Xin-xing Wan

Regulatory effects of resveratrol on antioxidant enzymes: A mechanism of growth inhibition and apoptosis induction in cancer cells

Resveratrol (RSV) is a natural polyphenol that is known as a powerful chemopreventive and chemotherapeutic anticancer molecule. This study focused on the effects of RSV on the activities and expression levels of antioxidant enzymes in the cancer cells. Prostate cancer PC-3 cells, hepatic cancer HepG2 cells, breast cancer MCF-7 cells and the non-cancerous HEK293T kidney epithelial cells were treated with a wide range of RSV concentrations (10-100 μM) for 24–72 h. Cell growth was estimated by trypan blue staining, activities of the antioxidant enzymes were measured spectrophotometrically, expression levels of the antioxidant enzymes were quantified by digitalizing the protein band intensities on Western blots, and the percentage of apoptotic cells was determined by flow cytometry. Treatment with a low concentration of RSV (25 μM) significantly increased superoxide dismutase (SOD) activity in PC-3, HepG2 and MCF-7 cells, but not in HEK293T cells. Catalase (CAT) activity was increased in HepG2 cells, but no effect was found on glutathione peroxidase (GPX) upon RSV treatment. RSV-induced SOD2 expression was observed in cancer cells, although the expression of SOD1, CAT and GPX1 was unaffected. Apoptosis increased upon RSV treatment of cancer cells, especially in PC-3 and HepG2 cells. Together, our data demonstrated that RSV inhibits cancer cell growth with minimal effects on non-cancerous cells. We postulate that the disproportional up-regulation of SOD, CAT and GPX expression and enzymatic activity in cancer cells results in the mitochondrial accumulation of H2O2, which in turn induces cancer cell apoptosis.

Genetic polymorphisms of metabolic enzymes—CYP1A1, CYP2D6, GSTM1, and GSTT1, and gastric carcinoma susceptibility

Genetic polymorphisms in metabolic enzymes are associated with numerous cancers. In this study, the relationships between genetic polymorphisms of phase I metabolic enzymes including cytochrome P450 1A1 (CYP1A1), CYP2D6 and phase II metabolic enzymes such as glutathione S-transferase M1 (GSTM1) and GSTT1 and gastric carcinoma susceptibility were investigated. Genomic DNA was isolated from the peripheral blood of 129 healthy controls and 123 gastric carcinoma patients from Han ethnic group of Hunan Province located in Central South China. The genetic polymorphisms of the above mentioned enzymes were analyzed using PCR-RFLP techniques. There was no significant difference among the frequencies of CYP1A1 and/or CYP2D6 gene’s wild type, heterozygous or homozygous mutations between the gastric carcinoma group and control group. But the differences among the frequencies of GSTM1 and GSTT1 null genotype between the gastric carcinoma and control group were significant (both P < 0.05). Also there were significant differences in the frequencies of GSTM1 null in high/high–middle differentiated, middle differentiated, middle–low differentiated and low differentiated gastric tumor separately. GSTM1 null showed an increased risk in middle–low differentiated and low differentiated gastric carcinoma type, but GSTT1 null was not a risk factor for the four pathological types of gastric carcinoma mentioned above. We report here that the genotypes of CYP1A1 and CYP2D6 are not associated with gastric carcinoma risk; GSTM1 null, but not GSTT1 null inheritably increases risk of some pathological types of gastric carcinoma in Han ethnic population of Hunan Province.

