Preclinical perspectives on platinum resistance

In the 30 years since the introduction of cisplatin into the clinic, laboratory studies have provided considerable information as to both how the drug exerts its antitumour effects and how some tumours are, or become, resistant. Once inside a cell, the chlorine groups of cisplatin are exchanged for water (aqua) species, which are more chemically reactive. The intracellular target for cisplatin is DNA, where a variety of adducts are formed, some on the same strand of DNA (intrastrand adducts) and others between strands (interstrand adducts). Of the 4 bases, guanine is the preferred site for binding and the most common adduct involves linkages on 2 adjacent guanines on the same strand of DNA. It remains uncertain which of the various types of adduct is the most important in terms of producing antitumour effects. Resistance to cisplatin has been studied extensively using tumour cells repeatedly exposed to the drug in vitro. In these cell models, resistance is generally due to a combination of mechanisms, some resulting in reduced damage to DNA and others following DNA damage. Resistance due to inadequate binding to DNA has been shown to be caused by reduced drug uptake (influx rather than efflux) and inactivation by thiol-containing species such as glutathione and metallothioneins. Resistance occurring post-DNA binding may be due to changes in DNA repair pathways [an increase in nucleotide excision repair (NER) or a loss of DNA mismatch repair (MMR)]. Conversely, the hypersensitivity of some cell lines to cisplatin has been shown to be due to defective NER, through loss or reduced expression of NER proteins such as XPG and XPA. Resistance may also be mediated through alterations in proteins involved in programmed cell death (apoptosis) such as p53 and the BCL2 family. A basic understanding of cisplatin resistance pathways has made a major impact in the development of new platinum analogues capable of circumventing resistance. Examples (which are now undergoing clinical trial) include ZD0473 (which, relative to cisplatin, possesses a reduced reactivity towards inactivating thiol-containing molecules) and the trinuclear platinum BBR3464 (which has markedly different DNA binding properties compared with cisplatin).     

Recognition and incision of gamma-radiation-induced cross-linked guanine-thymine tandem lesion G[8,5-Me]T by UvrABC nuclease

Nucleotide excision repair (NER) plays an important role in maintaining the integrity of DNA by removing various types of bulky or distorting DNA adducts in both prokaryotic and eukaryotic cells. In Escherichia coli, the excision repair proteins UvrA, UvrB, and UvrC recognize and incise the bulky DNA damages induced by UV light and chemical carcinogens. In this process, when a putative lesion in DNA is identified initially by UvrA, a subsequent strand opening is carried out by UvrB that not only ensures that the distortion is indeed due to a damaged nucleotide but also recognizes the chemical structure of the modified nucleotides with varying efficiencies. UvrB also recruits UvrC that catalyzes both the 3'- and the 5'-incisions. Herein, we examined the interaction of UvrABC with a DNA substrate containing a single G[8,5-Me]T cross-link and compared it with T[6,4]T (the 6-4 pyrimidine-pyrimidone photoproduct) and the C8 guanine adduct of N-acetyl-2-aminofluorene (AAF). The intrastrand vicinal cross-link G[8,5-Me]T containing a covalent bond between the C8 position of guanine and the 5-methyl carbon of the 3'-thymine is formed by X-radiation, while T[6,4]T is a vicinal cross-link induced by the UV light. We also selected the AAF adduct for comparison because it represents a highly distorting monoadduct containing a covalent linkage at the C8 position of guanine. The dissociation constants (K(d)) for UvrA protein binding to DNA substrates containing the G[8,5-Me]T, T[6,4]T, and AAF adducts, as determined by gel mobility shift assays, were 3.1 +/- 1.3, 2.8 +/- 0.9, and 8.2 +/- 1.9, respectively. Although UvrA had a considerably higher affinity for G[8,5-Me]T than for the AAF adduct, the G[8,5-Me]T intrastrand cross-link was incised by UvrABC much less efficiently than the T[6,4]T intrastrand cross-link and the AAF adduct. Similar incision results also were obtained with the DNA substrates containing the adducts in a six-nucleotide bubble, indicating that the inefficient incision of G[8,5-Me]T cross-link by UvrABC was probably due to the lack of efficient recognition of the adduct by UvrB at the second step of DNA damage recognition in the E. coli NER. Indeed, as compared to T[6,4]T and AAF substrates, which clearly showed UvrB-DNA complex formation, very little UvrB complex was detectable with the G[8,5-Me]T substrate. Our result suggests that G[8,5-Me]T intrastrand cross-link is more resistant to excision repair in comparison with the T[6,4]T and AAF adducts and thus will likely persist longer in E. coli cells.     

