Evaluation of P53 and BAX gene expression and induction of apoptosis and necrosis by the cis-Pt(II) complex of 3-aminoflavone in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) in human lymphocytes

Currently cis-diamminedichloroplatinum(II) (cis-DDP) is one of the most commonly applied compounds in chemotherapy of many types of cancer. However, a drawback is that its effectiveness presents with many side effects. Therefore, human normal lymphocytes were chosen as a model system to study cis-bis(3-aminoflavone)dichloroplatinum(II) (the cis-Pt(II) complex of 3-aminoflavone) in comparison with cis-DDP. We examined the effect of both tested compounds on cell viability and induction of apoptosis and necrosis. Trypan blue and acridine orange/ethidium bromide staining were carried out, as well as quantitative analysis of the apoptotic signal of P53 and BAX induction caused by the cis-Pt(II) complex of 3-aminoflavone in comparison with cis-DDP. cis-DDP induced a decrease of cell viability and led to a higher increase in necrosis and apoptosis than did the cis-Pt(II) complex of 3-aminoflavone. Moreover, at the molecular level cis-DDP increased P53 and BAX expression in comparison with the other tested compound. The cis-Pt(II) complex of 3-aminoflavone showed a weaker genotoxic effect in normal lymphocytes in comparison with cis-DDP, which was a stronger inducer of apoptosis and necrosis.     

Induction of apoptosis and necrosis in A549 cells by the cis-Pt(II) complex of 3-aminoflavone in comparison with cis-DDP

Non-small cell lung cancer (NSCLC) includes a group of tumors that respond poorly to drugs. cis-Diamminedichloroplatinum(II) (cis-DDP) toxicity still remains problematic, and not completely solved by the improvement of supportive care. Therefore, the cis-Pt(II) complex of 3-aminoflavone was selected from cis-DDP analogues with a more favourable toxic profile towards normal cells and at least similar or better anti-tumor activity in comparison with cis-DDP. The aim of this research is to compare the ability of the cis-Pt(II) complex of 3-aminoflavone and cis-DDP to induce apoptosis and necrosis in the human non-small cancer cell line A549. Trypan blue dye exclusion, fluorochrome staining (acridine orange/ethidium bromide double staining), MTT and TUNEL (TdT-mediated dUTP Nick-End Labeling) assays were used. The results obtained show that the cis-Pt(II) complex of 3-aminoflavone is more active in inducing apoptosis and necrosis and in decreasing viability in A549 cells than cis-DDP, which suggests that it could be a potential chemotherapeutic drug.     

Genotoxicity of cis-PtII complex of 3-aminoflavone in comparison with cis-DDP in A549 cells evaluated by comet assay

Cis-diamminedichloroplatinum(II) (cis-DDP) is one of the most successful antineoplastic drugs. However, besides effectiveness it gives many side effects. Therefore, current studies are concentrated on searching for new analogs equally effective in chemotherapy but less toxic. Comparison of genotoxic properties of cis-Pt(II) complex of 3-aminoflavone and cis-DDP in a comet assay with and without H2O2 application was performed in A549 cell line. The higher tail moment values were noticed for the former compound contrary to the latter one in both variants. It suggests mainly DNA breaks (besides cross-links) appearance after cis-Pt(II) complex of 3-aminoflavone application and might indicate DNA degradation in comparison with cis-DDP.     

Evaluation of the genotoxicity of cis-bis(3-aminoflavone)dichloroplatinum(II) in comparison with cis-DDP

Short-term tests that detect genetic damage have provided information needed for evaluating carcinogenic risks of chemicals to man. The mutagenicity of cis-bis(3-aminoflavone)dichloroplatinum(II) (cis-[Pt(AF)2Cl2]) in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) was evaluated in the standard plate-incorporation assay in four strains of Salmonella typhimurium: TA97a, TA98, TA100 and TA102, in experiments with and without metabolic activation. It was shown that cis-[Pt(AF)2Cl2] acts directly and is mutagenic for three strains of S. typhimurium: TA97a, TA98 and TA100. In comparison with cis-DDP this compound showed a weaker genotoxicity. Contrary to cis-DDP it has not shown toxic properties in the tester bacteria. The genotoxicity of both tested compounds was evaluated using chromosomal aberration, sister chromatid exchange and micronucleus assays, without and with metabolic activation, in human lymphocytes in vitro. The inhibitory effects of both compounds on mitotic activity, cell proliferation kinetics and nuclear division index were also compared. In all test systems applied, cis-[Pt(AF)2Cl2] was a less effective clastogen and a weaker inducer of both sister chromatid exchanges and micronuclei in comparison with cis-DDP, with and without metabolic activation. cis-[Pt(AF)2Cl2] has a direct mechanism of action and is less cytostatic and cytotoxic than the other compound. These results provide important data on the genotoxicity of cis-[Pt(AF)2Cl2] and indicate its beneficial properties as a potential anticancer drug, especially in comparison with cis-DDP.     

