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Cytochrome P450 is a set of heme protein super family. They get the name as their conjugation with carbon monoxide has a characteristic absorption in 450 nm. They are distributed in all kinds of tissues and organs in the human body, mainly involved in drug metabolism and bioactivation, accounting for about 75% of the total number of different metabolic reactions. In recent years, the researches on CYP450 genotype and phenotype correlation have drawn more and more attentions, people tried to find CYP450 gene polymorphism and the relationship between the cancer susceptibility. From aspect of clinical rational drug use, people want to utilise genotype to understand individual enzyme activity in drug metabolism, expect to improve the level of drug treatment and reduce the occurrence of adverse reactions in the same time.
Key words: Heme protein; Cytochrome P450; drug target
At present, the standard treatments provided to patients with cancer are surgery, radiotherapy and chemotherapy. Each of these methods has its own advantages and disadvantages. Although quite a few efforts and money have been spent in the development of novel cytotoxic drugs, the first choice of anticancer drugs in the clinical management for most types of caner has remained more almost the same in the last decade. Under normal circumstances, the main limitation of anticancer drugs provided in the current clinical practice is lack of tumor selectivity. Typically, these anticancer drugs have a narrow therapeutic index, which make cancer chemotherapies are always associated with side effects. Also, in the anticancer drug development programs, successful examples are few and far between. Therefore, it is high time to find innovative strategies, especially to find previously unconfirmed drug targets for cancer therapies. The enzymes in Cytochrome P450 play a vital role in the biotransformation of a wide range of substrates, including anticancer drugs. Over recent years, some important discoveries have identified Cytochrome P450 as a potential drug target in cancer therapies.
1.1 Heme Protein
Heme protein is a metalloprotein that contains one or more hemes. It usually acts as an enzyme and plays a vital role in oxidative metabolism and electron transport systems, including microsomal and mitochondrial electron transport, removal of toxic peroxides and the oxygenation and hydroxylation of important metabolically organic molecules. Its diverse functional process can be concluded in two steps. First, the enzyme itself stores oxidizing equivalents derived from either molecular oxygen or hydroperoxides by cleaving the O-O bond and releasing a water or alcohol molecule. Next, the enzyme utilises these stored equivalents to oxidise other cellular components, either small organic molecules or another redox protein .
1.2 Cytochrome P450
Among all the diverse kinds of heme proteins, Cytochrome P450 (CYP450) is not only the most abundant and widespread, but also the protein which has the most aboard substrate spectrum. Moreover, CYPs are the major enzymes involved in drug metabolism and bioactivation. Cytochrome P450 superfamily contains a series of enzymes which are similar in function and structure. They can be found in all kinds of vertebrate and invertebrate animals, plants, fungi and prokaryotes. In human, there are 57 genes and more than 59 pseudogenes distributed among 18 families are identified by The Human Genome Project. Principally, cytochrome P450 enzymes are present in liver. According to the differences in metabolites, CYPs enzymes can be divided into two types, one is to metabolise endogenous compounds and the other type is to metabolise exogenous compounds. Therefore, they metabolise thousands of endogenous and exogenous chemical compounds, which play a key role in hormone synthesis and breakdown, cholesterol synthesis, vitamin D metabolism, toxic compounds metabolism.
Along with the development of molecular epidemiology, some researches have found that there is a possible connection between the mutated gene of CYPs and the susceptibility of cancer. However, for a long time, the research upon CYPs metabolism focused only in liver, rather than in tumor tissues. With the rapid development of modern molecular biology and bioinformatics, the utilisation of technology of homologous modeling, molecular dynamics stimulation, some requisite progresses has been made in the CYPs` crystal structure, gene expression distribution and mechanism of activation of cancerogens[2-4]. Recent researches demonstrate that CYPs can not only metabolise activated cancerogens but also act a key role in the development of cancer. Therefore, comprehension of the relationship between tumor specific expression of CYPs and development of cancer has a significant meaning in anticancer drug development. This article is a review of recent researches on anticancer drug using Cytochrome P450 as a drug target.
