Oleanolic acid inhibits colon cancer cell stemness and reverses chemoresistance by suppressing JAK2/STAT3 signaling pathway

Background:Oleanolic acid(OA),a pentacyclic triterpenoid exhibiting specific anti-cancer properties and highly effective antioxidant activity,was isolated from traditional Chinese medicinal herbs.Conversely,the OA that impacts colon cancer(CC)cells and its underlying mechanisms remain poorly understood.Methods:The cytotoxic effect of OA alone or OA-5-Fluorouracil(5-FU)combination on normal and CC cells was analyzed by methyl thiazolyl diphenyl-tetrazolium bromide(MTT).Then,the impact of OA on CC cell lines(LoVo and HT-29)proliferation and stemness were measured using colon formation and tumorsphere formation assays.Octamer-binding transcription factor 4(Oct4),Prominin-1(CD133),Nanog,and transcription factor SOX-2(SOX2)are cell stemness-related indicators whose expression was assessed usingfluorescence qPCR assay,Western blotting,and immunohistochemistry.The effect of OA on the proliferative potency of CC cells was evaluated using an in vivo model.Results:The stem-like characteristics and clone production of colon cancer cells were markedly reduced by OA alone or in combination with OA-5-FU.Moreover,OA increases the susceptibility of CC cells to 5-FU by blocking the cell stemness-related markers(CD133,Nanog,SOX2,and Oct4)expression levels both in vitro and in vivo,as well as by inactivating the activator of transcription 3(STAT3 signaling)and Janus kinase 2/signal transducer(JAK2).Conclusion:Thesefindings imply that oleanolic acid,both in vitro and in vivo,suppresses the JAK2/STAT3 pathway,which in turn reverses chemoresistance and decreases colon cancer cell stemness.Therefore,by reducing the recommended amount of 5-FU,this strategy may improve chemotherapeutic effectiveness and minimize undesired side effects.

CK2αcauses stemness and chemotherapy resistance in liver cancer through the Hedgehog signaling pathway

Background:Liver cancer is one of the major causes of cancer-related deaths globally.Cancer cell stem-ness and chemotherapy resistance contribute to the high mortality.Although evidence indicates that the alpha subunit of protein kinase 2(CK2α)is involved in several human cancers,its function in liver cancer remains unknown.In the present study,we aimed to elucidate the role of CK2αin liver cancer.Methods:We examined the role of CK2αregulation in stemness and chemotherapy resistance capacity of liver cancer cells.MTT assays,tumor sphere formation assays,RT-PCR,flow cytometry,Western blotting assay,clonogenicity assay,matrigel invasion assay and bioinformatics were conducted in this study.Results:CK2αexpression in the liver cancer tissues was notably upregulated compared with that in the corresponding non-tumorous tissues.The overexpression of CK2αpromoted tumor sphere formation,increased the percentage of CD133(+)and side population cells,caused the resistance of liver cancer cells to 5-FU treatment,increased the expression levels of NANOG,OCT4,SOX2,Gli1 and Ptch1,and enhanced the ability of CD133(+)cell clone formation and invasion.Consistently,the downregulation of CK2αhad the opposite effects.CK2αsilencing inhibited the Hedgehog pathway by reducing the expression of Gli1 and Ptch1.Mechanistically,CK2αregulation on liver cancer cell stemness and chemotherapy resistance was found to be involved in the Hedgehog signaling pathway.Conclusions:Our study may bring some new insights into the occurrence of liver cancer.Furthermore,these findings suggest that targeting CK2αmay be a novel therapeutic strategy for patients with liver cancer.

