Correction: Enterovirus 71 Protease 2Apro Targets MAVS to Inhibit Anti-Viral Type I Interferon Responses.

[This corrects the article DOI: 10.1371/journal.ppat.1003231.].

Polyphyllin VII as a potential medication for targeting epithelial mesenchymal transitionin in thyroid cancer.

The need for novel anti-thyroid cancer (TC) medications is urgent due to the rising incidence and metastatic rates of malignant TC. In this study, we investigated the effect of Polyphyllin VII (PPVII) to TC cells, and explored their potential mechanism. B-CPAP and TPC-1 cells, were used to analyze the antitumor activity of PPVII by quantifying cell growth and metastasis as well as to study the effect on epithelial mesenchymal transition (EMT). The results showed that PPVII dramatically reduced the capacity of B-CPAP and TPC-1 cells to proliferate and migrate in a dose-response manner. Following PPVII treatment of TC cells, the expression levels of E-cadherin progressively increased and were higher than the control group, while the expression levels of EMT-related genes Vimentin, N-cadherin, Slug, Zeb-1, and Foxe1 gradually declined and were lower than the control group. It was proposed that PPVII might prevent TC from undergoing EMT. The Foxe1 gene was shown to be significantly expressed in TC, and a statistically significant variation in Foxe1 expression was observed across clinical stages of the disease, according to a bioinformatics database study. There was a strong link between the expression of the Foxe1 gene and the EMT-related gene. In the meantime, TC cells' expression of Foxe1 can be inhibited by PPVII. In conclusion, our results showed that PPVII may as a potential medication for targeting EMT in thyroid cancer.

LncRNA H19 Participates in Leukemia Inhibitory Factor Mediated Stemness Promotion in Colorectal Cancer Cells.

Colorectal cancer (CRC) is a common human malignancy and the third leading cause of cancer-related death worldwide. Cancer stem cells (CSCs) were considered to play important roles in the genesis and development of many tumors. In recent years, it has been observed that leukemia inhibitory factor (LIF) might be involved in the regulation of stemness in cancer cells. In this study, we observed that LIF could increase the spheroid formation and stemness marker expression (inculding Nanog and SOX2) in CRC cell lines, such as HCT116 and Caco2 cells. Meanwhile, we also observed that LIF could upregulate LncRNA H19 expression via PI3K/AKT pathway. Knockdown of the expression of LncRNA H19 could decrease the spheroid formation and SOX2 expression in LIF-treated HCT116 and Caco2 cells, and thereby LncRNA H19 knockdown could compensate for the stemness enhancement effects induced by LIF. Our results indicated that LncRNA H19 might participate in the stemness promotion of LIF in CRC cells.

Baricitinib relieves DSS-induced ulcerative colitis in mice by suppressing the NF-κB and JAK2/STAT3 signalling pathways.

Ulcerative colitis (UC) is a relapsing inflammatory disease with a unique aetiology. The treatment of UC is challenging, and the current clinical therapeutics for colitis have limited efficacy. Thus, finding new and effective treatment options remains urgent. Baricitinib, an inhibitor of Janus kinase (JAK), has been clinically used to treat rheumatoid arthritis (RA). However, its potential effects on UC have not been fully elucidated. In this study, we aimed to explore the effects of baricitinib on UC and its underlying mechanism. Dextran sulphate sodium (DSS)-induced murine model of chronic colitis was used to investigate the intervention efficacy following oral administration of baricitinib. The levels of key cytokines, such as IL-6, IFN-γ and IL-17A, were determined. Moreover, western blotting for IκBα, p-IκBα, JAK2, p-JAK2, STAT3 and p-STAT3 protein expression was performed to investigate the associated signalling pathways. Our findings demonstrated that baricitinib can significantly relieve DSS-induced UC in mice. After baricitinib intervention, IL-6, IFN-γ and IL-17A levels were decreased both in vitro and in vivo. Moreover, the elevated expression levels of p-IκBα, p-JAK2, and p-STAT3 were significantly reduced after treatment. Collectively, these results suggest that baricitinib is a potential therapeutic agent for alleviation of DSS-induced colitis. This study provides a method for subsequent investigations on potential curative drugs development of the for colitis.

MCP-1/CCR2 axis is involved in the regulation of γδT cells in lupus nephritis.

