Activation of the sonic hedgehog (SHh) pathway is required for the growth of numerous tissues and organs and recent evidence indicates that this pathway is often recruited to stimulate growth of ...cancer stem cells (CSCs) and to orchestrate the reprogramming of cancer cells via epithelial mesenchymal transition (EMT). The objectives of this study were to examine the molecular mechanisms by which (‐)‐epigallocatechin‐3‐gallate (EGCG), an active compound in green tea, inhibits self‐renewal capacity of pancreatic CSCs and synergizes with quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables. Our data demonstrated that EGCG inhibited the expression of pluripotency maintaining transcription factors (Nanog, c‐Myc and Oct‐4) and self‐renewal capacity of pancreatic CSCs. Inhibition of Nanog by shRNA enhanced the inhibitory effects of EGCG on self‐renewal capacity of CSCs. EGCG inhibited cell proliferation and induced apoptosis by inhibiting the expression of Bcl‐2 and XIAP and activating caspase‐3. Interestingly, EGCG also inhibited the components of SHh pathway (smoothened, patched, Gli1 and Gli2) and Gli transcriptional activity. Furthermore, EGCG inhibited EMT by inhibiting the expression of Snail, Slug and ZEB1, and TCF/LEF transcriptional activity, which correlated with significantly reduced CSC's migration and invasion, suggesting the blockade of signaling involved in early metastasis. Furthermore, combination of quercetin with EGCG had synergistic inhibitory effects on self‐renewal capacity of CSCs through attenuation of TCF/LEF and Gli activities. Since aberrant SHh signaling occurs in pancreatic tumorigenesis, therapeutics that target SHh pathway may improve the outcomes of patients with pancreatic cancer by targeting CSCs.
Triple-negative breast cancer (TNBC) is a subtype of breast cancer with high mortality and poor prognosis. Meanwhile, doxorubicin, a chemotherapeutic agent for triple-negative breast cancer, has poor ...sensitivity. The objective of this study was to examine the effect of cordycepin on doxorubicin sensitivity and efficacy in the TNBC xenograft model and explore the relevant molecular pathways. The combination of the drugs in nude mice carrying MDA-MB-231 xenografts significantly reduced the volume, size, and weight of xenografts and improved the tumor inhibition rate. The drug combination was significantly more effective than cordycepin or doxorubicin alone, reflecting the fact that cordycepin enhanced the anti-tumor effects of doxorubicin in MDA-MB-231 xenografts. At the same time, the monitoring of several biological parameters failed to detect any obvious side effects associated with this treatment. After predicting the importance of the TNF pathway in inhibiting tumor growth using network pharmacology methods, we verified the expression of TNF pathway targets via immunohistochemistry and quantitative PCR. Furthermore, a TNF-α inhibitor was able to abrogate the beneficial effects of cordycepin and doxorubicin treatment in MDA-MB-231 cells. This clearly indicates the role of TNF-α, or related molecules, in mediating the therapeutic benefits of the combined treatment in animals carrying TNBC xenografts. The observations reported here may present a new direction for the clinical treatment of TNBC.
Recent evidence from in vitro and in vivo studies has demonstrated that aberrant reactivation of the Sonic Hedgehog (SHH) signaling pathway regulates genes that promote cellular proliferation in ...various human cancer stem cells (CSCs). Therefore, the chemotherapeutic agents that inhibit activation of Gli transcription factors have emerged as promising novel therapeutic drugs for pancreatic cancer. GDC-0449 (Vismodegib), orally administrable molecule belonging to the 2-arylpyridine class, inhibits SHH signaling pathway by blocking the activities of Smoothened. The objectives of this study were to examine the molecular mechanisms by which GDC-0449 regulates human pancreatic CSC characteristics in vitro.
GDC-0499 inhibited cell viability and induced apoptosis in three pancreatic cancer cell lines and pancreatic CSCs. This inhibitor also suppressed cell viability, Gli-DNA binding and transcriptional activities, and induced apoptosis through caspase-3 activation and PARP cleavage in pancreatic CSCs. GDC-0449-induced apoptosis in CSCs showed increased Fas expression and decreased expression of PDGFRα. Furthermore, Bcl-2 was down-regulated whereas TRAIL-R1/DR4 and TRAIL-R2/DR5 expression was increased following the treatment of CSCs with GDC-0449. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GDC-0449-treated pancreatic CSCs. Thus, activated Gli genes repress DRs and Fas expressions, up-regulate the expressions of Bcl-2 and PDGFRα and facilitate cell survival.
These data suggest that GDC-0499 can be used for the management of pancreatic cancer by targeting pancreatic CSCs.