MIR-448 Regulates MAGEA6/AMPK Signaling Pathway in Hepatocellular Carcinoma Tumor Stem Cells

Objective: To explore the role of miR-448 in regulating MAGEA6/AMPK signaling pathway in the biological study of hepatocellular carcinoma (HCC) tumor stem cells. Methods: Using the database, the hepatocellular carcinoma related expression chips were obtained and the regulatory mirnas of candidate genes were predicted, and the predicted results were analyzed. The effects of miR-448 and MAGEA6 on the pellet formation rate and clone formation rate of hepatocellular carcinoma stem cells were detected by immunofluorescence identification of stem cell markers and light microscope counting method. The effects of miR-448 and MAGEA6 on migration and invasion of hepatocellular carcinoma stem cells were detected by scratch and Transwell assay. Dual luciferase reporter assay to verify whether miR-448 targets MAGEA6. The expression and influence of miR-448 on MAGEA6 and AMPK pathway were detected by qRT-PCR and Western blot. Results: It was found that miR-448 may directly regulate the expression of MAGEA6. Overexpression of miR-448 inhibited the characteristics, proliferation, migration, and invasion of hepatocellular carcinoma stem cells in vitro, as well as the ability of xenograft tumor formation in vivo. However, inhibition of miR-448 showed opposite results. In addition, miR-448 directly targets MAGEA6 and regulates AMPK signaling. Silencing MAGEA6 and adding AMPK activator further verified that miR-448 activated AMPK signaling pathway by targeting MAGEA6, thus affecting characteristics, proliferation, migration and invasion of hepatoma stem cells. Conclusions: Our results reveal that miR-448 activates AMPK signaling pathway by targeting MAGEA6, thereby affecting characteristics, proliferation, migration and invasion of hepatoma stem cells. It is suggested that overexpression of miR-448 may be a new therapeutic strategy for hepatocellular carcinoma.

Metformin targets multiple signaling pathways in cancer

Metformin,an inexpensive and well-tolerated oral agent commonly used in the first-line treatment of type 2 diabetes,has become the focus of intense research as a candidate anticancer agent.Here,we discuss the potential of metformin in cancer therapeutics,particularly its functions in multiple signaling pathways,including AMP-activated protein kinase,mammalian target of rapamycin,insulin-like growth factor,c-Jun N-terminal kinase/mitogen-activated protein kinase(p38 MARK),human epidermal growth factor receptor-2,and nuclear factor kappaB pathways.In addition,cutting-edge targeting of cancer stem cells by metformin is summarized.

Inhibition of self-renewal and differentiation of HT-29 cells-derived cancer stem-like cells by scutellarin via Hedgehog signaling pathway

OBJECTIVE To investigate the inhibitory effect of scutellarin on the self-renewal and differentiation of HT-29 cells-derived cancer stem-like cells(HT-29CSC)in vitro and in vivo,and to explore its mechanism.METHODS The effect of scutellarin on the growth of HT-29CSC was determined by 3D Culture assay.The effect of scutellarin on growth and transformation of HT-29CSC was probed by soft agar colony formation assay.The effect of scutellarin on the differentiation of HT-29CSC was determined by serum induction differentiation assay in vitro.The effects of scutellarin on the expressions of marker gene Lgr5,target gene c-Myc,proliferation gene CK20 and Nanog gene were measured by quantitative real-time RT-PCR.Investigate the effect of scutellarin on the expression of c-Myc,Gli1,and Lgr5 protein by Western blotting.A subcutaneous xenograft model of colon cancer in nude mice was established and administered by intraperitoneal injection.The change of body weight and tumor size of nude mice were observed every two days.Investi⁃gate the effects of scutellarin on the growth of xenograft tumors in nude mice.The expression of CD133,Lgr5,Gli1,Ptch1,c-Myc,Ki67,CK20,Nanog gene in tumors were measured by quantitative real-time RT-PCR.The expression of c-Myc,Gli1,Lgr5,CD133,Ki67 protein were measured by Western blotting.RESULTS Scutellarin can inhibit the growth of HT-29CSC in 3D culture.Compared with the solvent control group,scutellarin can significantly inhibit the growth and transformation and differentiation of HT-29CSC in vitro(P<0.01).The expression levels of marker genes Lgr5,target gene c-Myc,proliferation gene CK20 and Nanog in HT-29CSC were down-regulated by scutellarin.Scutellarin can reduce the expression of c-Myc,Gli1,and Lgr5 protein in HT-29CSC.Scutellarin can inhibit the growth of colon cancer xenografts,lower CD133,Lgr5,Gli1,Ptch1,c-Myc,Ki67,CK20,and Nanog mRNA level of xenograft tumors,reduce the expression of c-Myc,Gli1,Lgr5,CD133,and Ki67 protein of xenograft tumors in nude mice.CONCLUSION Scutellarin,which is the main component of scutellaria barbata,can inhibit the differentiation of HT-29CSC and the mechanism is to inhibit the activity of Hedgehog signaling pathway.

