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Xikun Zhou,
Jing Li,
Zhen Wang,
Zhongwen Chen, Ji Qiu,
Yinbing Zhang,
Wei Wang,
Yu Ma,
Nongyu Huang,
Kaijun Cui,
Jiong Li,
Yu-Quan Wei
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ABSTRACT: Epidermal growth factor receptor (EGFR) is overexpressed in a variety of human malignancies, including pancreatic cancer, breast cancer, colon cancer, and non-small cell lung cancer. Overexpression of EGFR is a predictive marker of therapeutic response and several lines of evidence suggest that EGFR is an excellent target for tumor therapy. However, the effective antitumor capacity of EGFR-specific T cells against EGFR-overexpressing tumor cells has not been fully elucidated. In our previous study, we identified an anti-EGFR single-chain variable fragment (scFv) with specific and high affinity after screening by ribosome display. In this study, the anticancer potential of anti-EGFR scFv was investigated on the basis of cell-targeted therapy. A chimeric antigen receptor (CAR) targeting EGFR was constructed and expressed on the cell membrane of T lymphocytes. These CAR-modified T cells demonstrated antitumor efficacy both in vitro and in vivo. In addition, the safety evaluation showed that CAR-modified lymphocytes have no or very minimal acute systemic toxicity. Taken together, our study provided the experimental basis for clinical application of genetically engineered lymphocytes; moreover, we also evaluate a new and interesting cell therapy protocol.
Neoplasia (New York, N.Y.) 05/2013; 15(5):544-53. · 5.48 Impact Factor
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Xikun Zhou, Ji Qiu,
Zhen Wang,
Nongyu Huang,
Xiaolei Li,
Qian Li,
Yinbing Zhang,
Chengjian Zhao,
Can Luo,
Nannan Zhang,
Xiu Teng,
Zhongwen Chen,
Xiao Liu,
Xianlian Yu,
Wenling Wu,
Yu-quan Wei,
Jiong Li
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ABSTRACT: Epidermal growth factor receptor (EGFR) plays an important role in the growth and metastasis of many solid tumors. Strategies that target EGFR hold promising therapeutic potential for the treatment for non-small cell lung cancer (NSCLC), as EGFR is normally overexpressed in these tumors. This study was designed to determine whether an anti-EGFR immunotoxin has anti-tumor activity against NSCLC, and if so, to further investigate the possible mechanisms of cytotoxicity.
A fusion protein of anti-EGFR single-chain variable fragment (anti-EGFR scFv) and the plant toxin gelonin (rGel) was constructed, expressed in bacteria, and purified to homogeneity. Cytotoxicity of anti-EGFR scFv/rGel (E/rG) immunotoxin was assessed on A549, HCC827, and H1975 cells (EGFR-overexpressing NSCLC-derived cell lines) and A549 xenografts in nude mice.
Cytotoxicity experiments using E/rG on A549, HCC827, and H1975 cells demonstrated that E/rG can specifically inhibit proliferation of these cells, whereas it did not affect the proliferation of Raji cells that do not express EGFR. Treatment for A549 xenografts in nude mice with E/rG resulted in significant suppression of tumor growth compared to controls. Immunofluorescence in frozen tissue sections confirmed that E/rG could specifically bind to tumor tissues in nude mice bearing A549 tumor xenografts, while rGel alone showed no binding activity. Furthermore, E/rG inhibited the growth of A549 cells by cytotoxic effects that blocked tumor proliferation, and the immunotoxin-induced cell death may be mediated by autophagy.
These results showed that E/rG might have significant potential as a novel clinical therapeutic agent against human NSCLC.
Journal of Cancer Research and Clinical Oncology 03/2012; 138(7):1081-90. · 2.56 Impact Factor
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ABSTRACT: To investigate the therapeutic effect of the plasmid pcDNA3.1-IL15 complexed with cationic liposome (CL-IL15) in the B16-F10 melanoma lung metastasis model.
A plasmid with high secretive efficiency of IL-15 was constructed and the optimum mix ratio was determined to formulate cationic liposome-plasmid complex with the optimal encapsulation. The CHO-K1 cell line was transfected by CL-IL15. The secretion of transfected IL-15 gene was detected by Western blot and its biological function was measured through the proliferation response of CTLL-2 cytotoxic T cell line of murine by MTT assay. The C57BL/6 mice were inoculated intravenously (i.v.) with B16-F10 melanoma lung metastasis cells then treated (i.v.) by CL-IL15 in a therapeutic setting to derermine the tumorigenesis and research the corresponding mechanisms.
The pcDNA3.1-IL15 plasmid was successfully constructed and the mass-ratio of optimal condition of cationic liposome-plasmid with perfect entrapment was 1:5 (plasmid: cationic liposome). Western blot analysis displayed the detection of IL-15 both in the medium and the pcDNA3.1-IL15 transfected cells. MTT assay showed that CTLL-2 cells could proliferate with the medium obtained from CHO-K1 cells transfected by CL-IL15. And the administration of CL-IL15 complexes led to the significant inhibition lung metastasis of malignant melanoma (P<0.05).
