Targeting STAT3 with SH-4-54 suppresses stemness and chemoresistance in cancer stem-like cells derived from colorectal cancer

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.

Sphere-forming-like cells(squamospheres) with cancer stem-like cell traits from VX2 rabbit buccal squamous cell carcinoma

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.

ATM Activation is Key in Vasculogenic Mimicry Formation by Glioma Stem-like Cells

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.

Limonin inhibits the stemness of cancer stem-like cells derived from colorectal carcinoma cells potentially via blocking STAT3 signaling

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.

Cancer stem-like cells directly participate in vasculogenic mimicry channels in triple-negative breast cancer

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.

Sox2 enhances the tumorigenicity and chemoresistance of cancer stem-like cells derived from gastric cancer

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.

Xiaotan Sanjie decoction attenuates tumor angiogenesis by manipulating Notch-1-regulated proliferation of gastric cancer stem-like cells

AIM: To determine the underlying mechanisms of action and influence of Xiaotan Sanjie (XTSJ) decoction on gastric cancer stem-like cells (GCSCs).

Hallmarks in colorectal cancer:Angiogenesis and cancer stem-like cells

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.

MiR-210 expression reverses radioresistance of stem-like cells of oesophageal squamous cell carcinoma

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 cell survival after radiotherapy(P < 0.05). Twenty-four hours after radiation, the proportion of pmiR-210 cells in S phase was increased(vs control cells, 30.4% ± 0.4%, and vs untreated TE-1R cells, 23.3% ± 0.7%, P < 0.05 for both). The levels of DNA-PKcs(0.21 ± 0.07) and ATM(0.12 ± 0.03, P < 0.05) proteins were significantly lower in the PmiR-210 cells than in control cells, but no differences were found in the levels of the corresponding mR NAs in the two cell types(P > 0.05 for all). Exogenous mi R-210 expression decreased the diameter of pmi R-210 cell spheres(vs control cells, 0.60 ± 0.14, P < 0.05).CONCLUSION: Mi R-210 expression is negatively correlated with the pathological type and the local survivalrate after radiotherapy, and high expression of miR-210 may reverse the radioresistance of ESCC stem-like cells.

Brain tumors:Cancer stem-like cells interact with tumor microenvironment

Cancer stem-like cells(CSCs)with potential of self-renewal drive tumorigenesis.Brain tumor microenvironment(TME)has been identified as a critical regulator of malignancy progression.Many researchers are searching new ways to characterize tumors with the goal of predicting how they respond to treatment.Here,we describe the striking parallels between normal stem cells and CSCs.We review the microenvironmental aspects of brain tumors,in particular composition and vital roles of immune cells infiltrating glioma and medulloblastoma.By highlighting that CSCs cooperate with TME via various cellular communication approaches,we discuss the recent advances in therapeutic strategies targeting the components of TME.Identification of the complex and interconnected factors can facilitate the development of promising treatments for these deadly malignancies.

Carbon-Ion Beams Efficiently Induce Cell Killing in X-Ray Resistant Human Squamous Tongue Cancer Cells

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.

Isolation and Identification of Cancer Stem-Like Cells from Murine Melanoma Cell Lines

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.

A novel mouse model of human breast cancer stem-like cells with high CD44^＋CD24^–/lower phenotype metastasis to human bone

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.

KLF4 is a tumor suppressor in anaplastic meningioma stem-like cells and human rneningiomas

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.

Near infrared light triggered nitric oxide releasing platform based on upconversion nanoparticles for synergistic therapy of cancer stem-like cells

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.

Drug resistance mechanisms of cancer stem-like cells and their therapeutic potential as drug targets

Despite of recent advances in cancer research and development of new anti-cancer drugs,tumor patients’prognoses have not yet been improved well enough.Treatment failure of tumors is highly attributed to the drug resistance of a small population of cancer cell known as cancer stem-like cells(CSCs).CSCs also have the self-renewal activity and differentiation potency,conferring strong tumorigenicity on them.Therefore,development of CSC targeting therapy is urgently needed in order to overcome possible recurrence and metastasis by them after therapy.CSCs show some characteristic features that are not observed in other differentiated cancer cells,which give them higher resistance against conventional chemotherapy or radiotherapy.Targeting such specific features could be useful for CSC eradication.This review will summarize the recent advances in the study of CSC characteristics along with the promising therapeutic strategies targeting them.

Conversion of female germline stem cells from neonatal and prepubertal mice into pluripotent stern cells

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.

Discovery and characterization of novel small-molecule inhibitors targeting nicotinamide phosphoribosyltransferase

Aim Nicotinamide phosphoribosyltransferase （NAMPT） plays an important role in cardiocerebro-vascu- lar physiopathological process. It is also a promising anticancer target. It is highly desirable to discover novel NAMPT inhibitors as anticancer drug candidates and understand their action mode. Methods We carried out a high throughput screening system on a chemical library of 24434 small-molecules. Anti-proliferative activity were further studied on active compounds. Isothermal titration calorimetry and cellular thermal shift assay were used to confirm the target specificity. Molecular modeling and site-directed mutagenesis studies were taken to investigate the binding mode of NAMPT inhibitor. Results Using high throughput screening system targeting NAMPT, we ob- tained a potent NAMPT inhibitor MS0 （China Patent ZL201110447488.9 ） with excellent in vitro activity （IC50 = 9.87 ± 1.15 nmol · L^-1 ） and anti-proliferative activity against multiple human cancer cell lines including stem-like cancer cells. Structure-activity relationship studies yielded several highly effective analogues. These inhibitors spe- cifically bound NAMPT, rather than downstream NMNAT. We provided the first chemical case using cellular ther- mal shift assay to explain the difference between in vitro and cellular activity; MS7 showed best in vitro activity （ IC50 = 0.93 ± 0.29 nmol · L^-1 ） but worst cellular activity due to poor target engagement in living cells. Site-di- rected mutagenesis studies identified important residues for NAMPT catalytic activity and inhibitor binding. Con- clusions The present study provides a class of novel NAMPT inhibitors for future development of anticancer a- gents. Our findings also contribute to deep understanding the action mode of NAMPT inhibitors and NAMPT basic research in cardiocerebro-vascular system.

Tumor growth and metastasis can be inhibited by maintaining genomic stability in cancer cells

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.

B7-H3 confers stemness characteristics to gastric cancer cells by promoting glutathione metabolism through AKT/pAKT/Nrf2 pathway

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 only GSH metabolism but also GC stemness.mIHC showed that B7-H3 was highly expressed in GC tissues and was positively correlated with the expression of CD44 and Nrf2.Importantly,GC patients with high expression of B7-H3,CD44,and Nrf2 had worse prognosis(P=0.02).Conclusions:B7-H3 has a regulatory effect on GC stemness and the regulatory effect is achieved through the AKT/Nrf2/GSH pathway.Inhibiting B7-H3 expression may be a new therapeutic strategy against GC.