Zonglou Guo

Microcystin-LR promotes proliferation by activating Akt/S6K1 pathway and disordering apoptosis and cell cycle associated proteins phosphorylation in HL7702 cells

Our previous studies had shown that MC-LR inhibited PP2A activity and hyperphosphorylated PP2A substrates at 24 h exposure in HL7702 cells. Although the cytoskeleton was rearranged, the cellular effects were not observed. The purpose of the present study with HL7702 cell exposed to MC-LR for 1-72 h was to further uncover the adverse effects of MC-LR comprehensively. The results showed that there were no obvious difference in apoptosis rate and cell-cycle distribution but the cell proliferation was changed since 36 h exposure while the uptake of MC-LR and its binding to PP2A/C kept unchanged since 1h exposure. PP2A activity had not manifested continued decline compare to 24h exposure and PP2A regulator α4 was found to release its associated PP2A/C since 1h exposure. The increasing of p-Akt-T308, p-Akt-S473, p-S6K1, p-S6, and p-4E-BP1 since 1h MC-LR exposure indicated that Akt/S6K1 cascade had been activated as early as 1h MC-LR treatment. And, PI3K/Akt inhibitor (LY294002) blocked MC-LR-induced Akt/S6K1 activation and proliferation. Besides, MC-LR also led to hyperphosphorylation of c-Myc, c-Jun, Bcl-2 and Bad and activation of Cdk1. Our study indicated that MC-LR exposure promoted HL7702 cell proliferation and the main mechanism was the activation of Akt/S6K1 cascade. Meanwhile, hyperphosphorylation of Bcl-2, Bad, c-Myc and c-Jun might also be involved. And, the inhibition of PP2A was the major reason for these molecular changes.

Microcystin-LR induces protein phosphatase 2A alteration in a human liver cell line.

Microcystin‐LR (MC‐LR) is a potent inhibitor of protein phosphatases 1 and 2A, and has potent hepatotoxicity and tumor promotion activity. Numerous studies on MC‐LR toxicity have been conducted in rat hepatocytes, but few studies of the effects of microcystins on human hepatocytes have been done. In this study, HL7702 cells (a human normal liver cell line) were incubated in MC‐LR for 24 h. The existence of MC‐LR in HL7702 cells was confirmed. Furthermore, PP2A activity and the alteration of PP2A subunits were assessed. The results show that PP2A activity decreased from the concentration of 1 μM MC‐LR, showing a concentration‐dependent decline, to about 34% at 10 μM MC‐LR. This activity undergone opposite change with alternations of phosphorylated Y307‐PP2A/C and PP2A/C subunit but showed same change with the alteration of the ratio of methylated L309‐PP2A/C to PP2A/C. B55α, a regulatory subunit of PP2A, was slightly increases in cells treated with the highest concentration of MC‐LR (10 μM), and colocalized increasedly with rearranged‐microtubules after 1 μM MC‐LR exposure. However, the proportion of early apoptotic cells did not show any change at various concentration of MC‐LR for 24 h. To our knowledge, this is the first report showing MC‐LR‐induced alteration of PP2A phosphatase in human cultured hepatocytes, and the mechanism of action seems to be similar as described before in vitro. The alteration of PP2A and microtubule seems to be the early event induced by MC‐LR exposure.

Microcystin-LR-Caused ROS generation involved in p38 activation and tau hyperphosphorylation in neuroendocrine (PC12) cells