CL-IL15 could inhibit the metastasis of malignant melanoma and the cationic liposome delivered plasmid pcDNA3.1-IL-15 complexes may be an efficient therapeutic strategy for the treating of lung metastasis. And the effective splenic cell-mediated cytotoxicity and the obvious NK cells recruitment may be involved.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 02/2012; 28(2):148-52.
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Nan N Zhang,
Nong Y Huang,
Xi K Zhou,
Xiao L Luo,
Chang Y Liu,
Yan Zhang, Ji Qiu,
Yin B Zhang,
Xiu Teng,
Can Luo,
Xian C Chen,
Bing Kan,
Yong Q Mao,
Ai P Tong,
Yu Q Wei,
Jiong Li
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ABSTRACT: Mice injected with Bacillus Calmette-Guérin (BCG) were challenged with lipopolysaccharide (LPS) to induce inflammatory liver injury. This study was performed to explore the protective effects of interleukin (IL)-4 against liver injury induced by BCG and LPS in mice.
Mice injected with BCG (125 mg/kg) were challenged with LPS (10 μg/kg) to induce the model of inflammatory liver injury. Half an hour after injection of LPS, mice were subcutaneously administered rmIL-4 at 5 and 0.5 μg/kg, respectively. Liver injury was evaluated by serum transaminase assay and H & E staining. Liver cytokine concentrations were determined by enzyme-linked immunosorbent assay, and intrahepatic cytokine and iNOS mRNA levels by reverse transcriptase polymerase chain reaction. Intrahepatic apoptosis was evaluated by terminal deoxynucleotidyl transferase mediated nick end labeling. NF-κB p65 and ERK signal pathway was detected by Western-blotting. NF-κB signal pathway was also detected by electrophoretic mobility shift assay.
IL-4 reduced the serum ALT, AST and LDH, alleviated the inflammatory cells infiltration, down regulated the expression of TNF-α, IL-1β, IFN-γ, IL-6 and iNOS mRNA in liver, and alleviated hepatic glutathione depletion (GSH). In addition, IL-4 displayed inhibition of extracellular signal-regulated kinase phosphorylation and NF-κB activation.
IL-4 may protect mice against BCG/LPS-induced immune liver injury, besides ERK and NF-κB signal pathways were involved in the effects.
Agents and Actions 09/2011; 61(1):17-26. · 1.59 Impact Factor
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ABSTRACT: To construct a prokaryotic expressing plasmid for recombinant immunotoxin which fused anti-EGFR scFv together with gelonin toxin, express and verify its function.
The gene fragments coding anti-EGFR single chain fragment were amplified with PCR and cloned into pET32a vector which contains gelonin toxin. The new plasmid was transformed into BL21 (DE3) cells. The induced inclusion bodies were denatured, refolded and purified through SP Sepharose Fast Flow Column. The purified immunotoxin rEG was identified by western blot analysis, and the bioactivity was identified using cell immnuohistochemistry and MTT assay.
The expressing vector pET32a-rEG has been constructed correctly, confirmed by restriction endonuclease digestion and sequencing. The recombinant immunotoxin rEG was purified after denaturing the inclusion bodies, refolding and cationic exchange chromatograph. The purified protein rEG had the right immunology specificity, rEG could efficiently target to EGFR positive cells identified by cell immnuohistochemistry. And the result of MTT assay showed rEG could specifically kill EGFR positive cells.
The recombinant immunotoxin rEG with high purity and biologic activity was prepared in this study, which would become the basic for the further study of the biologic function of rEG.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 09/2011; 42(5):621-4.
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Xikun Zhou,
Xiaolei Li,
Maling Gou, Ji Qiu,
Jing Li,
Chuanjiang Yu,
Yinbin Zhang,
Nannan Zhang,
Xiu Teng,
Zhongwen Chen,
Can Luo,
Zhen Wang,
Xiao Liu,
Guobo Shen,
Li Yang,
Zhiyong Qian,
Yuquan Wei,
Jiong Li
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ABSTRACT: Gene therapy shows promising application in cancer therapy, but the lack of an ideal gene delivery system is still a tough challenge for cancer gene therapy. Previously, we prepared a novel cationic nanogel, heparin-polyethylenimine (HPEI), which had potential application in gene delivery. In the present study, we constructed a plasmid with high expression efficiency of interleukin-15 (IL15) and investigated the effects HPEI-plasmid IL15 (HPEI-pIL15) complexes on the distribution level of the lung. We then evaluated the anticancer effect of HPEI-pIL15 complexes on lung metastases of B16-F10 melanoma and CT26 colon carcinoma. These results demonstrated that intravenous injection of the HPEI-pIL15 complex exhibited the highest plasmid distribution level in the lung compared with that of PEI2K-pIL15 and PEI25K-pIL15, and mice treated with HPEI-pIL15 had a lower tumor metastasis index compared with other treatment groups. Moreover, the number of natural killer cells, which were intermingled among the tumor cells, and the level of tumor necrosis factor-α and interferon-γ in the serum also increased in the pIL15-treated mice. Furthermore, the cytotoxic activity of spleen cells also increased significantly in the HPEI-pIL15 group. In addition, induction of apoptosis and inhibition of cell proliferation in lung tumor foci in the HPEI-pIL15 group was observed. Taken together, treating lung metastasis cancer with the HPEI nanogels delivered by plasmid IL15 might be a new and interesting cancer gene therapy protocol.