Mutations in the genes encoding nuclear factor (erythroid-derived 2)-like 2 (NRF2), Kelch-like ECH-associated protein 1 (KEAP1), and cullin 3 (CUL3) are commonly observed in human esophageal squamous ...cell carcinoma (ESCC) and result in activation of the NRF2 signaling pathway. Moreover, hyperactivity of the transcription factor Nrf2 has been found to cause esophageal hyperproliferation and hyperkeratosis in mice. However, the underlying mechanism is unclear. In this study, we aimed to understand the molecular mechanisms of esophageal hyperproliferation in mice due to hyperactive Nrf2. Esophageal tissues were obtained from genetically modified mice that differed in the status of the Nrf2 gene and genes in the same pathway (Nrf2−/−, Keap1−/−, K5Cre;Pkm2fl/fl;Keap1−/−, and WT) and analyzed for metabolomic profiles, Nrf2 ChIP-seq, and gene expression. We found that hyperactive Nrf2 causes metabolic reprogramming and up-regulation of metabolic genes in the mouse esophagus. One of the glycolysis genes encoding pyruvate kinase M2 (Pkm2) was not only differentially up-regulated, but also glycosylated and oligomerized, resulting in increased ATP biosynthesis. However, constitutive knockout of Pkm2 failed to inhibit this esophageal phenotype in vivo, and this failure may have been due to compensation by Pkm1 up-regulation. Transient inhibition of NRF2 or glycolysis inhibited the growth of human ESCC cells in which NRF2 is hyperactive in vitro. In summary, hyperactive Nrf2 causes metabolic reprogramming in the mouse esophagus through its transcriptional regulation of metabolic genes. Blocking glycolysis transiently inhibits cell proliferation and may therefore have therapeutically beneficial effects on NRF2high ESCC in humans.
We previously found that cordycepin inhibits the growth and metastasis formation of MDA-MB-231 cells through the Hedgehog pathway but has not validated this in vivo. In this study, we confirmed ...cordycepin’s anti-triple-negative breast cancer (TNBC) effect in nude mice and documented its mechanism. We found that cordycepin reduced the volume and weight of MDA-MB-231 xenografts and affected the expression of proliferation-, apoptosis-, epithelial–mesenchymal transition-, and matrix metalloproteinase-related proteins without side effects. RNA sequencing screening, pathway enrichment, and the protein network interaction analysis revealed enriched pathways and targets mainly concentrated on the Hedgehog pathway and its core components of SHH and GLI2. This indicates that the Hedgehog pathway plays a central role in the cordycepin-mediated regulation of growth and metastasis formation in TNBC. The database analysis of the Hedgehog pathway markers (SHH, PTCH1, SMO, GLI1, and GLI2) revealed that the Hedgehog pathway is activated in breast cancer tissues, and its high expression is not conducive to a patient’s survival. Finally, we verified that cordycepin effectively inhibited the Hedgehog pathway in TNBC through Western blotting and immunohistochemistry. This study found that cordycepin could regulate the growth and metastasis formation of TNBC through the Hedgehog pathway in vivo, which provides new insights for targeting and treating breast cancer.
Dysregulation of the sonic hedgehog (Shh) signaling pathway has been associated with cancer stem cells (CSC) and implicated in the initiation of pancreatic cancer. Pancreatic CSCs are rare tumor ...cells characterized by their ability to self-renew, and are responsible for tumor recurrence accompanied by resistance to current therapies. The lethality of these incurable, aggressive and invasive pancreatic tumors remains a daunting clinical challenge. Thus, the objective of this study was to investigate the role of Shh pathway in pancreatic cancer and to examine the molecular mechanisms by which sulforaphane (SFN), an active compound in cruciferous vegetables, inhibits self-renewal capacity of human pancreatic CSCs. Interestingly, we demonstrate here that Shh pathway is highly activated in pancreatic CSCs and plays important role in maintaining stemness by regulating the expression of stemness genes. Given the requirement for Hedgehog in pancreatic cancer, we investigated whether hedgehog blockade by SFN could target the stem cell population in pancreatic cancer. In an in vitro model, human pancreatic CSCs derived spheres were significantly inhibited on treatment with SFN, suggesting the clonogenic depletion of the CSCs. Interestingly, SFN inhibited the components of Shh pathway and Gli transcriptional activity. Interference of Shh-Gli signaling significantly blocked SFN-induced inhibitory effects demonstrating the requirement of an active pathway for the growth of pancreatic CSCs. SFN also inhibited downstream targets of Gli transcription by suppressing the expression of pluripotency maintaining factors (Nanog and Oct-4) as well as PDGFRα and Cyclin D1. Furthermore, SFN induced apoptosis by inhibition of BCL-2 and activation of caspases. Our data reveal the essential role of Shh-Gli signaling in controlling the characteristics of pancreatic CSCs. We propose that pancreatic cancer preventative effects of SFN may result from inhibition of the Shh pathway. Thus Sulforaphane potentially represents an inexpensive, safe and effective alternative for the management of pancreatic cancer.
