Broadening the chemotherapeutic arsenal depends on understanding existing agents with a view toward developing new modes of attack. In the 35 years since the discovery of the anti-tumor activity of cisplatin, laboratory studies have provided considerable information as to how the drug exerts its antitumor effects and how some tumors are, or become, resistant to this drug. Although a number of features of the mechanism underlying the anti-tumor effects of cisplatin have been thoroughly described, some still remain to be disclosed. There is a large body of experimental evidence that the success of platinum complexes in killing tumor cells mainly results from their ability to form various types of adducts with DNA.10 Hence, intensive research has focused on DNA modifications by platinum anti-tumor drugs and how these modifications modulate the 'downstream effects' such as specific protein recognition by DNA-processing enzymes and other cellular components (for a review, see for instance Ref. 11).
Cisplatin reacts with DNA in the cell nucleus, where the concentration of chloride is markedly lower than in extracellular fluids. The drug loses its chloride ligands in media containing low concentrations of chloride to form positively charged mono- and di-aqua species (Figure 25.2). It has been shown that only these aquated forms bind to DNA. Bifunctional cisplatin binds to DNA in a two-step process first forming mono-functional adducts preferentially at the guanine residues, which subsequently close to major intrastrand cross-links between adjacent purine residues (1,2-GG or 1,2-AG intrastrand cross-links). Other minor adducts are 1,3-GXG intrastrand cross-links (X = A, C, T), interstrand cross-links and mono-functional lesions. In all adducts, cisplatin is coordinated to the N7 atom of purine residues. The percentage of the 1,2-intrastrand adducts formed by cisplatin is larger than statistically expected so that this cross-link has generally been assumed to be the important adduct correlated with anti-cancer activity and was therefore most extensively investigated.
The adducts formed by cisplatin in DNA affect the DNA's secondary structure.11 The formation of major intrastrand cross-links of cisplatin leads to marked conformational alterations in DNA. These adducts induce a roll between the platinated purine residues, displacement of the platinum atom from the planes of the purine rings, a directional and rigid bend of the helix axis toward the major groove and a local unwinding. In addition, severe
perturbation of hydrogen bonding within the 50-coordinated GC base pair, widening and flattening of the minor groove opposite the cisplatin adduct, creation of a hydrophobic notch, global distortion extending over 4 5 base pairs and additional helical parameters characteristic of the A-form of DNA have also been reported. Other minor adducts of cisplatin also induce several irregularities in DNA, the details of which have been reviewed elsewhere.11
Cisplatin inhibits DNA and RNA synthesis by prokaryotic and eukaryotic DNA and RNA polymerases, respectively both in vitro and in vivo.12,13 Inhibition of DNA or RNA synthesis occurs mainly at GG sites, consistent with the fact that these sites are preferential binding sites of cisplatin.
The limitations of using cisplatin in cancer chemotherapy are also associated with intrinsic and acquired resistance of tumor cells to this drug.14,15 Resistance to cisplatin is multi-factorial and, in general, may consist of mechanisms either limiting the formation of DNA adducts and/or operating downstream of the interaction of cisplatin with DNA to promote cell survival. The formation of DNA adducts by cisplatin can be limited by reduced accumulation of the drug, enhanced drug efflux and cisplatin inactivation by coordination to sulfur-containing biomolecules including metallothioneins whose production may be increased as a consequence of cisplatin treatment. The second group of mechanisms includes enhanced repair of DNA adducts of cisplatin and increased tolerance of the resulting DNA damage.
In human cells, cisplatin intrastrand adducts are removed from DNA mainly by the nucleotide excision repair (NER) system.11 It has been found using cellfree extracts or a reconstituted NER system that intrastrand cross-links of cisplatin are efficiently repaired. Importantly, this repair of 1,2-, but not 1,3-intrastrand cross-links is blocked upon addition of an HMG-domain protein (HMG = high mobility group) (discussed later). There is also evidence that other cellular repair mechanisms, such as recombination or mismatch repair, can affect anti-tumor efficiency of cisplatin.11 Recent observations16 support the view that mismatch repair mediates the cytotoxicity of cisplatin in tumor cells and that dysfunction of this type of DNA repair may result in the resistance of tumor cells to cisplatin or in drug tolerance.
It is generally believed that anti-tumor activity of cisplatin is mediated by the recognition of its DNA adducts by cellular proteins.11,17 Several classes of these proteins have been identified and mechanisms have been proposed to explain how they mediate anti-tumor effects of cisplatin (discussed later). The greatest attention has been paid to the studies of recognition of platinated DNA by HMGB1 and HMGB2 proteins which belong to architectural chromatin proteins and play some kind of structural role in the formation of functional higher order protein-DNA or protein-protein complexes or as signaling molecules in genetically regulated repair pathways. These structure-specific proteins bind selectively to the 1,2-GG or 1,2-AG adducts of cisplatin, but not to its 1,3-intrastrand cross-links. Extensive reviews addressing these questions have been recently published.11,17
It is generally believed that the key intracellular pharmacological target for platinum compounds is DNA.10 The adducts formed by cisplatin distort the DNA conformation. DNA adducts of cisplatin inhibit replication and transcription, but they are also bypassed by DNA or RNA polymerases. In addition, cisplatin adducts are removed from DNA mainly by NER. They are, however, also recognized by a number of proteins which could block DNA adducts of cisplatin from damage recognition needed for repair (Figure 25.2). In this way these adducts can persist for sufficient time, which potentiates the anti-tumor effect of the drug. The other hypothesis based on the observation that a number of various proteins recognize cisplatin-modified DNA is that cisplatin-DNA adducts hijack proteins away from their normal binding sites, thereby disrupting fundamental cellular processes (Figure 25.2). Experimental support for these hypothetical aspects of the mechanism underlying anti-tumor activity of cisplatin or resistance of some tumors to this drug has been thoroughly reviewed recently.11,17 Initially, inhibition of DNA replication by cisplatin adducts was considered to be a process very likely relevant to its anti-tumor efficiency.18 However, other studies have indicated that cisplatin inhibits tumor-cell growth at doses which are considerably lower than those needed to inhibit DNA synthesis.19 Subsequent observations have revealed that cisplatin can trigger G2 cell-cycle arrest and programmed cell death (apoptosis),20 exposing another mechanism of cytotoxicity of this drug. However, since apoptosis is a very complex process, a number of possible pathways still have to be explored for a complete understanding of the mechanism by which cisplatin triggers apoptosis.21
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