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The Effect of hIL-2-Luffin P1 Immunotoxin Combining with Arotinoid Ethylester on Hut-78 Cells and Jurkat Cells: An Experimental Study

Author: LiuShuLei
Tutor: HeWei
School: Third Military Medical University
Course: Dermatology and Venereology
Keywords: immunotoxin ribosome inactivating protein (RIP) interleukin 2 receptor prokaryotic expression CTCL ATCL Hut-78 cells Jurkat cells MTT apoptosis cell cycle
CLC: R739.5
Type: PhD thesis
Year: 2008
Downloads: 99
Quote: 0
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Abstract


BackgroundCutaneous T cell lymphomas (CTCL) and acute T cell leukemia originate from T cells. The morbidity and mortality of CTCL and acute T cell leukemia have increasing trend, which greatly threatening human health. Conventional radiotherapy or chemotherapy show poor anti-tumor and significant toxicity and side effects. Long-term use of nonspecific immunosuppressants obviously impair the immunity and results in many complications.Immunotoxins, also called biological missiles, comprise a targeting molecule (vehicle) and a cytotoxic component. They specificly kill target cells Immunotoxins have two major advantages:①. the killing effects of immunotoxins on tumor cells does not depend on the host’s immune system, but depend on toxins carried by targeting vectors;②. immunotoxins do not kill normal cells, but they can specifically kill target cells after enter the target tumor cells. Hence, immunotoxins are ideal agent for tumor therapy. DAB389IL-2, i.e, denileukin diftitox, was the first immunotoxin approved by FDA for the treatment of CTCL, and is now under phase III clinical study. However, there are some problems about DAB389IL-2. The major problems about immunotoxins include: high immunogenicity of heterologous antibodies which are used as targeting vectors in current immunotoxins, and poor tissue permeability due to large molecular weight of immunotoxin. To enhance the killing effect of immunotoxins on target cells, the following should be considered:①to seek specific targets on target cells and enhance the effect of immunotoxins through up regulating the expression of cell surface targets;②to reduce immunotoxin antigenicity by modifying vectors and toxins, reduce their molecular weight or select human-derived vectors and toxin molecules. Accordingly, To miniaturize immunotoxins and choses human-derived immunotoxins are the two major solutions in the field of constructing immunotoxins.T cell associated tumors and a small number of B cell associated tumors highly express CD25. CD25 (αchain of IL-2 receptor) and IL-2 receptorβandγchains constitute high-affinity IL-2 receptor. In theory, low concentrations of human IL-2 fusion protein can selectively act on these lymphoma cells. Hence, IL-2 R can be an ideal therapeutic target on these tumor cells, which mediates T lymphoma cell suppression or killing. Human IL-2 is characterized by good targeting performance, low immunogenicity, long half-life time, and applicability in animals.Plant-derived ribosomes inactivate proteins (RIPs) are a kind of toxins with strong toxicity, and they directly act on ribosomes to inactivate 60S subunit, thus strongly suppressing protein synthesis activity and inducing apoptosis. The Luffin family consists of RIPs extracted from the seeds of Luffa cylindrica. Luffin P1, a recently discovered member of the Luffin family, is the smallest RIP yet known (molecular weight, 5.2kD), and it exhibits high activity on suppressing protein synthesis. Due to its low molecular weight, Luffin P1 can enter target cells easily, and help to reduce immunogenicity and side effect of immunotoxins, as well as increases therapeutic effect.Retinoic acid (RA) regulates epithelial cell differentiation and growth, maintains normal keratinization of epithelial tissue, and prevents tumorigenesis; hence, retinoic acid has been used to induce the differentiation of various tumors. In addition, retinoic acid upregulates the expression of IL-2 Rα,βchains, so it can regulate the expression of IL-2 receptor on target cell surface and promote the action of immunotoxins. In preliminary clinical studies, the combination of retinoic acid and immunotoxin increases the remission rate of CTCL, and does not increase toxicity and side effects.Accordingly, we constructed a new immunotoxin hIL-2-Luffin P1 by gene engineering, so as to (1) allow human IL-2 to specifically recognize IL-2 receptors on tumor cells and toxin molecules to enter target cells to exert their anti-tumor effect; (2) upregulate IL-2 receptor expression by retinoic acid to increase