Research Progress of Bioactive Components in Sanghuangporus spp.

The species in Sanghuangporus are a group of edible mushrooms with a long history of oral use in East Asia as a health-improvement method. They should be classified under the genus Sanghuangporus rather than mistakenly in Phellinus or Inonotus. The major components in this genus consist of polysaccharides, polyphenols, triterpenoids, and flavonoids, all of which exist in the fruiting bodies and mycelia. For extraction, studies have shown methods using hot water, ethanol, DES solvent, and alkaline, followed by purification methods including traditional anion column, Sevag solution, macroporous resin, and magnetic polymers. Proven by modern medical technology, these components possess promising anti-inflammatory, antioxidative, antitumor, and immunoregulation effects; additionally, they have health-improving effects including pulmonary protection, hypoglycemic properties, sleep improvement, gout mitigation, antiaging, neuroprotection, and muscle-strengthening abilities. Several toxicity studies have revealed their safety and recommend a dose of 1 g/kg for mice. As a newly emerged concept, functional food can provide not only life-sustaining nutrients but also some health-improving effects. In conclusion, we substantiate Sanghuang as a functional food by comprehensively presenting information on extraction and purification methods, component medical and structural properties, and nontoxicity, hoping to benefit the development of Sanghuang species as a group of functional food.

CD36: The Bridge between Lipids and Tumors.

It has been found that the development of some cancers can be attributed to obesity, which is associated with the excessive intake of lipids. Cancer cells undergo metabolic reprogramming, shifting from utilizing glucose to fatty acids (FAs) for energy. CD36, a lipid transporter, is highly expressed in certain kinds of cancer cells. High expressions of CD36 in tumor cells triggers FA uptake and lipid accumulation, promoting rapid tumor growth and initiating metastasis. Meanwhile, immune cells in the tumor microenvironment overexpress CD36 and undergo metabolic reprogramming. CD36-mediated FA uptake leads to lipid accumulation and has immunosuppressive effects. This paper reviews the types of FAs associated with cancer, high expressions of CD36 that promote cancer development and progression, effects of CD36 on different immune cells in the tumor microenvironment, and the current status of CD36 as a therapeutic target for the treatment of tumors with high CD36 expression.

Silencing LY6D Expression Inhibits Colon Cancer in Xenograft Mice and Regulates Colon Cancer Stem Cells' Proliferation, Stemness, Invasion, and Apoptosis via the MAPK Pathway.

This study explored the role of lymphocyte antigen 6 family member D (LY6D) in colon cancer stem cells' (CCSCs) proliferation and invasion. LY6D was knocked down using siRNA, and the down-regulation of LY6D was verified using Western blotting. After LY6D knockdown, CCSCs' proliferation, stemness, and invasion were suppressed, whereas apoptosis was increased. Gene Ontology (GO) enrichment analysis revealed that the differentially expressed genes (DEGs) between siLY6D and the negative control groups were significantly enriched in the cell-substrate adherens junction, focal adhesion, and cell-substrate junction terms. Meanwhile, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the DEGs were significantly enriched in the MAPK pathway. In addition, Western blotting results showed that pBRAF and pERK1/2, cascade kinases of the MAPK pathway, were significantly down-regulated after LY6D knockdown. In addition, nude mice xenograft experiments showed that the siLY6D treatment decreased tumor sizes and weights and improved tumor-bearing mice survival rates compared with the control group. In conclusion, these findings indicate that LY6D, which is highly expressed in CCSCs, is a key factor involved in tumor growth and development and might be a potential cancer marker and therapeutic target for colon cancer.

Current status and influencing factors of self-management in knee joint discomfort among middle-aged and elderly people: a cross-sectional study.

