Downregulation Of Β-catenin Research Articles

ATP7A-dependent copper sequestration contributes to termination of β-CATENIN signaling during early adipogenesis

ObjectivesAdipocyte fate determination is tightly regulated by extrinsic signaling pathways and intrinsic metabolic and morphologic changes that maintain adipose tissue function. Copper (Cu) homeostasis is required for the normal metabolism of mature adipocytes, whereas the role of Cu in adipogenesis is unclear. MethodsTo determine the role of Cu is adipocytes differentiation, we used 3T3-L1 adipocytes, immunocytochemistry, X-ray fluorescence, mass-spectrometry, pharmacological treatments, and manipulations of copper levels. ResultsIn differentiating 3T3-L1 cells, adipogenic stimuli trigger the upregulation and trafficking of the Cu transporter Atp7a, thus causing Cu redistribution from the cytosol to vesicles. Disrupting Cu homeostasis by the deletion of Atp7a results in Cu elevation and inhibition of adipogenesis. The upregulation of C/EBPβ, an initial step of adipogenesis, is not affected in Atp7a−/− cells, whereas the subsequent upregulation of PPARγ is inhibited. Comparison of changes in the Atp7a−/− and wild type cells proteomes during early adipogenesis revealed stabilization of β-catenin, a negative regulator of adipogenesis. Cu chelation, or overexpression of the Cu transporter ATP7B in Atp7a−/− cells, restored β-catenin down-regulation and intracellular targeting. ConclusionsCu buffering during early adipogenesis contributes to termination of β-catenin signaling. Abnormal upregulation of β-catenin was also observed in vivo in the livers of Atp7b−/− mice, which accumulate Cu, suggesting a tissue-independent crosstalk between Cu homeostasis and the Wnt/β-catenin pathway. These results point to a new regulatory role of Cu in adipocytes and contribute to better understanding of human disorders of Cu misbalance.

Suppressive Potential of Rosmarinus officinalis L. Extract against Triple-Negative and Luminal A Breast Cancer.

Rosemary is an aromatic plant with ancient and modern applications as a spice and herbal remedy. Due to the strong antioxidant potential of rosemary, the present study investigated the anti-proliferative andpro-apoptotic characteristics of rosemary on luminal A and triple-negative breast cancer cells. The effect of rosemary extract on the WNT10B and β-Catenin genes was also evaluated. The WNT10B and β-Catenin expression were measured by real-time PCR. The outcomes of the MTT assay and AnnexinV/PI flow cytometry assay showed that exposure of MCF-7 and MDA-MB-231 cells to rosemary reduced cell viability in a dose-time-dependent routine and promoted apoptosis in breast cancer cells. It was revealed that the extract could exert cytotoxic and apoptotic effects by downregulation of WNT10B and β-Catenin. Our results suggest rosemary as a promising complementary herbal medicine for breast cancers, without the adverse effects of chemotherapy drugs.

Knockdown of LRP5 Promotes Proliferation and Invasion of Tongue Squamous Cell Carcinoma through Compensatory Activation of Akt Signaling.

The role of LRP5, a critical receptor in the Wnt signaling pathway, remains unexplored in tongue squamous cell carcinoma (TSCC). This study investigates the impact of LRP5 knockdown on the biological behaviors of TSCC cell lines both in vitro and in vivo. Our findings indicate that LRP5 knockdown significantly enhances cell proliferation, migration, and invasion in CAL27 and SCC25 cell lines. RNA-seq analysis reveals compensatory activation of the Akt pathway, with 119 genes significantly upregulated post-LRP5 knockdown. Elevated MMP1 expression suggests its potential involvement in TSCC progression. Western blot analysis demonstrates increased Akt phosphorylation, upregulated proliferation-related PCNA, and downregulated apoptosis-related caspase-3 after LRP5 knockdown. Down-regulation of E-cadherin and β-Catenin, proteins associated with cell adhesion and invasion, further elucidates the molecular mechanism underlying increased cell migration and invasion. Our study concludes that compensatory Akt pathway activation is essential for the LRP5 knockdown-induced migration and proliferation of CAL27 and SCC25 cells. These results highlight LRP5 as a potential therapeutic target for TSCC. Simultaneous inhibition of Wnt and Akt signaling emerges as a promising approach for TSCC treatment.

Sinensetin suppresses breast cancer cell progression via Wnt/β-catenin pathway inhibition.