Hedgehog signaling pathway as a new therapeutic target in pancreatic cancer

Pancreatic cancer is one of the most aggressive and difficult cancers to treat.Despite numerous research efforts,limited success has been achieved in the therapeutic management of patients with this disease.In the current review,we focus on one component of morphogenesis signaling,Hedgehog(Hh),with the aim of developing novel,effective therapies for the treatment of pancreatic cancer.Hh signaling contributes to the induction of a malignant phenotype in pancreatic cancer and is responsible for maintaining pancreatic cancer stem cells.In addition,we propose a novel concept linking Hh signaling and tumor hypoxic conditions,and discuss the effects of Hh inhibitors in clinical trials.The Hh signaling pathway may represent a potential therapeutic target for patients with refractory pancreatic cancer.

The p38/MAPK pathway as a therapeutic target to prevent therapeutic escape of breast cancer stem cells

Cancer stem cells(CSCs)play an important role in metastasis development,tumor recurrence,and treatment resistance,and are essential for the eradication of cancer.Currently,therapies fail to eradicate CSCs due to their therapeutic stress-induced cellular escape,which leads to enhanced aggressive behaviors compared with CSCs that have never been treated.However,the underlying mechanisms regulating the therapeutic escape remain unknown.To this end,we established a model to isolate the therapeutic escaped CSCs(TSCSCs)from breast CSCs and performed the transcription profile to reveal the mechanism.Mechanistically,we demonstrated that the behavior of therapeutic escape was regulated through the p38/MAPK signaling pathway,resulting in TSCSCs exhibiting enhanced motility and metastasis.Notably,blocking the p38/MAPK signaling pathway effectively reduced motility and metastasis ability both in vitro and in vivo,which were further supported by downregulated motility-related genes and epithelial-mesenchymal transition(EMT)-related proteins vimentin and N-cadherin.The obtained findings reveal the p38/MAPK pathway as a potential therapeutic target for TSCSCs and would provide profound implications for cancer therapy.

Targeting cancer stem cells in drug discovery: Current state and future perspectives

In recent decades,cancer stem cells(CSCs)have been increasingly identified in many malignancies.CSC-related signaling pathways and their functions provide new strategies for treating cancer.The aberrant activation of related signaling pathways(e.g.,Wnt,Notch,and Hedgehog pathways)has been linked to multiple types of malignant tumors,which makes these pathways attractive targets for cancer therapy.CSCs display many characteristic features,such as self-renewal,differentiation,high tumorigenicity,and drug resistance.Therefore,there is an urgent need to develop new therapeutic strategies to target these pathways to control stem cell replication,survival,and differentiation.Notable crosstalk occurs among different signaling pathways and potentially leads to compensatory escape.Therefore,multitarget inhibitors will be one of the main methods to overcome the drug resistance of CSCs.Many small molecule inhibitors of components of signaling pathways in CSCs have entered clinical trials,and some inhibitors,such as vismodegib,sonidegib,and glasdegib,have been approved.Tumor cells are susceptible to sonidegib and vismodegib resistance due to mutations in the Smo protein.The signal transducers and activators of transcription 3(STAT3)inhibitor BBI608 is being evaluated in a phase III trial for a variety of cancers.Structural derivatives of BBI608 are the main focus of STAT3 inhibitor development,which is another strategy for CSC therapy.In addition to the potential pharmacological inhibitors targeting CSCrelated signaling pathways,other methods of targeting CSCs are available,such as nano-drug delivery systems,mitochondrion targeting,autophagy,hyperthermia,immunotherapy,and CSC microenvironment targeting.In addition,we summarize the latest advances in the clinical development of agents targeting CSC-related signaling pathways and other methods of targeting CSCs.