Different features of the DNA binding mode of antitumor cis-amminedichlorido(cyclohexylamine)platinum(II) (JM118) and cisplatin in vitro

cis-Amminedichlorido(cyclohexylamine)platinum(II) (JM118) is an antitumor Pt(II) analogue of cisplatin exhibiting considerably higher activity than cisplatin in human tumor cells. JM118 is also the major metabolite of the first orally administered Pt(IV) drug satraplatin. In an effort to design improved platinum antitumor agents, it is important to elucidate the biochemical factors that affect the cytotoxic properties of existing platinum drugs. Since DNA is considered the major pharmacological target of platinum drugs, the objective in the present work was to understand more fully the DNA binding mode of antitumor JM118. We examined the rate of aquation of the first chloride of bifunctional JM118 and found that it was considerably lower than that of cisplatin; consequently, the rate of the reaction of JM118 with DNA was lower compared to cisplatin. The influence of global modification by JM118 and its major site-specific adducts on DNA conformation by biochemical methods was investigated as well. While examination of the global modification revealed in several cases no substantial differences in the lesions induced by JM118 and cisplatin, DNA bending due to the 1,2-GG intrastrand adduct of JM118 was lower than that of cisplatin. The bending angles afforded by the adducts of JM118 were only slightly affected by the orientation of the cyclohexylamine ligand toward the 3' or 5' direction of the duplex. We also used in vitro assays that make it possible to monitor DNA repair synthesis by cell-free extracts and DNA-protein cross-linking to probe properties of DNA adducts of JM118. These results showed a higher DNA-protein cross-linking efficiency of JM118 and a less efficient removal from DNA of the adducts of JM118 in comparison with cisplatin. Thus, the results of the present work provide additional evidence that DNA binding of JM118 is in several aspects different from that of conventional cisplatin.     

DNA repair mechanisms involved in gemcitabine cytotoxicity and in the interaction between gemcitabine and cisplatin

The influence of DNA repair mechanisms on the interaction between gemcitabine and cisplatin was studied using a panel of Chinese hamster ovary (CHO) cell lines deficient in one of the following repair pathways: base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR) and non-homologous end joining (NHEJ). NER and HR are known to be involved in platinum-DNA adduct repair. Single agent experiments demonstrated that each of the repair deficient cell lines had a similar sensitivity towards gemcitabine as the parental cell lines, whereas the NER- and HR-deficient lines showed increased sensitivity towards cisplatin. Furthermore, in the parental cell lines, the administration sequence cisplatin followed by gemcitabine was synergistic, whereas the reversed schedule showed additivity and simultaneous administration revealed antagonistic cytotoxicity. In the repair deficient cell lines, using this synergistic schedule of cisplatin followed by gemcitabine, loss of synergy was observed in the NER- and HR-deficient cell lines. However, the magnitude of the effect in the NER-deficient cells was small. The sensitivity to the combination of cisplatin and gemcitabine shown by the BER- and NHEJ-deficient cell lines did not differ significantly from that of the parental cell line. Cellular accumulation of platinum as well as the formation of GG- and AG-intrastrand adducts in the parental line and in the HR-deficient line were not affected by gemcitabine.In conclusion, our results indicate that BER, NER, HR, and NHEJ are most likely incapable of modulating the cytotoxicity of gemcitabine, and that HR is involved in the synergistic interaction between cisplatin and gemcitabine in our cell system.     

