Junming Guo

Anticancer effect of aloe-emodin on cervical cancer cells involves G2/M arrest and induction of differentiation.

AbstractAim:The aim of this study was to investigate the effects of aloe-emodin, a natural compound from the root and rhizome of Rheum palmatum, on the growth of human cervical cancer cells, HeLa.Methods:HeLa cells were treated with various concentrations of aloe-emodin for 1-5 d, and cell growth was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. The long-term growth effect was investigated by crystal violet assay. The distributions of the cell cycle and apoptosis were analyzed by flow cytometry. The alkaline phosphatase (ALP) activity was analyzed by a chemical analyzer. Finally, Western blotting was used to indicate the abundant changes of protein kinase C (PKC), c-myc, cyclins, cyclin-dependent kinases (CDK), and proliferating cell nuclear antigen (PCNA).Results:Aloe-emodin inhibited the growth of HeLa cells in a dose-dependent manner at concentrations ranging between 2.5 and 40 μmol/L. The flow cytometric analysis showed that HeLa cells were arrested at the G2/M phase. This effect was associated with the decrease in cyclin A and CDK2, and the increase in cyclin B1 and CDK1. More importantly, the ALP activity was found to be increased by aloe-emodin treatment, and accompanied by the inhibition of PCNA expression. In addition, aloe-emodin suppressed the expression of PKCα and c-myc.Conclusion:These findings provide a possible mechanistic explanation for the growth inhibitory effect of aloe-emodin on HeLa, which includes cell cycle arrest and inducing differentiation.

Lin-28 reactivation is required for let-7 repression and proliferation in human small cell lung cancer cells

The let-7 family of microRNAs (miRNAs) are known to act as tumor suppressors and down-regulated in lung cancer. Recently, the RNA-binding protein Lin-28 was demonstrated to inhibit biogenesis of let-7 miRNAs by blocking both Drosha- and Dicer-mediated cleavage and accelerating decay of let-7 precursors. We selected NCI-H446 lung small cell lung cancer cell to determine whether it is broadly representative that Lin-28 can promote cell proliferation and affect cell cycle through negatively regulating let-7 biogenesis. Here, we showed that Lin-28 mRNA was up-regulated in NCI-H446 cell with a high c-Myc state. The result of real-time RT-PCR further indicated that pri-let-7a-1/7g and mature let-7g were remarkably down-regulated. The expression of lin-28 was down-regulated while the mature let-7g transcript was up-regulated inversely. The MTT assay indicated that the proliferation of lung cancer cells with lin-28 inhibition was signally impaired. The cells with lin-28 knockdown revealed a higher proportion of cells at G1/G0 phase and less at S phase. The results presented here demonstrate that induction of Lin-28 could mediate repression of let-7 family members, promote cell cycle progression and suppress cell proliferation.

Suppression of C-myc Expression Associates with Anti-Proliferation of Aloe-Emodin on Gastric Cancer Cells

Aloe-emodin is a hydroxyanthraquinone found in Aloe vera, as well as in leaves and roots of other plants. The mechanisms of its anticancer effect are largely unknown. The present study investigated its molecular mechanisms. Crystal violet assay showed that aloe-emodin had a long-term anti-proliferation effect on human gastric cancer MGC-803 and SGC-7901 cells. Scratch wound-healing motility assays indicated its anti-migration effect. Aloe-emodin arrested SGC-7901 cells at G2/M phase. More importantly, aloe-emodin inhibited the expressions of protein kinase C and c-myc. In conclusion, the anticancer effect of aloe-emodin on gastric cancer cells involves suppression of c-myc expression.

Growth inhibitory effects of gastric cancer cells with an increase in S phase and alkaline phosphatase activity repression by aloe-emodin.

Aloe-emodin is a novel active compound found in the root and rhizome of Rheum palmatum. To investigate the effects and mechanisms of aloe-emodin on human gastric cancer, MGC-803 cells were treated with 2.5, 5, 10, 20, and 40 μM aloe-emodin for 1- 5 d. The results showed that aloe-emodin inhibited the growth of cancer cells in a dose-dependent manner with an increase in S phase and in the proportion of cells cycling at a higher ploidy level (>G2/M). Moreover, the alkaline phosphatase (ALP) activity, an indicator of cell differentiation, was found decreased. This is one of the first to focus on the effect of ALP activity in human gastric carcinomas cells treated by aloe-emodin. These results indicate that aloe-emodin has a potential value for the treatment of gastric cancer and its mechanisms are by means of cell cycle interruption and induce differentiation.

Cloning, expression, purification and characterization of the cholera toxin B subunit and triple glutamic acid decarboxylase epitopes fusion protein in Escherichia coli

Induction of specific immunological unresponsiveness by oral autoantigens such as glutamic acid decarboxylase 65 (GAD65) is termed oral tolerance and may be a potential therapy for autoimmune diabetes. However, the requirement for large amounts of protein will limit clinical testing of autoantigens, which are difficult to produce. Mucosal adjuvants such as cholera toxin B subunit (CTB) may lower the level of autoantigens required. Here we describe cloning, expression, purification and identification study of the CTB and triple GAD(531-545) epitopes fusion gene. The fusion gene was ligated via a flexible hinge tetrapeptide and expressed as a soluble protein in Escherichia coli BL21 (DE3) driven by the T7 promoter. We purified the recombination protein from the cell lysate and obtained approximately 2.5mg of CTB-GAD((531-545)3) per liter of culture with greater than 90% purity by a Ni-NTA resin column. The bacteria produced this protein as the pentameric form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB and GAD65. Further studies revealed that oral administration of bacterial CTB-GAD((531-545)3) fusion protein showed the prominent reduction in pancreatic islet inflammation in non-obese diabetic mice. The results presented here demonstrate that the bacteria bioreactor is an ideal production system for an oral protein vaccine designed to develop immunological tolerance against autoimmune diabetes.

Glutamic acid decarboxylase epitope protects against autoimmune diabetes through activation of Th2 immune response and induction of possible regulatory mechanism

Oral tolerance mediated by autoantigens has been applied successfully as a potential therapeutic strategy for preventing and treating autoimmune diseases. We previously showed cholera toxin B subunit (CTB) is an efficient mucosal carrier molecule for induction of systemic T cell tolerance to linked insulin antigens. In this study, we used an oral antigen consisting of a fusion protein composed of CTB and triple copies of glutamic acid decarboxylase 65 (GAD65) peptides 531-545 (3p531) to test its in vivo effect and investigate the mechanism of immune tolerance. Non-obese diabetic mice fed microgram quantities of the CTB-3p531 fusion protein showed a prominent reduction in pancreatic islet inflammation and a delay in the development of diabetes. Increased anti-GAD65 IgG1, serum IgA and unchanged IgG2a antibodies titers; together with an increase of IL-4, IL-10 production and a decrease of IFN-gamma production suggested possible activation of GAD65-specific Th2 immune responses. Adoptive transfer of splenocytes indicated oral administration of CTB-3p531 fusion protein generated potent regulatory cells that can suppress diabetogenic T cells. This study demonstrates the CTB-3p531 fusion protein protects against autoimmune diabetes by generation of regulatory T cells and induction of immunological tolerance.