Meeting report on 8th International Symposium on Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy

The platinum-based drugs, cisplatin and carboplatin, represent major agents in the chemotherapeutic treatment of a variety of types of cancer. Novel, "third-generation" agents aimed at broadening the clinical activity of this class of drug are currently undergoing clinical evaluation. These include oxaliplatin, ZD0473 and BBR3464. Clinical trials and preclinical studies are also being conducted with liposomal (SPI-077 and L-NDDP) and polymeric platinum complexes (linked to HPMA or albumin). Combination studies of cisplatin/carboplatin with other anticancer drugs such as gemcitabine and UCN-01 (7-hydroxystaurosporine) and agents designed to reduce platinum drug toxicities (e.g., BNP-7787, DIMESNA) are ongoing. Preclinically, there is interest in trans platinum complexes, terpyridine platinum(II) complexes and other metal-containing agents (ruthenium and gold).     

Cellular pharmacology of cis and trans pairs of platinum complexes in cisplatin-sensitive and -resistant human ovarian carcinoma cells

The cellular pharmacology of two pairs of cis and trans platinum complexes has been studied in three human ovarian carcinoma cell lines, a parental relatively cisplatin-sensitive line (CH1), a subline possessing acquired cisplatin resistance (3-fold; CH1cisR) and an intrinsically cisplatin resistant line (13-fold; SKOV-3). Growth inhibition studies showed that both JM335 [trans ammine (cyclohexylaminedichloro dihydroxo) platinum(IV)] and its platinum(II) dichloro homolog JM334 were relatively less cross-resistant against both acquired and intrinsic cisplatin resistant cells. In contrast, resistance circumvention was not apparent in these cell lines with their cis isomeric counterparts (JM149 for JM335 and JM118 for JM334). The trans compound JM335 was more potent than its cis isomer against all three cell lines. There was no clear correlation between intracellular accumulation following 2 h exposure to each compound and resulting DNA platination or growth inhibition. The selective activity of the trans platinum complexes against the SKOV-3 cell line correlated with a deficiency in the repair of adducts within a fragment of the N-ras gene induced by trans compounds whereas adducts induced by the cis counterparts, and cisplatin, were repaired. The CH 1 parental line appeared repair deficient at the gene-specific level to adducts induced by both cis (including cisplatin) and trans compounds. Resistance in CH1cisR was associated with a lack of gene-specific repair of lesions formed by JM118 and JM149. All four compounds induced apoptosis in all three cell lines, as measured by fluorescent microscopy and field inverted gel electrophoresis, although the kinetics of apoptosis was markedly faster for the trans versus cis compounds. In summary, the trans platinum complexes JM335 and JM334 possess unique cellular properties compared to their cis counterparts particularly with respect to gene specific repair of DNA adducts and the rate of induction of apoptosis.     

Determination of the platinum drug cis-amminedichloro(2-methylpyridine)platinum(II) in human urine by liquid chromatography-tandem mass spectrometry

A validated stable isotope dilution liquid chromatography-tandem mass spectrometry assay for the novel platinum drug cis-amminedichloro(2-methylpyridine)platinum(II) (ZD0473) in human urine has been developed. This method uses selected reaction monitoring on the transition of m/z 393 [M+NH(4)](+) to m/z 304 [M+NH(4)-NH(3)-2 x H(35)Cl](+) for ZD0473, and m/z 400 [M+NH(4)](+) to m/z 310 [M+NH(4)-NH(3)-H(35)Cl-(2)H(35)Cl](+) for the internal standard [(2)H(7)]ZD0473. Standard curves were prepared over the range from 0.15 to 50 microg/ml. The lower limit of quantitation was 0.2 microg/ml for 100 microl of urine. This simple, rapid, reliable, and sensitive method of quantitation displayed acceptable accuracy and precision over the 3 days of assay validation. A novel platinum adduct was formed during the storage of ZD0473 in human urine. The adduct did not correspond to any of the typical sulfhydryl adducts that have been identified previously for platinum drugs. Formation of the adduct was prevented by the addition of 50% (w/v) sodium chloride to the urine. The assay can be used to quantify intact ZD0473 in the urine of subjects dosed with this new platinum drug.     

