Baicalin Antagonizes Prostate Cancer Stemness via Inhibiting Notch1/NF-κB Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the molecular mechanisms underlying the effect of baicalin on prostate cancer (PCa) progression both in vivo and in vitro.@*METHODS@#The in situ PCa stem cells (PCSCs)-injected xenograft tumor models were established in BALB/c nude mice. Tumor volume and weight were respectively checked after baicalin (100 mg/kg) treatment. Hematoxylin-eosin (HE) staining was used to observe the growth arrest and cell necrosis. mRNA expression levels of acetaldehyde dehydrogenase 1 (ALDH1), CD44, CD133 and Notch1 were determined by reverse transcription-polymerase chain reaction. Protein expression levels of ALDH1, CD44, CD133, Notch1, nuclear factor κB (NF-κB) P65 and NF-κB p-P65 were detected by Western blot. Expression and subcellular location of ALDH1, CD44, CD133, Notch1 and NF-κB p65 were detected by immunofluorescence analysis. In vitro, cell cycle distribution and cell apoptosis of PC3 PCSCs was assessed by flow cytometry after baicalin (125 µmol/L) treatment. The migration and invasion abilities of PCSCs were assessed using Transwell assays. Transmission electron microscopy scanning was utilized to observe the structure and autophagosome formation of baicalin-treated PCSCs. In addition, PCSCs were infected with lentiviruses expressing human Notch1.@*RESULTS@#Compared with the control group, the tumor volume and weight were notably reduced in mice treated with 100 mg/kg baicalin (P<0.05 or P<0.01). Histopathological analysis showed that baicalin treatment significantly inhibited cell proliferation and promoted cell apoptosis. Furthermore, baicalin treatment reduced mRNA and protein expression levels of CD44, CD133, ALDH1, and Notch1 as well as the protein expression of NF-κB p-P65 in the xenograft tumor (P<0.01). In vitro, the cell proliferation of PCSCs was significantly attenuated after treatment with 125 µmol/L baicalin for 72 h (P<0.01). The cell migration and invasion rates were decreased following treatment with baicalin for 48 and 72 h (P<0.01). Baicalin notably induced cell apoptosis and seriously damaged the structure of PCSCs. The mRNA and protein expressions of CD133, CD44, ALDH1 and Notch1 in PCSCs were significantly downregulated following baicalin treatment (P<0.01). Importantly, the inhibitory effects of baicalin on PCa progression and stemness were reversed by Notch1 overexpression (P<0.05 or P<0.01).@*CONCLUSION@#Mechanistically, baicalin exhibited a potential therapeutic effect on PCa via inhibiting the Notch1/NF-κB signaling pathway and its mediated cancer stemness.

Tangeretin inhibits tumor stemness of non-small cell lung cancer by regulating PI3K/AKT/mTOR signaling pathway / 中国胸心血管外科临床杂志

@#Objective    To study the effect of Tangeretin on non-small cell lung cancer (NSCLC) and the tumor stemness, and to find the molecular mechanism of its effect. Methods    We used cell counting and cell cloning experiments to study the effect of Tangeretin on the proliferation of NSCLC cells in vitro. The effect of Tangeretin on the invasion of NSCLC cells was detected by transwell assay. We detected the effect of Tangeretin on the proliferation of NSCLC cells in vivo by nude mouse tumor-bearing experiment. The effect of Tangeretin on tumor stemness of NSCLC cells was detected by self-renew assay, and CD133 and Nanog protein expressions. The expressions of PI3K/AKT/mTOR signaling pathway-related proteins were detected by Western blotting (WB). Results    Tangeretin had a good inhibitory effect on the proliferation of NSCLC cells in vivo and in vitro. Cell counting experiment, clonal formation experiment and nude mouse tumor-bearing experiment showed that Tangeretin could inhibit the proliferation activity, clonal formation ability, and tumor size of NSCLC cells in vivo. Self-renew experiments showed that Tangeretin could inhibit the self-renew ability of NSCLC cells. WB experiments showed that Tangeretin inhibited the expressions of tumor stemness markers CD133 and Nanog in NSCLC cells. Tangeretin could inhibit the activation of PI3K/AKT/mTOR signaling pathway-related proteins in NSCLC cells, and the activation of PI3K/AKT/mTOR signaling pathway could partially remit the inhibitory  effect of Tangeretin on tumor stemness of NSCLC cells. Conclusion    Tangeretin can inhibit the tumor stemness of NSCLC cells, which may be related to the regulation of PI3K/AKT/mTOR signaling pathway.

Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/β-catenin pathway under hypoxia / 中西医结合学报

OBJECTIVE@#Jiedu Recipe (JR), a Chinese herbal remedy, has been shown to prolong overall survival time and decrease recurrence and metastasis rates in patients with hepatocellular carcinoma (HCC). This work investigated the mechanism of JR in HCC treatment.@*METHODS@#The chemical constituents of JR were detected using liquid chromatography-mass spectrometry. The potential anti-HCC mechanism of JR was screened using network pharmacology and messenger ribonucleic acid (mRNA) microarray chip assay, followed by experimental validation in human HCC cells (SMMC-7721 and Huh7) in vitro and a nude mouse subcutaneous transplantation model of HCC in vivo. HCC cell characteristics of proliferation, migration and invasion under hypoxic setting were investigated using thiazolyl blue tetrazolium bromide, wound healing and Transwell assays, respectively. Image-iT™ Hypoxia Reagent was added to reveal hypoxic conditions. Stem cell sphere formation assay was used to detect the stemness. Epithelial-mesenchymal transition (EMT) markers like E-cadherin, vimentin and α-smooth muscle actin, and pluripotent transcription factors including nanog homeobox, octamer-binding transcription factor 4, and sex-determining region Y box protein 2 were analyzed using Western blotting and real-time polymerase chain reaction. Western blot was performed to ascertain the anti-HCC effect of JR under hypoxia involving the Wnt/β-catenin pathway.@*RESULTS@#According to network pharmacology and mRNA microarray chip analysis, JR may potentially act on hypoxia and inhibit the Wnt/β-catenin pathway. In vitro and in vivo experiments showed that JR significantly decreased hypoxia, and suppressed HCC cell features of proliferation, migration and invasion; furthermore, the hypoxia-induced increases in EMT and stemness marker expression in HCC cells were inhibited by JR. Results based on the co-administration of JR and an agonist (LiCl) or inhibitor (IWR-1-endo) verified that JR suppressed HCC cancer stem-like properties under hypoxia by blocking the Wnt/β-catenin pathway.@*CONCLUSION@#JR exerts potent anti-HCC effects by inhibiting cancer stemness via abating the Wnt/β-catenin pathway under hypoxic conditions. Please cite this article as: Guo BJ, Ruan Y, Wang YJ, Xiao CL, Zhong ZP, Cheng BB, Du J, Li B, Gu W, Yin ZF. Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/β-catenin pathway under hypoxia. J Integr Med. 2023; 21(5): 474-486.

Jianpi Yangzheng Xiaozheng Decoction Affect Proliferation and Stemness of Gastric Cancer HGC-27 Cells by Inhibiting Aerobic Glycolysis / 中国实验方剂学杂志

ObjectiveTo observe the effect of Jianpi Yangzheng Xiaozheng decoction （JYXD） on the proliferation and stemness of the human gastric cancer （GC） cell line HGC-27 by inhibiting aerobic glycolysis， and explore the underlying mechanism. MethodMethyl thiazolyl tetrazolium （MTT） assay was employed to determine the survival rate and chemotherapy sensitivity of HGC-27 cells treated with JYXD （0.25， 0.5， 1， 2， 4， 8， 16， 32 g·L-1）. Colony formation assay was employed to detect the effect of JYXD （2， 4， 8 g·L-1） on the colony formation of the cells. The aerobic glycolysis level of HGC-27 cells after treatment with JYXD was measured by glucose assay kit and lactic acid assay kit. The proportion of stem cell subsets in HGC-27 cells was detected by flow cytometry. Western blot was employed to determine the expression of glycolysis-associated proteins such as lactate dehydrogenase （LDH）， hexokinase 2 （HK2）， glucose transporter 1 （GLUT1）， and pyruvate kinase isozyme M2 （PKM2）， and the expression of stemness-associated proteins such as octamer-binding transcription factor 4 （OCT4）， SRY-box transcription factor 2 （SOX2）， and Nanog. ResultJYXD （0.5， 1， 2， 4， 8， 16， 32 g·L-1） inhibited the activity of HGC-27 cells （P<0.05， P<0.01）， with the inhibitory concentration 50（IC50） of 4.83 g·L-1， and it improved the sensitivity of HGC-27 cells to cisplatin chemotherapy. Compared with the control group， JYXD （2， 4， 8 g·L-1） reduced the colony formation number of HGC-27 cells （P<0.01） in a concentration-dependent manner. Flow cytometry showed that compared with that in the control group， the proportion of CD44+CD24+ALDH+ population in the cells treated with JYXD （2， 4， 8 g·L-1） decreased （P<0.05）. In addition， JYXD （2， 4， 8 g·L-1） inhibited the glucose uptake and lactic acid production of HGC-27 cells. Western blot showed that compared with the control group， JYXD （2， 4， 8 g·L-1） down-regulated the expression levels of SOX2， Nanog， OCT4， PKM2， LDH， GLUT1， and HK2 （P<0.05， P<0.01） in a concentration-dependent manner. ConclusionJYXD may inhibit the proliferation and reduce the stemness of HGC-27 cells by regulating the aerobic glycolysis.

Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway / 中西医结合学报

OBJECTIVE@#Physical exercise, a common non-drug intervention, is an important strategy in cancer treatment, including hepatocellular carcinoma (HCC). However, the mechanism remains largely unknown. Due to the importance of hypoxia and cancer stemness in the development of HCC, the present study investigated whether the anti-HCC effect of physical exercise is related to its suppression on hypoxia and cancer stemness.@*METHODS@#A physical exercise intervention of swimming (30 min/d, 5 d/week, for 4 weeks) was administered to BALB/c nude mice bearing subcutaneous human HCC tumor. The anti-HCC effect of swimming was assessed in vivo by tumor weight monitoring, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) detection of proliferating cell nuclear antigen (PCNA) and Ki67. The expression of stemness transcription factors, including Nanog homeobox (NANOG), octamer-binding transcription factor 4 (OCT-4), v-Myc avian myelocytomatosis viral oncogene homolog (C-MYC) and hypoxia-inducible factor-1α (HIF-1α), was detected using real-time reverse transcription polymerase chain reaction. A hypoxia probe was used to explore the intratumoral hypoxia status. Western blot was used to detect the expression of HIF-1α and proteins related to protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway. The IHC analysis of platelet endothelial cell adhesion molecule-1 (CD31), and the immunofluorescence co-location of CD31 and desmin were used to analyze tumor blood perfusion. SMMC-7721 cells were treated with nude mice serum. The inhibition effect on cancer stemness in vitro was detected using suspension sphere experiments and the expression of stemness transcription factors. The hypoxia status was inferred by measuring the protein and mRNA levels of HIF-1α. Further, the expression of proteins related to Akt/GSK-3β/β-catenin signaling pathway was detected.@*RESULTS@#Swimming significantly reduced the body weight and tumor weight in nude mice bearing HCC tumor. HE staining and IHC results showed a lower necrotic area ratio as well as fewer PCNA or Ki67 positive cells in mice receiving the swimming intervention. Swimming potently alleviated the intratumoral hypoxia, attenuated the cancer stemness, and inhibited the Akt/GSK-3β/β-catenin signaling pathway. Additionally, the desmin+/CD31+ ratio, rather than the number of CD31+ vessels, was significantly increased in swimming-treated mice. In vitro experiments showed that treating cells with the serum from the swimming intervention mice significantly reduced the formation of SMMC-7721 cell suspension sphere, as well as the mRNA expression level of stemness transcription factors. Consistent with the in vivo results, HIF-1α and Akt/GSK-3β/β-catenin signaling pathway were also inhibited in cells treated with serum from swimming group.@*CONCLUSION@#Swimming alleviated hypoxia and attenuated cancer stemness in HCC, through suppression of the Akt/GSK-3β/β-catenin signaling pathway. The alleviation of intratumoral hypoxia was related to the increase in blood perfusion in the tumor. Please cite this article as: Xiao CL, Zhong ZP, Lü C, Guo BJ, Chen JJ, Zhao T, Yin ZF, Li B. Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway. J Integr Med. 2023; 21(2): 184-193.

Multiple characteristic alterations and available therapeutic strategies of cellular senescence / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Given its state of stable proliferative inhibition, cellular senescence is primarily depicted as a critical mechanism by which organisms delay the progression of carcinogenesis. Cells undergoing senescence are often associated with the alteration of a series of specific features and functions, such as metabolic shifts, stemness induction, and microenvironment remodeling. However, recent research has revealed more complexity associated with senescence, including adverse effects on both physiological and pathological processes. How organisms evade these harmful consequences and survive has become an urgent research issue. Several therapeutic strategies targeting senescence, including senolytics, senomorphics, immunotherapy, and function restoration, have achieved initial success in certain scenarios. In this review, we describe in detail the characteristic changes associated with cellular senescence and summarize currently available countermeasures.

Effect of Reduning Injection， Shenfu Injection Combined with Gefitinib on Proliferation， Stemness Characteristics and Metabolism of Non-small Cell Lung Cancer Cells / 中国实验方剂学杂志

ObjectiveTo compare and observe the effect of Reduning injection （mainly clearing heat）， Shenfu injection （mainly warming Yang） combined with gefitinib on the proliferation， apoptosis， stemness characteristics and metabolism of lung cancer cells. MethodDifferent non-small cell lung cancer （NSCLC） cell lines were selected and intervened with gefitinib （5， 10， 20 μmol·L-1）， Reduning injection （0.6%， 0.9%）， Shenfu injection （0.6%， 0.9%）， gefitinib combined with Reduning injection， and gefitinib combined with Shenfu injection. Cell proliferation in each group was detected by cell counting kit-8 （CCK-8） assay， and cell apoptosis was detected by flow cytometry. The mRNA and protein expressions of lung cancer stem cell markers sex determining region Y-box 2 （Sox2） and aldehyde dehydrogenase family 1 member A1 （ALDH1A1） were determind by real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. The redox ratio of lung cancer cells was observed by femtosecond label-free imaging （FLI） and energy metabolism instrument was used to determine the glycolysis level in cells. ResultCompared with the blank group， Reduning injection reduced the survival rate of lung cancer cells （P<0.05）， increased the apoptosis rate （P<0.05）， down-regulated the mRNA and protein expressions of Sox2 and ALDH1A1 （P<0.05）， and up-regulated the redox ratio of cells （P<0.05）， while Shenfu injection exerted no remarkable effect on the above indexes. In addition， compared with gefitinib alone， Reduning injection combined with gefitinib inhibited the survival rate of lung cancer cells （P<0.05）， promoted the cell apoptosis （P<0.05）， down-regulated the mRNA and protein expressions of Sox2 and ALDH1A1 （P<0.05）， up-regulated the redox ratio of cells （P<0.05）， and lowered the proton efflux rate of glycolysis （P<0.05）， while Shenfu injection combined with gefitinib failed to affect these indexes of lung cancer cells significantly. ConclusionReduning injection may inhibit stemness characteristics of tumor cells by regulating their metabolism to enhance the proliferation-inhibiting and pro-apoptotic effects of gefitinib on lung cancer cells， while Shenfu injection had no significant enhancing effect on gefitinib. This indicates that epidermal growth factor receptor tyrosine kinase inhibitors （EGFR-TKIs） should be used in combination with heat-clearing Chinese medicines.

