Development of an environmentally sensitive fluorescent peptide probe for MrgX2 and application in ligand screening of peptide antibiotics.

Mast cells (MCs) are primary effector cells involved in immediate allergic reactions. Mas-related G protein-coupled receptor-X2 (MrgX2), which is highly expressed on MCs, is involved in receptor-mediated drug-induced pseudo-anaphylaxis. Many small-molecule drugs and peptides activate MrgX2, resulting in MC activation and allergic reactions. Although small-molecule drugs can be identified using existing MrgX2 ligand-screening systems, there is still a lack of effective means to screen peptide ligands. In this study, to screen for peptide drugs, the MrgX2 high-affinity endogenous peptide ligand substance P (SP) was used as a recognition group to design a fluorescent peptide probe. Spectroscopic properties and fluorescence imaging of the probe were assessed. The probe was then used to screen for MrgX2 agonists among peptide antibiotics. In addition, the effects of peptide antibiotics on MrgX2 activation were investigated in vivo and in vitro. The environment-sensitive property of the probe was revealed by the dramatic increase in fluorescence intensity after binding to the hydrophobic ligand-binding domain of MrgX2. Based on these characteristics, it can be used for in situ selective visualization of MrgX2 in live cells. The probe was used to screen ten types of peptide antibiotics, and we found that caspofungin and bacitracin could compete with the probe and are hence potential ligands of MrgX2. Pharmacological experiments confirmed this hypothesis; caspofungin and bacitracin activated MCs via MrgX2 in vitro and induced local anaphylaxis in mice. Our research can be expected to provide new ideas for screening MrgX2 peptide ligands and reveal the mechanisms of adverse reactions caused by peptide drugs, thereby laying the foundation for improving their clinical safety.

Hyaluronic acid-based M1 macrophage targeting and environmental responsive drug releasing nanoparticle for enhanced treatment of rheumatoid arthritis.

Herein, hyaluronic acid (HA) and ß-cyclodextrin (ß-CD) is used to form targeted drug delivery platform HCPC/DEX NPs with previously prepared carbon dots (CDs) as cross-linker, dexamethasone (DEX) is loaded for rheumatoid arthritis (RA) treatment. The drug loading capacity of ß-CD and M1 macrophage targeting of HA were utilized for efficient delivery of DEX to the inflammatory joints. Because of the environmental responsive degradation of HA, DEX can be released in 24 h and inhibit the inflammatory response in M1 macrophages. The drug loading of NPs is 4.79 %. Cellular uptake evaluation confirmed that NPs can specifically target to M1 macrophages via HA ligands, the uptake of M1 macrophages is 3.7 times that of normal macrophages. In vivo experiments revealed that NPs can accumulate in RA joints to alleviate inflammation and accelerate cartilage healing, the accumulation can be observed in 24 h. The cartilage thickness increased to 0.45 mm after HCPC/DEX NPs treatment, indicating its good RA therapeutic effect. Importantly, this study was the first to utilize the potential acid and reactive oxygen species responsiveness of HA to release drug and prepare M1 macrophage targeting nanodrug for RA treatment, which provides a safe and effective RA therapeutic strategy.

Imperatorin derivative OW1, a new vasoactive compound, attenuates cell proliferation and migration by inhibiting Nox1-mediated oxidative stress.

OBJECTIVES: Reactive oxygen species (ROS) are involved in the structural remodelling of vascular segments and vascular beds. We identified a new imperatorin derivative, OW1, which has significant effects on vasodilation and inhibits vascular remodelling in hypertensive rats. In this study, we investigated whether OW1 inhibits vascular cell proliferation and migration by attenuating Nox1-ROS signalling. METHODS: Vascular smooth muscle cells (VSMCs) were treated with OW1 (1, 3 and 10 µmol/L) for 24 h incubation, and it has been analysed for proliferation and peroxidation levels. Moreover, the mRNA and protein levels of nicotinamide adenine dinucleotide phosphate oxidase (Noxs) were measured by RT-PCR and western blot. Furthermore, Nox1-ROS-MAPK/MMP mediated cell proliferation was detected by western blot. KEY FINDINGS: Ang II-induced increases in the levels of peroxidation and Noxs in VSMCs were also inhibited by OW1. OW1 attenuates cell proliferation and migration through the MAPK pathway and MMPs. OW1 treatment had no significant effects on cell migration, ROS levels, or the expression of phosphorylated MAPKs in VSMCs when Nox1 was knocked down. OW1 reduced ROS levels and expression of phosphorylated MAPKs in NIH3T3 cells with a Nox1 overexpression plasmid. CONCLUSION: OW1 may inhibit vascular remodelling by downregulating the Nox1-ROS-MAPK/MMP signalling pathway.

