Synthesis and evaluation of a DHA and 10-hydroxycamptothecin conjugate

We have synthesized a conjugate of cis-4,7,10,13,16,19-docosahexenoic acid (DHA) and 10-hydroxycamptothecin (HCPT), DHA-HCPT. The antitumor activity of DHA-HCPT was evaluated in vitro against L1210 leukemia cells and in experimental animal tumor models including L1210 leukemia, Lewis lung carcinoma, and colon 38 adenocarcinoma. DHA-HCPT showed a greatly improved antitumor efficacy compared to HCPT.     

Identification of effector T cells responsible for enhanced antitumor immunity induced by tumor vaccine and pyran copolymer

Summary The combination of concanavalin A-bound, but not concanavalin A-free, L1210 murine leukemia vaccine and pyran copolymer produced an enhanced therapeutic response in L1210-bearing mice. Immunoprophylactic experiments showed that the effective combination produced enhanced antitumor immunity as determined by refractoriness to the inoculation of live L1210 cells. In vitro antiproliferation and intraperitoneal adoptive transfer tests showed that antitumor effector cells were detected in the spleen and peritoneal cavity of primed mice after, but not before, live L1210 cell inoculation. These effector cells were identified as T cells on the basis of their non-adherence to plastic flasks and sensitivity to treatment with anti-mouse brain-associated T cell antigen antisera and complement. Although non-T cell populations, including macrophages of mice primed with L1210 vaccine and pyran copolymer, inhibited the in vitro proliferation of L1210 cells, we obtained evidence suggesting that they were not the primary antitumor effector cell populations responsible for the in vivo elimination of the inoculated L1210 cells.     

Synthesis and antitumor activity of N-substituted iminodiacetato(1,1-bis[aminomethyl]cyclohexane)platinum(II) complexes

A number of water-soluble N-substituted iminodiacetato(1,1-bis[aminomethyl]cyclohexane)platinum(II) complexes have been synthesized, and their mode of coordination characterized by elemental analysis and infrared data. Preliminary in vitro screening test for antitumor activity of these complexes against L1210 murine leukemia cells were performed. The results indicate that these complexes have an acceptable in vitro cytotoxicity against L1210 leukemia.     

Synthesis, characterization, and antitumor activity of new chloroethylamine platinum complexes.

A series of cis-bis-(2-chloroethylamine)platinum(II) and platinum(IV) complexes were synthesized and characterized by elemental analysis, IR, and 1H and 195Pt NMR spectroscopic techniques. Complexes were tested in vitro against murine L1210 leukemia and human ovarian A2780 cell lines and in vivo against the L1210 leukemia model. Some of these complexes showed excellent antitumor activity in both systems. However, all were inactive against cisplatin-resistant A2780/CP cells.     

Disruption of DNA synthesis and structure of mouse L1210 leukemia cells, sensitive and resistant to 1-methyl-1 nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea in vivo

Leukemia L1210 cells with acquired resistance to 1-methyl-1-nitrosourea (MNU) (L1210/MNU) and 1.3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (L1210/BCNU) were developed from leukemia L1210 cells sensitive to these drugs (L1210/0). The modal chromosome number of leukemia L1210/MNU and L1210/BCNU cells increases from 40 (L1210/0) to 41. It was shown that in leukemia L1210/MNU cells the inhibition of DNA synthesis after MNU administration in a therapeutic dose (80 mg/kg) is lasted within 24 hours, while that in leukemia L1210/0 cell--within 96 hours. After administration of BCNU (20 mg/kg) inhibition of DNA synthesis in leukemia L1210/BCNU cells reached of 50% of control in comparison with practically complete inhibition of DNA synthesis in leukemia L1210/0 cells. Centrifugation on alkaline sucrose density gradients revealed no differences in the rate of sedimentation of leukemia L1210/0, L1210/MNU and L1210/BCNU cell lysates. After 1 hour treatment with MNU of mice bearing L1210/MNU and L1210/0 leukemia cells single-strand breaks in DNA were determined. After 4 hours these strand-breaks retained in leukemia L1210/0 cells, but were eliminated in leukemia L1210/MNU cells. Administration of BCNU to mice with leukemia L1210/0 and L1210/BCNU cells resulted in both cases in the production of DNA aggregates. There is no complete cross-resistance between MNU and BCNU which allows a substitution of these drugs providing for the increase in their therapeutic efficiency.     

