Is epithelial-mesenchymal transition related to the biological behavior of salivary gland neoplasms?

OBJECTIVE: To evaluate and compare the expression of E-cadherin, Snail1 and Twist1 in pleomorphic adenomas (PAs), adenoid cystic carcinomas (AdCCa) and carcinoma ex-pleomorphic adenomas (CaexPA) of salivary glands, as well as investigate possible associations with clinicopathological parameters. STUDY DESIGN: E-cadherin, Snail1 and Twist1 antibody immunostaining were analyzed semiquantitatively in 20 PAs, 20 AdCCas and 10 CaexPAs. Cases were classified as low and high expression for analysis of the association with clinicopathological parameters. RESULTS: Compared to PAs, AdCCas and CaexPAs exhibited higher nuclear expression of Snail1 (p = 0.021 and p = 0.028, respectively) and Twist1 (p = 0.009 and p = 0.001). Membranous and cytoplasmic expression of E-cadherin were positively correlated in PAs, AdCCas and CaexPAs (r = 0.645, p = 0.002; r = 0.824, p < 0.001; r = 0.677, p = 0.031). In PAs, positive correlation was found between nuclear expression of Snail1 and membrane expression of E-cadherin (r = 0.634; p = 0.003), as well as between nuclear expression of Snail1 and Twist1 (r = 0.580; p = 0.007). Negative correlations were detected between membrane expression of E-cadherin and cytoplasmic expression of Snail1 in AdCCas (r = - 0.489; p = 0.029). CONCLUSIONS: E-cadherin, Twist1, and Snail1 may participate in modulating events related to cell differentiation and adhesion in PAs and to biological behavior in AdCCas and CaexPAs, which indicates the involvement of EMT in these processes. Furthermore, the expression of these proteins in these carcinomas may reflect the plasticity feature of EMT.

CRISPR-Cas9 knockout of membrane-bound alkaline phosphatase or cadherin does not confer resistance to Cry toxins in Aedes aegypti.

The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins.

Pseudogene CSPG4P12 inhibits colorectal cancer progression by attenuating epithelial-mesenchymal transition.

Colorectal cancer is one of the most common malignant cancers. Pseudogenes have been identified as oncogenes or tumor suppressor genes in the development of various cancers. However, the function of pseudogene CSPG4P12 in colorectal cancer remains unclear. Therefore, the aim of this study was to investigate the potential role of CSPG4P12 in colorectal cancer and explore the possible underlying mechanism. The difference of CSPG4P12 expression between colorectal cancer tissues and adjacent normal tissues was analyzed using the online Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database. Cell viability and colony formation assays were conducted to evaluate cell viability. Transwell and wound healing assays were performed to assess cell migration and invasion capacities. Western blot was used to measure the expression levels of epithelial-mesenchymal transition-related proteins. Colorectal cancer tissues had lower CSPG4P12 expression than adjacent normal tissues. The overexpression of CSPG4P12 inhibited cell proliferation, invasion, and migration in colorectal cancer cells. Overexpressed CSPG4P12 promoted the expression of E-cadherin, whereas it inhibited the expression of vimentin, N-cadherin, and MMP9. These findings suggested that CSPG4P12 inhibits colorectal cancer development and may serve as a new potential target for colorectal cancer.

Regulation of cellular and molecular markers of epithelial-mesenchymal transition by Brazilin in breast cancer cells.

Breast cancer is the most common invasive neoplasm and the leading cause of cancer death in women worldwide. The main cause of mortality in cancer patients is invasion and metastasis, where the epithelial-mesenchymal transition (EMT) is a crucial player in these processes. Pharmacological therapy has plants as its primary source, including isoflavonoids. Brazilin is an isoflavonoid isolated from Haematoxilum brasiletto that has shown antiproliferative activity in several cancer cell lines. In this study, we evaluated the effect of Brazilin on canonical markers of EMT such as E-cadherin, vimentin, Twist, and matrix metalloproteases (MMPs). By Western blot, we evaluated E-cadherin, vimentin, and Twist expression and the subcellular localization by immunofluorescence. Using gelatin zymography, we determined the levels of secretion of MMPs. We used Transwell chambers coated with matrigel to determine the in vitro invasion of breast cancer cells treated with Brazilin. Interestingly, our results show that Brazilin increases 50% in E-cadherin expression and decreases 50% in vimentin and Twist expression, MMPs, and cell invasion in triple-negative breast cancer (TNBC) MDA-MB-231 and to a lesser extend in MCF7 ER+ breast cancer cells. Together, these findings position Brazilin as a new molecule with great potential for use as complementary or alternative treatment in breast cancer therapy in the future.