DNA polymerase ζ is a major determinant of resistance to platinum-based chemotherapeutic agents

Oxaliplatin, satraplatin, and picoplatin are cisplatin analogs that interact with DNA forming intrastrand and interstrand DNA cross-links (ICLs). Replicative bypass of cisplatin DNA adducts requires the cooperative actions of at least three translesion DNA synthesis (TLS) polymerases: Polη, REV1, and Polζ. Because oxaliplatin, satraplatin, and picoplatin contain bulkier chemical groups attached to the platinum core compared with cisplatin, we hypothesized that these chemical additions may impede replicative bypass by TLS polymerases and reduce tolerance to platinum-containing adducts. We examined multiple responses of cancer cells to oxaliplatin, satraplatin, or picoplatin treatment under conditions where expression of a TLS polymerase was limited. Our studies revealed that, although Polη contributes to the tolerance of cisplatin adducts, it plays a lesser role in promoting replication through oxaliplatin, satraplatin, and picoplatin adducts. REV1 and Polζ were necessary for tolerance to all four platinum analogs and prevention of hyperactivation of the DNA damage response after treatment. In addition, REV1 and Polζ were important for the resolution of DNA double-stranded breaks created during replication-associated repair of platinum-containing ICLs. Consistent with ICLs being the predominant cytotoxic lesion, depletion of REV1 or Polζ rendered two different model cell systems extremely sensitive to all four drugs, whereas Polη depletion had little effect. Together, our data suggest that REV1 and Polζ are critical for promoting resistance to all four clinically relevant platinum-based drugs by promoting both translesion DNA synthesis and DNA repair.     

High accumulation of platinum-DNA adducts in strial marginal cells of the cochlea is an early event in cisplatin but not carboplatin ototoxicity

Ototoxicity is a typical dose-limiting side effect of cancer chemotherapy with cisplatin but much less so with carboplatin. To elucidate the underlying molecular pathological mechanisms, we have measured the formation and persistence of drug-induced DNA adducts in the nuclei of inner ear cells of guinea pigs after short-term exposure to either cisplatin or carboplatin using immunofluorescence staining and quantitative image analysis. After application of carboplatin, all cells of the cochlea exhibited a similar burden of guanine-guanine intrastrand cross-links in DNA. In contrast, we observed a pronounced 3- to 5-fold accumulation of this cytotoxic adduct exclusively in the marginal cells of the stria vascularis between 8 and 48 h after treatment with cisplatin. In the kidney, the other critical target tissue of cisplatin toxicity, a similar high preferential formation of cytotoxic DNA adducts was measured in the tubular epithelial cells but not in other renal cell types. As for the ear, this excessive formation of DNA damage in a particular cell type was seen in animals treated with cisplatin but not those treated with carboplatin. Because cisplatin ototoxicity is often attributed to oxidative stress mediated by the generation of radical oxygen species (ROS), we have measured in parallel the levels of the lead DNA oxidation product 8-oxoguanine (8-oxoG) in cochlear cryosections. Compared with basal levels in untreated control cochleas, no additional formation of 8-oxoG was detectable up to 48 h after cisplatin treatment in the DNA of either inner-ear cell type. This suggests that the generation of ROS may be a secondary event in cisplatin ototoxicity.     

Synthesis, cytotoxicity, cell uptake and DNA interstrand cross-linking of 4,4'-dipyrazolylmethane-linked multinuclear platinum anti-cancer complexes

Two cationic multinuclear platinum complexes linked with the 4,4'-dipyrazolylmethane (dpzm) ligand, trans-[[Pt(NH3)2Cl]2-mu-dpzm]Cl2 (di-Pt) and trans-[trans-[Pt(NH3)2Cl]2[trans-[Pt(NH3)2(mu-dpzm)2]]]Cl4 (tri-Pt), have been synthesized. Both complexes show activity in the murine leukaemia cell line L1210 (IC50 = 3.8 and 2.5 microm, respectively) and the cisplatin-resistant subline L1210/DDP (8.8 and 3.6 microM), and in the human ovarian carcinoma 2008 (2.5 and 17.8 microM) and its cisplatin-resistant subline C13*5 (20.9 and 37.7 microM). Both complexes show high levels of uptake into 2008 cells, when administered at 100 microM, but significantly reduced uptake in the cisplatin-resistant cell line C13*5 (di-Pt, 66% decrease; tri-Pt, 42%; cisplatin, 86%). Both complexes form very high levels of DNA interstrand cross-links in vitro, with 50% interstrand cross-linking observed at far lower concentrations (di-Pt, 12 nM; tri-Pt, 22 nM) than cisplatin (450 nM). It is proposed that the higher extent of interstrand cross-linking may be due to the rigid nature of the dpzm linking ligand, which prevents the complexes from forming short-range intrastrand adducts, like the GpG adduct formed by cisplatin. The results of this study indicate the importance of the flexibility of the linking ligand for the cytotoxicity of di- and trinuclear platinum anti-cancer complexes.     