Although there are many treatments for breast cancer, such as surgery, radiotherapy, chemotherapy, estrogen receptor antagonists, immune checkpoint inhibitors and so on. However, safer and more effective therapeutic drugs for breast cancer are needed. Sinensetin, a safer therapeutic drugs, come from citrus species and medicinal plants used in traditional medicine, while its role and underlying mechanism in breast cancer remain unclear. Our study aimed to investigate the role and mechanism of sinensetin in breast cancer. Cell Counting Kit-8 (CCK-8) was used to determine the safe concentration of sinensetin in MCF-10A, MCF7 and MDA-MB-231 cells; 120 μM sinensetin was used in subsequent experiments. Real time polymerase chain reaction (RT-PCR), Western blotting, Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay, Transwell invasion assay and Clone formation assay were used in this study to determine cell viability, mRNA expression, protein levels, apoptosis, proliferation, invasion and so on. Herein, our results showed that 120 μM sinensetin suppressed the cell viability and promoted apoptosis of MCF7 and MDA-MB-231 cells. Treatment with 120 µM sinensetin for 24 h showed no significant toxicity to normal mammary cells; 120 μM sinensetin decreased cell proliferation, invasion, and epithelial-mesenchymal transition (EMT), and downregulated β-catenin, lymphatic enhancing factor 1 (LEF1), T-cell factor (TCF) 1/TCF7, and TCF3/TCF7L1 expression in MCF7 and MDA-MB-231 cells. The Wnt agonist SKL2001 reversed the inhibitory effect of sinensetin on cell survival, metastasis, and EMT. Sinensetin-induced downregulation of β-catenin, LEF1, and TCF1/TCF7 expression were upregulated by SKL2001 in MCF7 and MDA-MB-231 cells. In summary, sinensetin suppressed the metastasis of breast cancer cell via inhibition of Wnt/β-catenin pathway and there were no adverse effects on normal breast cells. Our study confirmed the role of sinensetin in breast cancer cells and provided a better understanding of the underlying mechanism.

Zerumbone (ZER), a Potential Anticancer for Breast Mediates Cancer Cell Death Through Targeting β-catenin Signaling Pathway in Tumor Regression in Sprague Dawley Rat Mammary Gland Tumors

Background & Objective: Canonical WNT/Wingless pathway regulates expression of target genes by modulating intracellular β-catenin leading to cancer growth and survival. Here, we studied inhibition of β-catenin using zerumbone (ZER) or β-catenin-siRNA in Sprague-Dawley rat cancer model. Methodology: Rat mammary gland tumor model was induced with the wild-type LA7 breast cancer cell line or β-catenin-knockdown-LA7 cells. LA7-rats were treated with ZER or β-catenin-siRNA as a positive control and the levels of β-catenin expression and its target gene were assessed using real-time PCR, immunohistochemistry and TUNEL assay. Results: We found good correlation between β-catenin inhibition and induction of apoptosis when using ZER or β-catenin-siRNA. In this aspect, ZER and β-catenin-siRNA inhibited cellular proliferation as reflected by reduced growth of breast cancer and apoptosis induction in rat mammary gland tumor. Further, our studies demonstrate that treatment with ZER and β-catenin-siRNA affected β-catenin-dependent gene expression. β-catenin downregulation by ZER or knockdown by β-catenin-siRNA improves the morphological and histological feature of the breast tissues as confirmed by subtle changes of the genes involved in cell-apoptotic pathways. Conclusion: This indicates that ZER targets similarly to β-catenin-siRNA and possibly be a useful anti-breast cancer by affecting expression of down-stream targets that are key components in cancer development. ZER can be a promising anticancer candidate for treatment of breast.

CRISPR/Cas9-Induced Fam83h Knock-out Leads to Impaired Wnt/β-Catenin Pathway and Altered Expression of Tooth Mineralization Genes in Mice.

