Biomedical subjects

Gary Boyd

Publications and source records attributed to Gary Boyd.

16 recordsLinked to original sources

PubMed2004

Glucuronidation as a mechanism of intrinsic drug resistance in colon cancer cells: contribution of drug transport proteins.

We have recently shown that drug conjugation catalysed by UDP-glucuronosyltransferases (UGTs) functions as an intrinsic mechanism of resistance to the topoisomerase I inhibitors 7-ethyl-10-hydroxycamptothecin and NU/ICRF 505 in human colon cancer cells and now report on the role of drug transport in this mechanism. The ability of transport proteins to recognise NU/ICRF 505 as a substrate was evaluated in model systems either transfected with breast cancer-resistance protein 1 (Bcrp1), multidrug-resistance protein 2 (Mrp2) or Mrp3, or overexpressing MRP1 or P-170 glycoprotein. Results from chemosensitivity assays suggested that NU/ICRF 505 was not a substrate for any of the above proteins. In drug accumulation studies in human colon cancer cell lines NU/ICRF 505 was taken up avidly and retained in cells lacking UGTs (HCT116), whereas, following equally rapid uptake, it was cleared rapidly from cells displaying UGT activity (HT29) as glucuronide metabolites. HT29 cells were shown to express MRP1 and 3, but not P-170 glycoprotein, MRP2 or breast cancer-resistance protein. The major glucuronide of NU/ICRF 505 inhibited ATP-dependent transport of estradiol 17-beta-glucuronide in Sf9 insect cell membrane vesicles containing MRP1 or MRP3, while co-incubation of HT29 cells with the MRP antagonist, MK571, significantly restored intracellular concentrations of NU/ICRF 505. These data lead us to conclude that the presence of a glucuronide transporter is essential for glucuronidation to represent a major de novo resistance mechanism and that UGTs will contribute more as a primary resistance mechanism when the parent drug (e.g. NU/ICRF 505) is not itself recognised by transport proteins.

Anthraquinones↗

PubMed2003

Glucuronidation as a mechanism of intrinsic drug resistance in human colon cancer: reversal of resistance by food additives.

Colon cancer exhibits inherent insensitivity to chemotherapy by mechanisms that are poorly characterized. We have shown that human colon cancer cells are efficient in drug conjugation catalyzed by UDP-glucuronosyltransferases (UGTs) and now report on the role of glucuronidation in de novo resistance to two topoisomerase I inhibitors. Identification of the UGT responsible for glucuronidation of SN-38 and the anthraquinone NU/ICRF 505 was achieved by first using a panel of human cDNA-expressed isozymes to measure conjugating activity. HT29 colon cancer cells were then probed by reverse transcriptase-PCR, Western Blot analysis, and liquid chromatography with mass spectrometry for their profile and activity of UGT isozymes and screened for effective inhibitors of glucuronidation. Expression analysis was also conducted in colon cancer biopsies and paired adjacent normal colon specimens. UGT1A9 was identified as the isozyme catalyzing biotransformation of the two compounds in HT29 cells and propofol as an effective competitive inhibitor of this metabolism. Inhibition of glucuronidation resulted in up to a 5-fold enhancement in drug activity. The majority of colon cancer biopsies studies expressed UGT protein at levels greater than in HT29 cells but with marked interpatient variations and proficiently glucuronidated the two anticancer drugs. A range of UGT aglycones were capable of modulating glucuronidation in the biopies with octylgallate being 10-fold more potent (ID(50) 24 microM) than propofol. In a subset of tumors (33%), UGT protein levels and activity exceeded that of paired normal colon. Glucuronidation may represent a mechanism of intrinsic drug resistance in colon cancer open to modulation by a range of food additives and proprietary medicines.

Anthraquinones↗

PubMed2002

Enhanced clearance of topoisomerase I inhibitors from human colon cancer cells by glucuronidation.

As part of a program to identify novel mechanisms of resistance to topoisomerase I (topo I) inhibitors, the cellular pharmacology of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of clinically used irinotecan (CPT-11) and NU/ICRF 505, an anthraquinone-tyrosine conjugate, has been investigated in two human colorectal cancer (CRC) cell lines. Two novel metabolites of NU/ICRF 505 (M1 and M2) and a single metabolite of SN-38 (M1) were detected by high performance liquid chromatography in the culture medium of HT29 cells but were absent in HCT116 cells. Identities of all three metabolites were established by a combination of biochemical and physicochemical techniques. M1 of SN-38 was the C10-(beta)-glucuronide of the parent lactone while M1 of NU/ICRF 505 was the C4-O-glucuronide and M2 the tyrosine-O-glucuronide, both of the parent compound. Drug transport studies revealed that by 24hr HT29 cells had effectively cleared 82.5% of NU/ICRF 505 (10 microM) into the culture medium as the two glucuronides. In contrast, intracellular concentrations of NU/ICRF 505 were maintained in HCT116 cells in the absence of glucuronidation at a level 550 times greater than in HT29 cells. HT29 cells cleared 40.9% of SN-38 (1 microM) as the glucuronide to the culture medium, while the parent drug was maintained at a level 2-fold greater in HCT116 cells. Enhanced drug clearance due to glucuronidation may contribute to intrinsic drug resistance of human CRC.

Anthraquinones↗

PubMed2002

Factors influencing the cellular accumulation of SN-38 and camptothecin.

PURPOSE: The influence of biophysical factors (drug metabolism, transport proteins, and chemical stability) on the cellular accumulation of camptothecin (CPT) and SN-38 was examined. METHODS: Drug transporter RNA transcript levels were measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Intracellular drug accumulation, metabolism, and drug stability studies were all performed by HPLC. RESULTS: A panel of three human cell lines exhibiting different drug resistant phenotypes was investigated. HT29 colon cells glucuronidated SN-38 but did not express P-gp or MRP1 or 2. HCT116 colon cells expressed P-gp and MRP2 but did not catalyse conjugation. A2780 ovarian cells neither catalysed drug metabolism nor contained these drug transporters. In all lines, SN-38 lactone was rapidly taken up achieving peak concentrations at the earliest time point studied (5 min, 3.3-4.1 ng/10(6) cells). Subsequently, a fall in intracellular lactone concentration occurred, stabilising after 4 h at 0.48-1.18 ng/10(6) cells. No significant differences in intracellular levels of lactone were observed between the three cell lines with one exception: a twofold increase in HCT116 cells at 24 h. Stability studies in culture medium revealed that SN-38 lactone concentrations disappeared at the same rate regardless of whether cells were present, initially falling to reach equilibrium with the hydroxy acid by 4 h. Indeed, changes in intracellular lactone concentrations followed closely chemical stability profiles in media. Similar patterns of cellular retention and chemical degradation were observed with CPT. CONCLUSION: The major determinant of drug accumulation in three diverse cell line phenotypes was lactone chemical stability in culture medium.

Adenocarcinoma↗

PubMedDate not supplied