Mechanisms drug resistance to cancer chemotherapy

Mechanisms medicine defense to malignant neoplastic disease chemotherapyIntroductionCancer is oneness of the major causes of death in the real world and statistics show that one in three people will be diagnosed with crab louse during their lifetime 1. Cancers ar crabby personous tumors and bath be distinguished from normal booths by four typicals uncontrolled proliferation, dedifferentiation and loss of serve, invasiveness, and ability to metastasise 2. These characteristics are ca utilise by altered cistron facial gesture, as a result of genetic mutations that inactivate tumour suppressor genes and / or activate oncogenes.Most crabby person chemotherapeutic medicines equal only one characteristic aspect, which is uncontrolled proliferation 3. In many cases the antiproloferation body process is cause by damage to desoxyribonucleic acid, which initiates apoptosis and cell death 4. As their main target is cell division, they affect all(prenominal) rapidly dividin g cells, including normal cells. This produces general toxic kernels, much(prenominal) as myelosuppression, alopecia, damage to gastrointestinal epithelium, sterility and severe nausea and vomiting.Besides the toxic cores of chemotherapy, other major problem is chemo apology 5. Resistance to chemotherapy is when the malignant neoplastic disease cells do not respond to the drugs. It discharge be inherented, as a genetic mutation, or it can be acquired, as a cellular response to drug exposure. Mechanisms of resistance include change magnitude efflux or lessen influx of cytotoxic drugs insufficient activating of the drug increased inactivation of the drug increased concentration of target enzyme rapid repair of deoxyribonucleic acid lesions or mutations in various genes. When patients develop resistance, ten-fold drugs with different pathways of entry and different cellular targets are used. However, crabmeat cells can become multidrug resistant, a phenomenon due to cells e xpressing apparatuss that cause simultaneous resistance to many different, structurally and functionally, unrelated drugs 6.Multidrug resistance, generally, results from over expression of ATP-dependent efflux pumps 5. These pumps fuddle spacious drug specificity and belong to a family of ATP- adhere cassette (ABC) transporters, of which P-glycoprotein (PGP) is one of the most important shares. Increased drug efflux, via these transporters, lowers intracellular drug concentration, allowing cancer cells to escape the toxic effects of the drugs. PGP inhibitors are being developed to overcome multidrug resistance and two that ready reached clinical trials are varapamil, a calcium transmission channel blocker, and cyclosporin A, an immunosuppressant 7.The remainder of this review will focus on the different chemotherapeutic agents currently being used for the treatment of cancer and their mechanism of action. as well the main mechanism of resistance to these drugs will be explore d, particularly focusing on the role of P-glycoprotein and how it can be modulated to stop drug resistance. doses used in cancer chemotherapyDrugs used in the treatment of cancer are summarised in tcapable 2. They are grouped into cytotoxic drugs, which preferentially but not exclusively target rapidly dividing cancer cells hormone therapy, which is a more specific form of treatment used for tumours derived from hormone bare-assed tissues and miscellaneous agents, which include a heel of recently developed drugs such as monoclonal antibodies.Cytotoxic drugsCytotoxic drugs can be further split up into the following alkylating agents, which act by forming covalent bonds with deoxyribonucleic acid and impeding replication antimetabolites, which block one or more of the metabolic pathways dissembled in deoxyribonucleic acid synthesis cytotoxic antibiotics, which are of microbial origin and prevent cell division by directly acting on desoxyribonucleic acid and engraft derivert ives, which affect microtubule function and hence the formation of the mitotic spindle.Alkylating agentsAlkylating agents form carbonium ions, which are highly reactive and interact instantaneously with nucleophilic sites such as N7 of guanine in desoxyribonucleic acid 8. They are bifunctional, which means they have two alkylating groups, and can cause intra- or inter-chain cross-linking between deoxyribonucleic acid strands. This prevents strand separation for DNA synthesis or transcription. They can overly cause udder mispairing between strands, which interferes with the progression of the replication fork 3. These actions block DNA synthesis, causing a block at