Reconstitution of acid secretion in digitonin-permeabilized rabbit gastric glands. Identification of cytosolic regulatory factors.

When isolated rabbit gastric glands were permeabilized with digitonin, they lost their ability to secrete acid, as monitored by [14C]aminopyrine accumulation, and they never recovered by supplement with cytosol prepared from gastric mucosa. However, the permeabilized glands elicited acid secretion when brain cytosol was supplemented. Fractionation of gastric cytosol by gel filtration revealed that the fraction at 30 kDa stimulated permeabilized glands by itself, whereas the 200-kDa fraction potently inhibited brain cytosol-stimulated acid secretion. Brain cytosol contained only the former stimulatory factor. With further gel filtration, the 30-kDa activator was separated into two components, 20 kDa (peak 1) and 1.8 kDa (peak 2), both of which are necessary for full activity. We purified peak 1 from bovine brain, and phosphatidylinositol transfer protein (PITP) was identified as the main component of the activity. The stimulating activity in brain and gastric mucosa correlated with the contents of PITP, and recombinant PITP mimicked the effect of peak 1, suggesting that PITP is one of the essential components in gastric acid secretion. When gastric glands were stimulated, the inhibitory activity, but not stimulatory activity, in the cytosol was increased. This suggests a regulatory mechanism such as stimulation translocates the inhibitory component from the secretory site on the membrane to cytosol. These results demonstrate a high degree of usefulness for our present model, the reconstituted digitonin-permeabilized gastric glands.

Green tea catechins enhance tumor development in the colon without effects in the lung or thyroid after pretreatment with 1,2-Dimethylhydrazine or 2,2'-dihydroxy-di-n-propylnitrosamine in male F344 rats.

Modifying effects of green tea catechins (GTCs) on the post-initiation stage of colon, lung and thyroid carcinogenesis were examined in F344 male rats. Groups of 20 animals were given subcutaneous injections of 40 mg/kg body wt of 1,2-dimethylhydrazine twice a week for 2 weeks or oral administration of 0.1% 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN) in the drinking water for 2 weeks for initiation. They then received diet containing 1 or 0.1% green tea catechin or basal diet alone for 33 weeks. Histopathological examination after final sacrifice showed that although total incidence and multiplicity of colon tumors were not significantly different from controls, values for colon adenomas were decreased while those for carcinomas and the average size of tumors were significantly increased in the 0.1% GTC group. A similar tendency was observed for the 1% GTC group. Incidences and/or multiplicity of lung hyperplasia and tumors, and thyroid lesions did not significantly vary among the DHPN-treated groups. These results indicate that GTCs do not inhibit, but rather may enhance colon carcinogenesis, while not influencing lung and thyroid carcinogenesis under the present experimental conditions.

Relation of transmembrane potential to cell survival following hyperthermia in HeLa cells.

Hyperthermia induces cell death and the usual endpoint to study this is the ability of the cells to form colonies. Hyperthermia is also known to alter membrane characteristics, especially transmembrane potential and this has been correlated with duration and degree of heating. The aim of the present study was to see the correlation between changes in membrane potential and clonogenic ability of HeLa cells after heat treatment of 41-44 degrees C. Membrane potential was measured by the fluorescence polarization of the plasma membrane probe 3,3'-dipentyloxacarbocyanine by flow cytometry. Cell survival was assessed by colony formation assay. The fluorescence intensity increased and cell survival decreased with an increase in temperature. The fall in survival following heat treatment closely paralleled the increase in fluorescent intensity, especially heat treatments of 60 min or more. After 2 h of heating at 44 degrees C, the surviving fraction decreased to 1% and the fluorescence intensity increased to 154.84% of the unheated controls. This study suggests that measurement of membrane potential by flow cytometry may potentially be an alternative to colony forming assay for assessing cell survival. Since the results of membrane potential measurements are available immediately, this has implications for its potential use as a predictive assay of thermosensitivity.

Indomethacin reduces the skin thermal damage in hyperthermic treatment of experimental malignant tumors.

