF-R in breast cancer cell growth and that the EGF-R overexpression is indicative of a poor prognosis.