Specificity of platinum-DNA adduct repair

Cell lines with resistance to cisplatin and carboplatin often retain sensitivity to platinum complexes with different carrier ligands (e.g., oxaliplatin and JM216). HeLa cell extracts were shown to excise cisplatin, oxaliplatin, and JM216 adducts with equal efficiency, suggesting that nucleotide excision repair does not contribute to the carrier-ligand specificity of platinum resistance. We have shown previously that the extent of replicative bypass in vivo is influenced by the carrier ligand of the platinum adducts. The specificity of replicative bypass may be determined by the DNA polymerase complexes that catalyze translesion synthesis past Pt-DNA adducts, by the mismatch-repair system that removes newly synthesized DNA opposite Pt-DNA adducts, and/or by DNA damage-recognition proteins that bind to the Pt-DNA adducts and block translesion synthesis. Primer extension on DNA templates containing site-specifically placed cisplatin, oxaliplatin, or JM216 Pt-GG adducts revealed that the eukaryotic DNA polymerases beta, zeta, gamma and HIV-1 RT had a similar specificity for translesion synthesis past Pt-DNA adducts (oxaliplatin > or = cisplatin > JM216). In addition, defects in the mismatch-repair proteins hMSH6 and hMLH1 led to increased replicative bypass of cisplatin adducts, but not of oxaliplatin adducts. Finally, primer extension assays performed in the presence of HMG1, which is known to recognize cisplatin-damaged DNA, revealed that inhibition of translesion synthesis by HMG1 also depended on the carrier ligand of the Pt-DNA adduct (cisplatin > oxaliplatin = JM216). These studies show that DNA polymerases, the mismatch-repair system and damage-recognition proteins can all impart specificity to replicative bypass of Pt-DNA adducts. Replicative bypass, in turn, may influence the carrier-ligand specificity of resistance.     

From bench to bedside--preclinical and early clinical development of the anticancer agent indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A)

Indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A) is just the second ruthenium-based anticancer agent after NAMI-A which was developed to the stage of clinical trials. Important steps in the mode of action of KP1019 are thought to be the binding to the serum protein transferrin and the transport into the cell via the transferrin pathway. Additionally, the selective activation by reduction in the tumor might contribute to the low side effects observed in in vivo studies. Apoptosis is induced at non-toxic levels via the mitochondrial pathway. These features distinguish it from the established platinum anticancer drugs and suggest that different types of cancer might be treatable with this drug. Indeed, promising activity against certain types of tumors, which are not successfully treatable with cisplatin, and only a very low incidence of acquired resistance has been observed in in vitro and in vivo studies. Recently, a clinical phase I trial was finished in which none of the treated patients experienced serious side effects, while disease stabilization in five of six evaluable patients was achieved. In this review, the preclinical and early clinical development of KP1019 - from bench to bedside - is recapitulated.     

Metal Complexes in Cancer Treatment: Journey So Far

Metal complexes in cancer therapy have attracted much interest mainly because metals exhibit unique characteristics, such as redox activity, metal-ligand interaction, structure and bonding, Lewis acid properties etc. In 1965, Barnett Rosenberg serendipitously discovered the metal-based compound cisplatin, an outstanding breakthrough in the history of metal-based anticancer complexes and led to a new area of anticancer drug discovery. Many metal-based compounds have been studied for their potential anticancer properties. Some of these compounds have FDA approval for clinical use, while others are now undergoing clinical trials for cancer therapy and detection. In the present study, we have highlighted the primary mode of action of metallic complexes and all FDA-approved/under clinical trial drugs with reference to cancer treatment. This review also focuses on recent progress on metal-based complexes such as platinum, ruthenium, iron, etc. with potential anticancer activities.     

Contributions of the Epidermal Growth Factor Receptor to Acquisition of Platinum Resistance in Ovarian Cancer Cells

Acquisition of platinum resistance following first line platinum/taxane therapy is commonly observed in ovarian cancer patients and prevents clinical effectiveness. There are few options to prevent platinum resistance; however, demethylating agents have been shown to resensitize patients to platinum therapy thereby demonstrating that DNA methylation is a critical contributor to the development of platinum resistance. We previously reported the Epidermal Growth Factor Receptor (EGFR) is a novel regulator of DNA methyltransferase (DNMT) activity and DNA methylation. Others have shown that EGFR activation is linked to cisplatin treatment and platinum resistance. We hypothesized that cisplatin induced activation of the EGFR mediates changes in DNA methylation associated with the development of platinum resistance. To investigate this, we evaluated EGFR signaling and DNMT activity after acute cisplatin exposure. We also developed an in vitro model of platinum resistance to examine the effects of EGFR inhibition on acquisition of cisplatin resistance. Acute cisplatin treatment activates the EGFR and downstream signaling pathways, and induces an EGFR mediated increase in DNMT activity. Cisplatin resistant cells also showed increased DNMT activity and global methylation. EGFR inhibition during repeated cisplatin treatments generated cells that were more sensitive to cisplatin and did not develop increases in DNA methylation or DNMT activity compared to controls. These findings suggest that activation of EGFR during platinum treatment contributes to the development of platinum resistance. Furthermore, EGFR inhibition may be an effective strategy at attenuating the development of platinum resistance thereby enhancing the effectiveness of chemotherapeutic treatment in ovarian cancer.     