Synthesis and characterization of complexes of p-isopropyl benzaldehyde and methyl 2-pyridyl ketone thiosemicarbazones with Zn(II) and Cd(II) metallic centers. Cytotoxic activity and induction of apoptosis in Pam-ras cells.

It is known that metallic complexes of methyl 2-pyridyl ketone thiosemicarbazone (HL1) and p-isopropyl benzaldehyde thiosemicarbazone (HL2) may have potential antitumor activity. We have prepared complexes of HL1 and HL2 with Zn(II) and Cd(II). The cytotoxic activity shown by these compounds against cell lines sensitive and resistant to cis-diamminedichloroplatinum(II) (cis-DDP) indicates that coupling of HL1 and HL2 to Zn(II) and Cd(II) centers may result in metallic complexes with important biological properties since they display IC50 values in a microM range similar to that of the antitumor drug cis-DDP. Moreover, it is interesting to note that the Zn/HL2 complex exhibits specific cytotoxic activity against Pam-ras cells (cis-DDP resistant cells which over-express the H-ras oncogene) with an in vitro therapeutic index of 3.26 versus 0.78 for cis-DDP. Treatment of Pam-ras cells with the IC50 value of the Zn/HL2 compound induces a 'DNA ladder' (fragmentation of genomic DNA in nucleosome units) indicative of apoptosis in this ras-transformed cell line. In contrast, a 'DNA smear' (non-specific fragmentation of genomic DNA) is observed in Pam 212 normal cells treated with the IC50 of this compound. The analysis by circular dichroism (CD) spectroscopy of the interaction of the Zn/HL2 compound with calf thymus DNA (CT DNA) indicates that it produces stronger alterations on the double helix conformation than cis-DDP. So, these results suggest that Zn/HL2 may be considered a potential antitumor agent.     

Platinum complexes with anticancer potential and their evaluation by a colorimetric lambda prophage induction assay

A simple biochemical phage induction assay (BIA) showed significant activity with 90% of the antitumor platinum compounds tested and lack of activity for all Pd(II) compounds and Pt(II) cationic complexes, compounds that are expected to be inactive. Structure-activity relationships for a large number of chemicals can be studied simultaneously by this simple, rapid, inexpensive and quantitative biochemical assay. Fifty-three platinum complexes were tested, including a number of ethylenediamines synthesized for this work. The magnitude of inducing activity varied over a 25-fold range; differences among analogs reflected structural differences in a chemically consistent manner. Seven platinum complexes showed greater activity than that of cis-diamminedichloroplatinum(II) (cisplatin, cis-DDP), while other compounds appeared to be substantially less toxic. The assay was predictive for most compounds with very high or very low activity in vivo against L1210. For compounds with intermediate levels of activity, no correlation between inducing and antitumor activity was observed.     

Reactions of the UVRABC excision nuclease with DNA damaged by diamminedichloroplatinum(II).

Mutants of Escherichia coli, which are blocked in excision repair (uvrA6, uvrB5, or uvrC34) are exceptionally sensitive to the antitumor drug cis-Pt(II)(NH3)2Cl2 (cis-DDP) but not the trans isomer. Plasmid DNA, damaged by either the cis or trans compound and treated with the UVRABC excision nuclease was cut as shown by conversion of supercoiled DNA to relaxed forms. All three protein products of the uvrA, uvrB, and uvrC genes were required for incision. End-labeled fragments damaged with cis-DDP and reacted with the UVRABC nuclease were cut at the 8th phosphodiester bond 5' and at the 4th phosphodiester bond 3' to adjacent GG's. DNA treated with trans-DDP was not cut appreciably at adjacent GG's by the repair enzyme as subsequent analysis of reaction products after enzyme digestion gave a pattern similar to those obtained with control untreated fragments. The results indicate that the UVRABC nuclease may promote cell survival by the removal of adjacent GG's which are crosslinked by cis-Pt(II)(NH3)2Cl2.     

