Cisplatin (cis-diamminediachloroplatium) is a widely used anti-cancer drug.
Since its approval in 1970’s for the treatment of genitourinary tumors, cisplatin has become one of the most widely used and successful drugs for the treatment of a variety of cancers, including ovarian, head, neck, bladder, cervical, and small cell lung cancers (Loehrer, 1984).
1.3.7.1 Anti-cancer effects of cisplatin and mechanisms of action of p53
. Once cisplatin enters a cell, its chloride ligand will be replaced by water molecules and form positively charged species that can react with nucleophilic sites on intracellular macromolecules such as protein, RNA and DNA (Dijt, 1988). It is generally accepted that the anti-cancer effect of cisplatin is mainly mediated by its interaction with DNA to form DNA adducts, which induce DNA damage and activate several signaling transduction pathways including ATR, ATM, p53, and MAPK (Siddik, 2003).
The interaction between cisplatin and DNA not only inhibits DNA replication and cell division, but also leads to apoptosis (Gonzalez et al., 2001). Tumor suppressor p53 activation is one of major factors responsible for apoptosis induced by cisplatin. It has been observed that cisplatin treatment can cause apoptosis in wide type p53 cancer cell but not in p53 deficient or mutant cancer cells (Song et al., 1998;
Kanata et al., 2000; Tang and Grimm, 2004; Beuvink et al., 2005), suggesting that p53 is the key regulator for cisplatin-mediated apoptosis in cancer cells.
p53 is a tumor suppressor protein (Ko, 1996; Levine, 1997), which is readily activated by DNA damage as well as other stimuli. Activation of p53 contributes to the tumor suppression either by inducing cell cycle arrest, possibly providing opportunity for the cells to repair damaged DNA, or by inducing apoptosis in the injured cells. So, p53 is playing a critical role in avoiding genetic instability and acts as tumor suppressor protein. It is believed that the loss of p53 activity promotes malignant transformation, leading to the high incidence of p53 mutations in a wide spectrumof human cancer (Hollstein, 1991; Levine et al., 1991).
At present, the molecular mechanisms controlling p53 activation have been
to control the function of p53: p53 transcriptional activation and p53 stability (Kubbutat et al., 1997; Ashcroft and Vousden, 1999). The stability of p53 protein, a short-lived protein, is mainly regulated by its interaction with its transcriptional target mouse double minute 2 (MDM2), which act as an ubiquitin E3 ligase and promotes p53 ubiquitination and proteasomal degradation (Kubbutat, 1997). Meanwhile, MDM2 is a transcriptional target of p53 and expression of MDM2 will promote p53 degradation to maintain the negative feedback loop. Upon DNA damage or other stimuli, p53 is up-regulated by transcriptional activation as well as via a number of mechanisms that disrupt the interaction between MDM2 and p53 and thus to increase p53 stability. Modifications on either p53 or MDM2 may affect their interaction. For instance, phosphorylation of p53 affects its interaction with MDM2 or its binding to DNA or its transcriptional activity (Steegenga et al., 1996). It is known that DNA damage-activated ATM, ATR and DNA-PK can phorphorylate p53 on Ser 15 and Ser 37 (Shieh, 1997), Chk2 is among the kinases that contributeto phosphorylation of p53 on serine 20 (Shieh, 1999), whereas JNK phosphorylates p53 on tyrosine 81 (Buschmann et al., 2001). Any of above phosphorylations on p53 may affect its interaction with MDM2 and finally affect its stability. On the other hand, post- translational modifications on MDM2 or inhibition of MDM2 activity or decrease of MDM2 protein can disrupt the interaction between p53 and MDM2 and promote the rapid accumulation of p53 (Ryan et al., 2001). MDM2 protein is also controlled by ubiquitination and proteasomal degradation (Chang, 1998).