Polymorphisms of DNA repair genes XPD , XRCC1 , and OGG1 , and lung adenocarcinoma susceptibility in Chinese population

Lung adenocarcinoma (ADC) is one of the major histological types of lung cancer. Genetic polymorphism in DNA repair genes and lung ADC susceptibility is well documented. In this case–control study, the association between the polymorphic sites of DNA repair genes XPD-751, XRCC1-399, and OGG1-326, and lung ADC susceptibility in ethnic Han Chinese population has been investigated. Genomic DNA was isolated from the peripheral blood of 201 healthy controls and 82 lung ADC patients from the people of Hunan Province, China. Polymorphisms of the investigated genes were analyzed by using polymerase chain reaction–restriction fragment length polymorphism. There was no significant difference between the samples from lung ADC patients and healthy controls about the genotype frequencies of XPD-751, XRCC1-399, and OGG1-326 sites. However, multifactor dimensionality reduction analysis showed that the genetic polymorphisms of the three-loci models of DNA repair genes (XPD-751/XRCC1-399/OGG1-326) are associated with lung ADC. Thus, this study reveals that a three-order interaction among the polymorphic sites of XPD-751, XRCC1-399, and OGG1-326 is associated with lung ADC risk in the studied population, although polymorphism in individual gene was not associated.

miR‑204-5p promotes the adipogenic differentiation of human adipose-derived mesenchymal stem cells by modulating DVL3 expression and suppressing Wnt/β-catenin signaling

MicroRNAs (miRNAs or miRs) play an important regulatory role during adipogenesis, and have been studied extensively in this regard. Specifically, the switch between the differentiation of mesenchymal stem cells (MSCs) towards adipogenic vs. osteogenic lineages is regulated by miR-204 which controls the expression of Runx2. However, the association between miR-204-5p and the Wnt/β-catenin signaling pathway during adipogenesis has not yet been clarified. In the present study, we demonstrate that miR-204-5p regulates the in vitro adipogenesis of human adipose-derived mesenchymal stem cells (hADSCs). The level of miR-204-5p was shown to be gradually upregulated during adipocytic differentiation, together with the mRNA expression of the critical adipogenic transcription factors, cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT) enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ), and the mature adipogenic marker, fatty acid binding protein 4 (FABP4). We further demonstrate that while the overexpression of miR-204-5p promotes adipogenesis, its knockdown causes the inhibition of this process. We then used bioinformatics tools and luciferase reporter assay to establish that dishevelled segment polarity protein 3 (DVL3), a key regulator of the Wnt/β-catenin signaling pathway, is a direct target of miR-204-5p. In addition, the overexpression of DVL3 led to an increase in β-catenin and cyclin D1 (CCND1) expression and, by contrast, the knockdown of DVL3 led to a decrease in the expression of β-catenin and CCND1. The knockdown of DVL3 significantly promoted adipogenesis. Finally, we demonstrated that the overexpression of miR-204-5p induced the downregulation of β-catenin and the canonical Wnt target gene, CCND1, in mature adipoctyes, while its knockdown led to their upregulation. Taken together, our data suggest that miR-204-5p regulates adipogenesis by controlling DVL3 expression and subsequently inhibiting the activation of the Wnt/β-catenin signaling pathway.

miR-125a-3p and miR-483-5p promote adipogenesis via suppressing the RhoA/ROCK1/ERK1/2 pathway in multiple symmetric lipomatosis

Multiple symmetric lipomatosis (MSL) is a rare disease characterized by symmetric and abnormal distribution of subcutaneous adipose tissue (SAT); however, the etiology is largely unknown. We report here that miR-125a-3p and miR-483-5p are upregulated in the SAT of MSL patients, promoting adipogenesis through suppressing the RhoA/ROCK1/ERK1/2 pathway. TaqMan microRNA (miR) array analysis revealed that 18 miRs were upregulated in the SAT of MSL patients. Transfection of human adipose-derived mesenchymal stem cells (hADSCs) with the individual agomirs of these 18 miRs showed that miR-125a-3p and miR-483-5p significantly promoted adipogenesis. A dual-luciferase assay showed that RhoA and ERK1 were the targets of miR-125a-3p and miR-483-5p, respectively. Moreover, transfection of hADSCs with mimics of miR-125a-3p and miR-483-5p resulted in a pronounced decrease of ERK1/2 phosphorylation in the nucleus; conversely, transfection of hADSCs with inhibitors of miR-125a-3p and miR-483-5p led to a significant increase of ERK1/2 phosphorylation in the nucleus. Most importantly, we found that miR-125a-3p and miR-483-5p promoted de novo adipose tissue formation in nude mice. These results demonstrated that miR-125a-3p and miR-483-5p coordinately promoted adipogenesis through suppressing the RhoA/ROCK1/ERK1/2 pathway. Our findings may provide novel strategies for the management and treatment of MSL or obesity.