CD44 related stemness maneuvers oral squamous cell carcinoma biology

Introduction: Despite the commendable advancements in oral squamous cell carcinoma (OSCC) diagnostics and therapeutics, it remains a considerable medical challenge. Recent evidence suggests that small populations of stem-like cancer cells are responsible for tumor initiation, progression and metastasis. These cancer stem cells (CSCs) have been identified and characterized in various types of cancers, including OSCCs. CSC hypothesis has been supported by the expression of CD44, CD133, ALDH1 and ABCG2. Amongst them, CD44 (a transmembrane glycoprotein), is the most reported CSC marker in OSCCs. The increasing incidence of OSCC combined with its poor survival rates motivates a need for research into the expression of adhesion molecules and may play a pivotal role in studying tumor biology related to invasion and distant metastasis. Objective: To quantify the expression of CD44 in the different grades of OSCC and to correlate the expression of CD44 with clinicopathological parameters. Method: A total of 20 formalin-fixed paraffin-embedded tissues of OSCC were retrieved from department archives. Immunohistochemical staining was performed using anti-CD44 antibody (Biogenex). The expression was assessed semi-quantitatively in varying histopathological grades of OSCC and were correlated with tumor, node, metastasis (TNM) staging which were obtained from the department records. The results were statistically evaluated. Result: Overexpression of CD44 was detected in 48% of well-differentiated OSCCs followed by a linear decrease in moderately differentiated and poorly differentiated OSCCs and the expression correlated with the tumor size (T) in 23% cases and with lymph node metastases (N) in 42% of cases (P ?0.05). Conclusion: The results of the present study suggested an altered expression of CD44 in OSCC. This depicts an association of CD44 with tumor aggressiveness and Epithelial Mesenchymal Transition (EMT) related to loss of cell adhesion in a subset of OSCC—clearly stating tumor cell stemness as a key factor in malignant potential of OSCC.

Wiskot-Aldrich syndrome-like lower expressed in colon cancer tissues and inhibited the stemness of human colon cancer / 解剖学报

Objective To explore the expression of Wiskot￣Aldrich syndrome-like(WASL)in colon cancer tissues and its function on colon cancer stemness. Methods The UALCAN database was used to analyze the expression of WASL in colon cancer tissues and its correlation among the gender, lymphnode stage and metastasis in colon cancer patients. Immunohistofluorescence and immunocyte fluorescence were used to verify the date from UALCAN. Western blotting was used to detect the expression and transfection efficiency of WASL in human normal colorectal mucosal cells (FHC cells) and colon cancer cells HCT-116, SW-480. Sphere formation assay and colony formation were used to detect the effect of WASL on the stemness of colon cancer. Hypoxia assay, serum deprivation, drug resistance assay were used to detect the stemness of colon cancer cells. Results WASL was lower expressed in colon cancer tissues (P<0. 05). WASL was lower expressed in colon cancer tissues than peri-cancer tissues. WASL was obviously lower expressed in HCT-116 and SW-480 than FHC cells. WASL overexpression stable cells were constructed suscessfully in HCT-116 and SW-480.Real-time PCR showed WASL obviously down-regulated the expression of cancer stemcells (CSCs) related markers [octamer binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), Lin28, krüppel-like factor (KLF)]. Sphere formation assay and colony formation assay showed WASL could inhibited the sphere formation and colony formation. Hypoxia assay and serum deprivation showed WASL inhibited the resistance of cancer cells to hypoxia and serum deprivation. Drug resistance assay showed WASL inhibit the resistance of cancer cells to 5-fluorouracil(5-FU). Conclusion WASL is lower expressed in colon cancer tissues and cells, and is related with poor prognosis of colon cancer. Overexpression of WASL can inhibit the stemness of colon cancer cells.