γδT cells are important innate immune cells that are involved in the occurrence and development of autoimmune diseases such as systemic lupus erythematosus (SLE). Lupus nephritis (LN) is a serious complication of SLE, characterized by the accumulation of immune cells (including γδT cells) in the target organs to participate in the disease process. Therefore, clarifying how γδT cells chemotactically migrate to target organs may be a key to developing therapeutic methods against LN. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of chemokines in LN patients and healthy controls. Real-time polymerase chain reaction (RT-PCR) and flow cytometry were used to measure the expression of chemokine receptors on the surface of γδT cells. The chemotactic migration ability of γδT cells was detected by Transwell assay. Signalling pathway activation of γδT cells was detected by Automated Capillary Electrophoresis Immunoassay and flow cytometry. The serum levels of chemokines, including monocyte chemoattractant protein-1 (MCP-1) in LN patients, were significantly increased. CCR2, the receptor of MCP-1, was also highly expressed on the surface of peripheral γδT cells in LN patients. In addition, the exogenous addition of MCP-1 can enhance chemotactic migration of γδT cells in LN patients. MCP-1 could activate STAT3 signalling in LN patients' peripheral γδT cells. γδT cells might participate in the pathogenesis of LN through MCP-1/CCR2 axis. This finding provides new opportunities for developing treatment methods against LN by targeting MCP-1/CCR2 axis.

Leukemia Inhibitory Factor Impairs the Function of Peripheral γδT Cells in Patients with Colorectal Cancer.

Immunotherapeutic strategies are recognized as promising treatment methods for colorectal cancer (CRC). αßT cell-mediated cytotoxicity is tolerated by cancer cells with low MHC class I expression; therefore, γδT cell-based cancer immunotherapy has generated increasing interest as a potential treatment option. To enhance the potency of γδT cell-based immunotherapy, the key factors involved in the regulation of γδT cells in CRC need to be identified along with devising ways to overcome potential hurdles. In this study, we observed that leukemia inhibitory factor (LIF), the serum level of which was highly increased in those with solid tumors, could specifically attenuate the cytotoxic function of peripheral γδT cells in patients with CRC. We observed that in patients with CRC, the expression levels of perforin and granzyme were significantly decreased in the recombinant human LIF (rhLIF)-treated peripheral γδT cells, whereas that of the LIF receptor (LIFR) was higher. The regulation of the cytotoxic function of the γδT cells by rhLIF was effected mainly through the STAT3 signaling pathway, which caused an increase in the expression levels of interleukin (IL)-17, COX-2, and prostaglandin E2 (PGE2). Our results revealed that rhLIF could impair the function of γδT cells in CRC patients by reducing the cytotoxic function and increasing the expression of tumor-promoting molecules, such as IL-17, COX-2, and PGE2.

Trim28 acts as restriction factor of prototype foamy virus replication by modulating H3K9me3 marks and destabilizing the viral transactivator Tas.

BACKGROUND: Prototype foamy virus (PFV) is nonpathogenic complex retroviruses that express a transcriptional transactivator Tas, which is essential for the activity of viral long terminal repeat (LTR) promoter and internal promoter (IP). Tripartite motif-containing protein 28 (Trim28) is well known as a scaffold protein normally enriched in gene promoter region to repress transcription. We sought to determine if whether Trim28 could be enriched in PFV promoter region to participate the establishment of PFV latency infection. RESULTS: In this study, we show that Trim28 restricts Tas-dependent transactivation activity of PFV promoter and negatively regulates PFV replication. Trim28 was found to be enriched in LTR instead of IP promoter regions of PFV genome and contribute to the maintenance of histone H3K9me3 marks on the LTR promoter. Furthermore, Trim28 interacts with Tas and colocalizes with Tas in the nucleus. Besides, we found that Trim28, an E3 ubiquitin ligase, binds directly to and promotes Tas for ubiquitination and degradation. And the RBCC domain of Trim28 is required for the ubiquitination and degradation of Tas. CONCLUSIONS: Collectively, our findings not only identify a host factor Trim28 negatively inhibits PFV replication by acting as transcriptional restriction factor enriched in viral LTR promoter through modulating H3K9me3 mark here, but also reveal that Trim28 mediated ubiquitin proteasome degradation of Tas as a mechanism underlying Trim28 restricts Tas-dependent transcription activity of PFV promoter and PFV replication. These findings provide new insights into the process of PFV latency establishment.

Identification of new potential antigen recognized by γδT cells in hepatocellular carcinoma.

