Encyclopedia of Cancer

Living Edition
| Editors: Manfred Schwab


Living reference work entry
DOI: https://doi.org/10.1007/978-3-642-27841-9_7069-3


Protein Phosphatase Okadaic Acid Microcystis Aeruginosa Round Form Primary Human Fibroblast 
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Cyanobacterial hepatotoxin, a potent tumor promoter; there are three forms of microcystins:
  • Microcystin-LR is cyclo(-D-Ala-L-Leu-D-erythro-beta methylisoaspartic acid-(Masp)- L-Arg-(2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-(4E,6E)-dienoic acid (Adda)-D-isoGlu-N-methyldehydroalanine (Mdha)-).

  • Microcystin-YR (microcystin containing tyrosine instead of the leucine in microcystin-LR).

  • Microcystin-RR (microcystin containing arginine instead of the leucine in microcystin-LR) (Fig. 1a).
    Fig. 1

    Structures of microcystins. (a) Microcystin-LR, microcystin-YR, and microcystin-RR. (b) Geometrical isomers (6Z)-Adda microcystin-LR and microcystin-RR


Microcystin-LR is a hepatotoxin, one of the microcystins isolated from cyanobacteria (blue-green algae), which include Microcystis aeruginosa, Microcystis viridis, Microcystis wesenbergii, Microcystis flos-aquae, and Oscillatoria agardhii. Microcystin-LR is found in waterblooms of toxic cyanobacteria and in eutrophic freshwater municipal and residential water supplies; it is associated with increasing environmental hazards in various areas of the world.

Toxins of cyanobacteria are grouped into cytotoxin, neurotoxin, and hepatotoxin. The hepatotoxins are microcystin-LR, microcystin-YR, and microcystin-RR, and there are 50 microcystin derivatives. Microcystin-LR is a tumor promoter in rat liver, a potent inhibitor of serine/threonine protein phosphatase 1 and 2A (PP1 and PP2A) (IC50 values 0.1 nM and 0.1 nM, respectively). Protein dephosphorylation by PP1 and PP2A is an opposite biochemical reaction of protein phosphorylation by protein kinases. Thus, inhibition of protein phosphatase causes accumulation of phosphoproteins in the cells. The World Health Organization (WHO) International Agency for Research on Cancer (IARC), Lyon, in 2006 assessed the carcinogenicity of microcystin-LR for humans, based on three perspectives: carcinogenicity in rodents, epidemiological evidence, and unique mechanisms of action of the compound; WHO concluded that microcystin-LR is “possibly carcinogenic to humans” (group 2B).

Tumor-Promoting Activity in Rat Liver

Liver organotropy of microcystins is unique because intraperitoneal administration of [3H]dihydromicrocystin-LR results in the highest uptake into the liver of mice – 17.0 ± 4.1 % of the total administered radioactivity after 5 min and 71.5 ± 6.9 % after 1 h – whereas oral administration results in 0.68 % of the total administered amount in the liver from 6 to 19 h after treatment. After a single intraperitoneal injection of 1.5 μg microcystin-YR, i.e., 75 μg/kg body weight, the liver became dark red and the mouse died within 2 h. The histological examination revealed massive intrahepatic hemorrhages resulting in acute death of liver cells by necrosis.

Two-stage carcinogenesis of microcystin-LR in the liver of male F344 rats showed tumor-promoting activity. Initiation was done with a single administration of diethylnitrosamine (DEN) 200 mg/kg. Tumor promotion was achieved by repeated intraperitoneal administrations of microcystin-LR 25 μg/kg, twice a week, a total of 20 times for 10 weeks (initiation and promotion). As an indicator for detecting tumor-promoting activity, autopsied liver sections were stained by the avidin-biotin-peroxidase complex method for immunohistochemistry of glutathione S-transferase placental form (GST-P) positive foci and neoplastic nodules, and only those foci larger than 50 μm in diameter were counted (Fig. 2). GST-P is a biochemical marker detecting neoplastic change, and the GST-P positive foci in the liver were assessed by the number of foci/cm2 of the liver, area of foci/liver (mm2/cm2), and volume of foci/liver (v/v%). Group 4, treated with initiator, DEN plus microcystin-LR, had larger numbers, areas, and volumes of foci per liver than did the control groups. Group 3, treated with microcystin-LR alone, did not induce any foci.
Fig. 2

Induction of glutathione S-transferase placental form (GST-P) positive foci. (a) The liver treated with a single administration of DEN. (b) The liver treated with DEN plus microcystin-LR 50 μg/kg body weight together 20 times for 10 weeks

Microcystins are not mutagenic in the Ames test with Salmonella typhimurium. Thus, microcystin-LR has a tumor-promoting activity in rat liver and does not have any initiating activity. Microcystin-YR and microcystin-RR also are assumed to be liver tumor promoters, because they have the same specific activity in various biochemical assays as does microcystin-LR.

Inhibition of Specific [3H]okadaic Acid Binding

The tertiary structure of microcystin-LR is similar to that of okadaic acid, since okadaic acid is thought to have a flexible cavity formed by an intramolecular hydrogen bond between C-1 carbonyl and C-24 hydroxyl groups. Microcystin-LR, microcystin-YR, and microcystin-RR inhibited the specific [3H]okadaic acid binding to cytosolic fraction with the same potencies. Thus, microcystins bind to the okadaic acid receptors PP1 and PP2A as strongly as okadaic acid does.

