Shinji Nishiwaki

Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR

SummaryCertain waterblooms of toxic cyanobacteria (blue-green algae) are a health threat because of their production of toxic peptides, termed microcystins, which cause liver damage in wild and domesticated animals. The most widely studied microcystin is microcystin-LR, a heptapeptide containing the twol-amino acids, leucine and arginine. The inhibition of protein phosphatase type 1 and type 2A activities by microcystin-LR is similar to that of the known protein phosphatase inhibitor and tumor promoter okadaic acid. We show in this report that microcystin-LR, applied below the acute toxicity level, dose-dependently increases the number and percentage area of positive foci for the placental form of glutathioneS-transferase in rat liver, which was initiated with diethylnitrosamine. The result was obtained independently through two animal experiments. This observation indicates that microcystin-LR is a new liver tumor promoter mediated through inhibition of protein phosphatase type 1 and type 2A activities. This provides further evidence that the okadaic acid pathway is a general mechanism of tumor promotion in various organs, such as mouse skin, rat glandular stomach and rat liver.

Structure‐Function Relationships of Microcystins, Liver Tumor Promoters, in Interaction with Protein Phosphatase

Microcystins, isolated from toxic blue‐green algae, are potent inhibitors of protein phosphatases 1 and 2A. Recently, we have reported that mierocystin LR has a potent tumor‐promoting activity on rat liver initiated with diethylnitrosamine. The structure of microcystins is unique in having an unusual ammo acid, 3‐amino‐9‐methoxy‐10‐phenyl‐2,6,8‐trimethyl‐deca‐4(E),6(E)‐dienoic acid (Adda), which is thought to be significant for the activity. Geometrical isomers at C‐7 in the Adda portion of microcystins, 6(Z)‐Adda microcystins LR and RR, have been isolated from cyanohacteria. To estimate their tumor‐promoting activities and to understand the importance of the Adda portion for activity, the maternal microcystins LR and RR and their isomers were subjected to examination of their interaction with protein phosphatases 1 and 2A and the release of glutamic pyruvic transaminase from rat liver. 6(Z)‐Adda microcystins LR and RR bound to protein phosphatases 1 and 2A, inhibited their activities and released glutamic pyruvic transaminase from rat liver into serum, ten to one hundred times more weakly than the maternal microcystins LR and RR. These results indicated that the conjugated diene with 4(E),6(E) geometry in the Adda portion is important in the interaction with protein phosphatases.

Structurally different members of the okadaic acid class selectively inhibit protein serine/threonine but not tyrosine phosphatase activity

The relative potencies of four main types of okadaic acid class compounds as inhibitors of the catalytic subunits of protein serine/threonine phosphatases 1 and 2A and the protein tyrosine phosphatase 1 were determined. These four types of compounds are okadaic acid, calyculin A, microcystin-LR, and tautomycin, which are isolated from different natural sources, a black sponge Halichondria okadai, a marine sponge Discodermia calyx, a blue-green alga Microcystis aeruginosa, and Streptomyces spirover ticillatus, respectively. While okadaic acid was a more effective inhibitor of protein phosphatase 2A (IC50, 0.07 nM) than protein phosphatase 1 (IC50, 3.4 nM), other compounds of the okadaic acid class were equally effective against the two protein serine/threonine phosphatases. The order of potency was microcystin greater than calyculin A greater than tautomycin, and the IC50S ranged from 0.1 to 0.7 nM. None of the okadaic acid class compounds inhibited protein tyrosine phosphatase 1 activity at concentrations up to 0.01 mM. These results indicate that the compounds of the okadaic acid class are selective inhibitors of protein serine/threonine but not tyrosine phosphatases.