Indomethacin (Ind), an inhibitor of prostaglandin synthesis, was previously shown to increase the thermosensitivity of murine tumors. The potency of drug to modify the thermal response of murine skin has been evaluated in mice heated in water bath at 44 degrees C for 30, 60 and 90 min. Ind was administered subcutaneously (s.c.) at dose of 5 mg/kg body weight (b.w.) 1.5 h before heating. The mouse foot skin reactions (FSR) were assessed using the scoring system of Urano et al (1979). The severity of skin thermal damage was decreased markedly by Ind. At the time when heating of the control group at 44 degrees C for 60 min resulted in the irreversible FSR in some mice, pretreatment with Ind before heating lead to the complete recovery from the heat damage in all mice. Similarly, after heating at 44 degrees C for 90 min, the degree of FSR was diminished by Ind from score 4.5 to score 2. It is concluded that Ind selectively protected normal skin during the hyperthermic treatment. Further clinical study is warranted.

Enhancement of antitumor effect of hyperthermia with glucose administration in murine mammary carcinoma.

Antitumor effects of hyperthermia are enhanced by lowering the pH in the tumor tissue with administration of glucose. This decreased pH in the tumor tissue with glucose administration was determined using mouse experimental tumors. 31P-MRS microelectrodes were used for the measurement of pH. By using these two measurement methods, time course change in the tumor tissue was determined in the controls and the groups treated with 6 g/kg of intraperitoneal glucose. The determination of pH with 31P-MRS was calculated from the chemical shift of the peak of creatine phosphate (Pcr) and that of inorganic phosphate (Pi). Following glucose administration, the tumor tissue showed a decrease of 0.3 pH units with the microelectrode method, but did not show any significant decrease in pH with the MRS determination. This finding suggested that 31P-MRS showed intracellular pH (pHi) due to the localization of Pi and that the microelectrode indicated interstitial or extracellular pH (pHe). The ATP/Pi ratio obtained in tumor tissue 24 h after heat treatment (with, without glucose) was correlated with tumor inhibition.

Relevance of a new impedance matching, or subtrap, method for the reduction of pain during hyperthermia.

Capacitive heating is widely used in hyperthermic treatment of human malignancies. However, the pain on the body surface or thermoesthesia in the subcutaneous fatty layer may prevent an elevation of temperature in the tumors. Impedance matching is improved by a subtrap method entailing the application of two copper plates (10 x 850 x 0.06 mm) as a subtrap circuit to each of two capacitive electrodes. In a clinical trial the Tmax, Tave, Tmin for the subtrap method were all higher in comparison with those for the conventional technique (42.5 +/- 0.7 degrees C, 41.9 +/- 1.0 degrees C, 41.3 +/- 1.1 degrees C vs. 41.1 +/- 1.5 degrees C, 40.6 +/- 1.3 degrees C, 40.0 +/- 1.3 degrees C). Although the maximal radiofrequency (RF) power applied to patients was higher with the subtrap method (875 +/- 189 W vs. 763 +/- 200 W), the incidence of surface pain was reduced dramatically. It is concluded that the subtrap method substantially improves the RF capacitive heating of deep-seated tumors.

Effect of hyperthermia on transmembrane potential of HeLa cells--a flow cytometric analysis.

The effect of hyperthermia on transmembrane potential was studied in HeLa cells in vitro using a 3',3'-dipentyl oxacarbocyanine [Di-0-C5(3)], a lipophilic cation probe that equilibrates across the plasma membrane according to the transmembrane potential. Uptake of the fluorescent probe was measured by flow cytometry. The flourescent intensity (FI) increased with increase in temperature, and the increase was statistically significant when the duration of heat treatment was 30 minutes or more. At each temperature studied the depolarization was higher after longer duration of heat treatment (p value: 41 degrees C < 0.05; 42 degrees C < 0.005; 43 degrees C < 0.001 and 44 degrees C < 0.001, respectively). The lack of significant depolarization after shorter duration of heating, particularly at lower temperatures could be due to the repair of membrane damage that could have occurred in the holding interval between heating and measurement. The results suggest that depolarization of membrane potential, i.e. increase in the intracellular cation concentration, can be considered as an indicator of cell injury by hyperthermia and may be mechanistically related to cell death by heat treatment. The technique may be suitable for studying repair of damage after hyperthermia.

