Toxicity of para-aminobenzhydrazide and its influence on nucleic acid biosynthesis in cultures of normal and tumor cells

We have investigated cytotoxic action of p-aminobenzhydrazide and its influence on biosynthesis of nucleic acids in cultures of intact cells, tumor cells and intact cells stimulated by phytohemagglutinin. p-Aminobenzhydrazide is considered as a representative of hydrazine's derivatives (in particular, of hydrazine sulphate). We compare its action with that of a typical cytotoxic agent such as iododeoxyuridine. We have found that p-aminobenzhydrazide influences biosynthesis of nucleic acids in the same way as iododeoxyuridine. However it acts toxically on tumor cells though it is not toxic for intact cells so that its action is different as compared to that of cytotoxic agents. Specific toxic action of aminobenzhydrazide on tumor cells may be due to the enhancement of antitumor activity substances of this compound and absence of such enhancement of side toxic effects.     

Cytotoxic and antitumor activity of a soluble fraction of Streptococcus pyogenes against S180 sarcoma cells

A fraction (60F) having cytotoxic and antitumor activities was obtained from cell-free extract of group A Streptococcus pyogenes by precipitating with 50% to 60% saturated ammonium sulfate. 60F showed cytotoxic activity inhibiting the uptake of 3H-thymidine by S180 sarcoma cells and enhancing 51Cr-release from 51Cr-labeled cells. 60F showed also antitumor activity, depressing tumor growth and prolonging lives of mice bearing S180 sarcoma cells.     

铜绿假单胞菌分泌性蛋白的抗肿瘤效应

目的 探讨3株不同来源的铜绿假单胞菌分泌性蛋白的抗肿瘤活性。方法 将3株不同来源的铜绿假单胞菌经LB培养基对其过夜静置培养,离心获得上清液,采用硫酸铵盐析沉淀蛋白质,再经PBS透析除盐。然后将蛋白作用于人肝癌细胞Hep-G2、宫颈癌细胞HeLa、肺癌细胞A549和人永生化表皮细胞HaCaT,CCK-8检测其对细胞的毒性作用,吉姆萨染色观察凋亡细胞形态变化。结果 SDS-PAGE证实成功获得不同分子量的分泌蛋白,CCK-8结果显示混合蛋白对3种不同肿瘤细胞的生长均有不同程度的抑制作用,且其抑制作用存在一定的时间和浓度依赖性,但对人正常细胞无明显抑制作用。吉姆萨染色初步显示细胞形态为凋亡状态。结论 初步证实该3株铜绿假单胞菌产生的分泌性蛋白具有不同程度的抗肿瘤作用,为进一步分离纯化具有抗肿瘤活性的单一蛋白质及研究其抗肿瘤机制提供依据。     

Synthesis and biological validation of novel pyrazole derivatives with anticancer activity guided by 3D-QSAR analysis

Using literature data on anticancer activity of pyrazole derivatives, 3D-QSAR models were developed and 3D-QSAR analysis was performed. The 3D-QSAR analysis enabled identification of molecular properties that have the highest impact on antitumor activity against lung cancer cells. The results of 3D-QSAR analysis were taken into account while new compounds were designed. Obtained 3D-QSAR models were used for prediction of activity of new compounds. In this way, design of new compounds was guided by 3D-QSAR analysis which was performed on literature data. Ten new pyrazole derivatives were synthesised and their antitumor activities against A549 and NCIH23 lung cancer cells were validated. In order to obtain full profile of anticancer activity, cells viability (MTS) assays were combined with cell proliferation (BrdU) assays which measure actively dividing cells in treated sample. Experimental measurements showed good agreement between predicted and measured activities for majority of compounds. Also, anticancer activities of new pyrazole derivatives pointed to the chemical groups that can be useful in designing antitumor molecules. Substitution of hydrazine linker with rigid, 1,2,4-oxadiazole moiety resulted in compound 10, which has low (if any) cytotoxic activity and high potential cytostatic activity. Therefore, compound 10 presents a good starting point for design of new, more potent and safer anticancer therapeutics.     