Dental enamel formation is a complex process that is regulated by various genes. One such gene, Family With Sequence Similarity 83 Member H (Fam83h), has been identified as an essential factor for dental enamel formation. Additionally, Fam83h has been found to be potentially linked to the Wnt/β-catenin pathway. This study aimed to investigate the effects of the Fam83h knockout gene on mineralization and formation of teeth, along with mediators of the Wnt/β-catenin pathway as a development aspect in mice. To confirm the Fam83h-KnockOut mice, both Sanger sequencing and Western blot methods were used. then used qPCR to measure the expression levels of genes related to tooth mineralization and formation of dental root, including Fam20a, Dspp, Dmp1, Enam, Ambn, Sppl2a, Mmp20, and Wnt/β-catenin pathway mediators, in both the Fam83h-Knockout and wild-type mice at 5, 11 and 18 days of age. also the expression level of Fgf10 and mediators of the Wnt/β-catenin pathway was measured in the skin of both Knockout and wild-type mice using qPCR. A histological assessment was then performed to further investigate the results. A significant reduction in the expression levels of Ambn, Mmp20, Dspp, and Fgf10 in the dental root of Fam83h-Knockout mice compared to their wild-type counterparts was demonstrated by our results, indicating potential disruptions in tooth development. Significant down-regulation of CK1a, CK1e, and β-catenin in the dental root of Fam83h-Knockout mice was associated with a reduction in mineralization and formation-related gene. Additionally, the skin analysis of Fam83h-Knockout mice revealed reduced levels of Fgf10, CK1a, CK1e, and β-catenin. Further histological assessment confirmed that the concurrent reduction of Fgf10 expression level and Wnt/β-catenin genes were associated with alterations in hair follicle maturation. The concurrent reduction in the expression level of both Wnt/β-catenin mediators and mineralization-related genes, resulting in the disruption of dental mineralization and formation, was caused by the deficiency of Fam83h. Our findings suggest a cumulative effect and multi-factorial interplay between Fam83h, Wnt/Β-Catenin signaling, and dental mineralization-related genes subsequently, during the dental formation process.

Tumor-Promoting Role of GNA14 in Colon Cancer Development.

Recent studies have shown that mutations in members of the G-protein α family contribute to the onset and progression of cancer. However, the role of GNA14 in CRC remains unknown. In this study, we examined the effect of GNA14 on CRC through genetic approaches in vitro and in vivo. We found that GNA14 knockdown by small interfering RNA (siRNA) inhibited the proliferation of CRC cells SW403 and HT29. Gna14 knockout mice developed normally without obvious abnormalities. However, the number of polyps in the small intestine was significantly reduced in Gna14 knockout mice compared to control mice after mating with ApcMin mice, a representative CRC mouse model. In particular, deletion of the Gna14 inhibited polyp growth, especially in the distal end of the small intestine. Histological examination showed that Gna14 knockout mice suppressed malignant tumor progression due to decreased proliferation and increased apoptosis in polyps compared to controls. In addition, GNA14 knockdown in CRC cells resulted in downregulation of ERK phosphorylation and β-catenin and β-catenin phosphorylation at S675. Similarly, ERK phosphorylation and phospho-β-catenin phosphorylation at S675 were decreased in polyps of Gna14 knockout mice. Collectively, these analyses show that GNA14 may accelerate CRC cell proliferation and malignant tumor progression through ERK and β-catenin pathways.

Chemoprevention of Colon Cancer by DFMO, Sulindac, and NO-Sulindac Administered Individually or in Combinations in F344 Rats.

Non-steroidal anti-inflammatory drugs (NSAIDs) are promising colorectal cancer (CRC) chemopreventive drugs; however, to overcome NSAIDs' associated side effects, there is a need to develop safer and efficacious approaches. The present study was designed to evaluate (i) the efficacy of nitric-oxide releasing (NO)-Sulindac as compared to Sulindac; (ii) whether NO-Sulindac is superior to Sulindac in enhancing low-dose difluoromethylornithine (DFMO)-induced chemopreventive efficacy, and (iii) assessing the key biomarkers associated with colon tumor inhibition by these combinations. In F344 rats, colonic tumors were induced by azoxymethane (AOM). At the adenoma stage (13 weeks post AOM), groups of rats were fed the experimental diets containing 0 ppm, 500 ppm DFMO, 150 ppm Sulindac, and 200 ppm NO-Sulindac, individually or in combinations, for 36 weeks. Colon tumors were evaluated histopathologically and assayed for expression levels of proliferative, apoptotic, and inflammatory markers. Results suggest that (except for NO-Sulindac alone), DFMO, Sulindac individually, and DFMO combined with Sulindac or NO-Sulindac significantly suppressed AOM-induced adenocarcinoma incidence and multiplicities. DFMO and Sulindac suppressed adenocarcinoma multiplicity by 63% (p < 0.0001) and 51% (p < 0.0011), respectively, whereas NO-Sulindac had a modest effect (22.8%, p = 0.09). Combinations of DFMO plus Sulindac or NO-Sulindac suppressed adenocarcinoma incidence (60%, p < 0.0001; 50% p < 0.0004), and multiplicity (81%, p < 0.0001; 62%, p < 0.0001). Rats that were fed the combination of DFMO plus Sulindac showed significant inhibition of tumor cell proliferation and induction of apoptosis. In addition, enhancement of p21, Bax, and caspases; downregulation of Ki-67, VEGF, and β-catenin; and modulation of iNOS, COX-2, and ODC activities in colonic tumors were observed. These observations show that a lower-dose of DFMO and Sulindac significantly enhanced CRC chemopreventive efficacy when compared to NO-Sulindac alone, and the combination of DFMO and NO-Sulindac was modestly efficacious as compared to DFMO alone.