Microcystin‐LR (MC‐LR), a potent specific hepatotoxin produced by cyanobacteria, has recently been reported to show neurotoxicity. Our previous study demonstrated that MC‐LR caused the reorganization of cytoskeleton architectures and hyperphosphorylation of the cytoskeletal‐associated proteins tau and HSP27 in neuroendocrine PC12 cell line by direct PP2A inhibition and indirect p38 mitogen‐activated protein kinase (MAPK) activation. It has been shown that oxidative stress is extensively associated with MC‐LR toxicity, mainly resulting from an excessive production of reactive oxygen species (ROS). However, the mechanisms by which ROS mediates the cytotoxic action of MC‐LR are unclear. In the present study, we investigated whether ROS might play a critical role in MC‐LR‐induced hyperphosphorylation of microtubule‐associated protein tau and the activation of the MAPKs in PC12 cell line. The results showed that MC‐LR had time‐ and concentration‐dependent effects on ROS generation, p38‐MAPK activation and tau phosphorylation. The time‐course studies indicated similar biphasic changes in ROS generation and tau hyperphosphorylation, which started to increase within 1 h and reached the maximum level at 3 h followed by a decrease after prolonged treatment. Furthermore, pretreatment with the antioxidants, N‐acetylcysteine and vitamin C, significantly decreased MC‐LR‐induced ROS generation and effectively attenuated p38‐MAPK activation as well as tau hyperphosphorylation. Taken together, these findings suggest that ROS generation triggered by MC‐LR is a key intracellular event that contributes to an induction of p38‐MAPK activation and tau phosphorylation, and that blockade of this ROS‐mediated redox‐sensitive signal cascades may attenuate the toxic effects of MC‐LR.

Protective effect of green tea polyphenols on tributyltin-induced oxidative damage detected by in vivo and in vitro models

The current study investigated the protective effects of green tea polyphenols (GTPP) on TBT‐induced oxidative damage. The results showed that reactive oxygen species (ROS) production and malondialdehyde content of the liver in mice exposed to TBT were reduced in the GTPP‐treated group compared to the untreated group. The intracellular ROS level was elevated in TBT‐treated human FL cells in a time‐dependent manner. Comet assay data demonstrated that the number of cells with damaged DNA in untreated mice was found to be significantly higher compared to GTPP‐treated mice. Damage to the nuclei and mitochondria observed in TBT‐treated mice were alleviated in mice treated with both TBT and GTPP. The results represent the first observation that GTPP were effective in reducing TBT‐induced oxidative damage both in vivo and in vitro. The possible protective mechanism may be due to the powerful ability of GTPP to scavenge ROS and prevent DNA breaks. We conclude that GTPP could be an effective agent or food supplement to reduce the cytotoxicity of TBT.

Protein phosphatases 2A as well as reactive oxygen species involved in tributyltin-induced apoptosis in mouse livers

Tributyltin (TBT), a highly toxic environmental contaminant, has been shown to induce caspase‐3‐dependent apoptosis in human amniotic cells through protein phosphatase 2A (PP2A) inhibition and consequent JNK activation. This in vivo study was undertaken to further verify the results derived from our previous in vitro study. Mice were orally dosed with 0, 10, 20, and 60 mg/kg of body weight TBT, and levels of PP2A, reactive oxygen species (ROS), mitogen‐activated protein kinase (MAPK), Bax/Bcl‐2, and caspase‐3 were detected in the mouse livers. Apoptosis was also evaluated using the TUNEL assay. The results showed that PP2A activity was inhibited, ROS levels were elevated, and MAPKs including ERK, JNK, and p38 were activated in mouse livers treated with the highest dose of TBT. Additionally, the ratio of Bax/Bcl‐2 was increased, caspase‐3 was activated, and apoptosis in mouse livers could be detected in the highest dose group. Therefore, a possible signaling pathway in TBT‐induced apoptosis in mouse livers involves PP2A inhibition and ROS elevation serving a pivotal function as upstream activators of MAPKs; activation of MAPKs in turn leads to an increase in the Bax/Bcl‐2 ratio, ultimately leading to the activation of caspase‐3. The results give a comprehensive and novel description of the mechanism of TBT‐induced toxicity.