Cinnamaldehyde inhibits pancreatic cancer cell proliferation and sternness via modulation of CD44s/STAT3 signaling pathway / 中国药理学通报

Aim To explore the effects of cinnamaldehyde on the proliferation and stemness of pancreatic cancer PANC-1 cells, and its possible mechanism of action.Methods CCK8 assay was used to detect the effect of cinnamaldehyde treatment on cell viability at different concentrations(0, 5, 15, 20, 30, 50, 70, 100, 150 μmol·L-1)and different intervention time(24, 48, 72 h).CFSE proliferation assay was used to detect the inhibitory effects of cinnamaldehyde on PANC-1 cells.Colony formation assay was employed to determine the colony-forming ability of PANC-1 cells after cinnamaldehyde treatment.The sphere formation assay was employed to detect the effects of cinnamaldehyde on sphere-forming ability in PANC-1 cells.Western blot analysis and qRT-PCR analysis were applied to determine the expression levels of Nanog, Sox-2 and Oct-4.Flow cytometry was used to detect the percentage of CD44+CD24+ cells and ALDH+ cells in cinnamaldehyde treated and untreated PANC-1 cells.Western blot analysis was used to detect the effects of cinnamaldehyde on the expression of CD44s, p-STAT3 and STAT3 in PANC-1 cells.Results Cinnamaldehyde suppressed cell viability in a dose- and time-dependent manner, and inhibited tumor-cell proliferation and colony forming ability significantly in a dose-dependent manner in PANC-1 cells.Sphere-forming assay showed that cinnamaldehyde could significantly inhibit sphere-forming ability in suspension culture of PANC-1 cells.The mRNA and protein expression levels of three stemness-related genes were down-regulated after cinnamaldehyde treatment.In addition, cinnamaldehyde treatment significantly decreased the proportion of CD44+CD24+ cells and ALDH+ cells.Western blotting showed that cinnamaldehyde inhibited the expression of CD44s and p-STAT3, while it had no effect on the expression of STAT3.With the addition of STAT3 activator(Colivelin TFA), the inhibition of cinnamaldehyde on proliferation and tumor-cell stemness in PANC-1 cells was partially rescued.Conclusions Cinnamaldehyde significantly inhibits the proliferation and tumor-cell stemness of pancreatic cancer PANC-1 cells, and the mechanism could be related to the modulation of CD44s/STAT3 signaling pathway.

Effects of Cholesterol-lowering Agents on Proliferation, Invasion and Neutrophil Extracellular Trap Formation in Liver Cancer Cells / 肿瘤防治研究

Objective To investigate the effects of cholesterol-lowering agents on the proliferation, stemness characters, migration, invasion, and neutrophil extracellular traps formation (NETs) formation in liver cancer cells. Methods ASPP2 or HMGCR gene was knocked down in mouse liver cancer cell Hepa1-6 to establish cells with high or low cholesterol, respectively. Simvastatin and berberine were used to reduce cholesterol synthesis. CCK-8 and plate cloning assays were conducted to detect the proliferation ability of liver cancer cells. Sphere formation assay and qRT-PCR were used to analyze the stemness character and expression of related genes. Wound-healing assay and Transwell assay were used to analyze the ability of cell migration and invasion. Immunofluorescence staining was carried out to analyze the effect of lipid-lowering agent on NETs formation. Results Cholesterol-lowering agents significantly inhibited the proliferation and stemness-related gene expression of Hepa1-6 cells (P < 0.001), significantly inhibited the migration and invasion of Hepa1-6 cells (P < 0.001), and significantly inhibited the neutrophil-induced invasion and formation of NETs (P < 0.001). Conclusion Cholesterol-lowering agents suppress the proliferation and invasion via inhibiting the stemness characters and NETs formation in liver cancer cells. It is a potential strategy for the treatment of liver cancer metastasis.

Effects of N6-methyladenosine-induced LncRNA PVT1 targeting MYC on the stemness of ketamine-treated breast cancer cells / 中华内分泌外科杂志

Objective:To investigate the effect of ketamine on dryness maintenance of breast cancer (BC) cells by regulating LncRNA PVT1/MYC axis.Methods:BC cell line MCF-7 was treated with different concentration of ketamine (0, 5, 10 or 20 g/ml) or treated with 20g/ml ketamine for different periods (0, 24, 48 or 72h) . Furthermore, the expression of METTL3, PVT1 and MYC in MCF-7 cells was interfered and MCF-7 cells were divided into different groups.Western blot was used to detect the expression levels of stem cell characteristic related molecules (OCT4 and SOX2) . The expression level of PVT1/MYC in each group was detected by qRT-PCR. MeRIP analysis was used to detect THE m6A methylation level of PVT1.Results:Ketamine treatment significantly reduced the number of BC globules and inhibited the protein expression of OCT4 and SOX2 in a dose-and time-dependent manner (all P<0.05) . Ketamine regulated m6A level of METTL3-mediated PVT1. Compared with ketamine+pcDNA3.1 group (207±11) , the number of globules formed in ketamine+PVT1 group (311±15) was significantly increased ( t=12.06, P<0.001) , and the protein expression levels of OCT4 and SOX2 were increased ( t=9.68, P<0.001; t=11.50, P<0.001) . MYC was a downstream regulatory gene of PVT1. Compared with ketamine+PVT1+ Si-NC group, ketamine+PVT1+si-MYC group significantly reduced the number of spheroid formation ( t=0.54, P=0.005) and the expression levels of OCT4 and SOX2 proteins ( t=5.98, P=0.004) ( t=7.33, P=0.002) . Conclusion:Ketamine mediates the expression of PVT1 and its downstream gene MYC by inhibiting THE m6A level of PVT1, thus inhibiting the stem cell-like characteristics of BC cells.