One important role of p53 as a tumor suppressor is its involvement in apoptosis. p53 activates the caspase cascade and apoptosis mainly via an intrinsic pathway that involves mitochondria, a central regulator of apoptosis. The integrity of outer mitochondrial membrane is tightly regulated by Bcl-2 family proteins. Pro-
apoptotic members of the Bcl-2 family, such as Bax, Bak and Bid, form channels in membranes and to regulate preexisting channels. Anti-apoptotic members of the family, such as Bcl-2 and Bcl-XL, tend to have the opposing effects on membrane channel formation. (Kelekar and Thompson, 1998). The pro-apoptotic functions of p53 can be mediated through a transcription-dependent pathway, which involves the activation of pro-apoptotic genes. It was shown that p53 can regulate the transcription of a group of pro-apoptotic proteins such as Bax (Miyashita and Reed, 1995), Noxa (Oda et al., 2000a), PUMA (Nakano and Vousden, 2001; Chipuk et al., 2005), DR5 (Wu, 1997), BID (Sax et al., 2002), and CD95 (Muller et al., 1998), which elicit caspase cascade and apoptosis.
In addition to transactivation of target genes, evidence has also implicated that p53 can induce apoptosis via a transcription–independent way. For example, the apoptosis induced by p53 may be through increasing surface Fas by transporting from the Golgi complex (Bennett et al., 1998), or require FADD-independent activation of caspase-8 (Ding et al., 2000). Further, it was found that p53 protein can translocate to mitochondria, form complexes with Bcl-XL and Bcl-2 proteins and directly induce cytochrome c release (Mihara et al., 2003). Recently, it has been demonstrated that the apoptosis induced by p53 is mediated by Bax mitochondrial translocation and activation (Chipuk et al., 2003; Erster et al., 2004). In non-stimulated cells, Bax exists as a monomer either in the cytosol or loosely attached to the outer mitochondrial membrane. Upon stimulation, the cytosolic Bax translocates to mitochondria and inserts into the membrane. Bax oligomerizes into large complexes which are believed to be crucial to mitochondrial membrane permeabilization (Goping et al., 1998). The activated Bax on mitochondria can be distinguished by a conformational change in the
N-terminus that exposes the formerly buried 6A7 epitope (Desagher et al., 1999;
Nechushtan et al., 1999)
1.3.7.2 Regulation of cisplatin-induced apoptosis
Despite the success against testicular cancer, the use of cisplatin against other cancers is limited due to acquired or intrinsic resistance. For example, cisplatin has minimal activity against some common cancer types, such as colorectal cancer (Natoli et al., 2000). Thus, resistance is the major constraint that undermines the curative potential of cisplatin.
Efforts have been made to define the cellular and molecular mechanisms responsible for cisplatin resistance (Kartalou and Essigmann, 2001). The resistance may be through either limiting the extent of cisplatin-induced damage, for example, alterations in cellular pharmacology, including decreased drug accumulation, increased cellular thiol levels and increased repair of platinum–DNA damage. In addition, alterations in the cellular response to the damage also contribute to the resistance. Since p53 is a major mediator of the apoptosis induced by cisplatin, mutation of p53 or alteration of expression level of Bcl-2, Bcl-XL, Bax or MDM2 may affect the responses to cisplatin. For example, MDM2 overexpression confers the cancer cells to be resistant to cisplatin (Kondo et al., 1995).
Therefore, cisplatin is usually not used alone for cancer therapy. Clinically, it is used in combination with other anti-cancer drugs, such as etoposide (Kovnar et al., 1990; Ardizzoni et al., 1999), bleomycin (Behnia et al., 2000) and irinotecan (Sandler, 2002). Recently, new strategies to enhance the cytotoxicity of cisplatin has been investigated (Duan et al., 2001; Iwase et al., 2003; Kim et al., 2003a; Fulda and Debatin, 2005; Mohanty et al., 2005). For instance, downregulation of MDM2 using
MDM2 antisense oligonucleotides or RNA interference can enhance the sensitivity to cisplatin (Yu et al., 2006).