Snail Protein Research Articles

The Physicochemical Properties of Flavor Enhancer Made from Different Types of Snail Protein Hydrolysates

Golden apple snails, apple snails, and freshwater snails are mollusks found in shallow waters with mud on the bottom, such as rice fields, swamps, and riverbanks. Snail has a high protein content of 14%-19%, but its utilization is still limited. In addition, certain types of snails, such as golden apple snails and apple snails, are pests to rice plants, so they have not been used optimally. One alternative is using snail as a flavor enhancer. Flavor enhancers can be made through enzymatic hydrolysis using bromelain enzymes. This study aimed to determine the effect of snail type and hydrolysis time on the physicochemical properties of flavor enhancer made of snail protein hydrolysate. This experimental study used a completely randomized design with two factors. Factor I was the type of snail (golden apple snail, apple snail, and freshwater snail), and factor II was the hydrolysis time (3 hours, 6 hours, and 9 hours). Data were analyzed using ANOVA with α 5%. When significant difference was identified, DMRT test at 95% significance level will be conducted. The result has showed that the flavor enhancer made of snail protein hydrolysate is influenced by different snail types and hydrolysis time. The apple snail with 9-hour hydrolysis produces the best flavor enhancer characterized by 3.96% water content, 14.73% yield, 92.29% water solubility, oil absorption of 1.15 ml/g, glutamic acid content of 107.23 ppm, and brownish yellow tone.

Physicochemical Properties of Apple Snail Protein Hydrolysate (Pila ampullacea) and its Potential as Flavor Enhancer

ple snail is a source of animal protein that has a high enough protein. Protein can be hydrolyzed by natural proteases, which one is bromelain. Enzymatic hydrolysis can produce hydrolysates containing peptides and amino acids that contribute to umami taste. The purpose of this study was to determine the effect of hydrolysis time and bromelain concentration on the physicochemical properties of apple snail hydrolysate. The optimal result in this study were used as natural flavor enhancer. This study used a completely randomized design with two factors. Factor I was the hydrolysis time (6 hours, 12 hours, and 18 hours) and factor II was the concentration of the bromelain (5%, 10%, and 15%). Based on the results showed that apple snail hydrolysate was influenced by hydrolysis time and bromelain concentration. The optimal result in this study was 18 hour hydrolysis treatment with 15% bromelain enzyme concentration had a yield 68.16%, degree of hydrolysis 72.09%, soluble protein 9.03%, total peptide 10.84 mg/mL, and glutamic acid 107.47 ppm. The application of apple snail protein hydrolysate give characteristics of flavor enhancers as follows: soluble protein 7.76%, glutamic acid 99.42 ppm, solubility 94.79%, water absorption 5.80 mL/g, hedonic test color 4.10 (neutral), aroma 5.05 (like slightly), and taste 5.10 (like slightly).

Prediction of Antibacterial Peptides against Propionibacterium acnes from the Peptidomes of Achatina fulica Mucus Fractions.

Acne vulgaris is a common skin disease mainly caused by the Gram-positive pathogenic bacterium, Propionibacterium acnes. This bacterium stimulates the inflammation process in human sebaceous glands. The giant African snail (Achatina fulica) is an alien species that rapidly reproduces and seriously damages agricultural products in Thailand. There were several research reports on the medical and pharmaceutical benefits of these snail mucus peptides and proteins. This study aimed to in silico predict multifunctional bioactive peptides from A. fulica mucus peptidome using bioinformatic tools for the determination of antimicrobial (iAMPpred), anti-biofilm (dPABBs), cytotoxic (ToxinPred) and cell-membrane-penetrating (CPPpred) peptides. Three candidate peptides with the highest predictive score were selected and re-designed/modified to improve the required activities. Structural and physicochemical properties of six anti-P. acnes (APA) peptide candidates were performed using the PEP–FOLD3 program and the four previous tools. All candidates had a random coiled structure and were named APAP-1 ori, APAP-2 ori, APAP-3 ori, APAP-1 mod, APAP-2 mod, and APAP-3 mod. To validate the APA activity, these peptide candidates were synthesized and tested against six isolates of P. acnes. The modified APA peptides showed high APA activity on three isolates. Therefore, our biomimetic mucus peptides could be useful for preventing acne vulgaris and further examined on other activities important to medical and pharmaceutical applications.