Cancer stem cells: Involvement in pancreatic cancer pathogenesis and perspectives on cancer therapeutics

Pancreatic cancer is one of the most aggressive and lethal malignancies. Despite remarkable progress in understanding pancreatic carcinogenesis at the molecular level, as well as progress in new therapeutic approaches, pancreatic cancer remains a disease with a dismal prognosis. Among the mechanisms responsible for drug resistance, the most relevant are changes in individual genes or signaling pathways and the presence of highly resistant cancer stem cells(CSCs). In pancreatic cancer, CSCs represent 0.2%-0.8% of pancreatic cancer cells and are considered to be responsible for tumor growth, invasion, metastasis and recurrence. CSCs have been extensively studied as of late to identify specific surface markers to ensure reliable sorting and for signaling pathways identified to play a pivotal role in CSC self-renewal. Involvement of CSCs in pancreatic cancer pathogenesis has also highlighted these cells as the preferential targets for therapy. The present review is an update of the results in two main fields of research in pancreatic cancer, pathogenesis and therapy, focused on the narrow perspective of CSCs.

Emerging agents that target signaling pathways to eradicate colorectal cancer stem cells

Colorectal cancer(CRC)represents the third most commonly diagnosed cancer and the second leading cause of cancer death worldwide.The modern concept of cancer biology indicates that cancer is formed of a small population of cells called cancer stem cells(CSCs),which present both pluripotency and self-renewal properties.These cells are considered responsible for the progression of the disease,recurrence and tumor resistance.Interestingly,some cell signaling pathways participate in CRC survival,proliferation,and selfrenewal properties,and most of them are dysregulated in CSCs,including the Wingless(Wnt)/β-catenin,Notch,Hedgehog,nuclear factor kappa B(NF-κB),Janus kinase/signal transducer and activator of transcription(JAK/STAT),peroxisome proliferator-activated receptor(PPAR),phosphatidyl-inositol-3-kinase/Akt/mechanistic target of rapamycin(PI3K/Akt/mTOR),and transforming growth factor-β(TGF-β)/Smad pathways.In this review,we summarize the strategies for eradicating CRC stem cells by modulating these dysregulated pathways,which will contribute to the study of potential therapeutic schemes,combining conventional drugs with CSC-targeting drugs,and allowing better cure rates in anti-CRC therapy.

γ-Secretase Inhibitor, DAPT Inhibits Self-renewal and Stemness Maintenance of Ovarian Cancer Stem-like Cells In Vitro

Objective: The Notch signaling pathway plays an important role in the stem cell signaling network and contributes to tumorigenesis. However, the functions of Notch signaling in ovarian cancer stem cells (OCSCs) are not well understood. We aimed to investigate the effects of Notch blockade on self-renewal and stemness maintenance of OCSCs. Methods: Ovarian cancer stem-like cells were enriched from ovarian cancer cell lines in serum-free medium. A γ-secretase inhibitor, (DAPT), was used to block Notch signaling. MTT assays were performed to assess self-renewal and proliferation inhibition, flow cytometry was performed to analyze cell surface marker and immunofluorescence, Western Blot and Real-time RT-PCR assays were performed to detect Oct4 and Sox2 protein and mRNA expression of the Ovarian cancer stem-like cells treated with DAPT. Results: Notch blockade markedly inhibits self-renewal and proliferation of ovarian cancer stem-like cells, significantly downregulates the expression of OCSCs-specific surface markers, and reduces protein and mRNA expression of Oct4 and Sox2 in OCSC-like cells. Conclusion: Our results suggest that Notch signaling is not only critical for the self-renewal and proliferation of OCSCs, but also for the stemness maintenance of OCSCs. The γ-secretase inhibitor is a promising treatment targeting OCSCs.