CYPs and cancer
The genes of CYPs have polymorphism, which has been proved in CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and so on. Some CYPs are polymorphic due to single nucleotide polymorphisms, gene duplications and deletions. As genotyping procedures have become available for most CYPs in human body, many outstanding association studies about cancer risk with CYP polymorphism have been done. Researches carried out in the last decade demonstrated the associated relationship between CYP polymorphisms and breast, lung, head and neck, and liver cancer.
Moreover, CYP enzymes catalyse the bioactivation of several procarcinogens and protoxins, producing strong electrophilic intermediates or productions, which can react with biomacromolecules in cells, like DNA, RNA and proteins. These reactions can destroy the cellular structure, make enzymes inactivate or abnormal, induce gene mutation or inhibit some gene expression, cause cell damage, and finally induce apoptosis or tumor.
Also, CYP enzymes can activate many external procarcinogens. For instance, CYP1A1 is able to activate so many kinds of polycyclic aromatic hydrocarbon, which have strong carcinogenicities after activation. Benzopyrene is one of PAHs, it is first epoxidised by CYP1A1, and then hydrolysed by epoxide hydrolase to form dihydroxy alcohol which is epoxidesed by CYP1A1 again to produce diol epoxides. The final production has a significant carcinogenic and mutagenic effect. Based on Ma`s research(æ’å…¥-çŒ® ç»†èƒžè‰²ç´ p450åœ¨è‚¿ç˜¤æ²»--ä¸çš„ç ”ç©¶è¿›å±•4), 90% of known procarcinogens are activated by CYP1A1 and CYP2A2. As a result, many cancer researchers tend to set the ability to induce CYP1A1 as a crucial indicator of the evaluation of carcinogenicity. CYP3A4 participate in activation of aflatoxin B1 in human lungs. CYP2C9 is the most dynamic reactant in the formation of 3,4-dihydroxy alcohol which is a procarinogen. CYP1A1, CYP2B6 and CYP2C9 combined together in the generation of 11,12-dihydroxy alcohol. In some circumstance, carcinogens won't cause biological damage unless there are some specific CYP450 enzymes existing.
CYP1A1, CYP2A2 and CYP3A3 are the three expressions in human body. Among them, CYP1A1 is located in chromosome 15, which has 43064 bp including 7 exons and 6 introns. Principally, CYP1A1 has 4 types of mutation. Mutation types of m1 and m2 are more commonly occurring in human. CYP1A1 m1 is mutated by adding a restriction enzyme site, Msp I, in the non-coding area of DNA. And the mutation in m2 is accomplished by base replacement between A and G in 462 locus of the seventh exon. The enzyme activities expressed by the two types of mutation are both higher than wild type, thereby more and more attentions are focused on the relationship between cancer and polymorphism of CYP1A1 gene.
Previous research shows that polymorphism of CYP1A1 may increase the risk of breast cancer. In addition, this risk is more outstanding in premenopausal women.(æ’å…¥-çŒ®) Moreover, Belogubova`s research(æ’å…¥-çŒ®) of the mutation of CYP1A1 indicated that it is closely related to lung squamous carcinoma.
The gene of CYP2E1 is located in 10q24.3-qter of human chromosome, including 11413 base pairs and 9 exons. The enzymes encoded by this gene are mainly found in liver. Recently, this enzyme has been found outside the liver, like gastrointestinal tract, lung and kidney. It can be induced by ethanol and participates in metabolism of nitrosamines and halohydrocarbon. Research shows that the gene of CYP2E1 has 6 restriction fragment length polymorphisms. Rsa I is one of them and it has been proved that it have a significant effect upon transcription and expression of CYP2E1.
Recent research shows the polymorphism of CYP2E1 gene is related to genetic susceptibility of gastrointestinal tumor. (æ’å…¥-çŒ®)`s research showed ….
