Cytotoxicity of ketoconazole in malignant cell lines

Summary The cytotoxic effects of ketoconazole, an antifungal agent known to have some activity against human prostate cancer, adrenal cancer, and male metastatic breast cancer, were evaluated using colony-growth and clonogenic assays in eight malignant cell lines. The cytotoxicity of ketoconazole showed a dose-and time-dependent pattern, with the following concentrations, inhibiting 90% of the growing colonies (IC₉₀): MCF 7 (human breast cancer) 7.25 g/ml, T 47 D (human breast cancer) 9.0 g/ml, MiaPaCa (human pancreatic carcinoma) 10.0 g/ml, COLO 357 (human pancreatic carcinoma) 9.5 g/ml, HCT 8 (human colonic adenocarcinoma) 27.1 g/ml, DU 145 (human prostatic cancer) 40.0 g/ml, AR 42 J (rat pancreatic carcinoma) 9.0 g/ml, and L1210 (murine leukemia) 8.6 g/ml. Since a concentration of 10 g/ml can be achieved in humans, the use of ketoconazole in human malignancies might be worthy of clinical evaluation.This investigation was supported in part by a grant from the German Volkswagen Foundation, Hannover, Federal Republic of Germany and by a gift from Dr Virgil Gianelli, Stockton, California     

Pharmacokinetic basis for the comparative antitumour activity and toxicity of chlorambucil,phenylacetic acid mustard and β,β-difluorochlorambucil (CB 7103) in mice

Summary This report describes the relationship between the pharmacokinetics, antitumour activity and toxicity of chlorambucil (CHL), phenylacetic acid mustard (PAAM) and ,-difluorochlorambucil (-F₂CHL) in mice. Pharmacokinetics were studied by HPLC, antitumour activity by a regrowth delay assay using the KHT murine sarcoma and toxicity by acute LD₅₀. For both antitumour activity and acute toxicity the order of potency was: PAAM>CHL>-F₂CHL. CHL and PAAM exhibited identical therapeutic indices, whereas that for -F₂CHL was somewhat improved. CHL is metabolized by mitochondrial -oxidation to the 3,4-dehydro derivative (DeHCHL) and PAAM, and the latter is further metabolized to its monodechloroethylated derivative DeC-PAAM, presumably by hepatic microsomal enzymes. Administered PAAM gave only one metabolite, DeC-PAAM. Unexpectedly, despite ,-disubstitution, -F₂CHL was also -oxidized to give DeHCHL and PAAM, but at reduced rates. Further, metabolic switching was demonstrated with the appearance in large amount of 2 new, unidentified metabolites, which may be dechlorethylation products. The pharmacokinetics of administered CHL, PAAM and -F₂CHL differ in that the plasma clearance was fastest for CHL, slowest for PAAM and intermediate for -F₂CHL. For the metabolites, CHL produced peak plasma concentrations of DeHCHL and PAAM, respectively, 7-fold and 2-fold greater than those produced by -F₂CHL. However, despite these differences, exposures to total bifunctional nitrogen mustards were similar following administration of the 3 drugs and therefore cannot account for their differential activity. In contrast, there was a good correlation between potency and PAAM exposure, which is highest after treatment with PAAM, intermediate after CHL and lowest after -F₂CHL. In plasma, 3.2% of PAAM is present as nonprotein-bound free drug, compared to 1.3% for DeHCHL, 0.9% for CHL and 0.45% for -F₂CHL. We propose the amount of free bifunctional nitrogen mustard, itself partly dependent on the extent of metabolism, to be of major importance for the in vivo potency of CHL analogues.     

START: A European state-of-the-art on-line instrument for clinical oncologists

START (State-of-the-Art Oncology in Europe), a freely available resource on the Internet, is a European information base of current clinical approaches to human tumours. Its aim is to help clinical oncologists make appropriate clinical decisions by providing them with updated information reflecting state-of-the-art cancer treatment as perceived by the European oncology community. It is based upon contributions from authors and internal reviewers from all over Europe as selected by START's editorial board under the supervision of an advisory board and a scientific committee. Close collaborations with the main European cancer societies are ongoing. An external feedback process augments the mechanisms for rendering START a truly European instrument. START is concerned with evidence-based cancer medicine, and the main clinical options are thus codified and their bases indicated in accord with a scale worked out from the perspective of clinical decision-making. Therapeutic options may be standard, investigational, or suitable for individual clinical use (within the context of a decision made jointly by the patient and the physician). The goal of instruments such as START is to improve the quality of patient care. In addition, START hopes to make contributions to the methodology by which medical research is transformed into clinical decisions.     