BACKGROUND: This study aims to identify the current status and factors influencing self-management of knee discomfort in middle-aged and elderly people in China. METHODS: A stratified multistage cluster sampling method was used to select participants from communities in China from January 15 to May 31, 2020. A cross-sectional survey was conducted using the general information questionnaire and the Knee Joint Discomfort Self-management Scale. Univariate analysis and a generalized linear model were used to analyze the factors influencing self-management. RESULTS: The prevalence of knee discomfort was 77%. Moderate to severe discomfort accounted for 30.5%. The average item score of self-management in 9640 participants was 1.98 ± 0.76. The highest and lowest levels were: 'daily life management' and 'information management'. Gender, ethnicity, education level, economic source, chronic disease, knee pain in the past month, and the degree of self-reported knee discomfort were significant predictors of self-management. CONCLUSION: The self-management of knee discomfort in middle-aged and elderly people is poor, and the degree of discomfort is a significant predictor. Healthcare providers should consider socioeconomic demographic and clinical characteristics to help these individuals improve their self-management skills. Attention should also be given to improving their ability to access health information and making them aware of disease risks.

Correlation between PTSD and sleep quality in community-dwelling elderly adults in Hunan province of China.

Background: To understand the occurrence of post-traumatic stress disorder (PTSD) and the current status of sleep quality among community-dwelling elderly adults in Hunan Province of China, to explore the correlation between the two, and to analyze the trend of sleep disorders in PTSD elderly adults. Methods: A simple random sample containing 1,173 community-dwelling elderly adults in Hunan Province was established between March and May 2022, and an on-site face-to-face survey was administered using the PTSD Checklist-Civilian Version (PCL-C) with good reliability and validity, the Pittsburgh Sleep Quality Index (PSQI) scale, and a self-designed general condition questionnaire. Results: The incidence of PTSD in the 1,173 participants was 14.3% (168/1,173). The total incidence of sleep disorders was 40.9% (480/1,173); more specifically, the incidence of sleep disorders in participants with no PTSD symptom, in participants with mild-to-moderate PTSD symptoms, and in participants with severe PTSD symptoms was 36.3, 69.8, and 66.7%, respectively. The Spearman's rank correlation analysis showed that the total PTSD score and the scores of each dimension (i.e., re-experiencing symptom cluster, avoidance symptom cluster and hypervigilance symptom cluster) were positively correlated with the total PSQI score and its dimension scores (i.e., sleep quality, time to fall asleep, sleep duration, sleep efficiency, sleep disturbance, hypnotic medication, and daytime function) (P < 0.05). The correlation coefficients ranged from 0.013 to 0.495. For all PSQI dimensions, the differences across participants with different degrees of PTSD were statistically significant (P < 0.05). Conclusions: The overall status of PTSD and sleep quality in community-dwelling elderly adults in Hunan Province was not optimistic. The elderly with PTSD were more prone to sleep disorders, and the more severe the symptoms of PTSD, the poorer the sleep quality was. However, differences were observed in the scores of each dimension of sleep across participants with different degrees of PTSD. Regardless of the degree of PTSD symptoms, the sleep quality of the elderly is severely affected, and the occurrence rate is not unlimited.

Salinomycin-Loaded High-Density Lipoprotein Exerts Promising Anti-Ovarian Cancer Effects by Inhibiting Epithelial-Mesenchymal Transition.

Background: Effective treatments for ovarian cancer remain elusive, and survival rates have long been considered grim. Ovarian cancer stem cells (OCSCs) and epithelial-mesenchymal transition (EMT) are associated with cancer progression and metastasis, as well as drug resistance and eventual treatment failure. Salinomycin (Sal) has an extensive effect on a variety of cancer stem cells (CSCs); however, its poor water solubility and toxicity to healthy tissues at high doses limit further research into its potential as an anti-cancer drug. We proposed a therapeutic strategy by constructing a tumor-targeting carrier that mimics high-density lipoprotein (HDL) to synthesize salinomycin-loaded high-density lipoprotein (S-HDL). This strategy helps reduce the side effects of salinomycin, thereby improving its clinical benefits. Methods: OCSCs were isolated from ovarian cancer cells (OCCs) and the uptake of HDL nanoparticles was observed using laser confocal microscopes. After the cell viability analysis revealed the inhibitory effect of S-HDL on OCCs and OCSCs, the main biological processes influenced by S-HDL were predicted with a transcriptome sequencing analysis and verified in vitro and in vivo. Results: Cellular uptake analysis showed that the HDL delivery system was able to significantly enhance the uptake of Sal by OCCs, tentatively validating the targeting role of recombinant HDL, so that S-HDL could reduce the toxicity of Sal and increase its anti-ovarian cancer effects. Conversely, S-HDL could exert anti-ovarian cancer effects by inhibiting the proliferation of OCCs and OCSCs, promoting apoptosis, blocking EMT, and suppressing stemness and angiogenesis-related protein expression in vitro and in vivo. Conclusion: S-HDL had stronger anti-ovarian cancer effects than unencapsulated Sal. Thus, it may be a potential agent for ovarian cancer treatment in the future.

