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Biomedical subjects

M Kuwano

Publications and source records attributed to M Kuwano.

At least 253 records · Page 14Linked to original sources
PubMed1986

Thioridazine enhances lysosomal accumulation of epidermal growth factor and toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin.

Thioridazine, a phenothiazine calmodulin inhibitor, aggravated the cytotoxic effect of a conjugate (EGF-PE) of epidermal growth factor (EGF) coupled with Pseudomonas exotoxin against cultured HeLa cells. Other phenothiazine calmodulin inhibitors, trifluoperazine and chlorpromazine, also intensified the cytotoxic effect of EGF-PE, whereas N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) had no such effect. By using iodinated epidermal growth factor ( [125I]EGF), the effect of thioridazine on intracellular transport of EGF was examined. The release of radioactivity associated with [125I]EGF into medium was slow in the presence of thioridazine. The Percoll gradient centrifugation pattern showed that thioridazine delayed both the appearance of [125I]EGF in lysosomes and the disappearance of [125I]EGF from the lysosomes. The pH value in lysosomes was 5.28 in thioridazine-treated HeLa cells, while that in untreated cells was 5.15. Thioridazine was found to inhibit lysosomal enzyme activities of cathepsin B and acid phosphatase, but not beta-hexosaminidase when cell extracts were treated with the drug. Electron microscopy showed an increased number of electron-dense bodies, possibly autophagosomes/lysosomes in HeLa cells grown for 48 h with 3 micrograms/ml thioridazine. The potentiating action of EGF-PE by thioridazine is discussed in relation to the altered lysosomal function in treated cells.

ADP Ribose Transferases↗
PubMed1986

Lysosomotropic agents reverse multiple drug resistance in human cancer cells.

Chloroquine, a well-known lysosomotropic amine, partially reversed the resistance of multi-drug-resistant KB carcinoma cells to adriamycin, daunomycin, vincristine, vinblastine and actinomycin D. Other lysosomotropic amines, propranolol, atropine, amantadine and nicotine, also restored the sensitivity of multi-drug-resistant cells to the anticancer drugs. The increased rate of accumulation of [3H] daunomycin by chloroquine was more prominent in the resistant KB-ChR-24 cells than in the parental KB cells. Chloroquine inhibited the efflux of daunomycin from the resistant cells. Circumvention of multiple drug resistance in human KB carcinoma cells by the agents is discussed in relation to lysosomal function.

Antineoplastic Agents↗
PubMed1986

Human mammary cancer cell mutants with altered hormone receptor activity.

We have recently isolated retinoic acid-resistant clones U-2 and U-3 from human breast cancer cell line MCF-7 (Ueda et al. (1985) Cancer Res. 45, 3332-3338). Growth of MCF-7 cells was found to be stimulated by estradiol but that of U-2 or U-3 was not. Cytosol from U-2 or U-3 cells contained no detectable estradiol receptor activity, whereas that from the parental MCF-7 cells showed estradiol receptor activity of 32 fmol/mg cytosol protein with a Kd of 2.6 X 10(-10) M by Scatchard analysis. Sucrose gradient centrifugation analysis of the cytosol fraction confirmed the presence of estradiol receptor activity in MCF-7 but not in U-2. Cytosol from MCF-7 and U-2 cells showed progesterone receptor activities of 106 fmol/mg protein with a Kd of 7.4 X 10(-10) M and 13 fmol/mg protein with a Kd of 9.9 X 10(-10) M, respectively. Addition of estradiol to the culture medium of the cells increased the level of progesterone receptor about 2-fold in MCF-7, but not in U-2. U-2 or U-3 cells showed about 5-fold higher resistance to an antiestrogen, tamoxifen, than MCF-7, and they were also 300- to 1,000-fold more resistant to other antiestrogens, epitiostanol and medroxyprogesterone, than MCF-7. The altered cellular sensitivity of U-2 or U-3 to the hormone antagonists is discussed in relation to the absence or presence of hormone receptors.