Conversion of DNA adducts of antitumour cis-diamminedichloroplatinum(II). Immunochemical analysis.

Polyclonal antibodies that bind selectively to DNA modified by antitumour cisplatin and its analogues were isolated. The reactivity of the antibodies with the epitope was enhanced by thermal denaturation of DNA that had been modified by cisplatin before its denaturation. On the other hand, denaturation of DNA before its modification resulted in considerably less reaction of the antibodies. The conversion of monofunctional cisplatin-DNA adducts to bifunctional lesions increased the capability of the modified DNA to competitively inhibit the antibodies. The double-helical oligonucleotides containing a unique bifunctional adduct formed by cisplatin at the d(GG) site cross-reacted with the antibodies in contrast to the oligonucleotide containing a single monofunctional adduct formed at the d(G) site. In addition, poly(dG-dC) . poly(dG-dC) modified by cisplatin did not react with the antibodies. It was concluded that the antibodies recognized monodentate lesions, intrastrand cross-links between two purine nucleosides separated by one or more nucleosides and interstrand cross-links negligibly. The antibodies apparently recognized a chemical nature of the bifunctional adduct formed between two adjacent purines and not an unusual conformational feature of DNA resulting from the formation of this adduct. The antibodies were used to analyse the adducts formed by cisplatin on DNA of cultured cells exposed to this drug. During the subsequent incubation of the already exposed cells in the drug-free medium, a part of the bifunctional adducts of cisplatin was completely removed from DNA or transformed to the adducts not recognized by the antibodies.     

DNA interactions of new cytotoxic tetrafunctional dinuclear platinum complex trans,trans-[{PtCl2(NH3)}2(piperazine)

A new tetrafunctional dinuclear platinum complex trans,trans-[{PtCl2(NH3)}2(piperazine)] with sterically rigid linking group was designed, synthesized and characterized. In this novel molecule, the DNA-binding features of two classes of the platinum compounds with proven antitumor activity are combined, namely trans oriented bifunctional mononuclear platinum complexes with a heterocyclic ligand and polynuclear platinum complexes. DNA-binding mode of this new complex was analyzed by various methods of molecular biology and biophysics. The complex coordinates DNA in a unique way and interstrand and intrastrand cross-links are the predominant lesions formed in DNA in cell-free media and in absence of proteins. An intriguing aspect of trans,trans-[{PtCl2(NH3)}2(piperazine)] is that, using a semi-rigid linker, interstrand cross-linking is diminished relative to other dinuclear platinum complexes with flexible linking groups and lesions that span several base pairs, such as tri- and tetrafunctional adducts, become unlikely. In addition, in contrast to the inability of trans,trans-[{PtCl2(NH3)}2(piperazine)] to cross-link two DNA duplexes, the results of the present work convincingly demonstrate that this dinuclear platinum complex forms specific DNA lesions which can efficiently cross-link proteins to DNA. The results substantiate the view that trans,trans-[{PtCl2(NH3)}2(piperazine)] or its analogues could be used as a tool for studies of DNA properties and their interactions or as a potential antitumor agent. The latter view is also corroborated by the observation that trans,trans-[{PtCl2(NH3)}2(piperazine)] is a more effective cytotoxic agent than cisplatin against human tumor ovarian cell lines.     