Alterations of tau and VASP during microcystin‐LR‐induced cytoskeletal reorganization in a human liver cell line

Previously, we have reported alterations to HSP27 during Microcystin‐LR (MC‐LR)‐induced cytoskeletal reorganization in the human liver cell line HL7702. To further elucidate the detailed mechanism of MC‐LR‐induced cytoskeletal assembly, we focused on two cytoskeletal‐related proteins, Tau and VASP. These two proteins phosphorylated status influences their ability to bind and stabilize cytoskeleton. We found that MC‐LR markedly increased the level of Tau phosphorylation with the dissociation of phosphorylated Tau from the cytoskeleton. Furthermore, the phosphorylation of Tau induced by MC‐LR was suppressed by an activator of PP2A and by an inhibitor of p38 MAPK. VASP was also hyperphosphorylated upon MC‐LR exposure; however, its phosphorylation appeared to regulate its cellular localization rather than cytoskeletal dynamics, and its phosphorylation was unaffected by the PP2A activator. These data suggest that phosphorylated Tau is regulated by p38 MAPK, possibly as a consequence of PP2A inhibition. Tau hyperphosphorylation is likely an important factor leading to the cytoskeletal destabilization triggered by MC‐LR and the role of VASP alteration upon MC‐LR exposure needs to be studied further. To our knowledge, the finding that Tau is implicated in cytoskeletal destabilization in MC‐LR‐treated hepatocytes and MC‐LR‐induced VASPs alteration has not been reported previously.

Inhibition of PP2A and the consequent activation of JNK/c-Jun are involved in tributyltin-induced apoptosis in human amnionic cells

Tributyltin (TBT), a highly toxic environmental contaminant, has been shown to induce mitochondrial‐dependent apoptosis in several mammalian cells. However, the upstream signal transduction pathways involved in TBT‐induced apoptosis are still not fully elucidated. In this study, the protein phosphatase (PP) 2A, microtubule organization, and mitogen‐activated protein kinases (MAPKs), including JNK, p38 and their downstream transcription factors, c‐Jun and ATF‐2, respectively, were investigated in human amnionic cells treated by TBT. Furthermore, the activation of procaspase‐3 after blocking either one of these MAPK pathways was also observed. The results showed that TBT effectively induced apoptosis characterized by caspase‐3 activation. In apoptotic cells, the inhibition of PP2A activity and microtubule depolymerization was detected. Additionally, JNK and p38, as well as their downstream targets, c‐Jun and ATF‐2, were activated. Moreover, a JNK inhibitor, but not p38 inhibitor, significantly reduced caspase‐3 activation. It can be concluded that the inhibition of PP2A may (1) play as a role in the activation of JNK and c‐Jun and the concomitant promotion of microtubule depolymerization and (2) lead to the activation of caspase‐3 in TBT‐induced apoptotic cells. The results of this study suggest a critical role of PP2A in the TBT toxicity mechanism.

HEK293 cells exposed to microcystin‐LR show reduced protein phosphatase 2A activity and more stable cytoskeletal structure when overexpressing α4 protein

Microcystin‐LR (MC‐LR) is one of the most toxic members of microcystins released by freshwater cyanobacterial. The major mechanism of MC‐LR toxicity has been attributed to its inhibition of protein phosphatases 1 (PP1) and 2A (PP2A). In our prior research, α4 protein, a regulator of PP2A, was found not only crucial for PP2A regulation but also for the overall response of HEK 293 cells encountering MC‐LR. To explore the role of α4 in MC‐LR toxicity via PP2A regulation, here, HEK 293 cells overexpressing α4 protein were exposed to MC‐LR and PP2A, cytoskeletal organization, and cytoskeleton‐related proteins were investigated. The results showed that PP2A activity decreased and PP2A/C subunit expression and phosphorylation at Tyr307 increased significantly in the group exposed to high MC‐LR. Vimentin IF became concentrated and formed perinuclear bundles. However, the assembly of actin filament and microtubules remained unchanged and the expression and phosphorylation of the cytoskeleton‐related proteins HSP27 and VASP did not increase significantly. Some of these results differ from those of our previous study in which normal HEK293 cells were exposed to MC‐LR. Our results indicate that elevated α4 expression confers some resistance to MC‐LR‐induced cytoskeletal change These new findings provide helpful insights into the mechanism of MC‐LR toxicity and the role of α4 in regulating PP2A function.