Immunospecific protein of 34.5 kDa from DNA-protein cross-links induced by cis- and trans-diamminedichloroplatinum

Cis-diamminedichloroplatinum(II) (cis-DDP) is one of the most often used anticancer drugs. It is generally accepted that the antitumor activity of the drug results from its interactions with DNA. Trans-diamminedichloroplatinum(II) (trans-DDP) also binds to DNA effectively, but is clinically ineffective. In the present work the lymphocyte nuclear proteins that participate in DNA-protein cross-links induced by cis- and trans-DDP are investigated. In lymphocytes which are incubated without platinum compounds there are DNA-binding proteins in the range of 45-71 kDa. It is shown that additional proteins of 28, 30, 34.5, 45 and 120 kDa are cross-linked with DNA in lymphocytes after 2-h incubation with cis-DDP at concentrations of 0.1 and 0.5 mM. Trans-DDP does not bind additional proteins to DNA after the same incubation time. Electrophoretic analysis shows that trans-DDP binds much more of the same nuclear proteins to DNA than cis-DDP after 12-h incubation. In this study a test for the identification of 34.5 kDa protein is also undertaken. This protein appears in the samples obtained after 12-h incubation of lymphocytes with cis- and trans-DDP at 0.5 and 1 mM, especially. The protein of 34.5 kDa from cross-links induced by 1 mM trans-DDP is recognized by antibodies against the protein of the same molecular weight from the nuclear matrix of the lymphocytes. The results obtained here are discussed in relation to the biological activity of diamminedichloroplatinum isomers.     

Apoptosis induction and inhibition of H-ras overexpression by novel trans-[PtCl2(isopropylamine)(amine')] complexes

Hitherto, it has been generally accepted as a paradigm of the biochemical pharmacology of platinum antitumor drugs that a cis configuration of the leaving groups is necessary for antitumor activity of platinum compounds. However, it has been recently observed that certain trans-platinum complexes have both in vitro and in vivo antitumor activity. We previously reported the synthesis, characterization and cytotoxic activity against ras-transformed cells of several trans-[PtCl2LL'] complexes where L and L' are asymmetric aliphatic amines (L = dimethylamine and butylamine, L' = isopropylamine). The results reported in this paper show that the compounds trans-[PtCl2(isopropylamine)(dimethylamine)] and trans-[PtCl2(isopropylamine)(butylamine)] kill Pam 212-ras cisplatin resistant cells through apoptosis induction. Moreover, Western blot data show that both compounds inhibit overexpression of H-ras oncogene in Pam 212-ras cells. Altogether, these data indicate that, in contrast with cis-DDP, the apoptotic activity of these novel trans-Pt(II) compounds in ras-transformed cells is associated with their ability to abolish ras-overexpression.     

Effects of antimitotic agents either free or bound to DNA on mouse peritoneal macrophages cultivated in vitro

Mouse peritoneal macrophages were cultivated in vitro and treated with ethidium bromide (EB) or with cis-dichloro-diammine platinum (II) (cis-Pt). EB provokes strong cytological alterations and cell degeneration; cis-Pt was not toxic under our experimental contitions. EB-DNA complex penetrates into the macrophages, is liberated from DNA in vacuoles, then diffuses into the cell and is highly cytotoxic. Cis-Pt-DNA complex also penetrates into the cells, but cis-Pt cannot be released from DNA, cis-Pt-DNA complex accumulates inside cytoplasmic vacuoles but has no cytotoxic activity.     

DNA-platinum interactions in vitro with trans- and cis-Pt(NH3)2Cl2.

In the present study the nature and the hydrolysis of DNA-Pt complexes with the platinum compounds, [Pt(dien)Cl]Cl, trans- and cis-Pt(NH3)2Cl2, using potentiometric chloride determinations, have been investigated. The trans-Pt(NH3)2Cl2 and the [Pt(dien)Cl]Cl react with the GC planes at the N7(G) sites, while the cis-Pt(NH3)2Cl2 compound reacts with the GC planes and forms a chelate by using the N7(G) and O6(G) sites. The complex is a specific 1:1 Pt:DNA adduct. The platinum atom in cis-Pt(NH3)2Cl2 liberates both chlorine atoms on chelation. A mechanism for the in vivo antitumor activity of the cis-Pt(NH3)2Cl2 is proposed and the structure activity relationship is discussed.     

Palladium(II) salt and complexes of spermidine with a six-member chelate ring. Synthesis, characterization, and initial DNA-binding and antitumor studies.

