In current study, cancer stem-like cells in the murine melanoma B16F10 cells were investigated. CD phenotypes of the B16F10 cells were analyzed by flow cytometry, and the specific CD phenotype cells from the B16F10 ce...In current study, cancer stem-like cells in the murine melanoma B16F10 cells were investigated. CD phenotypes of the B16F10 cells were analyzed by flow cytometry, and the specific CD phenotype cells from the B16F10 cells were isolated by MACS. Then we used colony formation assay in soft agar media, the cell growth assay in serum-free culture media as well as the tumorigenicity investigation of the specific CD phenotype cells in C57BL/6 mice, respectively, to identify cancer stem-like cells in the B16F10 cells. The results showed that the B16F10 cells could form spherical clones in serum-free culture media, and the rate of clonegenesis of CD133^＋, CD44^＋ and CD44^＋CD133^＋ cells was higher than that of CD133^-, CD44^- and CD44^＋CD133^＋ cells in soft agar media, respectively. The tumorigenic potential of CD133^＋, CD44^＋, CD44^＋CD133^＋ cells and CD44^＋CD133^＋CD24^＋ cells was stronger than that of CD133^-, CD44^-, CD44^＋CD133^- cells and CD44^＋CD133^＋CD24^- cells in mice, respectively. In conclusion, the CD44^＋CD133^＋CD24^＋ cells have some biological properties of cancer stem-like cells or are highly similar to the characteristics of cancer stem cells （CSC）. These results provide an important method for identifying cancer stem-like cells in B16F10 cells and for further cancer target therapy. Cellular ＆ Molecular Immunology.展开更多
Gastric cancer stem-like cells（GCSCs） have been identified to possess the ability of self-renewal and tumor initi-ation.However,the mechanisms involved remain largely unknown.Here,we isolated and characterized the G...Gastric cancer stem-like cells（GCSCs） have been identified to possess the ability of self-renewal and tumor initi-ation.However,the mechanisms involved remain largely unknown.Here,we isolated and characterized the GCSCs by side population（SP） sorting procedure and cultured sphere cells（SC） from human gastric cancer cell lines SGC-7901,BGC-823,MGC-803,HGC-27 and MKN-28.The sorting and culture assay revealed that SP cells proliferated in an asymmetric division manner.In addition,SP cells exhibited a higher potential of spheroid colony formation and greater drug resistance than non-SP cells（NSP）.Moreover,the SC were found with enhanced capabilities of drug resistance in vitro and tumorigenicity in vivo.Sox2 mRNA and protein was highly and significantly overex-pressed in the SP cells and SC.Importantly,downregulation of Sox2 with siRNA obviously reduced spheroid colony formation and doxorubicin efflux,as well as increased apoptosis rate in sphere cells in vitro and suppressed tumori-genicity in vivo.These results suggest that both SP cells and cultured SC enrich with GCSCs and that Sox2 plays a pivotal role in sustaining stem cell properties and might be a potential target for gastric cancer therapy.展开更多
Background A satisfactory animal model of breast cancer metastasizing to bone is unavailable. In this study, we used human breast cancer stem-like cells and human bone to build a novel “human-source” model of human ...Background A satisfactory animal model of breast cancer metastasizing to bone is unavailable. In this study, we used human breast cancer stem-like cells and human bone to build a novel “human-source” model of human breast cancer skeletal metastasis. Methods Human breast cancer stem-like cells, the CD44^＋/CD24^-/lower subpopulation, was separated and cultured. Before injection with the stem-like cells, mice were implanted with human bone in the right or left dorsal flanks. Animals in Groups A, B, and C were injected with 1×10^5, 1×10^6 human breast cancer stem-like cells, and 1×10^6 parental MDA-MB-231 cells, respectively. A positive control group （D） without implantation of human bone was also injected with 1×10^6 MDA-MB-231 cells. Immunohistochemistry was performed for determination of CD34, CD105, smooth muscle antibody, CD44, CD24, cytokine, CXC chemokine receptor-4 （CXCR4）, and osteopontin （OPN）. mRNA levels of CD44, CD24, CXCR4, and OPN in bone metastasis tissues were analyzed by real-time quantitative polymerase chain reaction （PCR）.Results Our results demonstrated that cells in implanted human bones of group B, which received 1×10^6 cancer stem-like cells, stained strongly positive for CD44, CXCR4, and OPN, whereas those of other groups showed no or minimum staining. Moreover, group B had the highest incidence of human bone metastasis （77.8%, P=0.0230） and no accompaniment of other tissue metastasis. The real-time PCR showed an increase of CD44, CXCR4, and OPN mRNA in metastatic bone tissues in group B compared with those of groups C and D, however the expression of CD24 mRNA in group B were the lowest. Conclusions In the novel “human source” model of breast cancer, breast cancer stem-like cells demonstrated a higher human bone-seeking ability. Its mechanism might be related to the higher expressions of CD44, CXCR4, and OPN, and the lower expression of CD24 in breast cancer stem-like cells.展开更多
Objective: Vasculogenic mimicry(VM) channels that are lined by tumor cells are a functional blood supply in malignant tumors.However, the role of VM-initiating cells remains poorly understood. Cancer stem-like cells(C...Objective: Vasculogenic mimicry(VM) channels that are lined by tumor cells are a functional blood supply in malignant tumors.However, the role of VM-initiating cells remains poorly understood. Cancer stem-like cells(CSCs) are positively correlated with VM. In this study, triple-negative breast cancer(TNBC) enriched with CSCs was used to investigate the relationship between VM and CSCs.Methods: The expression of several CSC markers was detected by immunohistochemistry in 100 human breast cancer samples.The clinical significance of CSC markers and the relationship between VM, CSCs, breast cancer subtypes, and VM-associated proteins were analyzed. CD133+ and ALDH+ human and mouse TNBC cells were isolated by FACS to examine the ability of VM formation and the spatial relationship between VM and CSCs.Results: CSCs were associated with TNBC subtype and VM in human invasive breast cancer. CSCs in TNBC MDA-MB-231 cells formed more VM channels and expressed more molecules promoting VM than the non-TNBC MCF-7 cells in vitro. MDA-MB-231 cells that encircled VM channels on Matrigel expressed CD133. Moreover, CSCs were located near VM channels in the 3D reconstructed blood supply system in human TNBC grafts. The CD133+ and ALDH+ cells isolated from TA2 mouse breast cancer formed more VM channels in vivo.Conclusions: CSCs line VM channels directly. Additionally, CSCs provide more VM-related molecules to synergize VM formation. The signaling pathways that control CSC differentiation may also be potential treatment targets for TNBC.展开更多
HYD-PEP06,an endostatin-modified polypeptide,has been shown to produce effective anticolorectal carcinoma effects through inhibiting epithelial-mesenchymal transition(EMT).However,whether HYD-PEP06 has similar suppres...HYD-PEP06,an endostatin-modified polypeptide,has been shown to produce effective anticolorectal carcinoma effects through inhibiting epithelial-mesenchymal transition(EMT).However,whether HYD-PEP06 has similar suppressive effect on hepatocellular carcinoma(HCC) remained unknown.In this study,HYD-PEP06 inhibited metastasis and EMT but not proliferation in vitro.Cignal finder pathway reporter array and Western blot analysis revealed that HYD-PEP06 suppressed HCCLM3 cell metastasis and EMT by inhibiting the PI3 K/AKT pathway.Moreover,HYD-PEP06 exerted antimetastasis effects in HepG2 cancer stem-like cells(CSCs) via suppressing the WNT/β-catenin signaling pathway.Finally,in HCCLM3 tumor-bearing BALB/c nu/nu nude mice,HYD-PEP06 substantially suppressed tumor growth,lung metastasis and HCC progress.Our results suggest that HYD-PEP06 inhibits the metastasis and EMT of HCC and CSCs as well,and thus has the potential as an agent for HCC treatment.展开更多