A pilot study of neoadjuvant chemotherapy with mitomycin C,etoposide, cisplatin,and epirubicin for adenocarcinoma of the cervix

Background To evaluate the efficacy and toxicity of the combination of mitomycin C, etoposide, cisplatin, and epirubicin (MEPA) as neoadjuvant therapy for patients with cervical adenocarcinoma.Methods Fourteen patients with cervical adenocarcinoma received neoadjuvant MEPA therapy followed by radical hysterectomy. The International Federation of Gynecology and Obstetrics stage was: IB1 in 2 patients, IB2 in 5, and IIB in 7. The MEPA regimen consisted of mitomycin C (15mg/m²) on day 1, etoposide (70mg/m²) on days 1 to 3, cisplatin (15mg/m²) on days 1 to 5, and epirubicin (30mg/m²) on day 1, with this course being repeated every 4 weeks. After two or three courses of chemotherapy, all patients underwent radical hysterectomy. Postoperative radiotherapy was given to 6 patients who showed risk factors at surgery.Results Of the 14 patients, 7 had complete remission (CR) clinically, 6 had partial remission, and only 1 showed no change. Examination of surgical material revealed no residual disease in 6 patients, and microscopic residual disease (5mm) in 2 patients. The patients who had no residual disease or microscopic disease in their hysterectomy specimens showed a significantly longer survival than those with macroscopic residual disease (P = 0.012). The dose-limiting toxicity was myelosuppression. Of the 33 treatment cycles administered, leukopenia of grade 3 or more occurred in 70%, and thrombocytopenia of grade 3 or more occurred in 79%. There were no therapy-related deaths.Conclusion Although severe myelosuppression was also observed, there was a satisfactory response rate to MEPA therapy, which showed a good pathological CR rate.     

Noninvasive fluorine-19 NMR study of fluoropyrimidine metabolism in cell cultures of human pancreatic and colon adenocarcinoma

Summary Fluorine-19 NMR spectrometry was used to monitor the metabolism of two antineoplastic fluoropyrimidines, 5-fluorouracil (5FU) and 5-deoxy-5-fluorouridine (5dFUrd), in cell cultures of human pancreatic (Capan-1) and colon (HT-29) adenocarcinoma. The preliminary results showed, for the two tumor cell lines treated with 5FU, the presence in nonperfused cells of three signals corresponding to intracellular metabolites: 5FU, F-nucleotides and F-nucleosides. When the cells were perfused only the signals of F-nucleotides and 5FU were present. The F-nucleosides observed during the analysis of the nonperfused cells came from the conversion of F-nucleotides. During the NMR recording of Capan-1 cells at 37 °C the first metabolite of the catabolic pathway of 5FU, 5,6-dihydro-5-fluorouracil, occurred. At the beginning of the NMR recording of Capan-1 cells treated with 5dFUrd, two signals corresponding to F-nucleotides and F-nucleosides (consistent with 5dFUrd) were observed; during the analysis, a supplementary signal corresponding to 5FU appeared. Even after pretreatment with methotrexate the signal of 5FU incorporated into RNA was not detected. Our experiments, performed in attempts to observe the signal of the ternary complex between thymidylate synthetase (TS), 5-fluoro-2-deoxyuridine-5-monophosphate (FdUMP) and 5,10-methylene-tetrahydrofolate (5,10-CH₂FH₄), allowed detection in some cases of a broad signal, whose chemical shift was similar to that reported in the literature following incubation of TS with FdUMP and 5,10-CH₂FH₄, but our results were not always reproducible.This study was supported by grants from Université Paul Sabatier, Conseil Régional Midi-Pyrénées-CNRS and Caisse Nationale de l'Assurance Maladie des Travailleurs Salariés (INSERM)     

Gastroesophageal reflux disease, use of H2 receptor antagonists, and risk of esophageal and gastric cancer