Relation between intracellular accumulation and cytotoxic activity of cis-[((1R, 2R)-1, 2-cyclohexanediamine-N,N')bis(myristato)]platinum(II) suspended in Lipiodol

SM-11355, cis-[((1R, 2R)-1, 2-cyclohexanediamine-N, N')bis(myristato)]platinum(II), suspended in Lipiodol (SM-11355/Lipiodol) showed cytotoxic activity in hepatic tumor models in vivo and tumor cell lines in vitro. SM-11355/Lipiodol demonstrated selective retention in tumor tissue in vivo and high accumulation in tumor cells in vitro. This study was aimed to clarify the relation between the cytotoxicity of SM-11355/Lipiodol and intracellular platinum content. The cytotoxic activities were estimated by using WST-1 reagent. Intracellular platinum content and platinum-DNA adduct were estimated following exposure with SM-11355/Lipiodol when methionine was added. Methionine clearly inhibited the cytotoxic activities of SM-11355/Lipiodol. Moreover, intracellular platinum content and platinum-DNA adduct following exposure of SM-11355/Lipiodol decreased with increases in methionine concentration. The characteristic release of SM-11355/Lipiodol was not affected by addition of methionine. The present results suggested that one of platinum compounds exposed to cells following SM-11355/Lipiodol treatment is very similar in cytotoxic mechanism to cisplatin.     

Mutagenic spectrum of butadiene-derived N1-deoxyinosine adducts and N6,N6-deoxyadenosine intrastrand cross-links in mammalian cells

Reactive metabolites of 1,3-butadiene, including 1,2-epoxy-3-butene (BDO), 1,2:3,4-diepoxybutane (BDO(2)), and 3,4-epoxy-1,2-butanediol (BDE), form both stable and unstable base adducts in DNA and have been implicated in producing genotoxic effects in rodents and human cells. N1 deoxyadenosine adducts are unstable and can undergo either hydrolytic deamination to yield N1 deoxyinosine adducts or Dimroth rearrangement to yield N(6) adducts. The dominant point mutation observed at AT sites in both in vivo and in vitro mutagenesis studies using BD and its epoxides has been A --> T transversions followed by A --> G transitions. To understand which of the butadiene adducts are responsible for mutations at AT sites, the present study focuses on the N1 deoxyinosine adduct at C2 of BDO and N(6),N(6)-deoxyadenosine intrastrand cross-links derived from BDO(2). These lesions were incorporated site-specifically and stereospecifically into oligodeoxynucleotides which were engineered into mammalian shuttle vectors for replication bypass and mutational analyses in COS-7 cells. Replication of DNAs containing the R,R-BDO(2) intrastrand cross-link between N(6) positions of deoxyadenosine yielded a high frequency (59%) of single base substitutions at the 3' adducted base, while 19% mutagenesis was detected using the S,S-diastereomer. Comparable studies using the R- and S-diastereomers of the N1 deoxyinosine adduct gave rise to approximately 50 and 80% A --> G transitions with overall mutagenic frequencies of 59 and 90%, respectively. Collectively, these data establish a molecular basis for A --> G transitions that are observed following in vivo and in vitro exposures to BD and its epoxides, but fail to reveal the source of the A --> T transversions that are the dominant point mutation.     

A Cisplatin Derivative that Inhibits Collagen Fibril‐Formation in vitro

Using an in vitro random screening of small‐molecule compounds, we discovered cis‐diamminedichloroplatinum(II) (cisplatin), an anticancer agent, as a potential inhibitor of collagen fibril‐formation. The inhibitory effect was found only when cisplatin was dissolved in dimethylsulphoxide (DMSO), indicating that the active species were cisplatin derivatives formed in the DMSO solution. The cisplatin derivatives inhibited the formation of collagen fibrils in vitro without affecting the triple‐helical conformation of the collagen molecules. Incubation with the cisplatin solution in DMSO also inhibited in situ deposition of collagen fibrils in a human umbilical vein endothelial cell (HUVEC) culture. In addition, the derivatization of cisplatin in DMSO abolished the cytotoxicity of the original compound. The platinum complex was further revealed to interact with specific sites on the collagen triple helix, and the binding sites were suggested to contain His and/or Met residues. Mass spectrometry analysis of the cisplatin solution in DMSO and a structure–activity relationship study strongly suggested that the active compound is [Pt(NH₃)₂(Cl)(DMSO)]⁺. This platinum complex will be useful for investigating molecular mechanisms of collagen self‐assembly and for drug development for the treatment of fibrotic diseases.     