Fine particulate matter (PM2.5) promotes chemoresistance and aggressive phenotype of A549 lung cancer cells.

Lung cancer is one of the most aggressive malignancies with a high mortality rate. In large cities, particulate matter (PM) is a common air pollutant. High PM levels with aerodynamic size ≤2.5 µm (PM2.5) associates with lung cancer incidence and mortality. In this work, we explored PM2.5 effects on the behavior of lung cancer cells. To this, we chronically exposed A549 cells to increasing PM2.5 concentrations collected in México City, then evaluating cell proliferation, chemoresponse, migration, invasion, spheroid formation, and P-glycoprotein and N-cadherin expression. Chronic PM2.5 exposure from 1 µg/cm2 stimulated A549 cell proliferation, migration, and chemoresistance and upregulated P-glycoprotein and N-cadherin expression. PM2.5 also induced larger multicellular tumor spheroids (MCTS) and less disintegration compared with control cells. Therefore, these results indicate lung cancer patients exposed to airborne PM2.5 as urban pollutant could develop more aggressive tumor phenotypes, with increased cell proliferation, migration, and chemoresistance.

CLIC4 Function in the Epithelial-Mesenchymal Transition of Epithelial Odontogenic Lesions.

BACKGROUND: Odontogenic lesions constitute a heterogeneous group of lesions. CLIC4 protein regulates different cellular processes, including epithelial-mesenchymal transition and fibroblast-myofibroblast transdifferentiation. This study analyzed CLIC4, E-cadherin, Vimentin, and α-SMA immunoexpression in epithelial odontogenic lesions that exhibit different biological behavior. METHODS: It analyzed the immunoexpression of CLIC4, E-cadherin, and Vimentin in the epithelial cells, as well as CLIC4 and α-SMA in the mesenchymal cells, of ameloblastoma (AM) (n = 16), odontogenic keratocyst (OKC) (n = 20), and adenomatoid odontogenic tumor (AOT) (n = 8). Immunoexpressions were categorized as score 0 (0% positive cells), 1 (< 25%), 2 (≥ 25% - < 50%), 3 (≥ 50% - < 75%), or 4 (≥ 75%). RESULTS: Cytoplasmic CLIC4 immunoexpression was higher in AM and AOT (p < 0.001) epithelial cells. Nuclear-cytoplasmic CLIC4 was higher in OKC's epithelial lining (p < 0.001). Membrane (p = 0.012) and membrane-cytoplasmic (p < 0.001) E-cadherin immunoexpression were higher in OKC, while cytoplasmic E-cadherin expression was higher in AM and AOT (p < 0.001). Vimentin immunoexpression was higher in AM and AOT (p < 0.001). Stromal CLIC4 was higher in AM and OKC (p = 0.008). Similarly, α-SMA immunoexpression was higher in AM and OKC (p = 0.037). Correlations in these proteins' immunoexpression were observed in AM and OKC (p < 0.05). CONCLUSIONS: CLIC4 seems to regulate the epithelial-mesenchymal transition, modifying E-cadherin and Vimentin expression. In mesenchymal cells, CLIC4 may play a role in fibroblast-myofibroblast transdifferentiation. CLIC4 may be associated with epithelial odontogenic lesions with aggressive biological behavior.

CSChighE-cadherinlow immunohistochemistry panel predicts poor prognosis in oral squamous cell carcinoma.

Identifying marker combinations for robust prognostic validation in primary tumour compartments remains challenging. We aimed to assess the prognostic significance of CSC markers (ALDH1, CD44, p75NTR, BMI-1) and E-cadherin biomarkers in OSCC. We analysed 94 primary OSCC and 67 metastatic lymph node samples, including central and invasive tumour fronts (ITF), along with clinicopathological data. We observed an increase in ALDH1+/CD44+/BMI-1- tumour cells in metastatic lesions compared to primary tumours. Multivariate analysis highlighted that elevated p75NTR levels (at ITF) and reduced E-cadherin expression (at the tumour centre) independently predicted metastasis, whilst ALDH1high exhibited independent predictive lower survival at the ITF, surpassing the efficacy of traditional tumour staging. Then, specifically at the ITF, profiles characterized by CSChighE-cadherinlow (ALDH1highp75NTRhighE-cadherinlow) and CSCintermediateE-cadherinlow (ALDH1 or p75NTRhighE-cadherinlow) were significantly associated with worsened overall survival and increased likelihood of metastasis in OSCC patients. In summary, our study revealed diverse tumour cell profiles in OSCC tissues, with varying CSC and E-cadherin marker patterns across primary tumours and metastatic sites. Given the pivotal role of reduced survival rates as an indicator of unfavourable prognosis, the immunohistochemistry profile identified as CSChighE-cadherinlow at the ITF of primary tumours, emerges as a preferred prognostic marker closely linked to adverse outcomes in OSCC.

