PURPOSE: This study aimed to investigate the effect of adipose-derived stem cell (ADSC) transplantation on atherosclerosis (AS) and its underlying mechanisms. MATERIALS AND METHODS: In our study, rat AS model was established, and ADSCs were isolated and cultured. Atherosclerotic plaque and pathological symptoms of thoracic aorta were measured by Oil Red O staining and Hematoxylin-Eosin staining, respectively. Total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured by an automatic biochemical analyzer. Expressions of vascular endothelial growth factor (VEGF), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), aortic endothelin-1 (ET-1), interleukin-6 (IL-6), c-reactive protein (CRP), and tumor necrosis factor α (TNF-α) were measured by enzyme linked immunosorbent assay, VEGF, VCAM-1, ICAM-1, ET-1, respectively, and NF-κB p65 mRNA expressions were detected by quantitative real-time polymerase chain reaction. Protein expressions of VEGF, VCAM-1, ICAM-1, ET-1, NF-κB p65, p-NF-κB p65, and IκBα were measured by western blot. Moreover, NF-κB p65 expression was measured by immunofluorescence staining. RESULTS: ADSC transplantation alleviated the pathological symptoms of aortic AS. ADSC transplantation decreased the levels of TC, TG, and LDL-C and increased serum HDL-C level. Meanwhile, ADSC transplantation decreased the levels of IL-6, CRP, and TNF-α in AS rats. Moreover, the expressions of VEGF, ET-1, VCAM-1, and ICAM-1 were decreased by ADSC transplantation. ADSC transplantation inhibited phosphorylation of NF-κB p65 and promoted IκBα expression in AS rats. CONCLUSION: Our study demonstrated that ADSC transplantation could inhibit vascular inflammatory responses and endothelial dysfunction by suppressing NF-κB pathway in AS rats.
Animals ; Aorta, Thoracic ; Atherosclerosis ; Blotting, Western ; C-Reactive Protein ; Cholesterol ; Endothelin-1 ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique ; Intercellular Adhesion Molecule-1 ; Interleukin-6 ; Lipoproteins ; Phosphorylation ; Plaque, Atherosclerotic ; Rats ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Stem Cell Transplantation ; Stem Cells ; Triglycerides ; Tumor Necrosis Factor-alpha ; Vascular Cell Adhesion Molecule-1 ; Vascular Endothelial Growth Factor A

OBJECTIVE: To investigate the effects of ethanol extract of Patrinia scabiosaefolia (EEPS) on chemo-resistance of colorectal cancer cells (CRC) and explore the possible molecular mechanisms. METHODS: 5-fluorouracil (5-FU)-resistant human colorectal carcinoma cell line (HCT-8/5-FU) and its parental cells HCT-8 were treated with EEPS (0, 0.25, 0.50, 1 or 2 mg/mL), or 5-FU (0, 100, 200, 400, 800 or 1600 μmol/L). The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the cell viability. Cell density was observed by phase-contrast microscope, cell counting and colony formation assay were used to determine the cell proliferation of HCT-8/5-FU cells treated with 0, 0.5, 1 or 2 mg/mL EEPS. Cell apoptosis was determined by Hoechst staining. Western-blot was performed to detect the phosphorylation of AKT as well as the protein expression level of B-cell CLL/lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax). RESULTS: Compared with HCT-8 cells, MTT assay results indicated that HCT-8/5-FU cells were resistant to 5-FU treatment (P<0.05), and sensitive to EEPS treatment (P>0.05). Moreover, compared with untreated HCT-8/5-FU cells, 1 and 2 mg/mL of EEPS treatment significantly reduced cell density, cell number, inhibited cell survival (P<0.05), and induced apoptosis in HCT-8/5-FU cells. Furthermore, 1 and 2 mg/mL of EEPS significantly decreased the phosphorylation level of p-AKT and Bcl-2 protein expression, and increased the expression of Bax protein (P<0.05). CONCLUSION EEPS is a promising therapeutic agent that may overcome chemo-resistance in cancer cells, likely through suppression of the AKT pathway and promotion of cancer cell apoptosis.
Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Colorectal Neoplasms ; drug therapy ; pathology ; Drug Resistance, Neoplasm ; drug effects ; Fluorouracil ; pharmacology ; therapeutic use ; Humans ; Patrinia ; chemistry ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; drug effects ; Tumor Stem Cell Assay ; bcl-2-Associated X Protein ; metabolism