Antibiotic-Resistant Escherichia coli Strains Isolated from Captive Giant Pandas: A Reservoir of Antibiotic Resistance Genes and Virulence-Associated Genes.

Recent studies showed that Escherichia coli (E. coli) strains isolated from captive giant pandas have serious resistance to antibiotics and carry various antibiotic resistance genes (ARGs). ARGs or virulence-associated genes (VAGs) carried by antibiotic-resistant E. coli are considered as a potential health threat to giant pandas, humans, other animals and the environment. In this study, we screened ARGs and VAGs in 84 antibiotic-resistant E. coli strains isolated from clinically healthy captive giant pandas, identified the association between ARGs and VAGs and analyzed the phylogenetic clustering of E. coli isolates. Our results showed that the most prevalent ARG in E. coli strains isolated from giant pandas is blaTEM (100.00%, 84/84), while the most prevalent VAG is fimC (91.67%, 77/84). There was a significant positive association among 30 pairs of ARGs, of which the strongest was observed for sul1/tetC (OR, 133.33). A significant positive association was demonstrated among 14 pairs of VAGs, and the strongest was observed for fyuA/iroN (OR, 294.40). A positive association was also observed among 45 pairs of ARGs and VAGs, of which the strongest was sul1/eaeA (OR, 23.06). The association of ARGs and mobile gene elements (MGEs) was further analyzed, and the strongest was found for flor and intI1 (OR, 79.86). The result of phylogenetic clustering showed that the most prevalent group was group B2 (67.86%, 57/84), followed by group A (16.67%, 14/84), group D (9.52%, 8/84) and group B1 (5.95%, 5/84). This study implied that antibiotic-resistant E. coli isolated from captive giant pandas is a reservoir of ARGs and VAGs, and significant associations exist among ARGs, VAGs and MGEs. Monitoring ARGs, VAGs and MGEs carried by E. coli from giant pandas is beneficial for controlling the development of antimicrobial resistance.

Regulation of survivin and caspase/Bcl-2/Cyto-C signaling by TDB-6 induces apoptosis of colorectal carcinoma LoVo cells.

Background: Colorectal carcinoma (CRC) treatment remains severe. Survivin is aberrantly overexpressed in CRC tissues and might be a potential target for CRC treatment. TDB-6 is a new taspine derivative. The purpose of this study is to investigate the inhibitory effect of TDB-6 on CRC and its underlying mechanism. Methods: The MTT assay and xenograft model were utilized to investigate the inhibitory effect of TDB-6 on LoVo cells in vitro and in vivo. Hoechst staining and Annexin-V FITC/PI analysis were conducted to study the effect of TDB-6 on LoVo cell apoptosis. Mitochondrial membrane potential (Δψm) assay was conducted to demonstrated whether TDB-6 could induce mitochondrial-mediated apoptosis of LoVo cells. Western blotting was conducted to investigate the effect of TDB-6 on survivin protein and caspase/Bcl-2/Cyto-C signaling. Results: The results indicated that TDB-6 induced mitochondria-mediated apoptosis and inhibited the proliferation and growth of LoVo cells in vitro and in vivo. Mechanistic investigation utilizing western blotting indicated that TDB-6 inhibited survivin protein expression, and the inhibitory effect was augmented by TDB-6 and YM-155 co-administration, which revealed that TDB-6 might induce apoptosis of LoVo cells by targeted regulation of survivin. TDB-6 also regulated survivin downstream signaling. It significantly increased the protein level of cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, cleaved-PARP, and Cyto-C, and decreased the protein level of Bcl-2. Conclusions: TDB-6 might be a promising survivin inhibitor with great potential for CRC treatment.