G2 chassis and subsequently apoptotic cell death.Alkylating agents currently being used in chemotherapy primarily belong to the following families nitrogen mustards (Cyclophosphamide, Chlorambucil, Melphalan, Ifosfamide, Busulfan) nitrosoureas (Carmustine, Lomustine, Fotemustine) aziridines (Thiotepa) Dacarbazine and p latinum compounds (Cisplatin, Carboplatin, Oxaliplatin) 9. Nitrogen mustards, nitrosoureas and aziridines are believed to kill tumour cells by energisement DNA inter-strand cross-links, while platinum compounds induce intra- and inter-strand cross-links, as well as DNA-protein cross-links under certain circumstances 8. Resistance to these drugs can develop as a result of cancer cells rapidly repairing drug induced lesions 10, which will be discussed in detail later.AntimetabolitesAntimetabolites interfere with the metabolic pathways involved in DNA synthesis. An showcase of an antimetabolite is Methotrexate, which is a folate antagonist 11. Folates are essential for the synthesis of purine nucleotides and thymidylate, which in turn are essential for DNA synthesis and cell division. Folates are actively taken up into cells by the minify folate holder (RFC), where they are converted to polyglutamates. Polyglutamate folates are then reduced to tetrahydrofolate (FH4) by the enzyme dihydrofolate reductase (DHFR). Methotrexate exerts its action by being taken up into cells by the follate carrier, and like folate being converted to the polyglutamate form. It has a higher affinity for DHFR than the endogenous folate and thus inhibits the enzyme, depleting intracellular FH4, and therefore hindering DNA synthesis.Another shell of an antimetabolite is Fluorouracil, which is a pyrimidine analogue 12. It interferes with DTMP synthesis by forming a ternary composite plant with thymidylate synthetase (TS) the enzyme that produces DTMP. DTMP is ask for the synthesis of DNA and purines, so the irreversible inhibition of the enzyme by fluorouracil results in is inhibition of DNA but not RNA or protein synthesis. Fludarabine is a purine analogue, which is another group of antimetabolites 13. It is metabolised to its triphosphate form, which inhibits DNA polymerase. As well as the general side effects associated with chemotherapy, patients may develop resistance to antime tabolites due to a decreased amount of drug intake 14 or altered concentration of target enzymes 15, which will be discussed later.Cytotoxic antibioticsCytotoxic antibiotics, such as the anthracyclines (Doxorubicin, Idarubicin, Daunorubicin, Epirubicin, Aclarubicin, Mitoxantrone) bind to DNA and inhibit both DNA and RNA synthesis 16. Their main cytotoxic action is mediated through an inhibitory effect on topoisomerase II, the activity of which is markedly increased in proliferating cells. During DNA replication, reversible swivelling needs to take place around the replication fork in order to prevent the daughter DNA molecule becoming inextricably entangled during mitotic segregation 17. The swivel is produced by topoisomerase II, which nicks both DNA strands and subsequently reseals the breaks. Doxorubicin intercalates in the DNA, and its effect is in essence, to stabilise the DNA-topoisomerase II complex after the strands have been nicked, thus halting the process at this point 1 8. Dactinomycin is similarly a cytotoxic antibiotic, which intercalates in the minor groove of DNA, interfering with the movement of RNA polymerase on the gene and thus preventing transcription 19. Bleomycins are a group of metal-chelating glycopeptide antibiotics that degrade preformed DNA, causing chain fragmentation and release of free bases 20. This action is approximation to involve chelation of ferrous iron and interaction with oxygen, resulting in the oxidation of iron and generation of superoxide and/or hydroxyl radicals. They are most effective in the G2 phase of the cell cycle and mitosis, but are also active against non-dividing cells, that is cells in the G0 phase. This class of drugs cause resistance by altered activity of topoisomerase II, aswell as reduced white plague of the drugs 21.Plant derivativesOne sub group of plant derivatives is the vinca alkaloids, which includes Vincristine, Vinblastine, Vindesine and Vinorelbine 22. They bind to tubulin and inhibit it s polymerisation into microtubules. This prevents spindle formation in dividing cells, which causes arrest at metaphase. They also inhibit other cellular activities that involve microtubules, such as leucocyte phagocytosis, chemotaxis and axonal transport in neurons. They are relatively non-toxic in comparison to the antecedently mentioned cytotoxic drugs. Another