Oridonin Induced Multiple Myeloma Cells Apoptosis And Enhanced The Sensitivities Of MM Cells To Actived Allo-PBMCs Cytolytic Effects

Background and ObjectivesOridonin (Ori), a tetracycline diterpenoid isolated from the plant Rabdosia rubescens, has various biological, pharmaceutical, and physiological functions such as anti-inflammation, anti-bacteria activity and others. It was shown in China to be able to suppress disease progress, reduce tumor burden, alleviate syndrome, and prolong survival in patients with esophageal, gastric carcinoma or liver cancer. Recent studies showed that oridonin induced apoptosis in a variety of cancer cells including those from prostate, breast, non-small cell lung cancers, acute leukemia(NB4, HL-60 cells), gioblastoma multiforme, and human melanoma cells. In addition, oridonin could also increase lifespan of mice bearing P388 lymphocytic leukemia. However, though studies showed that caspase 3(casp 3), casp 8, p53, Bcl-2/Bax, cytochrome c (cyto c), and nuclear factor kappa B(NF-icB)were involved in apoptosis induced by oridonin, mechanisms underlying the anti-MM cells activity of oridonin remain largely unknown, and whether oridonin can find clinical application still needs more investigation.Natural Killer cells (NK cells) are important effectors of the innate immune system with a critical role in early host defense against invading pathogens and transformed cells. In allogeneic hematopoietic transplantation, donor-derived allo-reactive natural killer (Allo-NK) cells have shown improved cure rate in hematopoietic malignancies. Cytotoxicity of NK ells is regulated both by HLA-Killer Immunoglobulin-like Receptors (KIRs) (HLA-KIR) inhibitory signals and NKG2D ligand-NKG2D (NKG2DL-NKG2D) activating signals. Nevertheless, it is unknown whether NKG2D+ cells have an effect on human multiple myeloma ARH-77 cells.On the basis of origin, etiology and pathogenesis of multiple myeloma, we hypothesize that oridonin could induce apoptosis on human multiple myeloma ARH-77cells and enhance the sensitivity of ARH-77 cells to NKG2D+ cells cytolytic effects. In this study, we choose human multiple myeloma ARH-77 cells as the research object, and investigate the anti-tumor effects by oridonin in vitro and in vivo.MethodsPartⅠOridonin induced human multiple myeloma ARH-77 cells apoptosisThe growth inhibitions of ARH-77 cells by various concentrations of oridonin at different time were analyzed by MTT assay. ARH-77 cells were treated with various concentrations of oridonin(0,2.5,5,10μmol/L) for 24h, and then observed following results:the morphological changes of apoptotic cells were observed by phasecontrast microscope and Hoechst 33258 staining; the apoptosis rate, the cell cycle, the changes of mitochondrial membrane potential(Δψm), TRAIL receptor on cell surface and intracellular reactive oxygen species(ROX) were detected by flow cytometry (FCM); mRNA expressions of Bcl-2 family members(Bcl-2, Bcl-xl, Bax, Bid), p53, NF-κB andβ-actin were analyzed by RT-PCR; the changes of caspase 8 and caspase 9 were measured by spectrophotometry.PartⅡThe sensitivities of human multiple myeloma ARH-77 cells to actived Allo-PBMCs mediated cytotoxicity after oridonin treatedARH-77 cells were treated with 5μmol/L oridonin for 24h, then the differences between oridonin-treated and no oridonin-treated ARH-77 cells in sensitivity to NKG2D+ cells cytotoxicity were measured by LDH releasing assay; mRNA expressions of NKG2D ligands MICA, MICB, ULBP1, ULBP2 and ULBP3 were measured by RT-PCR; the changes of NKG2D ligands(MICA, MICB, ULBP1, ULBP2 and ULBP3) on cell surface were analyzed by flow cytometry (FCM).PartⅢThe ability of Oridonin to human multiple myeloma ARH-77 cells in vivoSelected BulB/C nu/nu nude mice as experimental animals, through the tail vein injection of ARH-77 cells establish bearing human multiple myeloma ARH-77 cells experimental model. The general conditions, pathology, immunohistochemistry were used to analyze mice tissue morphology and human-specific CD20, CD45 antigen expression, and then evaluate the success of the establishment of experimental model. The tumor-bearing mice were