Cancer Science 07/2011; 102(7):1403-9. · 3.33 Impact Factor
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ABSTRACT: Chemo-resistance to cisplatin-centered cancer therapy is a major obstacle to the effective treatment of human ovarian cancer. Previous reports indicated that arsenic trioxide (ATO) induces cell apoptosis in both drug-sensitive and -resistant ovarian cancer cells.
In this study, we determined the molecular mechanism of ATO-induced apoptosis in ovarian cancer cells. Our data demonstrated that ATO induced cell apoptosis by decreasing levels of phosphorylated AKT (p-AKT) and activating caspase-3 and caspase-9. Importantly, BIM played a critical role in ATO-induced apoptosis. The inhibition of BIM expression prevented AKT dephosphorylation and inhibited caspase-3 activation during cell apoptosis. However, surprisingly, gene silencing of AKT or FOXO3A had little effect on BIM expression and phosphorylation. Moreover, the activation of caspase-3 by ATO treatment improved AKT dephosphorylation, not only by cleaving the regulatory A subunit of protein phosphatase 2A (PP2A), but also by increasing its activation. Furthermore, our data indicated that the c-Jun N-terminal kinases (JNK) pathway is involved in the regulation of BIM expression.
We demonstrated the roles of BIM in ATO-induced apoptosis and the molecular mechanisms of BIM expression regulated by ATO during ovarian cancer cell apoptosis. Our findings suggest that BIM plays an important role in regulating p-AKT by activating caspase-3 and that BIM mediates the level of AKT phosphorylation to determine the threshold for overcoming cisplatin resistance in ovarian cancer cells.
PLoS ONE 01/2011; 6(5):e20586. · 4.09 Impact Factor
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Yan Zhang,
Jiong Li,
Chang-Yong Liu,
Xi-Kun Zhou, Ji Qiu,
Yin-Bing Zhang,
Nong-Yu Huang,
You Li,
Xiang-Jun Chen,
Xiao-Lei Li,
Yong-Sheng Wang,
Han-Suo Yang,
Xian-Cheng Chen,
Bing Kan,
Yong-Qiu Mao,
Hong-Xin Deng,
Li Yang,
Yan-Jun Wen,
Xia Zhao,
Yu-Quan Wei
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ABSTRACT: Psoriasis is a chronic and relapsing inflammatory skin disease associated with various immunologic abnormalities. Repeated subcutaneous injection of interleukin-4 (IL-4) has been established as an effective treatment to counteract psoriasis.
We investigated whether gene therapy using IL-4 expression plasmid (pIL-4) via transdermal delivery was an alternative treatment for psoriasis. In our experiment, dimethylsulfoxide (DMSO) was used as a penetration enhancer.
At first, the penetration efficiency of the complex of reporter plasmid accompanied by DMSO was investigated both in vitro and in vivo. Then, the antipsoriasis efficiency of the treatment with pIL-4-DMSO was tested in mice.
The expression of the reporter gene was detected in epidermis and dermis both in vitro and in vivo. More importantly, the psoriasis symptoms were relieved, and significant reductions in some psoriasis-associated factors were observed after pIL-4-DMSO treatment.
We conclude that the topical application of pIL-4-DMSO can treat psoriasis to a significant extent.
Dermatology 08/2010; 221(1):84-92. · 2.05 Impact Factor
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ABSTRACT: To construct Recombinant Mouse Interleukin 4 prokaryotic expressing plasmid, express it in E. coli strain BL21 (DE3), purify and identify the expressed cytokine.
The optimized mIL-4 cDNA fragment was cloned into the prokaryotic expressing vector pET-32a (+) to generate pET32/rmIL-4 and transformed into BL21 (DE3) cells. After induction, the expressed protein wasfound to be in the inclusion of E. coli cells. The induced product was purified through Q Sepharose Fast Flow Column and Gel Filtration Column under renaturing condition. The purified protein was identified by Western blot analysis, and the biologic activity was identified by the generation of mIL-4 dependence cell CTLL-2 and in vivo experiment of mouse psoriasis model.
The recombinant plasmid pET32/rmIL-4 has been constructed correctly. The inclusion body was washed with 3 mol/L guanidine hydrochloride and denaturized in 7 mol/L guanidine hydrochloride. Then, the denaturized protein was gradient dialysis in the condition of pH 9. 5. The protein we purified has the right immunology specificity and biologic activity.
The recombinant mouse interleukin-4 with high purity and biologic activity was prepared in this study,which will become the basis for the further study of the biologic activity of IL-4.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 07/2009; 40(4):579-83.