By reaction of spermidine trihydrochloride with K2PdCl4 and PdCl2 at different pH's, we have synthesized the [sperH3]2[PdCl4]3 (I), [PdCl2(sperH)]2[PdCl4] (II), and [(PdCl2)3(sper)2] (III) compounds. The structure of these compounds was studied by IR and 1H NMR; complex II was analyzed by x-ray diffraction. In this complex the spermidine is attached to the PdCl2 group forming a six-member chelate ring with a protonated terminal amine group. The crystal of [PdCl2(sperH)]2[PdCl4] x 2H2O (II) is monoclinic, P2(1)/n, with a = 7.023(1) A, b = 12.662(1) A, c = 18.435(3) A, and beta = 99.95(1) degrees, Z = 4, R = 0.051, and Rw = 0.058 on the basis of 2690 independent reflections. We have compared the antitumor activity in vitro against the isolated human breast carcinoma MDA-MB 468 cell line of compounds I, II, and III with that of cis-diamminedichloroplatinum(II), cis-DDP. The results show that compounds III and III have values of ID50 similar (0.74 microgram/ml) or even lower (0.56 microgram/ml) than cis-DDP (0.80 microgram/ml). We also observed that compounds I, II, and III have the ability to induce conformational changes in covalently closed circular (ccc) form of the pUC8 plasmid DNA. Compounds II and III also induce conformational changes in the open circular (oc) form of this plasmid.     

A cycloplatinated compound of p-isopropylbenzaldehyde thiosemicarbazone and its chloro-bridged derivative induce apoptosis in cis-DDP resistant cells which overexpress the H-ras oncogene.

cis-Diamminedichloroplatinum(II) (cis-DDP) is a widely used antitumour drug which produces important damage on the DNA inducing apoptosis in several cell lines. We have analyzed the cytotoxic activity of novel cyclometallated complexes of p-isopropylbenzaldehyde thiosemicarbazone (p-is.TSCN) and their dimeric chloro-bridged derivatives in murine keratinocytes transformed by the H-ras oncogene which are resistant to cis-DDP (Pam-ras cells). The data show that, in contrast with cis-DDP, the tetrameric cycloplatinated complex [Pt(p-is.TSCN)]4 and its dimeric chloro-bridged derivative [Pt(microCl)(p-is.TSCN)]2 have a good in vitro therapeutic index when comparing the cytotoxicity in Pam-ras cells to normal murine keratinocytes (Pam 212 cells) since they induce cell death in Pam-ras cells at drug concentrations significantly lower than those needed to kill Pam 212 cells. At equitoxic doses (IC90), both complexes produce characteristic features of apoptosis in Pam-ras cells together with a drastic decrease in levels of H-ras protein. These effects are not observed when the cells are treated with the IC90 of the cis-DDP drug nor the p-is.TSCN ligand. Altogether, these results suggest that the platinum compounds [Pt(p-is.TSCN)]4 and [Pt(microCl)(p-is.TSCN)]2 might have potential as antitumour agents in view of their specific induction of apoptosis in cis-DDP resistant cells.     

Repair of platinum-DNA lesions in E. coli by a pathway which does not recognize DNA damage caused by MNNG or UV light

The adaptive response is an inducible DNA-repair system which diminishes the mutagenic and toxic effects of alkylating agents. A mutant of E. coli constitutive for adaptative repair, BS21, has been isolated. A spontaneous revertant of this strain, BS23, lacks the adaptive response. When compared to its wild-type parent, mutant BS21 showed an increased resistance to the killing and mutagenic effects of a compound which is not a classical alkylating agent, the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP). However, this resistance to cis-DDP was also found in strain BS23 which lacks the adaptive response. cis-DDP bound to the DNA of all 3 strains with the same efficiency. In addition, we have investigated the effect of UV radiation and we failed to observe a significant difference in the survival and mutagenesis of these strains. This evidence suggests that the resistance of BS21 and BS23 strains to cis-DDP is not a consequence of the adaptive response or increased excision repair.     

Synthesis and characterization of Pd(II) and Pt(II) complexes of p-isopropylbenzaldehyde N-protected thiosemicarbazones. Cytotoxic activity against ras-transformed cells.

Cytotoxicity tests in tumor cells sensitive to cis-DDP (HL-60, JURKAT, Hela and 3T3) and in tumor cells transformed by ras oncogenes and resistant to cis-DDP (Pam 212-ras) show that cyclometallated complexes 1a [Pd(p-is.TSCN-NHMe)]4, 2b [Pt(p-is.TSCN-NMe2)]4 and 4a [Pd(p-is.TSCN-NHex)]4 may be endowed with specific cytotoxic properties. In fact, these three novel metal-thiosemicarbazone compounds kill Pam 212-ras cells through apoptosis induction. These results, together with others recently published, indicate that the design and synthesis of metallated-thiosemicarbazone compounds may lead to the discovery of novel antitumor agents able to circumvent cis-DDP resistance, in particular tumor cell lines.     

