Nanocrystalline cellulose-fullerene: Novel conjugates.

The covalent grafting of two amino-fullerene C60 derivatives (C60-LC-NH2 and C60-SC-NH2, LC=long chain and SC=short chain) onto the surface of TEMPO oxidized nanocrystalline cellulose (NCC-COOH) has been reported for the first time. These hybrids (NCC-LC-C60 and NCC-SC-C60) form stable colloidal suspensions at concentrations up to 0.5mg/mL and act as effective photosensitizers for singlet oxygen production as demonstrated by the oxidation of L-methionine-methyl ester to the corresponding sulphoxide. Using the same approach, in a one-pot reaction both a fluorescent target molecule (FITC-LC-NH2) and the C60-LC-NH2 derivative have been successfully attached covalently onto the NCC-COOH surface. These hybrids, which showed no cytotoxicity on MCF-7 human breast cancer cells could be good candidates in photodynamic cancer therapy.

The chlorophyll catabolite pheophorbide a as a photosensitizer for the photodynamic therapy.

Pheophorbide a is a clorophyll catabolite that recently has drawn the attention of several investigators for its potential in photodynamic therapy. In this review we summarize its photophysical properties, phototoxicity, cellular localization, biodistribution and PDT activity as a free or conjugated molecule.

Transcription inhibition of oncogenic KRAS by a mutation-selective peptide nucleic acid conjugated to the PKKKRKV nuclear localization signal peptide.

Mutations in the Kirsten ras (KRAS) gene are present in almost all pancreatic adenocarcinomas, and one common mutation is at codon 12: GGT (Gly) is transformed into GAT (Asp). In this work we have targeted the KRAS coding sequence embracing the GAT mutation with a sense PNA molecule (P14), with the aim of downregulating the expression of the mutant allele. P14 was designed with a 15-base sequence complementary to the antisense strand of KRAS at the GAT (Asp) mutation and conjugated to the nuclear localization signal peptide PKKKRKV. CD spectra as a function of temperature show that P14 (2 microM) binds to the antisense strand of the GAT target in the mutated allele with a T(M) of 78 degrees C and to the antisense strand of the GGT target in the wild-type allele with a T(M) of 69 degrees C, in 50 mM Tris-HCl, pH 7.4, and 1 M NaCl. Moreover, P14 showed a high capacity to enter and accumulate in the nuclei of pancreatic cells (Panc-1 and BxPC3), whereas the nonconjugated analogue did not. Quantitative RT-PCR showed that 1 microM P14 was able to specifically suppress KRAS transcription in Panc-1 cells, which harbor mutant KRAS, but not in BxPC3 cells, which contain only wild-type KRAS. However, P14 inhibited KRAS transcription also in BxPC3 cells when used at concentrations of 5 and 10 microM. Following a single PNA treatment, changes in protein level were evident only in Panc-1 cells. As we found that all three genes of the ras family are expressed in the pancreatic cells, we designed PNA-NLS conjugates (P16 and P17) to target also HRAS and NRAS. The binding of each PNA conjugate to the ras genes was assayed by electrophoresis, and their capacity to inhibit transcription was measured by RT-PCR. All of the data obtained, both in vivo and in vitro, are discussed in terms of sequence specificity in the binding between PNA-NLS molecules and genomic DNA.

Anti-gene strategies to down-regulate gene expression in mammalian cells.

A current goal in molecular medicine is the development of new strategies for the selective inhibition of cancer-critical genes. Triplex-forming oligonucleotides and peptide nucleic acids bind to the double helix of DNA in a sequence-specific manner and with great affinity. Because of these properties, these molecules have been proposed as anti-gene therapeutic drugs. This review summarizes recent results on the use of oligonucleotides and peptide nucleic acids to downregulate gene expression in cultured cells. The data are discussed from the perspective of the recent literature on new molecular strategies with potential therapeutic applications.

Site-directed inhibition of DNA replication by triple helix formation.

Sequence-specific DNA recognition can be achieved by the use of triplex-forming molecules, namely, oligonucleotides (TFO) and peptide nucleic acids (PNAs). They have been used to regulate transcription or induce genomic DNA modifications at a selected site in cells and, recently, in vivo. We have determined the conditions under which a triplex structure can inhibit DNA replication in cells. An oligopyrimidine.oligopurine sequence suitable for triplex formation was inserted in a plasmid on both sides of the SV40 origin of replication. This insert-containing plasmid was replicated in COS-1 cells together with the parent plasmid, and the ratio between the corresponding replicated DNAs was quantitated. Selective inhibition of replication of the insert-containing plasmid can be ascribed to ligand binding to the oligopyrimidine.oligopurine sequence. Inhibition of DNA replication was observed using triplex-forming molecules that induce either covalent binding at the double-stranded target sequence (with TFO-psoralen conjugate and irradiation) or noncovalent triplex formation after strand displacement (with bis-PNA). In contrast, in the absence of covalent cross-linking, TFOs (which have been shown to arrest transcription elongation) did not act on replication. These results open new perspectives for future design and use of specific inhibitors of intracellular DNA information processing.