Objective: The incidence of esophageal adenocarcinoma has risen rapidly in the past two decades, for unknown reasons. The goal of this analysis was to determine whether gastroesophageal reflux disease (GERD) or the medications used to treat it are associated with an increased risk of esophageal or gastric cancer, using data from a large population-based case–control study. Methods: Cases were aged 30–79 years, newly diagnosed with esophageal adenocarcinoma (n=293), esophageal squamous cell carcinoma (n=221), gastric cardia adenocarcinoma (n=261), or non-cardia gastric adenocarcinoma (n=368) in three areas with population-based tumor registries. Controls (n=695) were chosen by random digit dialing and from Health Care Financing Administration rosters. Data were collected using an in-person structured interview. Results: History of gastric ulcer was associated with an increased risk of non-cardia gastric adenocarcinoma (OR 2.1, 95% CI 1.4–3.2). Risk of esophageal adenocarcinoma increased with frequency of GERD symptoms; the odds ratio in those reporting daily symptoms was 5.5 (95% CI 3.2–9.3). Ever having used H₂ blockers was unassociated with esophageal adenocarcinoma risk (OR 0.9, 95% CI 0.5–1.5). The odds ratio was 1.3 (95% CI 0.6–2.8) in long-term (4 or more years) users, but increased to 2.1 (95% CI 0.8–5.6) when use in the 5 years prior to the interview was disregarded. Risk was also modestly increased among users of antacids. Neither GERD symptoms nor use of H₂ blockers or antacids was associated with risk of the other three tumor types. Conclusions: Individuals with long-standing GERD are at increased risk of esophageal adenocarcinoma, whether or not the symptoms are treated with H₂blockers or antacids.     

Detection of Adriamycin-induced cardiotoxicity in cultured heart cells with technetium 99m-SESTAMIBI

Adriamycin, a broad-spectrum cytotoxic agent useful in cancer chemotherapy, is limited by a dose-dependent cardiomyopathy mediated in part by disruption of mitochondrial energetics. Hexakis(2-methoxyisobutyl isonitrile)technetium(I)(^99mTc-SESTAMIBI) is a gammaemitting radiopharmaceutical with myocellular accumulation properties dependent on mitochondrial membrane potential. To test the hypothesis that^99mTc-SESTAMIBI could monitor Adriamycin-induced alterations in cardiac energetics, cultured chick heart cells were treated with Adriamycin and^99mTc-SESTAMIBI tracer kinetics were determined. Concentration- and time-dependent depression of^99mTc-SESTAMIBI accumulation was evident within 60 min of treatment. The apparentK _i for acute Adriamycin inhibition of tracer accumulation was 82 M. After 24 h of treatment, Adriamycin concentrations as low as 0.1 M demonstrated detectable inhibitory effects. The apparentK _i for this subchronic Adriamycin inhibition of^99mTc-SESTAMIBI accumulation was 18 M. Subchronic concentration-dependent increases in adriamycin-induced myocellular injury as reflected by lactate dehydrogenase (LDH) release correlated inversely with decreases in^99mTc-SESTAMIBI accumulation. These data further support a contribution from altered mitochondrial energetics to Adriamycin-induced injury and establish a pharmacological foundation for pursuing the possibility of noninvasive imaging of chronic Adriamycin cardiotoxicity in cancer patients using^99mTc-SESTAMIBI.     

Peripheral Neuropathy Due to Biweekly Paclitaxel,Epirubicin and Cisplatin in Patients with Advanced Ovarian Cancer

We assessed the peripheral neuropathic changes induced by biweekly combination chemotherapy including paclitaxel 100–165mg/m² (in a 3-h infusion), epirubicin 75mg/m² and cisplatin 50mg/m² (TEC) in patients with advanced ovarian cancer.Neurologic evaluation, including a standardized questionnaire, bed-side neurological examination, and quantitative determination of vibratory perception thresholds (VPT) and grip strength took place before therapy, after 3 and 6 cycles, and thereafter whenever possible. During chemotherapy all patients received granulocyte colony-stimulating factor from days 2 to 12. Pretreated patients received amifostine two times, before epirubicin and before cisplatin administration.Neuropathic symptoms developed in 11/13 non-pretreated patients and in 7/9 chemotherapy-pretreated patients. Neuropathic signs developed in all patients. Neuropathic symptoms and signs were predominantly sensory in character. VPT changes developed primarily in the feet. According to National Cancer Institute of Canada Common Toxicity Criteria, grade 3 peripheral neuropathy after 6 cycles developed in 1/6 and 2/4 non-pretreated patients who received TEC containing paclitaxel 150 and 165mg/m², respectively.We conclude that peripheral neuropathy is dose-limiting in chemonaïve patients treated with biweekly TEC combination chemotherapy, at paclitaxel dose level 165mg/m² in a 3-h intravenous administration.     