E-cadherin and NF-κB expression in the vagina after ovarian ischemia and reperfusion.

PURPOSE: To investigate inflammation and cell adhesion molecules in the vagina after ovarian ischemia-reperfusion (IR) injury. METHODS: 20 Wistar albino female rats were divided into two groups: control, and IR groups. In IR group, blood flow was restricted for 2 hours for ovarian ischemia. Then, tissues were re-blood 2 hours for reperfusion. Vagina tissues were excised and processed for histopathological analysis. Histopathological and biochemical follow-ups were performed. RESULTS: Both malondialdehyde and myeloperoxidase values were increased in IR group compared to control group. Glutathione content was decreased in IR group compared to control group. Epithelial degeneration, inflammation, dilatation, and nuclear factor-κB (NF-κB) expression were increased in IR group compared to control group. E-cadherin expression was significantly decreased in IR group. In the IR group, E-cadherin showed a positive reaction in adenomas, gland-like cryptic structures, cellular junctions with clustered inflammatory cells. In the IR group, NF-κB expression was increased in basement membrane, inflammatory cells, in blood vessels. CONCLUSIONS: Ovarian ischemia caused degeneration of epithelial cells in the vaginal region and disruptions in the cell junction complex, which leads to activation of E-cadherin and NF-κB signaling pathway and alterations in reproductive and embryonal development in the vaginal region.

Immunohistochemical expression of vimentin, E-cadherin, and CD45 in natural cases of canine cutaneous round tumors.

Round cell tumors are common cutaneous lesions in dogs, with increased occurrence percentages among different skin tumors. This study aimed to investigate the frequency as well as gross and pathological characteristics of round cell tumors in natural cases of tumorous dogs in relation to breed, sex, and age. Moreover, it aimed to evaluate the immunohistochemical expression of a panel of immunohistochemical stains, including vimentin, E-cadherin, and cluster of differentiation (CD45) as an adjunct technique for the differential diagnosis of cutaneous round cell neoplasm. Data were collected from 64 dogs of both sexes (36 females and 28 males), various breeds, and different ages (8 months to 7 years). The histopathological nature of neoplastic growth was reported, and neoplasm prevalence was classified using age, sex, breed, and site on the body. We observed 48 cases of transmissible venereal tumors, 12 cutaneous histiocytomas, and 4 histiocytic sarcoma. Immunohistochemical characterization revealed an intense positive immunoreactivity for vimentin in transmissible venereal tumor cells and moderate positive immunoreactivity for E-cadherin and CD45 in cutaneous histiocytoma and histiocytic sarcoma cells. In conclusion, the canine transmissible venereal tumor was the most frequent form of round cell tumor; thus, a definitive cutaneous neoplasm diagnosis should be based on histopathological morphology and immunohistochemical findings.

Early gonadogenesis in Columba livia (birds: Columbiformes): Migration, colonization, and differentiation of germ cells.

In birds, primordial germ cells (PGCs) use the bloodstream to travel to a specific region, where the cells undergo extravasation followed by intrastromal migration to the gonadal crest for further colonization. Currently, DDX4, SSEA1, and Oct4 are used to identify germ cells. Other germline cell-associated molecules are N-cadherin, GnRHR, and 3ß hydroxysteroid dehydrogenase (3ßHSD), which have been used in mice and birds during gonadal development; however, its role in early gonadogenesis in birds is poorly described. This study aimed to evaluate the differential immunodetection of N-cadherin binding molecule, Oct4 pluripotency protein, GnRHR receptor, and 3ßHSD enzyme in Columba livia embryos during migration colonization of PGCs in the gonadal crest and early gonadogenesis. These markers were revealed by immunohistochemistry in histological preparations of C. livia corresponding to stages (S)15 to S40. Immunodetection of N-cadherin, Oct4, GnRHR, and 3ßHSD in the germ line of C. livia allowed the identification of PGCs in the yolk sac membrane at the level of the splanchnic mesoderm during migration to the genital crest and its colonization. In the same way, it was possible to characterize and localize PGCs during early gonadogenesis. This study in C. livia demonstrates that Oct4, N-cadherin, GNRHR, and 3ßHSD are immunodetected in PGCs and could be used as potential germline cell markers during cell migration out of blood vessels, colonization in the genital crest, and early gonadogenesis. Furthermore, this study could be used as a novel general model to understand the early gonadogenesis in altricial species.

Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models.

Caveolin-1 (CAV1) is a membrane-bound protein that suppresses tumor development yet also promotes metastasis. E-cadherin is important in CAV1-dependent tumor suppression and prevents CAV1-enhanced lung metastasis. Here, we used murine B16F10 and human A375 melanoma cells with low levels of endogenous CAV1 and E-cadherin to unravel how co-expression of E-cadherin modulates CAV1 function in vitro and in vivo in WT C57BL/6 or Rag-/- immunodeficient mice and how a pro-inflammatory environment generated by treating cells with prostaglandin E2 (PGE2) alters CAV1 function in the presence of E-cadherin. CAV1 expression augmented migration, invasion, and metastasis of melanoma cells, and these effects were abolished via transient co-expression of E-cadherin. Importantly, exposure of cells to PGE2 reverted the effects of E-cadherin expression and increased CAV1 phosphorylation on tyrosine-14 and metastasis. Moreover, PGE2 administration blocked the ability of the CAV1/E-cadherin complex to prevent tumor formation. Therefore, our results support the notion that PGE2 can override the tumor suppressor potential of the E-cadherin/CAV1 complex and that CAV1 released from the complex is phosphorylated on tyrosine-14 and promotes migration/invasion/metastasis. These observations provide direct evidence showing how a pro-inflammatory environment caused here via PGE2 administration can convert a potent tumor suppressor complex into a promoter of malignant cell behavior.

Imunoexpressão de clic4, α-sma, e-caderina e vimentina em lesões odontogênicas epitelias benígnas / Immunoexpression of clic4, α-sma, e-cadherin and vimentin in benign epithelial odontogenic lesions

As lesões odontogênicas epiteliais benignas constituem um grupo heterogêneo de lesões. A proteína CLIC4 atua na regulação dos processos de parada de crescimento e apoptose, participando também do processo de transdiferenciação dos fibroblastos em miofibroblastos que passam a expressar α-SMA. Além disso, a expressão de CLIC4 pode interferir no processo de transição epitélio-mesenquima (TEM) em neoplasias. Este trabalho avaliou a imunoexpressão de CLIC4, α-SMA, E-caderina e Vimentina em ameloblastomas (AM) (n = 16), ceratocistos odontogênicos (n = 20) e tumores odontogênicos adenomatóides (TOA) (n = 8). A análise da expressão imunoistoquímica das proteínas CLIC4, E-caderina e vimentina no componente epitelial das lesões e de CLIC4 e α-SMA no tecido conjuntivo foi realizada de forma semi-quantitativa por um avaliador previamente calibrado. A expressão no componente epitelial de CLIC4 foi analisada separadamente no núcleo e no citoplasma, bem como a marcação de E-caderina que foi avaliada na membrana e no citoplasma. As comparações dos percentuais de imunorreatividade em relação aos grupos estudados foram realizadas por meio dos testes não paramétricos de Kruskal-Wallis e Mann-Whitney. Possíveis correlações entre a expressão de CLIC4, α-SMA, E-caderina e Vimentina foram avaliadas por meio do teste de correlação de Spearman. O nível de significância foi estabelecido em 5% (p < 0,05). Foram observados diferentes padrões de marcação entre os grupos analisados, observando-se que a imunoexpressão exclusivamente citoplasmática da CLIC4 no componente epitelial dos AM (p < 0,001) e TOA (p < 0,001) foi significativamente superior a dos CO, não demonstrarando significância estatística entre os AM e TOA. A imunoexpressão (nuclear e citoplasmática) da CLIC4 no revestimento epitelial CO foi significativamente superior à encontrada no componente epitelial dos AM (p < 0,001) e dos TOA (p < 0,001). A imunoexpressão estromal de CLIC4 foi significativamente superior nos AM (p = 0,009) e CO (p = 0,004) quando comparados aos TOA. A imunoexpressao de α-SMA significativamente maior em AM (p = 0,016) e CO (p = 0,034) quando comparados aos TOA. Para a imunoexpressão membranar da E-caderina em CO foi significativamente superior em comparação à encontrada nos AM (p = 0,009) e nos TOA (p = 0,024). Foi observada maior imunoexpressão de E-caderina (membranar e citoplasmática) nos COs, quando comparados aos AM (p < 0,001) e aos TOAs (p < 0,001). A expressão de Ecaderina citoplasmática foi significativamente maior nos AM e TOA (p < 0,001) quando comparados aos CO. Observou-se diferença estatisticamente significativa na imunoexpressão de vimentina entre os casos de AM e os casos de TOA (p = 0,038) e CO (p < 0,001), bem como entre o TOA e CO (p < 0,001). As correlações testadas entre os escores das proteínas estudadas evidenciou que no grupo dos AM foi possível evidenciar moderada correlação positiva e estatisticamente significativa (r = 0,527; p = 0,036) entre a expressão citoplasmática da CLIC4 e a expressão citoplasmática da E-caderina. Também foi verificada fraca correlação negativa e estatisticamente significativa (r = -0,499; p = 0,049) entre a expressão núcleo-citoplasmática da CLIC4 e a expressão citoplasmática da E-caderina nos AM. Além disso, uma moderada correlação positiva e estatisticamente significativa entre a expressão estromal da CLIC4 e a expressão da α-SMA nos AM (r = 0,648; p = 0,007) e nos CO (r = 0,541; p = 0,014). Foi observada forte correlação negativa e estatisticamente significativa (r = -0,813; p < 0,001) entre a expressão da E-caderina e a expressão da vimentina nos AM. Os resultados deste estudo sugerem um potencial envolvimento de CLIC4 no processo de transdiferenciação de miofibroblastos, e que a presença destas células é mais frequentemente associada a lesões de comportamento biológico mais agressivo como os AM e CO, além de uma possível atuação desta proteína na regulação do ciclo celular e na TEM nas lesões estudadas (AU).

