Chinese-German Journal of Clinical Oncology April 2012, Vol. 11, No. 4, P187–P191DOI 10.1007/s10330-011-0922-4
The monoclonal antibody against TNF-related apop-tosis-inducing ligand death receptor 5 (DR5), which has similarly biological effect to TNF-related apoptosis-induc-ing ligand (TRAIL), specially induces apoptosis in tumor cells without toxicity in hepatocytes [1]. It is anticipated that this monoclonal antibody will provide a new reagent for tumor therapy. At present, a novel murine anti-hu-man DR5 monoclonal antibody mDRA-6 has been gener-ated in our lab. The previous data identified that mDRA-6 is an agonistic antibody to DR5 and that it stimulates the
apoptosis pathway in human leukemia cell lines, includ-ing Jurkat, U937 and HL-60, whereas in human leukemia Raji and K562 cells it was not [2]. In this study, we treated Raji and K562 cells with Vitamin E Succinic Acid (VES), which is non-toxic to humans [3, 4]. It was our goals to de-tect whether VES could regulate the expression level of DR5 in the cells and further elucidate the potential mech-anisms involving that VES enhances the effect of mDRA-6 to eradicate leukemia Raji and K562 cells. It will bring about a great advance in clinical treatment to cancer pa-tients with mDRA-6.
Vitamin E Succinic Acid enhances the effect of mDRA-6 to eradicate leukemia cells by inducing apoptosis*Jun Zhang, Shulian Li, Jingfang Du, Yuanfang Ma
Key Laboratory of Cellular and Molecular Immunology, Institute of Immunology, Henan University, Kaifeng 475004, China
Abstract Objective: The aim of our study was to detect whether Vitamin E Succinic Acid (VES) could regulate the ex-pression level of DR5 in the cells and further elucidate the potential mechanisms involving that VES enhances the effect of mDRA-6 to eradicate leukemia Raji and K562 cells. Methods: MTT method was used to detect the growth inhibition of VES and mDRA-6 to Raji and K562 cells. Annexin V-FITC/PI double staining assay was used to analysis the apoptosis of leukemia cell. Flow cytometry was used to detect the cell surface DR5 expression. Immunoblotting technique was used to detect the DR5 protein expression. Results: MTT detection showed that 10 μmol/L mDRA-6 on the cell death rates of Raji and K562 cells were 21.98% and 5.23%, respectively. While increasing concentration of VES (5 μmol/L, 10 μmol/L, 20 μmol/L) and mDRA-6 both on the cell viability of Raji or K562 cells, the mortality of Raji cells elevated to 24.67%, 35.65% (P < 0.01) and 40.22% (P < 0.01), respectively. Similarly, the mortality of K562 cells increase to 6%, 7.89% (P < 0.01) and 8.67% (P < 0.01), respectively. To further specify the increased cell death rate induced by mDRA-6 and VES, the treated cells were analyzed by Annexin-V/PI staining assay. As shown in Fig. 1, the apoptosis rates of Raji and K562 cells treated with 2 μg/mL mDRA-6 for 12 h were 20.79% and 7.74%. Compared with this, the proportion of apoptotic cells increased upon exposure to 2 μg/mL mDRA-6 combination with 10 μmol/L VES, the apoptosis rates of Raji and K562 cells were 43.18% and 16.99%, respectively. To examine the anticancer effects of a combination strategy based on mDRA-6 and VES. We analyzed whether VES could elevated the expression level of DR5 on Raji and K562 cytomembrane by FACS. Interestingly, after treated with 10 μmol/L VES for 12 h, the expression level of DR5 on Raji and K562 cell surface increased from 50.66% to 70.08%, and 15.02% to 16.38%, respectively. Immune imprinting technology test showed that, different concentrations of VES could increase Raji and K562 cell DR5 protein expression. Conclusion: VES enhances the effect of mDRA-6 to eradicate leukemia Raji and K562 cells. The proper mechanism is VES could enhance the Raji and K562 cell membrane expression of DR5, and VES can also enhance the DR5 protein expression of cells.
Key words Vitamin E Succinic Acid; TNF-related apoptosis-inducing ligand; death receptor 5; monoclonal antibody; apoptosis
Correspondence to: Jun Zhang. Email: [email protected]*Supported by grants from the National “863 Plan” (No. 2006AA02A254) and Outstanding Talent Program of Henan Province (No. 074200510014).
188 www.springerlink.com/content/1613-9089
Materials and methods
MaterialsRaji and K562 cells were obtained from ShangHai
institutes for biological sciences, CAS. VAS and DMSO were purchased from Sigma (USA). 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Gibco (USA). Fetal calf serum was pur-chase from Tianjin Haoyang Biological Manufacture Co., Ltd (China). The Annexin V-FITC/ PI Kit was purchased from BD Pharmingen (USA). The murine anti-human DR5 monoclonal antibody mDRA-6 and the murine anti-human DR5 combination antibody 366EC were prepared by our lab. Goat anti-mouse IgG-FITC was purchased from Jackson ImmunoResearch (USA).
