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J F Smyth

Publications and source records attributed to J F Smyth.

At least 19 recordsLinked to original sources
PubMed1992

The consequences of doxorubicin quinone reduction in vivo in tumour tissue.

A clear role for quinone reduction in the mechanism of action of doxorubicin has still to be established. There are three possible outcomes of this form of doxorubicin metabolism: (1) drug free radical formation, redox cycling and generation of reactive oxygen species (ROS) resulting in lipid peroxidation and DNA damage; (2) covalent binding of reactive drug intermediates to DNA; and (3) formation of an inactive 7-deoxyaglycone metabolite. In this work, the occurrence of each of these pathways has been studied in vivo in a subcutaneously growing rat mammary carcinoma (Sp 107). Doxorubicin was administered by direct intratumoural injection either as the free drug or incorporated in albumin microspheres (10-40 microns diameter). There was no evidence of an increase in lipid peroxidation over background after either treatment at any time point studied. In fact, doxorubicin administration resulted in a statistically significant reduction in lipid peroxidation at the later time points studied compared to control (no drug treatment), e.g. 24 hr: control, 21.7 +/- 2.8 SD nmol malondialdehyde/g tissue; free doxorubicin (70 micrograms drug), 14.5 +/- 4.0 SD nmol/g (P < 0.01 Student's t-test) and doxorubicin microspheres (70 micrograms drug), 17.4 +/- 1.1 nmol/g (P < 0.05). Covalent binding to DNA was measured by a 32P-post-labelling technique. Low levels of four putative drug-DNA adducts were detected; however, there were no qualitative or quantitative differences in profiles between free drug and microspheres. High 7-deoxyaglycone metabolite concentrations comparable to the parent drug itself were detected after administration of microspheres (3.0 micrograms/g +/- 1.7 SD at 24 hr and 3.1 micrograms/g +/- 1.1 SD at 48 hr). In contrast, these metabolites were present at levels close to the limit of detection of our HPLC assay after free drug (0.04 microgram/g +/- 0.03 SD at 24 hr and 0.02 microgram/g +/- 0.03 SD at 48 hr). Thus, 7-deoxyaglycone metabolite formation can occur in tumour tissue (indicating active drug quinone reduction) without concomitant increases in the level of lipid peroxidation or the levels of drug-DNA adducts. In conclusion, the main biological consequence of doxorubicin quinone reduction in vivo in tumour tissue would appear to be drug inactivation to a 7-deoxyaglycone metabolite rather than drug activation to DNA reactive species or ROS.

Albumins↗
PubMed1992

The enzymology of doxorubicin quinone reduction in tumour tissue.

We have reported previously that enzymes present in the Sp 107 rat mammary carcinoma catalyse doxorubicin quinone reduction (QR) to 7-deoxyaglycone metabolites in vivo [Willmott and Cummings, Biochem Pharmacol 36: 521-526, 1987]. In order to provide insights into the role of QR in the antitumour mechanism of action of doxorubicin, we have attempted in this work to identify the enzyme(s) responsible. NAD(P)H: (quinone acceptor) oxidoreductase (DT-diaphorase) was the major quinone reductase in the tumour accounting for approximately 70% of all the activity measured in microsomes and cytosols (microsomal activity, 28.4 +/- 4.6 nmol/min/mg; cytosolic activity, 94.3 +/- 11.9 nmol/min/mg). Its presence was confirmed by western blot analysis. Low levels of NADH cytochrome b5 reductase (15.6 +/- 6.3 nmol/min/mg) and NADPH cytochrome P450 reductase (14.5 +/- 4.0 nmol/min/mg) were detectable in microsomes. The presence of the latter was confirmed by western blot analysis. Pretreatment of tumours with doxorubicin (48 hr) at a therapeutic dose decreased the level of activity of all the reductases studied by at least 2-fold (P < 0.01, Student's t-test). Doxorubicin was shown not to be a substrate for purified rat Walker 256 tumour DT-diaphorase with either NADH or NADPH as co-factor and utilizing up to 20,000 units of enzyme/incubation but was confirmed to be a substrate for purified rat liver cytochrome P450 reductase. 7-Deoxyaglycone metabolite formation by purified cytochrome P450 reductase had an absolute requirement for NADPH as co-factor, was inhibited by molecular oxygen and dicoumarol (IC50 approx. 50 microM), and modulated by specific reductase antiserum. Reductive deglycoslation of doxorubicin to 7-deoxyaglycones was localized to the microsomal fraction of the Sp 107 tumour, with negligible activity being found in cytosols (NADH, NADPH and hypoxanthine as co-factors) and mitochondria (NADH and NADPH). The tumour microsomal enzyme had an absolute co-factor requirement for NADPH, was inhibited by oxygen and dicoumarol, and modulated by cytochrome P450 reductase antiserum. These data indicate strongly that NADPH cytochrome P450 reductase is the principal enzyme responsible for catalysing doxorubicin QR in the Sp 107 tumour.