Occurrence, genetic diversity and zoonotic potential of Blastocystis sp. in forest musk deer (Moschus berezovskii) in Southwest China.

Blastocystis sp. is a common anaerobic protist with controversial pathogenicity that can infect various animals and humans. However, there are no reports of Blastocystis sp. infections in forest musk deer (Moschus berezovskii). The present study was designed to examine the occurrence, subtype distribution and genetic characterization of Blastocystis sp. in forest musk deer in southwestern China, and to assess the potential for zoonotic transmission. A total of 504 fresh stool samples were collected from captive forest musk deer in four distinct areas of southwestern China. Overall, 14.7% of the forest musk deer (74/504) were found to be infected with Blastocystis sp. The highest occurrence of Blastocystis sp. was observed in Dujiangyan (27.5%), followed by Maerkang (23.3%). The occurrence of Blastocystis sp. was 7.9% and 4.1% in Shimian and Hanyuan, respectively. Significant differences in the occurrence of Blastocystis sp. among different areas were observed (p < 0.05), while we did not observe significant differences among animals of different age and sex (p > 0.05). Two known zoonotic subtypes (ST1 and ST5) and three animal-predominant subtypes (ST10, ST13, and ST14) were identified, of which ST10 was the most common (36/74, 48.6%). Our findings highlight that forest musk deer may be potential reservoirs of zoonotic human Blastocystis sp. infections.

Title: Présence, diversité génétique et potentiel zoonotique de Blastocystis sp. chez le cerf porte-musc (Moschus berezovskii) dans le sud-ouest de la Chine. Abstract: Blastocystis sp. est un protiste anaérobie commun, de pathogénicité controversée, et qui peut infecter divers animaux et les humains. Cependant, aucun cas d'infection par Blastocystis sp. n'a été rapporté chez le cerf porte-musc (Moschus berezovskii). La présente étude a été conçue pour examiner la présence, la distribution des sous-types et la caractérisation génétique de Blastocystis sp. chez le cerf porte-musc du sud-ouest de la Chine et pour évaluer son potentiel de transmission zoonotique. Au total, 504 échantillons de selles fraîches ont été prélevés sur des cerfs porte-musc captifs dans quatre régions distinctes du sud-ouest de la Chine. Dans l'ensemble, 14,7 % (74/504) des cerfs porte-musc se sont avérés infectés par Blastocystis sp. La plus forte occurrence de Blastocystis sp. a été observée à Dujiangyan (27,5 %), suivi de Maerkang (23,3 %). La présence de Blastocystis sp. était respectivement de 7,9 % et 4,1 % à Shimian et Hanyuan. Des différences significatives dans la présence de Blastocystis sp. entre les différentes zones ont été observées (p < 0,05), alors que nous n'avons pas observé de différences significatives entre les animaux d'âge et de sexe différents (p > 0,05). Deux sous-types zoonotiques connus (ST1 et ST5) et trois sous-types à prédominance animale (ST10, ST13 et ST14) ont été identifiés, dont ST10 était le sous-type le plus courant (36/74, 48,6 %). Nos découvertes mettent en évidence que le cerf porte-musc forestier peut être un réservoir potentiel d'infections à Blastocystis sp.

Vandetanib drives growth arrest and promotes sensitivity to imatinib in chronic myeloid leukemia by targeting ephrin type-B receptor 4.