In recent years, the application of chimeric antigen receptor T-cell (CAR-T) therapy based on gamma delta T (γδT) cells in hepatocellular carcinoma (HCC) immunotherapy has attracted more and more attention. However, specific antigens recognized by γδT cells are rarely identified, which has become the main restriction on such therapeutic application of γδT cells. In this report, we identified a new peptide and protein antigen recognized by γδT cells in HCC using our previous established strategy. First, we investigated the diversity of the γ9/δ2 T-cell immunorepertoire by sequence analyses of the expressed complementarity-determining region 3 (CDR3) in HCC patients. Then, we constructed γ9/δ2 T-cell receptor (TCR)-transfected cell lines expressing significant HCC CDR3 sequence and identified a series of peptides capable of binding to γδT cells specifically. Next, we identified, further tested and verified the biological functions of these peptides and their matched protein by bioinformatics analysis. We identified that the new protein hepatocyte growth factor-like protein, also called as macrophage-stimulating protein (MSP), and peptide HP1, not only bound to HCC-predominant γδTCR but also effectively activated γδT cells isolated from HCC patients. Moreover, they could stimulate γδT cells in peripheral blood from HCC patients to produce cytokines, which contributed to inhibiting HCC and played an important role in mediating cytotoxicity to HCC cell lines. In conclusion, we identified MSP and HP1, which showed potential as candidates for antigens recognized by γδT cells in HCC.

CX3CR1 might be a promising predictor of systemic lupus erythematosus patients with pulmonary fibrosis.

The inflammatory process in systemic lupus erythematosus (SLE) affects many organs including the lungs. Chemokines are suggested to play important roles in the pathogenesis of SLE with pulmonary fibrosis (PF). In the present study, our objective is to evaluate the correlation between chemokines and PF in SLE patients. Transcriptome sequencing analysis was used to find the different expressed genes between SLE patients with PF and without PF. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of chemokines in SLE patients and healthy controls. Expression of CX3CR1 was measured by real-time polymerase chain reaction (PCR) and flow cytometer. Sixteen differentially chemokine genes were found to be associated to SLE with PF. Meanwhile, the upregulation of C-X3-C motif chemokine receptor 1 (CX3CR1) and its ligand, CX3C chemokine ligand 1 (CX3CL1) were observed in SLE patients with PF than that of SLE patients without PF and healthy control. Phenotypic analysis also showed that the surface expression of CX3CR1 increased in PBMCs from SLE patients with PF. Our observations indicated that CX3CL1/CX3CR1 axis is associated with PF in SLE. CX3CR1 might be a promising predictor of SLE with PF and the interactions between CX3CL1 and CX3CR1 might provide potential candidate target for the treatment of SLE with PF.

Higher expression of monocyte chemotactic protein 1 in mild COVID-19 patients might be correlated with inhibition of Type I IFN signaling.

BACKGROUND: Chemokine levels in severe coronavirus disease 2019 (COVID-19) patients have been shown to be markedly elevated. But the role of chemokines in mild COVID-19 has not yet been established. According to the epidemiological statistics, most of the COVID-19 cases in Shiyan City, China, have been mild. The purpose of this study was to evaluate the level of chemokines in mild COVID-19 patients and explore the correlation between chemokines and host immune response. METHODS: In this study, we used an enzyme-linked immunosorbent assay to detect serum levels of chemokines in COVID-19 patients in Shiyan City. Expression of chemokine receptors and of other signaling molecules was measured by real-time polymerase chain reaction. RESULTS: We first demonstrated that COVID-19 patients, both sever and mild cases, are characterized by higher level of chemokines. Specifically, monocyte chemotactic protein 1 (MCP-1) is expressed at higher levels both in severe and mild cases of COVID-19. The receptor of MCP-1, C-C chemokine receptor type 2, was expressed at higher levels in mild COVID-19 patients. Finally, we observed a significant negative correlation between expression levels of interferon (IFN) regulatory factor 3 (IRF3) and serum levels of MCP-1 in mild COVID-19 patients. CONCLUSION: Higher expression of MCP-1 in mild COVID-19 patients might be correlated with inhibition of IFN signaling. The finding adds to our understanding of the immunopathological mechanisms of severe acute respiratory syndrome coronavirus 2 infection and provides potential therapeutic targets and strategies.