Carcinogenesis is a multistep process that requires the accumulation of various genetic and epigenetic aberrations to drive the progressive malignant transformation of normal human cells.Two major hallmarks of carcino...Carcinogenesis is a multistep process that requires the accumulation of various genetic and epigenetic aberrations to drive the progressive malignant transformation of normal human cells.Two major hallmarks of carcinogenesis that have been described are angiogenesis and the stem cell characteristic of limitless replicative potential.These properties have been targeted over the past decade in the development of therapeutic treatments for colorectal cancer(CRC),one of the most commonly diagnosed and lethal cancers worldwide.The treatment of solid tumor cancers such as CRC has been challenging due to the heterogeneity of the tumor itself and the chemoresistance of the malignant cells.Furthermore,the same microenvironment that maintains the pool of intestinal stem cells that contribute to the continuous renewal of the intestinal epithelia also provides the necessary conditions for proliferative growth of cancer stem-like cells.These cancer stem-like cells are responsible for the resistance to therapy and cancer recurrence,though they represent less than 2.5%of the tumor mass.The stromal environment surrounding the tumor cells,referred to as the tumor niche,also supports angiogenesis,which supplies the oxygen and nutrients needed for tumor development.Anti-angiogenic therapy,such as with bevacizumab,a monoclonal antibody against vascular-endothelial growth factor,significantly prolongs the survival of metastatic CRC patients.However,such treatments are not completely curative,and a large proportion of patient tumors retain chemoresistance or show recurrence.This article reviews the current knowledge regarding the molecular phenotype of CRC cancer cells,as well as discusses the mechanisms contributing to their maintenance.Future personalized therapeutic approaches that are based on the interaction of the carcinogenic hallmarks,namely angiogenic and proliferative attributes,could improve survival and decrease adverse effects induced by unnecessary chemotherapy.展开更多
Pluripotent stem cells derived from neonatal or adult testes are a useful tool to examine the mechanisms of pluripotency and a resource for cell-based therapies. However, therapies usingthese cells will only benefit m...Pluripotent stem cells derived from neonatal or adult testes are a useful tool to examine the mechanisms of pluripotency and a resource for cell-based therapies. However, therapies usingthese cells will only benefit males but not females. Recently, female germline stem cells （FGSCs） were discovered in ovaries. Whether FGSCs can be converted into pluripotent stem cells, similar to spermatogonial stem cells, is unknown. Here, we demonstrate that female embryonic stem-like cells （fESLCs） can be generated within 1 month from the stably proliferating FGSCs cultured in embryonic stem cell （ESC） medium, fESLCs exhibit properties similar to those of ESCs in terms of marker expression and differentiation potential. Thus, our findings suggest that generation of patient-specific fESLCs is feasible and provides a foundation for personalized regenerative applications.展开更多
Background:Maintenance of cancer stem-like cell(CSC)stemness supported by aberrantly regulated cancer cell metabolism is critical for CSC self-renewal and tumor progression.As a key glycolytic enzyme,hexokinase 2(HK2)...Background:Maintenance of cancer stem-like cell(CSC)stemness supported by aberrantly regulated cancer cell metabolism is critical for CSC self-renewal and tumor progression.As a key glycolytic enzyme,hexokinase 2(HK2)plays an instrumental role in aerobic glycolysis and tumor progression.However,whether HK2 directly contribute to CSC stemness maintenance in small cell lung cancer(SCLC)is largely unclear.In this study,we aimed to investgate whether HK2 independent of its glycolytic activity is directly involved in stemness maintenance of CSC in SCLC.Methods:Immunoblotting analyses were conducted to determine the expression of HK2 in SCLC CSCs and their differentiated counterparts.CSC-like properties and tumorigenesis of SCLC cells with or without HK2 depletion or overexpression were examined by sphere formation assay and xenograft mouse model.Immunoprecipitation and mass spectrometry analyses were performed to identify the binding proteins of CD133.The expression levels of CD133-associated and CSC-relevant proteins were evaluated by immunoblotting,immunoprecipitation,immunofluorescence,and immunohistochemistry assay.RNA expression levels of Nanog,POU5F1,Lin28,HK2,Prominin-1 were analyzed through quantitative reverse transcription PCR.Polyubiquitination of CD133 was examined by in vitro or in vivo ubiquitination assay.CD133+cells were sorted by flow cytometry using an anti-CD133 antibody.Results:We demonstrated that HK2 expression was much higher in CSCs of SCLC than in their differentiated counterparts.HK2 depletion inhibited CSC stemness and promoted CSC differentiation.Mechanistically,nonmitochondrial HK2 directly interacted with CD133 and enhanced CD133 expression without affecting CD133 mRNA levels.The interaction of HK2 and CD133 promoted the binding of the deubiquitinase ubiquitin-specific protease 11(USP11)to CD133,thereby inhibiting CD133 polyubiquitylation and degradation.HK2-mediated upregulation of CD133 expression enhanced the expression of cell renewal regulators,SCLC cell stemness,and tumor grow展开更多
Meningiomas are the most common primary tumors in central nervous system. While recent studies have revealed genetic clues to lower grade human meningiomas, the molecular determinants driving the progression and recur...Meningiomas are the most common primary tumors in central nervous system. While recent studies have revealed genetic clues to lower grade human meningiomas, the molecular determinants driving the progression and recurrence of anaplastic meningi- oma, the most malignant subtype with a low prevalence but high morbidity, are still poorly understood. It has been proposed that high recurrence rates of malignant meningiomas are linked to cancer stem cells. Indeed, tumor stem-Uke cells have been iso- lated from various meningioma subtypes, but never been obtained from anaplastic meningioma, in this study, we successfully isolated stem-Uke cells from human anaplastic meningioma. These cells are capable of forming spheres and initiating xenograft tumors that recapitulate anaplastic meningioma phenotypes, and thus could serve as an in vitro model for malignant meningi- omas. KLF4, a transcription factor known for its role in sternness maintenance, was identified as one of the most frequently mutated genes in the benign secretory meningioma. Interestingly, we found that KLF4 is downregulated in anaplastic meningi- oma compared with low-grade meningioma subtypes. By manipulating KLF4 expression in anaplastic meningioma stem-like cells, we demonstrated that KLF4 acts as a tumor suppressor during malignant progression in meningioma, affecting apoptosis, prolif- eration, invasion, and cell cycle. These results suggest a potential therapeutic value of KLF4 for clinical intervention of anaplastic meningioma.展开更多
BACKGROUND Over the years,the numbers of treatment options for colorectal cancer(CRC)have increased,leading to notable improvements in the overall survival of CRC patients.Although therapy may initially yield positive...BACKGROUND Over the years,the numbers of treatment options for colorectal cancer(CRC)have increased,leading to notable improvements in the overall survival of CRC patients.Although therapy may initially yield positive results,the development of drug resistance can result in treatment failure and cancer recurrence.This resistance is often attributed to the presence of cancer stem cells(CSCs).These CSCs not only contribute to therapeutic resistance but also play crucial roles in the initiation and development of tumor metastasis.AIM To investigate the antitumor effects of SH-4-54,which are mediated by targeting CSCs relative to treatment outcomes.METHODS CSCs were enriched by culturing CRC cells in serum-free medium.Hallmarks of stemness and IL-6/JAK2/STAT3 signaling were detected by Western blotting.Indicators of CSC malignancy,including proliferation,invasion,and tumor formation,were measured.RESULTS In this study,we employed SH-4-54,which exhibits anticancer activity in solid tumors through targeting the SH2 domain of both the signal transducer and activator of transcription(STAT)3 and the STAT5,and evaluated its effects on stemness and chemoresistance in colorectal CSCs.As expected,SH-4-54 treatment inhibited the phosphorylation of STAT3(p-STAT3)and decreased the percentage of ALDH1A1-positive CRC cells.The addition of SH-4-54 dissociated colorectal spheroids and decreased the expression of stemness markers,including ALDH1A1,CD44 and Nanog.SH-4-54 treatment decreased IL-6/JAK2/STAT3 signaling by inhibiting p-STAT3 and thus inhibited spheroid formation by SW480 and LoVo cells.Moreover,SH-4-54 treatment inhibited indicators of malignancy,including cell proliferation,invasion,and tumor formation,in CSCs in vitro and in vivo.Notably,SH-4-54 treatment significantly increased chemosensitivity to oxaplatin.CONCLUSION Taken together,these results indicate that SH-4-54 is a promising molecule that exerts antitumor effects on colorectal CSCs by inhibiting STAT3 signaling.展开更多