The oncogenic role of ephrin type-B receptor 4 (EPHB4) has been reported in many types of tumors, including chronic myeloid leukemia (CML). Here, we found that CML patients have a higher EPHB4 expression level than healthy subjects. EPHB4 knockdown inhibited growth of K562 cells (a human immortalized myelogenous leukemia cell line). In addition, transient transfection of EPHB4 siRNA led to sensitization to imatinib. These growth defects could be fully rescued by EPHB4 transfection. To identify an EPHB4-specific inhibitor with the potential of rapid translation into the clinic, a pool of clinical compounds was screened and vandetanib was found to be most sensitive to K562 cells, which express a high level of EPHB4. Vandetanib mainly acts on the intracellular tyrosine kinase domain and interacts stably with a hydrophobic pocket. Furthermore, vandetanib downregulated EPHB4 protein via the ubiquitin-proteasome pathway and inhibited PI3K/AKT and MAPK/ERK signaling pathways in K562 cells. Vandetanib alone significantly inhibited tumor growth in a K562 xenograft model. Furthermore, the combination of vandetanib and imatinib exhibited enhanced and synergistic growth inhibition against imatinib-resistant K562 cells in vitro and in vivo. These findings suggest that vandetanib drives growth arrest and overcomes the resistance to imatinib in CML via targeting EPHB4.

ND-16: A Novel Compound for Inhibiting the Growth of Cutaneous T Cell Lymphoma by Targeting JAK2.

OBJECTIVE: Cutaneous T cell lymphoma (CTCL) is a kind of extranodal non-Hodgkin Tcell lymphoma without healable treatment in the clinic. JAK2 amplification in CTCL patients makes it a potential target for CTCL treatment. In the present study, we aimed to evaluate the anticancer effect of ND-16, a novel nilotinib derivate, on CTCL cells and the underlying mechanism targeting JAK2. METHODS AND RESULTS: We found that ND-16 was capable of regulating JAK2 and had a selective inhibitory effect on CTCL H9 cells. The surface plasmon resonance and molecular docking study indicated ND-16 bound to JAK2 with a high binding affinity. Further investigation revealed that ND-16 inhibited the downstream cascades of JAK2, including STATs, PI3K/AKT/mTOR, and MAPK pathways, followed by regulation of Bcl-2 family members and cell cycle proteins CDK/- Cyclins. Flow cytometry analysis confirmed these results that ND-16-treated H9 cells showed cell apoptosis and cell cycle arrest at S-phase. CONCLUSION: ND-16 may be of value in a potential therapy for the management of CTCL.

First Isolation and Multilocus Sequence Typing of Brucella canis from a Subclinically Infected Pet Dog in China.

Canine brucellosis, a worldwide zoonotic disease, is mainly caused by Brucella canis. In the present study, we isolated a Brucella strain (CD3) from a subclinically infected pet dog in Sichuan Province, Southwestern China. Classical biotyping methods and molecular biological tests (BCSP31 and BcSS PCR) proved that the strain belonged to B. canis. Furthermore, B. canis CD3 and another two B. canis strains (WJ5 and YA4), which were all isolated from pet dogs in Sichuan, were genotyped using multilocus sequence typing (MLST). Our results showed that the three B. canis strains were identified as the same sequence type (ST21). The present study is the first to report B. canis strain from a subclinically infected pet dog in China, indicating a potential threat to public health posed by subclinical infections in pet dogs. We suggest that screening for B. canis should be incorporated into routine medical examination of pet dogs and other companion animals in areas with a history of animal or human brucellosis.

Sanguinarine suppresses migration and metastasis in colorectal carcinoma associated with the inversion of EMT through the Wnt/ß-catenin signaling.

BACKGROUND: Unresectable lung or liver organ metastases of colorectal carcinoma (CRC) remain a major obstacle in clinical therapeutics. Epithelial to mesenchymal transition (EMT), a major cause of highly frequent metastasis in tumor, can be promoted by the Wnt/ß-catenin pathway that is aberrantly activated in approximately 90% of CRC. This research aimed to elucidate the antimetastatic potential of sanguinarine (SG) in CRC and the underlying molecular mechanism. METHODS: The in vitro anticancer effect of SG was determined via cell viability experiment and colony formation assay. Xenograft model of nude mice was used to confirm the antitumor effect of SG in vivo. The antimetastatic potential of SG was investigated by the metastasis model of nude mice, hematoxylin and eosin (H&E) staining, migration assay, and wound-healing analysis. Immunoblotting analysis, immunofluorescence staining, and immunohistochemistry assay were conducted to elucidate the molecular mechanism. RESULTS: In this study, we reported that SG has a selective inhibitory effect on LoVo cells with metastatic characteristics. Furthermore, our results showed attenuation in the migration and metastatic ability of SG-treated LoVo cells and also decreased metastatic nodules of liver and lung in mice metastasis model. This was also confirmed at the molecular level via H&E staining. Further study revealed that SG had negative impacts on the Wnt/ß-catenin pathway and EMT markers in LoVo cells both in vitro and in vivo. CONCLUSIONS: Taken together, the antimetastatic potential of SG attributed to the suppression of the Wnt/ß-catenin signaling, which further prevented EMT progression. SG may be of value in a potential therapy for the management of metastasis CRC.