Androstanols↗
PubMed1986

Circumvention of multiple-drug resistance in human cancer cells by thioridazine, trifluoperazine, and chlorpromazine.

Human KB carcinoma cells were selected in four sequential steps for increasing resistance to colchicine and were found to be cross-resistant to multiple drugs. Thioridazine, a phenothiazine calmodulin inhibitor, almost completely reversed the resistance of the multiple-drug-resistant cells to doxorubicin, vinblastine, dactinomycin, and daunorubicin, and partially reversed the resistance to colchicine and vincristine. Other phenothiazine calmodulin inhibitors, trifluoperazine and chlorpromazine, were also found to show similar but somewhat weaker effects on drug resistance. However, a well-known naphthalenesulfonamide derivative calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), could not reverse the drug resistance. Very low accumulation of vincristine or daunorubicin was observed in the multiple-drug-resistant KB cells in comparison with accumulation in the parental KB cells. Increased rate of accumulation of the drugs by thioridazine, trifluoperazine, and chlorpromazine was most prominent in the resistant KB-ChR-24 cells than in KB cells. We have observed enhanced efflux of the drugs from the resistant cells, and thioridazine inhibited this efflux. These studies suggest a role for increased drug efflux in the development of the multiple-drug resistance phenotype in human carcinoma cells.

Calmodulin↗
PubMed1986

Overcoming drug resistance in cancer cells with synthetic isoprenoids.

A cultured subline (P388/ADM) of mouse P388 leukemia resistant to doxorubicin, vinblastine, vincristine, dactinomycin, and daunorubicin became sensitive again when treated with noncytotoxic doses of either of two synthetic isoprenoids: N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine (SDB-ethylenediamine) and N-(p-methylbenzyl)decaprenylamine X HCI (PMB-decaprenylamine). The isoprenoids also reversed resistance to doxorubicin and vincristine in a cultured vincristine-resistant P388 leukemia subline (P388/VCR). Median lethal doses (LD50) for PMB-decaprenylamine and SDB-ethylenediamine administered ip were 123 and 350 mg/kg against mice, whereas the LD50 for verapamil, another modifier of cellular drug resistance, was about 7.6 mg/kg. In vivo experiments with P388/VCR-bearing mice showed that both SDB-ethylenediamine and verapamil overcame vincristine resistance, but PMB-decaprenylamine showed only slight activity. SDB-ethylenediamine was especially effective, overcoming the vincristine resistance at 1 mg drug/kg. Since the structure of SDB-ethylenediamine resembles that of verapamil, a calcium-blocking agent that overcomes drug resistance, it was checked for calcium-blocking activity. However, calcium channel-blocking activity was not observed with 20 micrograms isoprenoid/ml, whereas calcium channel activity was completely blocked by 1 microgram verapamil/ml.

Animals↗
PubMed1986

Reversal of multidrug resistance by synthetic isoprenoids in the KB human cancer cell line.

A colchicine resistant clone, Chr-24, derived from the human carcinoma KB cell line is extensively resistant to multiple drugs including vinblastine, vincristine, Adriamycin, actinomycin D, and daunomycin. In comparison with KB cells, very low accumulation of daunomycin or vincristine is observed in multidrug-resistant cells. Two isoprenoids with 9 to 10 isoprene chains (polyprenoids), N-(p-methylbenzyl)decaprenylamine and N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine overcame the multidrug resistance almost completely in cultured Chr-24, whereas they only slightly sensitized the parental KB cells to anticancer agents. Both isoprenoids enhance the accumulation of vincristine or daunomycin in Chr-24, possibly by inhibiting efflux and also by enhancing influx of anticancer agents. A verapamil-like structure of N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine is discussed in relation to its ability to overcome drug resistance.

Antineoplastic Agents↗
PubMed1986

Techniques to reverse or circumvent drug-resistance in vitro.