Effect of hydrazine, isonicotinic acid hydrazide, hydrazine sulfate, and dimethylhydrazine on guanylate cyclase activity.

The chemical carcinogen hydrazine is a potent stimulator of guanylate cyclase. In the present investigation we found that three chemical carcinogens structurally related to hydrazine, isonicotinic acid hydrazide, hydrazine sulfate, and dimethylhydrazine, decreased guanylate cyclase activity. It is of interest that hydrazine has been shown to increase DNA synthesis whereas isonicotinic acid hydrazide, hydrazine sulfate, and dimethylhydrazine decrease DNA synthesis. The relationship, if any, linking the guanylate cyclase-cyclic GMP system to DNA synthesis and carcinogenesis remains to be explored.     

Enhancement of mixed leukocyte reaction and cytotoxic antitumor responses by heparin

The immunomodulating effects of heparin and natural and synthetic heparinoids (which are now undergoing clinical trials for the treatment of AIDS) on cellular immunity (DNA synthesis and cytotoxic responses of mouse lymphocytes to allogeneic cells and histocompatible tumors) were studied. The results showed that (1) high and low m.w. heparin enhanced mouse antitumor and antiallogeneic cell responses in vitro; (2) other sulfated heparinoids did not have this enhancing activity and some of them (including dextran sulfate) totally suppressed generation of cytotoxic cells; (3) these immunomodulating activities of heparin and heparinoids did not correlate with their anticoagulant effects, degree of sulfation, and mitogenic activity; (4) heparin did not increase the production of IL-2 and did not enhance the action of IL-2 on the cells in MLC, heparin also had no effect on the growth-promoting activity of IL-2 on cloned cytotoxic T cells; (5) heparin had a synergistic enhancing effect with IL-1 on the generation of cytotoxic cells in MLC; and (6) heparin abolished endothelial cell growth factor-induced suppression of cytotoxic response. The latter two effects by themselves, however, could not fully explain the entire immunoenhancing activity of heparin. These results indicate that heparin and heparinoids have multiple effects on the immune system and that some of them can enhance, whereas others can suppress cell-mediated responses.     

Role of Corynebacterium parvum in the activation of peritoneal macrophages. II. Identification of distinguishable anti-tumor activities by macrophage subpopulations

Two different mechanisms of murine macrophage (MP) antitumor activity are described in this report. C. parvum-activated peritoneal MPs were tested for cytotoxic and cytostatic activity 4 days after ip immunization. Cytotoxic activity could be distinguished from cytostatic activity using two different assay protocols. When MPs were separated by 1g velocity sedimentation, cytotoxic MPs were confined to high velocity fractions. In contrast, cytostatic MPs were found in cell fractions with velocities as low as 5.2 mm/hr. These two MP activities were also distinguishable by culturing at 37 degrees C for 24 hr. Cytotoxicity was abrogated when MPs were incubated in MEM, or MEM supplemented with lymphokine (LK) or indomethacin. In contrast, cytostasis remained at high levels when the cells were incubated with LK or indomethacin. Cytotoxicity was not retained after overnight culture even if LPS was present, or if various spleen or non-adherent peritoneal exudate cells were cocultured with the cytotoxic effector cells. Assays done to determine the presence of suppressor cells failed to find any inhibitory cell type. The phagocytic index, acid phosphatase activity, and H2O2 secretion were also measured before and after overnight culture. Acid phosphatase and phagocytic activities did not decline whereas H2O2 secretion declined significantly. These data indicate that in response to C. parvum, at least two different effector cell types with distinct antitumor activities are generated. Cytotoxicity, like the ability of cells to secrete H2O2, is found to be a short-lived function of CP stimulated MPs. In contrast, cytostasis is a function retained longer by MPs in culture.     