To investigate the effect of manganese superoxide dismutase (MnSOD) silence on the in vitro tumorigenicity in human small cell lung cancer NCI-H446 cells and the underlying mechanisms.
 Methods: Sphere formation cells from NCI-H446 cells were obtained by suspension culture, while the expression of MnSOD and urokinase type plasminogen activator (uPAR) was analyzed by Western blot. Silence of MnSOD was performed by adenovirus infection in the second passage formation cells, and the effect of MnSOD silence on tumorigenicity in NCI-H446 cells was evaluated by sphere formation assay and soft-agar colony formation assay, while the expression of uPAR was analyzed by Western blot.
 Results: Compared with NCI-H446 cells, the sphere formation rate, colony formation rate, and the expression of MnSOD and uPAR were significantly increased in the second passage sphere formation cells in NCI-H446 cells (P<0.05). Silence of MnSOD inhibited the sphere formation rate, colony formation rate, and the expression level of uPAR in the second passage sphere formation cells in NCI-H446 cells.
 Conclusion: MnSOD may promote tumorigenicity in NCI-H446 cells by up-regulation of uPAR expression in vitro.
Adenoviridae ; Carcinogenesis ; Cell Line, Tumor ; Humans ; In Vitro Techniques ; Lung Neoplasms ; etiology ; metabolism ; RNA Interference ; Receptors, Urokinase Plasminogen Activator ; genetics ; metabolism ; Small Cell Lung Carcinoma ; etiology ; metabolism ; Spheroids, Cellular ; pathology ; Superoxide Dismutase ; genetics ; metabolism ; Tumor Stem Cell Assay ; Up-Regulation

OBJECTIVETo investigate whether SIS3, a specific inhibitor of Smad3 phosphorylation, can reverse the stemness of multidrug-resistant(MDR) hepatocellular carcinoma cells.

METHODSMDR HCC Huh7.5.1/ADM cell lines were developed by exposing parental cells to stepwise increasing concentrations of ADM. CCK-8 assay was used to determine the cellular sensitivity of various anticancer drugs. Flow cytometry (FCM) was used to analyze the expression level of cancer stem cell marker CD133. Clone formation assay and mouse subcutaneous xenograft tumors were used to investigate the tumorigenicity in vitro and in vivo. Western blotting (WB) was used to analyze the changes of expressions of CD133, Smad3, Bcl-2, Bax and p-Smad3 in different conditions.

RESULTSADM treatment of HCC cells in vitro resulted in a development of subline, Huh7.5.1/ADM cells, with CSC phenotypes: stable MDR phenotype (besides ADMc Huh7.5.1/ADM cells were also more resistant to some other anticancer drugs including VCR, MMC and CTX ) (IC50: 0.215 ± 0.018 vs. 0.123 ± 0.004, 0.145 ± 0.009 vs. 0.014 ± 0.002, 1.021 ± 0.119 vs. 0.071 ± 0.006, 27.007 ± 1.606 vs. 1.919 ± 0.032) (unit: µg/ml) (P<0.05). Huh7.5.1/ADM cells enriched the cancer stem-like cell fraction (CD133-positive subpopulation) (76.06 ± 2.948% vs. 25.38 ± 4.349%) (P<0.05), had stronger tumorigenicity in vivo and colony formation ability, and activated the Smad3 activity. Inhibition of Smad3 activity by SIS3 decreased stemness of the Huh7.5.1/ADM cells: CD133-positive subpopulation (48.49 ± 2.304% vs. 76.06 ± 2.948%) (P<0.05); ADM IC50: (0.112 ± 0.019 vs. 0.215 ± 0.018), VCR IC50 (0.065 ± 0.013 vs. 0.145±0.009), MMC IC₅₀ (0.749 ± 0.121 vs. 1.021 ± 0.119), CTX IC50 (10.576 ± 1.248 vs. 27.007 ± 1.606) (unit: µg/ml) (P<0.05), and decreased tumorigenicity and colony formation ability.

CONCLUSIONSIS3 as a specific inhibitor of Smad3 signal is involved in the stemness of multidrug resistant hepatocellular carcinoma cells.