The prostaglandin E2 increases the production of IL-17 and the expression of costimulatory molecules on γδ T cells in rheumatoid arthritis.

γδ T cells play important roles in the development of rheumatoid arthritis (RA) through their antigen-presenting capacity, release of pro-inflammatory cytokines, immunomodulatory properties, interaction with CD4+ CD25+ Tregs and promotion of antibody production by helping B cells. Although prostaglandin E2 (PGE2) was proved to have the ability to enhance the antigen-presenting function of dendritic cells and IL-17 production of CD4+ αß T cells in RA, the role of PGE2 in γδ T cells from RA disease has not yet been clarified. The goal of this study was to determine the role of PGE2 in γδ T cells in RA. We first demonstrated that the population of γδT17 cells increased in patients with RA compared to healthy controls. Then, IL-17A level in patients with RA was shown to increase compared to healthy controls. After adding PGE2 to γδ T cells from patients with RA, the IL-17A level increased accordingly, and the expression of the costimulatory molecules, CD80 and CD86, on these cells also increased. These results suggest that PEG2 can increase the production of IL-17A and the expression of CD80 and CD86 on γδ T cells in patients with RA. These findings will benefit to explore new therapeutic targets for RA disease.

Down-Regulation of Fibroblast Growth Factor 2 (FGF2) Contributes to the Premature Senescence of Mouse Embryonic Fibroblast.

BACKGROUND Freshly isolated mouse embryonic fibroblasts (MEFs) have great proliferation capacity but quickly enter senescent state after several rounds of cell cycle, a process called premature senescence. Cellular senescence can be induced by various stresses such as telomere erosion, DNA damage, and oncogenic signaling. But the contribution of other molecules, such as growth factors, to cellular senescence is incompletely understood. This study aimed to compare the gene expression difference between non-senescent and senescent MEFs to identify the key molecule(s) involved in the spontaneous senescence of MEFs. MATERIAL AND METHODS Primary MEFs were isolated from E12.5 pregnant C57/BL6 mice. The cells were continuously cultured in Dulbecco's Modified Eagle Medium for 9 passages. SA-ß-Gal staining was used as an indicator of cell senescence. The supernatant from primary MEFs (P1 medium) or Passage 6 MEFs (P6 medium) were used to culture freshly isolated MEFs to observe the effects on cell senescence state. Gene expression profiles of primary and senescent MEFs were investigated by RNA-Seq to find the key genes involved in cell senescence. Adipocyte differentiation assay was used to evaluate the stemness of MEFs cultured in FGF2-stimulated medium. RESULTS The senescence of MEFs cultured in the P1 medium was alleviated when compared to the P6 medium. Downregulation of FGF2 expression was revealed by RNA-Seq and further confirmed by real-time quantitative polymerase chain reaction and western blot. FGF2-stimulated medium also had anti-senescence function and could maintain the differentiation ability of MEFs. CONCLUSIONS The premature senescence of MEFs was at least partially caused by FGF2 deficiency. Exogenous FGF2 could alleviate the senescent phenotype.

Correction: Enterovirus 71 Protease 2Apro Targets MAVS to Inhibit Anti-Viral Type I Interferon Responses.

[This corrects the article DOI: 10.1371/journal.ppat.1003231.].

Expression, purification and characterization of active untagged recombinant human leukemia inhibitory factor from E.coli.

Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is considered to be a pleiotropic cytokine and functions in both cell proliferation and differentiation. It is widely used in the culture of mouse embryonic stem cells and is implicated in the implantation of mouse model and possibly in humans. Great efforts have been made on the efficient generation of LIF to meet the requirement of this cytokine in biomedical research. However, because of the low expression level in the eukaryotic system and poor purification yields, recombinant human LIF has usually been expressed either as inclusion body or as fusion protein in E. coli (Escherichia coli). Here we introduce a simple method to express hLIF in a soluble form in E. coli and a subsequent purification method. The expression of hLIF was induced at a low temperature (16 °C) and most of the expressed hLIF was observed to be in a soluble form. Then by using three steps of chromatography, which could be easily scaled-up for industrial purposes, active untagged hLIF was purified with similar bioactivity compared to that of the commercial product. The endotoxin level of purified hLIF protein in our method was determined to be lower than 1EU/µg, which was also comparable to the commercial products. Furthermore, as hLIF was expressed in a soluble form, there was no need to develop the denaturation and renaturation methods. The yield of hLIF protein was evaluated to be approximately 0.7 mg hLIF from 1 g wet weight of E.coli in our method.