Extracellular HspBP1 inhibits formation of a cytotoxic Tag7-Hsp70 complex in vitro and in human serum

Tag7 (PGRP-S) was described as an innate immunity protein. Earlier we have shown that Tag7 forms with Hsp70 a stable complex with cytotoxic and antitumor activity. The same complex is formed in and secreted by cytotoxic T-lymphocytes. We have also found that Hsp-binding protein HspBP1 incapacitates the Tag7-Hsp70 complex. Here we have studied the interaction of extracellular Tag7 and HspBP1. We have shown that HspBP1 binds Tag7 in the conditioned medium of tumor CSML0 cells, thereby preventing formation of the cytotoxic Tag7-Hsp70 complex. We have also found that Tag7, if present in serum (in every third donor on average), is always in complex with HspBP1. This may be a protective measure against indiscriminate attack of the cytotoxic complex on normal cells.     

Enzymatic sulfation of galactose residue of keratan sulfate by chondroitin 6-sulfotransferase

We have previously found that the purified chondroitin 6-sulfotransferase(C6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate(PAPS) to position 6 of N-acetylgalactosamine in chondroitin,catalyzed the sulfation of keratan sulfate, and that both theC6ST activity and the keratan sulfate sulfotransferase (KSST)activity were expressed in COS-7 cells when C6ST cDNA was transfected.In this report we describe some properties of the KSST activitycontained in the purified C6ST, and characterize the sulfatedproducts formed from keratan sulfate and partially desulfatedkeratan sulfate. Optimal pH, requirement for cationic activators,and K_m value for PAPS of the KSST activity were very similarto those of the C6ST activity. ³⁵S-Labeled glycosaminoglycansformed from keratan sulfate and partially desulfated keratansulfate were N-deacetylated by treatment with hydrazine/hydrazinesulfate and then cleaved with HNO₂ at pH 4, and the resultingproducts were reduced with NaB³H₄. Analysis of the degradationproducts with paper chromatography and high performance liquidchromatography provided evidence that C6ST transferred sulfateto position 6 of galactose residue which was glycosidicallylinked to N-acetylglucosamine 6-sulfate residue or to N-acetylglucosamineresidue. Northern blot analysis using poly (A)⁺ RNA from 12-d-oldchick embryos indicated that the message of C6ST was expressednot only in the cartilage but also in the cornea in which keratansulfate is actively synthesized. chondroitin sulfate keratan sulfate glycosaminoglycan sulfotransferase hydrazinolysis deaminative cleavage     

Induction of LAK-like cells in the peritoneal cavity of mice by inactivated Candida albicans

We have investigated the effect of multiple administrations of inactivated Candida albicans (CA) cells on induction of non-MHC-restricted antitumor cytotoxic responses both in normal and congenitally athymic (nude) mice. Intraperitoneal inoculation of CD2F1 mice with five doses of 2 x 10(7) CA cells over a 2-week interval was associated with the induction of peritoneal exudate cells (PEC) that mediated natural killer cell activity. These cells, in contrast to those elicited by a single dose of CA, killed both NK-sensitive and NK-resistant tumor target cells in vitro. This broad-spectrum, antitumor cytotoxicity peaked 1 day after the last injection of CA, and decreased to control values within 6 (NK-resistant) or 14 (NK-sensitive target cells) days. Cytotoxicity could be recalled to a high level by a boosting injection of CA or a major mannoprotein-soluble antigen (MP) from the Candida cell wall, given 30 days after multiple CA treatment. Upon a 24-hr in vitro incubation, CA-induced peritoneal immunoeffectors lost their killing activity unless human recombinant interleukin-2 (rIL-2) was added to cultures. The non-MHC-restricted cytotoxic PEC activity induced by CA was mainly associated with nonadherent, nonphagocytic large granular lymphocytes (LGL) which exhibited the following phenotypes: (i) asialo GM1+, Lyt 2.2-, and partially Thy 1.2+ (effectors active against NK-sensitive targets) and (ii) asialo GM1+, Lyt 2.2-, and Thy 1.2+ (effectors active against NK-resistant targets). Nude mice also responded to multiple CA inoculations by displaying high cytotoxic activity against NK-sensitive targets and significant cytotoxicity against NK-resistant targets. This cytotoxicity could be recalled on Day +30, and the cytotoxic effectors involved were highly sensitive to anti-asialo GM1 plus complement treatment. Overall, the results add further experimental evidence to the wide range of immunomodulatory properties possessed by C. albicans, and demonstrate that the majority of antitumor cytotoxic activity induced by fungal cells was due to lymphokine-activated killer (LAK)-like effectors.     