In this study, we report that membrane-active agents can reverse or circumvent multidrug resistance in tumor cells through alteration of membrane permeability. Two different cell lines are used to assay the drug-sensitivity: colchicine-resistant clone derived from human cancer KB cell line with multidrug resistance and P388 leukemia cells resistant to many anticancer agents such as adriamycin, vincristine and other agents. In the latter system, circumvention effect can be tested with mice bearing drug-sensitive or -resistant P388 leukemia in vivo. Thioridazine (calmodulin inhibitor), N-(p-methylbenzyl)decaprenylamine, N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine and amphotericin B (a sterol-binding polyene antibiotic) have been screened with these two drug-resistant cell systems. They overcome the multidrug resistance in Chr-24 as well as P388 leukemia cells resistant to adriamycin or vincristine. N-solanesyl-N,N'-bis(3,4-dimethoxylbenzyl)ethylenediamine can reverse the drug-resistance in vivo against mice bearing resistant leukemia. Mechanism to reverse the drug-resistance is to be discussed in relation with altered membrane permeability of the anticancer agents.

Amphotericin B↗
PubMed1985

Cellular vitamin A-binding proteins in liver tumors and adjacent tissues.

The levels of cellular retinol-binding protein (CRBP) and retinoic acid-binding protein (CRABP) were assessed in surgically resected ten human liver tumors and their adjacent tissues by sucrose density gradient centrifugation and Scatchard analysis. The tissues adjacent to hepatocellular carcinomas usually showed portal cirrhosis or hepatitis. There was no significant difference in the dissociation constant (Kd) values of binding proteins of CRBP or CRABP between tumors and adjacent tissues. Five of the ten liver tumors showed similar levels of CRBP as in their adjacent tissues whereas three of the ten liver tumors showed about 40% or less contents of CRBP in comparison with their adjacent tissues and two others had no CRBP activity. By contrast, CRABP activities were found in five liver tumors and in adjacent tissues of one case, whereas there were no detectable CRABP activities in the adjacent tissues except in one case.

Adult↗
PubMed1985

Defective endocytosis of low-density lipoprotein in monensin-resistant mutants of the mouse Balb/3T3 cell line.

Two monensin-resistant clones show similar low-density lipoprotein binding activity but less internalization or degradation of low-density lipoprotein than the parental Balb/3T3 or other resistant clone. Sterol synthesis from radioactive acetate in the resistant mutant, MO-5, is inhibited by more than 70% of control in the presence of tenfold higher amounts of low-density lipoprotein than the dose that inhibits the parental Balb/3T3 to similar level. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity of Balb/3T3 and MO-5 is inhibited by 48% and 27% of control, respectively, in the presence of 10 micrograms/ml of low-density lipoprotein. Colloidal silica gradient centrifugation shows that transport of low-density lipoprotein from the surface membrane to the lysosome is much slower in MO-5 cells than in Balb/3T3 cells. Down regulation of low-density lipoprotein receptors on the cell surface in Balb/3T3 is observed by exposing the cells to 5-15 micrograms/ml low-density lipoprotein, whereas only slight if any down regulation is observed when MO-5 cells are treated with low-density lipoprotein. The altered endocytosis of low-density lipoprotein behaves as a dominant trait in hybrids of MO-5 and THO2-2, a derivative of Balb/3T3 resistant to both ouabain and 6-thioguanine.

Animals↗
PubMed1985

The effect of calcium antagonists on the proteolytic degradation of low-density lipoprotein in HeLa cells.

The degradation of 125I-labelled low-density lipoproteins (LDL) in HeLa cells was significantly inhibited when the cells were incubated either with the calcium channel blocking agents D600 and verapamil, or with the lysosomotropic agent chloroquine. However, nifedipine, another blocker of Ca2+ channels, did not affect the degradation of 125I-labelled LDL. The association of 125I-labelled LDL with HeLa cells was increased in proportion to the concentration of D600, and 125I-labelled LDL was accumulated in lysosomal fractions as assessed by Percoll density gradient analysis. Some 80% of 125I-labelled LDL in lysosomes of HeLa cells treated with D600 was acid-insoluble. The rate of incorporation of [3H]acetate into digitonin-precipitable material was increased 4-fold in the cells treated with 40 micrograms/ml D600 compared with untreated cells, but that of [3H]mevalonate was not enhanced. About 8 h of preincubation of the cells with D600 or verapamil was required to inhibit the LDL degradation by 50% of the control activity. It was also found that the inhibitory action of D600 could be reversed by removal of D600 from the medium. The activities of lysosomal enzymes, cathepsin B, beta-hexosaminidase, and acid phosphatase, were significantly decreased when the cells were treated with D600 and chloroquine, but not with nifedipine. Blockers of Ca2+ channels which effect the activity of lysosomal enzymes, should be useful for the study of the lysosomal function.

