Experimental Study On Treatment Of Human Breast Carcinoma Cells With Combination Of TRAIL And Glutathione Peroxidase Mimics

Tumor necrosis factor(TNF)α–related apoptosis-inducing ligand(TRAIL)is a member of the TNF family of cytokines. It exerts cytotoxic effects on malignant cells without any harm to normal cells. To date,five members of the human TNF receptor(TNFR)superfamily have been identified that can bind TRAIL,including death receptors DR4(TRAIL-R1)and DR5(TRAIL-R2),decoy receptors DcR1(TRAIL-R3)and DcR2(TRAIL-R4),as well as soluble receptor osteoprotegerin(OPG). However,only death receptors contain cytoplasmic death domain that are required for transmitting a cytotoxic signal. Following TRAIL engagement with either DR4 or DR5,the ligated death receptors cluster and initiat the formation of the death-inducing signaling complex(DISC). The functional death-inducing signaling complex is minimally composed of death receptors,FADD and Caspase 8 or 10. Active Caspases 8 and 10 cleave and directly activate downstream effector Caspases(3,6,7),which could ultimately result in apoptosis. Meanwhile,TRAIL receptors signaling pathway also leads to the activation of the nuclear factor-κB(NF-κB),which operates as a negative regulator for DISC formation. The subunit of NF-κB could up-regulate anti-apoptotic genes,such as cellular c-FLIP and c-IAPs,which eventually thwart activation of Caspases.As a promising therapy agent for treatment of malignancies,TRAIL has been shown to induce apoptosis against breast carcinoma. Unfortunately,the majority of cell lines are insensitive to TRAIL-induced apoptosis,and the mechanism of the resistance has been attributed to dysfunction of different steps in the apoptosis pathways,as well as elevation of survival signals. The combination therapy was therefore envisaged,basing on the recent evidences that pretreatment of tumor cells with a chemotherapeutic agent could sensitize them to TRAIL. However,the combinations should be taken very carefully due to the potential toxicity to normal tissues. In this respect,it will be important to find a safe and effective agent to overcome TRAIL resistance in breast cancer therapy.Seleno-cyclodextrin(2-selenium-bridgedβ-cyclodextrin,2-SeCD),a synthetic seleno-organic compound with unique glutathione peroxidase(GPx)-like activity,has exhibited anti-oxidant,anti-inflammatory and anti-tumor properties with very low toxicity. It has been extensivelydemonstrated as a promising substitute for ebselen,a well known GPx mimic,with enhanced enzymatic activity and water solubility. 2-tellurium-bridgedβ-cyclodextrin(2-TeCD),another synthetic organotellurium compound,has exhibited anti-oxidant and anti-inflammatory activities in vitro. Furthermore,2-TeCD has been shown to exhibit anti-tumor effects on breast carcinoma through selective inhibiting of thioredoxin reductase(TrxR)which is overexpressed in tumor tissues. Considering the anti-tumor property of the two agents,we hypothesized that their combination with TRAIL could be a promising strategy in the treatment of refractory breast tumors.1. 2-SeCD sensitizes resistant human breast cancer cells to TRAIL-induced cytotoxicity in vitro.We examined MDA-MB-468 and T47D cells for TRAIL sensitivity using different concentrations of recombinant human soluble TRAIL and/or 2-SeCD. Cells did not exhibit growth arrest by TRAIL alone treatment,while significant decrease of cell viability was observed with a synergistic treatment of 2-SeCD and TRAIL. Using the accepted criterion that apoptotic cells are Annexin V–positive/Propidium iodide–negative,we found that more than 50 percents of cells were undergo apoptosis in both of the cancer cell lines,upon exposure to the combination of the two agents. This experiment suggests that the sensitization of TRAIL-induced cytotoxicity by 2-SeCD is associated with apoptosis.We next analyzed the Caspase-8 cleavage,a proximal event in the TRAIL-induced Caspase cascade. TRAIL alone failed to induce detectable Caspase-8 and Caspase-3 processing when compared to control. In contrast,cleaved-Caspases was clearly detected in both of the cancer cells when treated with the combination treatment of 2-SeCD