BMI-1 activates hepatic stellate cells to promote the epithelial-mesenchymal transition of colorectal cancer cells.

BACKGROUND: Activated hepatic stellate cells (aHSCs) are the major source of cancer-associated fibroblasts in the liver. Although the crosstalk between aHSCs and colorectal cancer (CRC) cells supports liver metastasis (LM), the mechanisms are largely unknown. AIM: To explore the role of BMI-1, a polycomb group protein family member, which is highly expressed in LM, and the interaction between aHSCs and CRC cells in promoting CRC liver metastasis (CRLM). METHODS: Immunohistochemistry was carried out to examine BMI-1 expression in LM and matched liver specimens of CRC. The expression levels of BMI-1 in mouse liver during CRLM (0, 7, 14, 21, and 28 d) were detected by Western blotting (WB) and the quantitative polymerase chain reaction (qPCR) assay. We overexpressed BMI-1 in HSCs (LX2) by lentivirus infection and tested the molecular markers of aHSCs by WB, qPCR, and the immunofluorescence assay. CRC cells (HCT116 and DLD1) were cultured in HSC-conditioned medium (LX2 NC CM or LX2 BMI-1 CM). CM-induced CRC cell proliferation, migration, epithelial-mesenchymal transition (EMT) phenotype, and transforming growth factor beta (TGF-ß)/SMAD pathway changes were investigated in vitro. A mouse subcutaneous xenotransplantation tumor model was established by co-implantation of HSCs (LX2 NC or LX2 BMI-1) and CRC cells to investigate the effects of HSCs on tumor growth and the EMT phenotype in vivo. RESULTS: Positive of BMI-1 expression in the liver of CRLM patients was 77.8%. The expression level of BMI-1 continued to increase during CRLM in mouse liver cells. LX2 overexpressed BMI-1 was activated, accompanied by increased expression level of alpha smooth muscle actin, fibronectin, TGF-ß1, matrix metalloproteinases, and interleukin 6. CRC cells cultured in BMI-1 CM exhibited enhanced proliferation and migration ability, EMT phenotype and activation of the TGF-ß/SMAD pathway. In addition, the TGF-ßR inhibitor SB-505124 diminished the effect of BMI-1 CM on SMAD2/3 phosphorylation in CRC cells. Furthermore, BMI-1 overexpressed LX2 HSCs promoted tumor growth and the EMT phenotype in vivo. CONCLUSION: High expression of BMI-1 in liver cells is associated with CRLM progression. BMI-1 activates HSCs to secrete factors to form a prometastatic environment in the liver, and aHSCs promote proliferation, migration, and the EMT in CRC cells partially through the TGF-ß/SMAD pathway.

Discrimination of Chinese teas with different fermentation degrees by stepwise linear discriminant analysis (S-LDA) of the chemical compounds.

This study aimed to construct objective and accurate analytical models of tea categories based on their polyphenols and caffeine. A total of 522 tea samples of 4 commonly consumed teas with different fermentation degrees (green tea, white tea, oolong tea, and black tea) were analyzed by high-performance liquid chromatography (HPLC) coupled with spectrophotometry, utilizing ISO 14502, as analytical tools. The content of polyphenols and caffeine varied significantly according to differently fermented teas, indicating that these active constituents may discriminate fermentation degrees effectively. By principal component analysis (PCA) and stepwise linear discriminant analysis (S-LDA), the vast majority of tea samples could be successfully differentiated according to their chemical markers. This study yielded three discriminant functions with the capacity to simultaneously discriminate the four tea categories with a 97.8% correct rate. In classification of oolong and other teas, there were one discriminant function and two equations with best discriminant capacity. Furthermore, the classification of different degrees of fermentation of oolong and external validation achieved the desired results. It is suggested that polyphenols and caffeine are the distinct variables to establish internationally recognized models of teas.

Computational identification of MicroRNAs in strawberry expressed sequence tags and validation of their precise sequences by miR-RACE.

