Morag C.E. McFadyen

Cytochrome P450 CYP1B1 protein expression: a novel mechanism of anticancer drug resistance

The overexpression of human cytochrome P450 CYP1B1 has been observed in a wide variety of malignant tumours, but the protein is undetectable in normal tissues. A number of cytochrome P450 enzymes are known to metabolise a variety of anticancer drugs, and the consequence of cytochrome P450 metabolism is usually detoxification of the drug, although bioactivation occurs in some cases. In this study, a Chinese hamster ovary cell line expressing human cytochrome P450 CYP1B1 was used to evaluate the cytotoxic profile of several anticancer drugs (docetaxel, paclitaxel, cyclophosphamide, doxorubicin, 5-fluorouracil, cisplatin, and carboplatin) commonly used clinically in the treatment of cancer. The MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay was used to determine the levels of cytotoxicity. The key finding of this study was that on exposure to docetaxel, a significant decrease in sensitivity towards the cytotoxic effects of docetaxel was observed in the cell line expressing CYP1B1 compared to the parental cell line (P = 0.03). Moreover, this difference in cytotoxicity was reversed by co-incubation of the cells with both docetaxel and the cytochrome P450 CYP1 inhibitor alpha-naphthoflavone. This study is the first to indicate that the presence of CYP1B1 in cells decreases their sensitivity to the cytotoxic effects of a specific anticancer drug.

IMMUNOHISTOCHEMICAL LOCALIZATION OF CYTOCHROME P450 CYP1B1 IN BREAST CANCER WITH MONOCLONAL ANTIBODIES SPECIFIC FOR CYP1B1

Cytochrome P450 CYP1B1 is a recently identified member of the CYP1 P450 family. We have shown that this P450 displays increased expression in several types of human cancer, indicating that CYP1B1 is a potential tumor biomarker. In this study we developed monoclonal antibodies (MAbs) to CYP1B1 that are effective on formalin-fixed, paraffin-embedded tissue sections and investigated the presence of CYP1B1 in a series of primary breast cancers. The MAbs were generated using a synthetic peptide coupled to carrier protein as the immunogen. The MAbs specifically recognized CYP1B1 and did not recognize either CYP1A1 or CYP1A2, related CYP1 forms. The MAbs were tested by immunohistochemistry and were found to be effective on formalin-fixed, paraffin-embedded tissue sections. The majority of breast cancers showed positive immunoreactivity for CYP1B1, and in each case CYP1B1 was specifically localized to tumor cells. The presence of CYP1B1 in breast cancer cells is likely to contribute to their metabolism of estradiol because CYP1B1 is a specific estradiol hydroxylase.

Cytochrome P450 CYP1B1 over-expression in primary and metastatic ovarian cancer.

Ovarian cancer is the most frequent cause of death from gynaecological malignancies world wide. Little improvement has been made in the long-term outcome of this disease, with the 5-year survival of patients only 30%. This poor prognosis is due to the late presentation of the disease and to the unpredictable response of ovarian cancer to chemotherapy. The cytochrome P450 enzymes are a superfamily of haemoproteins, known to be involved in the metabolic activation and/or detoxification of a number of anti-cancer drugs. CYP1B1 is a tumour-related form of cytochrome P450 which is over expressed in a wide variety of primary tumours of different histological type. The presence of CYP1B1 may be of importance in the modulation of these tumours to anti-cancer drugs. We have conducted a comprehensive immunohistochemical investigation, into the presence of cytochrome P450 CYP1B1 in primary and metastatic ovarian cancer. The key findings of this study are the increased expression of CYP1B1 in the majority of ovarian cancers investigated (92%), with a strong correlation demonstrated between CYP1B1 expression in both primary and metastatic ovarian cancer (P= 0.005 Spearman’s rank correlation test). In contrast no detectable CYP1B1 was found in normal ovary.

