Smad4 regulates TGF-ß1-mediated hedgehog activation to promote epithelial-to-mesenchymal transition in pancreatic cancer cells by suppressing Gli1 activity.

Pancreatic cancer (PC) is an aggressive and metastatic gastrointestinal tumor with a poor prognosis. Persistent activation of the TGF-ß/Smad signaling induces PC cell (PCC) invasion and infiltration via epithelial-to-mesenchymal transition (EMT). Hedgehog signaling is a crucial pathway for the development of PC via the transcription factors Gli1/2/3. This study aimed to investigate the underlying molecular mechanisms of action of hedgehog activation in TGF-ß1-triggered EMT in PCCs (PANC-1 and BxPc-3). In addition, overexpression and shRNA techniques were used to evaluate the role of Smad4 in TGF-ß1-treated PCCs. Our data showed that TGF-ß1 promoted PCC invasion and infiltration via Smad2/3-dependent EMT. Hedgehog-Gli signaling axis in PCCs was activated upon TGF-ß1 stimulation. Inhibition of hedgehog with cyclopamine effectively antagonized TGF-ß1-induced EMT, thereby suggesting that the hedgehog signaling may act as a downstream cascade signaling of TGF-ß1. As a key protein that assists the nuclear translocation of Smad2/3, Smad4 was highly expressed in PANC-1 cells, but not in BxPc-3 cells. Conversely, Gli1 expression was low in PANC-1 cells, but high in BxPc-3 cells. Furthermore, knockdown of Smad4 in PANC-1 cells by shRNA inhibited TGF-ß1-mediated EMT and collagen deposition. Overexpression of Smad4 did not affect TGF-ß1-mediated EMT due to the lack of significant increase in nuclear expression of Smad4. Importantly, Gli1 activity was upregulated by Smad4 knockdown in PANC-1 cells and downregulated by Smad4 overexpression in BxPc-3 cells, indicating that Gli1 may be a negative target protein downstream of Smad4. Thus, Smad4 regulates TGF-ß1-mediated hedgehog activation to promote EMT in PCCs by suppressing Gli1 activity.

S100A2 induces epithelial-mesenchymal transition and metastasis in pancreatic cancer by coordinating transforming growth factor ß signaling in SMAD4-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor and is associated with a poor prognosis. Treatment strategies for PDAC are largely ineffective primarily because of delay in its diagnosis and limited efficacy of systematic treatment. S100A2 is associated with the proliferation, migration, and differentiation of several tumors; however, its effects on PDAC and the associated molecular mechanisms remain to be explored. We studied the mechanisms underlying the effect of S100A2 on epithelial-mesenchymal transition (EMT) and metastasis in PDAC cells. We found that the level of S100A2 remarkably increased and was associated with poor PDAC prognosis. The overexpression of S100A2 in PANC-1 cells also induced EMT, in addition to increasing the invasion and migration of PDAC cells, whereas the knockdown of S100A2 markedly inhibited cell metastasis. Furthermore, S100A2 was found to enhance metastatic abilities in vivo. The overexpression of S100A2 increased SMAD4 expression, whereas the knockdown of S100A2 reduced SMAD4 expression. SMAD4 overexpression could effectively rescue the effects of S100A2 knockdown on EMT. S100A2 mechanistically activated the transforming growth factor (TGF)-ß/Smad2/3 signaling pathway, upregulated SMAD4 expression, induced EMT, and increased PANC-1 cell metastasis. In conclusion, the S100A2/SMAD4 axis modulates EMT to accelerate PDAC development. Our results supplement and enrich the understanding of the pathogenesis underlying PDAC and provide a new theoretical basis and strategy targeting S100A2 for the diagnosis and treatment of PDAC.

STAT3 inhibition enhances gemcitabine sensitivity in pancreatic cancer by suppressing EMT, immune escape and inducing oxidative stress damage.