Effects of melatonin on doxorubicin cytotoxicity in sensitive and pleiotropically resistant tumor cells.

Melatonin has been reported to attenuate the oxidative damage caused by doxorubicin on kidney, brain, heart and bone marrow, whereas the in vivo antitumor effects of doxorubicin were not attenuated. The effects of melatonin on doxorubicin cytotoxicity have, therefore, been examined on human normal mammary epithelium HBL-100, on mammary adenocarcinoma MCF-7, on colon carcinoma LoVo, and on mouse P388 leukemia cell lines, and on tumor cell sublines pleiotropically resistant to anthracyclines. Melatonin in the concentration range 10-2000 pg/mL causes an inhibition of the growth of the human cell lines examined which is not clearly dose-dependent and less than 25% when significant. Melatonin similarly causes minor effects on doxorubicin cytotoxicity either on the parental human cell lines or on their resistant sublines. On the contrary, 200-1000 pg/mL melatonin cause a significant and dose-dependent partial sensitization to doxorubicin of resistant P388 mouse leukemia (P388/ADR), which occurs also in vivo, as indicated by a significant increase in survival time of the hosts. Doxorubicin intracellular concentrations in P388/ADR cells are increased by melatonin, suggesting that melatonin might inhibit P-glycoprotein-mediated doxorubicin efflux from the cells. These results indicate that the use of melatonin in clinical cancer treatment should not pose the risk of an attenuation of the effectiveness of doxorubicin, and encourage the further examination of the possible reduction by melatonin of the host toxicity of antitumor chemotherapy.

Anti-gene effect in live cells of AG motif triplex-forming oligonucleotides containing an increasing number of phosphorothioate linkages.

The murine Ki-ras promoter contains a unique polypurine--polypyrimidine [poly(R.Y)] sequence between -290 and -320 from the 3' boundary of exon phi. Previously we demonstrated triplex formation and transcription inhibition promoted by GT and AG oligonucleotides directed against this site [Alunni-Fabbroni et al. (1996) Biochemistry 35, 16361--16369]. In this work, we have investigated triplex formation and anti-gene activity of five 20-mer AG motif triplex-forming oligonucleotides specific for the Ki-ras poly(R.Y) target, derived from 5'-AGGGAGGGAGGAAGGGAGGG (20AG) by replacing an increasing number of phosphodiester linkages with phosphorothioate linkages (S(i)-20AG; i = 2, 3, 4, 5, 19). Electrophoretic mobility-shift experiments (EMSA) showed that four thioate oligonucleotides, S(i)-20AG (i = 2, 3, 4, 5), recognized the Ki-ras target and exhibited dissociation constants similar to that of 20AG: K(d) = 12 +/- 2 nM, while the all-thioate S(19)-20AG exhibited a K(d) of 128 +/- 15 nM. Moreover, the binding between the Ki-ras promoter and oligonucleotides S(i)-20AG (i = 2, 3, 4, 5, 19) was characterized by DMS/piperidine and DNase I footprinting experiments. We observed that the introduction in the phosphodiester oligonucleotide 20AG of sulfur atoms reduced its aggregation significantly and increased its nuclease resistance. Transient transfection experiments using preformed triplexes with a recombinant plasmid containing the reporter chloramphenicol acetyltransferase (CAT) gene under the control of Ki-ras promoter showed that oligonucleotides S(i)-20AG (i = 2, 3, 4, 5, 19) promote a strong inhibition of up to 75% of the CAT expression when compared with control Ki-ras unspecific oligonucleotides. Taken together, these data provide a guideline for designing triplex-forming effector molecules capable of controlling Ki-ras expression in vivo.

Stress and chemotherapy. Combined effects on tumor progression and immunity in animal models.

