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. 2021 Nov 22;13(11):830.
doi: 10.3390/toxins13110830.

Crotoxin Modulates Events Involved in Epithelial-Mesenchymal Transition in 3D Spheroid Model

Ellen Emi Kato  1 , Sandra Coccuzzo Sampaio  1   2
Affiliations

Crotoxin Modulates Events Involved in Epithelial-Mesenchymal Transition in 3D Spheroid Model

Ellen Emi Kato et al. Toxins (Basel). .

Abstract

Epithelial-mesenchymal transition (EMT) occurs in the early stages of embryonic development and plays a significant role in the migration and the differentiation of cells into various types of tissues of an organism. However, tumor cells, with altered form and function, use the EMT process to migrate and invade other tissues in the body. Several experimental (in vivo and in vitro) and clinical trial studies have shown the antitumor activity of crotoxin (CTX), a heterodimeric phospholipase A2 present in the Crotalus durissus terrificus venom. In this study, we show that CTX modulates the microenvironment of tumor cells. We have also evaluated the effect of CTX on the EMT process in the spheroid model. The invasion of type I collagen gels by heterospheroids (mix of MRC-5 and A549 cells constitutively prepared with 12.5 nM CTX), expression of EMT markers, and secretion of MMPs were analyzed. Western blotting analysis shows that CTX inhibits the expression of the mesenchymal markers, N-cadherin, α-SMA, and αv. This study provides evidence of CTX as a key modulator of the EMT process, and its antitumor action can be explored further for novel drug designing against metastatic cancer.

Keywords: crotoxin; epithelial–mesenchymal transition; spheroid model; tumor stroma.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Myofibroblast differentiation with various stimulatory factors. (A) Representative immunofluorescent images of MRC-5 cells pretreated with CTX (12.5 nM) for 2 h and then incubated in DMEM (with 10% FBS), TGF-β1 (2 ng/mL), or tumor-conditioned media from A549 cells for 3 days. The control group of cells were untreated and grown in DMEM with 10% FBS only. (B) Quantification of α-SMA fluorescence intensity. Green fluorescence indicates α-SMA-containing stress fibers and blue fluorescence indicates the nuclei. Scale bar = 25 µm. The data are presented from three independent experiments.
Figure 2
Figure 2
Spheroid formation by MRC-5 cells alone or in combination with A549 tumor cells. (A) MRC-5 single spheroid formation by the hanging drop method. (B) MRC-5 single spheroid constitutively formed in the presence of 12.5 nM of CTX. (C) MRC-5/A549 spheroid formation by the hanging drop method. (D) A small number of cancer cells did not incorporate into the cell aggregates (white arrow) (E) MRC-5/A549 spheroid constituted in the presence of 12.5 nM CTX—after 24 h, cell aggregates formed compact structures. (F) A small number of cancer cells did not incorporate into the cell aggregates (white arrow). Images obtained from inverted microscope 4X. (E) Live (green)/Dead (red) image of MRC-5/A549 spheroids. Scale bar = 100 µm. (F) Quantitative analysis from live/dead assay measured by relative fluorescence intensity. All the data presented here are from three independent experiments.
Figure 3
Figure 3
Invasion area of spheroids in 3D collagen gels. Representative images are of invasion into type I collagen gel (1.2 mg/mL) (A) by cells of MRC-5/A549 spheroids or (B) MRC-5/A549 constituted with 12.5 nM of CTX. Cell invasion was photographed under phase-contrast microscopy at 48 h. (C) Cell invasion area was measured and analyzed on ImageJ software. * p < 0.05 compared to the control group. The data presented here are from three independent experiments (n = 5).
Figure 4
Figure 4
Expression of EMT-associated proteins. Quantitative analysis of expression of EMT markers. After three days in culture, MRC-5/A549 and MRC-5/Calu-3 spheroids were lysed, and Western blot analyses were conducted for E-cadherin, N-cadherin, α-SMA, integrin subunit αv. GAPDH served as the loading control. *** p < 0.001 compared to the control group. * p < 0.01 compared to the control group. The data presented are from three independent experiments (n = 4).
Figure 5
Figure 5
Effect of crotoxin on the production of MMP-9, MMP-13, cytokines, and chemokines in the 3D collagen gel. Monoculture of human lung fibroblasts MRC-5 and human lung adenocarcinoma cell line A549 incubated with 12.5 nM of CTX for three days; invasion of collagen gel by composite spheroids of MRC-5/A549 at 48 h. After this period, spent media were collected to quantify the volume of MMP-9 (A) and MMP-13 (B) by ELISA. * p < 0.05 compared to the control group. # p < 0.05 compared to the control group. ** p < 0.01 compared to the control group. (n = 6). In (C), cytokine array analysis using composite spheroid invaded gel of at 48 h. The graph indicates the decrease in the growth factors by more than 1.2-folds. A pool of n = 4 was used.
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References

    1. Kalluri R., Weinberg R.A. The Basics of Epithelial-Mesenchymal Transition. J. Clin. Investig. 2009;119:1420–1428. doi: 10.1172/JCI39104. - DOI - PMC - PubMed
    1. Kim K.K., Wei Y., Szekeres C., Kugler M.C., Wolters P.J., Hill M.L., Frank J.A., Brumwell A.N., Wheeler S.E., Kreidberg J.A., et al. Epithelial Cell Alpha3beta1 Integrin Links Beta-Catenin and Smad Signaling to Promote Myofibroblast Formation and Pulmonary Fibrosis. J. Clin. Investig. 2009;119:213–224. doi: 10.1172/JCI36940. - DOI - PMC - PubMed
    1. Thiery J.P. Epithelial–Mesenchymal Transitions in Tumour Progression. Nat. Rev. Cancer. 2002;2:442–454. doi: 10.1038/nrc822. - DOI - PubMed
    1. Zeisberg M., Neilson E.G. Biomarkers for Epithelial-Mesenchymal Transitions. J. Clin. Investig. 2009;119:1429–1437. doi: 10.1172/JCI36183. - DOI - PMC - PubMed
    1. Ding S., Chen G., Zhang W., Xing C., Xu X., Xie H., Lu A., Chen K., Guo H., Ren Z., et al. MRC-5 Fibroblast-Conditioned Medium Influences Multiple Pathways Regulating Invasion, Migration, Proliferation, and Apoptosis in Hepatocellular Carcinoma. J. Transl. Med. 2015;13:237. doi: 10.1186/s12967-015-0588-8. - DOI - PMC - PubMed

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