AC133 Antigen ; Animals ; Antibiotics, Antineoplastic ; pharmacology ; Antigens, CD ; metabolism ; Carcinoma, Hepatocellular ; drug therapy ; metabolism ; pathology ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Glycoproteins ; metabolism ; Heterografts ; Humans ; Isoquinolines ; pharmacology ; Liver Neoplasms ; drug therapy ; metabolism ; pathology ; Mice ; Neoplasm Proteins ; metabolism ; Neoplastic Stem Cells ; drug effects ; Peptides ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Pyridines ; pharmacology ; Pyrroles ; pharmacology ; Smad3 Protein ; antagonists & inhibitors ; metabolism ; Tumor Stem Cell Assay ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the expression of mitogen-activated protein kinase (MAPK) in the chronic periodontitis tissue-derived in the periodontal ligament stem cells (PDLSCs) and explore its effect on the osteogenic differentiation of human PDLSCs in inflammatory microenvironment.

METHODSPDLSCs were obtained from human healthy individuals (H-PDLSCs) and patients with periodontitis (P-PDLSCs). The tumor necrosis factor (TNF)-Α and interleukin (IL)-1Β secretion and mRNA expression levels of H-PDLSCs and P-PDLSCs were detected using enzyme-linked immunosorbent assay and real-time quantitative PCR. Immunofluorescence staining was used for determining the protein levels of p38 in PDLSCs. The levels of p38 and p-p38 following culture in osteogenic medium for 7 d of H-PDLSCs and P-PDLSCs were detected using Western blotting. After the PDLSCs were stimulated with SB-203580,the p38 MAPK specific inhibitor, for 30 min and then in osteogenic induction process for 7 days,the expression levels of the osteogenic gene Runx2 and alkaline phosphatase (ALP) were determined by real-time quantitative PCR, and bone formation ability of PDLSCs was tested by alizarin red (AR) staining.

RESULTSThe secretions of TNF-Α and IL-1Β were significantly higher in P-PDLSCs compared with H-PDLSCs (68.80 ± 6.70 vs. 34.10 ± 3.07,P=0.001;57.10 ± 4.23 vs. 26.90 ± 2.58,P=0.000). The same trend was seen in the gene expression levels of both TNF-Α and IL-1Β in PDLSCs (PTNF-Α=0.011,P IL-1Β=0.009). p38 was more strongly induced in P-PDLSCs cells than in H-PDLSCs.The basal level of p38 in H-PDLSCs was lower than that in P-PDLSCs cells cultured in the basic medium. However,the level of p-p38 was increased in H-PDLSCs than in P-PDLSCs under osteogenic condition. Treatment of PDLSCs with SB-203580 and then cultures under osteogenic differentiation lead to significantly decreased expressions of Runx2 and ALP in both H-PDLSCs and P-PDLSCs (H-PDLSCs:P(Runx2)=0.044, PALP=0.036;P-PDLSCs:P(Runx2)=0.017, PALP=0.004).

CONCLUSIONSp38 MAPK is involved in the inflammatory response of PDLSCs in the chronic inflammatory microenvironment. The inhibition of p38 by SB-203580 also remarkably suppresses the osteogenic differentiation of PDLSCs in a chronic inflammatory microenvironment.

Alkaline Phosphatase ; Anthraquinones ; Blotting, Western ; Cell Differentiation ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Imidazoles ; Inflammation ; Interleukin-1beta ; Osteogenesis ; Periodontal Ligament ; Pyridines ; Staining and Labeling ; Stem Cells ; Tumor Necrosis Factor-alpha ; p38 Mitogen-Activated Protein Kinases

Tumors are believed to consist of a heterogeneous population of tumor cells originating from rare cancer stem cells (CSCs). However, emerging evidence suggests that tumor may also originate from non-CSCs. To support this viewpoint, we are here to present definitive evidence indicating that the number of tumorigenic tumor cells is greater than that of CSCs in tumor, and tumor can also derive from non-CSCs. To achieve this, an idealized mathematical model was employed in the present study and theoretical calculation revealed that non-CSCs could initiate the occurrence of tumor if their proliferation potential was adequate. Further, experimental studies demonstrated that 17.7%, 38.6% and 5.2% of tumor cells in murine B16 solid melanoma, H22 hepatoma and Lewis lung carcinoma, respectively, were potentially tumorigenic. Thus, based on the aforementioned findings, we propose that the scarce CSCs, if exist, are not the sole source of a tumor.
Algorithms ; Animals ; Carcinogenesis ; pathology ; Carcinoma, Lewis Lung ; pathology ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Liver Neoplasms, Experimental ; pathology ; Melanoma, Experimental ; pathology ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Models, Biological ; Neoplasms, Experimental ; pathology ; Neoplastic Stem Cells ; pathology ; Time Factors ; Tumor Stem Cell Assay ; methods

OBJECTIVETo determine the antiproliferative activity of Rubus parvifolius L. (RP) extract, its medicinal serum and RP total saponins (RPTS) against K562 cells in vitro and in vivo.