Synthesis, characterization and antitumor activity of copper(II) complex with nicotinamido-4-bis(2-chloroethyl)aminobenzaldimine.

Nicotinamido-4-bis(2-chloroethyl)aminobenzaldimine (NBAB) was synthesized and characterized by elemental analysis, IR and 1H NMR spectra. The complex of Cu(NBAB)2(NO3)2 was prepared in ethanol and characterized by elemental analysis, conductivity, cyclic voltammetry, IR, UV-Vis, fluorescence, CD and EPR spectra. The characteristic data suggest that the complex has an elongated octahedral structure, and NBAB behaves as bidentate in the keto form. The antitumor activities of NBAB and the complex against L1210 murine leukemia and K562 were investigated with both the MTT method and the colony formation test. The results in vitro indicate that antitumor activities of NBAB are superior to 2,2'-chlorodiethylamine hydrochloride (nitrogen mustard) for L1210, and inferior to nitrogen mustard for K562, but the antitumor activities of the complex for both cell lines are superior to nitrogen mustard.     

Preparation, characterization, and antitumor activity of water-soluble aminoalkanol platinum(II) complexes

A new series of highly water-soluble aminoalkanol platinum(II) complexes have been synthesized and characterized by elemental analysis, conductance, IR, and 195Pt NMR. Preliminary in vitro and in vivo screening tests for antitumor activities of these complexes against L1210 murine leukemia were performed. In general, these compounds were far less cytotoxic than cisplatin and possessed only a moderate degree of antitumor activity.     

Synthesis, cytotoxicity and antitumor activity of platinum(II) complexes of cyclopentanecarboxylic acid hydrazide

New platinum(II) complexes of cyclopentanecarboxylic acid hydrazide (cpcah) were prepared, characterized by elemental analysis, IR and 1H NMR spectra, and evaluated for in vitro cytotoxicity in Friend leukemia (FL) and A2780 ovarian tumor cells, induction of apoptosis in FL cells, as well as for in vivo antitumor activity toward murine L1210 leukemia and Lewis lung carcinoma. The spectral analyses indicated a cis-square planar structure of the complexes with hydrazide ligand coordinated via the NH2 group. The compounds exerted significantly lower in vitro and in vivo toxicities as compared with those of cisplatin (cis-diamminedichloroplatinum(II), DDP). On the other hand, the complex [Pt(NH3)(cpcah)Cl2] exhibited antitumor activity against L1210 leukemia in mice comparable to that of cisplatin, resulting at a dose of 42 mg/kg (administered 3 times) in a T/C (mean survival time) of 280%. This compound displayed an in vitro macromolecular synthesis inhibition pattern similar to that of DDP. At concentrations close to the cytostatic ones (10-20 microM) this complex, as well as DDP, was able to induce apoptosis in FL cells as shown by neutral comet assay and morphological analysis. We concluded that there is a correlation between the ability of platinum complexes to induce apoptosis and their antitumor activity.     