Pancreatic cancer (PC) is a highly-malignant tumor of the digestive system with a very poor prognosis and high mortality. Chemotherapy and PD-1/PD-L1 immune checkpoint blockade are important treatment strategies for advanced PC. However, chemotherapy resistance and poor therapeutic effect of immune checkpoint inhibitors is are the main clinical problems to be solved urgently at present. The effects of combined application of gemcitabine and STAT3 inhibition on the proliferation, apoptosis, migration, and invasion of PC cells (PCCs) were investigated. In addition, oxidative stress (OS), ferroptosis, immune escape, and the epithelial-mesenchymal transition (EMT) were evaluated. STAT3 inhibition with Stattic enhanced the inhibitory activity of gemcitabine on PCC proliferation by regulating the cell cycle. STAT3 inhibition enhanced mitochondrial-dependent apoptosis in gemcitabine-treated PCCs, but did not induce autophagy and ferroptosis. Further study showed that the anti-proliferative and pro-apoptotic effects may be associated with increased OS damage by inactivating Nrf2-HO-1 signaling, as well as DNA damage by inducing the imbalance between ATM andATR-Chk1 pathway. In addition, STAT3 inhibition strengthened gemcitabine-mediated suppression in PCC invasion and migration by antagonizing Smad2/3-dependent EMT. Moreover, the anti-tumorimmuneresponse of gemcitabine was upregulated by Stattic through reducing the expression of PD-L1 and CD47. Mechanistically, combined application of gemcitabine and Stattic suppressed the phosphorylation and nuclear expression of STAT3. Interestingly, the activities of AKT and ß-catenin signaling were also regulated, suggesting that drug combination has a broad-spectrum signal regulation effect. STAT3 inhibition enhanced the sensitivity of PCCs to the chemotherapy drug gemcitabine by suppressing EMT and immune escape and inducing OS damage.

Diagnostic model constructed by five EMT-related genes for renal fibrosis and reflecting the condition of immune-related cells.

Background: Renal fibrosis is a physiological and pathological characteristic of chronic kidney disease (CKD) to end-stage renal disease. Since renal biopsy is the gold standard for evaluating renal fibrosis, there is an urgent need for additional non-invasive diagnostic biomarkers. Methods: We used R package "limma" to screen out differently expressed genes (DEGs) based on Epithelial-mesenchymal transformation (EMT), and carried out the protein interaction network and GO, KEGG enrichment analysis of DEGs. Secondly, the least absolute shrinkage and selection operator (LASSO), random forest tree (RF), and support vector machine-recursive feature elimination (SVM-RFE) algorithms were used to identify candidate diagnostic genes. ROC curves were plotted to evaluate the clinical diagnostic value of these genes. In addition, mRNA expression levels of candidate diagnostic genes were analyzed in control samples and renal fibrosis samples. CIBERSORT algorithm was used to evaluate immune cells level. Additionally, gene set enrichment analysis (GSEA) and drug sensitivity were conducted. Results: After obtaining a total of 24 DEGs, we discovered that they were mostly involved in several immunological and inflammatory pathways, including NF-KappaB signaling, AGE-RAGE signaling, and TNF signaling. Five genes (COL4A2, CXCL1, TIMP1, VCAM1, and VEGFA) were subsequently identified as biomarkers for renal fibrosis through machine learning, and their expression levels were confirmed by validation cohort data sets and in vitro RT-qPCR experiment. The AUC values of these five genes demonstrated significant clinical diagnostic value in both the training and validation sets. After that, CIBERSORT analysis showed that these biomarkers were strongly associated with immune cell content in renal fibrosis patients. GSEA also identifies the potential roles of these diagnostic genes. Additionally, diagnostic candidate genes were found to be closely related to drug sensitivity. Finally, a nomogram for diagnosing renal fibrosis was developed. Conclusion: COL4A2, CXCL1, TIMP1, VCAM1, and VEGFA are promising diagnostic biomarkers of tissue and serum for renal fibrosis.

Sophocarpine inhibits tumor progression by antagonizing the PI3K/AKT/mTOR signaling pathway in castration-resistant prostate cancer.

Objective: The objective of this study was to investigate the inhibitory effect of sophocarpine on the progression of castration-resistant prostate cancer (CRPC) and the underlying molecular mechanism. Methods: DU145 and PC3 cells (two CRPC cell lines), incubated with different concentrations of sophocarpine, were used. Cell Counting Kit-8 assay, real-time cellular analysis, and colony formation assay were conducted to evaluate the proliferation of CRPC cells. Cytometry flow analysis was performed to evaluate the apoptosis rate of CRPC cells. Wound healing and Transwell invasion assays were performed and the levels of the epithelial-mesenchymal transition (EMT)-related proteins were determined to analyze cell migration and invasion abilities. A xenografted tumor model of nude mice was used to examine the anti-cancer effect of sophocarpine on CRPC. Western blotting was performed to evaluate the activities of the PI3K/AKT/mTOR signaling pathway both in cells and tumor tissues. Results: In vitro tests showed that sophocarpine suppressed the proliferation of CRPC cells, reduced the migration and invasion abilities, and increased the apoptosis rate. In vivo, sophocarpine decreased the weight and volume of tumor tissues. Mechanically, sophocarpine exerted its anti-cancer effects by inactivating PI3K/AKT/mTOR signaling. Conclusion: Sophocarpine inhibited the progression of CRPC by downregulating the PI3K/AKT/mTOR signaling pathway and showed a potential to be an anti-cancer agent against CRPC.