treated with an intraperitoneal injection of oridonin 15mg/kg), control mice received injection with PBS, observe of the above-mentioned targets as drug therapy effectiveness.Statistical analysis The analysis was performed using SPSS 16.0 software package. The data was represented as the mean±standard deviation (x±s).Comparisons of means among groups were performed using one-way analysis of variance (ANOVA), independent-samples t-test, factorial design analysis of variance, repeated measures, Kaplan-Meier. If variance were homogenous among groups, the SNK method was used for multiple comparisons, otherwise Dunnett's T3 method was used, P<0.05 were considered to be statistically significant. ResultsPartⅠOridonin induced human multiple myeloma ARH-77 cells apoptosis1.1 The inhibition to ARH-77 cells with oridoninMTT results showed that:oridonin significantly inhibited the growth of ARH-77cells, at the same time point, different concentrations of the oridonin on the ARH-77 cells, the inhibition rates were significantly different; at the same concentration, with time, the growth inhibition of oridonin were markedly enhanced. Oridonin obviously inhibited the growth of ARH-77 cells in a time- and dose-dependent manner.1.2 To investigate the apoptosis of ARH-77 cells induced by oridonin(1) Oridonin induced ATH-77 cells apoptosisAfter treated with 10μmol/L Ori for 24h, the cells shrank, cell debris increased, with vacuolus in it, Hoechst 33258 staining showed that some cells after treatment appeared in nuclear enrichment, aggregation, and some apoptotic bodies can be seen. After 0,2.5,5,10μmol/L oridonin induced, ARH-77 cells apoptosis, FCM showed that, the apoptosis ratios of each groups are (5.27±1.46)%, (15.07±0.78)%, (21.00±1.49)% and (27.60±1.77)%, respectively, and significantly difference at all the concentration (F=133.331, P=0.000).Oridonin induced ARH-77 cells apoptosis in a dose-dependent manner.(2) Influence of ARH-77 cell cycle induced by oridoninAfter treated with different concentration oridonin, ARH-77 cellcycles were changed,mainly of G0/G1 phase cells gradually increased, while decreasing the S phase cells, at low concentrations (2.5μmol/L) this trend does not maintain statistics significance, while the G2/M phase cells have no obvious change, indicating that ARH-77 cells were arrested in G1 phase by oridonin. cells Sub-diploid were also increased, compared with the control group (F=114.958, P=0.000).(3) In vitro colony formation of ARH-77 cells treated with oridoninIn vitro colony assays indicated, cells could shape clone at Day 14. Contrast with control group, low concentration(2.5μmol/L) of Ori in some extent, could stimulate ARH-77 cell clones formation, with oridonin concentration increased, the number of cell clones drastically reduced, when the oridonin increased 10μmol/L, no clone exists, that indicated human multiple myeloma ARH-77 cells have the ability to form clones in vitro, while the oridonin can inhibit the clone form ability. The numbers of Cloning for each groups compared with the control group were statistically significant (F=952.968, P=0.000)1.3 The mechanism of ARH-77 cells aopotosis induced with oridonin(1) Changes in intrinsic apoptosis pathwayRT-PCR results showed that:oridonin could significantly down-regulated the expressions of Bcl-2, Bcl-xl, mild up-regulated the expressions of Bax, and no effect on Bid. Indicate Oridonin induced ARH-77 cells apoptosis through regulated Bcl-2 family members. In addition, oridonin also have capable of raising the expressions of p53 mRNA and NF-κB mRNA expressions, both moleculars were involved in the ARH-77 cells apoptosis induced by Oridonin.Flow results manifested that, ARH-77 cells were treated with different concertions of oridonin(0,2.5,5,10μmol/L) for 24 h, then cells mitochondrial membrane potential were degreaded, indicating that with the increase of drug concentrations, mitochondrial membrane depolarization occurred (F=252.847, P= 0.000), and there were were differences among the each groups.To test cytochrome c by ELISA found, with the increased concentration of Ori, the total concentration of cytochrome c in ARH-77 