Effects of Triptolide on Cell Proliferation and CXCR4 Expression in Burkitt’s Lymphoma Raji Cells In Vitro

Objective： To investigate the inhibitory effects of triptolide on cell proliferation and CXCR4 expression in Burkitt＇s lymphoma cell line Raji cells. Methods： The effects of triptolide on the growth of Raji cells were studied by 3-（4, 5-Dimethyl-2-thiazolyl）-2, 5-diphenyl-2H-tetrazolium（MTT） assay. The effects of triptolide on CXCR4 expression on Raji cells were studied by flow cytometric analysis. Chemotaxis assays were performed to observe the effects of triptolide on migration of Raji cells towards recombinant human SDF-1α （rhSDF-1α） in vitro. Results： Triptolide inhibited the proliferation of Raji cells in a dose- and time-dependent way with a 24-h IC50 value of 43.06 nmol/L and a 36-h IC50 value of 25.08 nmol/L. Triptolide could downregulate the CXCR4 expression on Raji cells in a dose-dependent manner. Furthermore, chemotaxis assays showed that triptolide could block the migration of Raji cells to rhSDF-1α in vitro, and the inhibition was dose-dependent. Conclusion： Triptolide could inhibit the proliferation and migration of Raji cells in vitro. The underlying anti-tumor mechanism of triptolide might be related to the anti-proliferative effect and the blockage of SDF-1/CXCR4 axis.

Small hairpin RNA against Bcl-2 increases MTX-induced apoptosis in Raji cells

Objective:The aim of this study was to study the effect of Bcl-2 small hairpin RNA(shRNA) enhancing methotrexate(MTX)-induced apoptosis of Raji cells.Methods:Expression plasmid with Bcl-2 shRNA was transfected into Raji cells by Lipofectmine 2000 and then treated with MTX.At 48 h of transfection,the expression level of Bcl-2 mRNA and protein was evaluated by RT-PCR and immunofluorescence.MTT assay was used to analyze cell proliferation at 24,48 and 72 h.Apoptosis was detected by Giemsa staining and flow cytometric cell cycle analysis.Results:After transfection with Bcl-2 shRNA,the expression levels of Bcl-2 mRNA and protein in Raji cells decreased(P < 0.05).Using Giemsa staining,cells transfected with Bcl-2 shRNA combined with MTX at 48 h displayed changes of apoptosis.MTX significantly inhibited the growth of cells after transfected with Bcl-2 shRNA(P < 0.05).Apoptotic rates of the Raji cells treated with Bcl-2 shRNA combined with MTX significantly increased(P < 0.05),compared with either control shRNA/MTX combination or MTX-treatment cells alone.Conclusion:Our results suggest the shRNA against Bcl-2 mRNA could increase MTX-induced apoptosis of Raji cells.

Combined transfection of Bcl-2 siRNA and miR-15a oligonucleotides enhanced methotrexate-induced apoptosis in Raji cells

Objective: B-cell lymphoma 2 (Bcl-2) is an important member of the Bcl-2 family of proteins that regulate the induction of apoptosis. This study aims to investigate whether Bcl-2 small interfering RNA (siRNA) combined with miR-15a oligonucleotides (ODN) could enhance methotrexate (MTX)-induced apoptosis in Raji cells. Methods: Chemically synthesized miR-15a ODN and Bcl-2 siRNA were transfected in Raji cells by using a HiPerFect Transfection Reagent and then combined with MTX. Expression levels of Bcl-2 protein were detected by Western blot. Cell proliferation was determined by CCK8 assay. The rate of cell apoptosis was determined by Annexin V/PI double staining. The morphology of apoptotic cells was observed by Hoechst-33 258 staining. Results: After the cells were transfected with miR-15a ODN combined with Bcl-2 siRNA, Bcl-2 protein levels were evidently decreased. CCK8 assay showed that cell proliferation was significantly decreased and was significantly lower in miR-15a ODN combined with Bcl-2 siRNA plus MTX group than in miR-15a ODN with methotrexate group, Bcl- 2 siRNA with MTX group, and single MTX group (P<0.05). Hoechst 33258 staining revealed numerous apoptotic cells. AnnexinV/PI double staining showed that the apoptotic rates were (13.13±1.60)%, (34.47±2.96)%, (32.87±3.48)%, and (45.47±2.16)% in MTX, Bcl-2 siRNA plus MTX, miR-15a ODN plus MTX, and miR-15a ODN combined with Bcl- 2 siRNA plus MTX groups, respectively. Among these groups, the apoptotic rate of miR-15a ODN combined with Bcl-2 siRNA plus MTX group was the highest; this apoptotic rate was also significantly different from that of miR-15a ODN or Bcl-2 siRNA plus MTX (P<0.05). Conclusions: Bcl-2 siRNA combined with miR-15a ODN could enhance MTX-induced apoptosis in Raji cells. Bcl-2 siRNA and miR-15a combined with MTX may be a useful approach to improve the treatment effects on lymphoma.