group of plant derivatives is the taxanes, which include Paclitaxel and Docetaxel 23. They act on microtubules by stabilising them, in effect freezing them in the polymerised state, which achieves a analogous effect to that of the vinca alkaloids. Campothecins is another group of plant derivatives and include Irinotecan and Topotecan 24. They bind to and inhibit topoisomerase I high levels of which occur throughout the cell cycle.Hormone therapyTumours derived from hormone excitable tissues may be hormone dependent 25. This is due to the presence of steroid receptors in the malignant cells. Their growth can be inhibited by agents with apposing actions, hormone antagonists or drugs that inhibit the endogenous hormone synthesis. The most important group of drugs used to treat cancer are the steroids, namely the glucocorticoids (Prednisolone and Dexamethasone), oestrogens (Diethylstilbestrol and Ethinyloestradiol) and gonadotrophin-releasing hormone analogues (Octreotide and Lanreotide), as well as agents that antagonise hormone action (Tamoxifen, Toremifene and Fulvestrant). Such drugs rarely act as a cure but do mitigate the symptoms of the cancer and thus fulfill an important part in the clinical management of sex-hormone-dependant tumours.Miscellaneous agentsCrisantaspaseCrisantaspase is a preparation of the enzyme asparaginase and therefore, like asparaginase, can break muckle asparagine to aspartic acid and ammonia 26. It is active against tumour cells, such as those of acute lymphoblastic leukaemia, which have lost the capacity to synthesise asparagine and therefore require an exogenous sourc e. As most normal body cells are able to synthesise asparagine, the drug has a fairly selective action and very little suppressive effect on the bone marrow, the mucosa of the gastrointestinal tract or hair follicles.Monoclonal AntibodiesAntibodies are immunoglobulins that react with defined target proteins expressed on cancer cells. This activates the hosts immune response, which kills cancer cells by complement-mediated lysis or by killer cells. Monoclonal antibodies can also attach to and activate growth component part receptors on cancer cells, thus inhibiting the survival pathway and promoting apoptosis. Rituximab is a monoclonal antibody that is licensed (in combination with other chemotherapeutic agents) for treatment of certain types of lymphomas 27. It lysis B lymphocytes by binding to the calcium- channel forming CD20 protein and activating completment. It also sensitises resistant cells to other chemotherapeutic drugs.Trastuzumab (Herceptin) is a humanised murine monoclo nal antibody that binds to a protein termed HER2 (the human epithelial growth factor receptor 2) a receptor with integral tyrosine kinase activity 28. It induces the host immune response as well as inducing the cell cycle inhibitors p21 and p27.Imatinib MesylateImatinib is an inhibitor of signalling pathway kinases 29. It inhibits the platelet-derived growth factor (PDGF) a receptor tyrosine kinase, and the Bcr/Abl kinase a cytoplasmic kinase. These are considered to be unique factors in the pathogenesis of chronic myeloid leukaemias. Imatinib is licensed for the treatment of this tumour when it has proved to be resistant to other therapeutic strategies, as well as for the treatment of some gastrointestinal tumours that are not susceptible to surgery.Resistance to Anticancer DrugsAs mentioned previously patients can develop resistance to many chemotherapeutic agents. This can be caused by a number of mechanisms, which are summarised in figure 1.A decrease in the amount of drug taken up by the cellResistance can develop as a result of decreased drug uptake. This can be due to the loss of transporter function, for example RFC 30. Decreased influx of Methotrexate in tumour cells has been widely associated with decreased RFC gene expression. Down-regulation of the transporter protein is due to alterations in the transcription and translation factors. Transcriptional factors, such as the Sp1 family, CREB (cyclic AMP-response element binding protein) and p53, regulate RFC gene expression 31. Therefore loss of function of these transcription factors cause silencing of the RFC gene, which results in reduced protein level. Also post-translational modifications of transcription factors alter phosphorylation patterns, which abolishes Sp1 and CREB function thereby resulting in loss of RFC gene expression and subsequently resistance 32. revolutions in the human RFC gene can also decrease drug influx. Jensen et al (1998) have reported a mutation that causes marked changes i n the kinetic properties of RFC mediated transport