cells were decreasd (F=4.909, P=0.047), there were no dignificant statistics between 10μmol/L Ori-treated group and control group,2.5μmol/L Ori-treated group.Spectrophotometric detection of caspase 9 activity results showed that, after treatment with different concentrations of oridonin, compared with the control group, the activities of caspase 9 significant changed (F=2275, P=0.000). Comparison between each group, the activitiy of caspase 9 in low concentrations group was no significant change, after treated with 5,10μmol/L oridonin the activities of caspase 9 were significantly higher than the control group. These findings indicated oridonin could activate caspase 9 in extrinsic apoptosis pathway.(2) Changes in extrinsic apoptosis pathwayOn the ARH-77 cell surface, regardless of death receptors (DR4, DR5) or decoy receptors (DcR1, DcR2) expressions were lower, co-incubated with low concentration of Ori (2.5μmol/L) for 24h, the death receptors DR4 expressions was increased, but the proportion of DR5 was decreased, whereas the expression of two decoy receptors had a slight up-grade. Treated with moderate concentrations of oridonin, the death receptor expressions increased mainly about DR5, while the decoy receptors were less obvious; but close to IC50 concentration, decoy receptors DcR1 on ARH-77 cell surface were significantly higher than control group, meantime the expressions of death receptor DR5 were reduced.10ng/ml TRAIL can promote ARH-77 cells growth, 100ng/ml had no obvious affected to cells, the ATH-77 cells were strongly inhibited by 1000ng/ml TRAIL.5μmol/L Ori-treated group had the same trends with control group, in 10ng/ml and 100ng/ml Ori could make TRAIL more easy to enhance of the cells growth and 1000ng/ml TRAIL also could suppresse the ARH-77cells more significantly. therefore, the effects of Ori to the expressions of TRAIL-R in ARH-77 cells were more complex. the cells expressed the DR, transmitted apoptosis signals,meantime they adaptated accommodation to agonistic drugs. (3) The changes of Reactive Oxygen Species(ROX) on ARH-77 cellsFCM results showed that, after treated with low concentrations of oridonin (2.5μmol/L), the levels of ROS to ARH-77 cells were up-regulated, then increased the concentration of oridonin (5μmol/L) can quickly enhance the production of intracellular ROS, with the continued increase of oridonin concentration (10μmol/ L), the intracellular ROS further increased, the difference was statistically significant (F=3966, P=0.000), and the peak apparent left shift. So, these indicated the generation of ROS by ARH-77 cells were associated with the oridonin concentration.PartⅡThe sensitivities of human multiple myeloma ARH-77 cells to Allo-PBMCs-mediated cytotoxicity after oridonin treated(1) Measurement of Allo-PBMCs cytotocixityPeripheral blood mononuclear cells co-culture with rhIL-15, then measured the cytocixity of Allo-PBMCs against multiple myeloma and ARH-77 cells, which before and after treated oridonin 5μmol/L in vitro. With the effective target(E:T) ratios increased, the cytocixity of the experimental groups and control groups were enhanced, respectively, the difference was statistically significant (t=-4.048, P= 0.016; t=-7.152, P=0.002); while at the same E:T ratio, the cytocixities of Allo-PBMCs against the treated ARH-77 cells were stronger than the role of the former, the difference was statistically significant (t=-10.642, P=0.009; t=-6.458, P=0.003).ARH-77 were low sensititive to Allo-PBMCs cells cytolytic effects, at the E:T ratio of 10:1, cytotoxic activity was only (7.45±0.39). The results suggested that: ARH-77 cells could be considered to be the low degree of sensitive to Allo-PBMCs, moderate concentration of oridonin can enhance the cytocixity of Allo-PBMCs against multiple myeloma and ARH-77 cells. (2) The mRNA expression of NKG2D ligands on ARH-77 cells before and after treatment with oridoninRT-PCR results showed that:two groups were amplified by five fragments consistent with the expected fragment size. ARH-77 cells themselves expressed MICA, ULBP2, while MICB, ULBP1 and ULBP3 expression lower; compare with control group, the expressions of NKG2D ligands mRNA on experimental group treated with oridonin were:MICA, ULBP1 and ULBP3 expressions changed little, mRNA expressions of MICB were increased, the mRNA level of ULBP2 decreased.