and TRAIL. The immunoblotting results were further substantiated by Caspase activity assay. Compared to agent-alone group,the combination group significantly increased Caspase-3 and Caspase-8 activity in both of the MDA-MB-468 and T47D cells to about 4~6 times.The activation of the Caspase-8 in combined treatment suggests that 2-SeCD targets an early step in the TRAIL-induced apoptosis pathway; we therefore evaluated the effects of 2-SeCD on expression of TRAIL receptor DR4 and DR5. Using Western bolting,flow cytometric analysis,RT-PCR reactions , as well as iRNA techniques we extensively showed that 2-SeCD dose-dependently induced the expression of TRAIL receptors DR5,but not DR4 on both mRNA and protein levels. These data suggest that up-regulation of DR5 by 2-SeCD is essential for the ability of TRAIL to induce apoptosis in resistant breast cancer cells.2-SeCD treatment also suppressed TRAIL-induced nuclear factor-κB(NF-κB)prosurvival pathways by preventing cytosolic IκBαdegradation,as well as p65 nuclear translocation. Since traditional selenium compounds were not reported to target NF-κB activity in sensitizing TRAIL-resistant cancers,we hypothesize their unique GPx -like activity would be an attractive explanation. Therefore,inhibition of NF-κB nuclear translocation by 2-SeCD also contributes to its sensitization of TRAIL-induced apoptosis in breast cancer cells.2. 2-SeCD and TRAIL results in tumor growth inhibition and apoptosis in vivo.Based on the above findings,we next sought to evaluate whether the effect of 2-SeCD alone and in combination with TRAIL could inhibit tumor growth in vivo. MDA-MB-468 cells were subcutaneous injected into the right axilla of the female Balb/c nu/nu mice. TRAIL was ineffective in inhibiting the tumor growth; however,the combination of 2-SeCD and TRAIL exhibited significant inhibitory effects compared with other treatment groups. Immunohistochemical examination of tumor tissues revealed that 2-SeCD alone inhibited cell proliferation [proliferating cell nuclear antigen(PCNA)and Ki-67 staining] and induced apoptosis(TUNEL staining)in xenograft tumors. On the contrary,TRAIL had no significant effects compared with control group. Importantly,the combination of 2-SeCD and TRAIL were more effective in inhibiting tumor cell proliferation and inducing apoptosis than single agent alone. Finally,xenograft sections from each of the treatment groups were immunostained with anti-p65 and anti-DR5 antibodies. Treatment of mice with a combination of 2-SeCD and TRAIL significantly showed more expression of DR5 and less expression of p65-NF-κB proteins than that of mice treated with vehicles or TRAIL.For the first time,we present clear evidences that 2-SeCD,a GPx mimic,can overcome TRAIL resistance in vitro and in vivo; therefore this combination provides a powerful therapeutic option for human breast cancer treatment.3. Low doses of 2-TeCD could sensitizes resistant human breast cancer cells to TRAIL induced cytotoxicity in vitro.2-tellurium-bridgedβ-cyclodextrin(2-TeCD)shares the unique chemical characteristics with its selenium counterparts(2-SeCD),but with a much better GPx mimic activity. Despite the antioxidants properties,2-TeCD has been recently shown to exhibit anti-tumor effects on breast carcinoma with a very low concentration. However,the application of 2-TeCD should be taken very carefully due to potential toxic effects of tellurium on kidney,hepatic and nervous system. In this study,we reported that 2-TeCD,in a low dose,could convert the TRAIL resistant breast cancer cells to TRAIL sensitive. Importantly,by using a sequential treatment protocol,where cells were first treated with 2-TeCD for 24 hours followed by TRAIL treatment,we could reduce the doses of 2-TeCD for TRAIL-sensitization in the submicromolar range. Using the Annexin V/ propidium iodide staining and Caspase activity assay , we then reavealed that the sensitization of TRAIL-Induced cytotoxicity by 2-TeCD is associated with apoptosis. Finally,the mechanism of sensitization could also attribute to the fact that 2-TeCD dose-dependently induced the expression of TRAIL receptors DR5,as well as down-regulated the activity of NF-κB.