VAMP8 suppresses the metastasis via DDX5/β-catenin signal pathway in osteosarcoma

ABSTRACT Osteosarcoma is a highly metastatic malignant bone tumor, necessitating the development of new treatments to target its metastasis. Recent studies have revealed the significance of VAMP8 in regulating various signaling pathways in various types of cancer. However, the specific functional role of VAMP8 in osteosarcoma progression remains unclear. In this study, we observed a significant downregulation of VAMP8 in osteosarcoma cells and tissues. Low levels of VAMP8 in osteosarcoma tissues were associated with patients’ poor prognosis. VAMP8 inhibited the migration and invasion capability of osteosarcoma cells. Mechanically, we identified DDX5 as a novel interacting partner of VAMP8, and the conjunction of VAMP8 and DDX5 promoted the degradation of DDX5 via the ubiquitin-proteasome system. Moreover, reduced levels of DDX5 led to the downregulation of β-catenin, thereby suppressing the epithelial–mesenchymal transition (EMT). Additionally, VAMP8 promoted autophagy flux, which may contribute to the suppression of osteosarcoma metastasis. In conclusion, our study anticipated that VAMP8 inhibits osteosarcoma metastasis by promoting the proteasomal degradation of DDX5, consequently inhibiting WNT/β-catenin signaling and EMT. Dysregulation of autophagy by VAMP8 is also implicated as a potential mechanism. These findings provide new insights into the biological nature driving osteosarcoma metastasis and highlight the modulation of VAMP8 as a potential therapeutic strategy for targeting osteosarcoma metastasis.

Azithromycin induces read-through of the nonsense Apc allele and prevents intestinal tumorigenesis in C3B6F1 ApcMin/+ mice

Therapeutic strategies that promote read-through of a mutant gene have proved effective for certain non-neoplastic diseases. However, the efficacy of this approach is unproven regarding neoplastic diseases with germline nonsense mutations, including familial adenomatous polyposis. Here we examined the cancer-preventive efficacy of the macrolide antibiotic azithromycin, with a reported read-through effect, on intestinal tumorigenesis in C3B6F1 ApcMin/+ mice harboring a nonsense Apc mutation resulting in a truncated Apc protein. Mice were given drinking water lacking azithromycin or containing 0.0125–0.2 mg/mL azithromycin from 3 weeks of age. The small intestine and cecum were analyzed for pathological changes and alterations of intestinal flora. Azithromycin suppressed the number of tumors and the proportion of adenocarcinomas, with the most effective drinking-water concentration being 0.0125 mg/mL. Furthermore, azithromycin recovered the cellular level of full-length Apc, resulting in downregulation of β-catenin and cyclin D1. Conversely, the effect of azithromycin on the diversity of the intestinal microbiota depended on the drinking-water concentration. These results suggest that the balance between azithromycin-mediate read-through of mutant Apc mRNA and antibacterial effects influences intestinal tumorigenesis. Thus, azithromycin is a potential anticancer agent for familial adenomatous polyposis patients harboring nonsense mutations.

RETRACTED: Zhou et al. Pyrvinium Treatment Confers Hepatic Metabolic Benefits via β-Catenin Downregulation and AMPK Activation. Pharmaceutics 2021, 13, 330.

The authors and the journal retract the article, 'Pyrvinium Treatment Confers Hepatic Metabolic Benefits via β-Catenin Downregulation and AMPK Activation' [...].