The Deubiquitinase USP29 Promotes SARS-CoV-2 Virulence by Preventing Proteasome Degradation of ORF9b.

Ubiquitin signaling is essential for immunity to restrict pathogen proliferation. Due to its enormous impact on human health and the global economy, intensive efforts have been invested in studying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its interactions with hosts. However, the role of the ubiquitin network in pathogenicity has not yet been explored. Here, we found that ORF9b of SARS-CoV-2 is ubiquitinated on Lys-4 and Lys-40 by unknown E3 ubiquitin ligases and is degraded by the ubiquitin proteasomal system. Importantly, we identified USP29 as a host factor that prevents ORF9b ubiquitination and subsequent degradation. USP29 interacts with the carboxyl end of ORF9b and removes ubiquitin chains from the protein, thereby inhibiting type I interferon (IFN) induction and NF-κB activation. We also found that ORF9b stabilization by USP29 enhanced the virulence of VSV-eGFP and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). Moreover, we observed that the mRNA level of USP29 in SARS-CoV-2 patients was higher than that in healthy people. Our findings provide important evidence indicating that targeting USP29 may effectively combat SARS-CoV-2 infection. IMPORTANCE Coronavirus disease 2019 (COVID-19) is a current global health threat caused by SARS-CoV-2. The innate immune response such as type I IFN (IFN-I) is the first line of host defense against viral infections, whereas SARS-CoV-2 proteins antagonize IFN-I production through distinct mechanisms. Among them, ORF9b inhibits the canonical IκB kinase alpha (IKKɑ)/ß/γ-NF-κB signaling and subsequent IFN production; therefore, discovering the regulation of ORF9b by the host might help develop a novel antiviral strategy. Posttranslational modification of proteins by ubiquitination regulates many biological processes, including viral infections. Here, we report that ORF9b is ubiquitinated and degraded through the proteasome pathway, whereas deubiquitinase USP29 deubiquitinates ORF9b and prevents its degradation, resulting in the enhancement of ORF9b-mediated inhibition of IFN-I and NF-κB activation and the enhancement of virulence of VSV-eGFP and SARS-CoV-2 trVLP.

Relationship between knee joint discomfort, self-management behavior, and quality of life in the middle-aged and elderly people in China: A cross-sectional study.

Background: The aim of this study was to describe the knee joint discomfort, self-management behavior, and quality of life (QoL) in the middle-aged and elderly people in China and to clarify the relationship between the knee joint discomfort, self-management behavior, and QoL. Methods: It is a cross-sectional study and in this study, a stratified multistage random sampling method was used to collect data on the three factors such as knee joint discomfort, self-management behavior, and QoL among the middle-aged and elderly people in the Hunan Province. Spearman's correlation analysis was used to test the relationship between the knee joint discomfort, self-management behavior, and QoL. Results: The results of the present study showed that among them, the prevalence of knee pain was the highest (52.1%), followed by knee weakness (42.5%), numbness (41.8%), cold feeling (40.0%), tenderness (38.3%), and distension feeling (37.5%). Average score of self-management of knee joint discomfort in the middle-aged and elderly people was 2.14 ± 0.67. The level of self-management in each dimension ranged from high to low as emotional management, daily management, symptoms management, and information management. The average scores of physical component summary (PCS) and mental component summary (MCS) were 42.85 ± 5.34 and 43.62 ± 8.43, respectively. The occurrence, frequency, and severity of discomfort symptoms were positively correlated with the symptoms management, daily management, information management, and self-management behaviors, and negatively correlated with the emotional management, PCS and MSC, except for the occurrence of discomfort symptoms (P < 0.05). Conclusion: Knee joint discomfort was prevalent in the middle-aged and elderly people. In addition, they displayed a low level of self-management behavior and poor QoL. The middle-aged and elderly people faced knee discomfort symptoms, the more frequent and severe symptoms, the higher level of symptom management, daily management, and information management, and the lower level of emotional management and QoL.

