| Objective:T-cell NHL is one of hematologic malignant tumor, which is source from lymph tissue and absorbent gland. T-cell lymphoma has a high incidence in China, approximately composes about 25% in all non-Hodgkin's lymphoma. At present, neither domestic nor abroad has ideal treatment, the efficiency of conventional therpy of the CHOP or COP regimen can be recently, but long-term effect and prognosis of patients are very poor. Thus it is urgently for us to seek effective anti-cancer drugs or combined regimen, which can improve the effect and the prognosis of patients.The study showed that many genes with high methylation in promoter were associated with lymphoma,These genes are involved in many of the known cell signaling pathway. In the earlier stage of our research, we found that5-aza-CdR possessed demethylation properties, and induced apoptosis of lymphoma cells. SHP-1 is a negative control factor to JAK-STAT signaling pathway and plays an important role in regulating cell growth, proliferation, differentiation. It is a potential tumor-suppressor factor. The methylation in promoter CpG island of SHP-1 is closely related to the low expression of SHP-1, and maybe methylation is the mechanism of silencing of SHP-1 in our former research. The silencing of this gene can lead to the activity of relevant signal transduction molecules so as to promote the malignant tumor cell proliferation.The proteasome plays and essential role in the targeted degradation of such proteins and is therefore involved in the activation and inactivation of many cellular processes. In fact, studies using proteasome inhibitors have been shown that the proteasome is responsible for the elimination of more than 80% of all cellular proteins. The proteasome has been identified as an excellent target for cancer therapy because of its critical metabolic function. As we know, with lots of new chemotherapeutics drugs coming out, BTZ (bortezomib, PS-341, VELCADE) was the first-in-class proteosome inhibitor on the clinical research, and it has been extensively used for multiple myeloma and some B cell NHL patients.BTZ mainly decreases the level of NFκB or regulates cyclin and the signal apoptosis of tumor cell, and the anti-tumor effects of T-cell lymphoma is not very clear. This experiment detected the anti-tumor effects of bortezomib on T cell NHL(Jurkat cell line) in vitro, and observe whether the combination of bortezomib and 5aza-CdR have synergistic or supermposed effect on Jurkat lymphoma cells. Analysis the effect of proliferation and apoptosis induced by their combination, and its possible mechanism preliminary. Then can provide a theoretical guide to help us choose the better treatment regimen of T-cell lymphoma.Methods: 1 First of all, we used MTT assay to examine The effects of BTZ or 5-aza-CdR with different concentrations and their combinations on the growth of Jurkat cells were examined by MTT assay.2 Then, in order to investigate whether apoptosis is associated with the antitumor activity of BTZ and combing with 5-aza-CdR in Jurkat cells, we observed the cell morphology using Wright-Giemsa staining, evaluated the exposure of phosphatidylserine(PS) on Jurkat cells after double staining with fluorescein isothiocyanate (FITC)-labeled annexin-V and propidium iodide(PI).3 We further examined whether BTZ could enhance the growth inhibitory effect of 5-aza-CdR. Jurkat cells were treated for 24 hours by different concentrations of 5-aza-CdR combined with 10 nmol/L BTZ. The growth-inhibitory rate was analyzed by MTT assay. The cell apoptosis rate was analyzed by flow cytometry analysis (annexin V/PI staining) and the RT-PCR to assay the mRNA expression of the SHP-1 single and DNMTs family members could show whether BTZ could inhance the effects of 5-aza-CdR.4 Synergetic effects of BTZand 5-aza-CdR was analyzed by median-effect principle.Results: 1 Effect of BTZ alone on the proliferation of Jurkat cells Dates from Wright-Giemsa staining reveal that the number of cells in different concentration has gradually decreased in a dose- and time- manner. Dates from MTT assays show that 10~50 nmol/L BTZ treated cells, inhibition rates enhanced in a time- and dose-dependently manner, 50 nmol/L btz can best inhibit the proliferation activity of Jurkat cells when incubated for 72 hours, with the IC50 dose 28.95 nmol/L.2 Effect of BTZ alone on the apoptosis of Jurkat cells Normal cells and apoptotic cells could be distinguished under the microscope after staining with Wright-Giemsa. Normal nuclei kept intact and stained evenly. Apoptotic cells show strong chromatin condensation and nuclear fragmentation. After detecting the apoptosis of Jurkat cells with flow cytometry, there were apoptotic cells in BTZ with different concentrations treating groups. 10 nmol/L BTZ treating for 24h provide apoptosis rate 7.8%, while 30 nmol/L treating group with 15.96%, 50 nmol/L treating group with 29.3%,which is significant higher than 10 nmol/L group(P﹤0.05). Prolong the treating time to 48h, the apoptosis rate in 10 or 30 nmol/L group increased to 16.9%, 36.05% and 45.16%, respectively, which were significant higher than those in the same concentration of 24 h(P﹤0.05).3 Effect of 5-aza-CdR alone on the proliferation and apoptosis of Jurkat cells The result of MTT show that treating Jurkat cells with 1-5μmol/L 5-aza-CdR can suppress cell proliferation obviously, the growth inhibition ratio is in a direct ratio with the time to be spent and the density of 5-aza-CdR. Treating Jurkat cells with 3μmol/L 5-aza-CdR for 24～72 h, the percentage of early stage apoptotic cells(Annexin V positive, PI negative) are relevant 5.2%, 8.6%, 15.8%.4 Effect of BTZ in combination with 5-aza-CdR on the proliferation and apoptosis of Jurkat cells 5-aza-CdR alone can also inhibit the proliferation of Jurkat cells. When cultured for 24~72 h, 3μmol/L 5-aza-CdR brought inhibition rates of5.2%, 8.6%, 15.8%, respectively. 10 nmol/L BTZ which is combined with 1~5μmol/L of 5-aza-CdR can inhibit the proliferation of Jurkat cells, and the dual inhibition of BTZ plus 5-aza-CdR is significant higher than that of either above. Flow cytometry show that there were apoptotic cells in all groups. After they were cultured for 24~72h, the apoptotic rates of the agents combined groups were higher than the single-agent groups. After the cells had been cultured for 48 hours, the apoptotic rates of BTZ plus 5-aza-CdR group was 25.6%, which had significant enhancement then 5-aza-CdR alone(P﹤0.05).5BTZ could significantly enhance the demethylation of 5-aza-CdR in Jurkat cells Different concentrations 5-aza-CdR combined with 10 nmol/L BTZ for 24 h~72 h, the expression of DNMT1 and DNMT3A mRNA reduced with times going, butthe expression level of SHP-1 mRNA increased. The expression levels of DNMT1 and DNMT3A after the Jurkat cells treated with combination group reduced more than treated with both of 5-aza-CdR alone(P﹤0.05). The expression of SHP-1 after the Jurkat cells treated with combination group increased more than treated with 5-aza-CdR or BTZ alone(P﹤0.05).Conclusions: 1 Bortezomib has a dose- and time- dependent antiproliferation and proapoptotic effect on Jurkat lymphoma cells in vitro. With prolongation of time and increase of concentration of btz, the effect has gradually increased.2 5-aza-CdR also have a dose dependent antiproliferaiton and proapoptic effects on Jurkat cells, while combined with bortezomib, the effect is obviously improved when compared with 5aza-CdR used alone.3 Bortezomib in combination with 5-aza-CdR has antiproliferaiton on Jurkat cells in vitro. Compared with the effect of which drugs used alone, the effect of bortezomib in combination 5-aza-CdR group is superior to the effects of either group which drugs used alone. |
PDF Full Text Request