Inhibition of STAT3Y705 phosphorylation by Stattic suppresses proliferation and induces mitochondrial-dependent apoptosis in pancreatic cancer cells.

Patients with pancreatic cancer (PC) show dismal prognosis and high mortality. The development of PC is associated with the overactivation of STAT3. Here, we have determined that the non-peptide small molecule Stattic inhibits PC development by targeting STAT3. In vitro, Stattic treatment time- and dose-dependently inhibited proliferation of pancreatic cancer cells (PCCs) by reducing c-Myc expression and enhancing p53 activity. Consequently, p-Rb, cyclin D1, Chk1, and p21 (cell cycle proteins) were downregulated, and PCCs were arrested at the G1 phase, which was also confirmed by decreased Ki67 expression and unaltered PCNA expression. In addition, Stattic-induced mitochondrial-dependent apoptosis by elevating cleaved caspase-3, and Bax, cytochrome C levels, while reducing expression of Bcl-2, which may be regulated by reduced survivin expression. Further studies showed that Stattic exerts its anti-tumor effect via inhibition of STAT3Y705 phosphorylation and nuclear localization in PCCs. In a nude mouse tumorigenesis model, Stattic inhibited PC growth by antagonizing STAT3Y705 phosphorylation. Interleukin-6 used as a molecule agonist to activate STAT3, as well as overexpression of STAT3, could partially reverse Stattic-mediated anti-proliferation and pro-apoptotic effects of PCCs. Thus, these findings indicate that inhibition of STAT3Y705 phosphorylation by Stattic suppresses PCC proliferation and promotes mitochondrial-mediated apoptosis.

The anthelmintic drug niclosamide induces GSK-ß-mediated ß-catenin degradation to potentiate gemcitabine activity, reduce immune evasion ability and suppress pancreatic cancer progression.

Niclosamide, a cell-permeable salicylanilide, was approved by the Food and Drug Administration for its anthelmintic efficiency. A growing body of evidence in recent years suggests that niclosamide exhibits potential tumor-suppressive activity. However, the role and molecular mechanism of niclosamide in pancreatic cancer remain unclear. In this study, niclosamide inhibited proliferation of pancreatic cancer cells (PCCs), induced apoptosis via the mitochondrial-mediated pathway, and suppressed cell migration and invasion by antagonizing epithelial-to-mesenchymal transition. Also, niclosamide inhibited tumor growth and metastasis in pancreatic cancer xenograft mouse models. Mechanistically, niclosamide exerted these therapeutic effects via targeting ß-catenin. Niclosamide did not reduce ß-catenin mRNA expression in PCCs, but significantly downregulated its protein level. Moreover, niclosamide induced ß-catenin phosphorylation and protein degradation. Interestingly, niclosamide also induced GSK-3ß phosphorylation, which is involved in the ubiquitination degradation of ß-catenin. Pharmacological activation of ß-catenin by methyl vanillate and ß-catenin overexpression abolished the inhibitory effects of niclosamide. Furthermore, niclosamide potentiated the antitumor effect of the chemotherapy drug gemcitabine and reduced the ability of cancer immune evasion by downregulating the expression levels of PD-L1, which is involved in T cell immunity. Thus, our study indicated that niclosamide induces GSK-ß-mediated ß-catenin degradation to potentiate gemcitabine activity, reduce immune evasion ability, and suppress pancreatic cancer progression. Niclosamide may be a potential therapeutic candidate for pancreatic cancer.

The isoflavone puerarin exerts anti-tumor activity in pancreatic ductal adenocarcinoma by suppressing mTOR-mediated glucose metabolism.