Expression of domains for protein–protein interaction of nucleotide excision repair proteins modifies cancer cell sensitivity to platinum derivatives and genomic stability

Nucleotide excision repair (NER) is involved in the repair of DNA damage caused by platinum derivatives and has been shown to decrease the cytotoxic activity of these drugs. Because protein–protein interactions are essential for NER activity, we transfected human cancer cell lines (A549 and HCT116) with plasmids coding the amino acid sequences corresponding to the interacting domains between excision repair cross‐complementation group 1 (ERCC1) and xeroderma pigmentosum, complementation group A (XPA), as well as ERCC1 and xeroderma pigmentosum, complementation group F (XPF), all NER proteins. Using the 3‐(4,5‐dimethyl‐2 thiazoyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay and annexin V staining, we showed that transfected A549 cells were sensitized 1.2–2.2‐fold to carboplatin and that transfected HCT116 cells were sensitized 1.4–5.4‐fold to oxaliplatin in vitro. In addition, transfected cells exhibited modified in vivo sensitivity to the same drugs. Finally, in particular cell models of the interaction between ERCC1 and XPF, DNA repair was decreased, as evidenced by increased phosphorylation of the histone 2AX after exposure to mitomycin C, and genomic instability was increased, as determined by comparative genomic hybridization studies. The results indicate that the interacting peptides act as dominant negatives and decrease NER activity through inhibition of protein–protein interactions.     

Decreased cisplatin damage-dependent DNA synthesis in cellular extracts of mismatch repair deficient cells

The proficiency of both nucleotide excision repair (NER) and DNA mismatch repair (MMR) influences cellular sensitivity to cisplatin (cis-diamminedichloroplatinum). To gain further insight into how MMR may influence platinum drug sensitivity, the effect of loss of MMR on repair synthesis was measured in vitro by a commonly used method that relies on whole-cell extracts to drive [alpha-32P]dATP incorporation into cisplatin-damaged plasmid DNA. Extracts evaluated include those from cells with or without functional hMLH1 (HCT116+ch2 versus HCT116+ch3, respectively) and hMSH2 (HEC59 versus HEC59+ch2, respectively). Loss of MMR in the HCT116 system was associated with a 2.8-fold reduction in cisplatin damage-specific DNA synthesis, whereas it was associated with a 3.0-fold reduction in the HEC59 system, suggesting that a decrease in the ability to repair cisplatin-damaged DNA accompanies loss of MMR. An in vitro DNA excision assay that utilized a substrate containing a site-specific cisplatin adduct was performed. Using this highly NER-specific assay, no significant difference was apparent between the extracts derived from NER-proficient versus -deficient cells. These and other data lead us to suggest that the increase in apparent repair synthesis in platinum-damaged plasmids by extracts from MMR-proficient versus -deficient cellular extracts may reflect a distinct and possibly adverse DNA synthetic process rather than productive NER.     

Drug discovery research on in vivo antitumor-active azolato-bridged dinuclear Pt(II) complexes

Cis-diamminedichloridoplatinum(II) (cisplatin), which was first introduced as a clinical anticancer agent in the 1970s, is still among the most-utilized agents in current cancer chemotherapy. The discovery of cisplatin antitumor activity has catalyzed drug discovery research on antitumor platinum coordination compounds with improved efficacy. Some of new compounds show fewer side effects or expanded clinical applications. Apart from some clinical inconveniences, such as side effects, the high therapeutic efficacy of platinum-based agents implies that further modifications may lead to more effective anticancer platinum drugs which are effective against cancers that are typically resistant to chemotherapy, such as pancreatic cancer, and platinum-refractory cancer. Most of the cisplatin analogs cause cross-resistance to cisplatin, probably because of the similar biological consequences. It is suggested that platinum complexes which interact with DNA; the most probable target molecule, through a mechanism different from that of cisplatin can provide unique anticancer spectra required for next-generation anticancer drugs. Therefore, we synthesized a series of azolato-bridged dinuclear Pt(II) complexes with a general formula, [{cis-Pt(NH(3))(2)}(2)(μ-OH)(μ-azolato)](2+), which can form 1,2-intrastrand crosslinks with a minimal DNA distortion, whereas clinical platinum-based drugs provide 1,2-intrastrand crosslink with severe DNA distortion. Indeed, they exhibit much higher in vitro cytotoxicity than cisplatin, and we have recently found one of the dinuclear Pt(II) complexes exhibits markedly high in vivo antitumor efficacy against pancreatic cancer. Here, I update our drug-discovery research on the series of azolato-bridged dinuclear Pt(II) complexes that may be more effective and safer than current anticancer chemotherapeutic agents.     