Overexpression of dual-specificity phosphatases 4 and 13 attenuates transforming growth factor β1-induced migration and drug resistance in A549 cells in vitro

Transforming growth factor-beta (TGFβ) proteins induce an epithelial-mesenchymal transition (EMT) programme that is associated with increased invasive and drug-resistant phenotype of carcinoma cells. In addition to the canonical pathway involving SMAD proteins, the mitogen-activated kinase (MAPK) pathway via extracellular signal-regulated kinases ½ (ERK1/2) is also involved in promoting and maintaining a mesenchymal phenotype by tumor cells following TGFβ signal activation. As dual-specificity phosphatases (DUSPs) regulate ERK1/2 activity by dephosphorylation, we aimed to examine DUSPs’ expression upon TGFβ stimulation and whether DUSPs play a role in the EMT and related phenotypes promoted by TGFβ1 in A549 cells. We found that TGFβ1 stimulation led to marked changes in several DUSP proteins, including significant decreases in DUSP4 and DUSP13 expressions. We then showed that the ectopic co-expression of DUSP4/13 suppresses TGFβ1-induced ERK1/2 phosphorylation and protein levels of the EMT transcription factors Snail and Slug proteins. We then demonstrated that DUSP4/13 co-expression partially inhibited TGFβ1-promoted migration, invasion, and chemoresistance in A549 cells. Collectively, this report provides data for the involvement of DUSP4/13 in malignant phenotypes regulated by TGFβ1 in A549 cells.

The antibiotic drug trimethoprim suppresses tumour growth and metastasis via targeting Snail

The zinc finger transcription factor Snail is aberrantly activated in many human cancers and strongly associated with poor prognosis. As a transcription factor, Snail has been traditionally considered an 'undruggable' target. Here, we identified a potent small-molecule inhibitor of Snail, namely trimethoprim, and investigated its potential antitumour effects and the underlying mechanisms. The inhibitory action of trimethoprim on Snail protein and the related molecular mechanisms were revealed by molecular docking, biolayer interferometry, immunoblotting, immunoprecipitation, qRT-PCR, pull-down and cycloheximide pulse-chase assays. The anti-proliferative and anti-metastatic effects of trimethoprim via targeting Snail were tested in multiple cell-based assays and animal models. This study identified trimethoprim, an antimicrobial drug, as a potent antitumour agent via targeting Snail. Molecular modelling analysis predicted that trimethoprim directly binds to the arginine-174 pocket of Snail protein. We further discovered that trimethoprim strongly interrupts the interaction of Snail with CREB-binding protein (CBP)/p300, which consequently suppresses Snail acetylation and promotes Snail degradation through the ubiquitin-proteasome pathway. Furthermore, trimethoprim sufficiently inhibited the proliferation, epithelial-mesenchymal transition (EMT) and migration of cancer cells in vitro via specifically targeting Snail. More importantly, trimethoprim effectively reduced Snail-driven tumour growth and metastasis to vital organs such as lung, bone and liver. These findings indicate, for the first time, that trimethoprim suppresses tumour growth and metastasis via targeting Snail. This study provides insights for a better understanding of the anticancer effects of trimethoprim and offers a potential anticancer therapeutic agent for clinical treatment.

Thrombospondin-2 holds prognostic value and is associated with metastasis and the mismatch repair process in gastric cancer