Synthesis and Characterization of Salinomycin-Loaded High-Density Lipoprotein and Its Effects on Cervical Cancer Cells and Cervical Cancer Stem Cells.

BACKGROUND: Cervical cancer stem cells (CCSCs), a small part of tumor population, are one of the important reasons for metastasis and recurrence of cervical cancer. Targeting CCSCs may be an effective way to eliminate tumors. Salinomycin (Sal) has been proved to be an effective anticancer drug in many studies, especially for cancer stem cells (CSCs). However, the cytotoxicity of salinomycin limits its further research as an anticancer drug. High-density lipoprotein (HDL) nanoparticles are an excellent drug carrier, which can reduce the toxicity of Sal, have a certain targeting effect and improve the clinical benefit of Sal. METHODS: Salinomycin-loaded high-density lipoprotein (S-HDL) was synthesized and characterized by various analytical techniques. CD44highCD24low CCSCs were isolated from HeLa cells by magnetic separation. The uptake of HDL nanoparticles was observed by laser confocal microscopy, and the effect of S-HDL on the proliferation of CCCs and CCSCs was detected by cell viability analysis. Genome-wide analysis was used to analyze the effects of S-HDL on the biological processes of CCCs and then cell apoptosis, cell cycle and cell migration were selected for verification. RESULTS: S-HDL had a particle size of 38.98 ± 1.78 nm and an encapsulation efficiency of 50.73 ± 4.29%. Cell uptake analysis showed that HDL nanoparticles could enhance the drug uptake of CCCs and CCSCs and may target CCCs and CCSCs. In cell viability analysis, CCCs and CCSCs showed high sensitivity to S-HDL. S-HDL can more efficiently prevent CCSCs from developing tumorspheres than Sal in tumorsphere formation study. S-HDL had stronger ability to induce cell cycle arrest, promote cell apoptosis and inhibit cell migration compared with free Sal, which was consistent with the results of Genome Wide analysis. CONCLUSION: S-HDL can effectively target and eliminate CCCs and CCSCs, which is a potential drug for the treatment of cervical cancer.

The Molecular Mechanism of Multiple Organ Dysfunction and Targeted Intervention of COVID-19 Based on Time-Order Transcriptomic Analysis.

Coronavirus disease 2019 (COVID-19) pandemic is caused by the novel coronavirus that has spread rapidly around the world, leading to high mortality because of multiple organ dysfunction; however, its underlying molecular mechanism is unknown. To determine the molecular mechanism of multiple organ dysfunction, a bioinformatics analysis method based on a time-order gene co-expression network (TO-GCN) was performed. First, gene expression profiles were downloaded from the gene expression omnibus database (GSE161200), and a TO-GCN was constructed using the breadth-first search (BFS) algorithm to infer the pattern of changes in the different organs over time. Second, Gene Ontology enrichment analysis was used to analyze the main biological processes related to COVID-19. The initial gene modules for the immune response of different organs were defined as the research object. The STRING database was used to construct a protein-protein interaction network of immune genes in different organs. The PageRank algorithm was used to identify five hub genes in each organ. Finally, the Comparative Toxicogenomics Database played an important role in exploring the potential compounds that target the hub genes. The results showed that there were two types of biological processes: the body's stress response and cell-mediated immune response involving the lung, trachea, and olfactory bulb (olf) after being infected by COVID-19. However, a unique biological process related to the stress response is the regulation of neuronal signals in the brain. The stress response was heterogeneous among different organs. In the lung, the regulation of DNA morphology, angiogenesis, and mitochondrial-related energy metabolism are specific biological processes related to the stress response. In particular, an effect on tracheal stress response was made by the regulation of protein metabolism and rRNA metabolism-related biological processes, as biological processes. In the olf, the distinctive stress responses consist of neural signal transmission and brain behavior. In addition, myeloid leukocyte activation and myeloid leukocyte-mediated immunity in response to COVID-19 can lead to a cytokine storm. Immune genes such as SRC, RHOA, CD40LG, CSF1, TNFRSF1A, FCER1G, ICAM1, LAT, LCN2, PLAU, CXCL10, ICAM1, CD40, IRF7, and B2M were predicted to be the hub genes in the cytokine storm. Furthermore, we inferred that resveratrol, acetaminophen, dexamethasone, estradiol, statins, curcumin, and other compounds are potential target drugs in the treatment of COVID-19.