GROWTH-INHIBITORY EFFECTS OF CURCUMIN ON Raji CELLS AND ITS MECHANISMS

Objective： To study the growth-inhibitory effects of curcumin on B-NHL cell line Raji cells in vitro and its molecular mechanisms. Methods： The growth inhibition rates of Raji cells, after being treated with 6.25μmol/L - 50μmol/L curcumin for 12 h - 48 h, were examined by MTT assay. The apoptosis rate was detected by flow cytometry （FCM）, the protein expression levels of bcl-2 and p53 in Raji cells were examined by SP immunohistochemistry. The expression of p53 in Raji cell were checked by RT-PCR. Results： After being treated by various concentrations of curcumin, the growth of Raji cells was inhibited significantly. The rates of apoptosis were 11.8% -79.7% （P〈0.01）, the down regulation of p53 expression was observed within 24 h after the treatment of curcumin by RT-PCR. The expression of bcl-2 and p53 was decreased, which depended on the action time. Conclusion： Curcumin could significantly inhibit the growth of Raji cells. The induction of apoptosis by down-regulating the expression of bcl-2 and p53 was probably one of its molecular mechanisms.

The study of miR-15a oligonucleotide inhibiting cell growth and enhancing Ara-C-induced apoptosis in Raji cells

Objective:The aim of the research was to study whether microRNA-15a(miR-15a) oligonucleotide could inhibit cell growth and enhance cytarabine(Ara-C)-induced apoptosis in Raji cells.Methods:Transfecting miR-15a oligonucleotide into Raji cells with LipofectamineTM 2000,and then combined with Ara-C.IC50 value and cell proliferation were detected by CCK8 assay;the expression levels of Bcl-2 mRNA and protein were evaluated by RT-PCR and indirect immuno-fluorescence.The apoptotic cells were observed by Hoechst Dyeing;AnnexinV/PI double dyeing method was used to detect the cell apoptotic rate by Flow Cytometry(FCM).Results:After Raji cells were transfected with miR-15a oligonucleotide for 48 h,Bcl2 protein expression levels obviously decreased,however,there was no difference in Bcl-2 mRNA levels,as compared with the control group and blank group(P < 0.05).CCK8 assay showed that miR-15a oligonucleotide decreased the cell growth at 24,48 and 72 h,moreover,miR-15a oligonucleotides combined with Ara-C obviously decreased the cell growth than miR-15a group,Ara-C group and scrambled oligonucleotides(SODN) + Ara-C group.Meanwhile,miR-15a oligonucleotides combined with Ara-C significantly decreased IC50 of Ara-C(10.41 μg/mL),which were obviously lower than those of Ara-C group(15.43 μg/mL) and SODN plus Ara-C group(14.92 μg/mL).Plenty of apoptotic cells could be seen with Hoechst dyeing.AnnexinV/PI double dying assays by FCM indicated that the cell apoptotic rates in earlier period and late period of miR-15a + Ara-C group were 20.93% and 25.27%,respectively,which were obviously higher than those of miR-15a group,Ara-C group and SODN plus Ara-C group.Conclusion:miR-15a oligonucleotides can inhibit cell growth and enhance Ara-C-induced apoptosis in Raji cells.