Inhibition of Protein Phosphatases 1 and 2A

Microcystin-LR inhibits the catalytic subunit of PP1 purified from rabbit skeletal muscle (IC50 value 0.1 nM) and that of PP2A purified from human erythrocytes (IC50 value 0.1 nM). Microcystin-LR inhibited both PP1 and PP2A with the same potencies, but it did not inhibit protein tyrosine phosphatase. Microcystin-LR, microcystin-YR, and microcystin-RR inhibited protein phosphatase 2A, with the similar potencies, and the results correlate well with inhibition of specific [3H]okadaic acid binding.

Cellular Biochemical Response

Microcystin-LR and okadaic acid have different effects on primary human fibroblasts. Microcystin-LR at concentrations up to 9.6 μM did not induce any biochemical or biological effects on primary human fibroblasts, but 250 nM okadaic acid was enough to induce morphological changes in cells, from spindle-like to a round form, within 2 h of incubation. A solution of microcystin-YR at a very high concentration of 670 μM was injected into primary human fibroblasts resulted in morphological changes from spindle-like to a round form 45 min after microinjection. Obviously, there are differences in tissue specificity between microcystins and okadaic acid.

Structure-Function Relationship of Microcystins

The long hydrophobic portion of microcystins (Adda) is significant in their activity. Two geometrical isomers at C-7 in the Adda portion of microcystins, (6Z)-Adda microcystin-LR and microcystin-RR, were isolated from cyanobacteria (Fig. 1b). The specific [3H]dihydromicrocystin-LR binding to the cytosolic fraction was inhibited by microcystin-LR and microcystin-RR (IC50 values 0.38 and 0.42 nM, respectively) and by (6Z)-Adda microcystin-LR and microcystin-RR (IC50 values 32 and 52 nM, respectively). Thus, (6Z)-Adda microcystin-LR and microcystin-RR are 100 times weaker than their maternal (6E)-Adda microcystins, and the conjugated diene with (4E,6E) geometry in the Adda portion is essential in the interaction with protein phosphatases.

Furthermore, microcystin-LR and microcystin-LA have the same specific activity on the inhibitions of PP1 and PP2A and specific [3H]okadaic acid binding, suggesting that the arginine residue in microcystin-LR does not significantly contribute to its biochemical activity.

Crystal Structure of PP1-Microcystin-LR Complex

The crystal structure of PP1-microcystin-LR complex at 2.1 Å was initially reported in 1995. Microcystin-LR binds to catalytic subunit of PP1 through interaction with the metal-binding site, the hydrophobic groove, and the edge of the C-terminal groove near the active site (Fig. 3). The covalent linkage of microcystin-LR to PP1 was noted previously, but it is not essential for the inhibition of the enzyme by microcystin-LR.
Fig. 3

Electron density map of microcystin-LR binding to PP1 catalytic subunit (From: Goldberg et al. 1995)



  1. Carmichael WW (1992) Cyanobacteria secondary metabolites – the cyanotoxins. J Appl Bacteriol 72:445–459CrossRefPubMedGoogle Scholar
  2. Farber E, Solt D (1978) A new liver model for the study of promotion. Slaga TJ, Sivak A, Boutwell RK Eds, Carcinogenesis 2:443–451Google Scholar
  3. Fujiki H, Suganuma M (2010) Tumor promoters – microcystin-LR, nodularin and TNF-α and human cancer development. Anticancer Agents Med Chem 11:4–18CrossRefGoogle Scholar
  4. Goldberg J, Huang H-b, Kwon Y-g, Greengard P, Nairn AC, Kuriyan J (1995) Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1. Nature 376:745–753CrossRefPubMedGoogle Scholar
  5. Grosse Y, Baan R, Straif K, Secretan B, Ghissassi FEI, Cogliano V (2006) Carcinogenicity of nitrate, nitrite, and cyanobacterial peptide toxins. Lancet Oncol 7:628–629CrossRefPubMedGoogle Scholar
  6. Harada K-I, Ogawa K, Matsuura K, Murata H, Suzuki M, Watanabe MF, Itezono Y, Nakayama N (1990) Structural determination of geometrical isomers of microcystins LR and RR from cyanobacteria by two-dimensional NMR spectroscopic techniques. Chem Res Toxicol 3:473–481CrossRefPubMedGoogle Scholar
  7. Matsushima R, Yoshizawa S, Watanabe MF, Harada K-I, Furusawa M, Carmichael WW, Fujiki H (1990) In vivo and in vitro effects of protein phosphatese inhibitors, microcystins and nodularin, on mouse skin and fibroblasts. Biochem Biophys Res Commun 171:867–874Google Scholar
  8. Nishiwaki-Matushima R, Ohta T, Nishiwaki S, Suganuma M, Kohyama K, Ishikawa T, Carmichael WW, Fujiki H (1992) Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. J Cancer Res Clin Oncol 118:420–424CrossRefGoogle Scholar
  9. Sato K, Kitahara A, Satoh K, Ishikawa T, Tatematsu M, Ito N (1984) The placental form of glutathione S-transferease as a new marker protein for preneoplasia in rat chemicall heaptocarinogenesis Gann 75:199–202Google Scholar
  10. Suganuma M, Suttajit M, Suguri H, Ojika M, Yamada K, Fujiki H (1989) Specific binding of okadaic acid, a new tumor promoter in mouse skin. FEBS Lett 250:615–618Google Scholar

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© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Department of Clinical Laboratory Medicine, Faculty of MedicineSaga UniversitySaga 849-8501Japan