Animals↗
PubMed1992

Studies on the molecular pharmacology of GR63178A. A novel pentacyclic pyrolloquinone anticancer drug.

GR63178A (NSC D611615) is the second pentacyclic pyrolloquinone to be evaluated clinically as an anticancer drug. Its mechanism of action is unknown but may be related either to its quinone group or planar ring system. In this report we have investigated the ability of GR63178A to bind non-covalently to DNA, inhibit topoisomerase II and undergo reduction to reactive free radical species. Using two DNA duplexes, a 12-mer oligonucleotide which is a preferred sequence for minor groove binders and a hexamer which is a preferred sequence for intercalators, no evidence of significant binding with GR63178A was found. Neither GR63178A nor GR54374X (its 9-hydroxy metabolite) inhibited purified human topoisomerase II in a decatenation assay. Free radical chemistry was studied by both pulse radiolysis and ESR spectroscopy as well as by in vitro drug incubations with NADPH-fortified rat liver microsomes and purified cytochrome P450 reductase. The one-electron reduction potential of GR63178A was -207 mV +/- 10 which is much more positive than other quinone-containing anticancer drugs such as doxorubicin, mitomycin C and mitozantrone. GR63178A underwent enzyme-catalysed quinone reduction more readily than doxorubicin but produced significantly fewer reactive oxygen species. No evidence was detected of drug-induced, radical-mediated DNA damage in vitro using pBR322 plasmid DNA. Disproportionation of the GR63178A semi-quinone free radical proceeded with a rate constant of 1 x 10(9) M-1 sec-1 under anaerobic conditions, one order of magnitude faster than doxorubicin. The preferential disproportionation of the semi-quinone may explain our inability to detect a free radical signal by ESR. The hydroquinone of GR63178A was stable and exhibited strong visible absorption with a bathochromic shift of 120 nm over the parent drug. These unusual properties may be due to the hydroquinone undergoing a form of keto-enol tautomerization. Thus, GR63178A free radical formation does not appear to result in significant drug activation. In conclusion, GR63178A is unlikely to mediate its antitumour activity by DNA binding, topoisomerase II inhibition or free radical formation in direct contrast to similar anthracycline- and anthraquinone-based anticancer drugs.

Animals↗
PubMed1992

Rapid and selective isolation of radiolabelled inositol phosphates from cancer cells using solid-phase extraction.

A method is described for rapid and selective determination of radiolabelled inositol phosphates in cancer cells using solid-phase extraction with Bond Elut strong anion-exchange minicolumns. The inositol phosphates IP1, IP2 and IP3 are selectively eluted with 0.05, 0.3 and 0.8 M ammonium formate-0.1 M formic acid, respectively. Cancer cells are extracted with 10% perchloric acid which is then neutralised prior to loading samples on to the minicolumns. Recovery is 54.1, 66.6 and 61.3% for IP1, IP2 and IP3 with between-day coefficients of variation of 7.6, 6.8 and 1.9%, respectively. When the method was applied to cancer cells high-performance liquid chromatographic analyses confirmed both the identity of the IP1, IP2 and IP3 fractions and showed that there was no detectable cross contamination of these inositol phosphates with each other.

Chromatography, High Pressure Liquid↗
PubMed1992

Expression of interferon-gamma receptors on bladder cancer cells: does it correlate with biological response?

Previously we have shown a differential biological response of three human bladder cancer cell lines (RT4, RT112 and MGH-U1) to gamma interferon (IFN-gamma). The present study examines the relationship between the biological response and the expression of the interferon-gamma receptor on the tumour cell surface. Using a competitive radioligand binding assay and Scatchard analysis, we measured the number and affinity of the IFN-gamma receptors on each of the above cell lines. Individual cells from each line expressed large numbers (29,100-41,800) of high-affinity receptors (kd = 2.4-3.9 x 10(10) M). There was no statistically significant difference in either of these parameters between the three lines. We therefore conclude that the biological response of these bladder lines to IFN-gamma does not relate to the number or affinity of its receptor on the plasma membrane of these tumour cells.

Carcinoma, Transitional Cell↗
PubMed1992

A phase I and pharmacology study of GR63178A, a water-soluble analogue of mitoquidone.