Studies on hyperthermia combined with arterial blockade for treatment of tumors: (Part I) effectiveness of hyperthermia combined with arterial ligation.

Arterial ligation was combined with hyperthermia in rabbits with VX2 tumors implanted in the leg. For seven days after arterial ligation, blood flow was decreased and the pH was low in both normal muscle and tumor tissue. The temperature of normal muscle and tumor tissue increased faster and reached a higher level on heating immediately after ligation than without ligation. The antitumor effect of hyperthermia was stronger immediately after ligation than two or seven days afterwards. However, damage to normal muscle was severe with this combination therapy, so a better method of therapeutic arterial blockade is needed.

Studies on hyperthermia combined with arterial therapeutic blockade for treatment of tumors: (Part III) effectiveness of hyperthermia combined with arterial chemoembolization using degradable starch microspheres on advanced liver cancer.

Arterial chemoembolization using degradable starch microspheres and adriamycin was combined with hyperthermia to treat advanced liver cancer. The prolonged peak adriamycin level in hepatic venous blood suggested that the drug persisted for longer in the liver after injection containing microspheres. Heating efficiency was increased more in tumor tissue than in normal liver tissue after embolization. This combined therapy was performed in eight patients with advanced liver cancer and was effective in three (complete or partial remission). The mean survival time was 25 weeks and there were no severe side effects. This combined therapy may be useful for liver cancer.

Studies on hyperthermia combined with arterial blockade for treatment of tumors: (Part II) effectiveness of hyperthermia combined with arterial embolization using degradable starch microspheres.

The efficacy of temporary arterial embolization using degradable starch microspheres combined with hyperthermia was investigated in rabbits bearing VX2 tumors. Microsphere injection caused a marked decrease of tumor blood flow and pH. During heating, there was a marked increase of the maximum temperature in tumor tissue compared with normal muscle. Tumor growth was suppressed 330% times at 3 weeks after hyperthermia alone and 270% times following combined treatment with microspheres and hyperthermia. Damage to normal muscle tissue was mild. In conclusion, this combination therapy may be useful for causing selective tumor damage and reducing the effect on normal tissues.

Role of intracellular calcium concentration on tumor cell death from hyperthermia.

The intracellular calcium ion concentration of SCK cells was measured after the cells were heated to determine the relationship between thermotolerance and intracellular calcium concentration. SCK cells, which are breast cancer cells that spontaneously arise in A/J mice, were used. Exponentially growing cells were trypsinized and incubated for 60 min with fura-2/AM at a concentration of 10 M. The incubation was carried out at a density of 1x106 cells/ml in serum-free RPMI medium. Thermotolerance was induced by incubating cells for 5 h at 37yC after heating them at 43yC for 30 min. When heated at 44yC in a calcium containing buffer, the intracellular calcium concentration of non-thermotolerant and thermotolerant cells increased continuously as the cells were heated for longer periods of time. The same increase in intracellular calcium concentration was seen in these two cell lines when heated at 43yC in a calcium-free buffer. Thus, our data indicated that intracellular calcium ion increased during heating due to an influx into the cytoplasm from both internal stores and the extracellular medium.

Enhancement of the effect of an angiogenesis inhibitor on murine tumors by hyperthermia.

We investigated the effect of angiogenesis inhibitors (FR118487 and TNP-470) on two murine tumors (FM3A and SCC-VII) with and without hyperthermia. When angiogenesis inhibitors were administered immediately after tumor implantations there was no tumor growth. As the tumor diameter increased, the antitumor effect of the inhibitors decreased. When FR118487 was combined with hyperthermia, the antitumor effect was potentiated. This study suggested that angiogenesis inhibitors show their maximal effect against small tumors and have increased activity when combined with hyperthermia.