MESP1-knockdown inhibits the proliferation and epithelial mesenchymal transition of hepatocellular carcinoma and enhances the tumor-suppressive effect of 5-fluorouracil

Primary liver hepatocellular carcinoma (HCC) is the third most deadly malignancy worldwide,in part, because it is often diagnosed at an advanced stage. Thus, molecular markers are needed to aid in the early diagnosis and treatment of HCC. Expression of abnormal mesoderm posterior-1 (MESP1) promotes tumorigenesis; however,its role in the regulation of HCC proliferation, apoptosis,and invasion is unknown. Here,we analyzed data in The Cancer Genome Atlas (TCGA)and Genotype Tissue Expression (GTEx) databases on the pan-cancer expression of MESP1 and its relationship with clinical characteristics and prognosis of patients with HCC. The expression of MESP1 was measured in 48 HCC tissues using immunohistochemical staining,and the results were correlated with clinical stage, tumor differentiation, tumor size,and metastasis. MESP1 expression was downregulated using small interfering RNA (siRNA) in the HCC cell lines HepG2 and Hep3B,and cell viability, proliferation,cell cycle, apoptosis,and invasion were analyzed. Finally,we also evaluated the tumor suppression effect of MESP1 downregulation combined with 5-fluorouracil (5-FU) treatment. Our results showed that MESP1 is a pan-oncogene associated with poor prognosis in patients with HCC. siRNA-induced downregulation of MESP1 expression in HepG2 and Hep3B cells exhibited downregulation of β-catenin and GSK3β expression 48h after transfection, along with an increase in apoptosis rate, arrest in the G1-S phase,and a decrease in mitochondrial membrane potential. Moreover,the expression levels of c-Myc, PARP1, bcl2, Snail1, MMP9, and immune checkpoint genes (TIGIT, CTLA4,LAG3,CD274,and PDCD1) were downregulated, while those of caspase3 and E-cadherin were upregulated. Tumor cells also showed decreased migration ability. Furthermore, siRNA interference of MESP1 expression combined with 5-FU-treatment of HCC cells significantly enhanced the G1-S phase block and apoptosis. MESP1 showed an aberrant high expression in HCC and was associated with poor clinical outcomes; therefore, MESP1 may be a potential target for the diagnosis and treatment of HCC.

Inhibition of stemness and EMT by taxifolin ruthenium-p-cymene complex via downregulating the SOX2 and OCT4 expression on lung cancer

Lung carcinoma is perhaps the most often reported cancer incidence throughout the world. The flavonoid metal complexes have exhibited a potential chemotherapeutic effect. This study investigated the chemotherapeutic effect of taxifolin ruthenium-p-cymene complex against lung carcinoma through MTT assay, transwell migration assay, sphere formation assay, western blot, histopathology, immunohistochemistry, and TUNEL assay. The in silico study determined the target proteins of the complex. The synthesized organometallic complex was characterized via various spectroscopical techniques. The complex exhibited strong binding affinity for PI3K, EGFR, and β-catenin. The complex promoted apoptosis and inhibited the colony formation of the cancer cells. Moreover, the cancer cell migration and cancer stemness were reduced with decreased SOX2 and OCT4 expression. The complex induced cell cycle arrest at sub G0/G1 phase, S phase and G2/M phase and promoted caspase-3 mediated apoptosis. The in vivo study demonstrated successful restoration of lung tissue due to the treatment with the complex in benzo-α-pyrene induced lung cancer model. Additionally, the expression of p53 and caspase-3 was increased with significant downregulation of Akt, mTOR and β-catenin. In conclusion, the complex inhibited cancer cell viability, stemness, and EMT in both in vitro and in vivo model systems through the alteration of PI3K/Akt/mTOR/EGFR pathway and expression of stem cell markers including SOX2 and OCT4 that eventually abrogated the EMT mediated metastasis of cancer cells.

Endothelial TREM-1 receptor regulates the blood-brain barrier integrity after intracerebral hemorrhage in mice via SYK/β-catenin signaling.