GR63178A is a water-soluble analogue of mitoquidone, a pentacyclic pyrroloquinone. This group of drugs exhibit a novel structure and activity against several murine solid tumours and xenografts. In the present phase I study the toxicity and pharmacokinetics of GR63178A given on 5 consecutive days of a 21-day cycle were examined. A total of 24 patients presenting with a wide range of tumours were treated at 5 doses escalated to reach the maximal tolerated dose (MTD). Linear pharmacokinetics was documented over the dose range studied, and there was no difference in parent drug handling between day 1 and day 4 of dosing. A number of metabolites were detected. The toxicity profile was unusual in that pain occurred in 20/24 patients, most often at the site of known disease. This was the dose-limiting toxicity. Other side effects included nausea and vomiting (23/24), phlebitis at the infusion site (6/24) and headache (7/24). No treatment response was seen in this study. The MTD was demonstrated to be 160 mg/m2 daily (total, 800 mg/m2 per treatment cycle). The drug has now entered phase II trials at 120 mg/m2 daily x 5, repeated every 21 days.

Adult↗
PubMed1992

The long-term retention of platinum in human tissues following the administration of cisplatin or carboplatin for cancer chemotherapy.

Mass spectrometry has been used to study the distribution and retention of platinum in the tissues of patients following the administration of cisplatin or carboplatin. Blood platinum was measured up to 2 years and renal excretion up to 5 years after treatment. Platinum concentrations in plasma and red cells fell according to a power function, approximately as the inverse square of the time after administration. The concentration in the urine fell more slowly. Necropsy samples were used to examine the distribution of platinum in various human organs up to 17 months after treatment. The highest concentrations were found in the liver, which retained approximately 2% of the dose. Although the results were scattered between patients, there was little loss of platinum after about 1 month. The prolonged retention of platinum may be relevant to long-term toxicity.

Adolescent↗
PubMed1992

The inhibitory effects of interferon gamma on the growth of bladder cancer cells.

The inhibitory effect of interferon-gamma on the growth of three human bladder cancer cell lines, RT4, RT112 and MGH-U1, representing tumour grades 1, 2 and 3 respectively, was studied. The effects of 10, 100 and 1000 Uml.-1 of interferon-gamma on cell numbers and thymidine incorporation were measured at 24 hour intervals up to a maximum of seven days. Morphological appearances were also studied. Each line was susceptible to the growth inhibitory effects of interferon-gamma and this was both dose and time dependent. The effects of interferon-gamma, on the RT4 and RT112 cells were apparent from 24 hours, and were both cytostatic and cytotoxic in nature, whereas the effects on MGH-U1 cells were seen from 48 hours onwards and were only cytostatic. Cytological changes occurred in all three cell lines, being most pronounced in RT112. The growth of bladder cancer cells was inhibited by interferon-gamma, and in this study high grade tumour cells were least sensitive.

Carcinoma, Transitional Cell↗
PubMed1992

Intravesical Evans strain BCG therapy: quantitative immunohistochemical analysis of the immune response within the bladder wall.

Previous studies have demonstrated that is is the local immune response which is of importance for the anti-tumour activity of BCG therapy. We have investigated this by quantitative immunohistochemical analysis of serial bladder mucosal biopsies taken before, during and after an eight week course of intravesical Evans strain BCG therapy and three monthly thereafter in 16 patients (15 extensive CIS and one extensive G2pTa papillary tumour). This particular group of patients had a 67% complete response rate at six months post-treatment. The main findings on immunohistochemical analysis were the universal induction of MHC Class II antigens by urothelial cells which was statistically significant up to 6 months after completion of therapy, coupled with a T cell dominated cystitis. Increases in CD3+ T cell infiltration of the lamina propria and that of the CD4+ "Helper" subset which predominated were significant up to 3 months post-therapy and these cells showed evidence of increased immunological activation as shown by increased interleukin-2 receptor and MHC Class II antigen expression. There were also significant increases in CD68+ macrophage and the incidence of CD22+ B cell aggregates but CD57+ NK cells were sparse both before and after therapy. The degree of mononuclear cell infiltration for all markers examined (except CD57) was significantly greater in those biopsies in which the urothelial cells expressed MHC Class II antigens than in those that did not. Also the degree of T cell infiltration (CD3, CD4 and CD8) was significantly greater in the eight patients deemed to have had a complete response compared to those seven with a partial response or treatment failure. These results are discussed in terms of possible mechanisms of action for BCG therapy and in particular the role of enhanced antigen presentation by tumour cells.