New technique for mediastinal temperature measurement in hyperthermic cancer treatment: balloon catheter in the azygos vein.

The temperature in the mediastinum during hyperthermia is difficult to determine accurately. We measured the temperature in the azygos vein, using a new technique, and compared the measurements with temperatures in the oesophagus. Eight patients with mediastinal tumours resulting from lung cancer or oesophageal cancer were given hyperthermo-radiotherapy. The temperatures in the azygos vein and in the oesophagus were measured before and during blockage of the blood flow of the azygos vein using an angiographic balloon catheter. None of the patients had complications as a result of these procedures, and hyperthermia by capacitative heating was safely performed. The temperature in the azygos vein increased by a mean of 1.7 degrees C (0.2-2.8 degrees C) after blockage of the blood flow. The temperature in the oesophagus was 0.83 +/- 1.09 degrees C (mean +/- SD) higher than that in the azygos vein. Measurement of the temperature in the azygos vein gives a more accurate estimate of mediastinal temperature than does oesophageal temperature but it is an invasive procedure.

Hypothalamic pilocytic astrocytoma presenting with intratumoral and subarachnoid hemorrhage.

A 45-year-old male presented with sudden onset of severe headache. Computed tomography and magnetic resonance imaging demonstrated an irregularly enhanced suprasellar mass with intratumoral and subarachnoid hemorrhage. The mass was removed in two operations. Histological examination of the tumor revealed pilocytic astrocytoma. The relatively rich vascularity and perivascular tumor cell proliferation observed in this benign lesion were probably the causes of this extremely rare association.

The use of a hypoxic cell radiosensitizer AK-2123 gave no improvement in thermoradiotherapy combined with hydralazine.

An electron-affinic compound, AK-2123, and the anti-hypertensive agent, hydralazine, were combined with radiation and hyperthermia for treatment of murine SCC-VII tumours. Hydralazine markedly decreased tumour perfusion while AK-2123 had no influence on it. Hydralazine enhanced the tumouricidal effects of hyperthermia alone and in combination with radiation. AK-2123 provided a radiosensitization which was significant only in tumours irradiated without supplementary hyperthermia. The greatest tumour response was achieved when thermoradiotherapy was combined with hydralazine alone; the additional use of AK-2123 with this treatment combination did not further increase the effect. It is concluded that hydralazine plus heat virtually eliminated a hypoxia-related radioresistance in tumours, thus removing the requirement for AK-2123 administration.

Measurement of intratumor pH by pH indicator used in 19F-magnetic resonance spectroscopy. Measurement of extracellular pH decrease caused by hyperthermia combined with hydralazine.

RATIONALE AND OBJECTIVES: Without affect of metabolic changes, the authors measured intratumor pH by using 19F-magnetic resonance spectroscopy (MRS) with a fluorine compound (ZK-150471) on the basis of a calibration curve by pH electrode. METHODS: Using the 4.7-tesla magnetic resonance apparatus, a fluorine compound that had acid-base equilibrial change and was impermeable within cell membranes was used. The fluorine compound was injected intravenously. The signal was obtained from mouse mammary cancer by creating an experimental tumor on the leg of mice. And the tumors, which were heated with and without hydralazine. The pH evaluated from chemical shift of the fluorine compound. The pH data was obtained from an electrode for reference. RESULTS: The pH of nontreated tumors (n = 25) were 6.94 + 0.091. The pH decreased to 6.772 + 0.169 at 20 minutes even after 20 minutes of heating, and decreased to < 6.71 at 40 minutes after every heating time. The pH decreased to 6.456 at 20 minutes after 15 minutes of heating combined with hydralazine, and to 6.416 at 40 minutes after same treatment. CONCLUSIONS: It is possible to measure the extracellular pH by 19F-MRS with the fluorine compound noninvasively in vivo, even after heating.

Lack of chemoprevention effects of the monoterpene d-limonene in a rat multi-organ carcinogenesis model.