Development of host-dependent high-grade tumor-specific immunity through a novel mechanism triggered by the Lyt-2+ tumor-specific T cell clone (K7L) that induces temporal growth of L1210 leukemia-K7L-variant

When a murine leukemia L1210-specific Lyt-2+ T cell clone, K7L, was injected i.p. into CD2F1 mice together with L1210, the normal growth of L1210 in the peritoneal cavity of the mice at the early stage (days 0 to 5) was strongly inhibited, but L1210 grew progressively at the middle-stage (days 5 to 10), and then was rejected at the late stage (days 10 to 20). The mice thus survived for long times (more than 60 days), whereas the normal control injected with L1210 alone died within 14 days. The L1210 that grew at the middle stage in mice initially inoculated with L1210 together with K7L was a K7L-insensitive (K7L-) variant. All of eight tumor clones established from L1210-K7L- by limiting dilution was insensitive to the antitumor activity of K7L, and this property of tumor clones was stable after repeated in vitro passage. The initial depression of the L1210 growth by K7L followed by growth and rejection of the variant L1210-K7L- by the host T cell activity was then found to prepare a strong, long-lasting (more than 3 mo) immunity to protect mice against the high-dose (10(7) cells per mouse) challenge of original L1210. Corresponding to this result, definite tumor (L1210)-specific cytotoxic T lymphocyte (CTL) activity against both variant and original L1210 targets was developed by antigen (L1210) restimulation in the culture of spleen cells from these mice, but was not increased to a detectable level before L1210-K7L- variant started to grow. It was suggested that the 1210-K7L- variant and the original L1210 should have the common tumor-specific antigen that was independent of the K7L-reactive antigen, and that original L1210, whose growth was retarded by K7L, primed the host with the common antigen to be enormously boosted by the subsequently growing L1210-K7L- variant.     

Synthesis and study of spin-labeled nitrosoureas

For the first time we have synthesized spin-labeled nitrosoureas and have studied their properties--reduction of the iminoxyl group by vitamin C leading to the formation of the corresponding hydroxylamine derivatives and degradation in the presence of an aminoradical, leading to biradicals. The ESR spectra of biradicals in methanol have nine hyperfine resonance lines. The spin-labeled nitrosoureas have shown a high antitumor activity against the L 1210 lymphoid leukemia and P 388 lymphocytic leukemia in BDF1 mice. A study of a broad range of transplantable tumors is in progress.     

Synthesis, characterization, and antitumor activity of 1,2-bis(diphenylphosphino) ethane platinum(II) and palladium(II) complexes

A number of 1,2-bis(diphenylphosphino)ethane monomeric platinum(II) and palladium(II) complexes have been synthesized in light of their potential antitumor activity. The metal center is coordinated with a number of carboxylate anions in the cis-configuration. These complexes have been characterized by elemental analysis, conductivity measurement, and various spectroscopic techniques [IR and 195Pt NMR]. In vivo screening tests for activity of these complexes were performed against the L1210/0 murine leukemia cancer model, but none displayed a significant level of antitumor activity.     

Synthesis and antitumor activity of a series of 1,2-diaminocyclohexanepalladium(II) dicarboxylate complexes

A series of complexes of the type [Pd(O O(DACH)] (O O = dicarboxylate ligand, DACH = 1,2-diaminocyclohexane) has been prepared. These complexes have been characterized by elemental analysis and infrared spectroscopy. The complexes have been screened in vivo for antitumor activity against the L1210 leukemia cell line. These palladium complexes lack antitumor activity, which may be due to (1) lack of solubility and/or (2) lack of stability of the complexes in solution.     

The role of N-nitrosourea-induced changes in the nucleoid structure and activity of repair enzymes in the development of drug resistance in mice with leukemia L1210