Effect of Xuanfu Daizhe decoction on stemness of esophageal cancer cells / 中国应用生理学杂志

Objective: To investigate the effect of Xuanfu Daizhe decoction on the stemness of esophageal cancer cells. Methods: The BALB/c nude mice were randomly divided into the control group and experimental group, 5 mice in each group, which were continuously administered with normal saline and Xuanfu Daizhe decoction (9.89 g/kg) by gastrogavage, respectively. Human esophageal carcinoma cells ECA-109 (5×106) were subcutaneously injected into the mice on the 8th day. Tumor volume was measured twice a week. The mice were sacrificed 4 weeks after injection, and the tumor tissue and mouse serum were collected. The expressions of the major stemness-regulating transcription factors, i.e., NANOG, OCT4 and SOX2, were detected by RT-qPCR, Western Blot and immunohistochemistry. ECA-109 cells were treated with 10% fetal bovine serum and serum from the above two groups of mice for 48 hours respectively, and three replicate wells were set in each group, and the expressions of NANOG, OCT4, SOX2 and the levels of AKT and p-AKT were detected by RT-qPCR and Western Blot, respectively. ALDH activity in tumor cells was detected by flow cytometry; the number of spheroids of tumor cells was detected by the spheroidization experiment. Results: Compared with the control group, the growth and size of esophageal cancer tumors were significantly inhibited by Xuanfu Daizhe Decoction; the expressions of NANOG, OCT4, SOX2, the ALDH activity, the number of spheroids, and the levels of AKT and phosphorylated AKT (p-AKT) in esophageal cancer cells were significantly reduced by Xuanfu Daizhe Decoction both in vivo and in vitro. Conclusion: Xuanfu Daizhe Decoction inhibits the stemness of esophageal cancer cells, it may be a potentially effective drug for the treatment of esophageal cancer and provides a theoretical basis for the exploration of new effective drugs for the treatment of esophageal cancer.

Molecular markers and mechanisms for stemness maintenance of liver cancer stem cells: a review / 生物工程学报

Primary liver cancer (PLC) is an aggressive tumor and prone to metastasize and recur. According to pathological features, PLC are mainly categorized into hepatocellular carcinoma, intrahepatic cholangiocarcinoma, mixed hepatocellular cholangiocarcinoma, and fibrolamelic hepatocellular carcinoma, etc. At present, surgical resection, radiotherapy and chemotherapy are still the main treatments for PLC, but the specificities are poor and the clinical effects are limited with a 5-year overall survival rate of 18%. Liver cancer stem cells (LCSCs) are a specific cell subset existing in liver cancer tissues. They harbor the capabilities of self-renewal and strong tumorigenicity, driving tumor initiation, metastasis, drug resistance and recurrence of PLC. Therefore, the identification of molecular markers and the illustration of mechanisms for stemness maintenance of LCSCs can not only reveal the molecular mechanisms of PLC tumorigenesis, but also lay a theoretical foundation for the molecular classification, prognosis evaluation and targeted therapy of PLC. The latest research showed that the combination of 5-fluorouracil and CD13 inhibitors could inhibit the proliferation of CD13+ LCSCs, thereby reducing overall tumor burden. Taken together, LCSCs could be the promising therapeutic targets of PLC in the future. This review summarizes the latest progress in molecular markers, mechanisms for stemness maintenance and targeted therapies of LCSCs.

Dysregulation of MAD2L1/CAMK2A/PTTG1 Gene Cluster Maintains the Stemness Characteristics of Uterine Corpus Endometrial Carcinoma / 中国医学科学院学报

Objective To study the stemness characteristics of uterine corpus endometrial carcinoma(UCEC)and its potential regulatory mechanism.Methods Transcriptome sequencing data of UCEC was obtained from The Cancer Genome Atlas.Gene expression profile was normalized by edgeR package in R3.5.1.A one-class logistic regression machine learning algorithm was employed to calculated the mRNA stemness index(mRNAsi)of each UCEC sample.Then,the prognostic significance of mRNAsi and candidate genes was evaluated by survminer and survival packages.The high-frequency sub-pathways mining approach(HiFreSP)was used to identify the prognosis-related sub-pathways enriched with differentially expressed genes(DEGs).Subsequently,a gene co-expression network was constructed using WGCNA package,and the key gene modules were analyzed.The clusterProfiler package was adopted to the function annotation of the modules highly correlated with mRNAsi.Finally,the Human Protein Atlas(HPA)was retrieved for immunohistochemical validation.Results The mRNAsi of UCEC samples was significantly higher than that of normal tissues(