Benign epithelial odontogenic lesions constitute a heterogeneous group of lesions. the CLIC4 protein acts in the regulation of growth arrest and apoptosis processes, also participating in the process of transdifferentiation of fibroblasts Into myofibroblasts that begin to express α-SMA. Furthermore, CLIC4 expression can interfere with the epithelialmesenchymal transition (EMT) process in neoplasms. This work evaluated the immunoexpression of CLIC4, α-SMA, e-cadherin and vimentin in ameloblastomas (AM) (n = 16), odontogenic keratocysts (OK) (n = 20) and adenomatoid odontogenic tumors (AOT) (n = 8). The analysis of the immunohistochemical expression of the proteins CLIC4, ecadherin and vimentin in the epithelial component of the lesions and of CLIC4 and α-SMA in the connective tissue was carried out in a semi-quantitative way by a previously calibrated evaluator. Expression in the epithelial component of CLIC4 was analyzed separately in the nucleus and cytoplasm, as well as e-cadherin labeling, which was evaluated in the membrane and cytoplasm. Comparisons of the percentages of immunoreactivity in relation to the studied groups were carried out using the nonparametric kruskal-wallis and mann-whitney tests. Possible correlations between the expression of CLIC4, α-SMA, e-cadherin and vimentin were evaluated using the spearman correlation test. The significance level was set at 5% (p < 0.05). Different staining patterns were observed between the groups analyzed, observing that the exclusively cytoplasmic immunoexpression of CLIC4 in the epithelial component of AM (p < 0.001) and AOT (p < 0.001) was significantly higher than that of OK, not demonstrating statistical significance between the AM and AOT. The immunoexpression (nuclear and cytoplasmic) of CLIC4 in the co epithelial lining was significantly higher than that found in the epithelial component of AM (p < 0.001) and AOT (p < 0.001). Stromal CLIC4 immunoexpression was significantly higher in AM (p = 0.009) and OK (p = 0.004) when compared to AOT. The immunoexpression of α-SMA is significantly higher in AM (p = 0.016) and OK (p = 0.034) when compared to AOT. For e-cadherin membrane immunoexpression in co was significantly higher compared to that found in AM (p = 0.009) and AOT (p = 0.024). Greater immunoexpression of e-cadherin (membrane and cytoplasmic) was observed in OK, when compared to AM (p < 0.001) and AOT (p < 0.001). Cytoplasmic ecadherin expression was significantly higher in AM and AOT (p < 0.001) when compared to OK. A statistically significant difference in vimentin immunoexpression was observed between cases of AM and cases of AOT (p = 0.038) and OK (p < 0.001), as well as between AOT and OK (p < 0.001). The correlations tested between the scores of the proteins studied showed that in the am group it was possible to demonstrate a moderate positive and statistically significant correlation (r = 0.527; p = 0.036) between the cytoplasmic expression of clic4 and the cytoplasmic expression of e-cadherin. A weak and statistically significant negative correlation (r = -0.499; p = 0.049) was also found between the nucleus-cytoplasmic expression of clic4 and the cytoplasmic expression of e- cadherin in AM. Furthermore, a moderate positive and statistically significant correlation between the stromal expression of CLIC4 and the expression of α-SMA in AM (r = 0.648; p = 0.007) and OK (r = 0.541; p = 0.014). Additionally, a strong negative and statistically significant correlation (r = -0.813; p < 0.001) was observed between the expression of ecadherin and the expression of vimentin in AM. The results of this study suggest a potential involvement of CLIC4 in the myofibroblast transdifferentiation process, and that the presence of these cells is more frequently associated with lesions with more aggressive biological behavior such as AM and OK, in addition to a possible role of this protein in the regulation of cell cycle and EMT in the lesions studied (AU).