Puerarin (8-(ß-D-glucopyranosyl)-4', 7-dihydroxyisoflavone), a natural flavonoid compound isolated from the traditional Chinese herb Radix puerariae, have been demonstrated has potential anti-tumor effects via induction of apoptosis and inhibition of proliferation. However, the effect and molecular mechanism of puerarin in pancreatic ductal adenocarcinoma (PDAC) remains unknown. In this study, the tumor-suppressive effects of puerarin were determined by both in-vitro and in-vivo assays. The effects of puerarin on the proliferation, apoptosis, migration and invasion of pancreatic cancer cells (PCCs), and tumor growth and metastasis in PDAC xenograft mouse model were performed. Puerarin treatment significantly repressed PCC proliferation. Puerarin induced the mitochondrial-dependent apoptosis of PCCs by causing a Bcl-2/Bax imbalance. Moreover, puerarin inhibited PCC migration and invasion by antagonizing epithelial-mesenchymal transition (EMT). In nude mouse model, PDAC growth and metastasis were reduced by puerarin administration. Mechanistically, puerarin exerted its therapeutic effects on PDAC by suppressing Akt/mTOR signaling. Importantly, puerarin bound to the kinase domain of mTOR protein, affecting the activity of the surrounding amino acid residues associated with the binding of the ATP-Mg2+ complex. Further studies showed that the inhibitory effects of puerarin on PCCs were abolished by a mTOR activator, indicating a crucial role of mTOR in anti-tumor effects of puerarin in PDAC. As a result, puerarin hindered glucose uptake and metabolism by downregulating the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) dependent upon HIF-1α and glucose transporter GLUT1. Therefore, these findings indicated that puerarin has therapeutic potential for the treatment of PDAC by suppressing glucose uptake and metabolism via Akt/mTOR activity.

Inhibition of JAK2/STAT3 signaling pathway by panaxadiol limits the progression of pancreatic cancer.

Pancreatic cancer is the fourth leading cause of cancer-related death with the characteristics of chemoresistance and early metastasis. Panaxadiol, a triterpenoid saponin extracted from the roots of American ginseng, has been proved to display anti-tumor activity in colon cancer. In this study, we found panaxadiol significantly inhibited proliferation, and induced apoptosis in human pancreatic cancer cell lines PANC-1 and Patu8988 in a dose-dependent manner. Furthermore, the expression of apoptosis-related proteins (Bax, Bcl2, Cleaved-caspase3) was detected via western blot and immunofluorescence staining. In addition, panaxadiol was also found to inhibit the migration of pancreatic cancer cells by wound healing and transwell assays. In vivo, the growth of xenograft pancreatic cancer models was also notably suppressed by panaxadiol compared to the control group. Moreover, the down-regulation of JAK2-STAT3 signaling pathway was responsible for the underlying pro-apoptosis mechanism of panaxadiol, and this result was in good agreement with molecular docking analysis between panaxadiol and STAT3. In conclusion, our work comprehensively explored the anti-tumor ability in PANC-1 and Patu8988 cells of panaxadiol and provided a potential choice for the clinical treatment of pancreatic cancer patients.

The anti-dysenteric drug fraxetin enhances anti-tumor efficacy of gemcitabine and suppresses pancreatic cancer development by antagonizing STAT3 activation.

Fraxetin, a natural product isolated and purified from the bark of Fraxinus bungeana A.DC., has anti-inflammatory, analgesic, and anti-dysenteric activities. This study aimed to investigate the anti-tumor effects of fraxetin in pancreatic ductal adenocarcinoma (PDA). The effects of fraxetin on the malignant biological behavior of PDA were evaluated. Besides, the effects of fraxetin on the sensitivity of PCCs to gemcitabine, angiogenesis, the epithelial-mesenchymal transition (EMT), glucose metabolism, reactive oxygen species (ROS), and STAT3 activity were analyzed. By reversing the EMT, fraxetin suppressed proliferation, invasion, and migration, and induced mitochondrial-dependent apoptosis in PCCs. Also, treatment with fraxetin inhibited PDA growth and metastasis in nude mouse models. Furthermore, fraxetin made PCCs more sensitive to the chemotherapy drug gemcitabine. Mechanically, fraxetin treatment suppressed oncogenic KRAS-triggered STAT3 activation in PCCs and PDA tissues. Fraxetin shows significant interactions with STAT3 Src Homology 2 (SH2) domain residues, thereby preventing its homo-dimer formation, which then blocks the activation of downstream signal pathways. The anti-tumor activity of fraxetin in PDA was functionally rescued by a STAT3 activator colivelin. As a result, fraxetin hindered hypoxia-induced angiogenesis by decreasing HIF-1α and VEGFA expression, controlled glucose metabolism by reducing GLUT1 expression, inhibited the EMT by blocking the Slug-E-cadherin axis, and drove ROS-mediated apoptosis by regulating the STAT3-Ref1 axis. In conclusion, fraxetin enhances the anti-tumor activity of gemcitabine and suppresses pancreatic cancer development by antagonizing STAT3 activation.