the targeting performance of the immunotoxin and enhance the killing effect of hIL-2-Luffin P1 on target cells.Objectives: To construct, express and purify hIL-2-Luffin P1 immunotoxin and Luffin P1 protein and observe the effects of hIL2-Luffin P1 and hIL2-Luffin P1 plus arotinoid ethylester on the proliferation and apoptosis of Hut-78 cells and Jurkat cells. Methods: (1) Target gene fragments (hIL2-Luffin P1, Luffin P1) were amplified by PCR and cloned into the expression vector pET32a (+). The recombinant plasmids were subjected to identification by enzymatic digestion and gene sequencing. The correctly constructed recombinant plasmids were used to transform BL21 bacteria, followed by induced expression. Western blotting was carried out to identify the expressed protein using mouse anti-human His and rabbit anti-human IL-2 polyclonal antibodies. The expressed protein was subjected to purification, renaturation, desalting, digestion with enterokinase, and re-purification, and Western blotting analysis of hIL2-Luffin P1 protein was performed using rabbit anti-human IL-2 polyclonal antibody. (2) CD25 expression on Hut-78 cells and Jurkat cells was analyzed immunocytochemically. Hut-78 cells and Jurkat cells were treated with hIL-2-Luffin P1, arotinoid ethylester, hIL-2-Luffin P1 plus arotinoid ethyl ester and Luffin P1 at different concentrations for different time periods. The cell growth inhibition rate was determined by MTT assay. Early apoptosis and cell cycle distribution were analyzed by flow cytometry.Results: (1) Prokaryotic expression plasmids PET32a(+)-hIL-2-Luffin P1 and PET32a(+)-Luffin P1 were constructed successfully. (2) hIL-2-Luffin P1 protein was mainly detected in inclusion bodies, and Luffin P1 protein was expressed in partial soluble form. After protein purification, digestion with enterokinase and identification, highly active hIL-2-Luffin P1 and Luffin P1 proteins were obtained. (3) Immunocytochemical analysis demonstrated high expression of CD25 on Hut-78 cells and Jurkat cells, with an expression rate of 79.32% and 54.47%, respectively. (4) MTT assay demonstrated that hIL-2-Luffin P1 protein, retinoic acid, or both suppressed the proliferation of Hut-78 cells and Jurkat cells in a dose and time-dependent manner. The suppression effect of hIL-2-Luffin P1 protein and arotinoid ethylester was significantly higher than that of hIL-2-Luffin P1 protein or arotinoid ethylester, and Luffin P1 protein had no obvious inhibitory effect on the proliferation of the two cell types. After treatment with hIL-2-Luffin P1 or hIL-2-Luffin P1 and arotinoid ethyl ester, the inhibition rate of Hut-78 cells were significantly higher than that of Jurkat cells. IC50 of hIL-2-Luffin P1 for Hut-78 cells and Jurkat cells were 27.361μg/ml and 39.634μg/ml, respectively. arotinoid ethyl ester significantly and similarly suppressed Hut-78 cells and Jurkat cells. (5) Flow cytometry demonstrated that hIL-2-Luffin P1, arotinoid ethyl ester or hIL-2-Luffin P1 and retinoic acid induced early apoptosis of Hut-78 cells and Jurkat cells. The apoptosis rate was significantly higher in cells treated with hIL-2-Luffin P1 and arotinoid ethyl ester than in those treated with arotinoid ethyl ester or hIL-2-Luffin P1. Luffin P1 did not show effect of inducting apoptosis. (6) Flow cytometry demonstrate that after treatment with hIL-2-Luffin P1 or hIL-2-Luffin P1 and arotinoid ethyl ester, Hut-78 cells and Jurkat cells were retained at G1 phase, suggesting that both hIL-2 -Luffin P1 and hIL-2-Luffin P1 plus arotinoid ethylester prevent cell cycle transition from G1 phase to S phase in both cell types.Conclusions: Recombinant hIL-2-Luffin P1 immunotoxin and Luffin P1 protein were successful constructed and expressed by gene engineering. hIL-2-Luffin P1 suppresses the proliferation of Hut-78 cells and Jurkat cells, promotes apoptosis of these two cell types in a dose-and time-dependent manner, and makes these cells arrested at G1 phase. In contrast, Luffin P1 protein does not influence the proliferation, apoptosis and cell cycle of these two cells types. It is suggested that hIL-2-Luffin P1 protein shows specific targeting killing effect on IL-2R expressing Hut-78 cells and Jurkat cells. The effect of hIL-2-Luffin P1 plus arotinoid ethylester is similar with that of hIL-2-Luffin P1 alone, but is stronger than that of hIL-2-Luffin P1 or arotinoid ethyl ester, suggesting the synergism of hIL-2-Luffin P1 and arotinoid ethylester in influencing Hut-78 cells and Jurkat cells. This study may be helpful for treating CTCL and acute T cell leukemia.

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