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Optimizing CAR structure to improve safety and efficiency of CAR-T cell therapy

Chinese Journal of Cancer Biotherapy.2018;25(12):1209-1217. doi:10.3872/j.issn.1007-385x.2018.12.001

CAR-T cell therapy has developed rapidly in recent years, and has achieved amazing results in the treatment of some malignant tumors of the blood system, but little progress has been made in the treatment of solid tumors. At present, the main problems to be solved in CAR-T cell therapy are: (1) enhancing the killing activity of CAR-T cells; (2) relieving the immunosuppressive state of tumors; (3) bringing CAR-T cells into solid tumors; (4) enhancing the safety of CAR-T cell therapy. By optimizing the structure of CAR, a series of defects in the CAR-T cell therapy can be overcome, and the curative effect of CAR-T can be enhanced and the complications can be alleviated. In this paper, some optimization and improvement measures and methods on the structure design of CAR in recent years are elaborated, and the effectiveness and safety of the CAR-T cell therapy are explored.

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Development and evaluation considerations of viral vector production for CAR-T cell products

XU Longchang ; WEI Wei ; LUO Jianhui

Chinese Journal of Cancer Biotherapy.2018;25(12):1218-1222. doi:10.3872/j.issn.1007-385x.2018.12.002

Due to the spectacular therapy results in hematologic tumors, chimeric antigen receptor T (CAR-T) cell therapy has been the research hot-spot in the field of cell-immunotherapy. Viral vectors, as the critical raw material in CAR-T cell manufacturing, are closely related to the safety, efficacy and quality control of CAR-T cell products, in the aspects of the structure design of CAR gene, refinement of production process, quality control and setting of characterization and specification etc. Based on the research progress in lentiviral and γ-retroviral vectors development and the evaluation experiences, this paper discusses some common problems that need to be focused on in the preparation of viral vectors, expecting to provide references for the development and the authorization applications of domestic related products in the future.

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Targeted antitumor effects of C-PC/CMC-CD55sp nanospheres on cervical carcinoma Caski cells

LIU Huihui ; JI Huanhuan ; ZHU Feng ; JIANG Liangqian ; LIU Guoxiang ; LI Bing

Chinese Journal of Cancer Biotherapy.2018;25(12):1223-1229. doi:10.3872/j.issn.1007-385x.2018.12.003

Objective: To prepare a new type of phycocyanin/carboxymethyl chitosan-CD55 ligand peptide (CPC/CMC-CD55sp) nanospheres, and to study its targeted therapeutic effect on cervical cancer Caski cells. Methods: The novel CPC/CMC-CD55sp nanospheres (CPC/CMC-CD55sp) were synthesized by ionic cross-linking method, and the properties of nanospheres were observed by transmission electron microscopy (DLS) and fourier transform infrared spectroscopy (FTIR). The expression of CD55 on the surface of Caski and fibroblast (L-929) cells was detected by Western blotting and flow cytometry. The effect of nanospheres on the proliferation of Caski cells was detected by CCK-8. Flow cytometry and fluorescence microscopy were used to detect the uptake of microspheres by Caski cells; Western blotting and flow cytometry were used to detect the effect of CPC/CMC-CD55sp on expressions of apoptosis-related proteins and apoptosis rate in Caski cells; the hemolysis test was used to determine the biological safety of the drug. Results: CPC/ CMC-CD55sp was successfully prepared with good morphology and uniform diameter; and CD55 was highly expressed on the surface of Caski cells but low expressed on the surface of L-929 cells (P<0.01). CPC/CMC-CD55sp could targeted and efficiently reach Caski cells and be ingested into the cells. It exhibited weak hemolysis effect on human peripheral blood, which was in the safe range. CPC/ CMC-CD55sp displayed obvious inhibitory effect on Caski cell proliferation, and could induce cell apoptosis (P<0.05 or P<0.01). Conclusion: The new CPC/CMC-CD55sp can targeted inhibit the growth of cervical cancer Caski cells via inducing its apoptosis and has good bio-safety, which provides a new idea for the research and development of anti-tumor marine drugs.