BackgroundThis study aims to investigate thrombospondin 2 (TSP2) expression levels in gastric cancer (GC) and determine the relationship between TSP2 and clinical characteristics and prognosis.MethodsThe online database Gene Expression Profile Interactive Analysis (GEPIA) was used to analyse TSP2 mRNA expression levels in GC. The Kaplan–Meier plotter prognostic analysis tool was used to evaluate the influence of TSP2 expression on clinical prognosis in GC patients. TSP2 expression levels were analysed in paraffin-embedded GC samples and adjacent normal tissues by immunohistochemistry. The relationship between the clinicopathological characteristics and prognosis of GC patients was assessed. Transwell experiments were used to evaluate the effect of TSP2 on HGC27 and AGS cell invasion and migration. The EdU experiment was used to detect the effect of transfection of TSP2 on cell proliferation, and the flow cytometry experiment was used to detect the effect of TSP2 on cell apoptosis and the cell growth cycle. Western blotting (Wb) technology was used to detect MMP, E-cadherin, N-cadherin, Vimentin, Snail, AKT, PI3K, and VEGF protein expression in HGC27 cells.ResultsCompared with normal tissues, TSP2 mRNA expression in GC was significantly upregulated and was closely related to the clinical stage of GC. High TSP2 expression significantly affected the OS, FP and PPS of patients with GC. Among these patients, TSP2 expression levels did not affect the prognosis of patients with GC in the N0 subgroup but significantly affected the prognosis of patients with GC in the N (1 + 2 + 3) subgroup. TSP2 protein expression levels were significantly higher in GC tissue compared with normal tissues (P < 0.01). The overall survival (OS) and relapse-free survival (RFS) of patients with high TSP2 expression were lower than those of patients with low TSP2 expression. Cells transfected with the TSP2-silencing sequence exhibited increased apoptosis and inhibition of proliferation, migration and invasion. AKT and PI3K expression in cells was significantly downregulated (P < 0.01). AKT, PI3K and VEGF expression in cells transfected with the TSP2 silencing sequence was significantly reduced. Proliferation, migration, invasion ability, and TSP2 expression levels significantly correlated with mismatch repair genes, such as PMS2, MSH6, MSH2, and MLH1 (P < 0.05).ConclusionTSP2 expression is significantly increased in GC. TSP2 expression is closely related to metastasis and the mismatch repair process in GC patients and affects GC patient prognosis. The mechanism may involve regulating gastric cancer cell proliferation and migration by modulating the VEGF/PI3K/AKT signalling pathway. TSP2 is a potential marker and therapeutic target for the prognosis of GC patients.

A CFH peptide-decorated liposomal oxymatrine inactivates cancer-associated fibroblasts of hepatocellular carcinoma through epithelial\u2013mesenchymal transition reversion

Cancer-associated fibroblasts (CAFs) deteriorate tumor microenvironment (TME) and hinder intra-tumoral drug delivery. Direct depleting CAFs exists unpredictable risks of tumor metastasis. Epithelial–mesenchymal transition (EMT) is a critical process of CAFs converted from hepatic stellate cells during hepatocellular tumorigenesis; however, until now the feasibility of reversing EMT to battle hepatocellular carcinoma has not been comprehensively explored. In this study, we report a CFH peptide (CFHKHKSPALSPVGGG)-decorated liposomal oxymatrine (CFH/OM-L) with a high affinity to Tenascin-C for targeted inactivating CAFs through reversing EMT, which is verified by the upregulation of E-cadherin and downregulation of vimentin, N-cadherin, and snail protein in vivo and in vitro. After the combination with icaritin-loaded lipid complex, CFH/OM-L obviously boosts the comprehensive anticancer efficacy in both 3D tumor spheroids and stromal-rich tumor xenograft nude mouse models. The combinational therapy not only effectively reversed the in vivo EMT process but also significantly lowered the collagen, creating favorable conditions for deep penetration of nanoparticles. More importantly, CFH/OM-L does not kill but inactivates CAFs, resulting in not only a low risk of tumor metastasis but also a reprogramming TME, such as M1 tumor-associated macrophages polarization and natural killer cells activation. Such strategy paves a moderate way to remold TME without depleting CAFs and provides a powerful tool to design strategies of combinational hepatocellular carcinoma therapy.Graphical

Epithelial-mesenchymal transition modulates lower lip carcinogenesis and promotes cancer progression

ObjectiveTo investigate and compare the immunoexpression of E-cadherin, α-SMA, TGF-β and Snail proteins between cases of actinic cheilitis (AC) and squamous cell carcinoma of the lower lip (LLSCC). Study designE-cadherin, α-SMA, TGF-β and Snail antibody immunostaining was analyzed semiquantitatively in 54 AC cases and in 49 LLSCCs. The cases were classified as low and high expression for analysis of the association with clinicopathological variables and overall survival (OS) and disease-free survival (DFS) rates. ResultsHigh expression of E-cadherin (cytoplasmic) (p = 0.001) and α-SMA (p < 0.001) was identified in LLSCCs, as well as low expression of TGF-β in LLSCCs (p < 0.001) and high expression of Snail in AC cases (p = 0.006). Survival analysis revealed that high expression of α-SMA at the tumor invasion front, a network immunostaining pattern of this protein, and high expression of TGF-β in tumor buds were significantly associated with poor OS (p < 0.05). There was a higher risk of death among LLSCC cases with high expression of α-SMA (HR = 5.90, p = 0.03). High expression of TGF-β in tumor buds was significantly associated with poor DFS (p = 0.007) and with a higher risk of negative outcomes for DFS (HR = 4.44, p = 0.014). ConclusionsThe present results suggest the potential involvement of dysregulation of proteins associated with epithelial-mesenchymal transition in the modulation of lip carcinogenesis and greater aggressiveness of LLSCC.

