Translated Abstract
Multiple myeloma is defined as a plasma cell malignancy characterized by the hyperplasia of plasma cells in the bone marrow this malignancy is usually associated with high levels of monoclonal (M) immunoglobulin in the blood and leads to pathological fracture, ostealgia, infection, hypocalcaemia and anemia. For more than half a century, multiple myeloma therapy has shown only limited success, as approximately one-third of patients do not respond to chemotherapy. and the remainder will eventually relapse if they do not succumb to other diseases.Oridonin is a diterpenoid compound isolated from the Chinese medicinal herb Rabdosia rubescens and has remarkable anti-tumor activities. Recently, accumulating evidence has suggested that oridonin is able to inhibit the progression of tumors, thereby alleviating the tumor burden and cancer syndrome. Oridonin has been reported to induce apoptosis and autophagy through the Fas/FasL-mediated signaling cascade. Additionally, oridonin has shown involvement in the suppression of cell cycle progression and/or induction of cancer cell death in various in vitro trials. Although oridonin is closely associated with the induction of apoptosis, the definitive systematic molecular mechanism of action of oridonin in multiple myeloma therapy remains unknown and awaits further investigations.Currently, a proteomic approach is being actively applied to the molecular analysis of various human cancers. However, there has been no systematic identification of the global proteome of oridonin-induced apoptosis in multiple myeloma LP-1 cell lines until now. To further understand the molecular mechanism of oridonin in multiple myeloma therapy, we performed a proteomic analysis using a two-dimensional gel electrophoresis (2-DE)-based system and mass spectrometry (MS).
Objectives:
1. To investigate the LP-1 cell growth after incubated with different concentration of oridonin for different time periods to explore the mechanism of apoptosis induced by oridonin.
2. To compare the protein expression profiles of LP-1 cells and the cells treated by oridonin. And to provide more theoretical foundations for the molecule mechanism of the oridonin induced apoptosis.
Methods:
1. LP-1 was incubated in vitro with different concentration of oridonin for different time periods and MTT bioassay was used to analyze the growth inhibitory effect of oridonin in LP-1.
2. Use FACS to test theapoptosis rate and autophagy rate、apoptosis rate and cell cycle distribution in LP-1 treated with varying concentration of oridonin for different time periods.
3. Use transmission electron microsope (TEM) to observe the change of ultramicrostructure in cells treated by oridonin.
4. Using real time PCR and western-blot to test the expressions of Beclin 1, Bcl-2, Bax, Caspase-3, NF-κB, I-κB,Becline 1 mRNA and protein in LP-1 cells incubated with different concentration of oridonin .
5. Total proteins of LP-1 cells were extracted by the method of cell disruption, liquid nitrogen freeze thawing and ultrasound fractured. Proteome expression maps of LP-1 cells treated with and without oridonin were established respectively by 2-DE techniques, and differentially expressed protein spots among the maps were screened by image analysis software and artificial comparison. These differential pots were digested respectively in gel by enzymes, then their peptides mixtures were analyzed by MALDI-TOF-MS and PMF was obtained. Finally, the differential expression proteins were searched and identified among NCBInr and SwissProt databases by Mascrot software.
6. Using real time PCR and western-blot to test the differentially expressed proteins.
Results:
1. Different concentration of oridonin can significantly inhibit the proliferation of LP-1 cells (P＜0.05), and in a certain range of concentration and time the inhibitory effect resulted in dose and time-dependent inhibition.
2. Along with the increasing of drug concentration and treated time, the autophagy rate was increasing (P＜0.05 vs control group) the cell percentage in the G1 phase decreased (P＞0. 05 vs control group), the S phase changed unsignificantly (P＜0.05 vs control group) and the G2/M phase increased (P＜0.05 vs control group). When 10, 20, 50μmol/L oridonin treated LP-1 cells for 24, 48 hours, the apoptosis rate increased (P＜0.05 vs control group), and resulted in dose and time-dependent.
3. TEM results revealed that there was typical apoptosis charactors in LP-1 treated with oridonin for 24 hours.
4. When 10, 20, 50μmol /L oridonin treated LP-1 for 24 hours, gene and protein expressions of Beclin 1, Caspase3, Bid, Bax, I-κB were upregulated, while gene and protein expressions of Bcl-2, NF-κB was down regulated by oridonin(P＜0.05).
5. The total proteins of LP-1 cells were extracted successfully. Proteome expression maps of LP-1 cells treated with and without oridonin were established respectively by 2-DE techniques. 1260±41 and 1168±33 spots were detected on 2D pages. There were 8 protein spots that the quantity of differentially expressed was more than two times (≥2 or ≤0.5) among 2-DE maps were screened by ImageMasterTM 2D Platinum software and 7 of them were identified by mass spectrography (MS). Among them, 6 proteins were down-regulated and 1 proteins was up –regulated. They were stathmin, DHFR, PDHB, TrxR, cofilin, Hsp, LAP. Stathmin、DHFR play important role in oridonin induced cell apoptosis.
6. Differentially expressed proteins were examed by western-blot and real time PCR.
Conclusion:
1. Oridonin with different concentration can inhibit LP-1cell growth signifcantly, the inhibition effect is dose and time dependent.
2. Oridonin can induce apoptosis of LP-1 cells, the apoptosis rate is time and dose dependent. The the mechanism may be associated with the upregulation of Caspase-3, Bax, Bid, I-κB and the downregulation of Bcl-2, NF-κB. Meanwhile, oridonin can induce autophagy as well as apoptosis in LP-1 cells, which may be related to the upregulatione of Becline1.
3. The technique platform for the proteome expression maps was initially established and improved, and the proteome expression maps of LP-1 cells was obtained. 24 differential expression proteins were iditenfied, in which 6 proterins were up-regulated and 18 protenis were down-regulated.
4. 7 differential expression proteins between oridonin treated LP-1 cells and none treated cells were successfully identified. They were stathmin, DHFR, PDHB, TrxR, cofilin, Hsp, LAP. Among them, stathmin and DHFR may be the key targets proteins in the apoptotic process of LP-1 cells induced by oridonin.
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