Replacement of an NH(3) by an iminoether in transplatin makes an antitumor drug from an inactive compound

To investigate the modifications of antitumor activity and DNA binding mode of transplatin after replacement of one nonleaving group NH(3) by an iminoether group, trans-[PtCl(2)(Z-HN=C(OMe)Me)(NH(3)] and trans-[PtCl(2)(E-HN=C(OMe)Me)(NH(3)] complexes (differing in the Z or E configuration of iminoether, and abbreviated mixed Z and mixed E, respectively), have been synthesized. In a panel of human tumor cell lines, both mixed Z and mixed E show a cytotoxic potency higher than that of transplatin, the mean IC(50) values being 103, 37, and 215 microM, respectively. In vivo mixed Z is more active and less toxic than mixed E in murine P388 leukemia and retains its efficacy against SK-OV-3 human cancer cell xenograft in nude mice. In the reaction with naked DNA, mixed Z forms monofunctional adducts that do not evolve into intrastrand cross-links but close slowly into interstrand cross-links between complementary guanine and cytosine residues. The monofunctional mixed Z adducts are removed by thiourea and glutathione. The interstrand cross-links behave as hinge joints, increasing the flexibility of DNA double helix. The mixed Z, transplatin, and cisplatin interstrand cross-links, as well as mixed Z monofunctional adducts are not specifically recognized by HMG1 protein, which was confirmed to be able to specifically recognize cisplatin d(GpG) intrastrand cross-links. These data demonstrate that the DNA interaction properties of the antitumor-active mixed Z are very similar to those of transplatin, thus suggesting that clinical inactivity of transplatin could not depend upon its peculiar DNA binding mode.     

Increased nucleotide excision repair in cisplatin-resistant ovarian cancer cells: role of ERCC1-XPF

Increased platinum-DNA adduct removal has been shown by several DNA repair assays to be associated with cisplatin resistance in the A2780/C-series human ovarian cancer model system. In the present study, we provide further evidence that the resistance phenotype of these cell lines is due, in part, to enhanced nucleotide excision repair (NER). Cisplatin resistance was found to be associated with increased UV resistance. Northern blot analysis revealed that increased expression of ERCC1 was also associated with cisplatin resistance in this panel. Several other NER genes were found to be constitutively overexpressed in the most resistant cell line, C200, as compared with the parental A2780 cells. A plasmid substrate containing a site-specific cisplatin adduct was used to measure the nucleotide excision activity of cell extracts prepared from cisplatin-sensitive and -resistant cells. Using this in vitro assay, extracts prepared from C200 cells exhibited approximately 3-fold more activity than extracts prepared from A2780 cells, similar to the difference in UV sensitivity. Complementation of A2780 extracts with ERCC1-XPF protein resulted in approximately 2-fold increased activity, but had little effect on excision in C200 extracts. Overall, these results support a role for the ERCC1-XPF endonuclease as a determinant of increased NER in this cisplatin resistance model.     