A non-catalytic scaffolding activity of hexokinase 2 contributes to EMT and metastasis

Hexokinase 2 (HK2), which catalyzes the first committed step in glucose metabolism, is induced in cancer cells. HK2’s role in tumorigenesis has been attributed to its glucose kinase activity. Here, we describe a kinase independent HK2 activity, which contributes to metastasis. HK2 binds and sequesters glycogen synthase kinase 3 (GSK3) and acts as a scaffold forming a ternary complex with the regulatory subunit of protein kinase A (PRKAR1a) and GSK3β to facilitate GSK3β phosphorylation and inhibition by PKA. Thus, HK2 functions as an A-kinase anchoring protein (AKAP). Phosphorylation by GSK3β targets proteins for degradation. Consistently, HK2 increases the level and stability of GSK3 targets, MCL1, NRF2, and particularly SNAIL. In addition to GSK3 inhibition, HK2 kinase activity mediates SNAIL glycosylation, which prohibits its phosphorylation by GSK3. Finally, in mouse models of breast cancer metastasis, HK2 deficiency decreases SNAIL protein levels and inhibits SNAIL-mediated epithelial mesenchymal transition and metastasis.

JNK-mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation-dependent activation of Snail.

Sirtuin 1 (SIRT1), an NAD+‐dependent histone/protein deacetylase, has multifaceted functions in various biological events such as inflammation, aging, and energy metabolism. The role of SIRT1 in carcinogenesis, however, is still under debate. Recent studies have indicated that aberrant overexpression of SIRT1 is correlated with metastasis and poor prognosis in several types of malignancy, including colorectal cancer. In the present study, we found that both SIRT1 and SIRT1 phosphorylated on serine 27 were coordinately upregulated in colon cancer patients’ tissues and human colon cancer cell lines. This prompted us to investigate a role of phospho‐SIRT1 in the context of colon cancer progression. A phosphorylation‐defective mutant form of SIRT1, in which serine 27 was substituted by alanine (SIRT1‐S27A), exhibited lower protein stability compared to that of wild‐type SIRT1. Notably, human colon cancer (HCT‐116) cells harboring the SIRT1‐S27A mutation showed decreased cell proliferation and reduced capability to form xenograft tumor in athymic nude mice, which was accompanied by diminished transcriptional activity of Snail. HCT‐116 cells carrying SIRT1‐S27A were less capable of deacetylating the Snail protein, with a concomitant decrease in the levels of interleukin (IL)‐6 and IL‐8 mRNA transcripts. Taken together, these observations suggest that SIRT1 stabilized through phosphorylation on serine 27 exerts oncogenic effects at least partly through deacetylation‐dependent activation of Snail and subsequent transcription of IL‐6 and IL‐8 in human colon cancer cells.

Type XXVIII Collagen Regulates Renal Interstitial Fibrosis and Epithelial-Mesenchymal Transition by SREBP1-Mediated HKDC1 Expression.

A novel collagen called type XXVIII collagen (COL28) is involved in cancer and lung fibrosis. Preliminary data showed that renal tubular epithelial cells could proliferate, migrate, and undergo an epithelial-mesenchymal transition (EMT) when COL28 was overexpressed; however, it is still unknown how this occurs and what the underlying mechanism is. We analyzed the differential expression of genes (DEGs) in the stable COL28 overexpression HK-2 cell lines by RNA-sequencing analysis, before which Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analyses were performed. Genes related to COL28 promoting HK-2 cell proliferation and EMT were screened and verified. By using western blot and immunofluorescence, the effects of COL28 on the expression of α-SMA, E-cadherin, Snail, HKDC1, and SREBP1 were detected. The effect of COL28 overexpression on renal fibrosis in unilateral ureteral obstruction (UUO) mice was detected by H&E and Masson staining. HKDC1 interference agent was synthesized and transfected into the HK-2 cell line stably overexpressing COL28. In HK-2 cells, the effects of HKDC1 interference on the expression of α-SMA, E-cadherin, and Snail were detected. We screened and verified that HKDC1 was related to COL28 and promoted HK-2 cell proliferation and EMT. WB showed that in HK-2 cells, COL28 overexpression increased α-SMA, Snail, HKDC1, and SREBP1 expressions and decreased E-cadherin expression. Overexpression of COL28 aggravated renal interstitial fibrosis in UUO mice; upregulated α-SMA, Snail, HKDC1, and SREBP1 expressions; and decreased the E-cadherin protein expression in UUO mice. Interference of HKDC1 expression promoted the E-cadherin protein expression while inhibiting α-SMA, Snail, HKDC1, and SREBP1 protein expressions. Overexpression of COL28 can aggravate renal interstitial fibrosis by encouraging renal tubular epithelial cells to undergo EMT, and interference with HKDC1 expression can alleviate fibrosis by reversing EMT induced by COL28 overexpression.