MicroRNAs (miRNAs) are small, endogenously expressed, nonprotein-coding RNAs that regulate gene expression at the post-transcriptional level in both animals and plants through repressing translation or inducing mRNA degradation. A comprehensive strategy to identify new miRNA homologs by mining the repository of available strawberry expressed sequence tags (ESTs) was developed. By adopting a range of filtering criteria, we identified 11 potential miRNAs belonging to 5 miRNA families from 47 890 Fragaria vesca EST sequences. Using 2 specific 5' and 3' miRNA RACE PCR reactions and a sequence-directed cloning method, we accurately determined both end sequences of 5 candidate miRNAs. Meanwhile, qRT-PCR was used to detect the expression of these 5 miRNAs in different strawberry organs and tissues at several growing stages. These newly identified F. vesca miRNAs (fve-miRNAs) and their expression information can improve our understanding of possible roles of fve-miRNAs in regulating the growth and development of F. vesca.

FaPYR1 is involved in strawberry fruit ripening.

Although the plant hormone abscisic acid (ABA) has been suggested to play a role in the ripening of non-climatic fruit, direct genetic/molecular evidence is lacking. In the present study, a strawberry gene homologous to the Arabidopsis ABA receptor gene PYR1, named FaPYR1, was isolated and characterized. The 627 bp cDNA includes an intact open reading frame that encodes a deduced protein of 208 amino acids, in which putative conserved domains were detected by homology analysis. Using tobacco rattle virus-induced gene silencing (VIGS), the FaPYR1 gene was silenced in strawberry fruit. Down-regulation of the FaPYR1 gene not only significantly delayed fruit ripening, but also markedly altered ABA content, ABA sensitivity, and a set of ABA-responsive gene transcripts, including ABI1 and SnRK2. Furthermore, the loss of red colouring in FaPYR1 RNAi (RNA interference) fruits could not be rescued by exogenously applied ABA, which could promote the ripening of wild-type fruits. Collectively, these results demonstrate that the putative ABA receptor FaPYR1 acts as a positive regulator in strawberry fruit ripening. It was also revealed that the application of the VIGS technique in strawberry fruit could be used as a novel tool for studying strawberry fruit development.

Berbamine induces Fas-mediated apoptosis in human hepatocellular carcinoma HepG2 cells and inhibits its tumor growth in nude mice.

Berbamine, a natural compound from the plant Berberis amurensis, is a traditional Chinese medicine mainly used in stimulating normal hematopoiesis in clinic. Our previous studies demonstrated that berbamine has anti-leukemia activity. In this study, we investigated the anticancer activity of berbamine against human hepatocellular carcinoma (HCC) HepG2 cells in vitro and in vivo. Berbamine treatment decreased the cell growth in a dose-dependent manner with an IC(50) value of 34.5 +/- 0.5 microM. Flow cytometric analysis of apoptosis using Annexin V/propidium iodide staining showed that the percentage of apoptotic cells was increased in a time-dependent manner. Berbamine treatment increased the expression level of Fas and P53, caused depolarization of mitochondrial membrane and decrease of membrane potential, and activated caspase-3, -8, and -9 in HepG2 cells. Berbamine-induced apoptosis could be blocked by the broad caspase inhibitor z-VAD-fmk. HepG2 human HCC xenograft mice treated with berbamine showed a significant reduction in tumor growth rates compared to saline-treated mice. These studies suggest that berbamine exerts anticancer effects on human HCC HepG2 cells in vivo and in vitro, the induction of p53 and the activity of the Fas apoptotic system may participate in the anticancer activity of berbamine in HepG2 cells.

Berbamine induces apoptosis in human hepatoma cell line SMMC7721 by loss in mitochondrial transmembrane potential and caspase activation.