In the super family of CYPs, the role of CYP3A is especially prominent. It is one of the first purified enzymes from human body and is the dominating limiting velocity enzyme in drug metabolism. The two main enzymes in the subfamily of CYP3A are CYP3A4 and CYPA5, which catalyse more than 50% of drug redox reactions. CYP3A4 is the main CYP protein in liver. CYP3A5 is mainly found in intestine, kidney, prostate and lung, but it has polymorphism in liver. The expression and activity of CYP3A5 have huge differences between individuals and races. CYP3A5 plays a vital role in the susceptibility of cancer as it catalyses a large amount of procarcinogens into genotoxic intermediates.
According to some researches, CYP3A5*1 positive in female can result in a earlier pubescence, thereby increasing the risk of breast cancer.(æ’å…¥-çŒ®). Dandara(æ’å…¥-çŒ®) found CYP3A5 can metabolise potential cancerogens of esophagus carcinoma. Also, CYP3A5 can activate the aflatoxin B1, which is the main risk factor of liver cancer.
CYPs and targeted drug
CYPs metabolising endogenous chemicals as drug target
3.1.1 Inhibitors of CYP19
CYP19 is an aromatase participating in the transformation of androgen into estrogen. They can catalyse the formation of oestrone and estradiol from androgen by aromatising the A ring in androgen, cleavaging C19 and transforming carbonyl group into hydroxyl. And CYP19 is the limiting velocity enzyme in this reaction(æ’å…¥-çŒ®7). Before the menopause, most of estrogens are synthesised inside the ovary. However, as the degradation of ovary after menopause, the synthesis of estrogens has to be done under the presence of CYP19, especially in the breast stromal cells, which is the pathogenesis of breast cancer. The traditional therapy is to use antiestrogen drug, Tamoxifen. This drug is able to prevent estrogens binding to estrogen receptor(ER). However, the efficacy of Tamoxifen is depending on the existence of ER, also 50% of ER positive patients will generate resistance(æ’å…¥-çŒ®). Therefore, reducing the formation of estrogens through inhibiting the activity of CYP19 is an ideal method to treat hormone-dependent breast cancer. The recent inhibitors including non-steroids drugs like Anastrozole(1) and Letrozole(2) and steroids drugs like Exemestane(3). In vitro laboratory experiments indicate all of the inhibitors can selective inhibit the activity of CYP19, reduce the transformation from androgens to estrogens, thereby lowering the level of estrogens. The order of inhibitors` activities from strong to weak is Letrozole, Anastrozole and Exemestane.
(1) (2) (3)
Fig. 1. The Structure of Anastrozole, Letrozole and Exemestane.
Letrozole is a synthetic derivant of benzotriazole, which the activity is 150~250 times stronger than the first generation inhibitor, Aminoglutethimide. The long term use of Letrozole will not compromise the function of thyroid or the formation of glucocorticoid and mineralocorticoid, which result in a higher therapeutic index. Thereforeï¼Œthe development of Letrozole is a successful example in the field of targeting anticancer drugs.(æ’å…¥-çŒ® 8)
4.1.2 Inhibitors of CYP17
CYP17 is a key factor in the formation of androgens inside of human body. It can not only hydroxylate the pregnenolone and progesterone but also catalyse the formation of dehydroepiandrosterone from 17α-hydroxypregnenolone. The inhibitors of aromatase used in the treatment of hormone-dependent breast cancer have provided references for the therapy of androgen-dependent prostatic cancer.(æ’å…¥-çŒ®9) Present clinical treatments are using Luteinizing Hormone Releasing Hormone (LHRH) agonist or Gonadotropin-Releasing Hormone (GnRH) agonist combined with antiandrogen drugs to treat prostatic cancer. However, agonists of LHRH and GnRH can not stop all but only 10% of the synthesis of testosterones in adrenal glands. Moreover, taking the antiandrogen drug as a weak agonist of androgen receptor is easier to get side effects. Although the present ketoconazoles can act as non-specific inhibitors of CYPs, the specific inhibitors of CYPs have apparently more advantages. For instance, the steroid drug, Abiraterone(4), which has entered the phase 2 clinical trial(æ’å…¥-çŒ®10). Besides, the chemical compound, VN / 124-1(5), designed by Bruno(æ’å…¥-çŒ®11) has a fine inhibiting effect towards CYP17. Based on the animal in vivo data, the efficacy of VN / 124-1(50 mg/kg, bid) is superior to the control group, inhibiting rate reaches as high as 93.8% (P = 0.00065).(æ’å…¥-çŒ®12)
Fig. 2. Structure of Abiraterone and VN / 124-1.