Administration, Intravesical↗
PubMed1992

Transforming growth factor-beta mRNA expression and growth control of human ovarian carcinoma cells.

The pattern of TGF beta expression and in vitro response to TGF beta has been defined in three ovarian carcinoma cell lines (PEO1, PEO4 and PEO14). Marked differences in both mRNA expression and growth responses were detected between the cell lines. All expressed mRNA for TGF beta 3, PEO1 and PEO4 but not PEO14 expressed mRNA for TGF beta 1, whereas PEO14 but not PEO1 and PEO4 expressed TGF beta 2. Growth of PEO14 cells in culture was markedly inhibited by both TGF beta 1 and beta 2. PEO1 cells were inhibited by TGF beta 1, but not TGF beta 2 whilst growth of PEO4 cells were not affected by exposure to either of these peptides. These data indicate that several elements of potential autocrine loops involving TGF beta's are present within ovarian cancer cells.

Cell Cycle↗
PubMed1992

Growth control by epidermal growth factor and transforming growth factor-alpha in human lung squamous carcinoma cells.

Although EGF receptor expression is generally elevated in human lung squamous carcinoma, the biological significance of this phenomenon and the role of EGF and TGF-alpha in this disease are poorly understood. We have investigated three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) and have shown, using an antibody (EGFR1) directed against the EGF receptor, that the majority of cells in all three lines express the EGF receptor. Using a ligand binding assay, Scatchard analysis indicated high concentrations (1,300-2,700 fmol mg-1 protein) of a single low affinity binding site (Kd = 3-5 nM) within these lines. Addition of EGF or TGF-alpha at concentrations greater than 0.1 nM resulted in growth inhibition of all three lines and this was associated with an accumulation of cells in the G2/M phase of the cell cycle. Growth inhibitory effects were not explained by an enhancement of cellular differentiation as monitored by involucrin expression and the ability to form cornified envelopes. While the presence of EGF could not be detected in medium conditioned by the NX002 cell line, mRNA for TGF-alpha was detected in all three lines suggesting the possibility of an autocrine loop. These results together with reports of growth inhibition by EGF and TGF-alpha in other systems suggest that EGF and similar molecules might have a growth regulatory role in lung cancer cells and modulation of such may have therapeutic potential.

Carcinoma, Squamous Cell↗
PubMed1992

Phase II trials of fosquidone (GR63178A) in carcinoma of the breast, head and neck, ovary and melanoma.

A total of 91 eligible patients with metastatic cancer have been treated in a series of phase II trials of the novel pentacyclic pyrroloquinone, fosquidone. Tumour types were breast (24), ovary (25), head and neck (21) and melanoma (21). All patients, except those with melanoma had received prior chemotherapy. The drug was given intravenously as a 20 min infusion, at the dose of 120 mg/m2 on days 1 to 5 of a 3 week cycle. Treatment was well tolerated; the only significant side-effects being mild headaches and generalised musculo-skeletal pains. Response was assessed after 2 cycles of therapy. Only one patient (with head and neck cancer) achieved an objective partial response, lasting 6 weeks. A total of 12 patients demonstrated stable disease for a median duration of 15 to 20 weeks. Using this schedule of administration, fosquidone has no significant antitumour activity in this group of tumours.

Adult↗
PubMed1992

A phase II study of sulofenur (LY186641) in gastric cancer.

Sulofenur is a novel diarylsulfonylurea with proven anti-tumor activity in murine tumor models. In this phase II study in patients with advanced gastric or gastroesophageal adenocarcinoma, 17 patients were treated with sulofenur orally at 700 mg/m2 for 14 days every 3 weeks. No tumor responses were seen. The main toxicities were anemia, methemoglobinemia and abnormalities in liver function tests. These toxicities precluded dose escalation. However, plasma levels of sulofenur, and its hydroxy and keto metabolites were probably insufficient to exert anti-tumor effect in comparison with data from murine studies. Further structure-activity studies are warranted.

Adenocarcinoma↗
PubMed1992

Doxorubicin-loaded casein microspheres: protein nature of drug incorporation.

We have studied incorporation of [14C]doxorubicin within protease-sensitive casein microspheres both by 14C-activity, measuring total drug, and HPLC, measuring free drug only. It was found that total drug content (27.7 micrograms mg-1) exceeded free drug content (3.2 micrograms mg-1) suggesting that the major portion of doxorubicin was incorporated via a covalent linkage to matrix protein. In-vivo drug disposition and activity studies suggested that this fraction of doxorubicin was the major species within tumour tissue (total vs free: 5 min, 14.3 micrograms g-1 vs 0.7 micrograms g-1; 24 h, 11.7 micrograms g-1 vs 1.1 micrograms g-1; 48 h, 11.2 micrograms g-1 vs 1.2 micrograms g-1; 72 h, 10.0 micrograms g-1 vs 0.8 micrograms g-1), did not exhibit a 'burst' effect, was slowly cleared (30% loss over 3 days), and was equiactive (growth delay = 12 days) compared with drug in solution (growth delay = 10 days). This work clearly implicates in-vivo microsphere matrix biodegradation in drug release and subsequent disposition and activity.