The ETS1-LINC00278 negative feedback loop plays a role in COL4A1/COL4A2 regulation in laryngeal squamous cell carcinoma.

The present study aimed to investigate LINC00278 expression in laryngeal squamous cell carcinoma (LSCC) and its involvement in the process of proliferation, migration, and invasion, providing a rationale for mining potential diagnostic and therapeutic targets of LSCC. Univariate and multivariate Cox regression analyses were performed to identify optimal prognostic lncRNAs. MTS, colony formation, wound healing, and Transwell invasion assays were used to determine the effects of LINC00278 overexpression on the proliferation, migration, and invasion of cancer cells. The expressions of signaling pathway-related proteins and epithelial-mesenchymal transition (EMT) marker proteins were detected using western blot. The chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were performed to demonstrate the binding of ETS proto-oncogene 1, transcription factor (ETS1), and LINC00278 promoter region. The molecular targets of LINC00278 were identified by RNA sequencing analysis and co-expression analysis. Kaplan-Meier analysis and CIBERSORT algorithm were used to analyze survival and immune cell infiltration based on LINC00278, COL4A1, and COL4A2. Multivariate Cox regression was used to establish a six-gene prognostic model. LINC00278 expression was low in LSCC tissues, and it was significantly associated with the TNM (tumors/nodes/metastases) stage (p<0.001), lymphatic metastasis (p<0.01), and pathological differentiation (p<0.01). LINC00278 overexpression significantly reduced LSCC cell proliferation, migration, and invasion in TU686, TU177, and AMC-HN-8 cell lines. E-cadherin protein expression was increased, while N-cadherin, Vimentin, Zeb1, and Snail protein expression was decreased in the LINC00278 group, compared to the pcDNA3.1 group. Additionally, in AMC-HN-8 and FaDu cell lines, the LINC00278-treated group had significantly lower p-AKT and p-mTOR protein levels than the control group. ETS1 is a direct transcriptional regulator of the LINC00278 gene based on luciferase reporter assays and ChIP experiments. Western blot analysis demonstrated that high LINC00278 expression inhibited both ETS1 expression and phosphorylation. COL4A1/COL4A2 were identified as potential downstream targets of LINC00278. Meanwhile, the LINC00278/COL4A1/COL4A2-dominated low-risk group showed higher antigen-presenting activity and a higher immune score than the high-risk group. The findings indicated that ETS1 upregulated LINC00278 expression on the Y chromosome, which in turn inhibited LSCC growth in vivo and in vitro by inhibiting the AKT/mTOR signaling pathway via downregulation of COL4A1/COL4A2.

Design and synthesis of dual inhibitors targeting snail and histone deacetylase for the treatment of solid tumour cancer

Snail and histone deacetylases (HDACs) have an important impact on cancer treatment, especially for their synergy. Therefore, the development of inhibitors targeting both Snail and HDAC might be a promising strategy for the treatment of cancers. In this work, we synthesized a series of Snail/HDAC dual inhibitors. Compound 9n displayed the most potent inhibitory activity against HDAC1 with an IC50 of 0.405 μM, potent inhibition against Snail with a Kd of 0.180 μM, and antiproliferative activity in HCT-116 cell lines with an IC50 of 0.0751 μM. Compound 9n showed a good inhibitory effect on NCI–H522 (GI50 = 0.0488 μM), MDA-MB-435 (GI50 = 0.0361 μM), and MCF7 (GI50 = 0.0518 μM). Docking studies showed that compound 9n can be well docked into the active binding sites of Snail and HDAC. Further studies showed that compound 9n increased histone H4 acetylation in HCT-116 cells and decreased the expression of Snail protein to induce cell apoptosis. These findings highlight the potential for the development of Snail/HDAC dual inhibitors as anti-solid tumour cancer drugs.

