Honggang Hu

Inhibitory effect and mechanism of mesenchymal stem cells on liver cancer cells

Mesenchymal stem cells (MSCs), with their capacity for self-renewal and differentiation into various cell types, are important seed cells for stem cell therapy. MSCs exhibit potent pathotropic migratory properties that make them attractive for use in tumor prevention and therapy. However, little is known about the underlying molecular mechanisms that link MSCs to the targeted tumor cells. This study investigated the inhibitory effect and mechanism of MSCs on human hepatoma HepG2 cells using co-culture and conditioned medium system and animal transplantation model. The HepG2 cells were co-cultured with MSCs or treated with conditional media derived from MSCs cultures in vitro. Results of methylthiazolyldiphenyl tetrazolium assay and flow cytometric assay showed that the proliferation and apoptosis of HepG2 cells decreased and increased, respectively. Reverse transcription polymerase chain reaction analysis showed that the expression levels of bcl-2, c-Myc, β-catenin, and survivin were downregulated. The results of enzyme-linked immunosorbent assay and Western blot proved that MSCs secreted Dkk-1 to inhibit the expression of Wnt signaling pathway-related factors (bcl-2, c-Myc, β-catenin, and survivin) in tumor cells, consequently inhibiting the proliferation and promoting the apoptosis of HepG2 cells. Animal transplantation experiment showed that tumor growth was significantly inhibited when HepG2 cells were co-injected with MSCs into nude mice. These results suggested that MSCs inhibited the growth and promoted the apoptosis of HepG2 cells in a dose-dependent manner. This study provided a new approach and experimental basis for cancer therapy. This study also proved that the Wnt signaling pathway may have a function in MSC-mediated tumor cell inhibition.

Administration of amyloid-β42 oligomer-specific monoclonal antibody improved memory performance in SAMP8 mice.

Amyloid-β peptide (Aβ) is recognized by many as the leading cause of Alzheimers disease (AD), and Aβ oligomers play a major role in the early-onset form of AD. Recently, the application of passive immunization targeting Aβ has been investigated as a potential method of AD immunotherapy. We used a strain of monoclonal antibody against Aβ42 oligomers, designated A8, as an Aβ inhibitor to suppress Aβ aggregation and Aβ-derived cell toxicity in vitro, and as a passive immunotherapy approach to treat SAMP8 (senescence accelerated mouse sub-line P8) mice, an animal model of AD, in vivo. First, our results showed that pre-incubation of A8 with Aβ oligomers inhibited both the maturation of Aβ fiber and Aβ oligomer toxicity on SH-SY5Y cells. Second, learning and memory was improved through intraperitoneal administration of A8 in SAMP8 mice. Third, Aβ pathology was ameliorated with decreased Aβ oligomers and phospho-tau levels in SAMP8 mice. Our data suggest that our monoclonal antibody A8 may be a candidate as a potential immunotherapeutic agent in AD.

A follow-up association study of two genetic variants for bone mineral density variation in Caucasians.

SummaryWe tested whether two genetic variants were associated with BMD at multiple clinically relevant skeletal sites in Caucasians. We found that variant rs7776725 is consistently associated with hip, spine, wrist and whole-body BMD, which highlights the potential importance of this variant or linked variants for osteoporosis.IntroductionA recent genome-wide association study identified two single nucleotide polymorphisms (SNPs), rs7776725 and rs1721400, that were associated with bone mineral density (BMD) variation at the radius, tibia and calcaneus in a Korean population. In this study, we aimed to test whether the association of these two genetic variants can be replicated in Caucasians and whether their association with BMD can be extended to other clinically relevant skeletal sites.MethodsWe performed this study in two large cohorts of unrelated US Caucasians. Area BMD at the hip, spine, wrist (ultra-distal radius) and whole body were measured with Hologic dual-energy X-ray absorptiometer. SNPs were genotyped with Affymetrix human genome-wide genotyping arrays. Association analyses were performed using PLINK.ResultsWe detected highly significant association (combined p = 1.42 × 10−16) of rs7776725 with wrist BMD but only borderline association signal (combined p = 0.017) for rs1721400 with wrist BMD. In addition, we found that rs7776725 was associated with BMD at the hip, spine and whole body. At the FAM3C gene locus where rs7776725 was located, we identified several other SNPs (rs4727922, rs1803389, rs718766 and rs7793554) that were also associated with BMD.ConclusionsThis is the first follow-up association study of rs7776725 and rs1721400 with BMD. The rs7776725 showed consistent association with BMD at multiple clinically important skeletal sites, which highlighted the potential importance of rs7776725 or linked SNPs for risk of osteoporosis. Further in-depth re-sequencing studies and functional assays are necessary to elucidate the underlying mechanisms.