Aberrant reactivation of the Sonic Hedgehog (SHH) signaling pathway promotes prostate cancer (PC) growth and progression by regulating cancer‐related genes through its downstream effectors GLI1 and ...GLI2. Therefore, targeting the SHH‐GLI pathway provides an alternative approach to avoid cancer progression. The aim of this study was to delineate the underlying molecular mechanisms by which GDC‐0449 (a SMO receptor inhibitor) and GANT‐61 (a GLI transcription factor inhibitor) regulate cellular proliferation and self‐renewal in human PC stem cells (ProCSCs). Inhibition of the SHH signaling pathway by GANT‐61 induced apoptosis with more efficacy than by GDC‐0449 in ProCSCs and PC cell lines. GLI1 and GLI2 expression, promoter‐binding activity and GLI‐responsive luciferase reporter activity were all decreased with either GDC‐0449 or GANT‐61 treatment. Expression of Fas, DR4, DR5, and cleavage of caspase‐3 and PARP were increased, whereas levels of PDGFR‐α and Bcl‐2 were reduced. Double knockout of GLI1 and GLI2 using shRNA abolished the effects observed with either GDC‐0449 or GANT‐61 treatment. Collectively, our results showed that GANT‐61 and GDC‐0449 induced ProCSC apoptosis by directly or indirectly inhibiting the activities of the GLI family transcription factors, may enhance the efficacy of PC treatment.
We demonstrated that GDC‐0449 and GANT‐61 inhibited prostate cancer stem cell (ProCSC) proliferation by inducing apoptotic genes through the SHH signaling pathway. Moreover, we showed that GLI1/GLI2 played a significant role in this regulation, as double knockdown of GLI1/GLI2 in ProCSCs almost completely eliminated the anti‐proliferative/pro‐apoptotic effects by GANT‐61 and GDC‐0449. Although GDC‐0449 has been approved by the FDA and EMA for treating breast cancer patients and has shown promising preclinical tumor suppressing effects in multiple other cancers, much less information has been reported for GANT‐61 function in cancer. Also, this is the first study demonstrating the effect of these two drugs on ProCSCs. In our study, GANT‐61 exhibited greater efficacy than GDC‐0449 in inhibiting proliferation and inducing apoptosis, providing an alternative targeting approach that may be more effective in therapeutic treatment.
Highlights ► Sonic hedgehog pathway regulates pancreatic cancer stem cell characteristics. ► Inhibition of Gli transcription by GANT-61 suppresses pancreatic cancer stem cell characteristics and ...tumor growth. ► GANT-61 inhibits pluripotency maintaining factors Nanog, Oct-4, Sox-2 and cMyc. ► GANT-61 can be used for the management of pancreatic cancer by targeting pancreatic cancer stem cells.
The forkhead transcription factors of the O class (FOXO) play a direct role in cellular proliferation, oxidative stress response, and tumorigenesis. The objectives of this study were to examine ...whether FOXOs regulate antitumor activities of resveratrol in pancreatic cancer cells in vitro and in vivo.
Pancreatic cancer cell lines were treated with resveratrol. Cell viability, colony formation, apoptosis and cell cycle were measured by XTT, soft agar, TUNEL and flow cytometry assays, respectively. FOXO nuclear translocation, DNA binding and transcriptional activities were measured by fluorescence technique, gelshift and luciferase assay, respectively. Mice were orthotopically implanted with PANC1 cells and orally gavaged with resveratrol. The components of PI3K and ERK pathways, FOXOs and their target gene expressions were measured by the Western blot analysis. Resveratrol inhibited cell viability and colony formations, and induced apoptosis through caspase-3 activation in four pancreatic cancer cell lines (PANC-1, MIA PaCa-2, Hs766T, and AsPC-1). Resveratrol induced cell cycle arrest by up-regulating the expression of p21/CIP1, p27/KIP1 and inhibiting the expression of cyclin D1. Resveratrol induced apoptosis by up-regulating Bim and activating caspase-3. Resveratrol inhibited phosphorylation of FOXOs, and enhanced their nuclear translocation, FOXO-DNA binding and transcriptional activities. The inhibition of PI3K/AKT and MEK/ERK pathways induced FOXO transcriptional activity and apoptosis. Furthermore, deletion of FOXO genes abrogated resveratrol-induced cell cycle arrest and apoptosis. Finally, resveratrol-treated mice showed significant inhibition in tumor growth which was associated with reduced phosphorylation of ERK, PI3K, AKT, FOXO1 and FOXO3a, and induction of apoptosis and FOXO target genes.
These data suggest that inhibition of ERK and AKT pathways act together to activate FOXO transcription factors which are involved in resveratrol-mediated pancreatic tumor growth suppression.
This study explored the therapeutic effects of Auricularia auricula melanin (AAM) on alcoholic liver damage in vitro and in vivo. Human normal liver L02 cells were pre-treated with ethanol and then ...treated with AAM to explore the therapeutic effect of AAM on ethanol-induced hepatocyte injury. The results show that AAM significantly elevated the cell viability, ameliorated the cell morphology, reduced the ROS and increased the GSH/GSSG of ethanol-pretreated L02 cells. Then, mice were administered with ethanol to induce acute alcoholic liver damage, and administered with AAM to further study the therapeutic effect of AAM on alcoholic liver damage in mice. As a result, AAM reduced the levels of ALT, AST, TG, and MDA, increased the levels of ADH, SOD, and CAT in liver damage mice. The therapeutic effect of AAM may be related to inhibition of CYP2E1 expression and activation of Nrf2 and its downstream antioxidase. The research enriched the bioactivity of AAM and provided some ideas for the development of melanin-related health foods.
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