Metabolism of taxol by human and rat liver in vitro: A screen for drug interactions and interspecies differences

Human liver slices, human liver microsomes, and rat liver microsomes were used to investigate the metabolism of³H-taxol. The effects of drugs frequently coadministered with taxol and the effects of several cytochrome P450 system probes were studied. In all, 16 compounds were screened. After incubation with liver slices or with microsomal protein,³H-taxol was converted into several radioactive species resolved by HPLC. There were qualitative and quantitative species differences in the metabolism of taxol. The pattern of metabolism was similar for both human-derived preparations, with 6-hydroxytaxol being the major metabolite peak. In drug interaction studies performed with human liver microsomes, cimetidine 80 M, and diphenhydramine 200 M, had little or no effect on 6-hydroxytaxol formation. Quinidine, ketoconazole, dexamethasone and Cremophor EL inhibited 6-hydroxytaxol formation with IC₅₀ values of 36 M, 37 M, 16 M and 1 l/ml, respectively, but these concentrations exceed the usual clinical range. Cremophor EL also inhibited microsomal metabolism of taxol, but at 2 l/ml it had little or no effect on 6-hydroxytaxol production by human liver slices. These results suggest that: (1) taxol is metabolized by the cytochrome P450 system; (2) taxol metabolism is different in humans than in rats; (3) taxol metabolism in humans is unlikely to be altered by cimetidine, dexamethasone, or diphenhydramine, drugs regularly coadministered with taxol; (4) taxol metabolism can be indirectly affected by Cremophor EL, the formulation vehicle; (5) taxol metabolism may be altered by concentrations of ketoconazole achievable in humans only at very high doses; and (6) taxol metabolism and drug interaction studies of clinical relevance can be performed in vitro with human liver microsomes and human liver slices, but not with rat liver preparations.Part of this work was presented in poster form at the 84th Annual Meeting of the American Association for Cancer Research, Orlando, Fl., USA, 1993     

Multiple actions of synthetic ‘progestins’ on the growth of ZR-75-1 human breast cancer cells: Anin vitro model for the simultaneous assay of androgen,progestin, estrogen,and glucocorticoid agonistic and antagonistic activities of steroids