MicroRNA-183 attenuates osteoarthritic pain by inhibiting the TGFα-mediated CCL2/CCR2 signalling axis.

AIMS: MicroRNA-183 (miR-183) is known to play important roles in osteoarthritis (OA) pain. The aims of this study were to explore the specific functions of miR-183 in OA pain and to investigate the underlying mechanisms. METHODS: Clinical samples were collected from patients with OA, and a mouse model of OA pain was constructed by surgically induced destabilization of the medial meniscus (DMM). Reverse transcription quantitative polymerase chain reaction was employed to measure the expression of miR-183, transforming growth factor α (TGFα), C-C motif chemokine ligand 2 (CCL2), proinflammatory cytokines (interleukin (IL)-6, IL-1ß, and tumour necrosis factor-α (TNF-α)), and pain-related factors (transient receptor potential vanilloid subtype-1 (TRPV1), voltage-gated sodium 1.3, 1.7, and 1.8 (Nav1.3, Nav1.7, and Nav1.8)). Expression of miR-183 in the dorsal root ganglia (DRG) of mice was evaluated by in situ hybridization. TGFα, CCL2, and C-C chemokine receptor type 2 (CCR2) levels were examined by immunoblot analysis and interaction between miR-183 and TGFα, determined by luciferase reporter assay. The extent of pain in mice was measured using a behavioural assay, and OA severity assessed by Safranin O and Fast Green staining. Immunofluorescent staining was conducted to examine the infiltration of macrophages in mouse DRG. RESULTS: miR-183 was downregulated in tissue samples from patients and mice with OA. In DMM mice, overexpression of miR-183 inhibited the expression of proinflammatory cytokines (IL-6, IL-1ß, TNF-α) and pain-related factors (TRPV1, Nav1.3, Nav1.7, Nav1.8) in DRG. OA pain was relieved by miR-183-mediated inhibition of macrophage infiltration, and dual luciferase reporter assay demonstrated that miR-183 directly targeted TGFα. CONCLUSION: Our data demonstrate that miR-183 can ameliorate OA pain by inhibiting the TGFα-CCL2/CCR2 signalling axis, providing an excellent therapeutic target for OA treatment. Cite this article: Bone Joint Res 2021;10(8):548-557.

Inhibition of microRNA-27b-3p relieves osteoarthritis pain via regulation of KDM4B-dependent DLX5.

Osteoarthritis (OA) represents a progressive degenerative disorder that predominantly affects the synovial membranes of joints. Recent studies have highlighted the significant role played by microRNAs (miRNAs) in OA development. The current study aimed to elucidate the underlying modulatory role of miR-27b-3p in the development of OA. The expression of miR-27b-3p in the OA patients and rat models post anterior cruciate ligament transection operation was measured using reverse transcription quantitative polymerase chain reaction, through which overexpressed miR-27b-3p was found in both of the samples. To further explore the miR-27b-3p functions in OA, western blot analysis, enzyme-linked immunosorbent assay, and ß-galactosidase activity assay were conducted with the results showing that knockdown of miR-27b-3p promoted expression of the osteogenic differentiation markers while inhibiting expression of the adipogenic differentiation markers, inflammatory factors, and cellular senescence of bone marrow mesenchymal stem cells (BMSCs). After that, the interactions between miR-27b-3p, lysine Demethylase 4B (KDM4B), and Distal-Less Homeobox 5 (DLX5) identified using dual-luciferase reporter gene assay and ChIP assay revealed that miR-27b-3p inhibited KDM4B and further reduced expression of DLX5. Finally, the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were assessed in rat models, and increased PWT and PWL were detected after miR-27b-3p silencing. In conclusion, suppression of miR-27b-3p could enhance KDM4B and DLX5 to alleviate OA pain, shedding light on a new potential therapeutic target for OA.