Intracerebral hemorrhage (ICH) is a high mortality and disability stroke subtype. Destruction of the blood-brain barrier (BBB) is a crucial contributor to brain edema and neurological deficit after ICH. Triggering receptor expressed on myeloid cells 1 (TREM-1) has been reported to be expressed in endothelial cells, but its role in ICH remains unclear. This study aims to evaluate the role of TREM-1 on BBB permeability after ICH in mice. Two hundred and forty-two CD1 mice were used in this study. The ICH model was established by collagenase injection. LP17 was administered intranasally at 2 or 8 h after ICH to inhibit TREM-1. To explore the underlying mechanism, SYK activation CRISPR was administered intracerebroventricularly with LP17, and Anti-mouse TREM-1 rat IgG2a (a specific TREM-1 agonist) was injected intracerebroventricularly with R406 (a specific SYK inhibitor) intraperitoneally. Neurobehavioral outcome, brain water content, BBB permeability, and protein expression were evaluated. The expression level of the TREM-1 receptor increased rapidly as early as 6 h after ICH, and it was mainly expressed on the endotheliocytes in the neurovascular unit. Early and delayed administration of LP17 significantly decreased brain edema and improved neurobehavioral outcomes at 24 h after ICH. LP17 reduced the BBB permeability by increasing β-catenin, claudin-5 and ZO-1 expression. Furthermore, SYK activation CRISPR abolished the beneficial effect of LP17 on the expression of the above junction molecules. Meanwhile, R406 reversed the impact of the TREM-1 activator on the downregulation of β-catenin, claudin-5 and ZO-1 expression. This study demonstrated that TREM-1 deteriorated BBB permeability via modulating the expression of interendothelial junction molecules after ICH, and this regulation is partly mediated by the SYK/β-catenin signaling pathway.

SH3 domain-binding kinase 1 promotes proliferation and inhibits apoptosis of cervical cancer via activating the Wnt/β-catenin and Raf/ERK1/2 signaling pathways.

SH3 domain-binding kinase 1 (SBK1), is a member of the serine/threonine protein kinases family, and was confirmed to be upregulated in cervical cancer in our previous study. Nonetheless, the role of SBK1 in regulating cancer occurrence and development is unclear. In this study, the stable SBK1-knockdown and -overexpressed cell models were constructed by plasmid transfection technology. Cell viability and growth were assessed through CCK-8, colony formation, and BrdU methods. Cell cycle and apoptosis were analyzed by flow cytometry. The JC-1 staining assay was used to explore mitochondrial membrane potential. The scratch and Transwell assays were used to evaluate the cell metastatic ability. The nude mice models were utilized to explore the SBK1 expression affecting tumor growth in vivo. Our research indicated a high expression of SBK1 both in tissues and cells of cervical cancer. The proliferative, migratory, as well as invasive capacities of cervical cancer cells, were suppressed, and apoptosis was enhanced after SBK1 silence, whereas SBK1 upregulation led to opposite results. In addition, Wnt/β-catenin and Raf/ERK1/2 pathways were activated by SBK1 upregulation. Furthermore, downregulation of c-Raf or β-catenin, reversed the proliferation promotion and apoptosis inhibition effects in SBK1-overexpressed cells. The same results were observed with the use of the specific Raf inhibitor. SBK1 overexpression also contributed to tumor growth in vivo. Overall, SBK1 played a vital role in cervical tumorigenesis via activating the Wnt/β-catenin and Raf/ERK1/2 pathways.

Galectin-3 Mediates Tumor Progression in Astrocytoma by Regulating Glycogen Synthase Kinase-3β Activity

Numerous studies have considered galectin-3 or Glycogen synthase kinase 3 beta (GSK3B) as a potential prognosis marker for various cancers. However, the correlation between the protein expression of galectin-3/GSK3B and the clinical parameters of astrocytoma has not been reported. This study aims to validate the correlation between the clinical outcomes and protein expression of galectin-3/GSK3B in astrocytoma. Immunohistochemistry staining was performed to detect galectin-3/GSK3B protein expression in patients with astrocytoma. The Chi-square test, Kaplan-Meier evaluation, and Cox regression analysis were used to determine the correlation between clinical parameters and galectin-3/GSK3B expression. Cell proliferation, invasion, and migration were compared between a non-siRNA group and a galectin-3/GSK3B siRNA group. Protein expression in galectin-3 or GSK3B siRNA-treated cells was evaluated using western blotting. Galectin-3 and GSK3B protein expression were significantly positively correlated with the World Health Organization (WHO) astrocytoma grade and overall survival time. Multivariate analysis revealed that WHO grade, galectin-3 expression, and GSK3B expression were independent prognostic factors for astrocytoma. Galectin-3 or GSK3B downregulation induced apoptosis and decreased cell numbers, migration, and invasion. siRNA-mediated gene silencing of galectin-3 resulted in the downregulation of Ki-67, cyclin D1, VEGF, GSK3B, p-GSK3B Ser9 (p-GSK3B S9), and β-catenin. In contrast, GSK3B knockdown only decreased Ki-67, VEGF, p-GSK3B S9, and β-catenin protein expression but did not affect cyclin D1 and galectin-3 protein expression. The siRNA results indicated that GSK3B is downstream of the galectin-3 gene. These data support that galectin-3 mediated tumor progression by upregulating GSK3B and β-catenin protein expression in glioblastoma. Therefore, galectin-3 and GSK3B are potential prognostic markers, and their genes may be considered to be anticancer targets for astrocytoma therapy.

