Cisplatin and beyond: molecular mechanisms of action and drug resistance development in cancer chemotherapy

The behaviour of cisplatin in aqueous solution is presented in Figure 1.

Figure 1

The chemistry of cisplatin is completely different from all other chemistries of typical organic anticancer drugs. In blood, the concentration of the chloride ion is about 105 mM and, according to Le Chatelier’s principle, the loss of the chloride ion from cisplatin is suppressed by the chloride ion in solution; the reaction shown in Figure 1 does not progress very far to the right (from 1 to 2). According to the first order kinetics for conversion from 1 to 2, the half-life for cisplatin at 35.5 ºC is 1.05 h. The binding of a water molecule to the Pt²⁺ ion makes water very acidic and monoaqua species 3 is dissociated in the monohydroxo complex 4. So in an aqueous solution with the high chloride concentration the forms 1, 2 and 4 predominate. In the cytoplasm, where chloride ion concentration is only 4 mM, the equilibria is shifted to the right and forms 3, 5 and 6 predominate. A common route of cisplatin administration is the infusion of the solutions such as Platinol^® and Plationol^®AQ, which contain 3.3 mM cisplatin (1 mg/ml) and 154 mM sodium chloride, NaCl (normal saline solution). Since the pH is adjusted to pH about 4 solution in Platinol contains mainly (95%) of species 1 and only smaller amounts of 3 and 4.

Since the concentration of chloride in blood is lower than in the infusion solution (105 versus 154 mM), the equilibria (Figure 1) is shifted to the right. Because the carbonate and phosphate ions react with aquated cisplatin forms and because some platinum binds to protein that is being eliminated in significant amounts via the urine during the infusion, it is impossible to predict the composition of species 1 – 6 according to the data in Figure 1. This system is far from the chemical equilibrium. A major part (68 – 98%) of cisplatin is bound to the blood plasma proteins, especially on human serum albumin (HSA)⁶. One molecule of HSA binds five molecules of cisplatin. cis-[PtCl(NH₃)₂]⁺ is bound to nitrogen atom from His105 and His288 and sulphur atoms from Met329, Met298 and Met548. Treatment of HSA with cisplatin also leads to the formation of adduct between cis-[Pt(NH₃)₂]²⁺ and His67 and His247. Since both histidine residues are involved in the transport of Zn²⁺ ions to the cell by HSA⁷, this may be the reason why some patients on therapy with cisplatin have zinc imbalance in their body.⁸ For study of binding of platinum compounds to human serum proteins a novel method for accessing of Pt using conjoint liquid chromatography has also been developed.⁹

The first step for platinum drugs to exert their therapeutic as well as toxic effects is to enter the cells. This process is complex and not completely understood. Cisplatin enters a cell mainly by passive diffusion. Uptake of cisplatin by cells was proportional to the total concentration of cisplatin in the cell culture medium up to 3 mM concentration and was not saturable.¹⁰ The most likely candidates for passive diffusion through hydrophobic lipid bilayer are neutral species without the charge such as cisplatin and monohydroxo form (1 and 4, respectively in Figure 1). But studies with the inhibitors of active transporters suggested that there must also be other uptake mechanisms.¹¹ Recently, a lot of attention has been given to the active modes of the uptake.¹² The most likely candidates for the transport of cisplatin into a cell by facilitated diffusion are the copper transporters Ctr1 (SLC31A1) and Ctr2 (SLC31A2, copper importer).¹³ This transmembrane protein has an extracellular domain that is rich in methionine and histidine residues. It has been suggested that both metals, platinum and copper, use Ctr1 as their entry route into the cell although the chemistry of these two metals is quite different. Switching between different oxidation states has pivotal role in the transport of copper. Whereas copper exists in two biological oxidation states as Cu⁺ and Cu²⁺ ions, the cisplatin exists only in one stable biologically accessible oxidation state with Pt²⁺ ion. Because platinum cannot switch between different oxidation states, the uptake of cisplatin into the cell by this protein might be blocked. An additional factor that throws doubt on Ctr (1 and 2) as a main transporter of cisplatin is the reaction of extracellular methionine residue with Pt²⁺ resulting in formation of a stable Pt²⁺-S bond, similar to the bond between Pt²⁺ and methionine residues in HSA. Since the transport of Pt²⁺ ion through Ctr would require a facile breaking and reforming of the Pt²⁺-S bond, the thermodynamic and kinetic parameters of this bond would exclude the Ctr as an entry route for cisplatin. Furthermore, cis-ammonia molecules would be lost during cisplatin transfer with Ctr due to trans effect. Additionally, excessive copper in extracellular fluid triggers an internalization and degradation of Ctr protein. This acute regulatory response has also been reported following exposure of human ovarian carcinoma cells to cisplatin, but such response was not observed in other types of cancer cells.¹⁴ Although many studies have shown that cisplatin can bind to Ctr, none has shown that Pt²⁺ ion can actually be transported into a cell by this mechanism.¹⁵ Arnesano and Natile¹⁶ hypothesized that the interaction of cisplatin with Ctr leads to pinocytosis and vesicular transport of cisplatin into the cell, thus protecting the drug from inactivation with glutathione (GSH) and metallothioneins (MT) which are present in cells.