Profiling Cytochrome P450 Expression in Ovarian Cancer: Identification of Prognostic Markers

Purpose: The cytochromes P450 are a multigene family of enzymes with a central role in the oxidative metabolism of a wide range of xenobiotics, including anticancer drugs and biologically active endogenous compounds. The purpose of this study was to define the cytochrome P450 profile of ovarian cancer and identify novel therapeutic targets and establish the prognostic significance of expression of individual cytochrome P450s in this type of cancer. Experimental Design: Immunohistochemistry for a panel of 23 cytochrome P450s and cytochrome P450 reductase was done on an ovarian cancer tissue microarray consisting of 99 primary epithelial ovarian cancers, 22 peritoneal metastasis, and 13 normal ovarian samples. The intensity of immunoreactivity in each sample was established by light microscopy. Results: In primary ovarian cancer, several P450s (CYP1B1, CYP2A/2B, CYP2F1, CYP2R1, CYP2U1, CYP3A5, CYP3A7, CYP3A43, CYP4Z1, CYP26A1, and CYP51) were present at a significantly higher level of intensity compared with normal ovary. P450 expression was also detected in ovarian cancer metastasis and CYP2S1 and P450 reductase both showed significantly increased expression in metastasis compared with primary ovarian cancer. The presence of low/negative CYP2A/2B (log rank = 7.06, P = 0.008) or positive CYP4Z1 (log rank = 6.19, P = 0.01) immunoreactivity in primary ovarian cancer were each associated with poor prognosis. Both CYP2A/2B and CYP4Z1 were also independent markers of prognosis. Conclusions: The expression profile of individual P450s has been established in ovarian cancer. Several P450s show increased expression in ovarian cancer and this provides the basis for developing P450-based therapeutics in ovarian cancer. Expression of CYP2A/2B or CYP4Z1 in primary ovarian cancer were independent markers of prognosis.

Regional Distribution of Individual Forms of Cytochrome P450 mRNA in Normal Adult Human Brain

The cytochromes P450 are a large family of haemoproteins which have a major role in the oxidative metabolism of a wide range of xenobiotics and some endogenous compounds. In this study the presence of individual members of the CYP1, CYP2 and CYP3 P450 families has been investigated by reverse transcriptase polymerase chain reaction in different regions of normal human brain consisting of frontal and temporal cortices, mid brain, cerebellum, pons and medulla. All the P450s were identified in specific regions of brain with CYP1A1 and CYP2C being the most frequently expressed forms of P450. Sequencing identified the CYP2C PCR product as CYP2C8. This study indicates that individual P450 mRNAs are present in human brain and are found in specific brain regions. The distribution of individual P450s in different regions of human brain is likely to be highly important in determining the response of the brain to toxic foreign compounds.

Cytochrome P450 CYP3A in human renal cell cancer

SummaryRenal cell cancer is the main malignant tumour of the kidney and has an increasing incidence. This type of tumour has a poor prognosis and shows intrinsic resistance to several anti-cancer drugs. The CYP3A P450 family, which consists of three closely related forms, is involved in the oxidative activation and deactivation of a variety of carcinogens and several anti-cancer drugs. In this study the presence and cellular localization of CYP3A has been investigated using a combination of immunohistochemistry, immunoblotting and reverse transcriptase polymerase chain reaction (RT-PCR) in renal cell cancer and corresponding normal kidney. CYP3A was consistently expressed in both renal call cancer and in normal kidney. In renal cell cancer, CYP3A was localized to tumour cells and in normal kidney the predominant cellular localization of CYP3A was to proximal tubular epithelial cells. RT-PCR showed that both CYP3A5 mRNA and CYP3A7 mRNA were consistently present in both tumour and normal samples, while CYP3A4 mRNA was present in 65% of tumours and 90% of normal samples. This study indicates that individual members of the CYP3A family are expressed in renal cell cancer. The presence of CYP3A in renal cell cancer might be important in the metabolic potentiation as well as the detoxification of chemotherapeutic agents used to renal cancer.