Synthesis, acute toxicities, and antitumor effects of novel 9-substituted beta-carboline derivatives

A series of novel 9-substituted beta-carboline derivatives was synthesized from harmine and l-tryptophan, respectively. Cytotoxic activities of these compounds in vitro were investigated. The results showed that most compounds of 9-substituted beta-carboline derivatives had more remarkable cytotoxic activities in vitro than their corresponding parent compounds. Acute toxicities and antitumor effects of the selected beta-carboline derivatives in mice were also examined. The results demonstrated that a short alkyl or benzyl substituent at position-9 increased the antitumor activities significantly and a ethoxycarbonyl or carboxyl substituent at position-3 reduced the acute toxicity and neurotoxicity of these beta-carboline derivatives dramatically. Moreover the compounds both with an alkoxycarbonyl or carboxyl substituent at position-3 and a short alkyl or benzyl substituent at positon-9 exhibited more significant antitumor activities and lower acute toxicities and neurotoxicities than the other compounds. The compound 8c, having an n-butyl and a carboxyl substituent at position-9 and 3, respectively, was found to have the highest antitumor effect and the lowest acute toxicity and neurotoxicity. These data suggested that (1) appropriate substituents at both position-9 and 3 of beta-carboline derivatives might play a crucial role in determining their enhanced antitumor activities and decreased acute toxicities and neurotoxic effects; (2) the beta-carboline derivatives have the potential to be used as antitumor drug leads.     

Synthesis and in Vitro Anti-Tumor Activity of A New Class of Acyclic Thioglycosides

The reaction of sodium 2-cyano-ethylene-1-thiolate salts with 2,3,4,6-tetra-O-acetyl-D-gluco- and D-galactopyranosyl bromides and with 2,3,4-tri-O-acetyl-D-xylo-. and L-arabinopyranosyl bromides, respectively, afforded new thioglycosides. Heating of the resultout glycosides with hydrazine produced pyrazole derivatives. The cytotoxicities toward the hepatoma cell line (HEPG2) of some synthesized compounds were tested. Some compounds showed high cytotoxic activity against (HEPG2) cell line. The OH moieties in the free glycosides were vital for potency. The synthesis procedures, spectroscopic data and antitumor activities for the prepared compounds are reported herein.     

Tumor-specific idiotype vaccines. II. Analysis of the tumor-related network response induced by the tumor and by internal image antigens (Ab2 beta)

In this study the tumor-specific immuneresponse induced by irradiated tumor cells (L1210/GZL) and by anti-idiotype antibodies was analyzed. The anti-idiotype antibodies (Ab2) were made against the paratope of a monoclonal antitumor antibody (11C1) that recognizes a tumor-associated antigen which cross-reacts with the mouse mammary tumor virus-encoded envelope glycoprotein 52. Two Ab2, 2F10 and 3A4, induced idiotypes expressed by the monoclonal antitumor antibodies 11C1 and 2B2. Cytotoxic T cells, generated by immunization with irradiated tumor cells, lyse 2F10 and 3A4 hybridoma cells. Furthermore, immunization with Ab2 induces tumor-specific cytotoxic T lymphocytes. The frequency of tumor-reactive cytotoxic T lymphocyte was found to be similar in mice immunized with Ab2 or irradiated tumor cells when examined at the precursor level. However, only 2F10 induces protective immunity against the growth of L1210/GZL tumor cells. The depletion of a L3T4+ T cell population from 2F10 immune mice was found to increase the effectiveness of transferred T cells to induce inhibition of tumor growth. The inability of 3A4 to induce antitumor immunity could be correlated with the presence of a population of Lyt2+ regulatory T cells. Collectively, these results demonstrate the existence of a regulatory network controlling the expression of effective tumor immunity. Our results demonstrate that selection of binding site-related Ab2 may not be a sufficient criteria for the development of an idiotype vaccine. A better understanding of the regulatory interactions induced by anti-idiotypes is needed for the design of effective antitumor immunotherapy.     