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Kaempferol inhibits invasion and migration of non-small cell lung cancer A549 cells by down-regulating ERRα expression

ZHANG Jing ; SHI Xiaoyu ; MENG Wei ; MA Feng ; ZHAO Lixia ; ZHAO Junfeng

Chinese Journal of Cancer Biotherapy.2018;25(12):1230-1236. doi:10.3872/j.issn.1007-385x.2018.12.004

Objective: To study the effects of kaempferol on invasion and migration of non-small cell lung cancer (NSCLC)A549 cells and the related mechanisms. Methods: CCK-8 was used to detect the effect of different concentrations of kaempferol on the proliferation of A549 cells. Transwell assay and wound healing assay were used to detect the ability of cell invasion and migration. The expressions of EMT-related proteins (N-cadherin, Snail-2 and E-cadherin) were detected by Western blotting. The effect of kaempferol on the mRNA and protein expressions of estrogen related receptor alpha (ERRα) was determined by qRT-PCR. ERRα over-expression vector (pLV-ERRα) was transfected intoA549 cells, and the cell invasion, migration and the expression of EMT related proteins were detected by Transwell assay, wound healing assay and Western blotting, respectively. Results: Kaempferol dose-dependently inhibited the proliferation of A549 cells, and kaempferol at concentrations of 5, 10 and 20 μmol/L was used in the following experiments. After the treatment with different concentrations of kaempferol, the number of invasive cells and wound closure rate, the expression of N-cadherin and Snail-2, and the mRNAand protein levels of ERRα decreased significantly, while the expression of E-cadherin increased significantly, (all P<0.01). After transfection with pLV-ERRα, the number of invasive cells, scratch closure rate and the expressions of N-cadherin and Snail-2 of A549 cells over-expressing ERRα significantly increased, while the expression of E-cadherin decreased significantly (all P<0.05 or P<0.01); and all these indices were attenuated after the treatment with kaempferol (all P<0.05 or P<0.01). Conclusion: Kaempferol inhibits the invasion and migration as well as EMT of non-small cell lung cancer A549 cells by down-regulating ERRα, which may provide experimental basis for the clinical treatment of lung cancer.

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Effect of miR-141-3p targeting TGF-β2 on malignant biological behaviors of human prostatic cancer C4-2B cells

WANG Yu ; QIU Mingxing ; XIONG Guobing

Chinese Journal of Cancer Biotherapy.2018;25(12):1237-1243. doi:10.3872/j.issn.1007-385x.2018.12.005

Objective: To investigate the relationship between miR-141-3p and transforming growth factorβ2 (TGF-β2), and its effects on the malignant biological behaviors of human prostate cancer cell line C4-2B. Methods:After the transfection of miR-141-3p mimic, the mRNAexpression of miR-141-3p and TGF-β2 in C4-2B cells was detected by qRT-PCR. Bioinformatics method validated the relationship between miR-141-3p and TGF-β2. miR-141-3p mimic alone or with TGF-β2 over-expression vector was transfected into C42B cells, and then Western blotting was used to detect the expression of TGF-β2 protein in C4-2B cells, Hochest33258 staining was used to detect cell apoptosis, and Transwell assay was used to detect the invasion ability of cells in each group. Results:After the transfection of C4-2B cells with miR-141-3p mimic, the level of miR-141-3p increased significantly, and the level of TGF-β2 mRNA decreased significantly (all P<0.01). The activity of luciferase was significantly reduced after the co-transfection with miR-141-3p mimic and wild type report plasmid (P<0.01); However, the activity of luciferase was not obviously changed after co-transfection with miR141-3p mimic and mutant type report plasmid (P>0.05).After co-transfection with miR-141-3p mimic and pc-TGF-β2, the proliferation of C4-2B cells decreased significantly, the number of apoptotic cells increased significantly, and the cell invasion ability decreased significantly (all P<0.01). Conclusion: miR-141-3p inhibits the proliferation and invasion of human prostate cancer C4-2B cells and induces cell apoptosis by targeting TGF-β2.

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Tanshinone IIA inhibits apoptosis, invasion and migration of esophageal cancer EC9706 and KYSE70 cells by regulating epithelial-mesenchymal transition

ZHOU Pengfei ; LI Juan

Chinese Journal of Cancer Biotherapy.2018;25(12):1244-1250. doi:10.3872/j.issn.1007-385x.2018.12.006

Objective: To investigate the effect of tanshinone IIA on the invasion and migration of esophageal cancer EC9706 and KYSE70 cells, and to explore the underlying mechanism. Methods: Esophageal cancer cells (EC9706 and KYSE70) were divided into 4 groups: control group, tanshinone IIA groups (2, 4, 6 μg/ml). Cell prliferation viability was measured by CCK-8; Apoptosis was detected by flow cytometry; Invasion was tested by Transwell assay; And migration was measured by Scratch assay. The mRNA and protein levels of E-cadherin, Snail-2, Vimentin and N-cadherin were tested by quantitative Real-time reverse transcription PCR (qRT-PCR) and Western blotting, respectively. Results: Tanshinone IIAat concentrations less than 6 μg/ml did not affect the cell viability of esophageal cancer EC9706 and KYSE70 cells. The apoptosis in tanshinone IIA (4, 6 μg/ml) groups was significantly higher than that in control group ( P < 0.01). The number of invasive cells per field and wound-healing rate in tanshinone IIA(2, 4, 6 μg/ml) groups were significantly lower than those in control group (all P <0.01). Moreover, the cell morphology was transformed from a spindle-shaped mesenchymal form into epithelial morphology after tanshinone IIA treatment. Compared with control group, the expression of E-cadherin in tanshinone IIA groups (2, 4, 6 μg/ml) was significantly up-regulated while the expressions of Snail-2, Vimentin and N-cadherin were significantly down-regulated (all P <0.01). Conclusion: Tanshinone IIApromotes apoptosis and attenuates the invasion and migration of esophageal cancer cells by inhibiting the epithelial-mesenchymal transition.