Cantharidin treatment inhibits hepatocellular carcinoma development by regulating the JAK2/STAT3 and PI3K/Akt pathways in an EphB4-dependent manner.

Hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options. The tyrosine kinase receptor EphB4 promotes oncogenesis and tumor development and progression. Its inhibition is regarded as an effective strategy for the treatment of solid tumors. In the present study, we identified cantharidin as a novel EphB4 inhibitor for HCC treatment and evaluated the underlying molecular pharmacological mechanisms of action. We observed increased expression levels of EphB4 in HCC patients and a positive correlation between EphB4 and p-JAK2 levels in HCC patient samples. Knockdown of EphB4 using small interfering RNA decreased the expression levels of p-JAK2 and p-STAT3 in HCC cells, suggesting JAK2/STAT3 being a novel downstream signaling target of EphB4. Cell viability experiments revealed that the anti-cancer effect of cantharidin was positively correlated with EphB4 expression levels in HCC cell lines. We confirmed the potent antiproliferative activity of cantharidin on HepG2 cells with high expression of EphB4 and tumor xenograft. Molecular docking assay, immunoblotting assay and quantitative reverse transcription PCR assay indicated that cantharidin bound to EphB4, and thereby resulted in EphB4 suppression at mRNA and protein levels. Hep3B and SMMC-7721 cells were with low expression of EphB4. In EphB4-/HepG2, EphB4+/HepG2, and EphB4+/Hep3B cells, EphB4 knockdown alleviated the cantharidin-induced decrease in cell viability and colony formation ability and increase in apoptosis in HepG2 cells, while its overexpression exacerbated these effects in Hep3B cells and increased the apoptosis of HepG2 cells. In nude mouse models, cantharidin suppressed tumor growth more effectively in EphB4+/SMMC-7721 xenografts than in wild-type SMMC-7721 xenografts. Underlying mechanistic study showed that by targeting EphB4, cantharidin blocked a novel target, the downstream JAK2/STAT3 pathway, and the previously known target, the PI3K/Akt signaling, resulting in intrinsic apoptosis. These results indicated that cantharidin may be a potential candidate for HCC treatment by regulating the EphB4 signaling pathway.

Homoharringtonine suppresses LoVo cell growth by inhibiting EphB4 and the PI3K/AKT and MAPK/EKR1/2 signaling pathways.

Colorectal cancer (CRC) remains one of the most common gastrointestinal tumors, characterized by a poor survival rate. Effects of single use of homoharringtonine (HHT), approved for the treatment of acute myelocytic leukemia (AML) and chronic myeloid leukemia (CML), on CRC, are unknown. According to the TCGA database, EphB4 is aberrantly overexpressed in CRC patients. Therefore, the purpose of this study was to investigate the inhibitory effect of HHT on CRC and its underlying mechanism. HHT significantly suppressed LoVo cell growth in vitro and in vivo, and induced apoptosis and cell cycle arrest at the S phase. Mechanistic investigation using western blotting revealed that HHT suppressed EphB4, and this suppression was augmented by both HHT and NVP-BHG712 co-administration and EphB4 overexpression, indicating that HHT targets EphB4 to suppress LoVo cell growth. HHT inhibited EphB4 downstream pathways such as PI3K/AKT and MAPK/EKR1/2, resulting in the regulation of cell cycle-related molecules (cyclinA2 and CDC2), and the molecules in the Bcl-2 mitochondrial apoptosis pathway including Bcl-2, Mcl-1, Bax, Bad, caspase-3, caspase-7, and caspase-9. HHT may therefore be a promising EphB4 inhibitor with great potential for CRC treatment.