Previous studies have demonstrated that spheroid type cells grown under suspension culture conditions have cancer stem cell（CSC） traits in a number of cancers, but this phenomenon has not yet been reported in the VX...Previous studies have demonstrated that spheroid type cells grown under suspension culture conditions have cancer stem cell（CSC） traits in a number of cancers, but this phenomenon has not yet been reported in the VX2 rabbit oral cancer model. Hence, this study aimed to study the spheroid cells from VX2 rabbit buccal squamous cell carcinomas（SCCs） and assess their CSC characteristics. Five adult male New Zealand white outbred rabbits were used to generate VX2 rabbit buccal SCC. Sphere-forming cell culture was performed for the VX2 rabbit buccal SCC specimens. The self-renewal capability; cluster of designation（CD） 44, CD133, acetaldehyde dehydrogenase 1（ALDH1）, B cell-specific Moloney murine leukemia virus integration site 1（Bmi-1）, Nestin, octamer-binding transcription factor 4（Oct4）and reduced expression protein-1（Rex-1） expression with reverse transcription-polymerase chain reaction（RT-PCR）; chemoresistance to cisplatin and 5-fluorouracil; and in vivo tumorigenicity of spheroid cell transplantation in nude mice were evaluated to determine the CSC characteristics of the resulting spheroid cells. We successfully obtained spheroid cells from the VX2 rabbit OSCC tissues. The spheroid cells exhibited CSC traits, including the expression of CSC and stem cell markers（CD44, Bmi-1, Nestin, Oct4 and Rex-1）, capacity to generate new spheroid colonies within 1 week of reseeding from single-dissociated spheroid cells, chemoresistance capacity and generation of tumour xenografts（with histological features resembling those of the original VX2 rabbit buccal SCC） from the transplantation of 103 undifferentiated spheroid cells into nude mice. In summary, we demonstrated that spheroid cells with CSC cell traits can be derived from VX2 rabbit buccal SCCs, indicating that this animal cancer model is applicable for studying CSCs in human oral cancers.展开更多
AIM: To investigate the expression of miR-210 and the role it plays in the cell cycle to regulate radioresistance in oesophageal squamous cell carcinoma(ESCC). METHODS: Mi R-210 expression was evaluated in 37 pairs of...AIM: To investigate the expression of miR-210 and the role it plays in the cell cycle to regulate radioresistance in oesophageal squamous cell carcinoma(ESCC). METHODS: Mi R-210 expression was evaluated in 37 pairs of ESCC tissues and matched para-tumorous normal oesophageal tissues from surgical patients who had not received neoadjuvant therapy, and in the cells of two novel radioresistant cell lines, TE-1R and Eca-109 R, using quantitative reverse transcription-polymerase chain reaction(q RT-PCR). The transient up-regulation of mi R-210 expression in TE-1R and Eca-109 R cells was studied using liposomes and was confirmed using qR T-PCR. The rate of cell survival after a series of radio-treatment doses was evaluated using the cloneformation assay. Flow cytometry was used to detect the changes to the cell cycle patterns due to radiation treatment. RT-PCR and Western blot were used to detect the expression of ataxia telangiectasia mutated(ATM) and DNA dependent protein kinase(DNA-PKcs) after irradiation, and the cell sphere formation assay was used to evaluate the proliferative ability of the cancer stem-like cells.RESULTS: The level of mi R-210 expression was significantly decreased, by 21.3% to 97.2%, with the average being 39.2% ± 16.1%, in the ESCC tissues of most patients(81.1%, 30 of 37 vs patients with high mi R-210 expression, P < 0.05). A low level of expression of miR-210 was correlated with a poorly differentiated pathological type(P < 0.01) but was not correlated with the T-stage or lymph node infiltration(both P > 0.05). Early local recurrences(< 18 mo, n = 19) after radiotherapy were significantly related with low miR-210 expression(n = 13, P < 0.05). The level of mi R-210 was decreased by approximately 73%(vs TE-1, 0.27 ± 0.10, P < 0.01) in the established radioresistant TE-IR cell line and by 52%(vs Eca-109, 0.48 ± 0.17, P < 0.05) in the corresponding Eca-109 R line. Transient transfection with a mi R-210 precursor increased the level of mi R-210 expression, leading to a significant increase in 展开更多
Objective Vasculogenic mimicry(VM)is a novel vasculogenic process integral to glioma stem cells(GSCs)in glioblastoma(GBM).However,the relationship between VM and ataxia-telangiectasia mutated(ATM)serine/threonine kina...Objective Vasculogenic mimicry(VM)is a novel vasculogenic process integral to glioma stem cells(GSCs)in glioblastoma(GBM).However,the relationship between VM and ataxia-telangiectasia mutated(ATM)serine/threonine kinase activation,which confers chemoradiotherapy resistance,remains unclear.Methods We investigated VM formation and phosphorylated ATM(pATM)levels by CD31/GFAPperiodic acid-Schiff dual staining and immunohistochemical staining in 145 GBM specimens.Glioma stem-like cells(GSLCs)derived from the formatted spheres of U87 and U251 cell lines and their pATM level and VM formation ability were examined using western blot and three-dimensional culture.For the examination of the function of pATM in VM formation by GSLCs,ATM knockdown by shRNAs and deactivated via ATM phosphorylation inhibitor KU55933 were studied.Results VM and high pATM expression occurred in 38.5% and 41.8% of tumors,respectively,and were significantly associated with reduced progression-free and overall survival.Patients with VM-positive GBMs exhibited higher pATM levels(r_(s)=0.425,P=0.01).The multivariate analysis established VM as an independent negative prognostic factor(P=0.002).Furthermore,GSLCs expressed high levels of pATM and formed vascular-like networks in vitro.ATM inactivation or knockdown hindered VM-like network formation concomitant with the downregulation of pVEGFR-2,VE-cadherin,and laminin B2.Conclusion VM may predict a poor GBM prognosis and is associated with pATM expression.We propose that pATM promotes VM through extracellular matrix modulation and VE-Cadherin/pVEGFR-2 activation,thereby highlighting ATM activation as a potential target for enhancing anti-angiogenesis therapies for GBM.展开更多
BACKGROUND Limonin is one of the most abundant active ingredients of Tetradium ruticarpum.It exerts antitumor effects on several kinds of cancer cells.However,whether limonin exerts antitumor effects on colorectal can...BACKGROUND Limonin is one of the most abundant active ingredients of Tetradium ruticarpum.It exerts antitumor effects on several kinds of cancer cells.However,whether limonin exerts antitumor effects on colorectal cancer(CRC)cells and cancer stem-like cells(CSCs),a subpopulation responsible for a poor prognosis,is unclear.AIM To evaluate the effects of limonin on CSCs derived from CRC cells.METHODS CSCs were collected by culturing CRC cells in serum-free medium.The cytotoxicity of limonin against CSCs and parental cells(PCs)was determined by cholecystokinin octapeptide-8 assay.The effects of limonin on stemness were detected by measuring stemness hallmarks and sphere formation ability.RESULTS As expected,limonin exerted inhibitory effects on CRC cell behaviors,including cell proliferation,migration,invasion,colony formation and tumor formation in soft agar.A relatively low concentration of limonin decreased the expression stemness hallmarks,including Nanog andβ-catenin,the proportion of aldehyde dehydrogenase 1-positive CSCs,and the sphere formation rate,indicating that limonin inhibits stemness without presenting cytotoxicity.Additionally,limonin treatment inhibited invasion and tumor formation in soft agar and in nude mice.Moreover,limonin treatment significantly inhibited the phosphorylation of STAT3 at Y705 but not S727 and did not affect total STAT3 expression.Inhibition of Nanog andβ-catenin expression and sphere formation by limonin was obviously reversed by pretreatment with 2μmol/L colievlin.CONCLUSION Taken together,these results indicate that limonin is a promising compound that targets CSCs and could be used to combat CRC recurrence and metastasis.展开更多