Pentoxifylline Inhibits TNF-α/TGF-ß1-Induced Epithelial-Mesenchymal Transition via Suppressing the NF-κB Pathway and SERPINE1 Expression in CaSki Cells.

Cervical cancer (CC) is one of the most common and deadly types of female cancer worldwide. Late diagnosis in CC increases the risk of tumor cells spreading to distant organs (metastasis). The epithelial-mesenchymal transition (EMT) is a fundamental process of cancer metastasis. Inflammation can lead to tumor progression, EMT induction, and metastasis. The inflammatory microenvironment is a potent inducer of EMT; inflammatory cytokines such as Tumor Necrosis Factor-alpha (TNF-α) and Transforming growth factor-beta (TGF-ß1) activate transcriptional factors such as STAT3, Snail, Smad, and the Nuclear Factor kappa light-chain-enhancer of activated beta cells (NF-κΒ), which drive EMT. Anti-inflammatory compounds may be an option in the disruption of EMT. PenToXifylline (PTX) possesses potent anti-inflammatory effects by inhibiting NF-κB activity. In addition, PTX exerts an anti-fibrotic effect by decreasing Smad2/3/4. We hypothesize that PTX could exert anti-EMT effects. CaSki human cervical tumor cells were exposed to TNF-α 10 ng/mL and TGF-ß1 alone or in combination for 5 days. Our results revealed that TNF-α and TGF-ß1 induced N-cadherin and Vimentin, confirming the induction of EMT. Furthermore, the combination of cytokines synergized the expression of mesenchymal proteins, enhanced IκBα and p65 phosphorylation, and upregulated Serpin family E member 1 (SERPINE1) mRNA. PTX pretreatment prior to the addition of TNF-α and TGF-ß1 significantly reduced N-cadherin and Vimentin levels. To our knowledge, this is the first time that this effect of PTX has been reported. Additionally, PTX reduced the phosphorylation of IκB-α and p65 and significantly decreased SERPINE1 expression, cell proliferation, migration, and invasion. In conclusion, PTX may counteract EMT in cervical cancer cells by decreasing the NF-κB and SERPINE1.

Expression of cytotoxic T lymphocyte-associated antigen 4, CD44, and E-cadherin in the microenvironment of breast carcinomas.

OBJECTIVE: The expression of cytotoxic T lymphocyte-associated antigen 4, E-cadherin, and CD44 in the area of tumor budding was investigated in breast carcinomas in our study. METHODS: Tumor budding was counted at the invasive margins in 179 breast carcinomas. To understand the microenvironment of tumor budding, we examined the expression status of the immune checkpoint molecules such as cytotoxic T lymphocyte-associated antigen 4, E-cadherin, and CD44. RESULTS: Tumors were separated into low (≤5) and high tumor budding groups (>5) based on the median budding number. Lymphovascular, perineural invasion, and the number of metastatic lymph nodes were significantly higher in high-grade budding tumors (p=0.001, p<0.001, and p=0.019, respectively). Tumor-infiltrating lymphocytes were significantly higher in tumors without tumor buddings (p<0.001). When the number of budding increases by one unit, overall survival decreases by 1.07 times (p=0.013). Also, it increases the risk of progression by 1.06 times (p=0.048). In high tumor budding groups, the cytotoxic T lymphocyte-associated antigen 4 staining percentage of lymphocytes was significantly higher (p=0.026). With each increase in the number of buds, an increase in the percentage of cytotoxic T lymphocyte-associated antigen 4 staining was seen in lymphocytes in the microenvironment of TB (p=0.034). CONCLUSION: Tumor budding could predict poor prognosis in breast carcinomas, and anti-cytotoxic T lymphocyte-associated antigen 4 immunotherapies may be beneficial in patients with high tumor budding tumors.

Cholesterol depletion affects caveolin-1 expression, migration and invasion of oral tongue squamous cell carcinoma cell lines.

