Vernodalol Enhances TRAIL-induced Apoptosis In Diffuse Large B-cell Lymphoma Cells

Background:Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) comprising 30% of NHL. The standard RCHOP chemotherapy leads to about 50%-60% long-term remission in this aggressive lymphoma. Still, about 40% of patients treated will relapse or have refractory disease and most of them will succumb to the disease. Thus, additional new targets and approaches to improve the efficacy of DLBCL therapy are urgently needed. Defects in apoptotic signaling correlate with the aggressive behavior of relapsed DLBCL and their resistance to chemotherapy.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent anti-tumor agent that triggers apoptosis in multiple carcinoma cells but not normal cells. Several pre-clinical trials have investigated the potential of TRAIL-based therapies for DLBCL. However, those therapies showed only modest activity as single-agents. DLBCL cells are insensitive to TRAIL due to different mechanisms. Like Baicaleinand Silibinin, manynutraceuticals derived from’Mother Nature’that can upregulate DRs and increase TRAIL induced cancer cellsapoptosis.Vernonia anthelmintica, an acknowledged anti-malarial plant. Its seeds contain multiple sesquiterpene lactones with medicinal potency such as vernodlin, vernodalol, and vernolic acid. This class of compounds has been reported to possess anti-fungi, anti-microbe, and anti-tumor activities.In this study, we observed that Vernodalol (Vern), a sesquiterpene lactone, can sensitize human DLBCL cells to TRAIL-induced apoptosis.Objective:The study is aimed to explore the effect of Vern to DCBCL cell lines proliferation, apoptosis, and find the combined effect of Vern with TRAIL. Then, the mechanisms of Vernodalol enhances TRAIL-induced apoptosis in diffuse large B-cell lymphoma cell lines will be discussed mainly focus on death receptor 5(DR5), C/EBP homologous protein (CHOP), Mcl-1 and JNK signaling pathway. The in vitro anti-myeloma effect of Vern, TRAIL alone or Vern combined with TRAIL will be confirmed in vivo by BALB/cnude mice.Methods:1. DLBCLcells Sudhl2, Sudhl4 and Sudhl10 were treated with various concentrations of TRAIL in the presence or absence of various concentrations of Vern for 24 h, cell viability was measured by the MTT assay; Flow cytometry of Annexin V/PI staining of apoptosis. Sudhl2 cells were treated with TRAIL with or without Vernfor 24 h. Cells were stained with Annexin V/PI and analyzed by FACS; After the treatment of Sudhl2 cells as described above. Quantitative results obtained using Annexin V/PI staining. Whole-cell extracts from treated cells were analyzed by Western blotting using the caspase antibodies.2. Sudhl2 cells were treated with various doses of Vern for 24 h. Whole-cell extracts were prepared and analyzed by western blotting; Cell surface expression of DR5 and DR4 in Sudhl2 cells treated with 20 μM Vern for 24 h was measured by flow cytometryanalysis; Sudhl2 cells were treated with various concentrations of Vern for 12h. Real-time RT-PCR analysis of DR5 and DR4 mRNA;Sudhl2 cells were transfected with DR5, DR4 and scamble siRNAs, cell extracts were prepared for western blot analysis. After transfection with siRNA, Cell viability was measured by MTT assay. siRNA-transfected cells were stained with Annexin V/ PI and then quantitativeanalyzed.3. Sudhl2 cells were treated with various doses of Vern for 24 h. Whole-cell extracts were prepared and analyzed by western blotting. GAPDH was used as a loading control; After the treatment, Real-time RT PCR analysis of CHOP mRNA; Sudhl2 cells pretreated with cycloheximide, or actinomycin D before incubation with 20μM Vern; Then whole-cell extracts were prepared and analyzed by western blotting. Cells were transfected with CHOP siRNAs and western blot analysis of DR5 and CHOP; After transfection with siRNA, cells were stained with Annexin V/PI and then quantitativeanalyzed. Cell viability was measured by MTT assay. siRNA-transfected cells were stained with Annexin/PI V and then quantitativeanalyzed.4. Sudhl2 cells were treated with 20μM Vern for various time. Whole-cell extracts were prepared, and Western blotting was used to analyze the extracts for indicated proteins; p-ERK1/2, phospho-ERK1/2; pJNK, phospho-JNK; p-p38, phospho-p38; We pretreated Sudhl2 cells with p38 MAPK inhibitor (SB202190), JNK inhibitor (SP600125), and ERK1/2 inhibitor(PD98059) for 1 h and then treated the cells with 20μM Vern for 24 h; Whole-cell extracts were then prepared, and Western blotting was used to analyze the expression CHOP of DR5.5. Western blotting of Sudhl2 lysates from cells treated with various concentrations of Vern; Sudhl2 cells were transfected with the control vector or the Mcl-1 over expression vector, the level of Mc-1 was assessed by Western blotting; After the transfection, the cells were treated with TRAIL (20 ng/ml) alone or in combination with Vern (20 μM) for 