of folates 14. The structurally altered RFC was functionally characterized by a 9- and 31-fold increased affinity for transport of reduced folate cofactors and folic acid, respectively. This allowed the accumulation of intracellular folates, which carry on cell growth and DNA replication, allowing cancer cells to escape the cytotoxic effects of antifolate drugs.Altered concentration of target enzymeIncreased expression of target enzyme is a green mechanism of acquired resistance. For example Methotrexate resistance can develop as a result of DHFR gene amplification and subsequent enzyme overexpression 15. Gene amplification is thought to occur as a consequent of antifolate inhibitors binding to DHFR, which causes a conformational change that alters the translational autoregulatary negative feedback mechanism, wherein DHFR protein specifically interacts with its own mRNA and negatively controls translational efficiency. The drug c oncentration will be limited to the dose administered, which will not be able to block the additional enzyme that is synthesised, resulting in cancer cells overcoming the inhibitory effect of the drug.Insufficient activation of the drugSome drugs require metabolic activation to manifest their antitumour activity for example Cytarabine has to undergo catalytic conversion, by the action of deoxycytidine kinase, to an active form 33. So under expression or mutation of this drug-metabolising enzyme can reduce drug efficacy and cause resistance. Another example of resistance due to insufficient activation of the drug is Mercaptopurine, which is a prodrug 34. Mercaptopurine is activated by hypoxanthine guanine phosphoribosyl transferase (HGPRT) and mutations that reduce the activity of this enzyme will allow the cancer cells to escape the toxic effects of the drug.Increase in inactivationResistant to Mercatopurine can also develop as a result of increased inactivation of the drug 35. The mechanism behind this is thiopurine s-methyltransferase (TPMT), which inactivates Mercaptopurine and thereby prevents the formation of the active drug. Mutations in the TPMT gene will alter its activity and may cause resistance. rapid repair of drug-induced lesionsPatients can develop resistance as a result of cancer cells recognizing DNA lesions and rapidly initiating repair pathways 9. This is the main cause of resistance to alkylating agents as their mechanism of action is DNA damage There are several repair pathways and include the Direct Repair (DR) pathway, Base Excision Repair (BER) pathway, theme Excision Repair (NER) pathway, Homologous Recombination (HR) pathway and Non-Homologous End Joining (NHEJ) pathway.The DR pathway is mainly mediated by the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) 36. AGT transfers the alkyl adducts from the nucleotides to the cysteine equipoise within its active site, independently from other proteins and without causing DNA strand breaks.The BER pathway recognizes and accurately removes bases that have been damaged by alkylation 37. A damaged base is removed by a damage-specific DNA glycosylase, leading to the formation of a potentially cytotoxic apurinic or apirimidinic site intermediate. This is then processed by an AP endonuclease (APE1), which generates a strand break that is further processed by Poly ADP-Ribose Polymerase (PARP), DNA polymerase b (Polb) and ligase III to restore the damage.The NER pathway deals with the repair of bulky DNA lesions formed by DNA-alkylating agents such as Cisplatin, which distort the DNA double helix and block DNA replication and transcription 38. Two major mechanisms of DNA repair have been recognized in this pathway the transcription-coupled repair, which specifically targets at and removes lesions that block the progression of RNA polymerase II, and the global genome repair, which deals with lesions in the rest of the genome. Generally, nucleotide repair is a com plex multi-step process that sequentially deploys a group of proteins to reorganize the lesion, remove the damage, and support new DNA synthesis.The HR and NHEJ pathways are involved in the repair of DNA double strand breaks, commonly considered to be the most lethal of all DNA lesions. Double strand breaks are induced by chemotherapeutic agents such Bleomycin, and Etoposide. In the HR pathway, ATM (ataxia talagiectasia mutated kinase) and its related ATR proteins sense the severe DNA lesions, and are mobilized to phosphorylate a wide range of substrate proteins 39. Also a number of regulative proteins, including BRCA1, BRCA2 and p53, are recruited to coordinate the DNA repair. The NHEJ pathway involves the alignment of the broken