NLRP3 inflammasome activation in mesenchymal stem cells inhibits osteogenic differentiation and enhances adipogenic differentiation

Osteoporosis is one of the most common skeletal disease featured by osteopenia and adipose accumulation in bone tissue. NLRP3 inflammasome activation is an essential player in aging-related chronic diseases like osteoporosis, particularly due to the causal caspase-1 activation and its correlation to adipose accumulation in bone tissue. Moreover, the expression of anti-aging/senescence SIRT1 was reported to decline along with aging. As the major cellular contributor of bone formation, mesenchymal stem cells (MSCs) are multipotent stem cells processing mutually exclusive differentiatability toward osteocytes or adipocytes. Therefore, we hypothesized that NLRP3 inflammasome activation promotes adipogenesis and repress osteogenesis in MSCs via inhibiting SIRT1 expression. We activated NLRP3 inflammasome in human MSCs via lipopolysaccharide and palmitic acid (LPS/PA) treatment for self-renewal maintenance, adipogenic differentiation or osteogenic differentiation. LPS/PA treatment significantly increased NLRP3 expression, decreased SIRT1 expression and promoted caspase-1 activity in MSCs. LPS/PA treatment also boosted adipogenesis of MSCs and suppressed osteogenesis. Moreover, inhibition of caspase-1 activity repressed adipogenic differentiation and partially improved osteogenic differentiation of MSCs with LPS/PA treatment. Our study demonstrated the pivotal roles of NLRP3 inflammasome and downstream mediator caspase-1 for the progress of osteo-differentiation MSCs, and offered novel therapeutic target of treatment for osteoporosis.

ISG15 Inhibits IFN-α-Resistant Liver Cancer Cell Growth

Hepatocellular carcinoma (HCC) is one of the most prevalent tumors worldwide. Interferon-α (IFN-α) has been widely used in the treatment of HCC, but patients eventually develop resistance. ISG15 ubiquitin-like modifier (ISG15) is a ubiquitin-like protein transcriptionally regulated by IFN-α which shows antivirus and antitumor activities. However, the exact role of ISG15 is unknown. In the present study, we showed that IFN-α significantly induced ISG15 expression but failed to induce HepG2 cell apoptosis, whereas transient overexpression of ISG15 dramatically increased HepG2 cell apoptosis. ISG15 overexpression increased overall protein ubiquitination, which was not observed in cells with IFN-α-induced ISG15 expression, suggesting that IFN-α treatment not only induced the expression of ISG15 but also inhibited ISG15-mediated ubiquitination. The tumor suppressor p53 and p21 proteins are the key regulators of cell survival and death in response to stress signals such as DNA damage. We showed that p53 or p21 is only up regulated in HepG2 cells ectopically expressing ISG15, but not in the presence of IFN-α-induced ISG15. Our results suggest that ISG15 overexpression could be developed into a powerful gene-therapeutic tool for treating IFN-α-resistant HCC.