Using centrifugation of the nucleoid in a neutral sucrose gradient, the damages in the secondary structure of DNA and the activity of repair enzymes, such as DNA-polymerases alpha and beta and poly(ADP-riboso) polymerase, induced by 1-methyl-nitrosourea (MNU) and 1.3-bis (2-chloroethyl)-1-nitrosourea (BCNU) injected at maximal nonlethal single doses to mice bearing parent leukemia cells (L1210/0) and resistant to MNU and BCNU leukemia L1210 cells (L1210/MNU and L1210/BCNU), were studied. The MNU-induced production of single-strand breaks in L1210/0 and L1210/MNU cells was more conspicuous in newly replicated DNA than in those in preexisting DNA. A more fast repair of the damages in newly replicated DNA was detected in L1210/BCNU and especially in L1210/MNU leukemia cells as compared with L1210/0 cells. The data obtained suggest that there are prone errors in the repair of DNA template, since most of the single-strand breaks were revealed in the newly replicated DNA synthesized on the repaired DNA. The repair of DNA damages in L1210/BCNU and especially in L1210/MNU cells was accompanied by the activation of DNA-polymerases alpha and beta and poly(ADP-riboso)polymerase. Both DNA-polymerases--alpha and beta--were shown to be involved in repair of DNA damages induced by MNU and only DNA-polymerase beta was involved in the repair of damages induced by BCNU.     

Comparative characteristics of the antitumor and immunodepressive activity of carminomycin on the L-1210 experimental model]

The antitumor activity of carminomycin was estimated by the number of lymphoma colonies formed in the spleen of DBA/2 mice on their inoculation with the bone marrow cells from mice with transplantable leukemia L-1210. The immunodepressive properties of carminomycin were determined by the number of the antibody forming cells in the spleen of CBA and DBA/2 mice with leukemia L-1210 after immunization with sheep red blood cells. It was found that in a single dose of 1.5 mg/kg carminomycin inhibited the lymphoma colonies by 50 per cent. The maximum immunodepressive effect was observed when carminomycin was used in a single dose of 1.5 mg/kg 48 hours after the antigen stimulation. In this case the number of the antibody forming cells in DBA/2 mice with leukemia L-1210 was lower than that in CBA mice without leukemia.     

Selective treatment of leukemia L1210 with combination of deoxycytidine and lethal doses of cytosine arabinoside]

Oral administration of deoxycytidine simultaneously with intraperitoneal injections of toxic doses of cytosine arabinosidetomice with advanced L1210 leukemia diminished the toxic effects preventing drug death of these mice. They developed a marked antitumor effect. The mean survival time of these mice was considerably extended as compared to that of untreated animals or those given one of these drugs alone. At the optimum schedule of treatment about 23% of the mice survived over 60 days. Deoxycytidine protection reduced the antileukemic effect of cytosine arabinoside administered in nontoxic doses. The deoxycytidine plus cytosine arabinose combination was ineffective in the treatment of transplantable myeloid leukemia in mice.     

An XPG DNA repair defect causing mutagen hypersensitivity in mouse leukemia L1210 cells.

One of the most widely used antitumor drugs is cis-diamminedichloroplatinum(II) (cisplatin), and mechanisms of cisplatin resistance have been investigated in numerous model systems. Many studies have used mouse leukemia L1210/0 as a reference wild-type cell line, and cisplatin-resistant subclones have been derived from it. Increased DNA excision repair capacity is thought to play a key role in the acquired cisplatin resistance, and this has influenced development of drugs for clinical trials. We report here that the L1210/0 line is in fact severely deficient in nucleotide excision repair of damaged DNA in vivo and in vitro. L1210/0 cell extracts could be complemented by extracts from repair-defective human xeroderma pigmentosum (XP) or rodent excision repair cross-complementing (ERCC) mutant cells, except for XPG/ERCC5 mutants. Purified XPG protein could restore repair proficiency to L1210/0 extracts. Expression of mouse XPG mRNA was similar in all L1210 lines studied, suggesting a point mutation or small alteration of XPG in L1210/0 cells. The DNA repair capacity of a cisplatin-resistant subline, L1210/DDP10, is similar to that of type culture collection L1210 cells and to those of other normal mammalian cell lines. Nucleotide excision repair of DNA is thus clearly important in the intrinsic cellular defense against cisplatin. However, in contrast to what is generally believed, enhancement of DNA repair above the normal level in these rodent cells does not appear to be a mechanism of acquired resistance to the drug.     