Cell viability assayThe effects of VES and mDRA-6 on Raji and K562
cells were performed by MTT assay. Briefly, 3 × 104 Raji or K562 cells were plated in 96 wells culture plates re-spectively, and treated with different concentrations of VES (0, 5 μmol/L, 10 μmol/L, 20 μmol/L) and 10 μmol/L mDRA-6, then incubated for 12 h at 37 ℃ in a humidified air atmosphere with 5% CO2. After treatment, 20 μL MTT (5 mg/mL) was added to each well and the plates were incubated at 37 °C for another 4 h. The purple formazan crystals were dissolved in 150 μL DMSO, and absorbance was determined at 570 nm using Automatic enzyme-linked immunosorbent assay systems. The cell viability was calculated as follows: Viability (%) = [1 – (OD value of the treated wells – OD value of the blank control wells)]/ (OD value of the control wells – OD value of the blank control wells) × 100%. Each concentration was tested in three different experiments run in triplicate.
Detection of apoptotic cellsThe cells at a concentration of 1 × 105 cells/well were
respectively treated with 10 μmol/L VES and/ or 2 μmol/L mDRA-6, and incubated for 12 h at 37 ℃ in a humidified air atmosphere with 5% CO2. After treated, the cells were collected and added in 100 μL binding buffer. Apopto-sis was determined by flow cytometric analysis using an Annexin V/PI apoptosis detection Kit and the data were analyzed by CellQuest software.
Flow cytometric examined the expression level of DR5 at the cell surface
The cells at a concentration of 2 × 105 cells/well were respectively treated with 10 μmol/L VES or not, and in-cubated for 12 h at 37 ℃ in a humidified air atmosphere with 5% CO2. After treated, the cells were collected and washed three times by cold PBS. 200 μL anti-human DR5 combination antibody 366EC (5 μg/L) were added in two test sample groups. At the same time, 200 μL mice IgG
or PBS were respectively added in two control sample groups. The cells were incubated for 45 min on ice and the washed three times by cold PBS again. The cells were stained with 100 μL of FITC-conjugated mouse IgG (1:100) for 40 min on ice. After washing twice by PBS, the cells were analyzed using flow cytometric. The results were analyzed by CellQuest software.
Western blotting detected total DR5 protein expressing in the cells
5 × 106 cells were treated with different concentrations of VES (0 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmol/L) for 12 h. The cell pellet was lysed with protein extraction re-agents. Following cell protein quantitation, 50 μg protein/sample were subjected to SDA-PAGE. After transferred to a nitrocellulose membrane, the membrane was blocked for 2 h at room temperature. The membrane was then incubated overnight at 4 ℃ with first antibody 366EC, which was against human DR5. After washing the mem-brane 3 times with TBST, incubation with secondary an-tibodies conjugated with horseradish peroxidase was sub-sequently followed. The membranes were washed 3 times with TBST. Immunoreactive bands were visualized by an enhanced chemiluminescence (ECL) system and the level of protein expression determined using Image Quant TL software.
Statistical analysisExperiments were repeated a minimum of three times.
Data were reported as the mean ± SD. The statistical anal-ysis was performed by SPSS 15.0, and P value < 0.05 was considered statistically significant.
Results
VES enhances the cytotoxic functional activity of mDRA-6 in human leukemia cell lines Raji and K562
Under in-vitro conditions, we have examined the cy-totoxic effects of mDRA-6 alone or combined with dif-ferent concentration of VES on Raji and K562 cells. The inhibitory effect of 10 μmol/L mDRA-6 and increasing concentration of VES (5 μmol/L, 10 μmol/L, 20 μmol/L) on the cell viability of Raji and K562 cells was determined by means of MTT assay. The results indicated that the mortality of Raji and K562 cells treated with 10 μmol/L mDRA-6 for 12 h were 22.56% and 5.18% respectively. While the same dose of mDRA-6 was used in combina-tion with increasing concentration of VES (5 μmol/L, 10 μmol/L, 20 μmol/L), the mortality of Raji cells elevated to 24.67%, 35.65% (P < 0.01) and 40.22% (P < 0.01), respec-tively. Similarly, the mortality of K562 cells increase to 6%, 7.89% (P < 0.01) and 8.67% (P < 0.01), respectively.
189Chinese-German J Clin Oncol, April 2012, Vol. 11, No. 4
Increased apoptosis rate induced by a combination of mDRA-6 and VES in Raji and K562 cells
To further specify the increased cell death rate induced by mDRA-6 and VES, the treated cells were analyzed by Annexin-V/PI staining assay. As shown in Fig. 1, the apoptosis rates of Raji and K562 cells treated with 2 μg/mL mDRA-6 for 12 h were 20.79% and 7.74%. Compared with this, the proportion of apoptotic cells increased upon exposure to 2 μg/mL mDRA-6 combination with 10 μmol/L VES, the apoptosis rates of Raji and K562 cells were 43.18% and 16.99%, respectively.