Calcium Channel Blockers↗
PubMed1985

Synergistic combination therapy of 5-fluorouracil, vitamin A, and cobalt-60 radiation for head and neck tumors--antitumor combination therapy with vitamin A.

Using cultured transformed fibroblasts, an antitumor agent, 5-fluorouracil (5-FU), was found to be potentiated in its inhibitory action on RNA formation by vitamin A. 5-FU in combination with vitamin A enhanced more effectively the cellular sensitivity to irradiation than when 5-FU was used alone. So we have applied a triple combination of 5-FU, vitamin A, and cobalt-60 radiation (FAR therapy) to clinical treatment of head and neck tumors since 1972. The treatment of 192 patients with cancer of the larynx, 60 patients with hypopharyngeal and cervical esophageal cancer, and 23 patients with epipharyngeal cancer with FAR therapy during 6 years from 1972 to 1977 resulted in highly effective synergism, and the improved points of this therapy were considered.

Adult↗
PubMed1985

Monensin-resistant mouse Balb/3T3 cell mutant with aberrant penetration of vesicular stomatitis virus.

A mutant (MO-5) resistant to monensin (an ionophoric antibiotic) derived from the mouse Balb/3T3 cell line, was a poor host for vesicular stomatitis virus (VSV) or semliki forest virus (SFV) multiplication. The yield of VSV particles in MO-5 is one 100-fold reduced as is VSV-dependent RNA synthesis. In contrast to a pH-remedial mutant, the abortive production of infectious VSV particles in MO-5 cells was not restored by low pH treatment. The pH values in the endosome and the lysosome of MO-5 cells were 5.2 and 5.4, respectively, values that were comparable to the pH value in Balb/3T3 cells. Assays with [3H]uridine-labeled VSV indicated similar binding of VSV in MO-5: percoll gradient centrifugation analysis of [35S]methionine-labeled VSV-infected Balb/3T3 showed accumulation of VSV in the lysosome fraction 20 min after VSV infection, whereas VSV can be found mainly in endosome/Golgi fraction of MO-5 cells after 40 to 60 min on the percoll gradients. Degradation of [35S]methionine-labeled VSV was observed at a significant rate in Balb/3T3 cells, but not in MO-5 cells. The monensin-resistant somatic cell may thus provide a genetic route to study the mechanism of endocytosis or transport of enveloped viruses.

Animals↗
PubMed1985

Cloning, sequence analysis, and expression of alteration of the mRNA stability gene (ams+) of Escherichia coli.

The ams+ gene, which influences the stability of mRNA in Escherichia coli was cloned in pBR322. The product of the gene, which is a 17,000-dalton protein, was expressed in expression vector pRC23, a derivative of pBR322. The molecular weight is consistent with sequencing analysis which shows that the gene contains 595 nucleotides and has an open reading frame of 149 amino acids. We discussed the possible role(s) of the ams+ gene product in affecting mRNA stability.

Base Sequence↗
PubMed1985

Effect of monensin on the synthesis, maturation and secretion of vesicular stomatitis virus proteins in a monensin-resistant Chinese hamster ovary cell line.