Elevated expression of stemness genes in adipose-derived mesenchymal stem cells cultured on fibrin scaffold

In regenerative medicine, MSCs need to be pluripotent for better results. In this study, the effect of fibrinscaffold on expression of stemness genes was examined. Adipose-derived MSCs were cultured in tissue cultureplates (2D) and 3-dimensional (3D) fibrin scaffolds. The effect of fibrin scaffold on proliferation of adiposederived MSCs was evaluated by MTT assay. The expression of stemness genes (OCT4 and SOX2) wereevaluated by qRT-PCR, and flow cytometry was done for Nanog protein level. Cultured MSCs on fibrinscaffold were able to proliferate according to data obtained by MTT assay. Expression of OCT4 and SOX2 hada significant increase in cells were cultured in 3D condition compared to 2D condition (P \0.05). Also,increased expression of Nanog protein in 3D culture was observed (P \0.05). OCT4 and SOX2 in 3Dcondition increased two-fold and three-fold respectively in 2D and 3D conditions. Moreover, expression ofNanog increased 30% more than in 2D condition. Evaluation of important pluripotency regulators such asOCT4, SOX2, and Nanog showed that fibrin scaffolds are useful instruments to maintain stemness of MSCs,which is essential in field of stem cell therapy and regenerative medicine.

Knocking down LSD1 inhibits the stemness features of colorectal cancer stem cells

As a top leading cause of cancer death in many countries, colorectal cancer (CRC) has drawn increasing attention to the study of the pathological mechanism. According to the "cancer stem cell hypothesis", malignancies originate from a small fraction of cancer cells that show self-renewal properties to initiate and sustain tumor growth and tumor metastasis. Therefore, these cancer stem cells (CSC) probably play important roles in tumor recurrence, metastasis, and drug resistance. Previous research reported that lysine-specific histone demethylase 1 (LSD1) maintains cancer stemness through up-regulating stemness markers SOX2 and OCT4. CD133 is believed to be the most robust surface marker for CRC stem cells, however the regulatory effect of LSD1 on stemness of CD133+ CRC has never been reported. In this study, our objectives included: 1) to isolate pure CD133+ and CD133− cells from SW620 cell line; 2) to investigate the effect of LSD1 on the characteristics of CD133+ stem cancer cells by knocking down the target gene. Results suggested that the SW620 cell line had both CD133+ and CD133− subsets. The CD133+ subset exhibited more CSC-like characteristics compared with the CD133− subset with higher viability, colony formation rate, migration and invasion rate, resistance to anti-cancer drugs, and apoptosis in vitro. The CD133+ also induced faster tumor formation and larger tumors in vivo. In the LSD1-knockdown CD133+ cells, the CSC-like characteristics had been all weakened. We conclude that LSD1 was important for CSCs to maintain their "stemness" features, which could be a potential therapeutic target of CRC.

Regulatory effect and mechanism of miR-9-5p on malignant biological behaviors of breast cancer / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To explore the role of miR-9-5p in the biological behaviors of breast cancer cells and its possible regulatory mechanism. Methods: online OncomiR database was used to analyze the differential expression of miR-9-5p in breast cancer tissues and normal breast tissues. qPCR was used to detect the miR-9-5p expression in breast cancer cell lines and normal breast cells. Based on target gene prediction software TargetScan, ONECUT2 (one cut homeobox 2) was predicted to be the target gene of miR-9-5p. Dual luciferase reporter system was used to validate the relationship between miR-9-5p and its promising target gene ONECUT2. MDA-231 cells were transfected with miR-9-5p mimic, ONECUT2 siRNAs as well as the corresponding control sequences. The protein and mRNA levels of stemness-associated gene NOTCH1, NANOG and SOX9 (SRY (sex-determing region of Y chromosome) -Box transcription Factor 9) were detected by WB and qPCR. The effect of transfection on proliferation, apoptosis and chemo-resistance of cells was detected by BrdU method, Annexin Ⅴ method and MTS Assay, respectively. The ALDEFLUOR experiment was used to detect the effects of miR-9-5p and its target gene ONECUT2 on tumor stemness. NSG mouse breast cancer chemotherapy model was established, and the in vivo experiments further verified the effect of ONECUT2 on tumor malignant biological behaviors, such as cell stemness and chemo-resistance. Results: miR-9-5p was highly expressed in breast cancer tissues (P=0.007) and breast cancer MDA-231 cell line (P=0.0005), and was positively correlated with the poor prognosis of breast cancer patients (P=0.0016). Compared to control group, miR-9-5p could target and negatively regulate ONECUT2 expression, further increase ALDH+ cell population (P=0.0006), as well as increase the expressions of stemness-associated genes NOTCH1, NANOG and SOX9. Besides, miR-9-5p increased the anti-apoptosis ability (P=0.0003) and chemo-resistance of MDA-231 cells; however, miR-9-5p/ONECUT2 exerted no significant effect on the proliferation ability of MDA-231 cells (P>0.05). Compared with the control group, the volume of xenografts in mice of MDA-231/ONECUT2 group after DTX chemotherapy was significantly lower than that in the control group (P<0.05), and the protein expressions of NOTCH1, SOX9 and the mRNA expression of ABC transporter in the transplanted tumor tissues were significantly reduced (P<0.05 or P<0.01). Conclusions: The highly expressed miR-9-5p in breast cancer induces tumor stemness and anti-apoptotic ability by targeting ONECUT2 and enhances its resistance to chemotherapy.