Abstract 2504: Antiarrhythmic drugs repositioning to counteract the activated phenotype of prostate cancer-associated fibroblasts

Abstract The interplay between cancer cells and adjacent stroma is fundamental to sustain carcinogenesis and promote the acquisition of additional malignant features. Cancer-associated fibroblasts (CAFs) have been shown to fuel prostate cancer (PCa) development and metastasis by mutually interacting with tumor cells. Comparative gene expression profile analysis of CAFs and normal fibroblasts, showed that CAFs are characterized by a positive enrichment of gene sets that regulate cytoskeleton remodeling and muscle contractility, including genes coding for calcium, potassium and sodium channels. Here, for the first time, we investigated the effects of cation channel inhibitors, currently used as antiarrhythmic drugs, on CAF-activated state and CAF-PCa cell interplay. Our results showed that antiarrhythmic drugs are able to interfere with crucial features of reactive prostate CAFs, as indicated by changes in cell morphology and modulation of specific fibroblast activation markers, such as α-SMA, fibroblast activation protein and collagen type I. In addition, although to a variable extent, antiarrhythmics reduced CAF motility and hindered CAF capability to remodel extracellular matrix, as assessed by 3D gel contraction and scratch assays. Interestingly, conditioned medium and co-culture experiments reveled that CAFs were able to promote cell growth and induce epithelial to mesenchymal transition (EMT) in PCa cells, as indicated by E-cadherin and β-catenin down-regulation and Snail and Vimentin up-regulation. Interestingly, antiarrhythmics partially reverted CAF-mediated protumoral effects, abrogating their ability to foster PCa cell proliferation and plasticity. Moreover, based on previous evidence indicating the involvement of matrix metalloproteinase (MMP) in CAF-induced EMT on PCa cells, we found that antiarrhythmics impaired the secretory phenotype of CAFs, as indicated by the reduction of MMP-2 releasing in the medium. Finally, gene expression analysis highlighted that antiarrhythmic-treated CAFs showed a gene expression pattern resembling that of normal fibroblasts. Consistent with our in vitro findings, in vivo experiments confirmed that intra-tumoral administration of medium form antiarrhythmic-treated CAF reduced tumor growth rate of PCa xenografts compared to medium form untreated CAFs. Overall, cation channels inhibition interferes with crucial features of prostate CAFs, affecting their activated state. Moreover, co-culture experiments indicate that antiarrhythmics are able to impair CAF-induced proliferative spur and EMT in PCa cells, and also induce tumor growth delay in vivo. Collectively, such data suggest new opportunities for the therapeutic repositioning of antiarrhythmic drugs with the aim of targeting CAFs and hence interrupting their supportive boost in PCa. Citation Format: Valentina Doldi, Monica Tortoreto, Stefano Percio, Paolo Gandellini, Nadia Zaffaroni. Antiarrhythmic drugs repositioning to counteract the activated phenotype of prostate cancer-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2504.

Pyrogallol from Spirogyra neglecta Inhibits Proliferation and Promotes Apoptosis in Castration-Resistant Prostate Cancer Cells via Modulating Akt/GSK-3β/β-catenin Signaling Pathway.