In mice bearing Lewis lung carcinoma, rotational and restraint stress specifically increases the formation of lung metastasis, and restraint stress markedly attenuates the antitumor effects of cyclophosphamide. The aim of this investigation was therefore to examine the effects of restraint stress on tumor metastasis in mice bearing MCa mammary carcinoma, and on the effectiveness of CCNU and DTIC. Restraint stress increases MCa mammary carcinoma metastasis, causes a marked reduction in cyclophosphamide activity, and a minor attenuation of the effects of CCNU and DTIC. The possible occurrence of seasonal factors, observed for the increase by rotational stress of Lewis lung carcinoma metastasis, was also determined for cyclophosphamide effectiveness. The survival time of control mice is longer in February than in June, and is not appreciably modified by rotational stress. The effects of cyclophosphamide are similar in both seasonal periods, and are similarly attenuated by rotational stress. The seasonal effects of rotational stress, and the reduction of the effects of cyclophosphamide caused by rotational stress, are accompanied by corresponding variations in the number of CD3+ and CD4+ splenic T-lymphocyte subsets and in the CD4+/CD8+ ratio, respectively. The reported effects of stress on tumor progression and on the effectiveness of cyclophosphamide thus appear to occur via modulation of immune responses of the host directed against the tumor. These data appear of interest for their experimental implications, and suggest the opportunity to consider the role that the stress during treatment may play in determining the effectiveness of clinical antitumor chemotherapy.

Melatonin administration in tumor-bearing mice (intact and pinealectomized) in relation to stress, zinc, thymulin and IL-2.

Melatonin (MEL) may counteract tumors through a direct oncostatic role. MEL is also an antistress agent with immunoenhancing properties against tumors due to a suppressive role of MEL on corticosterone release. Rotational stress (RS) (spatial disorientation) facilitates metastasis progression in mice. Also, MEL counteracts tumors because of its influence on immune responses via the metabolic zinc pool, which, is reduced in tumors and stress. Zinc is required for normal thymic endocrine activity (i.e. thymulin) and interleukin-2 (IL-2) production. Because in vivo data is still controversial, exogenous MEL treatment (22 days in drinking water) in both intact and pinealectomized (px) mice bearing Lewis lung carcinoma leads to significant decrements of metastasis volume, restoration of the negative crude zinc balance, recovery of thymulin activity and increment of IL-2 exclusively in intact and px tumor bearing mice subjected to RS. Significant inverse correlations are found in both stressed intact and px tumor bearing mice after MEL treatment between zinc and corticosterone (r = 0.78, P < 0.01; r = 0.80, P < 0.01, respectively). Positive correlations between zinc and IL-2 (r = 0.75, P < 0.01; r = 0.73, P < 0.01, respectively) or thymulin (r = 0.75, P < 0.01; r = 0.82, P < 0.01, respectively) are observed in same models of mice. These findings suggest a MEL action to decrease metastasis mediated by a possible interplay between zinc and MEL, via corticosterone, with consequent restoration of thymic efficiency and IL-2 production. MEL as an antistress agent with immunoenhancing properties in cancer deserves further consideration.nuclear factor-kb; POMC, proopiomelanocortin; Px, pinealectomized mice; RIA, radioimmunoassay; RS, rotational stress; SDI, stressed intact mice; SDPx, stressed pinealectomized mice; TNF-alpha, tumor necrosis factor-alpha; ZnFTS, active zinc-bound thymulin; ZnFTS + FTS, total thymulin.

Melatonin and oxidative damage in mice liver induced by the prooxidant antitumor drug, adriamycin.

Antioxidant properties have been attributed to melatonin; it seemed therefore worthwhile to determine its effects in relation to the prooxidant action of adriamycin, which contributes to its toxic and therapeutic effects. Melatonin effectively acts as a direct free radical scavenger in the concentration range of 20-100 microM as determined in vitro, using Fenton reaction as a source of free radicals that were determined by EPR using spin trapping method. Following the administration of a single i.v. dose of 28 mg/Kg or of 3 repeated i. p. doses of 5 mg/Kg adriamycin to CBA mice, glutathione levels in the liver cells were significantly reduced. When the treatment with adriamycin was preceded by the s.c. administration of 2 mg/Kg melatonin, the decrease in total and reduced glutathione concentrations was significantly prevented. A significant increase in lipid peroxidation was observed in liver cells after a single administration of adriamycin which was not attenuated by pretreatment with melatonin. These results indicate that further examination of the possible protective action of melatonin on the toxic effects of prooxidant antitumor drugs on normal and neoplastic tissues would be of interest also in relation to their chronotoxicological properties.

Melatonin decreases bone marrow and lymphatic toxicity of adriamycin in mice bearing TLX5 lymphoma.