Breast cancer is the most common cancer in females with extremely high lethality mainly due to the occurrence of metastasis,which is closely related to cancer stem-like cells(CSCs).It has been reported that CSC freque...Breast cancer is the most common cancer in females with extremely high lethality mainly due to the occurrence of metastasis,which is closely related to cancer stem-like cells(CSCs).It has been reported that CSC frequency in drug-resistant breast cancer and non-small cell lung cancer is reduced by activating dopamine D1 receptor(D1 DR).In the present study,we aimed to investigate the effect of a compound C17 that can be used orally for breast cancer metastasis as well as the underlying mechanism involving the activation of D1 DR.The confocal immunofluorescence analysis demonstrated that D1 DR was up-regulated by C17.The cell survival and colony formation were inhibited by C17 through the detection by Sulforhodamine B colorimetric(SRB)assay and colony formation assay,respectively.Results from both wound healing assay and transwell assay demonstrated that C17 inhibited the migration of 4T1 cells.Flow cytometry analysis indicated that C17 significantly reduced the CSC frequency.In addition,C17 could inhibit the lung metastasis in 4T1 orthotopic mouse model of breast cancer without obvious toxicity,and it could up-regulate the expression of intratumoral E-cadherin and down-regulate the expressions of Snail and N-cadherin in primary breast tumor,which might be related to the activation of D1 DR.Our findings provided a potential candidate compound for the treatment of metastatic breast cancer with good compliance and safety.展开更多
Near infrared(NIR) light-driven nitric oxide(NO) release nano-platform based on upconversion nanoparticles(UCNPs) and light sensitive NO precursor Roussin's black salt(RBS) was fabricated to generate NO upon 808 n...Near infrared(NIR) light-driven nitric oxide(NO) release nano-platform based on upconversion nanoparticles(UCNPs) and light sensitive NO precursor Roussin's black salt(RBS) was fabricated to generate NO upon 808 nm irradiation. The application of 808 nm laser as the excitation source could achieve better penetration depth and avoid overheating problem. The combination of UCNPs and RBS could realize the on-demand release of NO at desired time and location by simply controlling the output of NIR laser.Cellular uptake results showed that more nanoparticles were internalized in cancer stem-like cells(CSCs)rather than non-CSCs. Therefore, a synergistic cancer therapy strategy to eradicate both CSCs and nonCSCs simultaneously was developed. Traditional chemo-drug could inhibit non-CSCs but has low killing efficiency in CSCs. However, we found that the combination of NO and chemotherapy could efficiently inhibit CSCs in bulk cells, including inhibiting mammosphere formation ability, decreasing CD44^+/CD24^- subpopulation and reducing tumorigenic ability. The mechanism studies confirmed that NO could not only induce apoptosis but also increase drug sensitivity by declining drug efflux in CSCs. This UCNPsbased platform may provide a new combinatorial strategy of NO and chemotherapy to improve cancer treatment.展开更多
Background:Cancer stem-like cells(CSCs)are a small subset of cells in tumors that exhibit self-renewal and differentiation properties.CSCs play a vital role in tumor formation,progression,relapse,and therapeutic resis...Background:Cancer stem-like cells(CSCs)are a small subset of cells in tumors that exhibit self-renewal and differentiation properties.CSCs play a vital role in tumor formation,progression,relapse,and therapeutic resistance.B7-H3,an immunoregulatory protein,has many protumor functions.However,little is known about the mechanism underlying the role of B7-H3 in regulating gastric cancer(GC)stemness.Our study aimed to explore the impacts of B7-H3 on GC stemness and its underlying mechanism.Methods:GC stemness influenced by B7-H3 was detected both in vitro and in vivo.The expression of stemness-related markers was examined by reverse transcription quantitative polymerase chain reaction,Western blotting,and flow cytometry.Sphere formation assay was used to detect the sphere-forming ability.The underlying regulatory mechanism of B7-H3 on the stemness of GC was investigated by mass spectrometry and subsequent validation experiments.The signaling pathway(Protein kinase B[Akt]/Nuclear factor erythroid 2-related factor 2[Nrf2]pathway)of B7-H3 on the regulation of glutathione(GSH)metabolism was examined by Western blotting assay.Multi-color immunohistochemistry(mIHC)was used to detect the expression of B7-H3,cluster of differentiation 44(CD44),and Nrf2 on human GC tissues.Student’s t-test was used to compare the difference between two groups.Pearson correlation analysis was used to analyze the relationship between two molecules.The Kaplan-Meier method was used for survival analysis.Results:B7-H3 knockdown suppressed the stemness of GC cells both in vitro and in vivo.Mass spectrometric analysis showed the downregulation of GSH metabolism in short hairpin B7-H3 GC cells,which was further confirmed by the experimental results.Meanwhile,stemness characteristics in B7-H3 overexpressing cells were suppressed after the inhibition of GSH metabolism.Furthermore,Western blotting suggested that B7-H3-induced activation of GSH metabolism occurred through the AKT/Nrf2 pathway,and inhibition of AKT signaling pathway could suppress not o展开更多
The existence of cancer stem cells, stem-like cancer cells （SLCCs）, or tumor-initiating cells is considered as the cause of tumor formation and recurrence, indicating the importance of studying novel therapy that ta...The existence of cancer stem cells, stem-like cancer cells （SLCCs）, or tumor-initiating cells is considered as the cause of tumor formation and recurrence, indicating the importance of studying novel therapy that targets SLCCs. The origin of SLCCs is controversial because of two competing hypotheses： SLCCs are either transformed from tissue adult stem cells or dedifferentiated from transformed progenitor cells. Our previous research demonstrates that SLCCs are inducible by increasing genomic instability in cancer cells. In this study, to block the emergence of SLCCs, aminoethyl isothiourea （AET）, a compound that clears free radicals and is used to protect patients from radioactive exposure, was used as an agent that maintains genomic stability in combination with mitomycin C （MMC）, a commonly used chemotherapeutic drug that damages DNA. Using a rabbit tumor model with VX2 hepatic carcinoma, we found that MMC alone increased lung metastases and disadvantaged survival outcome, but the combination of MMC and AET reversed this effect and even prolonged overall survival. Moreover, in a VX2 xenograft model by immunocompromised mice, MMC alone enriched tumor-initiating cells, but the administration of MMC in combination with AET eliminated tumor cells effectively. Furthermore, MMC alone enhanced genomic instability, but MMC combined with AET attenuated the extent of genomic instability in primary VX2 tumor tissue. Taken together, our data suggest that the genomic protector AET can inhibit the induction of SLCCs, and this combination treatment by AET and cytotoxic agents should be considered as a promising strategy for future clinical evaluation.展开更多
In order to see whether carbon ion (C-ion) beams have a biological advantage over X-rays, studies were designed to examine the effects of C-ion beams on radiosensitivity in X-ray resistant cells. Clinically relevant X...In order to see whether carbon ion (C-ion) beams have a biological advantage over X-rays, studies were designed to examine the effects of C-ion beams on radiosensitivity in X-ray resistant cells. Clinically relevant X-ray resistant SAS-R cells derived from human tongue cancer SAS cells were used. The cells were exposed to X-rays or Spread-Out Bragg peak (SOBP) beam C-ions. Cell survival was measured using a modified high-density survival assay. Cell survival signaling and cell death signaling were analyzed using flow cytometry. The cells were labeled with putative cancer stem cell markers such as CD44 and CD326. SAS-R cells were 1.6 times more radioresistant than SAS cells after exposure to X-rays. Cell survival was similar in each cell line after exposure to C-ion beams. SAS-R cells displayed enhanced cell survival signaling when compared to SAS cells under normal conditions. On the other hand, the phosphorylation of AKT-related proteins decreased and polycaspase activities were enhanced when cells were irradiated with C-ion beams in both cell lines. More CD44 and CD326 positive cells were seen in SAS-R cells than in SAS cells. Moreover, the marker positive cell numbers significantly decreased after exposure to C-ion beams when compared to X-rays at iso-survival doses in SAS-R cells. C-ion beams efficiently induced cell killing in X-ray resistant cells which displayed activated cell survival signaling and contained more numerous cancer stem-like cells.展开更多
基金supported in part by National Natural Science Foundation of China(No.90406023)Science Foundation of Southeast University(No.9223001446)Science Foundation of Jiangsu Province Hygienical Division,China(No.16230005777).