Microcystin-LR induces a wide variety of biochemical changes in the A549 human non-small cell lung cancer cell line: Roles for protein phosphatase 2A and its substrates.

Our previous studies have described the toxic effects of microcystin‐LR (MC‐LR) in various normal cell lines and human hepatoma SMMC‐7721 cells, but the specific effects of MC‐LR in other types of cancer cells with respect to protein phosphatase 2A (PP2A) have not been fully elaborated. A549 human lung adenocarcinoma cells have been identified to express organic anion‐transporting polypeptides (OATP) involved in cellular uptake of MC‐LR, and thus probably make an appropriate in vitro model to assess MC‐LRs cytotoxicity. Hence, in our present study, A549 cells were treated with various concentrations of MC‐LR for 24 h. The presence of MC‐LR in A549 cells was confirmed, and PP2A activity, PP2A substrates, cytoskeleton, apoptosis, and proliferation were subsequently explored. The results showed that 5–10 μM MC‐LR inhibited PP2A activity significantly but 0.5–1 μM MC‐LR did not change PP2A activity dramatically. The inhibition could result from the hyperphosphorylation of PP2A/C at Tyr307, an elevation in the total PP2A/C expression and the dissociation of α4/PP2A/C complexes. Moreover, MC‐LR led to rearrangements of filamentous actin and microtubules, which might be correlated with the hyperphosphorylation of Ezrin, VASP and HSP27 due to PP2A inhibition and mitogen‐activated protein kinase (MAPK) activation. However, exposure to MC‐LR for 24 h failed to trigger either apoptosis or proliferation, which might be related to PP2A‐inhibition‐induced hyperphosphorylation of Bcl‐2 and Bad and the activation status of Akt. In conclusion, our data indicated that MC‐LR induced extensive molecular and cellular alterations in A549 cells through a PP2A‐centered pathway, which differed in some respects from our previous study in SMMC‐7721 cells. To our knowledge, this is the first report comprehensively demonstrating the effects of MC‐LR in A549 cells, and our findings provide insights into the mechanism of MC‐LR toxicity in cancer cells.

Protein phosphatase 2A inhibition and subsequent cytoskeleton reorganization contributes to cell migration caused by microcystin-LR in human laryngeal epithelial cells (Hep-2).

The major toxic mechanism of Microcystin‐LR is inhibition of the activity of protein phosphatase 2A (PP2A), resulting in a series of cytotoxic effects. Our previous studies have demonstrated that microcystin‐LR (MCLR) induced very different molecular effects in normal cells and the tumor cell line SMMC7721. To further explore the MCLR toxicity mechanism in tumor cells, human laryngeal epithelial cells (Hep‐2) was examined in this study. Western blot, immunofluorescence, immunoprecipitation, and transwell migration assay were used to detect the effects of MCLR on PP2A activity, PP2A substrates, cytoskeleton, and cell migration. The results showed that the protein level of PP2A subunits and the posttranslational modification of the catalytic subunit were altered and that the binding of the AC core enzyme as well as the binding of PP2A/C and α4, was also affected. As PP2A substrates, the phosphorylation of MAPK pathway members, p38, ERK1/2, and the cytoskeleton‐associated proteins, Hsp27, VASP, Tau, and Ezrin were increased. Furthermore, MCLR induced reorganization of the cytoskeleton and promoted cell migration. Taken together, direct covalent binding to PP2A/C, alteration of the protein levels and posttranslational modification, as well as the binding of subunits, are the main pattern for the effects of MCLR on PP2A in Hep‐2. A dose‐dependent change in p‐Tau and p‐Ezrin due to PP2A inhibition may contribute to the changes in the cytoskeleton and be related to the cell migration in Hep‐2. Our data provide a comprehensive exposition of the MCLR mechanism on tumor cells.