Evaluation of antioxidant and antiproliferative activities of 1,2‐bis (p‐amino‐phenoxy) ethane derivative Schiff bases and metal complexes

In this study, the effects of the two Schiff base derivatives and their metal complexes were tested for MDA concentration, which is an indicator of lipid peroxidation, antioxidant vitamin A, vitamin E, and vitamin C levels in cell culture. A comparison was performed among the groups and it was observed that MDA, vitamin A, vitamin E, and vitamin C concentrations were statistically changed. According to the results, all compounds caused a significant oxidative stress without Zn complexes. Moreover, Mn(II), Cu(II), Zn(II), and Ni(II) complexes of Schiff bases derived from a condensation of 1,2‐bis (p‐aminophenoxy) ethane with naphthaldehydes and 4‐methoxy benzaldehyde were examined in terms of antitumor activity against MCF‐7 human breast cancer and L1210 murine leukemia cells. Furthermore, the derivatives were tested for antioxidative and prooxidative effects on MCF‐7 breast cancer cells. The compounds which were tested revealed that there was an antitumor activity for MCF‐7 and L 1210 cancer cells. Also, some of the compounds induced oxidative harmful.     

A macromolecular prodrug of doxorubicin conjugated to a biodegradable cyclotriphosphazene bearing a tetrapeptide

A new biodegradable water-soluble phosphazene trimer-doxorubicin conjugate was synthesized, in which equimolar hydrophilic methoxy-poly(ethylene glycol) with a molecular weight of 350 (MPEG350) and a tumor-specific tetrapeptide (Gly-Phe-Leu-Gly) were grafted to cyclotriphosphazene. The present conjugate exhibited cytotoxicity lower than that of free doxorubicin (IC50=0.10 microM) but a reasonably higher in vitro cytotoxicity (IC50=1.1 microM) against the leukemia L1210 cell line probably due to its enzymatically controlled release.     

Effect of levamisole on cytotoxic T-cell-mediated immune resistance to L1210 murine leukemia in hyperimmune mice

Summary The effect of levamisole (LMS) on T-cell-mediated antitumor immunity was examined in adult and aged mice hyperimmune to L1210 leukemia. The immune resistance of aged mice was depressed compared with that of adult mice, which almost completely rejected 5×10⁷ L1210 cells inoculated IP. A significant level of tumor-specific cytotoxicity was detected in the spleen cells of adult hyperimmune mice by the ⁵¹Cr-release assay after in vitro sensitization with mitomycin C-treated L1210 cells. This was mediated by cytotoxic T cells, since in vivo administration of antithymocyte serum or in vitro treatment of the spleen cells with anti-Thy 1.2 antibody and complement abrogated the cytotoxicity completely. In aged mice, however, cytotoxic T-cell activity was lower although the animals were immune to L1210.Administration of LMS (0.38 mg/kg) restored the depressed cytotoxicity of aged mice to the level seen in adult mice. Furthermore, in adult hyperimmune mice LMS augmented T-cell-mediated cytotoxic activity and restored the reduced cytotoxicity caused by in vivo administration of antithymocyte serum. These results indicate that LMS was effective in augmenting T-cell-mediated tumor immunity in immunologically competent or deficient hosts.     

Antitumor activity and metal complexes of the first transition series. Trans-bis(salicylaldoximato)copper(II) and related copper(II) complexes,a novel group of potential antitumor agents

The antitumor activity of forty nine different metal complexes of the first transition series against mouse leukemia L 1210 cells and of two of the complexes against Ehrlich ascites carcinoma have been tested in vitro by the method described in this paper. Eight complexes showed a 50% inhibition of tumor cell division at concentration level 5–6 μg/ml of the complex for the former and two most effective complexes also for the latter. The trans-bis-(salicylaldoximato)copper(II) and trans-bis(resorcylaldoximato)copper(II) complexes were found to possess the highest antitumor activity.