Inhibitory Effects of (—)‐Epigallocatechin Gallate on Spontaneous Hepatoma in C3H/HeNCrj Mice and Human Hepatoma‐derived PLC/PRF/5 Cells

The inhibitory effect of (—)‐epigallocatechin gallate (EGCG), a main constituent of Japanese green tea, on spontaneous hepatoma in C3H/HeNCrj mice was investigated. A total of 72 mice were divided into three groups; the control group without EGCG, and two experimental groups receiving 0.05% (w/w) or 0.1% EGCG in drinking water. EGCG reduced the incidence of hepatoma‐bearing mice from 83.3% (control) to 56.0% (0.05% EGCG) and 52.2% (0.1% EGCG), and also reduced the average number of hepatomas per mouse from 1.83 (control) to 0.72 (0.05% EGCG) and 0.91 (0.1% EGCG) at week 65. Ridit analysis of the distribution of the number of hepatomas in each group revealed that EGCG significantly increased the rate of mice without hepatoma in the two EGCG groups as compared to the control. EGCG did not affect body weight gain, food consumption or any serum biochemical parameter. EGCG inhibited the growth and secretion of α‐fetoprotein by human hepatoma‐derived PLC/PRF/5 cells without decreasing their viability. These results indicate that EGCG may be a practical, nontoxic preventive agent against human hepatoma.

Suppression by carotenoids of microtenoids of microcystin-induced morphological changes in mouse hepatocytes

Microcystin-LR is a liver tumor promoter in the okadaic acid class, a group of potent inhibitors of protein phosphatases 1 and 2A. Because of inhibition of protein phosphatases, microcystin-LR induces hyperphosphorylation of cellular proteins, including cytoskeletal proteins—cytokeratins 8 and 18—and causes morphological changes in mouse hepatocytes in primary culture. We studied the effects of carotenoids to antagonize microcystin-LR-induced morphological changes in hepatocytes. β-Carotene (100 nM to 100 μM), suppressed the morphological changes induced by 100 nM microcystin-LR in a dose-dependent manner. Other carotenoids tested exerted similar suppressive effects, although retinoids, such as all-trans retinol, all-trans retinoic acid, and 9-cis retinoic acid, were only weakly suppressive. The relative potency of the suppression correlated significantly with the number of conjugated double bonds in thetrans configuration. β-Carotene strongly suppressed the hyperphosphorylation of cellular proteins induced by microcystin-LR without significant changes in the basal phosphorylation level. Other antioxidants, such as α-tocopherol, did not protect the cells against microcystin-LR. Taken together, the antagonistic effects of carotenoids against microcystin-LR are difficult to explain by their antioxidant or provitamin A activities. Suppression of the hyperphosphorylation of cellular proteins may be a novel mechanism by which carotenoids inhibit tumor promotion.

Aberrant metabolism of retinoid X receptor proteins in human hepatocellular carcinoma

A polyclonal antibody was raised against a recombinant ligand binding domain construct of the human retinoid X receptor (RXR) alpha. This antibody reacted with an endogenous 54 kDa nuclear protein from human hepatoma-derived HuH7 cells in immunoblot analyses. Immunoblotting of nuclear proteins from human hepatocellular carcinomas (HCCs) and their surrounding tissues revealed the presence of a 44 kDa RXR distinct from the 54 kDa RXR and a dramatic decrease in the relative amounts of 44 kDa RXR to 54 kDa RXR in all HCCs compared with normal tissue. In vitro shift and intracellular conversion from 54 kDa RXR to 44 kDa species were observed with the nuclear extracts of HuH7 cells. Furthermore, transfection of hRXR alpha cDNA into HuH7 cells resulted in the increase of 54 kDa RXR, whereas transfected mouse hepatocytes accumulated 44 kDa RXR. These results strongly indicated that 44 kDa RXR was a physiological proteolytic fragment of 54 kDa RXR and that post-translational metabolism of RXR was impaired in HCC and the HuH7 hepatoma-derived cell line.