Short Communication: Morphological diversity and the addition of golden snail protein: Its effect on flavonoid content on Echinacea purpurea

Abstract. Ferdyana WC, Widiyastuti Y, Pujiasmanto B, Sakya AT, Yunus A. 2021. Short Communication: Morphological diversity and the addition of golden snail protein: Its effect on flavonoid content on Echinacea purpurea. Biodiversitas 23: 62-66. Echinacea purpurea is a subtropical medicinal plant that contains flavonoids. Flavonoids function as antioxidants and immunomodulators. However, information on the method to increase flavonoid content in E. purpurea is still limited. The golden snail protein increases flavonoid content. The purpose of this study was to determine the best accession and optimal concentration of golden snail protein on biomass production and flavonoid content in E. purpurea. The study used a Factorial Completely Randomized Design (CRD). The first factor was three accessions of E. purpurea (A1, A2, A3); the second factor was 5 levels of the addition of golden snail protein (0 mL/L/week, 10 mL/L/week, 20 mL/L/week, 30 mL/L/week, and 40 mL /L/week). There were differences in morphological characters of three E. purpurea accessions, i.e., stem and the shape of the flower crown. Accession 2 had the highest flavonoid content except for the 10 mL/L/week treatment. The highest increase in flavonoid content in three accessions was obtained in the treatment of 10 mL/L/week gold snail protein by 19% flavonoid content compared to control treatment (0 mL/L/week). The concentration of 0 mL/l/week or control had the lowest flavonoid content.

Molecular Alterations Associated with Acquired Drug Resistance during Combined Treatment with Encorafenib and Binimetinib in Melanoma Cell Lines.

Simple SummaryMelanoma is the most aggressive, deadliest form of skin cancer. Combined BRAF-MEK inhibitor (BRAFi/MEKi) therapy was a breakthrough in the treatment of melanoma with BRAFV600-mutations. However, many patients frequently develop drug resistance to the combinatory treatment. The aim of our study was to characterize the molecular background behind acquired resistance to BRAFi/MEKi-s. After the successful development of resistant cell lines, we investigated the invasion properties, changes in gene and protein expressions, as well as the effect of the “drug holiday” of the resistant cell lines. Drug-resistant melanoma cells had a higher invasive potential and acquired a spindle-like structure, and many cancer-related proteins were overexpressed in the resistant cells. Furthermore, transcriptome analysis revealed that differentially expressed genes are functionally linked to a variety of biological functions that may lead to resistance to the inhibitors. These results may offer valuable insight into further understanding of BRAFi/MEKi resistance, as well as to the development of therapeutic tools to overcome drug resistance.Combination treatment using BRAF/MEK inhibitors is a promising therapy for patients with advanced BRAFV600E/K mutant melanoma. However, acquired resistance largely limits the clinical efficacy of this drug combination. Identifying resistance mechanisms is essential to reach long-term, durable responses. During this study, we developed six melanoma cell lines with acquired resistance for BRAFi/MEKi treatment and defined the molecular alterations associated with drug resistance. We observed that the invasion of three resistant cell lines increased significantly compared to the sensitive cells. RNA-sequencing analysis revealed differentially expressed genes that were functionally linked to a variety of biological functions including epithelial-mesenchymal transition, the ROS pathway, and KRAS-signalling. Using proteome profiler array, several differentially expressed proteins were detected, which clustered into a unique pattern. Galectin showed increased expression in four resistant cell lines, being the highest in the WM1617E+BRes cells. We also observed that the resistant cells behaved differently after the withdrawal of the inhibitors, five were not drug addicted at all and did not exhibit significantly increased lethality; however, the viability of one resistant cell line (WM1617E+BRes) decreased significantly. We have selected three resistant cell lines to investigate the protein expression changes after drug withdrawal. The expression patterns of CapG, Enolase 2, and osteopontin were similar in the resistant cells after ten days of “drug holiday”, but the Snail protein was only expressed in the WM1617E+BRes cells, which showed a drug-dependent phenotype, and this might be associated with drug addiction. Our results highlight that melanoma cells use several types of resistance mechanisms involving the altered expression of different proteins to bypass drug treatment.