Enterovirus 71 protease 2Apro targets MAVS to inhibit anti-viral type I interferon responses.

Enterovirus 71 (EV71) is the major causative pathogen of hand, foot, and mouth disease (HFMD). Its pathogenicity is not fully understood, but innate immune evasion is likely a key factor. Strategies to circumvent the initiation and effector phases of anti-viral innate immunity are well known; less well known is whether EV71 evades the signal transduction phase regulated by a sophisticated interplay of cellular and viral proteins. Here, we show that EV71 inhibits anti-viral type I interferon (IFN) responses by targeting the mitochondrial anti-viral signaling (MAVS) protein--a unique adaptor molecule activated upon retinoic acid induced gene-I (RIG-I) and melanoma differentiation associated gene (MDA-5) viral recognition receptor signaling--upstream of type I interferon production. MAVS was cleaved and released from mitochondria during EV71 infection. An in vitro cleavage assay demonstrated that the viral 2A protease (2A(pro)), but not the mutant 2A(pro) (2A(pro)-110) containing an inactivated catalytic site, cleaved MAVS. The Protease-Glo assay revealed that MAVS was cleaved at 3 residues between the proline-rich and transmembrane domains, and the resulting fragmentation effectively inactivated downstream signaling. In addition to MAVS cleavage, we found that EV71 infection also induced morphologic and functional changes to the mitochondria. The EV71 structural protein VP1 was detected on purified mitochondria, suggesting not only a novel role for mitochondria in the EV71 replication cycle but also an explanation of how EV71-derived 2A(pro) could approach MAVS. Taken together, our findings reveal a novel strategy employed by EV71 to escape host anti-viral innate immunity that complements the known EV71-mediated immune-evasion mechanisms.

Emodin is a Potential Drug Targeting CD44-positive Hepatocellular Cancer.

BACKGROUND: Liver cancer is one of the most prevalent forms of cancer of the digestive system in our country. The most common subtype of this disease is hepatocellular carcinoma (HCC). Currently, treatment options for HCC patients include surgical resection, liver transplantation, radiofrequency ablation, chemoembolization, and biologic-targeted therapy. However, the efficacy of these treatments is suboptimal, as they are prone to drug resistance, metastasis, spread, and recurrence. These attributes are closely related to cancer stem cells (CSCs). Therefore, the utilization of drugs targeting CSCs may effectively inhibit the development and recurrence of HCC. METHODS: HepG2 and Huh7 cells were used to analyze the antitumor activity of emodin by quantifying cell growth and metastasis, as well as to study its effect on stemness. RESULTS: Emodin effectively suppressed the growth and movement of HCC cells. Emodin also significantly inhibited the proliferation of CD44-positive hepatoma cells. CONCLUSION: Emodin shows promise as a potential therapeutic agent for HCC by targeting CD44-- positive hepatoma cells.

Polyphyllin VII as a Potential Drug for Targeting Stemness in Hepatocellular Cancer via STAT3 Signaling.

BACKGROUND: At present, the treatment of hepatocellular carcinoma (HCC) is disturbed by the treatment failure and recurrence caused by the residual liver cancer stem cells (CSCs). Therefore, drugs targeting HCC CSCs should be able to effectively eliminate HCC and prevent its recurrence. In this study, we demonstrated the effect of Polyphyllin VII (PP7) on HCC CSCs and explored their potential mechanism. METHODS: HepG2 and Huh7 cells were used to analyze the antitumor activity of PP7 by quantifying cell growth and metastasis as well as to study the effect on stemness. RESULTS: Our results demonstrated that PP7 promoted apoptosis and significantly inhibited proliferation and migration of both HepG2 and Huh7 cells. PP7 also inhibited tumor spheroid formation and induced significant changes in the expression of stemness markers (CD133 and OCT-4). These effects of PP7 were mediated by STAT3 signaling. CONCLUSION: PP7 can effectively suppress tumor initiation, growth, and metastasis and inhibit stemness through regulation of STAT3 signaling pathway in liver cancer cells. Our data would add more evidence to further clarify the therapeutic effect of PP7 against HCC.

The function of MSP-activated γδT cells in hepatocellular carcinoma.