When CBA male mice bearing TLX5 lymphoma were treated in the evening with a single i.v. dose of adriamycin (20-40 mg/Kg), the administration of a single pharmacological dose of melatonin (10 mg/kg s.c.) 1 hr earlier reduced the acute mortality from 10/24 to 2/24. The increase in survival time caused by adriamycin over drug untreated controls was not reduced by melatonin. The administration of melatonin alone did not cause any antitumor or evident toxic effect. Melatonin also attenuated the reduction caused by adriamycin in the number of bone marrow GM-CFU, and of CD3+, CD4+ and CD8+ splenic T-lymphocyte subsets. Reduced and total glutathione levels were decreased in the bone marrow and in the liver cells of the animals treated with adriamycin, and were significantly restored by melatonin. Moreover, lipid peroxidation by adriamycin was reduced by melatonin, as indicated by malondialdehyde measurement in the liver of the treated animals. These data indicate that the protective effects of melatonin against the host toxicity of the prooxidant antitumor drug, adriamycin, might be attributed at least partially to its antioxidant properties. These findings appear of interest in relation to the physiological rhythmic levels of endogenous melatonin and to the chronotoxicology of anthracyclines.

Seasonal effects of rotational stress on Lewis lung carcinoma metastasis and T-lymphocyte subsets in mice.

Rotational stress specifically increases the formation of spontaneous lung metastasis in mice bearing Lewis lung carcinoma, without significantly modifying the growth of primary tumor. The increase in metastasis number and volume caused by rotational stress varies in magnitude with a highly significant circannual rhythm; the acrophase approximately coincides with summer solstice. Rotational stress causes a significant reduction in the number of CD3+ and CD4+ T-lymphocyte subsets in summer, whereas in winter the number of CD3+ subset is significantly increased; the CD4+/CD8+ ratio and the number of NK 1.1 antigen positive cells are not significantly modified by rotational stress in both periods considered. The increase in metastasis formation by rotational stress thus appears to negatively correlate with the number of splenic CD3+ and CD4+ T-lymphocyte subsets. This seasonal behavior occurs in spite of the control of light cycle, temperature and humidity in the animal housing, suggesting the existence in the host of an endogenous oscillator with a circannual period. These data indicate the opportunity to consider endogenous rhythms within the host, as well as seasonal factors, in studies on stress and neuroimmunomodulation in experimental oncology.

Restraint stress reduces the antitumor efficacy of cyclophosphamide in tumor-bearing mice.

Treatment with the cytotoxic antitumor drug cyclophosphamide is highly effective in mice bearing Lewis lung carcinoma, causing the absence of macroscopically detectable tumors at necroscopy after sacrifice. When the effects of the treatment on survival are determined, a significant increase in survival time and in the proportion of long-term survivors is observed. When restraint stress is further applied, tumors develop in all of the mice treated with cyclophosphamide, and survival time and the fraction of long-term survivors are significantly reduced. Flow cytometry of splenic T-lymphocyte subsets in normal mice indicates a significant decrease in the number of CD3+, CD4+, and CD8+ subsets after treatment with cyclophosphamide and after application of restraint stress; the interaction of the two treatments is significant for CD3+ and marginally significant for CD4+ subsets. The attenuation by restraint stress which was observed for the effects of cyclophosphamide on the presence of tumors at necroscopy and for the survival of the treated mice might thus be interpreted as follows: restraint stress attenuates the immune functions of the host directed toward the weakly immunogenic tumor, an effect which, in the absence of restraint stress, interacts effectively with the cytotoxic action of cyclophosphamide toward tumor cells. The results obtained using this animal model thus indicate that experimental stress reduces the therapeutic efficacy of a cytotoxic antitumor drug; experimental and clinical implications are discussed.

Suppression of metastatic potential and up-regulation of gelatinases and uPA in LLC by protracted in vivo treatment with dacarbazine or razoxane.

Treatment of mouse Lewis lung carcinoma with razoxane or dacarbazine was protracted for 10 transplant generations. While the capacity of the treated tumors to grow locally in immuno-competent or in immuno-depressed hosts was retained and not significantly modified, the metastatic phenotype was eliminated when the treated tumor cells were transplanted into immuno-competent hosts. The reduction in metastatic potential was slightly less pronounced, in terms of both number and volume of metastases, when the treated tumor cells were transplanted into immuno-depressed hosts. These properties were retained after 3 transplant generations without treatment. Northern blotting and zymography of primary-tumor crude extracts revealed that treatment with either razoxane or dacarbazine for one generation approximately doubled the expression of MMP-2 and MMP-9, while lacking any effect on that of 1.0 and of 3.5 kb TIMP-2. When the treatment was maintained for 10 generations, the expression of MMP-2 and MMP-9 for both drugs showed up-regulation of approximately 10- and 2-fold respectively. TIMP-2 mRNA of 1.0 kb doubled its expression, while that of 3.5 kb registered just above the control. Dacarbazine doubled the expression of uPA after 10 generations, while razoxane boosted it approximately 3-fold after either 1 or 10 generations. The permanent loss of metastatic phenotype induced in Lewis lung carcinoma by dacarbazine and razoxane is thus attributable to biological mechanisms independent of down-regulation of expression and/or activation of the 2 gelatinases.