Deubiquitinase USP13 promotes the epithelial-mesenchymal transition and metastasis in gastric cancer by maintaining Snail protein

The dynamic balance between ubiquitination and deubiquitination is a key mechanism that regulates protein degradation and maintains cell protein homeostasis. Ubiquitin-specific peptidase 13 (USP13), a deubiquitinase (DUB), regulates various physiological and pathological processes, including cancer. A previous study reported that high USP13 mRNA expression confers poor prognosis in gastric cancer (GC). However, the biological function of USP13 in GC remains unknown. Here, we revealed that USP13 expression was upregulated in GC tissue samples compared to noncancerous tissues. USP13-positive expression was associated with poor differentiation, high invasiveness, and advanced tumor stage. Notably, upregulated USP13 expression was closely correlated with the reduced survival of GC patients. We also confirmed increased USP13 expression in GC cell lines. USP13 knockdown prominently suppressed MGC-803 cell migration and invasion. Conversely, USP13 overexpression markedly enhanced SGC-7901 cell motility. Furthermore, USP13 positively regulates the epithelial-mesenchymal transition (EMT) of GC cells. Interestingly, USP13 remarkably enhanced Snail protein expression but did not affect its mRNA levels in GC cells. We confirmed a positive correlation between USP13 and Snail expression in GC tissues. Mechanistically, USP13 knockdown promoted Snail degradation, which could be blocked by the proteasome inhibitor MG132. USP13 interacted with Snail to deubiquitinate and stabilize Snail in GC cells. Finally, Snail knockdown significantly blocked USP13-induced SGC-7901 cell migration and invasion. In conclusion, USP13 overexpression was frequently detected in GC and contributed to the EMT and metastasis of GC by stabilizing Snail.

Splice-Variant Knock-Out of TGFβ Receptors Perturbates the Proteome of Ovarian Carcinoma Cells.

The aim of this study was to analyze the biological role of different transforming growth factor-β (TGFβ) receptor splice variants in ovarian carcinoma (OC). Specific receptor variant knockouts (KO) were prepared using the CRISPR/Cas9 genome editing system in two OC cell lines, TβRI variant 1 (TβRIv1) KO in ES-2 cells and TβRII variant 1 (TβRIIv1) KO in OVCAR-8 cells. Control and KO cells were compared by proteomic analysis, functional tests, analysis of epithelial–mesenchymal transition (EMT) drivers, and Western blot of signaling proteins. Proteomic analysis revealed significant changes in protein pathways in the KO cells. TβRIv1 KO resulted in a significant reduction in both cellular motility and invasion, while TβRIIv1 KO significantly reduced cellular motility and increased Reactive Oxygen Species (ROS) production. Both receptor variant KOs reduced MET protein levels. Of the EMT drivers, a significant decrease in TWIST protein expression, and increase in SNAIL protein and MALAT1 mRNA levels were observed in the TβRIIv1 KO compared to control. A significant decrease in JNK1 and JNK2 activation was found in the TβRIv1 KO compared to control cells. These findings provide new insight regarding the biological role of the TGFβ receptor variants in the biology and potentially the progression of OC.

Omeprazole suppresses aggressive cancer growth and metastasis in mice through promoting Snail degradation.

Omeprazole is a proton pump inhibitor that has recently been reported to exhibit anticancer activity against several types of cancer. However, the anticancer mechanisms of omeprazole remain elusive. Snail is an oncogenic zinc finger transcription factor; aberrant activation of Snail is associated with the occurrence and progression of cancer. In this study, we investigated whether Snail acted as a direct anticancer target of omeprazole. We showed that omeprazole displayed a high binding-affinity to recombinant Snail protein (Kd = 0.076 mM), suggesting that omeprazole directly and physically binds to the Snail protein. We further revealed that omeprazole disrupted CREB-binding protein (CBP)/p300-mediated Snail acetylation and then promoted Snail degradation through the ubiquitin-proteasome pathway in HCT116 cells. Omeprazole treatment markedly suppressed Snail-driven epithelial-mesenchymal transition (EMT) in aggressive HCT116, SUM159, and 4T1 cancer cells in vitro and reduced EMT-associated tumor invasion and metastasis in cancer cell xenograft models. Omeprazole also inhibited tumor growth by limiting Snail-dependent cell cycle progression. Overall, this study, for the first time, identifies Snail as a target of omeprazole and reveals a novel mechanism underlying the therapeutic effects of omeprazole against cancer. This study strongly suggests that omeprazole may be an excellent auxiliary drug for treating patients with malignant tumors.