文摘In current study, cancer stem-like cells in the murine melanoma B16F10 cells were investigated. CD phenotypes of the B16F10 cells were analyzed by flow cytometry, and the specific CD phenotype cells from the B16F10 cells were isolated by MACS. Then we used colony formation assay in soft agar media, the cell growth assay in serum-free culture media as well as the tumorigenicity investigation of the specific CD phenotype cells in C57BL/6 mice, respectively, to identify cancer stem-like cells in the B16F10 cells. The results showed that the B16F10 cells could form spherical clones in serum-free culture media, and the rate of clonegenesis of CD133^＋, CD44^＋ and CD44^＋CD133^＋ cells was higher than that of CD133^-, CD44^- and CD44^＋CD133^＋ cells in soft agar media, respectively. The tumorigenic potential of CD133^＋, CD44^＋, CD44^＋CD133^＋ cells and CD44^＋CD133^＋CD24^＋ cells was stronger than that of CD133^-, CD44^-, CD44^＋CD133^- cells and CD44^＋CD133^＋CD24^- cells in mice, respectively. In conclusion, the CD44^＋CD133^＋CD24^＋ cells have some biological properties of cancer stem-like cells or are highly similar to the characteristics of cancer stem cells （CSC）. These results provide an important method for identifying cancer stem-like cells in B16F10 cells and for further cancer target therapy. Cellular ＆ Molecular Immunology.
基金supported in part by the Foundation of State Key Laboratory of Reproductive Medicine,the project funded by the Priority Academic Program Development(PAPD) of Jiangsu Higher Education Institutionsthe National Natural Science Foundation of China(No. 30930080 and 81161120537)
文摘Gastric cancer stem-like cells（GCSCs） have been identified to possess the ability of self-renewal and tumor initi-ation.However,the mechanisms involved remain largely unknown.Here,we isolated and characterized the GCSCs by side population（SP） sorting procedure and cultured sphere cells（SC） from human gastric cancer cell lines SGC-7901,BGC-823,MGC-803,HGC-27 and MKN-28.The sorting and culture assay revealed that SP cells proliferated in an asymmetric division manner.In addition,SP cells exhibited a higher potential of spheroid colony formation and greater drug resistance than non-SP cells（NSP）.Moreover,the SC were found with enhanced capabilities of drug resistance in vitro and tumorigenicity in vivo.Sox2 mRNA and protein was highly and significantly overex-pressed in the SP cells and SC.Importantly,downregulation of Sox2 with siRNA obviously reduced spheroid colony formation and doxorubicin efflux,as well as increased apoptosis rate in sphere cells in vitro and suppressed tumori-genicity in vivo.These results suggest that both SP cells and cultured SC enrich with GCSCs and that Sox2 plays a pivotal role in sustaining stem cell properties and might be a potential target for gastric cancer therapy.
基金This work was supported by grants from National Natural Science Foundation of China （No. 300740076）, Jiangsu Six Kinds of Outstanding Talents Foundation （No. 2006B070）, Jiangsu Science and Education for Health Foundation （No. RC2007054） and Jiangsu Province Post-doctor Foundation （No. 0601048B）.
文摘Background A satisfactory animal model of breast cancer metastasizing to bone is unavailable. In this study, we used human breast cancer stem-like cells and human bone to build a novel “human-source” model of human breast cancer skeletal metastasis. Methods Human breast cancer stem-like cells, the CD44^＋/CD24^-/lower subpopulation, was separated and cultured. Before injection with the stem-like cells, mice were implanted with human bone in the right or left dorsal flanks. Animals in Groups A, B, and C were injected with 1×10^5, 1×10^6 human breast cancer stem-like cells, and 1×10^6 parental MDA-MB-231 cells, respectively. A positive control group （D） without implantation of human bone was also injected with 1×10^6 MDA-MB-231 cells. Immunohistochemistry was performed for determination of CD34, CD105, smooth muscle antibody, CD44, CD24, cytokine, CXC chemokine receptor-4 （CXCR4）, and osteopontin （OPN）. mRNA levels of CD44, CD24, CXCR4, and OPN in bone metastasis tissues were analyzed by real-time quantitative polymerase chain reaction （PCR）.Results Our results demonstrated that cells in implanted human bones of group B, which received 1×10^6 cancer stem-like cells, stained strongly positive for CD44, CXCR4, and OPN, whereas those of other groups showed no or minimum staining. Moreover, group B had the highest incidence of human bone metastasis （77.8%, P=0.0230） and no accompaniment of other tissue metastasis. The real-time PCR showed an increase of CD44, CXCR4, and OPN mRNA in metastatic bone tissues in group B compared with those of groups C and D, however the expression of CD24 mRNA in group B were the lowest. Conclusions In the novel “human source” model of breast cancer, breast cancer stem-like cells demonstrated a higher human bone-seeking ability. Its mechanism might be related to the higher expressions of CD44, CXCR4, and OPN, and the lower expression of CD24 in breast cancer stem-like cells.
基金supported by the Student’s Platform for Innovation and Entrepreneurship Training Program, China (Grant No. 201510062001)
文摘Objective: Vasculogenic mimicry(VM) channels that are lined by tumor cells are a functional blood supply in malignant tumors.However, the role of VM-initiating cells remains poorly understood. Cancer stem-like cells(CSCs) are positively correlated with VM. In this study, triple-negative breast cancer(TNBC) enriched with CSCs was used to investigate the relationship between VM and CSCs.Methods: The expression of several CSC markers was detected by immunohistochemistry in 100 human breast cancer samples.The clinical significance of CSC markers and the relationship between VM, CSCs, breast cancer subtypes, and VM-associated proteins were analyzed. CD133+ and ALDH+ human and mouse TNBC cells were isolated by FACS to examine the ability of VM formation and the spatial relationship between VM and CSCs.Results: CSCs were associated with TNBC subtype and VM in human invasive breast cancer. CSCs in TNBC MDA-MB-231 cells formed more VM channels and expressed more molecules promoting VM than the non-TNBC MCF-7 cells in vitro. MDA-MB-231 cells that encircled VM channels on Matrigel expressed CD133. Moreover, CSCs were located near VM channels in the 3D reconstructed blood supply system in human TNBC grafts. The CD133+ and ALDH+ cells isolated from TA2 mouse breast cancer formed more VM channels in vivo.Conclusions: CSCs line VM channels directly. Additionally, CSCs provide more VM-related molecules to synergize VM formation. The signaling pathways that control CSC differentiation may also be potential treatment targets for TNBC.
基金the financial support by the National Natural Science Foundation of China (Nos. 81770281, 81730012, and 81861128022)。
文摘HYD-PEP06,an endostatin-modified polypeptide,has been shown to produce effective anticolorectal carcinoma effects through inhibiting epithelial-mesenchymal transition(EMT).However,whether HYD-PEP06 has similar suppressive effect on hepatocellular carcinoma(HCC) remained unknown.In this study,HYD-PEP06 inhibited metastasis and EMT but not proliferation in vitro.Cignal finder pathway reporter array and Western blot analysis revealed that HYD-PEP06 suppressed HCCLM3 cell metastasis and EMT by inhibiting the PI3 K/AKT pathway.Moreover,HYD-PEP06 exerted antimetastasis effects in HepG2 cancer stem-like cells(CSCs) via suppressing the WNT/β-catenin signaling pathway.Finally,in HCCLM3 tumor-bearing BALB/c nu/nu nude mice,HYD-PEP06 substantially suppressed tumor growth,lung metastasis and HCC progress.Our results suggest that HYD-PEP06 inhibits the metastasis and EMT of HCC and CSCs as well,and thus has the potential as an agent for HCC treatment.