ends followed by recruitment and activation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and DNA ligase IV to apprehend the ligation step 40.MutationsMutations in various genes can give rise to resistant target molecules, for example the p53 gene 41. The p53 protein is an important regulator of the cell cycle and is sensitive to any DNA damage caused during replication. Following DNA damage it will normally induce G1 arrest and/or apoptosis to prevent the production of big cells. Mutations in this gene will cause the loss of p53 function, which will allow cells with damaged DNA to continue replicating, resulting in resistance to DNA damaging drugs. opposite genes, such as h-ras and bcl-2/bax, involved in the apoptotic pathway, have also been implicated in resistance 42. Resistance due to mutations in genes will affect a wide range of anticancer drugs as all cells contain the same genetic material. It also potentially increases the proportion of surviving mutant cells, which leads to greater tumour heterogeneity.Increased expression of efflux pumpsResistance to natural hydrophobic drugs, such as vinca alkaloids and taxanes, as well as the cytotoxic antibiotics, such as anthracyclines and Dactinomycin, occurs du e to the over expression of ATP-dependent efflux pumps in cancer cells 5. These pumps belong to a family of ATP-binding cassette (ABC) transporters, which are divided into eight distinct subfamilies, shown in table 1. Of these subfamilies PGP, also known as MDR1, has a broad drug specificity, which explains the cross-resistance to several chemically unrelated compounds. It is a multidrug efflux pump that has 12 transmembrane regions, which bind hydrophobic drug substrates that are either neutral or positively charged 6. It also has two ATP-binding sites, as hydrolysis of two ATP molecules are needed for the transport one drug molecule 43. Binding of substrate to the transmembrane regions stimulates the ATPase activity of PGP, causing a conformational change that releases substrate to the extracellular space. Hydrolysis at the second ATP site is required to re-set the transporter so that it can bind substrate again, completing one catalytic cycle. Increased expression of the PGP tran sporter in cancer cells increases the amount of catalytic cycles that occur, which increases the amount of drug effluxed 5. This lowers the intracellular drug concentration below a cell-killing threshold, which results in resistance.Not all multidrug-resistant cancer cells express PGP. Resistance in these cells was find to be linked with the expression of the multidrug-resistance-associated protein 1 (MRP1) 44. MRP1 is similar to PGP in structure (table 1) but, unlike PGP, it recognizes neutral and negatively charged hydrophobic natural products, and transports glutathione and other conjugates of these drugs, or, in some cases, such as for Vincristine, co-transports unconjugated glutathione. Some anticancer drugs, such as Mitoxantrone, are poor substrates for PGP and MRP1. Mitoxantrone resistance is due to a more distant member of the ABC transporter family, MXR (Mitoxantrone-resistance gene) 45. This transporter is thought to be a homodimer of two half-transporters, each containin g an ATP-binding domain at the amino-terminal end of the molecule and six transmembrane segments (table 1).Resistance can also develop as a result of increased expression of ABC transporters in the apical membrane of the gastrointestinal tract 46. ABC transporters play a key physiological role, where they extrude toxins thus forming a protective mechanism and a first line of defense. Increased expression of these transporters decreases drug uptake and therefore decrease drug bioavailability. Examples of chemotherapeutic agents that develop resistance by this mechanism include antimetabolites, such as Methotrexate and Fluorouracil, and alkylating agents, such as Cisplatin. Also water-soluble drugs that piggyback on transporters and carriers or enter by means of endocytosis can fail to accumulate as they will not be able to enter the body. Additionally, PGP actively secretes intravenously administered drugs into the gastrointestinal tract 47. Resistance due to increased levels of PGP transporters in the gastrointestinal tract is illustrated by MDR1a/MDR1b-knockout mice, which have shown to have increased tissue concentrations of PGP substrates. Studies have also shown increased tissue absorption of PGP substrates, following oral administration, when co-administered with a PGP inhibitor.Reversal of drug resistance in cancer ways to overcome multidrug resistance due to the over expression of ABC transporters are