Estructura representacional: Teoría del Núcleo Central

One of the major forms of cancer treatment is chemotherapy. In general, cancers are either resistant to chemotherapy or obtain resistance during treatment, thereby leading to ineffective chemotherapy [118]. Several resistance mechanisms to topoisomerase I- targeted anti-cancer agents have been characterisedin vitro, however, their significance in the clinical setting has not been fully identified until now. These mechanisms involve pre- target events such as drug accumulation, metabolism, intracellular drug distribution, or drug-target interactions [67]. Post-target events, including DNA synthesis or repair, cell cycle progression as well as cell death regulation, have also been demonstrated to play a significant role in the sensitivity to these drugs [67].

Numerous mechanisms have been described in cancer cells able to reduce accumulation of cytotoxic drugs by up-regulating cell membrane located adenosine triphosphate (ATP)- dependent efflux pumps [119]. Examples of drugs to which resistance has been observed or for which resistance is acquired during treatment include topoisomerase inhibitors (e.g., TPT), anthracyclines (e.g., doxorubicin), the Vinca alkaloids (e.g., vincristine) and the taxanes (e.g., paclitaxel) [118]. The model in this thesis assumes that a resistance to TPT is a result of the over-expression of efflux transport proteins in the plasma membrane of cancer cells. Examples of such mechanisms include the two transmembrane xenobiotic transporter proteins, P-glycoprotein (P-gp) which has been reported in hamster ovarian cells [120, 121], and the multidrug resistance protein 1 (MRP1) [122]. More recently, other human ATP-binding cassette (ABC) transporters that are involved in the resistance of anti- cancer drugs have been discovered. The most important of these, a novel protein, is the breast cancer resistance protein (BCRP) [123] also known as the placenta-specific ABC protein (ABCP) [124] or mitoxantrone-resistance protein (MXR) [125].

Following immunoblotting analysis with BCRP-specific antibodies, it has been suggested that BCRP is a 72 kDa membrane protein [126, 127]. It has been shown from various

studies on drug efflux mechanisms that BCRP is mostly localised at the plasma membrane of the drug-resistant cells over-expressing the transporter instead of at internal vesicular membranes [123, 128, 129]. Therefore, BCRP is presumably involved in active transport from the cell rather than in transport into internal vesicles. Following experimental studies by Maliepaard et al. [130] and Ishii et al. [131] it has been observed that TPT accumulation in resistant cell lines was reduced by enhanced drug efflux. The actual efflux pumping mechanism is explained in detail in Chapter 4. Transfection studies conducted in various laboratories have verified that enforced expression of BCRP complementary DNA (cDNA) in different types of cells caused resistance to multiple anti-cancer drugs and reduced drug accumulation in the cell [128, 129, 132]. Such experiments provide strong evidence that BCRP is a main cause of drug resistance in tissue culture models for certain types of chemotherapeutic drugs including TPT. This protein is significantly expressed in organs central for absorption (the small intestine), distribution (the placenta and blood- brain barrier), and elimination (the liver and small intestine) [118]. Further investigations have come to the conclusion that BCRP plays a significant role in drug disposition.

The ABC transporter is the largest protein superfamily identified to date [133]. ABC transporters are widely spread in all organisms (including mammals and bacteria) and are responsible for transporting a wide range of compounds across the plasma membranes against concentration gradient (active pumping mechanism) with ATP hydrolysis as the source of energy for the process of substrate translocation. ABC transporters are involved in multiple physiological processes. Examples of this include, transporting drug (xenobiotics) or drug conjugates in addition to the excretion of endogenous metabolites or physiological substrates [134]. A number of human genetic diseases have been found to be associated with defects in ABC transporter genes such as CFTR in cystic fibrosis [135], MRP2 in Dubin-Johnson syndrome [136] and ABCR in Stargardt disease [137]. Typically, the structure (Figure 3.6) of the majority of mammalian ABC transporters contains two types of structural domains (homologous halves) each containing two parts as follows: the hydrophilic intracellular nucleotide binding domain (NBD) and the hydrophobic

membrane spanning domain (MSD) which is putatively arranged into six α-helices [118,

138]. P-gp is arranged in two tandem repeated halves of two domains: one hydrophobic MSD followed by one hydrophilic NBD [120, 134]. The two repeated halves are connected

by a polypeptide linker sequence. The great majority of ABC transporters have such a 4- domain structural organisation.

Figure 3.6: A membrane topology model of BCRP. BCRP contains one NBD followed by

one MSD with six predicted transmembrane α-helices. Two or three putative N-

glycosylation sites (N418, N557, or N596) are predicted to be in the extracellular loops as indicated (taken from Reference [118]).

BCRP belongs to the sub-family G of the large ABC transporter superfamily. BCRP, also known as ABCG2, is the second member of the sub-family G of the human ABC transporter. This transporter has been described in drug-resistant ovary, breast, colon, gastric cancer in addition to fibrosarcoma cell lines [123-125, 130, 139]. Moreover, BCRP has only one ATP-binding cassette and six putative transmembrane domains, suggesting that BCRP is a half-transporter, which may work as a homodimer or heterodimer [140, 141], unlike P-gp and MRP1 which are arranged in two repeated halves. Another unique feature is the configuration of the (BCRP/ABCG2) protein in which the NBD is followed by the MSD as illustrated in Figure 3.6, whereas P-gp and MRP1 have an opposite domain arrangement, in that the MSD precedes the NBD. Therefore, this unique domain organisation implies that the transport mechanism of ABCG proteins (including BCRP/ABCG2) may be different from those of other ABC transporters.

CPT derivatives are the second most important class of anti-cancer agents that are transported by the (BCRP/ABCG2) transporter [118]. A wide range of BCRP-over- expressing cell lines reveal resistance to CPT derivatives including TPT [131, 142]. The

study carried out by Rabindranet al.[143] demonstrates that MCF-7 cells transfected with BCRP cDNA display significant resistance to TPT in comparison to the vector control cells, this is additional evidence that CPT derivatives (including TPT) are BCRP substrates. In a study published by Rajendra et al. [144], it has been reported that the important factors for substrate recognition (i.e. effective efflux pumping) are the hydrophilic groups in the chemical structure of CPT. It has been shown by Yoshikawa et al. [145] that BCRP prefers to transport CPT derivatives with high polarity over the low polarity CPT analogues [118]. Accordingly, polarity is also essential for recognition of the CPT analogues by BCRP. All such information is essential in designing clinically useful CPT analogues that are not transported by BCRP/ABCG2. Based on the advice of the project collaborators at Cardiff University and according to these observations, it is assumed that in the drug kinetics model (see Chapter 4) the BCRP/ABCG2 transporter carries TPTH (high polarity) from the cytoplasm through the cell membrane to the

extracellular region irreversibly.