This study was designed to assess the multiple steroid receptor mediated activities of a series of synthetic progestins on breast cancer cell growth, using the human ZR-75-1 cell line which possesses functional estrogen (ER), androgen (AR), and glucocorticoid (GR) receptors as well as progesterone (PgR) receptors. Four 17-hydroxyprogesterone derivatives (chlormadinone acetate, CMA; cyproterone acetate, CPA; medroxyprogesterone acetate, MPA; and megestrol acetate, MGA) and two 19-nortestosterone derivatives (norethindrone, NRE, and norgestrel, NRG) were thus investigated.Based on the requirement of estrogens for PgR-mediated antiproliferative effects and the reversal of PgR-mediated action by insulin, it was found that although all progestins could inhibit ZR-75-1 cell growth through the PgR at low concentrations, the relative contribution of this receptor in cell growth control is highly variable between compounds. The quantitative importance of PgR-mediated inhibition of cell proliferation was inversely related to the amplitude of the androgenic effects induced by the compounds, the AR-mediated effects increasing in the order CPA < MGA < CMA < NRE < NRG < MPA. The specificity of these androgenic effects is further supported by their reversal upon addition of the antiandrogen hydroxyflutamide. In addition, the 17-hydroxyprogesterone derivatives, but not the 19-nortestosterone derivatives, had glucocorticoid activities at high (micromolar) concentrations, as shown by reversal of growth inhibition by the antagonist RU486 in the presence of saturating concentrations of 5-dihydrotestosterone. All progestins tested, except MPA and NRE, also had some antiglucocorticoid activity, NRG being the most potent in this respect. Finally, NRE and NRG exerted a marked mitogenic effect in estrogen-free medium which was clearly mediated through the ER as shown by the competitive reversal of their action by the steroidal antiestrogen EM-139.The present results show that growth measurements of the human breast cancer cells ZR-75-1 permit, with the appropriate steroid additions, the assay of progestin, androgen, estrogen, and glucocorticoid agonistic as vell as antagonistic activities of test compounds. The present study shows, somewhat surprisingly, that while the AR is almost completely responsible for the action of MPA at low concentrations, the majority of the action of NRE, NRG, and MGA is also exerted through AR, while the androgenic action of CPA plays a lower role in the growth inhibition induced by this compound. Such a model should be of great help in designing more specific steroid drugs and in better understanding the role of the different steroid classes which can be used to control the growth of hormone-sensitive cancer. The present data also indicate that progestin is an inappropriate name for MPA, NRE, NRG, MGA, CMA, and CPA, which all possess other and sometimes more potent steroidal activites than those related to interaction with the progesterone receptor.Abbreviations CMA chlormadinone acetate [17-acetoxy-6-chloropregna-4, 6-dien-3, 20-dione] - CPA cyproterone acetate [17-acetoxy-6-chloro-1,2-methylene-pregna-4, 6-dien-3, 20-dione] - DEX dexamethasone [9-fluoro-11, 17, 21-trihydroxy-16-methyl-pregna-1, 4-dien-3, 20-dione] - DHT 5-dihydrotestosterone [17-hydroxy-5-androstan-3-one] - E₂ estradiol [estra-1, 3, 5 (10)-trien-3, 17-diol] - EM 139 [N-n-butyl-N-methyl-11-(16-chloro-3, 17-dihydroxyestra-1, 3, 5 (10)-triene-7-yl) undecanamide] - MGA megestrol acetate [17-acetoxy-6-methylpregna-4, 6-dien-3, 20-dionel] - MPA medroxyprogesterone acetate [17-acetoxy-6-methylpregn-4-en-3, 20-dione] - NRE norethindrone [17-hydroxy-19-nor-17-pregn-4-en-20-yn-3-one] - NRG norgestrel [13-ethyl-17-hydroxy-18, 19-dinor-17-pregn-4-en-20-yn-3-one] - OHF hydroxyflutamide (SCH 16423) [, , -trifluoro-2-methyl-4-nitro-m-lactotoluidide] - R1881 methyltrienolone [17-hydroxy-17-methyl estra-4, 9, 11-trien-3-one] - R5020 promegestone [17, 21-dimethyl-19-norpregna-4, 9-dien-3, 20-dione] - RU486 [17-hydroxy-11-(4-dimethylaminophenyl)-17-(1-propynyl)-estra-4, 9-dien-3-one] - triamcinolone acetonide [9-fluoro-11, 21-dihydroxy-16, 17(1-methylethylidenebis oxy) pregna-1, 4-dien-3, 20-dione]     

Micromolar concentrations of 2-methoxyestradiol kill glioma cells by an apoptotic mechanism,without destroying their microtubule cytoskeleton

The purpose of this study was to investigate the potential effects of 2-methoxyestradiol, a natural mammalian steroid, in glioma cells, since antiproliferative effects of this compound had been shown earlier in several leukemia and carcinoma cell lines. The effects of 0.2, 2 and 20M concentrations of 2-methoxyestradiol were measured in three malignant human glioma cell lines (U87MG, U138MG, LN405) and one malignant rat glioma cell line (RG-2) using a microtiter-tetrazolium (MTT) assay. In all cell lines, a significant reduction of the viable cell number by more then 75% occurred ( P < 0.05) for concentrations of 2 and 20M 2-methoxyestradiol after 6days. A concentration of 0.2M had smaller effects (10–40% cell reduction), which were significant in two of the cell lines tested. The apoptotic nature of cell death was further analyzed in U87MG and RG-2 cells. Caspase-3 activity was significantly induced to levels between 3.4- and 23-fold after 4days for the two higher 2-methoxyestradiol concentrations (P < 0.05). In the cell line RG-2 nuclear fragmentation was visible in many nuclei, following stains with Hoechst H33258. A round cell morphology occurred in most treated cells, which was not accompanied by a complete destruction of the microtubule network, as it can be observed with other microtubule targeting drugs. K. Chamaon: These authors contributed equally to the work.J. Stojek: These authors contributed equally to the work.     