being researched. Some of the main approaches include developing PGP inhibitors, antibodies against the PGP transporter, antisense oligonucleotides and liposome-encapsulated drugs.Drugs that can reverse multidrug resistance, such as PGP inhibitors, could be useful interventions to improve bioavailability, by increasing oral uptake of anticancer drugs and decreasing drug excretion, thereby reducing dosing requirements 7. Two inhibitors that are used in the laboratory and in clinical trials that attempted to reverse drug resistance are the calcium channel bl ocker, verapamil and, the immunosuppressant, cyclosporin A. Another order that can be used to inhibit PGP is by competitive inhibition 48. PGP binds many different hydrophobic compounds so any drug that interacts with the substrate-binding region is seeming to be a competitive inhibitor of other drugs. Thus, two drugs that are transported by PGP will compete for this transport, resulting in increased oral absorption of both, decreased excretion, and redistribution. This bod of drug interaction can be used to inhibit the multidrug transporter, when the inhibitor drug has little or no other pharmacologic effect.Monoclonal antibodies (MAbs) against PGP have been used to kill multidrug resistant cells 49. MAbs are of therapeutic use as they can activate the immune response, which results in complement mediated lysis or antibody dependent cellular cytotoxicity of the cells. An example of a MAb is MRK-16, which has shown selective toxicity towards tumours that are over expressing PGP. Molecules, which are normally involved in signal transduction on T and B cells can also be targeted for antibody therapy 50. Such molecules include CD19, which is a membrane receptor involved in signal transduction and potentiates the response of B cells to antigens. MAbs directed against CD19 can induce cell-cycle arrest due to negative growth signals that cross-link immunoglobulin M and CD19.Antisense drugs work by down regulating gene expression 51. This occurs by sequence-specific conspicuous of either DNA or RNA, which inhibits transcription or translation, respectively. Different antisense-oligodeoxynucleotides have been reported to chemosensitize resistant tumour cells to anticancer drugs through down regulation of PGP expression and thus increasing the intracellular accumulation of anticancer drugs in the cancer cells. The efficiency of a synthetic oligodeoxynucleotide (ODN) in regulating gene expression in living cells depends on its thermodynamic stability, resistance tow ard nucleases and cellular uptake 52. A number of studies indicate that a synthetic ODN coupled with a DNA intercalator such as acridine, naphthyl imide, psoralen or pyrene might act to increase stability.Novel drug delivery systems such as liposome-encapsulated drugs have also been developed to overcome multidrug resistance 53. Liposome formulations contain a olive-sized fraction of polyethylene glycol (PEG)-derivatised phospholipid, which has been shown to dramatically alter the pharmacokinetic properties of certain anticancer drugs. These pharmacokinetic alterations include long elimination half-life and small volume of distribution. Another formulation developed to bypass PGP transporters is anionic liposomes, which are internalised by certain cells and are able to provide drug release in intracellular compartments.ConclusionCancer is prevalent in the western world and much research is dedicated to produce effective chemotherapy. Current chemotherapy includes alkylating agents, antimetabolites, cytotoxic antibiotics, plant derivertives, hormone therapy and monoclonal antibodies. However the efficacy of these chemptherapeutic agents is limited to patients developing multidrug resistance. This is mainly due to the over expression of ABC transporters, particularly the PGP transporter, as they have broad drug specificity so can bind many structurally unrelated drugs 5.Techniques to reverse multidrug resistance are being developed and include co-administration of PGP inhibitors, which prevent the binding of anticancer drugs the transporter 7, the use of antibodies, which kill cells over expressing the PGP transporter 49, antisense oligonucleotides that down regulate PGP expression 51 and liposome-encapsulated drugs, which alter the pharmacokinetic properties of anticancer drugs 53.A better understanding of the mechanism by which ABC transporters efflux chemotherapy and further analysis, in clinical trials, of known mechanisms of multidrug resistance would incr ease the development of agents that reverse multidrug resistance. 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