4.1.3 Inhibitors of CYP24A1
Vitamin D is the derived from sterol, which can control the physiological function though the absorption of calcium. And 1, 25- dihydroxycholecalciferol (1, 25-D3) is one of the vitamin D`s metabolites, it can inhibit proliferation of cancer cells in several tissues including colon and prostate, induce the apoptosis by promoting the differentiation of cancer cells. Therefore, 1, 25-D3 is considered to have anticancer effects, which is consistent with negative correlation between the morbidity of cancer and the sunlight exposure. CYP24A1 is a 25-OH-vitamin D3-24-hrdroxylase. Further research shows that CYP24A1 can catalyse the inactivation of 1, 25-D3 by hydroxylating C24.(æ’å…¥-çŒ®13-15), so inhibiting the activity of CYP24A1 will contribute to the anticancer activity of 1, 25-D3. VID400(6) is one of pyrrole chemicals, which can selective inhibit the activity of CYP24A1. In vitro experiments shows that VID400(200 nmol/L) can completely inhibit the catalytic reaction of 1, 25-D3 by CYP24A1.(æ’å…¥-çŒ®16-17)
Fig. 3. Structure of VID400.
Parise`s(æ’å…¥-çŒ®18) research found that the sulfone analogue of 1, 25-D3 is a brand new CYP24A1 inhibitor. Therefore, the leading compound(7) designed by this theory have a specific inhibiting effect towards CYP24A1 (IC50 = 7.4 nmol/L).
Fig. 4. Structure of sulfone analogue of 1, 25-D3.
4.2 CYPs metabolising exogenous chemicals as drug target
4.2.1 Inhibitors of CYP1A1
Phortress(8), is a selective targeting drug, which is used in the treatment of breast and ovarian cancer. At present, it is in the phase 1 clinical research sponsored by Cancer Research, UK. Phortress is a prodrug od benzothiazole, it can exert its cytotoxic effect through the activation of Aryl Hydrocarbon Receptor (AHR) and Phortress.
Fig. 5. Structure of Phortress.
Compared with Tamoxifen, the activation of Phortress can be done without the presence of estrogen receptor (ER). Therefore, Phortress can be used to treat breast cancer patients who are ER negative. Outside the cell, the hydrophilic Phortress hardly can hydrolyse. In contrast, it can rapidly conduct hydrolysis inside cell and form lipophilic compounds, 5F203 (9).
Fig. 6. Structure of 5F203.
5F203 can get into targeting cell and act as an agonist of AHR, inducing the gene of AHR to express CYP1A1 or CYP1B1. Then CYP1A1 can metabolise 5F203 to produce active substances with high eletrophilicity, thereby causing the DNA damage and cell apoptosis. In vitro experiments shows that Phortress (1μmol/L) can produce amount of DNA adducts inside the MCF-7 breast cancer cells. Whereas the concentration of Phortress is 10μmol/L, two main DNA adducts can be detected by chromatography inside the MCF-7 breast cancer cells. One of the adducts` chromatographic peak is consistent with the result achieved from 5F203. In vivo experiments indicates that Phortress can inhibit tumor growth in mice when dose them on the 1st and 7th day with the concentration of Phortress is 20mg/kg or dose them for four days continuously with the concentration is 4mg/kg. These experiment results demonstrate that the Phortress have a significant efficacy in the treatment of breast cancer and a remarkable clinical prospect.(æ’å…¥-çŒ® 1a1 å’Œ 1b1 9)
1c(c1), 1f(c2), 1g(c3) are all Aminoflavone(10) compounds, they have a prominent inhibiting effect to the cancer cells after activated by CYP1A1. Recently, following the step of Phortress, Aminoflavone has entered the phase 1 clinical trial.