Immunotherapeutic strategies targeting γδT cells are now recognized as a promising treatment method for hepatocellular carcinoma (HCC). To date, no specific antigen or antigenic epitope recognized by γδT cells has been identified, limiting their application in the field of HCC treatment. Previously, we used an established screening strategy to identify a novel HCC protein antigen recognized by γδT cells called MSP. In this study, we explored the function of MSP activated-γδT cells in HCC. Results demonstrated that the proportions of γδT cells in the peripheral blood of HCC patients and the level of IFN-γ in the serum were higher than in healthy controls. We also determined that γδT cells can bind MSP protein. MSP-activated γδT cells were shown to contain a specific CDR3δ2 sequence that supports the recognition of MSP by γδT cells. We determined that MSP is highly expressed in HCC, MSP-activated γδT cells in the peripheral blood of HCC patients express co-stimulatory molecules, and MSP-activated γδT cells directly killed HCC cells. In conclusion, we demonstrated that the novel protein ligand MSP activated γδT cells, leading to the killing of HCC cells through direct and indirect mechanisms. These findings could provide a potential new target for the clinical diagnosis and treatment of HCC and a foundation for clinical treatment strategies in HCC.

Flanking V and J sequences of complementary determining region 3 of T cell receptor (TCR) δ1 (CDR3δ1) determine the structure and function of TCRγ4δ1.

The γδ T cell receptor (TCR) differs from immunoglobulin and αß TCR in its overall binding mode. In human, genes δ1, δ2, and δ3 are used for TCRδ chains. Previously, we have studied antigen binding determinants of TCRδ2 derived from dominant γδ T cells residing in peripheral blood. In this study we have investigated the critical determinants for antigen recognition and TCR function in TCRδ1 originated from gastric tumor-infiltrating γδ T lymphocytes using three independent experimental strategies including complementary determining region 3 (CDR3) of TCRδ1 (CDR3δ1)-peptide mediated binding, CDR3δ1-grafted TCR fusion protein-mediated binding, and TCRγ4δ1- and mutant-expressing cell-mediated binding. All three approaches consistently showed that the conserved flanking V and J sequences but not the diverse D segment in CDR3δ1 determine the antigen binding. Most importantly, we found that mutations in the V and J regions of CDR3δ1 also abolish the assembly of TCR and TCR-CD3 complexes in TCRγ4δ1-transduced J.RT3-T3.5 cells. Together with our previous studies on CDR3δ2 binding, our finding suggests that both human TCRδ1 and TCRδ2 recognize antigen predominately via flanking V and J regions. These results indicate that TCRγδ recognizes antigens using conserved parts in their CDR3, which provides an explanation for a diverse repertoire of γδTCRs only recognizing a limited number of antigens.

Polyphyllin VII is a Potential Drug Targeting CD44 Positive Colon Cancer Cells.

BACKGROUND: Current therapies for colon cancer are hindered by treatment failure and recurrence, mainly due to colon cancer stem cells (CSCs). Thus, treatment using drugs targeting CSCs should be effective in eliminating colon cancer cells and impeding cancer recurrence. OBJECTIVE: This study aimed to test if PPVII can be a potent drug candidate for the treatment of colon cancer by targeting CD44 positive colon cancer cells. METHODS: In this study, we first demonstrated that CD44 is highly expressed in colon cancer tissues by TCGA/GTEX database analysis and immunohistochemical staining. RESULTS: In this study, we first demonstrated that CD44 is highly expressed in colon cancer tissues by TCGA/GTEX database analysis. CD44 had high accuracy as a diagnostic and predictive index for colorectal cancer through receiver operating characteristic curve (ROC) analysis. At the same time, survival curve analysis also showed that the high expression of CD44 was associated with poor prognosis in patients with colon cancer. CD44's higher expression in colon cancer tissues was further confirmed by immunohistochemical staining; the positive rate of CD44 expression was 87.95%. Then, one of the constituents that derives from the root of Paris polyphylla, Polyphyllin VII (PPVII), has been confirmed to inhibit the migration of colon cancer cells. Our results also demonstrated that PPVII could inhibit the sphere-forming ability of colon cancer cells. Further experiment results showed that PPVII could downregulate the expression of CD44 in colon cancer cells. In addition, PPVII was proved to have inhibitory effects against CD44 positive colon cancer cells. CONCLUSION: Therefore, PPVII might be a potent candidate reagent for the treatment of colon cancer by targeting CD44 positive colon cancer cells.