METHODSNude mice models bearing leukemia tumors were treated with different concentrations of RP extract. The size, weight and histopathological change of leukemic tumors were determined. Semi-solid agar culture and methylthiazolyl tetrazolium (MTT) assay were used to determine in vitro the inhibition of colony formation and proliferation of K562 cells respectively by different concentrations of RP medicinal serum and RPTS.

RESULTSRP extract had a tumor inhibition rate of 84.8% when administered to mice at a dose of 1.0 g/day of crude RP root equivalent. Semi-solid agar culture of K562 cells in the presence of 20% (v/v) of RP medicinal serum and 150 mg/L RPTS demonstrated a 50.8% and 100% inhibition of the colony forming unit (CFU)-K562, respectively. The same doses of RP medicinal serum and RPTS showed a proliferation inhibition of 31.4% and 86.3%, respectively against K562 cells in MTT assay.

CONCLUSIONRP extract and RPTS show effective antiproliferative activity against myeloid leukemia cells in vitro and in vivo.

Agar ; Animals ; Cell Proliferation ; drug effects ; Chromatography, High Pressure Liquid ; Humans ; K562 Cells ; Leukemia ; drug therapy ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Plant Extracts ; pharmacology ; therapeutic use ; Rosaceae ; chemistry ; Saponins ; pharmacology ; therapeutic use ; Subcutaneous Tissue ; pathology ; Tumor Stem Cell Assay ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the effects of serum containing Chinese medicine (CM) Sanpi Pingwei (, SPPW) formula on the proliferation and apoptosis of human SGC-7901 cells and the possible mechanism.

METHODSSerum containing CM SPPW formula (SPPW serum) was prepared by a serum pharmacology method. Human SGC-7901 cells were incubated with SPPW serum at three different concentrations and with the anticancer drug 5-fluorouracil (5-FU), respectively. Cell proliferation was assessed by MTT assay, and cell apoptosis was detected by flow cytometry assay. Real-time quantitative polymerase chain reaction (RT-PCR) and Western blot assay were employed to confirm the expressions of Bcl-2, Bax and p53 in SGC-7901 cells at mRNA and protein levels, respectively.

RESULTSSPPW serum suppressed the proliferation of SGC-7901 cells in a time- and dose-dependent manner. The colony forming rate of negative control was 48.2%, while those in the three SPPW serum groups and the 5-FU group decreased significantly (P<0.01). The number of colony forming units in the SPPW high dosage group was significantly smaller than that in the 5-FU group (P<0.01). MTT assay showed that SPPW serum restrained the proliferation of SGC-7901 cells, and the inhibition rate increased significantly in a dose-dependent manner. Annexin V/PI Assay suggested that SPPW serum induced the apoptosis of SGC-7901 cells significantly. RT-PCR and western blot assay indicated that SPPW serum upregulated the protein and mRNA expression levels of Bax and p53 in SGC-7901 cells, but downregulated the protein and mRNA expressions of Bcl-2.

CONCLUSIONSSPPW formula inhibits the proliferation of SGC-7901 cells in vitro and induces the cell apoptosis. It plays an anticancer role by regulating the expressions of Bax, p53 and Bcl-2 in SGC-7901 cells.

Animals ; Apoptosis ; drug effects ; Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Fluorouracil ; pharmacology ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Serum ; chemistry ; Tumor Stem Cell Assay ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; bcl-2-Associated X Protein ; genetics ; metabolism