OBJECTIVE: To investigate the effect of berbamine on human hepatoma cell line SMMC7721. METHODS: The effects of 24 h and 48 h incubation with different concentrations (0 to approximately 64 microg/ml) of the berbamine on SMMC7721 cells were evaluated using 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. Hoechst 33258 staining was conducted to distinguish the apoptotic cell, and the appearance of sub-G1 stage was determined by PI (propidium iodide) staining, the percentage of apoptotic cell was determined by flow cytometry following annexin V/PI staining. Flow cytometry was performed to analyze the cell cycle distribution and the mitochondrial membrane potential (psi(m)); the expression of activated caspase3 and caspase9 was analyzed by Western-blot. RESULTS: The proliferation of SMMC7721 was decreased after treatment with berbamine in a dose- and time-dependent manner. Berbamine could induce apoptosis in SMMC7721 cells and could cause cell cycle arrest in G0/G1 phase, to induce loss of mitochondrial membrane potential (psi(m)) and activate caspase3 and caspase9. Berbamine-induced apoptosis could be blocked by the broad caspase inhibitor z-VAD-fmk. CONCLUSION: Berbamine exerts antiproliferative effects on human hepatocellular carcinoma SMMC7721 cells. The anticancer activity of berbamine could be attributed partly to its inhibition of cell proliferation and induction of apoptosis in cancer cells through loss in mitochondrial transmembrane potential and caspase activation.

[The mechanism of apoptosis of chronic myeloid leukemia cells induced by the novel p210 bcr/abl inhibitor berbamine].

OBJECTIVE: To investigate the mechanism of apoptosis of chronic myeloid leukemia (CML) cells induced by the novel p210 bcr/abl inhibitor berbamine. METHODS: Human Ph+ CML leukemia K562 cells, which express endogenous p210 bcr/abl protein, were cultured in RPMI 1640 and treated with berbamine as indicated time and dose. Flow cytometry (FCM) and Annexin-V-Fluos/PI staining kit were used to evaluate the apoptosis of leukemic cells; FCM and cytoperm/cytofix plus Caspase-3-McAb-PE were employed to measure the leukemic cells with activated Caspase-3. Phosphorylation of p210 bcr/abl protein in the leukemic cells were assessed by a combination of immunoprecipitation (IP) with c-abl antibody and Western blotting with p-Tyr (pY99) antibody. The protein levels of p210 bcr/abl, Hsp90 and Hsp70 in the leukemic cells were determined by Western blotting with antibodies to c-abl, Hsp90, and Hsp70 respectively. RESULTS: After treatment with berbamine at 8 microg/ml for 48 h, the percentages of leukemic cells expressing activated caspase-3 and apoptotic cells were 45.69% and 48.43% respectively. IP and WB results showed that berbamine at low concentration markedly inhibited phosphorylation of p210 bcr/abl protein in the leukemia cells, and the amount of phosphorylated p210 bcr/abl in the leukemia cells exposed to berbamine at 8 microg/ml for 6 h were only 8.41% of that of untreated leukemia cells without the protein levels of p210 bcr/abl down-regulated. Significantly, berbamine also down-regulated chaperone Hsp90 protein, and the amount of Hsp90 protein in the leukemia cells treated with berbamine at 8 microg/ml for 48 h accounted for 18.37% of that of the untreated leukemia cells. Berbamine at 8 microg/ml had no obvious effect on chaperone Hsp70 protein expression associated with the resistance of leukemia cells to apoptosis. CONCLUSION: (1) Berbamine induces caspase-3-mediated apoptosis of Ph+ leukemia cells through inhibiting phosphorylation of p210 bcr/abl protein and down-regulating its chaperone Hsp90 protein. (2) Unlike Hsp90 inhibitor GA that upregulates Hsp70, berbamine has no obvious effect on chaperone Hsp70 protein expression in leukemia cells, suggesting that berbamine may be a novel class of Hsp90 inhibitor, and further study is required.

[Knockdown of PLK1 mRNA by special siRNA].