基金Supported by Grants from the University of Limoges,Limoges University Hospital,La Ligue Contre le Cancer and the Région Limousin,which was given financial by the ComitéOrientation Recherche Cancer(to Perraud A,Christou N and Akil H)
文摘Carcinogenesis is a multistep process that requires the accumulation of various genetic and epigenetic aberrations to drive the progressive malignant transformation of normal human cells.Two major hallmarks of carcinogenesis that have been described are angiogenesis and the stem cell characteristic of limitless replicative potential.These properties have been targeted over the past decade in the development of therapeutic treatments for colorectal cancer(CRC),one of the most commonly diagnosed and lethal cancers worldwide.The treatment of solid tumor cancers such as CRC has been challenging due to the heterogeneity of the tumor itself and the chemoresistance of the malignant cells.Furthermore,the same microenvironment that maintains the pool of intestinal stem cells that contribute to the continuous renewal of the intestinal epithelia also provides the necessary conditions for proliferative growth of cancer stem-like cells.These cancer stem-like cells are responsible for the resistance to therapy and cancer recurrence,though they represent less than 2.5%of the tumor mass.The stromal environment surrounding the tumor cells,referred to as the tumor niche,also supports angiogenesis,which supplies the oxygen and nutrients needed for tumor development.Anti-angiogenic therapy,such as with bevacizumab,a monoclonal antibody against vascular-endothelial growth factor,significantly prolongs the survival of metastatic CRC patients.However,such treatments are not completely curative,and a large proportion of patient tumors retain chemoresistance or show recurrence.This article reviews the current knowledge regarding the molecular phenotype of CRC cancer cells,as well as discusses the mechanisms contributing to their maintenance.Future personalized therapeutic approaches that are based on the interaction of the carcinogenic hallmarks,namely angiogenic and proliferative attributes,could improve survival and decrease adverse effects induced by unnecessary chemotherapy.
基金Funding This work was supported by National Basic Research Program of China （2013CB967401 and 2010CB945001）, and the National Nature Science Foundation of China （81370675 and 81121001）.
文摘Pluripotent stem cells derived from neonatal or adult testes are a useful tool to examine the mechanisms of pluripotency and a resource for cell-based therapies. However, therapies usingthese cells will only benefit males but not females. Recently, female germline stem cells （FGSCs） were discovered in ovaries. Whether FGSCs can be converted into pluripotent stem cells, similar to spermatogonial stem cells, is unknown. Here, we demonstrate that female embryonic stem-like cells （fESLCs） can be generated within 1 month from the stably proliferating FGSCs cultured in embryonic stem cell （ESC） medium, fESLCs exhibit properties similar to those of ESCs in terms of marker expression and differentiation potential. Thus, our findings suggest that generation of patient-specific fESLCs is feasible and provides a foundation for personalized regenerative applications.
基金Ministry of Science and Technology of the People’s Republic of China,Grant/Award Number:2020YFA0803300National Natural Science Foundation of China,Grant/Award Numbers:82188102,82030074,82122053,32100574+10 种基金Beijing Municipal Science&Technology Commission,Grant/Award Number:Z191100006619115R&D Program of Beijing Municipal Education commission,Grant/Award Number:KJZD20191002302CAMS Innovation Fund for Medical Science,Grant/Award Numbers:2021-1-I2M-012,2021-I2M-1-067Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences,Grant/Award Number:2021-PT310-001Key-Area Research and Development Program of Guangdong Province,Grant/Award Number:2021B0101420005Sanming Project of Medicine in Shenzhen,Grant/Award Numbers:SZSM201612097,SZSM201812062Aiyou Foundation,Grant/Award Number:KY201701Natural Science Foundation of Shandong Province,Grant/Award Number:ZR2020QH191Zhejiang Natural Science Foundation-Key Project,Grant/Award Number:LD21H160003Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang,Grant/Award Number:2019R01001Zhimin Lu is the Kuancheng Wang Distinguished Chair。
文摘Background:Maintenance of cancer stem-like cell(CSC)stemness supported by aberrantly regulated cancer cell metabolism is critical for CSC self-renewal and tumor progression.As a key glycolytic enzyme,hexokinase 2(HK2)plays an instrumental role in aerobic glycolysis and tumor progression.However,whether HK2 directly contribute to CSC stemness maintenance in small cell lung cancer(SCLC)is largely unclear.In this study,we aimed to investgate whether HK2 independent of its glycolytic activity is directly involved in stemness maintenance of CSC in SCLC.Methods:Immunoblotting analyses were conducted to determine the expression of HK2 in SCLC CSCs and their differentiated counterparts.CSC-like properties and tumorigenesis of SCLC cells with or without HK2 depletion or overexpression were examined by sphere formation assay and xenograft mouse model.Immunoprecipitation and mass spectrometry analyses were performed to identify the binding proteins of CD133.The expression levels of CD133-associated and CSC-relevant proteins were evaluated by immunoblotting,immunoprecipitation,immunofluorescence,and immunohistochemistry assay.RNA expression levels of Nanog,POU5F1,Lin28,HK2,Prominin-1 were analyzed through quantitative reverse transcription PCR.Polyubiquitination of CD133 was examined by in vitro or in vivo ubiquitination assay.CD133+cells were sorted by flow cytometry using an anti-CD133 antibody.Results:We demonstrated that HK2 expression was much higher in CSCs of SCLC than in their differentiated counterparts.HK2 depletion inhibited CSC stemness and promoted CSC differentiation.Mechanistically,nonmitochondrial HK2 directly interacted with CD133 and enhanced CD133 expression without affecting CD133 mRNA levels.The interaction of HK2 and CD133 promoted the binding of the deubiquitinase ubiquitin-specific protease 11(USP11)to CD133,thereby inhibiting CD133 polyubiquitylation and degradation.HK2-mediated upregulation of CD133 expression enhanced the expression of cell renewal regulators,SCLC cell stemness,and tumor grow
基金This study was supported by grants from the National Natural Science Foundation of China （81372707, 81072070）, Shanghai Committee of Sdence and Technology （15140902200, 16140903000）, and the Natural Science Foundation of Shanghai （15ZR1405600）.
文摘Meningiomas are the most common primary tumors in central nervous system. While recent studies have revealed genetic clues to lower grade human meningiomas, the molecular determinants driving the progression and recurrence of anaplastic meningi- oma, the most malignant subtype with a low prevalence but high morbidity, are still poorly understood. It has been proposed that high recurrence rates of malignant meningiomas are linked to cancer stem cells. Indeed, tumor stem-Uke cells have been iso- lated from various meningioma subtypes, but never been obtained from anaplastic meningioma, in this study, we successfully isolated stem-Uke cells from human anaplastic meningioma. These cells are capable of forming spheres and initiating xenograft tumors that recapitulate anaplastic meningioma phenotypes, and thus could serve as an in vitro model for malignant meningi- omas. KLF4, a transcription factor known for its role in sternness maintenance, was identified as one of the most frequently mutated genes in the benign secretory meningioma. Interestingly, we found that KLF4 is downregulated in anaplastic meningi- oma compared with low-grade meningioma subtypes. By manipulating KLF4 expression in anaplastic meningioma stem-like cells, we demonstrated that KLF4 acts as a tumor suppressor during malignant progression in meningioma, affecting apoptosis, prolif- eration, invasion, and cell cycle. These results suggest a potential therapeutic value of KLF4 for clinical intervention of anaplastic meningioma.
文摘BACKGROUND Over the years,the numbers of treatment options for colorectal cancer(CRC)have increased,leading to notable improvements in the overall survival of CRC patients.Although therapy may initially yield positive results,the development of drug resistance can result in treatment failure and cancer recurrence.This resistance is often attributed to the presence of cancer stem cells(CSCs).These CSCs not only contribute to therapeutic resistance but also play crucial roles in the initiation and development of tumor metastasis.AIM To investigate the antitumor effects of SH-4-54,which are mediated by targeting CSCs relative to treatment outcomes.METHODS CSCs were enriched by culturing CRC cells in serum-free medium.Hallmarks of stemness and IL-6/JAK2/STAT3 signaling were detected by Western blotting.Indicators of CSC malignancy,including proliferation,invasion,and tumor formation,were measured.RESULTS In this study,we employed SH-4-54,which exhibits anticancer activity in solid tumors through targeting the SH2 domain of both the signal transducer and activator of transcription(STAT)3 and the STAT5,and evaluated its effects on stemness and chemoresistance in colorectal CSCs.As expected,SH-4-54 treatment inhibited the phosphorylation of STAT3(p-STAT3)and decreased the percentage of ALDH1A1-positive CRC cells.The addition of SH-4-54 dissociated colorectal spheroids and decreased the expression of stemness markers,including ALDH1A1,CD44 and Nanog.SH-4-54 treatment decreased IL-6/JAK2/STAT3 signaling by inhibiting p-STAT3 and thus inhibited spheroid formation by SW480 and LoVo cells.Moreover,SH-4-54 treatment inhibited indicators of malignancy,including cell proliferation,invasion,and tumor formation,in CSCs in vitro and in vivo.Notably,SH-4-54 treatment significantly increased chemosensitivity to oxaplatin.CONCLUSION Taken together,these results indicate that SH-4-54 is a promising molecule that exerts antitumor effects on colorectal CSCs by inhibiting STAT3 signaling.