Cucurbit[7]uril encapsulated cisplatin overcomes cisplatin resistance via a pharmacokinetic effect

The cucurbit[n]uril (CB[n]) family of macrocycles has been shown to have potential in drug delivery where they are able to provide physical and chemical stability to drugs, improve drug solubility, control drug release and mask the taste of drugs. Cisplatin is a small molecule platinum-based anticancer drug that has severe dose-limiting side-effects. Cisplatin forms a host-guest complex with cucurbit[7]uril (cisplatin@CB[7]) with the platinum atom and both chlorido ligands located inside the macrocycle, with binding stabilised by four hydrogen bonds (2.15-2.44 ?). Whilst CB[7] has no effect on the in vitro cytotoxicity of cisplatin in the human ovarian carcinoma cell line A2780 and its cisplatin-resistant sub-lines A2780/cp70 and MCP1, there is a significant effect on in vivo cytotoxicity using human tumour xenografts. Cisplatin@CB[7] is just as effective on A2780 tumours compared with free cisplatin, and in the cisplatin-resistant A2780/cp70 tumours cisplatin@CB[7] markedly slows tumour growth. The ability of cisplatin@CB[7] to overcome resistance in vivo appears to be a pharmacokinetic effect. Whilst the peak plasma level and tissue distribution are the same for cisplatin@CB[7] and free cisplatin, the total concentration of circulating cisplatin@CB[7] over a period of 24 hours is significantly higher than for free cisplatin when administered at the equivalent dose. The results provide the first example of overcoming drug resistance via a purely pharmacokinetic effect rather than drug design or better tumour targeting, and demonstrate that in vitro assays are no longer as important in screening advanced systems of drug delivery.     

Conformation, recognition by high mobility group domain proteins, and nucleotide excision repair of DNA intrastrand cross-links of novel antitumor trinuclear platinum complex BBR3464

The new antitumor trinuclear platinum compound [(trans-PtCl(NH(3))(2))(2)mu-trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2)](4+) (designated as BBR3464) is currently in phase II clinical trials. DNA is generally considered the major pharmacological target of platinum drugs. As such it is of considerable interest to understand the patterns of DNA damage. The bifunctional DNA binding of BBR3464 is characterized by the rapid formation of long range intra- and interstrand cross-links. We examined how the structures of the various types of the intrastrand cross-links of BBR3464 affect conformational properties of DNA, and how these adducts are recognized by high mobility group 1 protein and removed from DNA during in vitro nucleotide excision repair reactions. The results have revealed that intrastrand cross-links of BBR3464 create a local conformational distortion, but none of these cross-links results in a stable curvature. In addition, we have observed no recognition of these cross-links by high mobility group 1 proteins, but we have observed effective removal of these adducts from DNA by nucleotide excision repair. These results suggest that the processing of the intrastrand cross-links of BBR3464 in tumor cells sensitive to this drug may not be relevant to its antitumor effects. Hence, polynuclear platinum compounds apparently represent a novel class of platinum anticancer drugs acting by a different mechanism than cisplatin and its analogues.     

MutS preferentially recognizes cisplatin- over oxaliplatin-modified DNA

Loss of mismatch repair leads to tumor resistance by desensitizing cells to specific DNA-damaging agents, including the anticancer drug cisplatin. Cisplatin analogs with a diamminocyclohexane (DACH) carrier ligand, such as oxaliplatin and Pt(DACH)Cl(2), do not elicit resistance in mismatch repair-deficient cells and therefore present promising therapeutic agents. This study compared the interactions of the purified Escherichia coli mismatch repair protein MutS with DNA modified to contain cisplatin and DACH adducts. MutS recognized the cisplatin-modified DNA with 2-fold higher affinity in comparison to the DACH-modified DNA. ADP stimulated the binding of MutS to cisplatin-modified DNA, whereas it had no effect on the MutS interaction with DNA modified by DACH or EN adducts. In parallel cytotoxicity experiments, methylation-deficient E. coli dam mutants were 2-fold more sensitive to cisplatin than DACH compounds. A panel of recombination-deficient mutants showed striking sensitivity to both compounds, indicating that both types of adducts are strong replication blocks. The differential affinity of MutS for DNA modified with the different platinum analogs could provide the molecular basis for the distinctive cellular responses to cisplatin and oxaliplatin.     