We compared the effects of the cationic ionophore, monensin, on the synthesis, maturation and release of vesicular stomatitis virus (VSV) in cultures of Chinese hamster ovary (CHO) cells and the monensin-resistant clone, MonR-31. Our results depended on the dose and time of the addition of monensin to the infected cells, from 1 h prior to VSV infection to 1 h after infection. VSV production was more resistant in MonR-31 than in CHO cells when the ionophore was added 1 h prior to VSV infection. Monensin added 1 h after VSV infection showed the opposite phenomenon; release of virus particles into the medium was 10- to 10(5)-fold less in MonR-31 cells than in CHO cells, and the intracellular virus number in the resistant cells was one-third to one-fourth of that in the parental CHO cells. Syntheses of all virus-associated G, N and M proteins were inhibited in both cell lines by monensin, but especially so in the MonR-31 cells. There were no marked qualitative changes in the biochemical properties of viral glycoprotein G in virus-infected CHO and MonR-31 cells treated with monensin after virus infection. An endoglycosidase H-resistant G with a molecular weight smaller than that of normal G and attachments of palmitate or fucose on the truncated G protein appeared. Alteration of the secretion of as well as the synthesis of the enveloped virus is discussed in relation to the monensin susceptibility of the resistant MonR-31 clone.

Animals↗
PubMed1985

[Pharmacological studies of N-(2-mercapto-2-methylpropionyl)-L-cysteine (SA 96). VI. Effects on vitamin B6, metals and skin collagen in rats].

N-(2-Mercapto-2-methylpropionyl)-L-cysteine (SA 96), an antirheumatic agent, and the main metabolite of SA 96, N-[2-methyl-2-(methylthio)propionyl]-L-cysteine (SA 679), were investigated for the effects on vitamin B6 (VB6), metals and skin collagen in rats in comparison with D-penicillamine (D-Pc). SA 96 had no effect on VB6 amount in serum and liver at doses of 30 mg/kg and 150 mg/kg, p.o., for 28 days, but SA 96 as well as SA 679 lowered the level in the liver slightly at a dose of 600 mg/kg. On the other hand, D-Pc lowered the VB6 level markedly both in serum and liver at doses of 150 mg/kg and 600 mg/kg, p.o., for 28 days. SA 96, SA 679 or D-Pc had no effect on urinary VB6 excretion. The degree of complexing of SA 96 as well as SA 679 with pyridoxal-5-phosphate in vitro was very slight as compared with D-Pc. SA 96 and D-Pc increased Cu and Zn excretion in urine, decreased Cu level both in serum and liver, and increased Zn level in serum. However, the degree of these effects of SA 96 on the metals was very slight as compared with D-Pc. SA 96 or SA 679 had no effect on skin collagen, neither soluble nor insoluble collagen, but D-Pc increased soluble collagen markedly. In addition, at a dose of 600 mg/kg, D-Pc decreased insoluble collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Potentiation by squalene of the cytotoxicity of anticancer agents against cultured mammalian cells and murine tumor.

Squalene (SQ) was examined for ability to potentiate the cytotoxicity and antitumor activity of anticancer agents. The dose-response curves of cell survival of cultured HeLa or V79 cells in the presence of various anticancer agents [adriamycin (ADM), 5-fluorouracil (5-FU), bleomycin (BLM) and cis-dichlorodiamminoplatinum (CDDP)] showed that SQ potentiated the cytotoxicity of these anticancer agents. SQ may enhance the cytotoxic effect of ADM by interfering with the efflux of ADM from the cells. The antitumor activities of these anticancer agents combined with SQ were tested against sarcoma 180 (S180) ascites cells. Some combinations were found to show synergistic antitumor effects: ADM, BLM or CDDP with SQ was effective against S180, but only slight if any synergistic effect was observed with the combination of 5-FU and SQ. The antitumor activity of BLM was most strongly potentiated by SQ, among the tested agents.

Animals↗
PubMed1985

[Somatic cell mutants with altered receptor and endocytosis activities].

Binding and endocytosis are responsible for cellular uptake of a wide variety of physiologically active ligands. We have selected somatic cell mutants with altered endocytosis activity for low-density lipoprotein, insulin, and epidermal growth factor. Of these mutants, the one with altered response to epidermal growth factor shows aberrant growth behavior in soft agar.

Animals↗