As forms 2, 3 and 5 shown in Figure 1 are cations, led investigators to examine the involvement of organic cation transporters (OCTs, SLC22A) in cisplatin transport across the membrane. This assumption was strengthened by the observation that OCTs are expressed in tissues sensitive to cisplatin toxicity. It was shown that both OCT1 (SLC22A1) and OCT2 (SLC22A2) are implicated in transport of cisplatin into the HEK 293 cells as well as the other cell lines.¹⁷ Under physiological conditions, cisplatin may also undergo a transformation in cisplatin carbonato complexes, which are anionic species and these forms may be transported into the cell by organic anion transporters (OATs, SLC22A). The transport of platinum drugs by OAT may also explain the nephrotoxicity of these substances.¹⁸ Both OCT and OAT mediated cisplatin nephrotoxicities are observed in 30% of patients. OCT2 is specifically expressed in the kidneys and for this reason it is a suitable target for the investigating of the protection against nephrotoxicity. The tissue specific expression of OCT may be also critical for the development of ototoxicity as well as peripheral neurotoxicity.¹⁹

Since the therapeutic range of cisplatin is narrow we cannot overcome the cell resistance simply by increasing the dose. Resistance to cisplatin is a consequence of the enhanced removal of the drug that enters a cell and the efficiency of DNA repair mechanisms, which remove lethal adducts between DNA and cisplatin. Another copper transporter, ATP7A and closely related ATP7B, which deliver copper into the organelles and are responsible for removing the excess copper out of a cell, may also be involved in the cisplatin-induced resistance.²⁰ Mutation in ATP7B gene produces accumulation of copper in the body, a state known as Wilson’s disease²¹, while the mutation in ATP7A gene has the opposite effect and is responsible for a copper deficiency, known as the Menkes disease.²² Both ATP7A and ATP7B can bind cisplatin at cysteine residue in their N-terminal metal binding domains forming a stable Pt²⁺-S bond. Elevated levels of these proteins correlate with a diminished accumulation of cisplatin and consequently with lower cytotoxicity.²³

Beside the copper-transporting proteins GSH and metallothioneins may also influence cisplatin transport. These cysteine rich, low molecular weight proteins are involved in intracellular inactivation of platinum and other similar drugs through coordination to thiol groups. An overexpression of metallothionein in the tumour cells is present in 70% of the patients with oesophageal cancer and it is correlated with resistance to cisplatin.²⁴ It was shown that GSH and cisplatin form anionic bis Pt²⁺-GSH complexes which can be refluxed from leukaemia cells in the presence of ATP.²⁵ Later it was shown that multidrug resistance-associated protein MRP2 (ABCC2) and OAT were responsible for the efflux of this complex.²⁶ Since the formation of the adduct between cisplatin and two deprotonated forms of GSH prevents the drug from reacting with DNA and other targets, MRP2 and OAT are important factors in detoxification of the cell from cisplatin. Due to combination of uptake and export transporters only 1% of administered drug reaches the site of action within the cells. Cellular traffic of cisplatin is summarized in Figure 2.

