Enhancement of camptothecin-induced cytotoxicity with UCN-01 in breast cancer cells: abrogation of S/G2 arrest

Purpose: To determine the ability of UCN-01 to abrogate the cell cycle arrest induced by camptothecin (CPT) in tumor cells that lack p53 function, and therefore enhance the cytotoxicity of CPT in these cells in relation to normal cells with wild-type p53. Methods: The responses of MDA-MB-231 and GI 101A breast cancer cells were compared to those of normal bovine endothelial cells. Cytotoxicity was assessed by the MTT assay, and the resulting data were modeled using median-effect analysis. Inhibition of DNA synthesis was determined by loss of [³H]thymidine incorporation, and cell cycle status was determined by flow cytometric analysis of propidium-iodide-stained nuclei. Results: UCN-01, a specific inhibitor of protein kinase C (PKC) presently in clinical trials, abrogated CPT-induced activation of S and G₂ checkpoints in human MDA-MB-231 and GI 101A breast carcinoma cells, both of which are mutants for the p53 gene. This abrogation occurred with the use of sublethal doses (100 nM) of UCN-01 and correlated with the enhancement of CPT-induced cytotoxicity. Median-effect analysis showed that synergistic cytotoxic interactions existed between CPT and UCN-01 against these tumor cells. In normal cells, however, abrogation of the S phase arrest caused accumulation in G₀/G₁ phase, perhaps by the presence of wild-type p53 activity, with no change in CPT-induced cytotoxicity. Conclusion: We have shown previously that the cytotoxicity of CPT is correlated with cell cycle response in normal and tumor cells. Low doses of CPT arrest cells in the G₂/M phase and inhibit DNA synthesis, but higher doses cause arrest of cells in S phase. Thus modulation of events at the S and G₂ checkpoints may provide an opportunity to enhance CPT-induced cytotoxicity in tumor cells. The results of this study indicate that UCN-01 enhances the progression of tumor cells through S phase thus greatly increasing CPT-induced cytotoxicity. Normal cells, however, are able to arrest in G₀/G₁ and thus avoid the increased toxicity induced by CPT. Our findings suggest potential usefulness of combining UCN-01 in topoisomerase I inhibitor-based drug therapy for the treatment of breast cancer with a dysfunctional p53 gene. Received: 25 February 1999 / Accepted: 4 October 1999     

Alterations of fast axoplasmic transport in experimental methyl n-butyl ketone neuropathy.

Methyl n-butyl ketone (MBK) is known to produce a giant axonal neuropathy in man and experimental animals characterized pathologically by a gradual increase in the number of neurofilaments which become associated with focal areas of axonal swelling and thinning of the myelin sheath. Fast axoplasmic transport was studied in rats exposed to MBK. In 10 severely paralyzed rats exposed to MBK there was a significant impediment of fast axoplasmic transport following dorsal root ganglion injections (x +/- S.D. = 283.2 +/- 20.34 mm/day) compared to normal controls (417.6 +/- 23.78 mm/day). In rats undergoing injections into the ventral horn of the spinal cord there was a gradual impairment of the mean down flow rate for transport of [3H]leucine which correlated with the severity of the MBK induced neuropathy. Quantitative morphological determinations showed that the total number of neurotubules per unit cross-sectional myelin area and the number of neurotubules associated with mitochondria in swollen axons was unchanged from normal. The total number of mitochondria in randomly sampled axons varied significantly from controls but the absolute number of mitochondria associated with neurotubules was unchanged from normal. The results of these studies suggest that the impediment of fast axoplasmic transport may be related to the increased neurofilaments producing focal areas of axonal blockage.     

δ-Aminovaleric acid antagonizes the pharmacological actions of baclofen in the central nervous system

Summary The action of -aminovaleric acid (AVA) on the muscle relaxant properties of baclofen, a GABA_B receptor agonist, was investigated in two experimental models: (1) the pathologically increased muscle tone of the gastrocnemius muscle in spastic mutant Han-Wistar rats and (2) the Hoffmann (H)-reflex recorded from plantar foot muscles after electrical stimulation of the tibial nerve in barbiturate (60 mg/kg) anaesthetized rats. In both paradigms coadministration of AVA (500 nmol/5 l) antagonized the muscle relaxant action of intrathecally applied baclofen (0.2–2 nmol), but failed to affect the muscle relaxant effects of intrathecally injected muscimol (2–20 nmol). In contrast, coadministration of bicuculline (1 nmol) did block the muscle relaxant action of muscimol, but failed to alter the effects of baclofen. When administered alone, bicuculline (1 nmol), or AVA (500 nmol–2 mol) were without intrinsic action in both paradigms. In an additional series of experiments we investigated the action of AVA on a supraspinal effect of baclofen. Coadministration of AVA (12.5 nmol/0.5 l) in the ventromedial thalamic nucleus antagonized the catalepsy induced by baclofen (ED₅₀ 10 pmol/0.5 l), as indicated by an increase in ED₅₀ of baclofen by a factor of 4.835 and a parallel shift of the probit-log dosage regression line to the right. The parallel shift seems to be consistent with a competitive mechanism of action of AVA. This study presents evidence that AVA antagonizes central pharmacological actions of baclofen at both spinal and supraspinal sites without affecting the actions of a GABA_A agonist, muscimol.     