Enhanced anticancer effect of oncostatin M combined with salinomycin in CD133+ HepG2 liver cancer cells.

Oncostatin M (OSM) induces the differentiation of liver cancer stem cells (LCSCs) and increases sensitivity to the chemotherapeutic agent 5-fluorouracil, whereas salinomycin (Sal) induces apoptosis in cancer stem cells and inhibits the proliferation of liver cancer cells. However, there have been no studies investigating the anticancer effects of combination treatment with OSM and Sal. In the present study, we investigated the synergistic effects of OSM and Sal on LCSCs, the CD133+ subpopulations from HepG2 human liver cancer cells. CD133+ LCSCs were isolated using an immunomagnetic bead technique and identified through colony formation. After incubating with OSM and Sal, the ability of LCSC proliferation and invasion, as well as apoptosis rates were evaluated, and the expression of stemness-related genes was examined by quantitative real-time polymerase chain reaction. Additionally, the secretion of α-fetoprotein (AFP) and albumin (ALB) were analyzed by enzyme-linked immunosorbent assay. Our results indicated that OSM combined with Sal significantly suppressed LCSC proliferation and invasion and induced apoptosis, as determined by flow cytometry and increases in cleaved caspase-3 levels detected by western blotting. The results of the JC-1 staining assay indicated that this effect involved the mitochondrial pathway. Moreover, combination treatment reduced the expression of CD133 in LCSCs and suppressed stemness-related gene expression. Furthermore, the LCSCs produced lower levels of AFP and higher levels of ALB following combination treatment. In all experiments, combination treatment elicited more efficient anticancer effects on LCSCs as compared with single-drug treatment; therefore, our results demonstrated that combined treatment with OSM and Sal inhibited proliferation and induced differentiation and apoptosis in LCSCs, suggesting combined use of OSM and Sal as a therapeutic strategy for liver cancer.

Preparation of Vitamin E-Containing High-Density Lipoprotein and Its Protective Efficacy on Macrophages.

Atherosclerosis is a major cause for cardiovascular diseases. High-density lipoprotein (HDL) may reduce atherosclerosis through several different mechanisms. HDL is composed of lipids, cholesterol, cholesteryl esters, triglycerides, and phospholipids, mainly phosphatidylcholine plus specialized proteins called apolipoproteins (apos). In this study, we prepared vitamin E containing HDL (VE-HDL) that contains egg phosphatidylcholine, cholesterol, vitamin E, and two kinds of recombinant human apolipoproteins (rhapo)-rhapoA-I and rhapoE in vitro by the facilitation of cholate. After that, we studied the effects of VE-HDL on foam cell formation, cellular cholesterol efflux, oxidative low-density lipoprotein (oxLDL)-stimulated oxidative stress, and apoptosis of macrophages to evaluate the protective efficacy of VE-HDL on macrophages. As the results showed, we prepared a new type of reconstituent HDL with apolipoproteins and vitamin E for the first time. VE-HDL has protective efficacy on macrophages. It has the prospect of becoming a therapeutic agent on atherosclerosis in the future.

Long non-coding RNAs on the stage of cervical cancer (Review).

Cervical cancer is one of most malignant gynecological tumors. However, effective means for diagnosing and treating cervical cancer have yet to be identified. A few decades ago, long non-coding RNAs (lncRNAs) were regarded as useless parts of the genome, however, increasing data have demonstrated the importance of lncRNAs in the diagnosis and treatment of cervical cancers. The aim of the present study is to summarize the role(s) of HOTAIR, MALAT1, CCAT2, SPRY4-IT1, RSU1P2, CCHE1, lncRNA-EBIC and PVT1. Approximately 14 lncRNAs are involved in cervical cancer and several important proteins, miRNAs and other molecules and play crucial roles in a few traditional signaling pathways that have been proven to be related to those lncRNAs. In conclusion, lncRNAs may be useful as exact treatment targets and diagnostic biomarkers for improving therapies in cervical cancer patients and lncRNAs may contribute to effective diagnosis and treatment methods for cervical cancer.

Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris.

Melittin is well known to possess cytolytic activity with wide-spectrum lytic properties and its potential use as an agent to treat several types of cancer has been tested. Due to the non-specific toxicity, melittin can impair not only cancer cells but also normal tissue. Thus, tumor-targeted toxins may be helpful for developing novel anticancer therapeutics. The urokinase-type plasminogen activator (uPA) plays a central role in tissue remodelling events occurring in normal physiology and in pathophysiology, including cancer invasion and metastasis. Heartening findings showed that uPA receptor is predominantly expressed on many types of cancer. Therefore, the amino-terminal fragment (ATF) of uPA which was able to identify and bond with cancer cells was used as the cell-targeting domain to make up tumor-targeted toxin in this study. In the present study, pPICZαC-ATF-melittin eukaryotic expression vector was successfully constructed. After transformed into P. pastoris and induced by methanol, rATF-mellitin was detected by SDS-PAGE and western blot analysis. After induction with methanol, the expression level of rATF-mellitin was 312 mg/l in 80-l fermentor. rATF­mellitin was purified to >95% purity using SP Sepharose ion exchange chromatography and source™ 30 RPC with 67.2% recovery. Cell proliferation assay showed that rATF-melittin inhibited growth of SKOV3 cells and had no cytotoxicity effect on normal cells. For the first time, we established a stable and effective rATF-mellitin P. pastoris expression system to obtain a high level of expression of secreted rATF-mellitin which was purified by a highly efficient purification procedure.

Expression and activity analysis of a new fusion protein targeting ovarian cancer cells.

The aim of the present study was to develop a new therapeutic drug to improve the prognosis of ovarian cancer patients. Human urokinase-type plasminogen activator (uPA)17-34-kunitz-type protease inhibitor (KPI) eukaryotic expression vector was constructed and recombinant human uPA17-34-KPI (rhuPA17-34-KPI) in P. pastoris was expressed. In the present study, the DNA sequences that encode uPA 17-34 amino acids were created according to the native amino acids sequence and inserted into the KPI-pPICZαC vector, which was constructed. Then, uPA17­34-KPI-pPICZαC was transformed into P. pastoris X-33, and rhuPA17-34-KPI was expressed by induction of methanol. The bioactivities of a recombinant fusion protein were detected with trypsin inhibition analysis, and the inhibitory effects on the growth of ovarian cancer cells were identified using the TUNEL assay, in vitro wound­healing assay and Matrigel model analysis. The results of the DNA sequence analysis of the recombinant vector uPA17-34-KPI­pPICZα demonstrated that the DNA­encoding human uPA 17-34 amino acids, 285-288 amino acids of amyloid precursor protein (APP) and 1-57 amino acids of KPI were correctly inserted into the pPICZαC vector. Following induction by methonal, the fusion protein with a molecular weight of 8.8 kDa was observed using SDS-PAGE and western blot analysis. RhuPA17-34-KPI was expressed in P. pastoris with a yield of 50 mg/l in a 50-ml tube. The recombinant fusion protein was able to inhibit the activity of trypsin, inhibit growth and induce apoptosis of SKOV3 cells, and inhibit the invasion and metastasis of ovarian cancer cells. By considering uPA17-34 amino acid specific binding uPAR as the targeted part of fusion protein and utilizing the serine protease inhibitor activity of KPI, it was found that the recombinant fusion protein uPA17-34-KPI inhibited the invasion and metastasis of ovarian tumors, and may therefore be regarded as effective in targeted treatment.

Preparation and functional characterization of human vascular endothelial growth factor-melittin fusion protein with analysis of the antitumor activity in vitro and in vivo.