文摘Previous studies have demonstrated that spheroid type cells grown under suspension culture conditions have cancer stem cell（CSC） traits in a number of cancers, but this phenomenon has not yet been reported in the VX2 rabbit oral cancer model. Hence, this study aimed to study the spheroid cells from VX2 rabbit buccal squamous cell carcinomas（SCCs） and assess their CSC characteristics. Five adult male New Zealand white outbred rabbits were used to generate VX2 rabbit buccal SCC. Sphere-forming cell culture was performed for the VX2 rabbit buccal SCC specimens. The self-renewal capability; cluster of designation（CD） 44, CD133, acetaldehyde dehydrogenase 1（ALDH1）, B cell-specific Moloney murine leukemia virus integration site 1（Bmi-1）, Nestin, octamer-binding transcription factor 4（Oct4）and reduced expression protein-1（Rex-1） expression with reverse transcription-polymerase chain reaction（RT-PCR）; chemoresistance to cisplatin and 5-fluorouracil; and in vivo tumorigenicity of spheroid cell transplantation in nude mice were evaluated to determine the CSC characteristics of the resulting spheroid cells. We successfully obtained spheroid cells from the VX2 rabbit OSCC tissues. The spheroid cells exhibited CSC traits, including the expression of CSC and stem cell markers（CD44, Bmi-1, Nestin, Oct4 and Rex-1）, capacity to generate new spheroid colonies within 1 week of reseeding from single-dissociated spheroid cells, chemoresistance capacity and generation of tumour xenografts（with histological features resembling those of the original VX2 rabbit buccal SCC） from the transplantation of 103 undifferentiated spheroid cells into nude mice. In summary, we demonstrated that spheroid cells with CSC cell traits can be derived from VX2 rabbit buccal SCCs, indicating that this animal cancer model is applicable for studying CSCs in human oral cancers.
基金Supported by National Natural Science Foundation of China,No.30972962
文摘AIM: To investigate the expression of miR-210 and the role it plays in the cell cycle to regulate radioresistance in oesophageal squamous cell carcinoma(ESCC). METHODS: Mi R-210 expression was evaluated in 37 pairs of ESCC tissues and matched para-tumorous normal oesophageal tissues from surgical patients who had not received neoadjuvant therapy, and in the cells of two novel radioresistant cell lines, TE-1R and Eca-109 R, using quantitative reverse transcription-polymerase chain reaction(q RT-PCR). The transient up-regulation of mi R-210 expression in TE-1R and Eca-109 R cells was studied using liposomes and was confirmed using qR T-PCR. The rate of cell survival after a series of radio-treatment doses was evaluated using the cloneformation assay. Flow cytometry was used to detect the changes to the cell cycle patterns due to radiation treatment. RT-PCR and Western blot were used to detect the expression of ataxia telangiectasia mutated(ATM) and DNA dependent protein kinase(DNA-PKcs) after irradiation, and the cell sphere formation assay was used to evaluate the proliferative ability of the cancer stem-like cells.RESULTS: The level of mi R-210 expression was significantly decreased, by 21.3% to 97.2%, with the average being 39.2% ± 16.1%, in the ESCC tissues of most patients(81.1%, 30 of 37 vs patients with high mi R-210 expression, P < 0.05). A low level of expression of miR-210 was correlated with a poorly differentiated pathological type(P < 0.01) but was not correlated with the T-stage or lymph node infiltration(both P > 0.05). Early local recurrences(< 18 mo, n = 19) after radiotherapy were significantly related with low miR-210 expression(n = 13, P < 0.05). The level of mi R-210 was decreased by approximately 73%(vs TE-1, 0.27 ± 0.10, P < 0.01) in the established radioresistant TE-IR cell line and by 52%(vs Eca-109, 0.48 ± 0.17, P < 0.05) in the corresponding Eca-109 R line. Transient transfection with a mi R-210 precursor increased the level of mi R-210 expression, leading to a significant increase in
基金supported by the Natural Science Foundation of Anhui Province(2208085MH250,2308085MH272)National Key Research and Development Program of China(2021YFF1201000)+2 种基金Natural Science Research Project of the Anhui Educational Committee(2023AH040404,2023AH053402)Anhui Provincial Health and Medical Research Project(AHWJ2023A10143)Research Funds of Centre for Leading Medicine and Advanced Technologies of IHM(2023IHM01043)。
文摘Objective Vasculogenic mimicry(VM)is a novel vasculogenic process integral to glioma stem cells(GSCs)in glioblastoma(GBM).However,the relationship between VM and ataxia-telangiectasia mutated(ATM)serine/threonine kinase activation,which confers chemoradiotherapy resistance,remains unclear.Methods We investigated VM formation and phosphorylated ATM(pATM)levels by CD31/GFAPperiodic acid-Schiff dual staining and immunohistochemical staining in 145 GBM specimens.Glioma stem-like cells(GSLCs)derived from the formatted spheres of U87 and U251 cell lines and their pATM level and VM formation ability were examined using western blot and three-dimensional culture.For the examination of the function of pATM in VM formation by GSLCs,ATM knockdown by shRNAs and deactivated via ATM phosphorylation inhibitor KU55933 were studied.Results VM and high pATM expression occurred in 38.5% and 41.8% of tumors,respectively,and were significantly associated with reduced progression-free and overall survival.Patients with VM-positive GBMs exhibited higher pATM levels(r_(s)=0.425,P=0.01).The multivariate analysis established VM as an independent negative prognostic factor(P=0.002).Furthermore,GSLCs expressed high levels of pATM and formed vascular-like networks in vitro.ATM inactivation or knockdown hindered VM-like network formation concomitant with the downregulation of pVEGFR-2,VE-cadherin,and laminin B2.Conclusion VM may predict a poor GBM prognosis and is associated with pATM expression.We propose that pATM promotes VM through extracellular matrix modulation and VE-Cadherin/pVEGFR-2 activation,thereby highlighting ATM activation as a potential target for enhancing anti-angiogenesis therapies for GBM.
文摘BACKGROUND Limonin is one of the most abundant active ingredients of Tetradium ruticarpum.It exerts antitumor effects on several kinds of cancer cells.However,whether limonin exerts antitumor effects on colorectal cancer(CRC)cells and cancer stem-like cells(CSCs),a subpopulation responsible for a poor prognosis,is unclear.AIM To evaluate the effects of limonin on CSCs derived from CRC cells.METHODS CSCs were collected by culturing CRC cells in serum-free medium.The cytotoxicity of limonin against CSCs and parental cells(PCs)was determined by cholecystokinin octapeptide-8 assay.The effects of limonin on stemness were detected by measuring stemness hallmarks and sphere formation ability.RESULTS As expected,limonin exerted inhibitory effects on CRC cell behaviors,including cell proliferation,migration,invasion,colony formation and tumor formation in soft agar.A relatively low concentration of limonin decreased the expression stemness hallmarks,including Nanog andβ-catenin,the proportion of aldehyde dehydrogenase 1-positive CSCs,and the sphere formation rate,indicating that limonin inhibits stemness without presenting cytotoxicity.Additionally,limonin treatment inhibited invasion and tumor formation in soft agar and in nude mice.Moreover,limonin treatment significantly inhibited the phosphorylation of STAT3 at Y705 but not S727 and did not affect total STAT3 expression.Inhibition of Nanog andβ-catenin expression and sphere formation by limonin was obviously reversed by pretreatment with 2μmol/L colievlin.CONCLUSION Taken together,these results indicate that limonin is a promising compound that targets CSCs and could be used to combat CRC recurrence and metastasis.