Primary tumors of the small intestine

In our department of surgery 28 patients with malignant and 10 with benign tumors of the small intestine were treated from 1940 to 1974. Fifteen patients with malignant and 7 with benign tumors underwent surgery with the intention to cure. Palliative operations or explorations were carried out on 13 patients with malignant tumors. Three patients with benign tumors were not operated on. The initial symptoms were vague: abdominal pain, nausea, anemia or bleeding in 75 and 80 percent of patients with malignant and benign tumors, respectively. The indication for operation in the malignant cases was, however, stenosis of the intestine or biliary tract or a palpable mass in 60 percent of the cases. At operation the tumors were thus in an advanced stage. Because the initial symptoms are vague, early diagnosis is difficult.The overall 5 year survival rate was 21 percent after surgery for malignant tumors. Among the patients considered by the surgeons to have had radical operations excluding cancer patients, 40 percent survived 5 years. The surgeon's opinion regarding “radical” operation as well as the presence or absence of metastases at microscopy were of limited prognostic value.     

The photocarcinogenic risk of narrowband UVB (TL-01) phototherapy: early follow-up data

BACKGROUND: Limited information is available on the carcinogenic risk associated with narrowband TL-01 UVB phototherapy in humans. OBJECTIVES: To determine the skin cancer incidence in a population treated with TL-01 phototherapy. PATIENTS AND METHODS: All TL-01-treated patients were identified from the departmental computerized database. Patients with malignant melanoma (MM), squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) were identified by record linkage with the Scottish Cancer Registry. The incidence of each was compared with the normal Scottish population matched for age and sex. RESULTS: Data were obtained from 1908 patients. The median follow-up duration was 4 years (range 0.04-13). The median cumulative number of TL-01 treatments and dose were 23 (1-199) and 13 337 (30-284 415) mJ cm(-2), respectively. No increased incidence of SCC or MM was observed. Ten patients developed BCC compared with an expected 4.7 in the Scottish population [standardized rate ratio 213 (95% confidence interval 102-391); P < 0.05]. CONCLUSIONS: A small but significant increase of BCC was detected in the TL-01 group. This could be explained by a number of factors, including ascertainment bias. To determine the true carcinogenic risk of TL-01 phototherapy, longer follow-up is essential.     

Effects of a Korean herbal formulation, Silsosangami, consisting of seven medicinal herbs, and its seven herbs on endotoxin-induced experimental thrombosis in rats

A traditional Korean medicine, Silsosangami (SSG), consisting of seven different herbs of Typhae Pollen, Pteropi Faeces, Paeoniae Radicis rubra, Cnidii Rhizoma, Persicae Semen, Carthami Flos and Curcumae Tuber, has been reported to have a hypolipidemic effect in human subjects. In the present study, the inhibitory effects of SSG on a thrombosis in rats, induced by endotoxin treatment were examined. The anti-thrombic properties of SSG were also investigated with respect to blood parameters. The extracts of SSG and five of the seven herbs, except Cnidii Rhizoma and Carthami Flos, inhibited both endotoxin-induced disseminated intravascular coagulation (DIC) and thrombosis in rats. The extract also inhibited the endotoxin-induced decrease in blood platelets and fibrinogen, and the endotoxin-induced increase in fibrin degradation products (FDP) on disseminated intravascular coagulation in normal rats. In conclusion, the artificially induced, protective effects of SSG on ischemic infarction might be related to their inhibitory effects on DIC, platelet coagulation and thrombotic action.     