ENHANCEMENT OF RADIATION-INDUCED APOPTOSIS IN RAJI CELL LINE BY BC1-2 ANTISENSE OLIGODEOXYNUCLEOTIDE

Objective: To investigate whether the Bc1-2 antisense oligonucleotide(ASODN) may enhance radiation-induced apoptosis in Raji cell line. Methods: Cell surviving fraction was determined using the trypan blue dye exclusion assay. The expression level of bc1-2 protein was assayed by immunofluorescence using fluoresce isothiocyanate label. Apoptosis was detected by Giemsa staining and flow cytomertric cell cycle analysis. Results: It was found that Bc1-2 ASODN combined with radiation had significantly reduced the number of viable cells (P<0.05). There was no difference on cell survival between mismatch Bc1-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone. Bc1-2 ASODN combined with radiation could significantly inhibit expression of Bc1-2 protein in Raji cells (P<0.05). Cells treated with Bc1-2 ASODN combined with radiation at 72 h displayed classic apoptotic changes. Apoptosis rates of Raji cells treated with Bc1-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone, respectively. Conclusion: Bc1-2 antisense oligonucleotide can enhance radiation-induced apoptosis in Raji cell line.

Anticancer Activities of Trichostatin A on Maligant Lymphoid Cells

The anticancer activity of trichostain A （TSA） on human B cell non-Hodgkin＇s lymphoma and its mechanism were explored, The effect of TSA on the growth of Raji cells and normal peripheral blood mononuclear cells （NPBMNC） was studied by MTr assay, The effect of TSA on the apoptosis of Raji cells and NPBMNC was studied by flow cytometry and TDT-mediated dUTP nick end labeling （TUNEL）. The effect of TSA on the cell cycle of Raji cells was studied by propidium iodide method. The results showed that TSA potently inhibited proliferation of Raji cells at microgram concentrations and induced apoptosis of Raji cells in a time-and concentration-dependent manner. Treatment with TSA induced accumulation of cells in G0/G1 or G2/M and a concomitant decrease of cell population in S phase. However, NPBMNC was less sensitive to the cytotoxic effect of TSA than Raji cells. It was concluded that TSA may inhibit the proliferation of Raji cells by regulating the cell cycle and inducing the cell apoptosis. Moreover, TSA demonstrates low toxicity in NPBMNC but selectively induces apoptosis of Raji cells.

Bcl-2 Small Interfering RNA Inhibits the Growth of Human Lymphoma Transplanted Subcutaneously in Nude Mice

OBJECTIVE To investigate whether small hairpin RNA (shRNA)targeting Bcl-2 mRNA could inhibit the growth of lymphomatransplanted subcutaneously in nude mice.METHODS Recombinant Bcl-2 shRNA expression vector withgreen fluorescence protein (GFP) gene was constructed andpreserved in our lab. We evaluated the antitumor effect of the Bcl-2shRNA in vivo which was the model of nude mice bearing Rajicells xenografts. Human Raji cells were injected subcutaneouslyinto nude mice to establish lymphoma models. When thediameters of tumor were above 0.5 cm after Raji cells injection,the mice bearing tumor were randomly divided into four groups:saline control group, negative shRNA group, plasmid vectorgroup, Bcl-2 shRNA group. The polyethylenimine (PEI) was usedto transfect shRNA into tumor. The mixed PEI and shRNA wasinjected into tumors. The growth and size of tumor were observed.Tissue was stained by H&E for its pathological morphology. Theexpression of Bcl-2 mRNA in the tumor mass was detected byreverse transcription polymerase chain reaction (RT-PCR).RESULTS A significant difference in median tumor weightwas observed in mice treated with Bcl-2 shRNA, compared withthose in the groups of negative shRNA or plasmid vector or salinesolution (P< 0.05). Pathological evaluation was completed in allexcised tumors from nude mice bearing Raji cells xenografts.The tumor tissue of the mice treated with Bcl-2 shRNA showedapoptosis, serious necrosis of the cells and inflammatory cellsinfiltration. There was no change in the morphology of cellsamong negative shRNA, plasmid vector and saline solution group.In the group of the Bcl-2 shRNA, the expression levels of Bcl-2mRNA of the tumor tissue were effectively inhibited (P < 0.05).CONCLUSION The shRNA targeting at the Bcl-2 mRNAcould inhibit the growth of human lymphoma transplantedsubcutaneously in nude mice.

