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

Abstract

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-LR's 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. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1065-1078, 2017.

Microcystin-LR Does Not Alter Cell Survival and Intracellular Signaling in Human Bronchial Epithelial Cells

“…Although these results suggest uptake of MC-LR and its interaction with cellular proteins, the viability assays did not reveal any major effects of MC-LR in both HBE1 and 16HBE14o-cells. The results of our study are mostly consistent with findings of Wang et al [25], who found no significant effect of 10 µM MC-LR on viability in A549 human non-small lung cancer cells following 24-h exposure. On the contrary, Li et al [29] found that 10 µM MC-LR significantly reduced (EC50) cell survival in HBE cells after 24 h, and concentrations ≥50 nM were cytotoxic to immortalized murine alveolar type II cell line [45].…”

“…To evaluate possible effects of a very abundant cyanotoxin MC-LR in the human respiratory system, we used two immortalized, non-cancerous, respiratory-relevant human bronchial epithelial cell lines. The concentrations of MC-LR in our experiments were chosen based on previous studies with human bronchial or alveolar cells [25,29], where MC-LR at relatively high concentrations (30-40 µM) nearly completely inhibited cell viability after 24-h exposure, while 1-20 µM significantly reduced cell viability to 90%-35% of the control [29]. Thus, in our prolonged exposure (48-96 h) experiments, we decided to test a concentration range of 1-20 µM MC-LR, while using 1 µM MC-LR to assess the effects of a toxicologically more feasible concentration on a sub-cellular level for mechanistic studies.…”

“…Intracellular signaling pathways, such as ERK1/2, p38, and JNK signaling, play a critical role in the control of inflammatory respiratory diseases and cell proliferation-related neoplastic disorders [71]. Disruption of MAPK signaling by MC-LR has been documented in several studies with multiple in vitro models [24][25][26]45,72] and might be a direct consequence of MC-LR-induced inhibition of PP1/PP2A. Although the MC-LR-positive protein bands were formed and accumulated in both HBE1 and 16HBE14o-cells, we observed only minor changes in the activation of ERK1/2 and p38 kinases, suggesting that non-cytotoxic concentrations of MC-LR did not significantly interfere with intracellular MAPK signaling in these cells.…”

“…It was documented that in vitro exposure of human bronchial epithelial cells to MC-LR resulted in decreased viability, determined by tetrazolium reduction assay, in a concentration-dependent manner [29]. Another study demonstrated that MC-LR is capable to inhibit PP2A, activate MAPK pathways (p38, ERK1/2) and anti-apoptotic genes (Akt, B-cell lymphoma 2), and induce cytoskeletal changes, but not to interfere with either apoptosis or proliferation in A549 human non-small lung cancer cells after 24-h exposure [25]. These findings suggest that MC-LR causes adverse effects in both lung tissue and respiratory system-relevant in vitro systems.…”

Cantharidic acid induces apoptosis of human leukemic HL‐60 cells via c‐Jun N‐terminal kinase‐regulated caspase‐8/‐9/‐3 activation pathway

“…Similar to CTD and NCTD, CAC has been recognized as a protein phosphatase 2A (PP2A) inhibitor . Interestingly, PP2A is thought to be a cancer suppressor, as inhibition of PP2A can induce phosphorylation and activation of substrate kinases, most of which can further accelerate growth . JNK and p38 MAPK have been reported to be the direct substrates of PP2A .…”