TPD7 inhibits the growth of cutaneous T cell lymphoma H9 cell through regulating IL-2R signalling pathway.

IL-2R pathway is a key regulator in the development of immune cells and has emerged as a promising drug target in cancer treatment, but there is a scarcity of related inhibitors. TPD7 is a novel biphenyl urea taspine derivate, which has been shown anti-cancer effect. Here, we demonstrated the anti-cancer activity of TPD7 in cutaneous T cell lymphoma and investigated the underlying mechanism of TPD7 through IL-2R signalling. The inhibitory effect of TPD7 on cell viability exhibited a strong correlation with the expression level of IL-2R, and cutaneous T cell lymphoma H9 and HUT78 cells were most sensitive to TPD7. TPD7 was nicely bound to IL-2R and down-regulated the mRNA and protein levels of IL-2R. Furthermore, TPD7 suppressed the downstream cascades of IL-2R including JAK/STAT, PI3K/AKT/mTOR and PLCγ/Raf/MAPK signalling, resulting in Bcl-2 mitochondrial apoptosis pathway and cell cycle proteins CDK/Cyclins regulation. And, these were verified by flow cytometry analysis that TPD7 facilitated cell apoptosis in H9 cells via mitochondrial pathway and impeded cell cycle progression at G2/M phase. TPD7 is a novel anti-cancer agent and may be a potential candidate for cutaneous T cell lymphoma treatment by regulating IL-2R signalling pathway.

Screening potential antagonists of epidermal growth factor receptor from Marsdenia tenacissima via cell membrane chromatography model assisted by HPLC-ESI-IT-TOF-MS.

Marsdenia tenacissima, or Tongguanteng in Chinese, is a traditional Chinese herb and has a broad application in clinical practice for its pharmacological effects of treating asthma, pneumonia, tonsillitis, pharyngitis tumors, etc. However, few studies have reported the screening of the active components of this medicine for tumor therapy. In this work, a two-dimensional analytical system was developed to screen antagonists of epidermal growth factor receptor (EGFR) from M. tenacissima. A fraction was retained on the EGFR cell membrane chromatography (CMC) column, separated and identified as tenacissoside G (TG), tenacissoside H (TH) and tenacissoside I (TI) by two-dimensional HPLC-IT-TOF-MS. Molecular docking and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay were carried out to assess the activity of TS (including TG, TH and TI). Molecular docking results showed that the binding mode of TS on EGFR is similar to that of gefitinib. The MTT assay demonstrated that gefitinib and TS (especially TI) could inhibit the growth of EGFR highly expressed cell lines in a dose-dependent manner in the range of 5-50 µmol/L. In conclusion, the two-dimensional EGFR/CMC-HPLC-IT-TOF-MS system could be a useful approach in drug discovery from traditional Chinese medicines for searching for potential antitumor candidates.

HMQ-T-B10 induces human liver cell apoptosis by competitively targeting EphrinB2 and regulating its pathway.

Hepatocellular carcinoma (HCC) is a highly prevalent cancer worldwide and it is necessary to discover and develop novel preventive strategies and therapeutic approaches for HCC. Herein, we report that EphrinB2 expression is correlated with liver cancer progression. Moreover, by using phosphorylated proteomics array, we reveal a pro-apoptosis protein whose phosphorylation and activation levels are up-regulated upon EphrinB2 knockdown. These results suggest that EphrinB2 may act as an anti-apoptotic protein in liver cancer cells. We also explored the therapeutic potential of HMQ-T-B10 (B10), which was designed and synthesized in our laboratory, for HCC and its underlying mechanisms in vitro and in vivo. Our data demonstrate that B10 could bind EphrinB2 and show inhibitory activity on human liver cancer cells. Moreover, induction of human liver cancer cell apoptosis by B10 could be augmented upon EphrinB2 knockdown. B10 inhibited HCC cell growth and induced HCC cell apoptosis by repressing the EphrinB2 and VEGFR2 signalling pathway. Growth of xenograft tumours derived from Hep3B in nude mice was also significantly inhibited by B10. Collectively, these findings highlight the potential molecular mechanisms of B10 and its potential as an effective antitumour agent for HCC.

