ABSTRACT
It has been reported that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. These evidences suggest that QM may play an essential role in cell differentiation before mineralization. However, our research results showed that QM overexpression in MC3T3-E1 enhanced cell differentiation and mineralization. In this study, alkaline phosphatase (ALP) activity and nodule mineralization were increased in MC3T3-E1 from QM overexpression cultures relative to normal expression QM cultures. RT-PCR revealed upregulation of the marker genes type I collagen, ALP, osteocalcin, osterix and BMP-2 and a slight decrease of a negative regulator osteopontin. These results suggest that the increasing of QM expression could stimulate osteoblast differentiation and mineralization in MC3T3-E1.
Subject(s)
Calcification, Physiologic , Cell Differentiation/genetics , Ribosomal Proteins/genetics , Tumor Suppressor Proteins/genetics , 3T3 Cells , Alkaline Phosphatase/metabolism , Animals , Base Sequence , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/metabolism , Collagen Type I/metabolism , DNA Primers , Mice , Osteocalcin/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribosomal Protein L10 , Transforming Growth Factor beta/metabolismABSTRACT
In this study, cycloheximide (CHX) and VP-16 alone and in combination (C&V) have been used to strongly trigger apoptosis in U937 cells. The presence of CHX markedly prevented VP-16-induced apoptosis, suggesting that in this process de novo protein synthesis is required. But interestingly, C&V had shown more similarities with CHX but not VP-16 alone, including the effects on cell cycle distribution and induction of apoptosis, which occurred more quickly and was steadily enhanced by increasing concentrations of CHX or by N-alpha-tosyl-L-lysyl-chloromethyl ketone (TLCK), a serine protease inhibitor. These results indicate that CHX, not VP-16, is indeed the dominant inducer of U937 apoptosis, when they are coadministered. In particular, VP-16 even promoted CHX-induced apoptosis, but did not alter its selection of cell types. In T-cells resistant to CHX (Molt-4), we have detected no apoptotic response to their combination. These findings may well explain why the inhibitory effects of CHX on apoptosis induced by the same stimuli are usually different according to the cell type used, and also suggest that CHX may have the potential to lower side effects and drug resistance of cancer therapy.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Apoptosis/drug effects , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/physiology , Cell Proliferation/drug effects , Cycloheximide/pharmacology , Dose-Response Relationship, Drug , Etoposide/pharmacology , Humans , U937 CellsABSTRACT
AIM: To study effects of chebulinic acid on erythroid and megakaryocytic differentiation in K562 cells. METHODS: The benzidine staining method was used to evaluate hemoglobin synthesis; the expression of erythroid specific glycophorin A (GPA) protein and megakaryocytic surface marker CD61 was determined by flow cytometry using fluorescence labeled antibodies; erythroid and megakaryocytic mRNA expression was analyzed by RT-PCR. RESULTS: During erythroid differentiation induced by butyric acid (BA) or hemin, chebulinic acid not only inhibited the hemoglobin synthesis of BA- and hemin-treated K562 cells in concentration-dependent manner with IC50 of 4 micromol/L and 40 micromol/L respectively, but also inhibited another erythroid differentiation marker acetylcholinesterase at the concentration of 50 micromol/L in the cells either treated or untreated with each erythroid differentiation inducers, whereas chebulinic acid 50 micromol/L did not change GPA protein expression in these cells significantly. When K562 cells were treated with TPA 50 microg/L for 72 h to induce megakaryocytic differentiation, the presence of chebulinic acid 50 micromol/L slightly provoked the decrease of GPA protein expression induced by TPA. Chebulinic acid did not change the TPA-induced CD61 expression at the same concentration. Chebulinic acid also reduced the mRNA levels of erythroid relative genes including gamma-globin, PBGD, NF-E2, and GATA-1 genes in K562 cells either treated or untreated with BA, whereas chebulinic acid upregulated the mRNA levels of GATA-2 transcription factor in these cells. CONCLUSION: Chebulinic acid had inhibitory effect on erythroid differentiation likely through changing transcriptional activation of differentiation relative genes, which suggests that chebulinic acid or other tannins might influence the efficiency of some anti-tumor drugs-induced differentiation or the hematopoiesis processes.
Subject(s)
Glycophorins/biosynthesis , Hydrolyzable Tannins , Integrin beta3/biosynthesis , Tannins/pharmacology , Acetylcholinesterase/biosynthesis , Acetylcholinesterase/genetics , Cell Differentiation/drug effects , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Erythrocytes/metabolism , Erythrocytes/pathology , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , GATA2 Transcription Factor , Gene Expression Regulation , Globins/biosynthesis , Globins/genetics , Glycophorins/genetics , Humans , Integrin beta3/genetics , K562 Cells , Megakaryocytes/metabolism , Megakaryocytes/pathology , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit , RNA, Messenger/genetics , Transcription Factors/biosynthesis , Transcription Factors/geneticsABSTRACT
Tannins are a group of widely distributed plant polyphenols, some of which are beneficial to health because of their chemopreventive activities. In the present study, we investigated the effects and action mechanisms of woodfordin I, a macrocyclic ellagitannin dimer, on human chronic myelogenous leukemia (CML) K562 cells. The results showed that woodfordin I was able to suppress the proliferation and induce apoptosis in K562 cells. Apoptosis was evaluated by cytomorphology, internucleosomal DNA fragmentation, and externalization of phosphatidylserine. Woodfordin I treatment caused a rapid and sustained loss of mitochondrial transmembrane potential (MMP), transient generation of reactive oxygen species (ROS), transient elevation of intracellular Ca2+ concentration, and cytosolic accumulation of cytochrome c. The activation of caspase-9 and 3, but not caspase-8, was also demonstrated, indicating that the apoptotic signaling triggered by woodfordin I was mediated through the intrinsic mitochondria-dependent pathway. Western blot and immunofluorescence analysis revealed that the anti-apoptotic Bcl-2 and Bcl-xL levels were downregulated, together with the pro-apoptotic Bax protein. Significantly, woodfordin I-induced apoptosis was associated with a decline in the levels of c-Abl, Bcr-Abl, and cellular protein tyrosine phosphorylation. Considering the consequence of all the events in the process of woodfordin I-induced apoptosis, the mitochondrial dysfunction is directly responsible for the pro-apoptotic effects on K562 cells. Furthermore, because CML is a malignancy of pleuripotent hematopoietic cells caused by the dysregulated tyrosine kinase activity of Bcr-Abl, these findings suggest that woodfordin I may be a potential lead compound against CML.