A genome-wide association analysis implicates SOX6 as a candidate gene for wrist bone mass

Osteoporosis is a highly heritable common bone disease leading to fractures that severely impair the life quality of patients. Wrist fractures caused by osteoporosis are largely due to the scarcity of wrist bone mass. Here we report the results of a genome-wide association study (GWAS) of wrist bone mineral density (BMD). We examined ∼500000 SNP markers in 1000 unrelated homogeneous Caucasian subjects and found a novel allelic association with wrist BMD at rs11023787 in the SOX6 (SRY (sex determining region Y)-box 6) gene (P=9.00×10−5). Subjects carrying the C allele of rs11023787 in SOX6 had significantly higher mean wrist BMD values than those with the T allele (0.485:0.462 g cm−2 for C allele vs. T allele carriers). For validation, we performed SOX6 association for BMD in an independent Chinese sample and found that SNP rs11023787 was significantly associated with wrist BMD in the Chinese sample (P=6.41×10−3). Meta-analyses of the GWAS scan and the replication studies yielded P-values of 5.20×10−6 for rs11023787. Results of this study, together with the functional relevance of SOX6 in cartilage formation, support the SOX6 gene as an important gene for BMD variation.

On-column refolding purification of DT389-hIL13 recombinant protein expressed in Escherichia coli.

Protein refolding is a bottleneck in the production of therapeutic proteins from inclusion bodies. In recent years, several studies have described on-column refolding of recombinant proteins. DT389-hIL13 is a recombinant protein that targets the glioma. In our study, the recombinant protein DT389-hIL13 was expressed in Escherichia coli (E. coli). The isolated inclusion bodies were refolded using size exclusion chromatography (SEC) and further purified using anion exchange chromatography. Three different methods of SEC on-column refolding were studied. In vitro tests on U251 cells showed that the recombinant protein could effectively inhibit the proliferation of U251 cells, especially the protein refolded by urea and pH gradient method. The half-maximal inhibitory concentration (IC50) of 0.887 nM was achieved with this new method, unlike an IC50 of 11.4 nM achieved in the non-gradient method.

The Inhibitory Effect of Mesenchymal Stem Cells with rAd-NK4 on Liver Cancer

Mesenchymal stem cells (MSCs) can migrate to the tumor site and integrate into the tumor tissue. As a delivery vehicle of antitumor factors, MSCs have been tried in various tumor therapies. NK4 can both inhibit the growth, metastasis, and invasion of tumor cells induced by hepatocyte growth factor (HGF) and suppress tumor angiogenesis that is independent on HGF/cellular-mesenchymal-to-transition factor pathway. Adenovirus can directly deliver NK4 for tumor treatment but may induce immunological rejection. We combined MSCs with an adenovirus vector to deliver NK4 for liver tumor treatment. This study detected the migration of MSCs to high metastasis liver carcinoma cells MHCC-97H in vitro, investigated the inhibitory effect of rAd-NK4-MSCs on the growth and metastasis of MHCC-97H cells, further explored the inhibitory mechanism of rAd-NK4-MSCs to MHCC-97H cell metastasis, and examined the inhibitory effect of rAd-NK4-MSCs on the migration of human umbilical vein endothelial cells (HUVECs) in vitro. In this study, migration experiment was used for the potential capacity of MSCs and inhibition on migration of rAd-NK4-MSCs. Western blot was used for detecting the inhibition mechanism of rAd-NK4-MSCs to MHCC-97H cells. And, animal transplantation experiment was used for the inhibition of rAd-NK4-MSCs in vivo. MSC migration assay showed MSCs can significantly migrate to MHCC-97H cells. Co-culture results indicated that rAd-NK4-MSCs significantly inhibited the proliferation and migration of MHCC-97H cells in vitro. Western blot results proved that rAd-NK4-MSCs inhibited MHCC-97H cell migration correlating with suppressing Erk1/2 phosphorylation. HUVEC migration experiment suggested that rAd-NK4-MSCs had a potential of inhibiting tumor angiogenesis. Animal transplantation experiment showed that the tumor growth was significantly inhibited in the rAd-NK4-MSC group. Taken together, this study proved that NK4-modified MSCs had an inhibitory effect on the growth and migration of MHCC-97H cells and tumor angiogenesis, which provided a new strategy for liver tumor target therapy.