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.     

The cellular basis of the efficacy of the trinuclear platinum complex BBR 3464 against cisplatin-resistant cells

Multinuclear platinum compounds have been designed to circumvent the cellular resistance to conventional mononuclear platinum-based drugs. In this study we performed a comparative study of cisplatin and of the triplatinum complex BBR 3464 in a human osteosarcoma cell system (U2-OS) including an in vitro selected cisplatin-resistant subline (U2-OS/Pt). BBR 3464 was extremely potent in comparison with cisplatin in U2-OS cells and completely overcame resistance of U2-OS/Pt cells. In both cell lines, BBR 3464 accumulation and DNA-bound platinum were higher than those observed for cisplatin. On the contrary, a low frequency of interstrand cross-links after exposure to BBR 3464 was found. Differently from the increase of DNA lesions induced by cisplatin, kinetics studies indicated a low persistence of interstrand cross-link formation for BBR 3464. Western blot analysis of DNA mismatch repair proteins revealed a marked decrease of expression of PMS2 in U2-OS/Pt cells, which also exhibited microsatellite instability. Studies on DNA mismatch repair deficient and proficient colon carcinoma cells were consistent with a lack of influence of the DNA mismatch repair status on BBR 3464 cytotoxicity. In conclusion, the cytotoxic potency and the ability of the triplatinum complex to overcome cisplatin resistance appear to be related to a different mechanism of DNA interaction (formation of different types of drug-induced DNA lesions) as compared to conventional mononuclear complexes.     

Effect of the antitumor drug cis-diamminedichloroplatinum(II) and related platinum complexes on eukaryotic DNA replication

An SV40-based in vitro replication system has been used to examine the effects of platinum compounds on eukaryotic DNA replication. Plasmid templates containing the SV40 origin of replication were modified with the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) or the inactive analogues [Pt(dien)Cl]+ and trans-DDP. The platinated plasmids were used as templates for DNA synthesis by the DNA polymerases present in cytosolic extracts prepared from human cell lines HeLa and 293. Bifunctional adducts formed by cis- and trans-DDP inhibited DNA replication by 95% at a bound drug to nucleotide ratio [(D/N)b] of less than 9 x 10(-4), in contrast to the monofunctional [Pt(dien)Cl]+ analogues, which required a (D/N)b of 3.4 x 10(-3) for 62% inhibition of DNA replication. An average of two platinum adducts per genome was sufficient for inhibition of DNA replication by cisplatin. When trans-DDP-modified, but not cis-DDP-modified, SV40 origin containing plasmids [(D/N)b = 1.7 x 10(-3)] were allowed to incubate in the 293 cytosolic extracts for 1 h prior to addition of T-antigen to initiate replication, DNA synthesis was restored to 30% of control. This result suggested the presence of an activity in the extracts that reactivates trans-DDP-modified DNA templates for replication. This hypothesis was confirmed by an in vitro nucleotide excision repair assay that revealed activity in 293 and HeLa cell extracts selective for trans-DDP-modified plasmid DNAs. Such selective repair of trans-DDP-damaged DNA in human cells would contribute to its lack of antitumor activity.     

Structure-functional organization of eukaryotic high-affinity copper importer CTR1 determines its ability to transport copper, silver and cisplatin

It was shown recently, that high affinity Cu(I) importer eukaryotic protein CTR1 can also transport in vitro abiogenic Ag(I) ions and anticancer drug cisplatin. At present there is no rational explanation how CTR1 can transfer platinum group, which is different by coordination properties from highly similar Cu(I) and Ag(I). To understand this phenomenon we analyzed 25 sequences of chordate CTR1 proteins, and found out conserved patterns of organization of N-terminal extracellular part of CTR1 which correspond to initial metal binding. Extracellular copper-binding motifs were qualified by their coordination properties. It was shown that relative position of Met- and His-rich copper-binding motifs in CTR1 predisposes the extracellular CTR1 part to binding of copper, silver and cisplatin. Relation between tissue-specific expression of CTR1 gene, steady-state copper concentration, and silver and platinum accumulation in organs of mice in vivo was analyzed. Significant positive but incomplete correlation exists between these variables. Basing on structural and functional peculiarities of N-terminal part of CTR1 a hypothesis of coupled transport of copper and cisplatin has been suggested, which avoids the disagreement between CTR1-mediated cisplatin transport in vitro, and irreversible binding of platinum to Met-rich peptides.     