The pharmacokinetics of vincristine in man:

Summary A radioimmunoassay has been used to investigate the pharmacokinetics of vincristine in 39 cancer patients who received between 0.4 and 1.54 mg vincristine/m² as part of standard treatment protocols. There was wide interindividual variation in both the terminal elimination half-life of vincristine (t_1/2) and the associated volume of distribution (Vd), resulting in an 11-fold range of dose-corrected area under the plasma concentration versus time curve values (AUC_0–). Elevated vincristine AUC_0– values were observed in those patients with raised serum alkaline phosphatase at the time of vincristine estimation. The t_1/2 was significantly longer in these patients than in those with serum alkaline phosphatase within normal limits, suggesting that biochemical evidence of cholestasis is associated with reduced clearance of vincristine. Evidence is also presented to suggest that the clearance of vincristine is dose-dependent within the therapeutic dose range. We observed a disproportionate rise in vincristine plasma concentration at doses exceeding 1 mg/m², due primarily to a lengthening of mean t_1/2 compared with that observed for patients receiving 1 mg vincristine/m² or less.     

Phase III trial of cyclophosphamide, epirubicin, fluorouracil (CEF) versus cyclophosphamide, mitoxantrone, fluorouracil (CNF) in women with metastatic breast cancer

Background: The mitoxantrone combination CNF and the epirubicin combination CEF have shown similar activity and less toxicity than the standard CAF combination in metastatic breast cancer (MBC). A prospective randomised study was started to compare safety and activity between CEF and CNF administered using a classical chemotherapeutic schedule in MBC.Patients and methods: From December 1987 to June 1993, 151 patients were randomised to receive cyclophosphamide (C) 100mgm^–2 p.o. days 1–14, fluorouracil (F) 500mgm^–2 i.v. days 1 and 8, and epirubicin (E) 30mgm^–2 i.v. days 1 and 8, or mitoxantrone (N) 6 mgm^–2 i.v. days 1 and 8, every 4 weeks. Seventythree patients were eligible for CEF and 72 for CNF.Results: Objective responses were observed in 61.6 of the CEF group and 44.4 in CNF group (p=0.004). The median duration of response was 64 weeks in CEF and 50 weeks in CNF group (p=0.02) and median time to progression was 51 and 33 weeks, respectively (p=0.0004). At the time of analysis, all except six patients (one in CNF and five in CEF) had died and the median survival time in the CEF group was longer than in CNF (74.4 weeks vs 51.4 weeks; log-rank ² test p=0.015). CNF produced more hematologic toxicity than CEF (WHO scale; grades 2–4): leucopenia 84% vs 68% (p=0.03) and trombocytopenia 17% vs 4.5% (p=0.01); CEF caused more grade 2 and 3 alopecia: 93% vs 70% (p=0.00 1).Conclusion: The combination CEF using this schedule and dosage in metastatic breast cancer is more effective with less toxicity than CNF, except for alopecia, and was associated with longer survival.     

Partial characterization of glioma-derived growth factor 2: A novel mitogenic activity from human cell line D-54 MG

Summary We have shown that several human malignant glioma cell lines are stimulated by bacterial lipopolysaccharide (E. coli 0111B4, 1 g/ml) to produce a high molecular weight (> 200 kD) growth activity for BALB 3T3, clone A31 cells [1, 2]. This glioma-derived growth factor (GDGF-2) acts like a competence factor. Malignant glioma cell line D-54 MG constitutively produced GDGF-2, which we have partially characterized from serumfree conditioned culture medium. GDGF-2 is resistant to heat (100° C, 5 min), acidic (pH 2, 2 hr) or reducing (0.5 M 2 ME, 30 min) conditions as well as exposure to RNases; however, it is sensitive to > 4 freeze-thaw cycles, alkaline (pH 11, 2 hr) conditions or pre-treatment with proteolytic enzymes. GDGF-2 had a pl of 6.8 determined by preparative isoelectric focusing, bound to DEAE, with elution at 35 and 185 mM NaCl and at 43% acetonitrile from a C4 reversed phase column. GDGF-2 activity was not neutralized by antibodies to TGF, TGF, PDGF, VEGF or TNF indicating that it is not immunochemically related to these growth factors. However GDGF-2 co-chromatographed on Superose 12 HPLC (250 × 9 mm; 5% isopropanol, 6 mM CHAPS in PBS) with a substance that suppressed growth of mink lung epithelial cells (Mv1Lu), but not BALB 3T3 cells, and could be neutralized by anti-TGF antibodies. GDGF-2 activity eluted from heparin columns in 0.6 M NaCl; thus, it is not a heparin binding growth factor. D-54 MG cell line produced alpha₂-macroglobulin (₂M), which is known to bind TGF; however, immunoprecipitation of ₂M did not deplete TGF or GDGF-2 activity. Further, neither GDGF-2 or TGF can be dissociated into lower molecular weight active components by chromatography in high salt (2 M NaCl) or 2-ME (0.5 M). GDGF-2 may be a novel autocrine or paracrine mitogen, stimulating mitotic division or interfering with normal cell growth regulation.     