To study knockdown effect of small interfering RNA (siRNA) to PLK1 (Polo-like kinase 1) mRNA in colorectal cancer cell line SW480 and its mitosis and growth was changed. Ten special siRNA molecules were designed targeting different sites of PLK1 mRNA sequence and chemically synthesized. The siRNA molecules were transfected into SW480 by Oligofectamine. The gene mRNA level was assayed by Real-Time PCR. The changes of PLK1 protein, SW480 cell cycle and survival percentage was checked by Western-blot, Flow cytometry and Cell counter assays respectively. All 10 siRNA molecules knocked PLK1 mRNA down in different level. Of them P1, P4 and P9 showed over 80% knockdown efficiency and the others had more than 20% knockdown efficiency to PLK1 mRNA. The best knockdown effect over 95% of all groups was at 25 nmol/L of a mixture with P1, P4 and P9 siRNA equally. In this situation the protein was very less and the cells were blocked at G2 phase of cell cycle. After 72 h cell survival percentages were consistent with PKL1 mRNA level change by siRNA gradient concentration. The results showed that siRNA targeting PLK1 mRNA had effectively knocked PLK1 mRNA down in SW480 cell line. And a blended siRNAs held the best knockdown effect. The cell was blocked on the mitosis and growth.

Evaluation of ST13 gene expression in colorectal cancer patients.

We identified a novel gene ST13 from a subtractive cDNA library of normal intestinal mucosa in 1993, more studies showed that ST13 was a co-chaperone of Hsp70s. Recently we detected the ST13 gene expression in tumor tissue and adjacent normal tissue of the same colorectal cancer patient and investigated if the ST13 gene expression might have any prognostic value. Analysis was performed at molecular level by reverse transcription-PCR using real-time detection method. We measured two genes simultaneously, ST13 as the target gene and glyceraldehydes-3-phosphate dehydrogenase as a reference gene, in primary colorectal tumor specimens and tumor-adjacent normal mucosa specimens from 50 colorectal cancer patients. The expression levels of the ST13 gene were significantly decreased in primary tumors compared with adjacent mucosa (P<0.05). But there were no significant differences in the expression of ST13 as compared with different Dukes' stage, tumor differentiation grade, invasion depth, lymph node metastasis and disease-specific survival.

[Effect of Kanglaite injection oncyclooxygenase activity in lung carcinoma A549 cell].

OBJECTIVE: To study the effect of Kanglaite injection on cyclooxygenase activity in lung carcinoma A549 cells. METHODS: The expression of mRNA of COX-1 and COX-2 were measured by RT-PCR. The expression of COX-2 protein was measured by Western-blot. RESULT: Kanglaite injection could selectively decreased COX-2 mRNA expression and protein expression while COX-1 mRNA expression was unchanged. CONCLUSION: Kanglaite injection is selectively inhibitory agent of COX-2, and possess inhibitory effects on cyclooxygenase 2.

[Study on effect of berbamine on multidrug resistance leukemia K562/Adr cells].

OBJECTIVE: To study the effect and mechanism of berbamine on the apoptosis of multidrug resistant leukemia K562/Adr cells and in reversing the drug resistance. METHODS: IC50 value of K562/Adr cell was determined with MTT method, cell apoptosis rate was analyzed by flow cytometry with Annexin V FITC-PI assay, with the peak and cell cycle detected by PI staining. At the same time, flow cytometry was also used in determining Caspase-3, P-GP protein expression and drug accumulating capacity in cells, and RT-PCR method was used to analyze the gene expression of mdr-1. RESULTS: Berbamine could inhibit human leukemia K562/Adr cell growth in dose-dependent manner, it could also induce cell apoptosis, increase the protein expression of Caspase-3 and the drug excretion capacity of cells, reduce the mRNA and protein expression levels of mdr-1 gene. CONCLUSION: Berbamine could activate Caspase-3 to induce human leukemia K562/Adr cell apoptosis, and by reducing mdr-1 gene expression to reverse its multidrug resistance.