Selective enhancement of bleomycin cytotoxicity by local anesthetics

The cytotoxic effect of the antitumor antibiotic bleomycin toward cultured mouse FM3A cells was greatly enhanced by exposure of the cells to local anesthetics either before or together with treatment with bleomycin. Such local anesthetics include dibucaine, tetracaine, butacaine, lidocaine and procaine. Dibucaine-induced cell sensitization to bleomycin cytotoxicity produced a decrease in cell survival that became dependent on dose and time of bleomycin treatment. This effect of local anesthetics seems to be selective to bleomycin, since dibucaine and lidocaine do not enhance the cytotoxic effect of other antitumor agents including adriamycin, mitomycin C and $\text{cis}$-diamminedichloroplatinum(II).     

Intratumoral injection of inactivated Sendai virus particles elicits strong antitumor activity by enhancing local CXCL10 expression and systemic NK cell activation

We have already demonstrated that inactivated, replication-defective Sendai virus particles (HVJ-E) have a powerful antitumor effect by both the generation of tumor-specific cytotoxic T cells and inhibition of regulatory T cell activity. Here, we report that HVJ-E also has an antitumor effect through non-T cell immunity. Microarray analysis revealed that direct injection of HVJ-E induced the expression of CXCL10 in established Renca tumors. CXCL10 was secreted by dendritic cells in the tumors after HVJ-E injection. Quantitative real-time RT-PCR and immunohistochemistry revealed that CXCR3+ cells (predominantly NK cells) infiltrated the HVJ-E-injected tumors. Moreover, HVJ-E injection caused systemic activation of NK cells and enhanced their cytotoxity against tumor cells. In an in vivo experiment, approximately 50% of tumors were eradicated by HVJ-E injection, and this activity of HVJ-E against Renca tumors was largely abolished by NK cell depletion using anti-asialo GM1 antibody. Since HVJ-E injection induced systemic antitumor immunity by enhancing or correcting the chemokine-chemokine receptor axis, it might be a potential new therapy for cancer.     

On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex

Camptothecin, a cytotoxic antitumor compound, has been shown to produce protein-linked DNA breaks mediated by mammalian topoisomerase I. We have investigated the mechanism by which camptothecin disrupts DNA processing by topoisomerase I and have examined the effect of certain structurally related compounds on the formation of a DNA-topoisomerase I covalent complex. Enzyme-mediated cleavage of supercoiled plasmid DNA in the presence of camptothecin was completely reversed upon the addition of exogenous linear DNA or upon dilution of the reaction mixture. Camptothecin and topoisomerase I produced the same amount of cleavage from supercoiled DNA or relaxed DNA. In addition, the alkaloid decreased the initial velocity of supercoiled DNA relaxation mediated by catalytic quantities of topoisomerase I. Inhibition occurred under conditions favoring processive catalysis as well as under conditions favoring distributive catalysis. By use of [3H]camptothecin and an equilibrium dialysis assay, the alkaloid was shown to bind reversibly to a DNA-topoisomerase I complex, but not to isolated enzyme or isolated DNA. These results are consistent with a model in which camptothecin reversibly traps an intermediate involved in DNA unwinding by topoisomerase I and thereby perturbs a set of equilibria, resulting in increased DNA cleavage. By examining certain compounds that are structurally related to camptothecin, it was found that the 20-hydroxy group, which has been shown to be essential for antitumor activity, was also necessary for stabilization of the covalent complex between DNA and topoisomerase I. In contrast, no such correlation existed for UV-light-induced cleavage of DNA by Cu(II)-camptothecin derivatives.     