Vascular endothelial growth factor and its tyrosine kinase receptors have been identified as key mediators of the regulation of pathologic blood vessel growth and maintenance in the promotion of angiogenesis and tumor growth. Therefore, an alternative approach to destroying tumor endothelium would be to make this tissue particularly sensitive to VEGF-mediated drug delivery. To verify this hypothesis, we generated a protein containing VEGF165 fused to melittin. Melittin is a small linear peptide composed of 26 amino acid residues that can exert toxic or inhibitory effects on many types of tumor cells. This protein is a cytolytic peptide that attacks lipid membranes, leading to significant toxicity. In the present study, the Pichia pastoris expression system was used to express the fusion protein. Under optimal conditions, stable VEGF165-melittin production was achieved using a series of purification steps. The activity of VEGF165-melittin fusion protein was compared with melittin for its ability to suppress the growth of tumor cell line in vitro. The fusion toxin selectively inhibited growth of human hepatocellular carcinoma HepG-2 cell line with high expression of VEGFR-2. We found that sensitivity of VEGFR-2 transfected 293 cells to VEGF165-melittin enhanced as the cellular VEGFR-2 density increased. In an in vivo initial experiment, the fusion protein inhibited tumor growth in xenografts assays. Furthermore, successful expression and characterization of the fusion protein demonstrated its efficacy for use as a novel treatment strategy for cancer.

Expression and anticancer activity analysis of recombinant human uPA1­43-melittin.

The present study is focused on expression of a target fusion protein which can be used in ovarian cancer target therapy. It aimed to construct human urokinase-type plasminogen activator (uPA)(1-43)-melittin eukaryotic expression vector to express recombinant human uPA(1-43)-melittin (rhuPA(1-43)-melittin) in P. pastoris and to detect its anticancer effects on ovarian cancer cells. The DNA sequences that encode uPA1-43 amino acids and melittin were synthesized according to its native amino acid sequences and consequently inserted into pPICZαC vector. Then uPA1-43-melittin -pPICZαC was transformed into P. pastoris X-33, and rhuPA(1-43)-melittin was expressed by methonal inducing. The bioactivities of recombinant fusion protein were detected with inhibition effects on growth of ovarian cancer cells, cell cycle detection and TUNEL assay. The results of DNA sequence analysis of the recombinant vector uPA(1-43)-melittin -pPICZαC demonstrated that the DNA encoding human uPA 1-43 amino acids and 1-26 amino acids of melittin was correctly inserted into the pPICZαC vector. After being induced by methonal, fusion protein with molecular weight 7.6 kDa was observed on the basis of SDS-PAGE and western blot analysis. The recombinant protein was able to suppress growth of SKOV3, induce cell cycle arrest and apoptosis of SKOV3 cells. The fusion protein does not have any obvious toxicity on normal tissues. RhuPA(1-43)-melittin was successfully expressed in P. pastoris. Taking uPA(1-43) amino acids specifically binding to uPAR as targeted part of fusion protein, and making use of antitumor activity of melittin, the recombinant fusion protein it was able to inhibit growth of ovarian tumors and to be applied for effective targeted treatment.

Expression and purification of recombinant human apolipoprotein A-II in Pichia pastoris.

Apolipoprotein A-II (ApoA-II) is the second most abundant protein constituent of high-density lipoprotein (HDL). The physiologic role of ApoA-II is poorly defined. ApoA-II may inhibit lecithin:cholesterol acyltransferase and cholesteryl-ester-transfer protein activities, but may increase the hepatic lipase activity. ApoA-II may also inhibit the hepatic cholesteryl uptake from HDL probably through the scavenger receptor class B type I depending pathway. Interpretation of data from transgenic and knockout mice of genes involved in lipoprotein metabolism has been often complicated as clinical implications because of species difference. So it is important to obtain human ApoA-II for further studies about its functions. In our studies, Pichia pastoris expression system was first used to express a high-level secreted recombinant human ApoA-II (rhApoA-II). We have cloned the cDNA encoding human ApoA-II and achieved its high-level secreting expression with a yield of 65 mg/L of yeast culture and the purification process was effective and easy to handle. The purified rhApoA-II can be used to further study its biological activities.