INTRODUCTION: Cholesterol is a key lipid molecule within cell membranes. This is especially true in cavelolas, invaginated membrane nanodomains, which present the protein caveolin-1 (CAV-1). It is important to note that this structure is involved in many cell signalling pathways. Additionally, high cholesterol is seen in different tumor types but little is known in regards to oral tongue squamous cell carcinoma (OTSCC). The aim of this study was to evaluate the influence of cholesterol depletion on primary (SCC-25) and metastatic (HSC-3) OTSCC cell lines. MATERIALS AND METHODS: Cell membrane fluidity, cell viability, gene and protein expression of CAV-1 and of epithelial-mesenchymal transition (EMT) markers, cell migration in Myogel and invasion-myoma assay were evaluated after cholesterol depletion with methyl-ß-cyclodextrin (MßCD - 7.5, 10 or 15 mM) RESULTS: Decreased cell viability and increased membrane fluidity of SCC-25 cells was seen with cholesterol depletion but cell viability was less affected and there was no effect on membrane fluidity in HSC-3. Cholesterol depletion also decreased CAV-1 at 6 h but increased it after 24 h.; both epithelial and mesenchymal EMT genes were upregulated after 6 h, followed by downregulation at 24 h in SCC-25. In HSC-3, CAV-1 was downregulated, and E-cadherin gene (ECAD) was upregulated at 6 h. Only the protein ß-catenin in SCC-25 was affected, and cell migration of both cell lines was decreased, affecting SCC-25 more intensely. The invasive capacity within human myoma organotypic model was increased in SCC-25 and decreased in HSC-3. CONCLUSION: Cholesterol depletion affects CAV-1 and ECAD inversely. This affect also depends on cell type since the invasive capacity was augmented in primary cells while decreased in metastatic cells.

Exogenous growth hormone promotes an epithelial-mesenchymal hybrid phenotype in cancerous HeLa cells but not in non-cancerous HEK293 cells.

In cancer, the Epithelial to Mesenchymal Transition (EMT) is the process in which epithelial cells acquire mesenchymal features that allow metastasis, and chemotherapy resistance. Growth hormone (GH) has been associated with melanoma, breast, and endometrial cancer progression through an autocrine regulation of EMT. Since exogenous and autocrine expression of GH is known to have different molecular effects, we investigated whether exogenous GH is capable of regulating the EMT of cancer cells. Furthermore, we investigated whether exogenous GH could promote EMT in non-cancerous cells. To study the effect of GH (100 ng/ml) on cancer and non-cancer cells, we used HeLa and HEK293 cell lines, respectively. We evaluated the loss of cell-cell contacts, by cell scattering assay and migration by wound-healing assay. Additionally, we evaluated the morphological changes by phalloidin-staining. Finally, we evaluated the molecular markers E-cadherin and vimentin by flow cytometry. GH enhances cell scattering and the migratory rate and promotes morphological changes such as cell area increase and actin cytoskeleton filaments formation on HeLa cell line. Moreover, we found that GH favors the expression of the mesenchymal protein vimentin, followed by an increase in E-cadherin's epithelial protein expression, characteristics of an epithelial-mesenchymal hybrid phenotype that is associated with metastasis. On HEK293cells, GH promotes morphological changes, including cell area increment and filopodia formation, but not affects scattering, migration, nor EMT markers expression. Our results suggest that exogenous GH might participate in cervical cancer progression favoring a hybrid EMT phenotype but not on non-cancerous HEK293 cells.

HIF-1α contributes to metastasis in choriocarcinoma by regulating DEC1 expression.

PURPOSE: To elucidate the underlying mechanism of HIF-1α in migration and invasion of choriocarcinoma. METHODS: Cell proliferation was determined by CCK-8 assay when cell invasion was detected by transwell assay. The protein expression was detected by western blotting, immunohistochemistry, and qPCR assay. RESULT: HIF-1α was shown to be strongly expressed in both clinical tumour tissues and cell lines in choriocarcinoma. When HIF-1α was efficiently knocked down in JEG3 cells, the proliferation rate was reduced by approximately 50% and the number of cells that migrated through the transwell insert was greatly decreased. The cell invasion rate was also significantly reduced. Moreover, typical markers of epithelial-mesenchymal transition such as E-cadherin, were increased, while vimentin and α-SMA were decreased after HIF-1α knockdown. In contrast, overexpression of DEC1 reversed the effects of HIF-1α knockdown. Cell proliferation, migration, and invasion were partially recovered. The level of E-cadherin was decreased, while the level of vimentin and α-SMA was increased. In addition, the level of ß-catenin and LEF1 was downregulated after HIF-1α knockdown. The expression of MMP2 and MMP9 also declined. However, overexpression of DEC1 after HIF-1α knockdown partially reversed the expression pattern of these molecules. CONCLUSION: HIF-1α contributed to EMT and metastasis through activation of canonical ß-catenin signalling in choriocarcinoma and this process was dependent on DEC1. This study provides a new mechanism of HIF-1α in choriocarcinoma and suggests that intervention with DEC1 might be a promising therapeutic choice for choriocarcinoma.