Fig. 7. The structure of Aminoflavone.
The metabolism of Aminoflavone is similar with 5F203. However, some researched indicates that the metabolism of Aminoflavone request the presence of sulfotransferase A1 (SULTA1)(æ’å…¥-çŒ® 1A1 12), so the mechanism of activating Aminoflavone is more complicate than Phortress. It can induce the expression of CYP1A1, CYP1B1 and SULTA1 in the MCF-7 tumor cells, thereby accelerating the process of self metabolic activation. Its activated production reacts with AHR to further induce the expression of CYPs. This positive feedback can enhance the activity of Aminoflavone.(æ’å…¥-çŒ®)
Gene-Directed Enzyme Prodrug Therapy (GDEPT) is to insert extrinsic enzyme gene into tumor cells, and achieve anticancer ability after the activation of prodrug.(æ’å…¥-çŒ®1a1 18) A GDEPT is reported by Yakkundi(æ’å…¥-çŒ®19), which is using CYP1A1 and AQ4N(11) to treat tumor. In anaerobic cells, AQ4N prodrug can be biological reduced into cytotoxic AQ4(12), through the metabolism of CYP1A1. And the cytotoxity will not affect normal cells, as AQ4N only reacts in the microenvironment of anaerobic or low oxygen, which is the environment of tumor cells. The early researches indicate that it can inhibit the growth of tumor cells after injecting AQ4N, CYP3A4 and CYP2B6 in tumor cells of low oxygen environment. However, through the research upon the ability of activation in the family of CYP enzymes, scientists find AQ4N can produce stronger anticancer drug by the metabolism of CYP1A1. Mitoxantrone(13) not only can competitive inhibit CYP1B1, but also can be metabolic activated by CYP1A1 to achieve antitumor ability.
Fig. 8. The structure of AQ4N, AQ4 and Mitoxantrone.
Tegafure(14), Difuradin(15) and Doxifluridine(16) are all furan derivatives of Fluorouracil(17). They are prodrugs, and can slowly release Fluorouracil inside body. The whole process is metabolic activated by CYP1A1. (æ’å…¥-çŒ® 1a1 14) Fluorouracil is a suicide inhibitor used in the treatment of cancer. It works through irreversible inhibition of thymidylate synthase.(æ’å…¥-çŒ® 1a1 15)
(14) (15) (16) (17)
Fig. 9 The structure of Tegafure, Difuradin, Doxifluridine and Fluorouracil.
4.2.2 Inhibitors of CYP1B1
Researches demonstrate high expression of CYP1B1 in many tumor tissues including bladder cancer, breast cancer and colon cancer. Also, in vitro experiments show that CYP1b1 can activate numerous procarcinogens. They prove that CYP1B1 have a close relationship with tumor. In the mean time, CYP1B1 can inactivate many anticancer drugs, like Docetaxel, Ellipticine, Mitoxantrone and Tamoxifen(æ’å…¥-çŒ®p450 23-24), which is the reason of drug resistance in tumors. Therefore, the development of inhibitors and prodrugs of CYP1B1 is urgent in the treatment of cancer.
Resveratrol(14) exists in many plant, it can inhibit the activity of CYP1A1 and CYP1B1.
Fig. 9. The structure of Resveratrol.
The hydrophenol group in Resveratrol is a strong electron-donating group, which makes the Resveratrol a strong antioxidant. Also this ability is related to the position and number of the hydrophenol group. Therefore, changing the number and position of hydrophenol group in the two benzene rings may also get the same pharmacological effects with Resveratrol. Piceatannol(15) is a hydroxy analog of Resveratrol, which is transformed inside the body through CYP1B1 by adding a hydroxyl group at C3 position. It has many pharmacological effects in treating cancer and preventing proliferation of cancer cells.(æ’å…¥-çŒ®6 1a1 and 1b1) Moreover, the derivants of Resveratrol can inhibit the activity of CYP1B1. For instance, 2, 4, 3`, 5`- tetram-ethoxystibene(16) is one of the strongest selective inhibitors of CYP1B1. Its IC50 for inhibiting CYP1B1, CYP1A1 and CYP1A2 is 6 nmol/L, 300 nmol/L and 3μmol/L respectively.(æ’å…¥-çŒ® p45025)
Fig. 10. The structure of Piceatannol and 2, 4, 3`, 5`- tetram-ethoxystibene.