基金Beijing Municipal Natural Science Foundation(Grant No.7192100)Innovation Team of Ministry of Education(Grant No.BMU2017TD003)
文摘Breast cancer is the most common cancer in females with extremely high lethality mainly due to the occurrence of metastasis,which is closely related to cancer stem-like cells(CSCs).It has been reported that CSC frequency in drug-resistant breast cancer and non-small cell lung cancer is reduced by activating dopamine D1 receptor(D1 DR).In the present study,we aimed to investigate the effect of a compound C17 that can be used orally for breast cancer metastasis as well as the underlying mechanism involving the activation of D1 DR.The confocal immunofluorescence analysis demonstrated that D1 DR was up-regulated by C17.The cell survival and colony formation were inhibited by C17 through the detection by Sulforhodamine B colorimetric(SRB)assay and colony formation assay,respectively.Results from both wound healing assay and transwell assay demonstrated that C17 inhibited the migration of 4T1 cells.Flow cytometry analysis indicated that C17 significantly reduced the CSC frequency.In addition,C17 could inhibit the lung metastasis in 4T1 orthotopic mouse model of breast cancer without obvious toxicity,and it could up-regulate the expression of intratumoral E-cadherin and down-regulate the expressions of Snail and N-cadherin in primary breast tumor,which might be related to the activation of D1 DR.Our findings provided a potential candidate compound for the treatment of metastatic breast cancer with good compliance and safety.
基金supported by the National Basic Research Program of China(2016YFA2021600,2016YFA0202104,and2015CB932104)the National Natural Science Foundation of China(31571015,11621505,and 21320102003)Chinese Academy of Sciences Youth Innovation Promotion Association(2013007)
文摘Near infrared(NIR) light-driven nitric oxide(NO) release nano-platform based on upconversion nanoparticles(UCNPs) and light sensitive NO precursor Roussin's black salt(RBS) was fabricated to generate NO upon 808 nm irradiation. The application of 808 nm laser as the excitation source could achieve better penetration depth and avoid overheating problem. The combination of UCNPs and RBS could realize the on-demand release of NO at desired time and location by simply controlling the output of NIR laser.Cellular uptake results showed that more nanoparticles were internalized in cancer stem-like cells(CSCs)rather than non-CSCs. Therefore, a synergistic cancer therapy strategy to eradicate both CSCs and nonCSCs simultaneously was developed. Traditional chemo-drug could inhibit non-CSCs but has low killing efficiency in CSCs. However, we found that the combination of NO and chemotherapy could efficiently inhibit CSCs in bulk cells, including inhibiting mammosphere formation ability, decreasing CD44^+/CD24^- subpopulation and reducing tumorigenic ability. The mechanism studies confirmed that NO could not only induce apoptosis but also increase drug sensitivity by declining drug efflux in CSCs. This UCNPsbased platform may provide a new combinatorial strategy of NO and chemotherapy to improve cancer treatment.
基金supported by Suzhou Special Project on Clinical Key Diseases Treatment Technology of Suzhou Commission of Health(No.LCZX201803)People’s Livelihood and Science and Technology project of Department of Science and Technology of Suzhou(No.SS2019059)+5 种基金Jiangsu Provincial Medical Key Discipline(No.ZDXK202246)Key Project of Jiangsu Provincial Health Commission(No.zd2021050)the Natural Science Foundation of the Jiangsu Higher Education Institutions of China(No.20KJA310005)Key Project of Medical Research of Jiangsu Commission of Health(No.ZDA2020008)National Natural Science Foundation of China(No.81802843)Social Development Project of Department of Science and Technology of Jiangsu Province(No.BE2019667)
文摘Background:Cancer stem-like cells(CSCs)are a small subset of cells in tumors that exhibit self-renewal and differentiation properties.CSCs play a vital role in tumor formation,progression,relapse,and therapeutic resistance.B7-H3,an immunoregulatory protein,has many protumor functions.However,little is known about the mechanism underlying the role of B7-H3 in regulating gastric cancer(GC)stemness.Our study aimed to explore the impacts of B7-H3 on GC stemness and its underlying mechanism.Methods:GC stemness influenced by B7-H3 was detected both in vitro and in vivo.The expression of stemness-related markers was examined by reverse transcription quantitative polymerase chain reaction,Western blotting,and flow cytometry.Sphere formation assay was used to detect the sphere-forming ability.The underlying regulatory mechanism of B7-H3 on the stemness of GC was investigated by mass spectrometry and subsequent validation experiments.The signaling pathway(Protein kinase B[Akt]/Nuclear factor erythroid 2-related factor 2[Nrf2]pathway)of B7-H3 on the regulation of glutathione(GSH)metabolism was examined by Western blotting assay.Multi-color immunohistochemistry(mIHC)was used to detect the expression of B7-H3,cluster of differentiation 44(CD44),and Nrf2 on human GC tissues.Student’s t-test was used to compare the difference between two groups.Pearson correlation analysis was used to analyze the relationship between two molecules.The Kaplan-Meier method was used for survival analysis.Results:B7-H3 knockdown suppressed the stemness of GC cells both in vitro and in vivo.Mass spectrometric analysis showed the downregulation of GSH metabolism in short hairpin B7-H3 GC cells,which was further confirmed by the experimental results.Meanwhile,stemness characteristics in B7-H3 overexpressing cells were suppressed after the inhibition of GSH metabolism.Furthermore,Western blotting suggested that B7-H3-induced activation of GSH metabolism occurred through the AKT/Nrf2 pathway,and inhibition of AKT signaling pathway could suppress not o
文摘The existence of cancer stem cells, stem-like cancer cells （SLCCs）, or tumor-initiating cells is considered as the cause of tumor formation and recurrence, indicating the importance of studying novel therapy that targets SLCCs. The origin of SLCCs is controversial because of two competing hypotheses： SLCCs are either transformed from tissue adult stem cells or dedifferentiated from transformed progenitor cells. Our previous research demonstrates that SLCCs are inducible by increasing genomic instability in cancer cells. In this study, to block the emergence of SLCCs, aminoethyl isothiourea （AET）, a compound that clears free radicals and is used to protect patients from radioactive exposure, was used as an agent that maintains genomic stability in combination with mitomycin C （MMC）, a commonly used chemotherapeutic drug that damages DNA. Using a rabbit tumor model with VX2 hepatic carcinoma, we found that MMC alone increased lung metastases and disadvantaged survival outcome, but the combination of MMC and AET reversed this effect and even prolonged overall survival. Moreover, in a VX2 xenograft model by immunocompromised mice, MMC alone enriched tumor-initiating cells, but the administration of MMC in combination with AET eliminated tumor cells effectively. Furthermore, MMC alone enhanced genomic instability, but MMC combined with AET attenuated the extent of genomic instability in primary VX2 tumor tissue. Taken together, our data suggest that the genomic protector AET can inhibit the induction of SLCCs, and this combination treatment by AET and cytotoxic agents should be considered as a promising strategy for future clinical evaluation.
文摘In order to see whether carbon ion (C-ion) beams have a biological advantage over X-rays, studies were designed to examine the effects of C-ion beams on radiosensitivity in X-ray resistant cells. Clinically relevant X-ray resistant SAS-R cells derived from human tongue cancer SAS cells were used. The cells were exposed to X-rays or Spread-Out Bragg peak (SOBP) beam C-ions. Cell survival was measured using a modified high-density survival assay. Cell survival signaling and cell death signaling were analyzed using flow cytometry. The cells were labeled with putative cancer stem cell markers such as CD44 and CD326. SAS-R cells were 1.6 times more radioresistant than SAS cells after exposure to X-rays. Cell survival was similar in each cell line after exposure to C-ion beams. SAS-R cells displayed enhanced cell survival signaling when compared to SAS cells under normal conditions. On the other hand, the phosphorylation of AKT-related proteins decreased and polycaspase activities were enhanced when cells were irradiated with C-ion beams in both cell lines. More CD44 and CD326 positive cells were seen in SAS-R cells than in SAS cells. Moreover, the marker positive cell numbers significantly decreased after exposure to C-ion beams when compared to X-rays at iso-survival doses in SAS-R cells. C-ion beams efficiently induced cell killing in X-ray resistant cells which displayed activated cell survival signaling and contained more numerous cancer stem-like cells.