Anticancer Effect of Curcumin on Human B Cell non-Hodgkin's Lymphoma

Summary： To explore the anticancer effect of curcumin on human B cell non Hodgkin＇s lymphoma and compare its effects on human B cell non Hodgkin＇s lymphoma cells and normal peripheral blood mononuclear cells （NPBMNCs）. MTT assay was used to study the effect of curcumin on the growth of Raji cells and NPBMNCs. The effect of curcumin on the apoptosis of Raji cells and NPBMNC were studied by flow cytometry and TDT-mediated dUTP nick and labeling （TUNEL）. The effect of curcumin on the cell cycle of Raji cells were examined by propidium iodide staining flow cytometry. The results showed that curcumin strongly inhibited proliferation of Raji cells, 24 h IC50, for Raji cells was 22.8±1.82μmol/l, and curcumin induced Raji cell apoptosis in a time-and dose-dependent manner. Raji cells treated with curcumin showed G0/G1 or G2/M phase increase and S phase decrease. However, curcumin did not demonstrate apparent proliferation inhibition and apoptosis induction in NPBMNCs. It was concluded that curcumin is able to inhibit the proliferation of Raji cells by regulating the cell cycle and inducing the cell apoptosis. Morever, curcumin has low toxicity on NPBMNCs but can selectively induce apoptosis in Raji cells.

Lanthanum chloride or citrate is absorbed mainly via M cells in gastrointestinal tracts with lanthanum phosphates as the transformed species

In the present study, we investigated the transformed species and the absorptive mechanism of rare earth elements（REEs） in gastrointestinal（GI） tract, using La Cl3 and La Cit as representative compounds. Artificial gastric and intestinal fluids were used to simulate the environment of the digestive tract in vivo. The inductively coupled plasma mass spectrometry（ICP-MS） result showed that more than 99.9% of La Cl3 and La Cit formed precipitation in artificial intestinal fluid, with the average size distribution of 200 nm（2-h incubation） increasing to 600 nm（24-h incubation） determined by dynamic light scattering（DLS）, indicating the aggregation of the particles. The Fourier transform infrared spectroscopy（FTIR） analysis demonstrated that the constituents of these particles were mainly in the form of lanthanum phosphates. To explore the transport mechanism of REEs in GI tract, the mice Peyer＇s patches（PPs） and intestinal epithelium were separated to evaluate the content of lanthanum by ICP-MS following oral administration with 2 or 100 mg/kg/day of La Cit for 7 d. The results showed that the amount of lanthanum phosphate particles absorbed by PPs was significantly greater than that of intestinal epithelium, indicating that lanthanum particles might be phagocytosed mainly by M cells located in the follicle-associated epithelium（FAE） overlying PPs. Furthermore, Caco-2 cell monoculture and Caco-2/Raji B cell coculture models were established to simulate intestinal epithelial cells and FAE, respectively. The result showed that the transport of lanthanum in Caco-2/Raji B coculture model was significantly higher than that in Caco-2 monoculture model（about 60 times higher）, and the level of lanthanum in the basal compartment of Caco-2 monoculture model was very low, supporting that M cells were the main route for lanthanum phosphate particles to be transported and absorbed. Taken together, these data suggested that La Cl3 and La Cit in GI tract were absorbed mainly via M cells with lanthanum phosphates as transformed species. The obtained results would provide the theoretical basis for the rational application of REEs in agriculture and medicine.