Chalcone is a flavonoid compound, and the centre core is formed by an aromatic ketone and an enone. The research by Potter(æ’å…¥-çŒ® p450 26) indicates that Chalcone achieves the anticancer ability through metabolised by CYP1B1. Therefore, Chalcone can be a anticancer prodrug. For example, a potential low activity prodrug 17, its IC50 towards MCF-7 cells can increase from 0.69 μmol/L to 0.65 nmol/L after metabolised by CYP1B1.
Fig. 11. The structure of prodrug 17.
Likewise, DMU-135(18), DMU-403(19), DMU-407(20) and DMU-419(21) all show the same progress of metabolism.(æ’å…¥-çŒ® 1a1 16) The report of Sale(æ’å…¥-çŒ® p450 27) says DMU-117(22) is a potential nonselective inhibitor of tyrosine kinase, which is formed from the metabolism of DMU-135 catalysed by CYP1B1.
Fig. 12. The structure of DMU-135, DMU-403, DMU-407, DMU-419 and DMU-117.
The development of CYPs targeted anticancer drugs is still in a discovering stage and numerous problems still need to be solved. Therefore, the further research in the physiological role and the metabolic mechanism of CYPs can undoubtedly provide new insights into the research of targeted anticancer drugs.The recent strategies of CYPs targeted anticancer therapies including: 1. CYPs targeted prodrug 2. CYPs inhibitor 3. Targeted enzyme immunotherapy 4. Gene therapy by expressing specific CYPs in tumor tissues.(æ’å…¥-çŒ® p450 1) At present, the first two strategies are most widely applied in cancer treatment.
Researches have found that, part of specific expression and overexpression of CYPs in tumor tissues are different from the expression in normal tissues. Therefore, with the protein structure and substrate character of CYPs, scientists are able to design anticancer prodrugs which are required to be activated by CYPs enzymes. This can make the drug therapies more targeting. And Phortress and AQ4N are the successful examples of prodrugs. Also, CYPs sensitisers can strengthen the activity of CYPs and accelerate the activation of prodrugs, thereby enhancing tumor treatment effect. So the sensitisers should be developed in the same pace.
Moreover, the CYPs inhibitors are plaing a crucial role in the prevention and treatment of cancer as well. Inhibiting some key CYPs` ability can reduce the activation of procarcinogens, in addition to inhibit the growth of tumor. And in some cancer therapies, some CYPs can catalyse the inactivation of anticancer drugs, which is the main mechanism of drug resistance in tumors. Therefore, the combination of sensitisers and inhibitors of CYPs can decrease the incidence of tumors and enhance the effect of anticancer drugs. Furthermore, some CYPs have specific physiological functions inside body. The disorder of their functions can be tumorigenic. So the inhibition and reduction of those CYPs` expression can achieve the purpose of the treatment of cancer.
However, there are still a lot of researches should be done when using CYPs as a target to develop new anticancer drugs.Use the development of prodrugs as an example. Initially, we should verify the CYPs in tumor tissues possess the catalytic activity, and express quantities are enough to activate prodrug to achieve anticancer purpose. Secondly, the prodrug must be enzymes` substrate, which has a high affinity of specific binding with CYPs and the metabolites must have strong activity. Then the drugs can have targeted treating effect. Finally, the catalysis rate of CYPs should meet the requirements, thereby producing enough amounts of cytotoxic substrates to cause the death of cancer cells.
In conclusion, further researches upon the process of CYPs gene regulation, physiological function of protein and the specificity of substrates can contribute to the development of specific anticancer drugs. Treating cancer by using CYPs targeted drugs can enhance people's acknowledgement of cancer in a unique angle. Therefore, this whole new treatments deserve more dedications in the battle of cancer.