Animals↗
PubMed1992

Malignant melanoma.

The incidence of malignant melanoma in some parts of the world continues to increase at a surprising rate. The medical profession is increasingly aware of the prognostic implications of tumour thickness, site and the potential value of early detection. Surgery for thin lesions remains overwhelmingly the most valuable therapy, and despite the very considerable endeavours of research groups around the world there is little sight of significant progress in increasing the survival of patients with more advanced disease. The reports summarised in this and previous annuals confirm the very disappointing results of cytotoxic drugs. The results referred to in this review establish that modern immunology is capable of devising much more specific immunotherapy than was tried in the early days of BCG and C. parvum; notwithstanding the considerable morbidity of some biological response modifications, it remains to be shown that the response rates achieved can provide useful extension of survival.

Chemotherapy, Cancer, Regional Perfusion↗
PubMed1991

Does dexamethasone enhance control of acute cisplatin induced emesis by ondansetron?

OBJECTIVE: To determine the contribution of dexamethasone to the efficacy of the 5-hydroxytryptamine antagonist ondansetron in control of cisplatin induced nausea and vomiting. DESIGN: Randomised double blind crossover study. SETTING: Two cancer centres in teaching hospitals, one in the United Kingdom and the other in Germany. SUBJECTS: 100 patients (53 men and 47 women) new to cisplatin chemotherapy, 84 of whom completed two consecutive courses of chemotherapy. INTERVENTIONS: Patients were given intravenous dexamethasone (20 mg) or physiological saline with intravenous ondansetron 8 mg before cisplatin, then ondansetron 1 mg/h for 24 hours. Oral ondansetron 8 mg was taken three times daily on days 2-6. MAIN OUTCOME MEASURES: Incidence of complete or major control of emesis (0-2 episodes in the 24 hours after chemotherapy). RESULTS: Complete or major control was obtained in 49 out of 71 (69%) of patients after receiving ondansetron plus dexamethasone compared with 40 out of 71 (56%) when they were given ondansetron alone (p = 0.012). This effect was most pronounced in the first 12 hours after chemotherapy. Patients receiving the combination also had significantly less nausea. Of the 53 patients who expressed a preference, 38 (72%) preferred the combination treatment (p = 0.002) to ondansetron alone. The effect of ondansetron on delayed emesis was less pronounced. CONCLUSIONS: Dexamethasone makes a significant contribution to the efficacy of ondansetron in the control of acute platinum induced emesis.

Acute Disease↗
PubMed1991

The role of protein kinase C and the phosphatidylinositol cycle in multidrug resistance in human ovarian cancer cells.

The present study aimed to investigate the role of protein kinase C (PKC), the phosphatidylinositol pathway (PI) and cytosolic calcium in multidrug resistance (MDR) in human ovarian carcinoma cells. Binding of the phorbol ester 13,14-dibutyrate (PDBu) was 3-fold higher in resistant A2780AD versus sensitive A2780 cells indicating increased PKC activity. However, when inositol phosphate production (IP) was measured in quiescent cells similar total IP release was seen in both lines suggesting no difference in the basal turnover of PI. Non-specific stimulation of the PI pathway was achieved with the calcium ionophore A23187 which increased IP production in a time- and dose-dependent fashion in both cell lines but was significantly less effective in A2780AD. The PI pathway was investigated further using the agonists aluminium fluoride, serum and bombesin but these agents failed to elicit a response. The effect of a wide range of Adriamycin concentrations on the PI cycle and cell growth was also studied. Intracellular calcium was measured with the fluorescent dye fura-2-pentaacetoxymethylester (Fura-2). A23187 produced a rise in cytosolic calcium in A2780 and A2780AD but from a level 3-fold lower in the unstimulated resistant cell line. The dose responsiveness of this effect was greater but irreversible in A2780AD cells. Collectively these results imply that alterations in PI turnover appear not to be responsible for the differences in PDBu binding and calcium handling observed between A2780 and A2780AD and suggests only a minor role for the PI cycle in the maintenance of MDR in human ovarian cancer cell lines.

Calcimycin↗