MicroRNAs (miRNAs) participate in diverse biological functions and carcinogenesis by inhibiting specific gene expression. We previously reported that suppression of adenine nucleotide translocase 2 (ANT2) by using the short hairpin RNA (shRNA) approach has an antitumor effect in several cancer cells. We here examined the influence of ANT2 on expression of miRNAs in hepatocellular carcinoma (HCC) to further elucidate the tumor-suppressive mechanism of ANT2 shRNA. We first carried out screening for miRNAs, whose expression is regulated by ANT2 suppression in the Hep3B HCC cell line using miRNA microarrays. Validation of candidate miRNAs was done by incorporating clinical samples, and their effects on the tumorigenesis of HCC were studied in vitro and in vivo. miR-636 was one of the miRNAs whose expression was highly upregulated by ANT2 suppression in miRNA microarray analysis, as confirmed by real-time reverse transcription-polymerase chain reaction. Notably, miR-636 was markedly downregulated in HCC tissues compared with matched non-neoplastic liver in clinical samples. Restoration of miR-636 in Hep3B cells led to significant reduction of cell proliferation and colony formation. miR-636 restoration resulted in a decreased level of Ras, one of the putative targets of miR-636, and inactivation of its signaling pathway. Moreover, tumorigenesis was efficiently suppressed by miR-636 in an in vivo tumor xenograft model of HCC. The data suggest that miR-636 might function as a tumor suppressor miRNA affecting HCC tumorigenesis via downregulation of Ras, and that ANT2 suppression by shRNA could exert an anticancer effect by restoring miR-636 expression in HCC.
Adenine Nucleotide Translocator 2/*metabolism ; Animals ; Carcinoma, Hepatocellular/*genetics/pathology ; Cell Line, Tumor ; Cell Proliferation ; Cell Transformation, Neoplastic/*genetics/pathology ; Down-Regulation/*genetics ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Humans ; Liver Neoplasms/genetics/pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; MicroRNAs/*genetics/metabolism ; Phosphatidylinositol 3-Kinases/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; RNA, Small Interfering/*metabolism ; Signal Transduction/genetics ; Transcription, Genetic ; Tumor Stem Cell Assay ; Up-Regulation/genetics ; ras Proteins/*genetics/metabolism

OBJECTIVETo investigate the effects of panax notoginseng saponins (PNS) on expression, regulation and phosphorylation of multiple protein kinases in mitogen activated protein kinase (MAPK) intracellular signal pathway and GATA transcription factors in hematopoietic cells, so as to explore its mechanism of proliferation and differentiation activity on hematopoiesis.

METHODSThe human granulocytic HL-60, erythrocytic K562, megakaryocytic CHRF-288 and Meg-01 cell lines were treated by PNS, the positive control of K562, CHRF-288 cells treated by recombination human erythropoietin (Epo) and thrombopoietin (Tpo) respectively. The total cell lysate and nuclei protein were extracted after being treated by PNS, subsequently, analyzed by both Western blot and immune-precipitation. Meanwhile, the nuclei extract was performed for electrophoretic mobility shift assay (EMSA) by using (32)P radio labeled double-stranded GATA consensus oligonucleotide.

RESULTSThe expression levels of kinase MEK-1, MEK-2, ERK-1, ERK-2, AKT-1, AKT-2 and PI-3K were increased by PNS treatment to different extent in four cell lines, depending on cellular heterogeneity and sensitivity to PNS, also phosphorylation of MEK-1, ERK-1 was differentially promoted by PNS respectively P<0.05, 0.01, 0.001). The expression levels of transcription factors GATA-1 and GATA-2 were increased, moreover, their DNA binding activities were raised dramatically in PNS treated K562, CHRF-288 and Meg-01 cells compared with the controls respectively (P<0.05, 0.01, 0.001). The positive control of K562, CHRF-288 cells treated by Epo or Tpo respectively also displayed up-regulation of protein kinases and GATA transcription factors respectively (P<0.05, 0.01, 0.001).

CONCLUSIONThe results indicated that intracellular signal pathway initiated by PNS was involved in MAPK pathway and transcription factors of GATA family in hematopoietic cells. PNS displayed the role to promote proliferation and differentiation, by means of increasing expression level and phosphorylation status of multiple protein kinases, also inducing synthesis of GATA transcription factors and upregulation its DNA binding activity.

Blotting, Western ; Cell Line, Tumor ; DNA ; metabolism ; Electrophoretic Mobility Shift Assay ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; GATA Transcription Factors ; metabolism ; Hematopoietic Stem Cells ; drug effects ; enzymology ; Humans ; Immunoprecipitation ; Mitogen-Activated Protein Kinase Kinases ; metabolism ; Panax notoginseng ; chemistry ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; drug effects ; Protein Binding ; drug effects ; Protein Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Saponins ; pharmacology ; Up-Regulation ; drug effects