The Role of Cytochrome P450 in Cytotoxic Bioactivation: Future Therapeutic Directions

The cytochrome P450s are an essential group of enzymes involved in metabolism of drugs, foreign chemicals, arachidonic acid, cholesterol, steroids and other important lipids. The cytochrome P450 enzyme system is responsible for much of the phase I metabolism of chemotherapeutic agents. At the simplest level the detoxification properties of the cytochrome P450s are used to help clear a cytotoxic before it results in serious irreversible toxicity to the patient while at other levels the cytochrome P450s are involved to varying extents in drug bioactivation. This metabolism primarily occurs in organs and tissues of the body known to express cytochrome P450 ubiquitously (i.e. liver and gastrointestinal tract), but there is also evidence to suggest that it occurs within the tumor microenvironment due to localized, tumor specific expression of certain P450 isoforms. Several of todays currently prescribed cytotoxics (e.g. cyclophosphamide and tamoxifen) undergo systematic bioactivation by cytochrome P450, which often results in toxicity to the patient. The realization that many tumors have differential cytochrome P450 expression when compared to the corresponding normal tissue has allowed the rational design of the next generation of cytotoxic around cytochrome P450 enzymology. Several new agents now entering clinical trials (e.g. Phortress and AQ4N) are specifically designed to exploit tumor cytochrome P450, resulting in local bioactivation of the cytotoxic at the tumor site. Specific activation of pro-drugs by isoforms whose expression or particular catalytic activity is limited to cancer cells offers the possibility of truly targeted chemotherapy with minimized systemic toxicity.

Cytochrome P450 1B1: a novel anticancer therapeutic target

Cytochrome P450 (CYP)1B1 is overexpressed in tumor cells and is also recognized as a biomarker of the tumor phenotype. This review highlights the tremendous potential of this enzyme as a novel cancer therapeutic target. The range of therapeutic strategies including immunotherapeutics, CYP1B1-activated prodrugs and CYP1B1 inhibitors, that are currently being developed to exploit the presence and activity of CYP1B1 in tumor cells is outlined. The therapeutic strategy, which is at the most advanced stage of development, is a CYP1B1-based vaccine which has already successfully completed a Phase I clinical trial.

Duodenal infusion of fat, cholecystokinin secretion and satiety in the pig

The influence of the cholecystokinin (CCK) antagonist L-364,718 (0.1 mg/kg) on short-term control of food intake was studied in 6 pigs. Arterial injection of L-364,718 abolished the inhibition of intake to CCK octapeptide infusion (4 micrograms/kg/hr; from 42% p less than 0.001, to 97% of control intake), but did not alter control intake (99%). Injection of L-364,718 also abolished the inhibition of intake to duodenal infusion of emulsified fat (12 g/hr; from 76% p less than 0.001 to 105%) and of monoglyceride (24 g/hr; from 64% p less than 0.001 to 101%), but did not alter the inhibition to oleic acid (60 g/hr; 48% p less than 0.01 and 61% p less than 0.02), to glycerol (127 g/hr; 84% p less than 0.05 and 89%) or to glucose (144 g/hr; 78% p less than 0.02 and 69% p less than 0.001). These results suggest that monoglyceride-induced CCK secretion is mainly responsible for the satiety to duodenal fat in the pig, but that there is also a CCK-independent effect via the fatty acid. The results further indicate that intake of a normal barley-based diet (2% fat) is controlled via CCK-independent mechanisms.

Differential expression of CYP1A1, CYP1A2, CYP1B1 in human kidney tumours.

The presence of mRNA of individual members of the CYP1 gene family in normal and neoplastic kidney has been investigated by RTPCR. CYP1B1 mRNA was consistently expressed in both normal and neoplastic kidney while CYP1A1 was present in the majority of normal and neoplastic whereas CYP1A2 was infrequently expressed. Expression of the Ah receptor and Arnt which are involved in the transcriptional activation of the CYP1 genes was also studied. The Ah receptor mRNA and Arnt mRNA were consistently expressed both in kidney tumours and normal kidney. These results indicate differential expression of individual members of the CYP1 gene family in normal and neoplastic kidney and suggest that several mechanisms including the Ah receptor complex could be involved in their regulation.