Redistribution of the Novel Clostridioides difficile Spore Adherence Receptor E-Cadherin by TcdA and TcdB Increases Spore Binding to Adherens Junctions.

Clostridioides difficile causes antibiotic-associated diseases in humans, ranging from mild diarrhea to severe pseudomembranous colitis and death. A major clinical challenge is the prevention of disease recurrence, which affects nearly ~20 to 30% of the patients with a primary C. difficile infection (CDI). During CDI, C. difficile forms metabolically dormant spores that are essential for recurrence of CDI (R-CDI). In prior studies, we have shown that C. difficile spores interact with intestinal epithelial cells (IECs), which contribute to R-CDI. However, this interaction remains poorly understood. Here, we provide evidence that C. difficile spores interact with E-cadherin, contributing to spore adherence and internalization into IECs. C. difficile toxins TcdA and TcdB lead to adherens junctions opening and increase spore adherence to IECs. Confocal micrographs demonstrate that C. difficile spores associate with accessible E-cadherin; spore-E-cadherin association increases upon TcdA and TcdB intoxication. The presence of anti-E-cadherin antibodies decreased spore adherence and entry into IECs. By enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and immunogold labeling, we observed that E-cadherin binds to C. difficile spores, specifically to the hairlike projections of the spore, reducing spore adherence to IECs. Overall, these results expand our knowledge of how C. difficile spores bind to IECs by providing evidence that E-cadherin acts as a spore adherence receptor to IECs and by revealing how toxin-mediated damage affects spore interactions with IECs.

TWIST1 regulates proliferation, migration, and invasion and is a prognostic marker for oral tongue squamous cell carcinoma.

BACKGROUND: Epithelial-mesenchymal transition is one of the main mechanisms for tumor progression and metastasis. Transcription factors such as TWIST1 are key regulators of the epithelial-mesenchymal transition and are regarded as potential therapeutic targets for the treatment of cancer. The purpose of this study was to examine TWIST1 as a possible epithelial-mesenchymal transition-related prognostic biomarker in oral epithelial dysplasia and oral tongue squamous cell carcinomas, as well as the biological behavior of TWIST1-silencing in oral tongue squamous cell carcinomas cell lines. METHODS: Immunohistochemical analysis of TWIST1, E-cadherin, and N-cadherin was carried out in 47 samples representing oral epithelial dysplasia and 41 oral tongue squamous cell carcinomas. The suppression of TWIST1 expression was performed using shRNA-expression vectors in HSC-3 and SCC-9 cells to investigate in vitro the impact of TWIST1 on proliferation, apoptosis, viability, migration, and invasion of SCC-9 and HSC-3 cells. RESULTS: The expression of nuclear TWIST1 was significantly higher in oral tongue squamous cell carcinomas than in oral epithelial dysplasis (p < 0.0001), whereas TWIST1 in the cytoplasm was more expressed in oral epithelial dysplasis (p = 0.012). The high cytoplasmic expression of TWIST1 was significantly associated with shortened overall survival (p < 0.05), and increased nuclear TWIST1 expression was significantly related to high risk of recurrence (p = 0.03). Knockdown of TWIST1 in oral tongue squamous cell carcinomas cells induced the expression of E-cadherin and inhibited N-cadherin, which were followed by decreased proliferation, migration, and invasion. CONCLUSIONS: Our research suggests that TWIST1 is linked to the development of oral tongue carcinogenesis and may be used as a prognostic indicator and therapeutic target for oral tongue squamous cell carcinomas patients.

Epithelial-mesenchymal transition inhibition by metformin reduces melanoma lung metastasis in a murine model.

Melanoma is an aggressive cancer with fast metastatic spread and reduced survival time. One common event during the neoplastic progression is the epithelial-mesenchymal transition (EMT), which enhances invasiveness, cell migration, and metastasis. In this study, we investigated the effects of metformin at EMT in melanoma cell lines B16-F10 and A-375, in vitro, and the impact of EMT downregulation on melanoma progression in vivo. The metformin cells treatment reduces the migration potential in vitro and reduced the development of pulmonary metastases and the expressions of N-cadherin, vimentin, ZEB1, and ZEB2 at the metastases site, in vivo. These results indicate that metformin can promote EMT downregulation impairing the metastatic potential of melanoma cells.