HMQ-T-F5 (1-(4-(2-aminoquinazolin-7-yl)phenyl)-3-(2­bromo­5- (trifluoromethoxy)phenyl) thiourea) suppress proliferation and migration of human cervical HeLa cells via inhibiting Wnt/ß-catenin signaling pathway.

BACKGROUND: Drug therapy plays an important role in the treatment of cervical cancer, which is one of the most common solid tumors in women. Therefore, it is important to seek more effective and less toxic therapies. PURPOSE: The aim of this study is to investigate the therapeutic potential of HMQ-T-F5 (1-(4-(2-aminoquinazolin-7-yl)phenyl)-3-(2­bromo­5-(trifluoromethoxy) phenyl)thiourea) (F5) for cervical cancer and explore the related mechanism. METHODS: By performing MTT assay, colony formation assay, flow cytometry, wound-healing assay, transwell assay, immunofluorescent staining and siRNA assay, we study the effect of F5 on human cervical HeLa cells. The mechanism of F5 was also investigated. RESULTS: We found that F5 significantly inhibited HeLa cell proliferation, led to accumulation of cells in the S phase, and induced apoptosis and inhibited migration. Mechanistically, F5 inhibited HeLa cell growth and migration through repressing the expression and nuclear translocation of ß-catenin, enhancing Axin expression, inhibiting the phosphorylation of LRP5/6 and GSK3ß, as well as downregulating the Wnt downstream targeted proteins. Knockdown of a checkpoint ß-catenin by siRNA significantly attenuated HeLa cell proliferation. Furthermore, XAV939, an inhibitor of ß-catenin, was used to treat HeLa cells and the results demonstrated that F5 inhibited proliferation and migration via the inhibition of the Wnt/ß-catenin pathway. CONCLUSION: Our findings demonstrated that F5 can target ß-catenin potentially and is useful in the treatment of cervical cancer.

Wnt/ß-catenin signaling pathway is involved in regulating the migration by an effective natural compound brucine in LoVo cells.

BACKGROUND: Colorectal cancer remains the third most common malignancies and migration is one of the main factors for its high mortality rate. Brucine, a natural plant alkaloid, has been proved to possess a variety of pharmacological functions including anti-tumor activities. PURPOSE: The aim of this study was to investigate the inhibitory effect of brucine on the colorectal cancer and the underlying mechanism. METHODS: In this study, colony formation assay and transwell assay were used to investigate the effect of brucine on LoVo cells viability and migration. Immunofluorescence assay, western blot assay and Gelatin zymography assay were used to study the mechanism of brucine. Xenograft model in nude mice was induced to investigate the in vivo effect of brucine on LoVo cells. RESULTS: Brucine could significantly decrease the viability, inhibit the colony formation and induce the apoptosis of LoVo cells. Brucine could also suppress the migration of LoVo cells in a dose-dependent manner. Western blot analysis elucidated that the inhibition of migration was associated with the decreasing expression of matrix metalloproteinases including MMP2, MMP3 and MMP9. Moreover, we found that treatment of brucine could downregulate the expression of Frizzled-8, Wnt5a, APC and GSNK1A1, and increase the expression of AXIN1. Meanwhile, brucine also decreased the phosphorylation level of LRP5/6 and GSK3ß, and increased the level of p-ß-catenin. Xenografted model in nude mice study also revealed that oral administration of brucine could inhibit the growth and migration of LoVo cells by activating the expression of AXIN1 and p-ß-catenin. CONCLUSION: Brucine could suppress the migration of the colorectal cancer in vitro and in vivo and the effect was associated with the inhibition of the Wnt/ß-catenin signaling pathway.