[Clinical experience from treatment of seven SARS patients].

OBJECTIVE: To summarize the clinical experience from treatment of patients with severe acute respiratory syndrome (SARS). METHODS: Retrospective analysis of seven patients with SARS in Ditan hospital treated since April 22 in 2004 was performed. RESULTS: In the 7 patients, 2 were male, 5 were female, and the average age was (35.3 plus/minus 11.3) years. The main clinical manifestations were fever, cough, minor or serious dyspnea, nausea, signs of injury to other organs, and so on. The treatment regiments included oxygen, small dosage and short period of methylprednisolone (1 to 2 mg/kg), use of ventilator, psychological intervention, and treatment of underlying diseases, after which, all the 7 patients recovered. CONCLUSION: Rational use of methylprednisolone and timely use of ventilator were the key steps of treatment.

Surgical management of transected injury to the pancreatic neck.

OBJECTIVE: To present a batch of data of transected pancreatic neck injuries and to sum up the experience in surgical interventions for the injuries. METHODS: We analysed 13 patients with a transected injury to the pancreatic neck from Jan. 1995 to Dec. 2000. External drainage was performed in all patients. Pancreatoduodenectomy was conducted in 2 patients with a transected injury to the pancreatic neck associated with duodenal ruptures, and TPN was administered immediately after operation. Proximal closure of the transected margin and distal pancreaticojejunostomy was performed in 4 patients. Proximal closure of the transected margin and distal pancreaticojejunostomy plus splenectomy was performed in 7 patients associated with contusion of pancreatic body or tail plus spleen rupture. RESULTS: 12 patients healed and one patient died of anesthetic accident during the course of restoration of the dislocation of his right hip joint. Complications occurred in 7 patients. CONCLUSIONS: The operation should be performed according to the degree of the injuries and associated duodenal injuries. Routine drainage and nutrient support should be recommended.

[Baicalein selectively induce apoptosis in human leukemia K562 cells].

AIM: To study the antitumor effect of baicalein on human leukemia K562 cell and its mechanism. METHODS: The IC50 value and cytotoxity of K562 cell were detected by MTT method. The apoptotic cell was analyzed by FCM using Annexin V FITC--PI staining method. Sub-G1 peak was also measured by FCM. Protein expressions of Bcl-2, Fas, Caspase 3 were evaluated with FCM. RESULTS: Baicalein was shown to significantly inhibit the proliferation of K562 cell in a dose-dependent manner and selectively induce apoptosis of human leukemia K562 cells. Flow cytometric analysis showed that baicalein arrested K562 cells in the S phase. In addition, protein expression of Fas, Caspase 3 of K562 cells increased after exposure to baicalein, but Bcl-2 was unchanged. CONCLUSION: Baicalein can selectively induce apoptosis of human leukemia K562 cell dose and time dependently through up-regulation of caspase-3 and fas gene expression level.

[Effects of sodium hyaluronate on growth and adhesion of two human colorectal cancer cell lines in vitro].

OBJECTIVE: To investigate the effects of sodium hyaluronate on the growth and adhesion of colorectal cancer cells. METHODS: Human colorectal cancer cell lines SW620 and Colo205 were treated with sodium hyaluronate (25 -2,500 microg/ml), and cancer cell proliferation was measured by MTT assay in vitro. Flow-cytometric analysis was applied to detect expression of CD44 on SW620 and Colo205 cells. RESULT: In vitro sodium hyaluronate enhanced proliferation of Colo205 cells, but it had no appreciable effect on SW620 growth under the same doses, Meantime, CD44 expression on cancer cells decreased compared with controls. CONCLUSION: In vitro sodium hyaluronate has different effects on growth of different colorectal cancer cell lines, but can inhibit CD44 expression of colorectal cancer cells and influence their ability of adhesion.