Unique DNA binding mode of antitumor trinuclear tridentate platinum(II) compound

The new trinuclear tridentate Pt(II) complex [Pt(3)Cl(3)(hptab)](3+) (1; hptab = N,N,N',N',N',N'-hexakis(2-pyridylmethyl)-1,3,5-tris(aminomethyl)benzene) exhibits promising cytotoxic effects in human and mouse tumor cells including those resistant to conventional cisplatin (Dalton Trans. 2006, 2617; Chem. Eur. J. 2009, 15, 5245). The present study is focused on the molecular pharmacology of 1, in particular on its interactions with DNA (which is the major pharmacological target of platinum antitumor drugs), to elucidate more deeply the mechanism underlying its antitumor effects. Results obtained with the aid of methods of molecular biophysics and pharmacology reveal new details of DNA modifications by 1. Complex 1 binds to DNA forming in the absence of proteins and molecular crowding agents mainly trifunctional intrastrand cross-links. In these DNA adducts all three Pt(II) centers of 1 are coordinated to DNA base residues, which leads to extensive conformational alterations in DNA. An intriguing aspect of the DNA-binding mode of this trinuclear Pt(II) complex 1 is that it can cross-link proteins to DNA. Even more interestingly, 1 can cross-link in the presence of molecular crowding agent, which mimics environmental conditions in cell nucleus, two DNA duplexes in a high yield--a feature observed for the first time for antitumor trinuclear platinum complexes. Thus, the concept for the design of agents capable of forming intramolecular tridentate DNA adducts, DNA-protein and interduplex DNA-DNA cross-links based on trinuclear tridentate Pt(II) complexes with semirigid aromatic linkers may result in new compounds which exhibit a variety of biological effects and can be also useful in nucleic acids research.     

Gemcitabine potentiates cisplatin cytotoxicity and inhibits repair of cisplatin-DNA damage in ovarian cancer cell lines

Synergistic cytotoxicity between cisplatin and the nucleoside analog gemcitabine was observed in a panel of cisplatin-sensitive (2008, A2780) and -resistant (2008/C13*5.25, A2780/CP70) human ovarian cell lines. Previous studies have suggested a role for DNA repair in the mechanism of synergy between the two drugs. We therefore further investigated the hypothesis that the synergistic cytotoxicity between gemcitabine and cisplatin in these cell lines may be caused by gemcitabine-mediated inhibition of cisplatin intrastrand adduct (IA) and interstand cross-link (ICL) repair. The effect of gemcitabine on the accumulation and repair of cisplatin IA and ICL in each cell line was then measured directly using gene-specific quantitative polymerase chain reaction and denaturation/renaturation techniques, respectively. Pretreatment of 2008 cells with 1 microM gemcitabine for 2 h before exposure to cisplatin for 7 h enhanced the accumulation of cisplatin IA and ICL by 50 and 40%, respectively (P < 0.05), above that induced by cisplatin alone. To investigate the possibility that the increased accumulation of cisplatin lesions was caused by inhibition of removal of cisplatin damage, 2008 cells were incubated with 200 microM cisplatin for 5 h in the presence and absence of gemcitabine and then a further 8 h in the absence of cisplatin. Only 57% IA were removed in the combination treated cells compared with 74% in cisplatin control cells. Similarly, repair of cisplatin ICL was inhibited in the gemcitabine-treated cells compared with the cells treated with cisplatin only (60 versus 72%). These findings demonstrate a direct inhibitory effect of gemcitabine on the repair of cisplatin IA and ICL and suggest a mechanistic basis for the cytotoxic synergy between the two drugs.     

切除修复交叉互补基因1与消化系统肿瘤铂类耐药的关系研究进展

消化系统肿瘤由于其相对较高的发病率及死亡率,危害日益突出,故对消化道肿瘤治疗的研究具有相当的意义。铂类药物广泛用于恶性肿瘤的化疗,是消化系统肿瘤化疗的传统药物之一,但耐药性的产生严重影响其疗效,目前认为核苷酸切除修复（nucleotide excision repair,NER）是铂类药物耐药的重要机制之一,因其导致耐药的主要机理为清除大规模铂类化合物所致DNA螺旋扭曲。其中核苷酸切除修复交叉互补基因1（excision repair cross comple／mentation group 1,ERCC1）在NER过程中发挥着重要作用,ERCC1在消化系统肿瘤外周血及组织中的表达差异与含铂方案化疗的疗效存在着密切的联系。本文就ERCC1的研究进展与消化系统肿瘤顺铂化疗疗效之间的关系进行综述。