VES elevated the expression level of DR5 on Raji and K562 cytomembrane
To examine the anticancer effects of a combina-tion strategy based on mDRA-6 and VES. We analyzed whether VES could elevated the expression level of DR5 on Raji and K562 cytomembrane by FACS. Interestingly, after treated with 10 μmol/L VES for 12 h, the expression level of DR5 on Raji and K562 cytomembrane increased from 50.66% to 70.08%, and 15.02% to 16.38%, respec-tively (Fig. 2).
VES elevated the total expression level of DR5 in Raji and K562 cells
To further identify the total expression level of DR5 in treated cells, we examined the DR5 protein expression level in Raji and K562 cells by Western blotting. In line with our expectation, the expression level of DR5 was gradually elevated by treated with increasing concentra-tion of VES (5 μmol/L, 10 μmol/L, 20 μmol/L).
Discussion
The tumor necrosis-related apoptosis inducing ligand (TRAIL) is a member of the TNF family capable of induc-ing apoptosis in a wide variety of cancer cells upon bind-ing to pro-apoptotic receptors, while having no effect on the majority of normal human cells tested [5]. This subset, which also includes Fas ligand and TNF-α, can activate the extrinsic apoptotic cell death pathway on binding to cognate death receptors 4 or 5 (DR4/5) at the cell surface. Upon binding to DR4 or DR5, TRAIL induces receptor trimerization to form a structure known as the death-in-ducing signaling complex (DISC), which activates caspase-8, leading directly to the activation of caspase-3 and sub-sequent apoptosis. However, some recombination TRAIL has a cytotoxic effect on normal cells. Our previous results indicated that the monoclonal antibody against DR4/5, which has similarly biological effect to TRAIL, specially induces apoptosis in tumor cells without toxicity in he-patocytes [1]. DR5 or DR4 expresses in leukemia cells and normal cells at different levels, but the expression level
of DR5 or DR4 significantly increased in leukemia cells compared with normal cells. Especially, the expression level of DR5 is higher than DR4 in leukemia cells [6], and many factors could regulate the expression level of DR5 [7]. In this context, it suggests that DR5 mediated signaling pathways played an important role in the progression of TRAIL inducing apoptosis, DR5 functional antibody may has a broad prospect of application.
A novel monoclonal antibody mDRA-6, which pre-pared from our lab, can specifically bind to human DR5 and is able to induce the apoptosis of tumor cells. Howev-er, this monoclonal antibody is similar to TRAIL, the pro-apoptotic effect of mDRA-6 is not the same in different tumor cells. Our previous study indicated that mDRA-6 can induce different kinds of leukemia cell lines apopto-sis, including Jurkat, U937 and HL-60, whereas in human leukemia Raji and K562 cells it was not [2]. In this context, how to enhance the lethality of mDRA-6 in tumor cells without normal cells is very important to broaden the clinical application of the therapy strategy. Accumulating evidence suggested that VES is a natural Vitamin E ana-logue, which inhibits tumor growth. It induces apoptosis, inhibits tumor cell proliferation and differentiation, ar-rests DNA synthesis, and blocks cell cycle progression in various cancer cell. In addition, VES selectively kills tu-mor cells without toxic effects on normal cells and tissues [8, 9], and some experimental data shown that TRAIL plays a role in the pathway of VES inducing tumor cells apop-tosis [10]. In order to observe whether VES could promote the proapoptotic activity of mDRA-6 in hyposensitivity tumor cells, we chose leukemia cell lines Raji and K562 as the cell model, and detected the lethality of mDRA-6 in the leukemia cells through the increasing concentration of VES. The current results showed that VES acts syner-gistically with mDRA-6 in two human leukemia cell lines. These results suggest that a combinatorial treatment con-sisting of VES and mDRA-6 can be more beneficial than the separated single treatments.
To examine the anticancer effects of a combina-tion strategy based on mDRA-6 and VES. We analyzed whether VES could elevated the expression level of DR5 on Raji and K562 cytomembrane by FACS and the to-tal expression level of DR5 in treated cells by Western blotting. The obtained data indicated that the expression level of DR5 was gradually elevated by treated with in-creasing concentration of VES (5 μmol/L, 10 μmol/L, 20 μmol/L) in Raji and K562 cells. This may be the potential mechanisms involving that VES enhances the effect of mDRA-6 to eradicate leukemia Raji and K562 cells. Many kinds of chemotherapeutics used as he approved therapy for cancer patients because they could up-regulate the ex-pression level of DR5 [11], then enhanced the proapoptotic activity of antibodies against DR5, but its response rates are disappointing and the effect is not sustained. VES is a
190 www.springerlink.com/content/1613-9089
natural Vitamin E analogue, which selectively kills tumor cells without toxic effects on normal cells and tissues. Our data show for the first time that a combinatorial treat-
ment of leukemia cells in vitro based on VES and mDRA-6 represents an effective antitumor strategy without de-tectable side effects.
Fig. 1 Synergistic affects of mDRA-6 and VES on Raji and K562 cells
Fig. 2 Expression of DR5 on cytomembrane
191Chinese-German J Clin Oncol, April 2012, Vol. 11, No. 4
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