Differential effects of chemotherapeutic agents on the Bcl‐2/Bax apoptosis pathway in human breast cancer cell line MCF‐7

The present study explored the effects of three commonly used chemotherapeutic agents on the Bcl2/Bax apoptosis pathway and the interaction of these chemotherapeutic drugs with the estradiolmediated regulation of this pathway. Our results showed that: (1) Treatment of MCF7 cells with Adriamycin resulted in time and concentrationdependent decreases in Bcl2 and increases in Bax mRNA and protein levels. (2) Camptothecin elicited similar trends on Bcl2 and Bax as Adriamycin, while etoposide, at 50–100 fold (1–5M) the effective concentration of Adriamycin and camptothecin, only resulted in an increase in Bax mRNA levels. (3) Adriamycin and camptothecin, but not etoposide, were effective in suppressing estradiolstimulated increases in Bcl2 mRNA levels. Our study provides evidence that the Bcl2/Bax apoptosis pathway may be differentially regulated by chemotherapeutic agents. In addition, interaction between these agents and estradiol on the Bcl2/Bax apoptosis pathway may also exist.     

Effect of Dose and Infusion Time on the Delivery of p-boronophenylalanine for Neutron Capture Therapy

Clinical trials of boron neutron capture therapy (BNCT) for glioblastoma multiforme are currently in progress using p-boronophenylalanine (BPA) as the 10B delivery agent. Enhancement of tumor boron uptake and/or the tumor-to-blood (T:B) boron concentration ratio would have the potential of significantly improving the therapeutic gain of BNCT. The effects of total dose, infusion time, and route of administration of BPA on tumor and blood boron concentrations were studied in rats bearing the 9L gliosarcoma. Increasing the total dose of BPA from 250 to 1000mg/kg, administered intravenously over a 2-h infusion period, resulted in an increase in tumor boron concentration from 30 to 70µg 10B/g, with a constant T:B boron concentration ratio of about 3.7:1. Similarly, extension of the infusion time from 2 to 6h, at a constant dose-rate of 125mg BPA/kg/h, resulted in an increase in tumor boron concentration from 30 to 80µg 10B/g, while, again, maintaining a constant T:B ratio of about 3.7:1. In contrast, intracarotid infusion of BPA for 1h at a dose rate of 125mg BPA/kg resulted in an increase in the tumor boron concentration from 26 to 38µg 10B/g with a corresponding increase in the T:B ratio from 3.5:1 to 5.0:1. The effects of these results on the therapeutic gain potentially achievable with BNCT are discussed.     

Glioma Cell Invasion: Regulation of Metalloproteinase Activity by TGF-β

Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases that selectively degrade components of the extracellular matrix. MMPs are implicated in tumor cell invasion because they mediate the breakdown of the basal membrane. In addition, they seem to be important for the creation and maintenance of a microenvironment that facilitates tumor cell survival. Among the essential characteristics of human malignant gliomas are infiltrative growth, angiogenesis and suppression of antitumor immune surveillance. Transforming growth factor-beta (TGF-) is intimately involved in the regulation of these processes. We have previously demonstrated that TGF- promotes the migration of LN-18 and LN-229 glioma cells via a process that may involve the upregulation of _V3 integrin expression. Furthermore, we have defined a novel pathway for hepatocyte growth factor (HGF)-induced glioma cell migration and invasion which requires the induction of TGF-₂ expression. Here, we demonstrate that TGF-₂ induces MMP-2 expression and suppresses tissue inhibitor of metalloproteinases (TIMP)-2 expression and that concentration-dependently promotes the invasion of U87MG and LN-229 glioma cells in a matrigel invasion assay. Similarly, ectopic expression of the anti-apoptotic BCL-x_L protein leads to enhanced matrigel invasion by LN-18 and LN-229 glioma cells. We outline the possible interrelations of TGF-, proteins of the BCL-2 family, integrins and metalloprotease activity. By virtue of its promotion of glioma invasion and its growth regulatory and immunomodulatory properties, TGF- continues to be one of the most promising targets for the experimental therapy of human malignant glioma.     