Binding of cis- and trans-diamminedichloroplatinum(II) to deoxyribonucleic acid exposes nucleosides as measured immunochemically with anti-nucleoside antibodies

We report the use of anti-nucleoside antibodies to probe for local denaturation of calf thymus DNA upon binding of the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, and the biologically inactive analogues trans-diamminedichloroplatinum(II), trans-DDP, and chloro(diethylenetriamine)platinum(II) chloride, [Pt(dien)Cl]Cl. These antibodies specifically recognize each of the four DNA nucleosides. They bind well to denatured DNA, but not to native DNA in which the bases are less accessible owing to Watson-Crick duplex structure. At relatively high levels of modification (D/N approximately 0.1), cis-DDP causes significant disruption of DNA base pairing as reflected by the increased binding of anti-cytidine, anti-adenosine, and anti-thymidine antibodies. At lower levels of platinum adduct formation, however, all four anti-nucleoside antibodies bind more to DNA modified with trans-DDP. This result indicates that adducts formed by trans-DDP disrupt the DNA structure to a greater extent than those formed by cis-DDP at low D/N ratios. Modification of DNA by the monofunctional complex [Pt(dien)Cl]Cl does not affect its recognition by anti-nucleoside antibodies, demonstrating that base pair disruption is a consequence of bifunctional binding. The relative anti-nucleoside antibody recognition of cis-DDP-modified DNA is anti-cytosine greater than anti-adenosine approximately anti-thymidine much greater than anti-guanosine, consistent with the major adduct being an intrastrand d(GpG) cross-link. These results reveal that base pair disruption in a naturally occurring DNA modified by either cis-DDP or trans-DDP is sufficient to be detected by protein (antibody) binding. The relevance of these findings to current ideas about the molecular mechanism of action of cis-DDP is discussed.     

Studies on the interaction between antitrypanosome cis-DDP analogs with DNA

The nature of the conformational changes produced in DNA by cis-DDP analogs has been studied by physiochemical techniques. The UV spectra showed that the DNA undergoes bathochromic shifts accompanied by hyperchromic effects in reaction with specific analogs (cis-Pt(DDH) (mucobromic)2, cis-Pt(tranilcypromine)2Cl2 and cis-Pt(DDH) Cl2), while a different series of analogs (cis-Pt(DDH) (metafluorobenzoic)2 and cis-Pt(pentamidine)Cl2) induce a significative decrease in the absorbance at 258 nm. Moreover one of these analogs (cis-Pt(pentamidine)Cl2) causes strong stabilization of the double helix to heat denaturation. The CD spectra indicate moreover that cis-Pt(pentamidine)Cl2 modifies the secondary structure of the DNA in a significant way with an increase of the positive band and a decrease of the ellipticity of the negative band. The antitrypanosome activity of cis-Pt(pentamidine)Cl2 is probably due to inhibition of the intracellular parasites division in parasitized cells.     

Uvr excision repair protein complex of Escherichia coli binds to the convex side of a cisplatin-induced kink in the DNA.

The Escherichia coli UvrABC endonuclease is capable of initiating the repair of a wide variety of DNA damages. To study the binding of the UvrAB complex to the DNA at the site of a lesion we have constructed a synthetic DNA fragment with a defined cis-diamminedichloroplatinum(II) (cis-Pt).GG adduct. The cis-Pt.GG is the major adduct after treatment of DNA with the antitumor agent cisplatin. Binding to the DNA at the site of the defined lesion was studied with DNase I and MPE.Fe(II) hydroxyl radical footprinting. The results indicate that the UvrAB complex binds to the convex side of the kink in the DNA caused by the cis-Pt.GG adduct. Concerted incisions of the damaged strand by the UvrABC endonuclease were at the 8th phosphodiester bond 5' to and at the 4th bond 3' of the adjacent guanines. An additional incision was found at the 15th phosphodiester bond 5' to the damaged site. This extra incision was stimulated by a high concentration of UvrC.     

Synthesis and preliminary cytotoxicity study of glucuronide derivatives of CC-1065 analogues

Glucuronide derivatives of CBI-bearing CC-1065 analogues have been synthesized, and their cytotoxicities tested against U937 leukemia cells. The new compounds show potent antitumor activity in vitro. Compounds 1 and 2, and their corresponding glucuronides 3 and 4 have IC(50) values of 0.6, 0.1, 1.4 and 0.6 nM, respectively. Glucuronide 3 is approximately 2-fold less toxic than its hydroxyl counterpart 1, and glucuronide 4 is approximately 6-fold less toxic than its hydroxyl counterpart 2. Glucuronides 3 and 4 may have limited use in the ADEPT approach. However, they may be used as antitumor agents in a conventional way.