Evaluation of the anticarcinogenic activity of Swertia chirata Buch.Ham, an Indian medicinal plant, on DMBA-induced mouse skin carcinogenesis model

Considerable attention has been focused on plants which are sources of natural anti-oxidant compounds, because most of them have a modulatory role on physiological functions and biotransformation reactions involved in the detoxification process. Such compounds are likely to afford protection from cytotoxic, genotoxic and metabolic actions of environmental toxicant thereby reducing the risk for cancer. The present study reports the anticarcinogenic activity of Swertia chirata Buch.Ham, an Indian medicinal plant. All the four detoxification enzymes studied viz, GST, GPx, SOD and CAT were found to be activated in different degrees following treatment with infusion of Swertia chirata, its crude extract and a purified 'Amarogentin' rich extract. The activation of the enzymes was accompanied by significant reduction in lipid peroxidation and inhibition of incidence as well as multiplicity of Dimethylbenz(a)anthracene (DMBA) induced papillomas. The effect of S.chirata on apoptosis and cell proliferation was also studied in mice skin exposed to DMBA. Both the crude and purified extracts significantly inhibited cell proliferation and induced apoptosis. This is the fi rst report of its kind and the observation suggests the chemopreventive potential of Swertia chirata.     

Antioxidant activity of supercritical extract of Melissa officinalis subsp. officinalis and Melissa officinalis subsp. inodora

The antioxidant activity of Melissa officinalis subsp. officinalis and of Melissa officinalis subsp. inodora extracts, obtained by using carbon dioxide under supercritical conditions was investigated. The samples were prepared in two steps. A preliminary extraction at 90 bar and 50 degrees C eliminated the essential oil, then a further extraction at 300 bar and 50 degrees C obtained the high molecular mass extract. These samples were tested for autoxidation and the iron or EDTA-mediated oxidation of linoleic acid at 37 degrees C in the absence of solvent, in in vitro systems. During linoleic acid autoxidation and its EDTA-mediated oxidation both M. officinalis and M. inodora extracts showed an antioxidant activity, and no significant differences in their efficacy were observed. None showed any prooxidant activity.     

In vivo amyloid imaging in Alzheimer’s disease

Targeted approaches to therapy for Alzheimers disease have evolved based on detailed understanding of the genetic, molecular biologic, and neuropathologic basis of the disease. Given the potential for greater treatment efficacy in the earlier stages of the disease, the notion of early diagnosis has become more relevant. Current clinical and imaging diagnostic approaches lack reliability in the preclinical and prodromal phases of the disease. We review emerging studies on imaging of the molecular substrate of the disease, most notably the amyloid peptide, which hope to increase early diagnostic efficacy. We offer a brief overview of the demographics, diagnostic criteria, and current imaging tests, followed by a review of amyloid biology and developments in cerebral amyloid imaging yielded by recent in vitro, in vivo and human studies.     

Determination of the rat tissue partitioning of endotoxin in vitro for physiologically-based pharmacokinetic (PBPK) modeling

The biosynthetically double-labeled lipopolysaccharide (LPS), containing (3)H-labeled on the fatty acyl-chains and (14)C-labeled on the glucosamine of Salmonella enterica serotype typhimurium, was isolated from bacteria grown in proteose peptone-beef extract (PPBE) medium in the presence of labeled precursors; 133 micro Ci/ml of [2-(3)H] acetate sodium salt and 0.167 micro Ci/ml of N-acetyl[D-1-(14)C]glucosamine. The LPS was extracted from the bacteria with 90% phenol/chloroform/petroleum ether, purified and stored in 0.1% (v/v) triethylamine/10 mM Tris HCl at -70 degrees C. Tissue slices and portions of the meninges were prepared and incubated in artificial cerebrospinal fluid (CSF) or Krebs phosphate buffer (Krebs) containing 150 ng/ml LPS with [(3)H] LPS (0.004 micro Ci/ml, sp. act. 28 micro Ci/mg LPS). The tissues were incubated under 95% oxygen/5% carbon dioxide at 37 degrees C with constant agitation until steady-state uptake was reached (60 min). At the end of the incubation period, tissues were processed for radioactivity measurement. The rat tissue partitioning of LPS in artificial CSF for brain and Krebs for other organs was measured by using the ratio of tissue to medium at the steady state in vitro. The following results were obtained from the study: Heart, 0.15; liver, 0.19; spleen, 0.12; kidney, 0.18; stomach, 0.17; small intestine, 0.18; brain stem, 0.10; cerebellum, 0.11; meninges, 0.77; hippocampus, 0.12; hypothalamus, 0.12; frontal cortex, 0.09 and caudate nucleus, 0.10. This information, along with plasma or blood/buffer partition coefficients, is a requisite for constructing a physiologically-based pharmacokinetic (PBPK) model of endotoxins for quantitative risk assessment.