Epithelial-Mesenchymal Transition and Breast Cancer Stem Cells in Breast Cancer Progression

Breast cancer stem cells (BCSCs) are a small subpopulation of cancer cells having the ability of self-renewing and multi-lineage differentiation, which have also been termed as “tumor-initiating cells”. And in recent years, the role of epithelial mesenchymal transition (EMT) in malignant tumors has been valued. This paper will briefly review and discuss the relationship between BCSCs and EMT.

Targeting stem cell signaling pathways for drug discovery:advances in the Notch and Wnt pathways

Signaling pathways transduce extracellular stimuli into cells through molecular cascades to regulate cellular functions.In stem cells,a small number of pathways,notably those of TGF-?/BMP,Hedgehog,Notch,and Wnt,are responsible for the regulation of pluripotency and differentiation.During embryonic development,these pathways govern cell fate specifications as well as the formation of tissues and organs.In adulthood,their normal functions are important for tissue homeostasis and regeneration,whereas aberrations result in diseases,such as cancer and degenerative disorders.In complex biological systems,stem cell signaling pathways work in concert as a network and exhibit crosstalk,such as the negative crosstalk between Wnt and Notch.Over the past decade,genetic and genomic studies have identified a number of potential drug targets that are involved in stem cell signaling pathways.Indeed,discovery of new targets and drugs for these pathways has become one of the most active areas in both the research community and pharmaceutical industry.Remarkable progress has been made and several promising drug candidates have entered into clinical trials.This review focuses on recent advances in the discovery of novel drugs which target the Notch and Wnt pathways.

Novel nervous and multi-system regenerative therapeutic strategies for diabetes mellitus with mTOR

Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy.The mechanistic target of rapamycin（m TOR） is a promising agent for the development of novel regenerative strategies for the treatment of DM.m TOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis,insulin resistance,insulin secretion,stem cell proliferation and differentiation,pancreatic β-cell function,and programmed cell death with apoptosis and autophagy.m TOR is central element for the protein complexes m TOR Complex 1（m TORC1） and m TOR Complex 2（m TORC2） and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase（PI 3-K）,protein kinase B（Akt）,AMP activated protein kinase（AMPK）,silent mating type information regulation 2 homolog 1（Saccharomyces cerevisiae）（SIRT1）,Wnt1 inducible signaling pathway protein 1（WISP1）,and growth factors.As a result,m TOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease.Future studies directed to elucidate the delicate balance m TOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.

Novel molecular targets in hepatocellular carcinoma

Globally,hepatocellular carcinoma(HCC)is a leading cause of cancer and cancerrelated deaths.The therapeutic efficacy of locoregional and systemic treatment in patients with advanced HCC remains low,which results in a poor prognosis.The development of sorafenib for the treatment of HCC has resulted in a new era of molecular targeted therapy for this disease.However,the median overall survival was reported to be barely higher in the sorafenib treatment group than in the control group.Hence,in this review we describe the importance of developing more effective targeted therapies for the management of advanced HCC.Recent investigations of molecular signaling pathways in several cancers have provided some insights into developing molecular therapies that target critical members of these signaling pathways.Proteins involved in the Hedgehog and Notch signaling pathways,Polo-like kinase 1,arginine,histone deacetylases and Glypican-3 can be potential targets in the treatment of HCC.Monotherapy has limited therapeutic efficacy due to the development of inhibitory feedback mechanisms and induction of chemoresistance.Thus,emphasis is now on the development of personalized and combination molecular targeted therapies that can serve as ideal therapeutic strategies for improved management of HCC.