Sanguisorba officinalis L. suppresses 5-fluorouracil-sensitive and-resistant colorectal cancer growth and metastasis via inhibition of the Wnt/β-catenin pathway

BackgroundColorectal cancer (CRC) is a widespread cancer with high morbidity and mortality. Chemoresistance and metastasis are the current challenges for CRC treatment. Sanguisorba officinalis Linn. (called DiYu in Chinese, DY) is a traditional Chinese medicine (TCM) whose root is long used as medicinal part. In our previous study, the aqueous extract of DY could inhibit the Wnt/β-catenin pathway and showed great antitumor effect against CRC. The Wnt/β-catenin pathway is involved in CRC chemoresistance and metastasis. However, there is little study on the antitumor and antimetastatic effects of DY on resistant CRC cells. The aim of this study is to explore the effect of aqueous extract of DY on the growth and metastasis of 5-fluorouracil (5-FU) sensitive and resistant CRC, and to elucidate the underlying molecular mechanism. MethodologyIn this study, cell viability, cell colony formation and apoptosis analyses were performed to verify the in vitro antitumor effect of DY on 5-FU-sensitive and -resistant CRC cells. Next, transwell assays were used to test the inhibition activity of DY on CRC migration and invasion. Western Blotting assays were carried out to identify the molecular mechanism underlying the efficacy of DY extract. Xenograft CRC nude mice model and tumor metastasis model were used to confirm the in vivo antitumor and antimetastatic effects of DY. ResultsDY inhibited cell proliferation and apoptosis via the upregulation of Bax, cleaved-caspase3 and cleaved-PARP proteins and downregulation of Bcl-2 protein. DY also inhibited cell migration and invasion via the downregulation of N-cadherin, vimentin and snail proteins and upregulation of E-cadherin protein, demonstrating that DY suppressed cell metastasis by reversing epithelial-to-mesenchymal transition (EMT) procession. Moreover, the protein expression levels of β-catenin in whole cell, cytoplasm and nucleus were decreased after DY treatment. Taken together, DY suppressed CRC cell growth and metastasis via inhibition of the Wnt pathway. Additionally, DY also demonstrated effective antitumor and anti-metastasis activities in vivo. ConclusionsIn conclusion, DY suppressed the growth and metastasis of 5-FU-sensitive and -resistant CRC via inhibition of the Wnt pathway, which indicated that DY could be a potential drug to treat CRC patients and improve clinic outcome.

Decreased exosome-delivered miR-486-5p is responsible for the peritoneal metastasis of gastric cancer cells by promoting EMT progress

BackgroundThe present study aims to investigate the preliminary mechanism underlying the peritoneal metastasis of gastric cancer cells.MethodsExosomes from GC9811 cells (Con-Exo) and from GC9811-P cells (PM-Exo) were extracted by ultracentrifugation, which were identified with transmission electron microscopy (TEM) and nanoparticle trafficking analysis, as well as the expression of CD9, CD63, and CD81 detected by Western blot assay. α-SMA expression was determined by immunofluorescence assay and Western blot assay. The levels of Snail1, E-cadherin, and Actin-related protein 3 (ACTR3) were evaluated by Western blot assay. MiRNA array was performed on exosomes to screen the differentially expressed miRNAs. The expressions of miRNAs, SMAD2, CDK4, and ACTR3 were determined by QRT-PCR. The delivery of miR-486-5p was confirmed by laser confocal detection.ResultsFirstly, TEM, nanoparticle trafficking analysis, and Western blot assays were used to confirm the successful extraction of Con-Exo and PM-Exo. The incubation of Con-Exo and PM-Exo could decrease E-cadherin expression and increase of α-SMA respectively in HMrSV5 cells, with the increased proportion of fusiform cells. More significant changes were observed in PM-Exo-treated HMrSV5 cells. Secondary, compared to Con-Exo, miR-486-5p and miR-132-3p were found downregulated, and miR-132-5p was found upregulated in PM-Exo. The transfection of miR-486-5p and miR-132-3p was observed to suppress EMT, and the transfection of miR-132-3p was observed to induce EMT. Laser confocal detection confirmed the delivery of miR-486-5p from gastric cancer cells to HMrSV5 cells through exosomes. Lastly, the expression of Mothers against decapentaplegic homolog 2 (SMAD2), cyclin-dependent kinase 4 (CDK4), and ACTR3 was found to be downregulated via miR-486-5p.ConclusionDecreased delivery of miR-486-5p via exosomes might be responsible for the peritoneal metastasis of gastric cancer cells by promoting epithelial-mesenchymal transition progress.