Treatment of plasma cell neoplasm with recombinant leukocyte A interferon and human lymphoblastoid interferon

Summary Thisty cases of plasma cell neoplasms (24 multiple myeloma, one plasma cell leukemia, and three primary macroglobulinemia) were treated with two kinds of highly purified -interferons, recombinant human leukocyte interferon (rIFN-A) (16 cases) and human lymphoblastoid interferon (HLBI) (14 cases). Partial remission (PR) was obtained in two of 16 evaluable cases treated with rIFN-A and in two of 12 evaluable cases treated with HLBI. If minor response (MR) was included, responses were observed in seven (31.3%) and six (50%), respectively. Response (PR+MR) was noted in 38% of 21 previously treated patients and 71% of seven previously untreated patients. Side-effects were noted in more than two-thirds of the patients. They included fever, malaise, nausea/anorexia and myelosuppression. Thus, these two kinds of highly purified -interferon were effective in plama cell neoplasm, producing unequivocal response in 14.3% of the cases without unacceptable side-effects.     

Influence of estrogen metabolism on proliferation of human breast cancer

The aromatase cytochrome P450 complex is responsible forthe in vivo conversion of androgens to estrogens.Although breast cancer epithelial cells have been reportedto have appreciable aromatase activity, its biologic significanceremains uncertain. To address this, the effect ofandrogens on the expression of the estrogen-regulated genepS2 in hormone-dependent human breast carcinoma cells invitro was examined.Steroid-deprived MCF-7 cells were exposed to varying concentrations(1 nM, 10 nM, and 100 nM ofandrostenedione or testosterone for 2, 4, and 6days. Baseline aromatase activity was 4.9 (± 3.1)fmol ³H₂O/hour/g DNA [34.3 (± 21.3) fmol/hr/10⁶ cells]and was not influenced by the androgens. Asan indication of estrogen biosynthesis, northern analysis wasperformed to quantitate pS2 mRNA expression. Although nosignificant pS2 induction was observed at 2 days,both 4 and 6 day exposure to 100nM testosterone resulted in a 3-fold increase inpS2 mRNA expression. 5-dihydrotestosterone (5-DHT) failed to elicita similar pS2 response. This testosterone-induced response wasinhibited with the aromatase inhibitor 7(4DV-amino) phenylthio-1,4-androstadiene-3,17-dione (7-APTADD)and with 10 M tamoxifen.MCF-7 breast cancer cells possess endogenous aromatase activityat high enough levels to convert androgens toestrogens and elicit an estrogen-induced response. The expressionof aromatase may offer a potential advantage tohormone-responsive cells, providing an additional autocrine growth pathwaywhich may be exploited.     

High-dose chemotherapy with autologous stem cell rescue for children with high risk and recurrent medulloblastoma and supratentorial primitive neuroectodermal tumors

Current treatment for high risk and recurrent medulloblastoma (MB) and supratentorial primitive neuroectodermal tumors (stPNET) has a very poor prognosis in children. High dose chemotherapy (HDCT) and autologous stem cell rescue have improved survival rates. We present 19 patients (thirteen classified in the high risk group and six patients with recurrent disease) that received HDCT and autologous stem cell rescue.In the high risk group [Med Pediatr Oncol 38 (2002) 83], all patients underwent neurosurgical debulking. Standard chemotherapy was prescribed in 10 patients. Radiotherapy was given to 4 patients (all older than 4years old). In the recurrence disease group [Childs Nerv Syst 15 (1999) 498], five patients underwent surgery. Radiotherapy was given to those who were not previously irradiated. The HDCT in twelve patients consisted of busulfan 4mg/kg/day, orally over 4days in 6-hourly divided doses and melphalan at a dose of 140mg/m²/day by intravenous infusion over 5min on day –1. Three patients additionally received thiotepa 250mg/m²/day intravenously over 2days and four patients additionally received topotecan 2mg/m²/day over 5days by intravenous infusion over 30min. The other seven patients received busulfan and thiotepa at the same doses.Patients stem cells were mobilized with granulocyte colony-stimulating factor at a dose of 12g/kg twice daily subcutaneously for four consecutive days. Cryopreserved peripheral blood progenitor cells were re-infused 48h after completion of chemotherapy. With a median follow-up of 34months (range 5–93) eight complete responses and one partial response were observed. Three patients died of treatment-related toxicities (15%). The 2 year event-free survival was 37.67±14% in all patients and 57±15% for the high risk group.Therefore we conclude that HDCT may improve survival rates in patients with high risk/recurrent MB and stPNET despite treatment toxicity.