Survival time in mice bearing TLX5 lymphoma subjected to rotational stress and chemotherapy with CCNU.

The effects of a psychological stress model rotational stress were examined in mice bearing TLX5 lymphoma. The survival time of the animals was determined as a function of tumor inoculum size and treatment with the antitumor drug, CCNU. Rotational stress significantly decreased the mean survival time of mice implanted with 10 or 10(2) tumor cells, and significantly increased tumor takes in mice implanted with 10 cells. Treatment with CCNU significantly prolonged the survival time of the treated animals; the application of rotational stress significantly attenuated the increase in survival time caused by CCNU. These results indicate that in mice with a limited tumor burden, psychological stress favors the progression of TLX5 lymphoma, and reduces the effectiveness of the antitumor drug, CCNU. Moreover, the experimental model employed may provide a tool useful for investigating the mechanisms involved in the sensitivity of lymphoma to psychosocial stress.

Blockers of adrenergic neurons and receptors, tumor progression and effects of rotational stress in mice.

The aim of this work was to determine the possible participation of adrenergic responses to the increase in tumor dissemination induced by rotational stress using drugs affecting monoaminergic function. The growth of the primary tumor and the formation of lung metastasis were determined in mice implanted with Lewis lung carcinoma, subjected to rotational stress and treated with the adrenergic neuron blocker reserpine, the alpha-receptor blocker phenoxybenzamine, and the beta-blocker propranolol. Treatment with reserpine markedly reduced the formation of spontaneous lung metastasis and completely abolished the increase in metastases caused by rotational stress without direct effect on tumor cells or blood vessels. Phenoxybenzamine and propranolol caused opposite effects on tumor progression. Previous immunosuppression by cyclophosphamide in mice with tumors reduced the antimetastatic effects of reserpine. These results suggesting the importance of the adrenergic modulation of immune resistance factors in controlling metastasis development and indicate a possible role in monitoring the use and effects of monoaminergic drugs in cancer patients.

Effects of prolonged treatment with decarbazine on tumor metastatic potential in mice bearing Lewis lung carcinoma.

The effects of decarbazine on tumour growth and metastatic dissemination upon treatment protracted for 10 tumour transplant generations were examined in mice bearing Lewis lung carcinoma. Primary tumour growth is unaffected by the drug, independently from the duration of the treatment. In contrast, dacarbazine significantly inhibits the formation of lung metastasis. The proportion of mice with metastasis decreases for an increasing number of transplant generations of treatment, and after 10 transplant generations of treatment metastatic capacity is completely lost in immunocompetent mice. The reduction in metastatic potential is relatively stable, being retained for three successive transplant generations without treatment. The metastatic potential of treated tumours in immunosuppressed mice is substantially similar to that in immunocompetent hosts, indicating that chemical xenogenization of tumour cells does not occur as reported for transplantable mouse leukaemias. The results obtained using clonally selected tumour lines with different metastatic potential rule out the selection by dacarbazine of tumour cell sublines with reduced metastatic potential as the mechanism of the drug's action. Upon prolonged treatment, dacarbazine appears to cause a rather stable and dramatic loss in metastatic potential, not accompanied by resistance, which might be attributed to genotypic alteration(s) of tumour cells, and which might participate into the clinical effects of the drug.

Metastasis and neuroendocrine system in stressed mice.

The effects of experimental stressors have been examined for their differential effects on primary tumor growth and spontaneous lung metastasis in syngeneic mice bearing the weakly immunogenic tumor Lewis lung carcinoma. The effects caused by the early weaning, physical restraint and foot-shock are in general small, and affected by a high variability. On the contrary, spatial disorientation reproducibly causes a specific increase in tumor metastases. The effects of spatial disorientation are sensitive to the inhibition by treatment with bromocryptine and guanethidine, and particularly by a central and peripheral adrenergic neuron blocking agent reserpine. The use of different lighting conditions and assay of urinary melatonin excretion reveal an association between pineal gland function and effects of spatial disorientation on metastasis.