(3) The expressions of NKG2D ligand on ARH-77 cell surface by FCMFCM tests showed:ARH-77 cells with low expressions of MICB (8.57±1.35), did not express MICA, ULBP1, ULBP2 and ULBP3; after treated with oridonin 5μmol/L for 24h, the expressions of MICB were significant increased (t=-6.833, P =0.002). The expressions of MICA, ULBP2 were also moderately enhanced (t=-2.828, P=0.047; t=-4.243, P=0.013), but The expressions of ULBP3 were decreased (t=2.889, P=0.045), and ULBP1 had no significant change.(4) The espressions of TRAIL,Fas,FasL in Allo-PBMCsAfter treated with IL-15, the expressions of Fas, FasL and TRAIL on cell surface were (86.40±14.71), (10.20±5.09), (1.00±0.14), there were almost no TRAIL on Allo-PBMCs. The findings indicated that Fas/FasL may play a role in induce apoptosis effects, but the TRAIL/TRAIL-R pathyway were not activated.PartⅢThe ability of Oridonin to human multiple myeloma ARH-77 cells in vivo(1) Establishment the experiment model of nude mice bearing human multiple myelomaVia the tail vein injection of ARH-77 myeloma cells for about 5 weeks, some mice started appear weight loss, be unwilling to move. to about 6 weeks or so, the model group mice died, the normal control group and cyclophosphamide pre-dealing group mice were in good conditions, did not appear weight loss. To further clarify the ARH-77 cells successfully transplanted into mice, at the same time the organization of the pathology and immunohistochemistry were analysized. The model mice liver and spleen were both with tumor cells infiltration, and immunohistochemistry results showed that infiltrated cells expressed human-specific CD45 and CD20 antigen, and the expressions of CD20 were higner than those of CD45.The remaining organs (lungs, kidneys, intestines, etc.) had no significant abnormalities. The normal control group mice showed, the structure of organs in microscope were tidy and no abnormal cell infiltration, cyclophosphamide pretreatment group results were in accordance with the normal control group.The findings suggested the infiltrated cells in mice were derive from human multiple myeloma ARH-77 cells.(2) The study of oridonin in vivoThe condition of model group of mice were the same as the forementioned model, in the first 5 weeks, using oridonin treated group mice does not appear the status of the model group, the growth were in good condition. To the 8 weeks, some mice appeared emaciated, loss of appetite and other behaviors. In order to compare with model group,1 mouse of the treated groupwas sacrificed at the same time with model group mice, in the concurrent analysis and comparison of pathology and immunohistochemistry. Model group mice liver and spleen had tumor cell infiltration, and immunohistochemistry showed that infiltration cells expressed human-specific CD45 and CD20 antigen, and CD20 expressions were higher than CD45. Oridonin treated group mice's livers had only a small amount of abnormal cells infiltrated, the structures of liver and spleen were basically normal.The apoptosis cells were observed in Ori-treated mouse by TUNEL detected. The studies indicated oridonin may inhibit the ARH-77 myeloma cell growth and infiltration in mice and prolong survival.Conclusions1. Oridonin can induce human multiple myeloma ARH-77 cell apoptosis in a time-dose-dependent manner;2. Oridonin induced ARH-77 cells apoptosis by regulating the Bcl-2 family members, the expression of cell surface death receptors, as well as changes in mitochondrial membrane permeability, stimulate intrinsic/extrinsic apoptosis pathway;3. Oridonin can lead to the changes of reactive oxygen species (ROS) on ARH-77 cells;4. Medium concentration Oridonin able to improve the expression of NKG2D ligands on ARH-77 cells surface, and thus enhance the cytotoxity sensitivity to Allo-PBMCs.5. Successful establishment of human multiple myeloma animal model, Oridonin can reduce the infiltration of tumor cells and prolong survival in vivo.