Castration-resistant prostate cancer (CRPC) is an advanced form of prostate cancer associated with poor survival rates. The high proliferation and metastasis rates have made CRPC one of the most challenging types of cancer for medical practitioners and researchers. In this study, the anti-cancer properties and inhibition of CRPC progression by S. neglecta extract and its active constituents were determined using two CRPC cell lines, DU145 and PC3. The ethyl acetate fraction of S. neglecta (SnEA) was obtained using a solvent-partitioned extraction technique. The active constituents of SnEA were then determined using the HPLC technique, which showed that SnEA mainly contained syringic acid, pyrogallol, and p-coumaric acid phenolic compounds. After the determination of cytotoxic properties using the SRB assay, it was found that pyrogallol, but not the other two major compounds of SnEA, displayed promising anti-cancer properties in both CRPC cell lines. SnEA and pyrogallol were then further investigated for their anti-proliferation and apoptotic induction properties using propidium iodide and Annexin V staining. The results showed that SnEA and pyrogallol inhibited both DU145 and PC3 cell proliferation by inducing cell cycle arrest in the G0/G1 phase and significantly decreased the expression of cell cycle regulator proteins (cyclin D1, cyclin E1, CDK-2, and CDK-4, p < 0.001). SnEA and pyrogallol treatments also promoted apoptosis in both types of CRPC cells through significantly downregulating anti-apoptotic proteins (survivin, Bcl-2, and Bcl-xl, p < 0.001) and upregulating apoptotic proteins (cleaved-caspase-9, cleaved-caspase-3 and cleaved-PARP-1, p < 0.001). Mechanistic study demonstrated that SnEA and pyrogallol inactivated the Akt signaling pathway leading to enhancement of the active form of GSK-3β in CRPC cell lines. Therefore, the phosphorylation of β-catenin was increased, which caused degradation of the protein, resulting in a downregulation of β-catenin (unphosphorylated form) transcriptional factor activity. The current results reflect the potential impact of S. neglecta extract and pyrogallol on the management of castration-resistant prostate cancer.

Glutaredoxin 1 protects lens epithelial cells from epithelial-mesenchymal transition by preventing casein kinase 1α S-glutathionylation during posterior capsular opacification

Oxidative stress drives protein S-glutathionylation, which regulates the structure and function of target proteins and is implicated in the pathogenesis of many diseases. Glutaredoxin 1 (Grx1), a cytoplasmic deglutathionylating enzyme, maintains a reducing environment within the cell under various conditions by reversing S-glutathionylation. Grx1 performs a wide range of antioxidant activities in the lens and prevents protein-thiol mixed disulfide accumulation, reducing protein–protein aggregation, insolubilization, and apoptosis of lens epithelial cells. Oxidative stress is related to epithelial–mesenchymal transition (EMT) during posterior capsular opacification (PCO). However, whether Grx1-regulated protein S-glutathionylation plays an essential role in PCO remains unclear. In this study, we revealed that Grx1 expression was decreased in mice following cataract surgery. Furthermore, the absence of Grx1 elevated oxidative stress and protein S-glutathionylation and aggravated EMT in both in vitro and in vivo models. Concurrently, these results could be reversed by Grx1 overexpression. Notably, liquid chromatography–tandem mass spectrometry results showed that casein kinase 1α (CK1α) was susceptible to S-glutathionylation under oxidative stress, and CK1α S-glutathionylation (CK1α-SSG) was mediated at Cys249. The absence of Grx1 upregulated CK1α-SSG, subsequently decreasing the CK1α-induced phosphorylation of β-catenin at Ser45. The consequential downregulation of degradative β-catenin and upregulation of its nuclear translocation activated the Wnt/β-catenin signaling pathway and aggravated EMT. In conclusion, the downregulated expression of Grx1 in mice following cataract surgery aggravated EMT by upregulating the extent of CK1α-SSG. To the best of our knowledge, our study is the first to report how S-glutathionylation regulates CK1α activity under oxidative stress.

Biosynthesized gold nanoparticles using leaf extract of Citrus medica inhibit hepatocellular carcinoma through regulation of the Wnt/β-catenin signaling pathway

Gold nanoparticle (AuNPs) influence the biomedical sciences owing to different potential characteristics and potential features. To date, the bioinspired fabrication of AuNPs is further recognized due to their safety and efficacy. In this study, we synthesized AuNPs using leaf extract of Citrus medica (C. medica) and well-characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and UV–visible analyses. Afterward the selective anticancer effects of biosynthesized AuNPs against HepG2 cells were evaluated by different techniques including MTT, oxidative markers, qPCR, and western blot assays.The results showed that the polyphenols of the C. medica leaf extract was responsible for the bioinspired reduction and stability of the AuNPs with a powder size in the range of 20–50 nm. Additionally, it was seen that biosynthesized AuNPs show selective inhibition on the proliferation of HepG2 (human liver cancer cell line) relative to THLE3 (normal adult liver epithelial cells) via generation of ROS and MDA, reduction of GSH level and SOD activity, and over expression of Bax/Bcl-2 and Caspase-3 mRNA. It was also shown that biosynthesized AuNPs regulate the Wnt signaling pathway through upregulation of phosphorylated GSK-3β known as the inactivated form of GSK-3β and downregulation of β-catenin and Cyclin-D1 at protein level. This study may hold great promise for development of NP-based anticancer agents, although it needs further investigations in vivo.