Cancer stem cells and HER2 positive breast cancer:The story so far

Human Epidermal Growth Factor Receptor type 2(HER2)gene amplification and/or protein overexpression is observed in patients suffering from HER2t breast cancer.This subtype of breast cancer has improved prognosis due to availability of anti-HER2 therapy.However,drug resistance and tumor recurrence still remains a major concern.Cancer Stem Cells(CSCs)are believed to constitute the subset of cell population that is resistant to drug treatment and possesses characteristics of stem cells.CSCs enable the tumors to thrive despite major insults.This review provides a comprehensive idea about the concept of CSCs in context of HER2t breast cancer by providing the description of the markers that are used for the identification of CSCs and by elucidating the signaling pathways that are associated with HER2t breast CSCs.Furthermore,the review also describes the interaction of HER2 with those signaling pathways and the future of targeting CSCs in HER2t breast cancer.

Manifestation of Pathological States of Numerous Diseases in the Largest Organ of the Human Body: (II) From Pancreatitis to Pancreatic Cancer Invasion, Formation of Stroma around the Primary Tumor in the Fascia, to Early Detection of Non-Coding microRNAs in Body Fluids and Development of Drugs to Treat Different Stages of Pancreatic Cancer

Patients suffering from pancreatic ductal adenocarcinoma (PDAC) have an average survival time of 4 - 6 months after confirmed diagnosis. The primary tumor is surrounded by a thick interstitial fluid with high pressure and dense distribution of collagen, forming a huge stroma, rendering the tumor resistant to chemo- and radiotherapy. From the genetic point of view, pancreatic carcinogenesis is driven by mutations, resulting in common activation of the oncogene KRAS, and/or inactivation of one or more of the tumor suppressor genes CDKN2A, TP53, SMAD4 [1]. The pancreas is a mixed exocrine and autocrine organ, with different cell types building up the organ. The pathogenesis involves more than 13 signaling pathways at different stages. Off-balance of the function of the proteins in these pathways due to the stated 4 plus other mutations could readily lead to carcinogenesis. We first present the basic mechanism of these 13 relevant pathways. We then provide a detailed analysis of the progression of this disease, from pancreatitis to tumor formation and metastasis, with special attention on the roles played by the newly discover calcium channel Piezo, stellate cells, stem-cell-like cells, and the concept invadopodium. Thirty potential drugs, based on in vitro and xenograft experiments from different groups, are discussed, including vitamins A, Tocotrienols-E, and D, chemical compounds, non-coding micro RNAs, circular RNA, piwi-interacting RNAs. The recent detection of exosomes enclosing many of these RNAs in body fluids gives us hope of developing early detection methodology because these RNAs carry messages for cell-cell communication at a distance. Delivery of potent drugs by nanoparticles gives us chance to send drugs through the stroma to target the tumor. Since body fluids form a circulating system, together with the connective tissues (where the tumor is associated) form the largest organ—the fascia, we conclude that manifestation of successive pathological states of pancreatic carcinogenesis can be found in compartments of the fascia. We present 17 figures, hoping to ease off the complexity of the pathogenesis of this most lethal cancer disease.

OCT4’s role and mechanism underlying oral squamous cell carcinoma

Oral squamous cell carcinoma(OSCC),a common malignancy of the head and neck,ranks sixth worldwide in terms of cancers with the most negative impact,owing to tumor relapse rates,cervical lymphnode metastasis,and the lack of an efficacious systemic therapy.Its prognosis is poor,and its mortality rate is high.Octamer-binding transcription factor 4(OCT4)is a member of the Pit-Oct-Unc(POU)family and is a key reprogramming factor that produces a marked effect in preserving the pluripotency and self-renewal state of embryonic stem cells(ESCs).According to recent studies,OCT4 participates in retaining the survival of OSCC cancer stem cells(CSCs),which has far-reaching implications for the occurrence,recurrence,metastasis,and prognosis of oral carcinogenesis.Therefore,we summarize the structure,subtypes,and function of OCT4 as well as its role in the occurrence,progression,and prognosis of OSCC.