Novel compounds TAD-1822-7-F2 and F5 inhibited HeLa cells growth through the JAK/Stat signaling pathway.

Cervical carcinoma remains the second most common malignancy with a high mortality rate among women worldwide. TAD-1822-7-F2 (F2) and TAD-1822-7-F5 (F5) are novel compounds synthesized on the chemical structure of taspine derivatives, and show an effective suppression for HeLa cells. Our study aims to confirm the potential targets of F2 and F5, and investigate the underlying mechanism of the inhibitory effect on HeLa cells. In this study, Real Time Cell Analysis and crystal violet staining assay were conducted to investigate the effect of F2 and F5 on HeLa cells proliferation. And the analytical methods of surface plasmon resonance and quartz crystal microbalance were established and employed to study the interaction between F2 and F5 and potential target protein JAK2, suggesting that both compounds have strong interaction with the JAK2 protein. Western blot analysis, immunofluorescence staining study and PCR was conducted to investigate the molecules of JAK/Stat signaling pathway. Interestingly, F2 and F5 showed diverse regulation for signaling molecules because of their different chemical structure. F2 increased the expression of JAK2 and downregulated the level of P-JAK1 and P-JAK2, and decreased P-Stat3 (Ser727). While F5 could increase the expression of JAK2 and naturally decrease the phosphorylation of JAK1 and Tyk2, and decreased the expression of P-Stat6. Moreover, F2 and F5 showed the same downregulation on the P-Stat3 (Tyr705). Therefore, F2 and F5 could target the JAK2 protein and prevent the phosphorylation of JAKs to suppress the phosphorylation of the downstream effector Stats, which suggested that F2 and F5 have great potential to be the inhibitors of the JAK/Stat signaling pathway.

HMQ-T-F2 exert antitumour effects by upregulation of Axin in human cervical HeLa cells.

Looking for novel, effective and less toxic therapies for cervical cancer is of significant importance. In this study, we reported that HMQ-T-F2(F2) significantly inhibited cell proliferation and transplantable tumour growth. Mechanistically, HMQ-T-F2 inhibited HeLa cell growth through repressing the expression and nuclear translocation of ß-catenin, enhancing Axin expression, as well as downregulating the Wnt downstream targeted proteins. Knock-down of a checkpoint ß-catenin by siRNA significantly attenuated HeLa cell proliferation. Furthermore, XAV939, an inhibitor of ß-catenin, was used to treat HeLa cells and the results demonstrated that HMQ-T-F2 inhibited proliferation and migration via the inhibition of the Wnt/ß-catenin pathway.

In vitro and in vivo study of pH-sensitive and colon-targeting P(LE-IA-MEG) hydrogel microspheres used for ulcerative colitis therapy.

Hydrocortisone sodium succinate (HSS) is an anti-inflammatory drug, but its application on ulcerative colitis (UC) treatment is limited by its associated side-effects. To solve this problem, a kind of pH-sensitive P(LE-IA-MEG) hydrogel microspheres (HMSs) were prepared as the drug carrier of hydrocortisone sodium succinate (HSS) for the treatment of UC. The P(LE-IA-MEG) HMSs were spherical in shape with good dispersion and the mean particle size was 34.87±0.90µm. HSS was successfully loaded into the P(LE-IA-MEG) HMSs. The in vitro release study of HSS-loaded HMSs (HSS-HMSs) revealed that the HSS-HMSs possessed desirable pH-sensitivity, the cumulative release rate was 4.07% and 94.64% in the solution with pH 1.2 and pH 7.4 solution during 12h, respectively. Furthermore, the study on pharmacokinetic, gastrointestinal drug residue and side-effects were conducted to evaluate the in vivo colon-targeting property of the HSS-HMSs. All the results showed that the HSS-HMSs could deliver HSS to the colon as well as reduce its premature absorption in the upper gastrointestinal tract. Finally, the HSS-HMSs showed better ameliorative effects and therapeutic effects on mice with experimental colitis as compared to HSS. In conclusion, the HSS-HMSs had great potential in the treatment of UC.