Figure 2

The inter-individual variability of the efficacy of platinum based chemotherapy as well as its toxicity

is the result of the genetic variability in genes involved in drug metabolism, drug transport and DNA repair. Therefore, the determination of genetic markers such as genetic polymorphisms in these genes may provide the hints about the optimal cisplatin regimens for personalized therapy.

Both uptake and efflux transporters are subject to genetic variability. Polymorphic transporters are involved in processes that increase the intracellular level of the drug: transport with copper transporters Ctr1 and 2 and organic cation transporters OCT 1 and 2. The same is true for the transporters that are involved in the efflux of cisplatin from the cell and resistance, copper-transporting ATPases ATP7A and ATP7B, organic anion transporters, OAT, glutathione and metallothionein, multidrug resistance-associated protein MRP2 as well as multidrug extrusion transporter-1 (MATE1).²⁷

The presence of single nucleotide polymorphisms in all transporters shown in Figure 2 influence the response of patients to platinum drugs in a great extent and this influence is dependent on both, the type of platinum drug and the type of cancer cells.²⁸ The data on these polymorphisms are summarized in the Table 1.

The main mechanism of resistance is a DNA-platinum drug adducts repairing system. Polymorphisms of DNA-adduct repair enzymes also play a role in sensitivity towards platinum-based chemotherapy (Table 2). X-ray repair cross-complementing group 1 (XRCC1), excision repair cross complementation 1 (ERCC1) and xeroderma pigmentosum complementary group (XPA, XPD and XPG) are enzymes that play the crucial role

in these processes. The polymorphic A1196G allele in XRCC1 gene is present in 20-38% of lung cancer patients.²⁹ A number of studies have been performed to investigate the association of XRCC1 gene polymorphisms with platinum drug efficacy but the results are inconsistent.

Recent studies also investigated Major Vault Protein (MVP) present as the main component of the vault in normal tissues as well as in malignant cells, including ovarian cancer, colon carcinoma and acute myeloid leukaemia.³⁰ Although MVP has been linked to the development of multidrug resistance in cancer cells, several studies have reported conflicting results.³¹ The association between polymorphisms in MVP gene and platinum resistance has not yet been confirmed probably due to the limited number of patients.³²

Although allergic reactions to cisplatin are rare, with later cisplatin derivatives, carboplatin and oxaliplatin the allergic reactions are more common. Still they are less frequent than with anticancer drugs which names end with “mab” and other drugs that contain proteins. Hypersensitivity reactions to platinum generally occur after multiple cycles of therapy – they are acquired and are consistent with type 1 IgE-mediated hypersensitivity. In patients presenting with severe hypersensitivity reactions to carboplatin it is feasible to replace it with cisplatin. Overall incidence of hypersensitivity to platinum agents is 5 – 20% for cisplatin and occurs mostly between 4^th-8^th course of infusion. The incidence is 1 – 44% for carboplatin and 10 – 20% for oxaliplatin.³³ The most striking difference between carboplatin hypersensitivity, compared to hypersensitivity to nonplatinum drugs, is that the cumulative risk of hypersensitivity reactions increases with the number of infusions and there is no evidence of plateau.³⁴ Hypersensitivity reactions occur more frequently in patients receiving certain drug combinations such as carboplatin – paclitaxel as compared to carboplatin in combination with pegylated lyposomal doxorubicin.³⁵

Since hyperthermia enhances cytotoxic effects of cisplatin, the trimodal combination of platinum drugs with hyperthermia and radiation can lead to potent synergistic interaction.³⁶ There is a synergistic effect of regional hyperthermia (39-43ºC) and cisplatin anti-tumour efficacy, if cisplatin is encapsulated in temperature-sensitive liposomes used for targeted drug delivery. It is hypothesized that hyperthermia increases cisplatin accumulation in part by increasing Ctr1 multimerization and thus greater cisplatin accumulation. Increased Ctr1 multimerization following hyperthermia treatment (41°C) in vitro, compared to normothermic controls (37°C), was observed suggesting that there may be a mechanism for an increased cisplatin uptake in heat-treated cells. Hyperthermia enhanced cisplatin-mediated cytotoxicity in wild type (WT) cells with a dose modifying factor (DMF) of 1.8 compared to 1.4 in Ctr1-/- cells because WT cells contained greater levels of platinum compared to Ctr1-/- cells.³⁷