Inhibitory effect of genetically engineered mesenchymal stem cells with Apoptin on hepatoma cells in vitro and in vivo

Hepatocellular carcinoma (HCC) is an aggressive tumor and has become one of the most frequent causes of cancer death in the world. The rate of post-operative recurrence and metastasis are still high even though after surgical resection. It is a difficult problem with extraordinary importance for the clinical treatment. So stem cell therapy becomes one of the anti-tumor biotherapy methods which is exploring. Due to the feature of homing to tumor site and immunosuppressive, mesenchymal stem cells (MSCs) have the capacity of gene treatment to tumor as a vehicle. Apoptin derived from chicken anemia virus is one kind of protein with an inherent ability to lyse cancer cells while leaving normal cells unharmed. Adenovirus (Ad) vectors can be modified to deliver therapeutic genes with the advantages of low toxicity and high transfer capacity. Now it has not been reported that combining MSCs and Adenovirus with Apoptin are used in HCC treatment. This study intends to construct recombinant adenovirus which expresses Apoptin and then infects human bone marrow MSCs, and explore the migration of MSCs to the hepatoma cells and inhibitory effect of genetically engineered mesenchymal stem cells with Apoptin on hepatoma cells in vitro and in vivo. Our research successfully established the recombinant Ad which was constructed by Ad system, and obtained MSCs which could secrete Apoptin. We found that both the modified MSCs with Apoptin and their conditional medium significantly inhibited the proliferation of liver cancer cells HepG2, which provided a novel means and experimental basis for stem cell treatment for HCC. This study tries to search for a stem cell therapy for cancers, which will provide a new approach and experimental basis for the clinical treatment of cancer. At the same time, this research will also provide experimental basis for a novel in vivo drug delivery system through stem cells as vehicle, which will resolve immune rejection induced by repeated applications of drug directly delivered by Ad vectors and reduce the high cost of a large-scale production and purification of exogenous drugs.

Progress in studies on the DEK protein and its involvement in cellular apoptosis

DEK protein is an ubiquitous phosphorylated nuclear protein. Specific binding of DEK to DNA could change the topology of DNA and then affect the gene activity of the underlying DNA sequences. It is speculated that there might be some potential relationship between the stress reaction of cells and DEK proteins. The phosphorylation status of DEK protein is altered during death-receptor-mediated cell apoptosis. Both phosphorylation and poly(ADP-ribosyl)ation could promote the release of DEK from apoptotic nuclei to extracellular environment, and in this case DEK becomes a potential autoantigen of some autoimmune diseases. The available evidence powerfully suggests that DEK protein is closely relevant to apoptosis. The overexpression of DEK protein has dual function in cell apoptosis, in terms of inhibiting or triggering cell apoptosis.

Proteins of the origin recognition complex (ORC) and DNA topoisomerases on mammalian chromatin

BackgroundThe process of DNA replication requires the separation of complementary DNA strands. In this process, the unwinding of circularly closed or long DNA duplices leads to torsional tensions which must be released by topoisomerases. So topoisomerases play an important role in DNA replication. In order to provide more information about topoisomerases in the initiation of mammalian replication, we investigated whether topoisomerases occur close to ORC in the chromatin of cultured human HeLa cells.ResultsWe have used different cell fractionation procedures, namely salt and nuclease treatment of isolated nuclei as well as formaldehyde-mediated cross-linking of chromatin, to investigate the distribution of topoisomerases and proteins of the origin recognition complex (ORC) in the chromatin of human HeLa cells. First we obtained no evidence for a physical interaction of either topoisomerase I or topoisomerase II with ORC. Then we found, however, that (Orc1-5) and topo II occurred together on chromatin fragments of 600 and more bp lengths. At last we showed that both topo II and Orc2 protein are enriched near the origin at the human MCM4 gene, and at least some of the topo II at the origin is active in proliferating HeLa cells. So taken together, topoisomerase II, but not topoisomerase I, is located close to ORC on chromatin.ConclusionTopoisomerase II is more highly expressed than ORC proteins in mammalian cells, so only a small fraction of total chromatin-bound topoisomerase II was found in the vicinity of ORC. The precise position of topo II relative to ORC may differ among origins.

CorrigendumCorrigendum to “Quantitative proteomics and integrative network analysis identified novel genes and pathways related to osteoporosis” [J. Proteomics 142 (16 June 2016) 45–52]

a College of Life Sciences and Bioengineering, Beijing Jiao Tong University, Beijing 100044, China b Center for Bioinformatics and Genomics, Department of Biostatistics and Bioinformatics, Tulane University, New Orleans 70112, LA, USA c College of Life Sciences, Hunan Normal University, Changsha 410081, Hunan, China d Laboratory of Proteins and Proteomics, Department of Epidemiology, Soochow University School of Public Health, Suzhou 205123, Jiangsu, China