Inhibition of Gastroesophageal Reflux by Semi-solid Nutrients in Patients With Percutaneous Endoscopic Gastrostomy

BACKGROUND Aspiration is one of the major complications after percutaneous endoscopic gastrostomy (PEG). The administration of semi-solid nutrients by means of gastrostomy tube has recently been reported to be effective in preventing aspiration pneumonia. The effects of semi-solid nutrients on gastroesophageal reflux, intragastric distribution, and gastric emptying were evaluated. METHODS Semi-solid nutrients were prepared by liquid nutrients mixed with agar at the concentration of 0.5%. The distribution of the administered radiolabeled liquid and semi-solid nutrients was monitored by a scintillation camera for 15 post-PEG patients. The percentage of esophageal reflux, the distribution of the proximal and distal stomach, and the gastric emptying time were evaluated. RESULTS The percentage of gastroesophageal reflux was significantly decreased in semi-solid nutrients (0.82 +/- 1.27%) compared with liquid nutrients (3.75 +/- 4.25%), whereas the gastric emptying time was not different. The distribution of semi-solid nutrients was not different from liquid nutrients in the early phase, whereas higher retention of liquid nutrients in the proximal stomach was observed in the late phase. CONCLUSIONS Gastroesophageal reflux was significantly inhibited by semi-solid nutrients. One of the mechanisms of the inhibition is considered to be an improvement in the transition from the proximal to distal stomach in semi-solid nutrients.

Rapid purification of protein phosphatase 2A from mouse brain by microcystin-affinity chromatography

Microcystin LR. which is a monocyclic heptapeptide containing two L‐amino acids, and leucine and arginine, is a new inhibitor of protein phosphatases 1 and 2A. Microcystin LR‐affinity chromatography was used to purify protein phosphatase 2A as a holoenzyme. Five mg of microcystin LR were immobilized to ECH Sepharose 4B by the carbodiimide coupling reaction. Following DEAE‐cellulose column chromatography, microcystin‐affinity chromatography, as the second step in the procedure, resulted in purification of protein phosphatase 2A in a pure form. The enzyme isolated from mouse brain consisted of two regulatory subunits of 67 kDa and 58 kDa and a catalytic subunit of 41 kDa. Microcystin‐affinity chromatography is useful for isolation of protein phosphatase 2A.

Activation of Protein Kinase C by Mycobacterial Cord Factor, Trehalose 6-Monomycolate, Resulting in Tumor Necrosis Factor-α Release in Mouse Lung Tissues

Cord factors are mycoloyl glycolipids in cell walls of bacteria belonging to Actinomycetales, such as Mycobacterium, Nocardia and Rhodococcus. They induce granuloma formation in the lung and interstitial pneumonitis, associated with production of macrophage‐derived cytokines. We studied how cord factors induce biological activities in the cells. Cord factors isolated from M. tuberculosis, trehalose 6‐monomycolate (mTMM) and trehalose 6,6′‐dimycolate (mTDM), enhanced protein kinase C (PKC) activation in the presence of phosphatidylserine (PtdSer), diacylglycerol and Ca2+, and mTMM activated PKCα more strongly than PKCβ or γ under the same assay conditions. Kinetic studies of mTMM in response to PKC activation revealed that mTMM increased the apparent affinity of PKC to Ca2+ in the presence of both PtdSer and diolein. Although this is similar to observations with unsaturated fatty acids, such as arachidonic acid, mTMM was synergistic with PtdSer for PKC activation, but arachidonic acid was not. mTMM was also different as regards PKC activation, as phorbol ester was. A single i.p. administration of mTMM to mouse induced tumor necrosis factor‐α (TNF‐α) in serum and in the lung, which is a unique target tissue of cord factors. Based on our recent finding that TNF‐α is an endogenous tumor promoter, the correlation between lung cancer and pulmonary tuberculosis is discussed.

Photoaffinity labeling of protein phosphatase 2A, the receptor for a tumor promoter okadaic acid, by [27-3H]methyl 7-O-(4-azidobenzoyl)okadaate

The interaction of okadaic acid and a protein phosphatase, which is the receptor for a tumor promoter, okadaic acid, was studied. [27-3H]Methyl 7-O-(4-azidobenzoyl)okadaate was synthesized and protein phosphatase 2A, which consists of two regulatory subunits of 65 kDa and 42 kDa and a catalytic subunit of 37 kDa, was purified from bovine brain. After UV irradiation of the reaction mixture, [3H]methyl 7-O-(4-azidobenzoyl)okadaate covalently bound to the catalytic subunit, but not to the two regulatory subunits. These results strongly suggest that okadaic acid, a potent inhibitor of protein phosphatases, binds specifically to the catalytic subunit rather than to the other regulatory subunits.