Inhibition of proliferation-linked signaling cascades with atractylenolide I reduces myofibroblastic phenotype and renal fibrosis.

Renal fibrosis is a frequent axis contributing to the occurrence of end-stage nephropathy. Previously, it has been reported that atractylenolide Ⅰ (ATL-1), a natural compound extracted from Atractylodes macrocephala, has anti-cancer and antioxidant effects. However, the renal anti-fibrotic effects of action remain unclear. In this study, the anti-fibrotic effects of ATL-1 were examined in fibroblasts, tubular epithelial cells (TECs) triggered by TGF-ß1 in vitro, and using a unilateral ureteral obstruction (UUO) mouse model in vivo. We found that ATL-1 represses the myofibroblastic phenotype and fibrosis development in UUO kidneys by targeting the fibroblast-myofibroblast differentiation (FMD), as well as epithelial-mesenchymal transition (EMT). The anti-fibrotic effects of ATL-1 were associated with reduced cell growth in the interstitium and tubules, leading to suppression of the proliferation-linked cascades activity consisting of JAK2/STAT3, PI3K/Akt, p38 MAPK, and Wnt/ß-catenin pathways. Besides, ATL-1 treatment repressed TGF-ß1-triggered FMD and the myofibroblastic phenotype in fibroblasts by antagonizing the activation of proliferation-linked cascades. Likewise, TGF-ß1-triggered excessive activation of the proliferation-linked signaling in TECs triggered EMT. The myofibroblastic phenotype was repressed by ATL-1. The anti-fibrotic and anti-proliferative effects of ATL-1 were linked to the inactivation of Smad2/3 signaling, partially reversing FMD, as well as EMT and the repression of the myofibroblastic phenotype. Thus, the inhibition of myofibroblastic phenotype and fibrosis development in vivo and in vitro through proliferation-linked cascades of ATL-1 makes it a prospective therapeutic bio-agent to prevent renal fibrosis.

Quercetin suppresses pancreatic ductal adenocarcinoma progression via inhibition of SHH and TGF-ß/Smad signaling pathways.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive type of malignant tumor with a poor prognosis and high mortality. Aberrant activation of hedgehog signaling plays a crucial role in the maintenance and progression of PDA. Here, we report that the dietary bioflavonoid quercetin has therapeutic potential for PDA by targeting sonic hedgehog (SHH) signaling. The effects of quercetin on the proliferation, apoptosis, migration, and invasion of pancreatic cancer cells (PCCs) and tumor growth and metastasis in PDA xenograft mouse models were evaluated. Additionally, SHH signaling activity was determined. Quercetin significantly inhibited PCC proliferation by downregulating c-Myc expression. In addition, quercetin suppressed epithelial-mesenchymal transition (EMT) by reducing TGF-ß1 level, which resulted in inhibition of PCC migration and invasion. Moreover, quercetin induced PCC apoptosis through mitochondrial and death receptor pathways. In nude mouse models, PDA growth and metastasis were reduced by quercetin treatment. Mechanically, quercetin exerts its therapeutic effects on PDA by decreasing SHH activity. Interestingly, quercetin-induced SHH inactivation is mainly dependent on Gli2, but not Gli1. Enhance SHH activity by recombinant Shh protein abolished the quercetin-mediated inhibition of PCC proliferation, migration, and invasion. Furthermore, Shh activated TGF-ß1/Smad2/3 signaling and promoted EMT by inducing the expression of Zeb2 and Snail1 that eventually resulted in a partial reversal of quercetin-mediated inhibition of PCC migration and invasion. We conclude that quercetin inhibited the growth, migration, and invasion and induced apoptosis of PCCs by antagonizing SHH and TGF-ß/Smad signaling pathways. Thus, quercetin may be a potential candidate for PDA treatment.