Cisplatin resistance and mechanism in a viral test system: SV40 isolates that resist inhibition by the antitumor drug have lost regulatory DNA

Isolates of SV40 that have enhanced ability to survive inhibition by the antitumor drug cisplatin were selected by serial drug challenge in vivo. These mutant viruses have acquired specific deletions within the repeated regulatory motif (GGGCGG)6 or GC box. This DNA element was shown previously to be a strong target of drug attack by cisplatin and other anticancer drugs in vitro and is an important viral and cellular DNA control sequence. Thus, drug resistance in this viral test system is dependent on the loss of important target DNA sequences. The results also indicate that drug efficacy may be related to the ability of certain anticancer drugs to attack regulatory DNA sequences containing strings of guanosines.     

Improving platinum(II)-based anticancer drug delivery using cucurbit[n]urils

Despite the synthesis of hundreds of new platinum(II) and platinum(IV)-based complexes each year as potential anticancer drugs, only three have received world-wide approval: cisplatin, carboplatin and oxaliplatin. The next big advance in platinum-based chemotherapy is not likely to come from the development of new drugs, but from the controlled and targeted delivery of already approved drugs or those in late stage clinical trials. Encapsulation of platinum drugs inside macromolecules has already demonstrated promise, and encapsulation within cucurbit[n]urils has shown particular potential. Partial or full encapsulation within cucurbit[n]urils provides steric hindrance to drug degradation by peptides and proteins, and the use of different sized cucurbit[n]urils allows for the tuning of drug release rates, cytotoxicity and toxicity.     

Protein interactions with platinum-DNA adducts: from structure to function

Because of the efficacy of cisplatin and carboplatin in a wide variety of chemotherapeutic regimens, hundreds of platinum(II) and platinum(IV) complexes have been synthesized and evaluated as anticancer agents over the past 30 years. Of the many third generation platinum compounds evaluated to date, only oxaliplatin has been approved for clinical usage in the United States. Thus, it is important to understand the mechanistic basis for the differences in efficacy, mutagenicity and tumor range between cisplatin and oxaliplatin. Cisplatin and oxaliplain form the same types of adducts at the same sites on DNA. The most abundant adduct for both compounds is the Pt-GG intrastrand diadduct. Cisplatin-GG adducts are preferentially recognized by mismatch repair proteins and some damage-recognition proteins, and this differential recognition of cisplatin- and oxaliplatin-GG adducts is thought to contribute to the differences in cytotoxicity and tumor range of cisplatin and oxaliplatin. A detailed kinetic analysis of the insertion and extension steps of dNTP incorporation in the vicinity of the adduct shows that both pol beta and pol eta catalyze translesion synthesis past oxaliplatin-GG adducts with greater efficiency than past cisplatin-GG adducts. In the case of pol eta, the efficiency and fidelity of translesion synthesis in vitro is very similar to that previously observed with cyclobutane TT dimers, suggesting that pol eta is likely to be involved in error-free bypass of Pt adducts in vivo. This has been confirmed for cisplatin by comparing the cisplatin-induced mutation frequency in human fibroblast cell lines with and without pol eta. Thus, the greater efficiency of bypass of oxaliplatin-GG adducts by pol eta is likely to explain the lower mutagenicity of oxaliplatin compared to cisplatin. The ability of these cellular proteins to discriminate between cisplatin and oxaliplatin adducts suggest that there exist significant conformational differences between the adducts, yet the crystal structures of the cisplatin- and oxaliplatin-GG adducts were very similar. We have recently solved the solution structure of the oxaliplatin-GG adduct and have shown that it is significantly different from the previously published solution structures of the cisplatin-GG adducts. Furthermore, the observed differences in conformation provide a logical explanation for the differential recognition of cisplatin and oxaliplatin adducts by mismatch repair and damage-recognition proteins. Molecular modeling studies are currently underway to analyze the mechanistic basis for the differential bypass of cisplatin and oxaliplatin adducts by DNA polymerases.