Synthesis and biological evaluation of indazolo[4,3-bc][1,5]naphthyridines (10-aza-pyrazolo[3,4,5-kl]acridines): a new class of antitumor agents

A series of potential DNA-binding antitumor agents, 2-[omega-(alkylamino)alkyl]-9-methoxy-5-nitro-2,6-dihydroindazolo[4,3-bc][1,5]naphthyridines (2a-f), 10-aza derivatives of PZA, has been prepared by condensation of 9-chloro-2-methoxy-6-nitro-5,10-dihydrobenzo[b][1,5]naphthyridin-10-one (6) with the appropriate (omega-aminoalkyl)hydrazine in tetrahydrofuran/methanol. Compound 6 was obtained by heating at 100 degrees C in H(2)SO(4)5, yielded by the condensation of 2,6-dichloro-3-nitrobenzoic acid (4) and 6-methoxy-3-pyridinamine (3). The non-covalent DNA-binding properties of 2 have been examined using a fluorometric technique. In vitro cytotoxic potencies of these derivatives against human hormone-refractory prostate adenocarcinoma cell line (PC-3) are described and compared to that of parent drug PZA. We selected the most cytotoxic target derivatives 2c,d, the in vitro inactive 2f, and reference compound PZA to investigate whether in vitro treatment with these drugs was able to induce necrotic and/or apoptotic cell death. To this purpose, we evaluated the percentage of apoptotic cells in PC-3 treated with the target compounds 2c,d,f and reference compound PZA, by Annexin V staining and Propidium iodide (PI)/Annexin V, biparametric flow cytometric analysis and agarose gel electrophoresis.     

Jacaric acid, a linolenic acid isomer with a conjugated triene system, has a strong antitumor effect in vitro and in vivo

In this study, we compared the cytotoxic effects of natural conjugated linolenic acids (CLnAs) on human adenocarcinoma cells (DLD-1) in vitro, with the goal of finding CLnA isomers with strong cytotoxic effects. The antitumor effect of the CLnA with the strongest cytotoxic effect was then examined in mice. The results showed that all CLnA isomers have strong cytotoxic effects on DLD-1 cells, with jacaric acid (JA) having the strongest effect. Examination of the mechanism of cell death showed that CLnAs induce apoptosis in DLD-1 cells via lipid peroxidation. The intracellular levels of incorporated CLnAs were measured to examine the reason for differences in cytotoxic effects. These results showed that JA was taken into cells efficiently. Collectively, these results suggest that the cytotoxic effect of CLnAs is dependent on intracellular incorporation and induction of apoptosis via lipid peroxidation. JA also had a strong preventive antitumor effect in vivo in nude mice into which DLD-1 cells were transplanted. These results suggest that JA can be used as a dietary constituent for prevention of cancer.     

Synthesis and antitumor activity of selected 7-alkylidene substituted cephems

Selected 7-alkylidene substituted cephems were synthesized and subjected to antitumor assay. The effect of substituents was examined to establish structure-activity relationships. It was found that the intensive intracellular generation of nitric oxide induced by tert-butyl 7-alkylidene cephalosporanate sulfones could be also regarded as an additional cytotoxic factor taking place both in vitro and in vivo experiments.     

Potentiation of the in vitro specific cytotoxic response to syngeneic lymphoma cells by PPD-stimulated tuberculin sensitive cells.

Spleen cells from rats immunized with the syngeneic (C58NT)D Gross virus-induced lymphoma have been shown to differentiate into cytotoxic effector cells following secondary in vitro stimulation with tumor cells. In the studies presented here, we evaluated whether cells specifically responding to PPD would increase the development of specific cytotoxic reactivity by a second cell population primed to lymphoma antigen. Mixtures of (C58NT)D-primed and BCG-primed responding cells generated cytotoxic activity to syngeneic lymphoma cells following cocultivation with mitomycin C-treated stimulating (C58NT)D cells; the addition of PPD to these mixtures produced a significant increase in cytotoxicity. The increased antitumor response resulted from an increase in specific cytotoxic activity from primed precursor cells. Responding cells activated with PPD alone in the absence of lymphoma antigen showed no lytic activity. Optimal numbers of tuberculin sensitive cells and concentration of PPD were determined. Evaluation of the kinetics of the generation of the cytotoxic response indicated that the addition of BCG-primed ceils and PPD increased the magnitude of cytotoxicity but did not alter the time course of the generation of cytotoxic activity. The addition of tuberculin sensitive cells and PPD to the in vitro secondary immune response also led to augmentation of generation of cells with antitumor activity detectable in vivo.