Study on the Effects of Calpeptin on Estrogen-induced Transformation and Stemness Markers Expression of Mammary Epithelial Cells MCF- 10A Based on Calpain-ERK Signaling Pathway / 中国药房

OBJECTIVE：To study the effects of Calpeptin inhibitor Calpeptin on the transformation and stemness markers expression induced by estradiol（E2），and to investigate its mechanism. METHODS ：Taking human mammary epithelial cells MCF-10A as research object ，transformed cells were induced by E 2 treatment. Cells were divided into control group （0.1％DMSO）， E2-transformed group （50 nmol/L），E2-transformed+Calpeptin group （50 nmol/L E 2+1 μmol/L Calpeptin），then continuously treated with corresponding drug-containing culture medium for 15 generations. Then ，MTT assay was used to determine the proliferation rate of cells （24，48 h）；plate colony test was used to detect the Clone formation rate of cells ；the number of sphere-forming cells was measured by suspension spheroidization test ；mRNA expressions of stemness marker （CD44，Nanog，OCT4）and extracellular sigal-regulated kinase （ERK）were detected by RT-qPCR ，and protein expressions of CD 44，Nanog，OCT4 ，ERK and p-ERK were detected by Western blotting assay. Another E 2-transformed cells were divided into control group （0.1％DMSO）and U0126 （ERK inhibitor ）group（10 μmol/L）. Clone formation rate ，the number of sphere-forming ，protein expressions of CD 44，Nanog， OCT4，ERK and p-ERK were determined with above methods ，and to validate the relationship of ERK inhibition with transformed cell behavior and the expression of stemness markers. RESULTS ：Compared with control group ，proliferation rate and clone formation rate of E 2 transformed group were increased significantly （P＜0.01），and the number of sphere-forming was increased significantly（P＜0.01）；mRNA expression levels of CD 44，Nanog，OCT4，ERK and protein expression levels of CD 44，Nanog， OCT4 and p-ERK in cells were increased significantly （P＜0.01）. Compared with E 2-transformed group ，proliferation rate （24，48 h）and clone formation rate of E 2-transformed + Calpeptin group were decreased significantly （P＜0.01），and the number of sphere-forming was decreased significantly （P＜0.05）；mRNA expression levels of CD 44，Nanog，OCT4 ，ERK and protein expression levels of CD 44，Nanog，OCT4，p-ERK in cells were decreased significantly （P＜0.05 or P＜0.01）. After treated with ERK inhibitor U 0126，clone formation rate of E 2-transformed cells ，the number of sphere-forming ，protein expression levels of CD44，Nanog，OCT4 and p-ERK were increased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：Calpeptin can inhibit the transformation and the expression of stemness markers of human mammary epithelial cells MCF- 10A，and the mechanism of it may be associated with inhibiting the activation of Calpain-ERK signaling pathway.

The Impact of IONPs and PBNPs Nanoparticles in Regulating Stemness of Ovarian Carcinoma Cells / 中国药学杂志

OBJECTIVE: To study the effects of iron oxide nanoparticles (IONPs) and prussian blue nanoparticles (PBNPs) on the stemness of epithelial ovarian cancer (EOC) cell lines, which may provide experimental basis and reference significance for the application of nanoparticles in the treatment of EOC. METHODS: SKOV3 and HO8910 cell lines were treated with IONPs and PBNPs respectively, then the drug-resistant genes in mRNA and protein levels were detected by RT-PCR and Western blot. The change of cell proliferation ability were detected by cell proliferation and clone formation experiments, and the expression of surface stemness-related molecules were detected by flow cytometer. RESULTS: The expression of ABCG2 and ALDH1A1 were both down regulated in SKOV3 and HO8910 cell lines treated with IONPs and PBNPs, respectively, while IONPs decreased the CD44+CD117+ subpopulation, and PBNPs increased the CD44+CD117+ subpopulation. Compared with the control cells, the proliferation of SKOV3 cells treated with IONPs and PBNPs were significantly reduced, however, there were no effects on HO8910 cell's proliferation after the cells treated with PBNPs. CONCLUSION: Drug-resistance genes and stem cell subpopulation of EOC SKOV3/HO8910 cell lines treated with IONPs were markedly reduced. However, PBNPs can improve the stem cell subpopulation and promote the cancer stem cell's characteristic of EOC cells but the drug-resistance genes were decreased.