24 h, cell viability was measured by the MTT assay; Sudhl2 cells were transfected with scramble siRNA or Mcl-1 siRNA, incubated for 24 h and further treated with or without TRAIL (20 ng/ml) for another 24 h, cell viability was measured by the MTT assay.6. Nude BALB/c mice bearing Sudhl2 cells were treated with vehicle, Vern (2 mg/kg), TRAIL (2 mg/kg)or combined Vern and TRAIL; Tumor volume, tumor weight and body weight were measured after treatment.Results:1. Vern enhances anti-tumor effect of TRAIL in DLBCL cells.Three DLBCL cell lines Sudhl2, Sudhl4 and Sudhl10 were treated with various concentrations of Vern in the presence or absence of different doses of TRAIL for 24 h. Vern in combination with TRAIL reduced the cell viability in a concentration-dependent manner in all of the three cell lines. Annexin V/PI assay was applied to measure apoptosis in Sudhl2 cells. Treatment of Vern or TRAIL resulted in about 5% and 10% apoptosis respectively; and the combination increased the apoptosis to around 30%. The combined treatment with TRAIL plus Vern strongly enhanced the expression of cleaved caspase-8, caspase-3 as well as PARP.2. Vern enhances expression of death receptor DR5 and contributes to the augment of TRAIL-mediated cell death.Incubation of Sudhl2 cells with Vern lead to an increased expression of DR5 but not DR4 in a dose-dependent manner. After treatment of Vern, the level of DR5 on the cellular surface was increased while the level of DR4 was not affected. We found that Vern noticeably increased DR5 mRNA level but not DR4 in a dose-dependent manner using real-time PCR, indicating that Vern regulates DR5 expression at the transcriptional level. Transfection of Sudhl2 cells with DR5 siRNA or DR4 siRNA resulted in significant down-regulation of DR5 and DR4, respectively. We found cytotoxicity as well as apoptosis induced by Vern plus TRAIL were effectively abolished in cells transfected with DR5 siRNA, whereas transfection with DR4 siRNA or scramble siRNA had little effect.3. Vern induces DR5 expression in a CHOP-dependent wayWe found that Vern increased the protein as well as mRNA expression of CHOP in a dose-dependent manner in Sudhl2 cells. Pretreatment of cells with inhibitors of transcriptional (actinomycin D, ACT) and translational (cycloheximide, CHX) suppressed vernodalol-induced CHOP expression.We used specific siRNA to silence CHOP. We observed that the up-regulation of DR5 was significantly abrogated after the silencing of CHOP while transfection with scramble siRNA had no effect.We found that the effect of Vern on TRAIL-mediated apoptosis was diminished in cells after the down-regulation of CHOP. Meanwhile, knock-down of CHOP significantly attenuated the cytotoxicity induced by the combined treatment of Vernodalol and TRAIL.4. Vern-induced up-regulation of DR5 is mediated through JNK activationActivation of ERK and JNK but not p38 was observed after the treatment of Vern. Specific inhibitors for JNK, ERK and p38 were utilized to investigate which MAPK is responsible for Vern-induced up-regulation of DR5. Pretreatment of Sudhl2 cells with JNK inhibitor SP600125 suppressed Vern-induced up-regulation of DR5 and CHOP in a dose-dependent manner. In contrast, ERK1/2 inhibitor PD98059 and p38 inhibitor SB202190 had no such inhibitory effect on the expression of DR5-induced by Vern.5. The down-regulation of Mcl-1 is associated with Vern and TRAIL-induced apoptosis.After the treatment of Vern, there is a slight up-regulation of Bad and down-regulation of Bcl-2. At the same time, the expression of Mcl-1 was significantly inhibited in a dose-dependent manner. Then we examined whether the suppression of Mcl-1 is vital for cytotoxicity following combined treatment of TRAIL plus Vern. When Mc-1 was over expressed, the synergistic anti-proliferation and apoptosis-inducing effect of TRAIL plus Vern was diminished. Sudhl2 cells were transiently transfected with specific Mcl-1 siRNA. The down-regulation of Mcl-1 expression by siRNA resulted in enhanced cytotoxicity of TRAIL. Taken together, these results indicate that the down-regulation of Mcl-1 has an important role in Vern-mediated TRAIL sensitization.6. Evaluation of combined effects of Vern and TRAIL in xenograft model.In the mouse model, administrated with combined Vern and TRAIL resulted in significant inhibition of tumor growth when compared with other groups. In the meantime, monitoring of the body weight showed no significant weight loss.Conclusions:1. Verncombined TRAIL could inhibit DLBCL cell lines (Sudhl2, Sudhl4 and Sudhl10) cells proliferation; Vern enhances TRAIL-induced apoptosis in Sudhl2 cells.2. Vern enhances TRAIL-induced apoptosis via down-regulation of Mcl-1 and up-regulation of DR5 which depends on the activation of JNK and induction of CHOP.3. Vern and TRAIL combination demonstrates potent anti-tumor effect against DLBL cells without noticeable side effects in BALB/c mice xenograft model.