Silibinin protects β cells from glucotoxicity through regulation of the Insig-1/SREBP-1c pathway

Exposure to high glucose may cause glucotoxicity, leading to pancreatic β cell dysfunction including cell apoptosis, impaired glucose‑stimulated insulin secretion (GSIS) and intracellular lipid accumulation. Sterol regulatory element binding protein-1c (SREBP-1c), a key nuclear transcription factor that regulates lipid metabolism, has been proven to play a role in insulin secretion. Insulin induced gene-1 (Insig-1) is an upstream regulatory factor of SREBP-1c. The overexpression of Insig-1 significantly inhibits SREBP-1c expression and thereby blocks the expression of downstream genes. It has been proven that silibinin, a natural flavanone, is involved in a variety of biological functions. In the present study, we examined whether silibinin protects high glucose-induced β cell dysfunction through the Insig-1/SREBP-1c pathway. Our data demonstrated that 30.0 µM of silibinin significantly improved cell viability (P<0.05) after rat insulinoma INS-1 cells were exposed to high glucose for 72 h. Silibinin partially attenuated GSIS following exposure to high glucose for either 24 or 72 h (both P<0.05). As shown by reverse transcription quantitative PCR, silibinin upregulated the mRNA expression of insulin secretion‑related genes [insulin receptor substrate 2 (IRS-2), pancreatic and duodenal homeobox 1 (PDX-1) and insulin], but downregulated uncoupling protein‑2 (UCP-2) expression. Silibinin inhibited intracellular lipid accumulation and free fatty acid (FFA) synthesis. Further experiments revealed that silibinin improved β cell function through the regulation of the Insig-1/SREBP-1c pathway. In conclusion, these results clearly suggest that the protection of β cells from glucotoxicity can be significantly enhanced through the regulation of the Insig-1/SREBP-1c pathway. Thus, silibinin may be a novel therapeutic agent for β cell dysfunction.

Identification of Heptapeptides Interacting with IFN-α-Sensitive CML cells

Background: Interferon-alpha (IFN-α) is the traditional therapeutic agent for chronic myeloid leukemia (CML). The molecular mechanism of IFN-α efficacy in the treatment of CML is not fully clear. Objectives: To identify the peptides and/or proteins that bind to the proteins specifically expressed on the surface of IFN-α-sensitive CML cells by using a phage display library. Design/methods: IFN-α-sensitive KT-1/A3 cells were used as the target, and IFN-α-resistant subline KT-1/A3R was used as absorber for phage display biopanning. The positive phage clones were identified by enzyme-linked immunosorbent assay and flow cytometry. The peptides were deduced from their DNA sequences. Results: Multiple clones showed high binding efficiency to KT-1/A3 cells compared with that of the other leukemia cells. One of the peptides, KLWVIPQ, has a partial amino acid sequence homology with the C-terminal domain of E3 ubiquitin-protein ligase. Conclusions: This study presents the identification of specific heptapeptides that bind to IFN-α-sensitive KT-1/A3 cells. The cancer-selective ligands provide novel strategies for early and differential diagnoses, as well as potential targeted drug delivery.

Effects of a particular heptapeptide on the IFN-α-sensitive CML cells

Using the phage display biopanning technique, we have previously identified a heptapeptide KLWVIPQ which specifically binds to the surface of the IFN-α-sensitive but not the IFN-α-resistant CML cells. The effects of this heptapeptide on the IFN-α-sensitive CML cells were investigated in the present study. IFN-α-sensitive KT-1/A3 and IFN-α-resistant KT-1/A3R CML cells were transfected by pEGFP-KLWVIPQ expression vector and/or induced by IFN-α. WST-1 cell proliferation assay, flow cytometry, and western blotting were performed to determine the effects of this heptapeptide and/or IFN-α on CML cells. The viability of the KT-1/A3 cells was inhibited and apoptosis was induced by either expression of the heptapeptide KLWVIPQ or IFN-α treatment with concurrent upregulation of P53 and downregulation of P210bcr/abl. However, these effects were not observed in the IFN-α-resistant KT-1/A3R cells. These results suggest that the heptapeptide KLWVIPQ shares a similar mechanism with IFN-α in the regulation of CML cell growth and apoptosis, implying that the heptapeptide KLWVIPQ could be a novel target to go further into mechanisms of IFN-α sensitivity and/or resistance in CML.