Since the atomic number of platinum is high, 78, it is possible to produce Auger electrons and/or Auger radiation upon treating the platinum drug with ionizing radiation. Treatment of cervical cancer with a conjunction of cisplatin and ionizing radiation increased survival and disease free intervals and became a part of standard care for the treatment of cervical cancers.³⁸ The »Trojan horse« treatment of glioblastoma involves gold (atomic number 79) nanoparticles and attached molecules of cisplatin. Treated cultured glioblastoma cells in preclinical studies absorb nanoparticles and DNA binds platinum attached to nanoparticles. After radiation, both gold and platinum serve as high atomic number radio sensitizers that emit Auger electrons and radiation. The resulting assembly of gold nanoparticles with attached cisplatin and antibodies after radiation exhibit both chemotherapeutic power to cancer cells as well as Auger-mediated secondary electron emission, which cause DNA double strand breaks adjacent to the cisplatin bound to DNA.³⁹ Binding cisplatin to gold nanoparticles is also a strategy to enhance the delivery of cisplatin through the blood brain barrier. A combination with a magnetic resonance-guided ultrasound intensifies glioblastoma treatment. It is demonstrated that the assembly of gold nanoparticles and cisplatin greatly inhibits the growth of glioblastoma cells compared to the free cisplatin and synergy with radiation therapy.⁴⁰ An important reactive oxygen species (ROS) scavenger DJ-1 protein (PARK7) modulates different oncogenic pathways that support the growth and invasion of ovarian cancer cells. This cancer targeted nanoplatform based on siRNA-mediated suppression of DJ-1 protein outperforms cisplatin alone. Three cycles of siRNA-mediated DJ-1 therapy combined with a low dose of cisplatin completely eradicated ovarian cancer tumours from the mice without recurrence during a 35-week long study.⁴¹

The next step in the research includes the attachment of the transport system for delivering nanoparticles with cisplatin to the target.⁴² Mice treated with hyaluronic-acid conjugated mesoporous silica nanoparticles carried TWIST-siRNA and cisplatin exhibited specific tumour targeting and reduction of tumour burden.⁴³

Another important aspect of novel approaches to cisplatin chemotherapy is to reduce cisplatin toxicity. The two other approved platinum-based chemotherapeutics, carboplatin and oxaliplatin exhibit improved nephrotoxicity⁴⁴ and ototoxicity⁴⁵ profiles, but are also less efficient than cisplatin. This challenge could be addressed by harnessing a nanotechnology-based strategy. An example of the cisplatin toxicity prevention on the reproductive system is the use of selenium nanoparticles (Nano-Se). Nano-Se particles, due to their strong antioxidant potential are suitable to prevent cisplatin induced gonadotoxicity. Co-administration with cisplatin significantly improves the sperm quality, serum testosterone and spermatogenesis in male rats.⁴⁶ Lipoplatin is another example of bias cisplatin toxicity. These nanoparticles of 110 nm average diameter are composed of lipids and cisplatin. After intravenous administration it escapes clearance from macrophages and the half-life of lipoplatin is 120 h.⁴⁷ Attachment of platinum drugs to nanoparticles passively targets solid tumours through the enhanced permeability. Lipoplatin exerted negligible nephrotoxicity, ototoxicity and neurotoxicity in Phase I human studies.⁴⁸

For poorly permeable platinum drugs such as cisplatin and similar low lipophilic analogues, a higher doses are needed to exert therapeutic effect and consequently toxicity is more pronounced. To mitigate this effect an enhanced influx into the cancer cells can be achieved with electroporation in the process of electrochemotherapy.^49,50,51 This method increases the cytotoxicity up to 80-fold in cisplatin-sensitive as well as cisplatin-resistant tumour types.⁵² Other approach include the synthesis of novel platinum compounds with more lipophilic leaving groups with potential antitumor effect. One such candidate is trans-[PtCl₂(3-hydroxymethylpyridine)₂] applied with electroporation as drug delivery method⁵³.