Modifying effects of dietary administration of the monoterpene d-limonene were examined using a multi-organ carcinogenesis model. Groups of twenty F344 male rats were treated sequentially with N-diethylnitrosamine (DEN, i.p.), N-methyl-N-nitrosourea (MNU, i.p.), 1,2-dimethylhydrazine (DMH, s.c.), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN, in drinking water) and dihydroxy-di-N-propylnitrosamine (DHPN, in drinking water) during the first 4 weeks (DMBDD treatment), and then d-limonene was administered in the diet, at the dose of 2.0, 1.0 or 0.5%. The maximal tolerable dose was 2.0% under the present conditions. Further groups were treated with DMBDD or 2.0% d-limonene alone as controls. All surviving animals were killed at week 28, and major organs were examined histopathologically for development of preneoplastic and neoplastic lesions. The incidences and/or multiplicities of renal atypical tubules and adenomas were increased in animals fed 2.0% d-limonene. The immunohistochemical reactivity for alpha2u-globulin in the proximal tubules was greater in rats fed d-limonene than in the carcinogen alone group. No enhancing or inhibitory effect was noted for tumor development in other organs. The present results indicate a lack of any chemopreventive effect of d-limonene in any organ of male rats under the present experimental conditions.

Withaferin A: a new radiosensitizer from the Indian medicinal plant Withania somnifera.

Withaferin A, a steroidal lactone isolated from the roots of the Indian medicinal plant Withania somnifera, reduced survival of V79 cells in a dose-dependent manner. LD50 for survival was 16 microM. One-hour treatment with a non-toxic dose of 2.1 microM before irradiation significantly enhanced cell killing, giving a sensitizer enhancement ratio (SER) of 1.5 for 37% survival and 1.4 for 10% survival. SER increased with drug dose, but at higher doses the increased lethality appears to be due to two effects-- drug toxicity and radiosensitization. The drug induced a G2/M block, with a maximum accumulation of cells in G2-M phase at 4 h after treatment with 10.5 microM withaferin A in 1 h. The applicability of this drug as a radiosensitizer in cancer therapy needs to be explored.

Inhibitory effects of 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), green tea catechins and other antioxidants on 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1)-induced rat hepatocarcinogenesis and dose-dependent inhibition by HTHQ of lesion induction by Glu-P-1 or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx).

The effects of 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), green tea catechins (GTC), alpha-tocopherol, beta-carotene, chlorophyllin, phenylethylisothiocyanate (PEITC), 3-O-ethylascorbic acid (EAsA), 3-O-dodecylcarbomethyl ascorbic acid (DAsA), n-tritriacontane-16,18-dione (TTAD) and d-limonene on 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1)- or dimethylnitrosamine (DMN)-induced hepatocarcinogenesis, and the dose dependence of HTHQ inhibition of Glu-P-1- or 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx)-influence on lesion development were examined in a rat medium-term liver bioassay system featuring diethylnitrosamine initiation and partial hepatectomy. At the end of week 8, the number and total area of glutathione S-transferase placental form (GST-P) positive liver foci in rats treated with 0.03% Glu-P-1 alone were increased significantly (46.8 +/- 11.0 and 12.0 +/- 5.6 respectively) as compared to the control values (3.8 +/- 1.6 and 0.4 +/- 0.2). Combined treatment with 1% HTHQ remarkably reduced both of these parameters (8.1 +/- 2.1 and 0.6 +/- 0.2). GTC (1%), PEITC (0.1%), beta-carotene (0.1%) and DAsA (1%) also demonstrated inhibition but less than HTHQ. On the other hand, these antioxidants did not influence development of foci initiated by 0.002% DMN. In the dose-response study, up to 0.125% HTHQ significantly reduced the effects of 0.02% Glu-P-1 or 0.03% MeIQx on the number and area of foci. These results indicate that several antioxidants exert chemopreventive effects against heterocyclic amine (HCA)-induced hepatocarcinogenesis, and particularly HTHQ which thus deserves further attention as a chemopreventor in the contest of the environmentally important HCA group of carcinogens.