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miR-429 promotes capecitabine-resistance in pancreatic cancer PANC-1 cells by down-regulating PTEN and activating PI3K/AKT signaling pathway

HE Ping ; WANG Ping ; XIONG Longxin

Chinese Journal of Cancer Biotherapy.2018;25(12):1251-1258. doi:10.3872/j.issn.1007-385x.2018.12.007

Objective: To explore the mechanism of miR-429 targeting PTEN to affect capecitabine-resistance in pancreatic cancer PANC-1 cells though the PI3K/AKT signaling pathway. Methods: Capecitabine-resistant pancreatic cancer cell line PANC-1/CAP was constructed, and the expression of miR-429 and PTEN were detected by quantitative Real-time polymerase chain reaction (qRT-PCR) and Western blotting. The effect of miR-429 knock-down on cell proliferation viability, apoptosis and capecitabine-resistance was measured by colony formation assay, CCK-8 assay andAnnexin V-FITC/PI double staining flow cytometry assay, respectively. Subsequently, dual luciferase reporter assay verified that PTEN was a target gene of miR-429. Furthermore, the effect of miR-429 on PTEN-PI3K/ AKT signaling pathway was measured by Western blotting. Results: miR-429 was found to be up-regulated in PANC-1 cells and PANC-1/CAP cells compared with the non-malignant pancreatic ductal cell line (HPDE6-C7) (P<0.05 or P<0.01). Moreover, silencing of miR-429 significantly decreased cell proliferation viability, capecitabine-resistance and enhanced apoptosis of PANC-1/CAP cells; additionally, dual luciferase reporter assay confirmed that PTEN was a target of miR-429 (P<0.05 or P<0.01). Suppression of miR-429 up-regulated PTEN and blocked the PI3K/AKT signaling pathway to decrease cell proliferation viability and further reduce the capecitabine-resistance of PANC-1/CAP cells (P<0.05 or P<0.01). Conclusion: miR-429/PTEN-PI3K/AKT signaling pathway plays a certain role in regulating the capecitabine-resistance of pancreatic cancer, and inhibition of miR-429 expression may reverse the resistance of PANC-1/CAPto capecitabine.

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Inhibitory effect of recombinant oncolytic adenovirus on luciferase-labeled and non-labeled human lung cancerA549 cells

MA Yizhen ; FAN Yuanyuan ; NIE Xi ; SUN Lili ; ZHU Yilong ; LI Yiquan ; LI Wenjie ; YIN Xunzhe ; LI Shanzhi ; ZHAO Jin ; LI Xiao ; GUO Yan ; JIN Ningyi

Chinese Journal of Cancer Biotherapy.2018;25(12):1264-1269. doi:10.3872/j.issn.1007-385x.2018.12.009

Objective: To investigate the inhibitory effect of recombinant oncolytic adenovirusAd-Apoptin-hTERTp-E1A(ATV) on luciferase-labeled human lung cancer cells (A549-luc) and human lung cancerA549 cells, and to compare the differences in the inhibitory effect on two cell lines. Methods:ATV was used to infectA549-luc cells andA549 cells respectively. WST-1 and crystal violet staining were used to determine the difference in the inhibitory effect of ATV. Hoechst and Annexin V-FITC/PI staining were used to verify the inhibition mode ofATV. Results: WST-1 and crystal violet staining showed thatATV had significant inhibitory effect on bothA549-luc and A549 cells ( P <0.05). ATV showed significant inhibitory effect on both cells at 24, 48 and 72 h ( P <0.05 or P <0.01), and reached the peak at 72 h; ATV at concentrations of 1, 10 and 100 MOI all showed inhibitory effect on both cells, and reached the peak at 100 MOI. Hoechst staining showed that A549-luc cells and A549 cells infected with ATV showed typical nuclear fragmentation and marginal set. The results of Annexin V-FITC/PI Flow cytometry showed that ATV infection resulted in apoptosis of A549-luc and A549 cells, which was in a time-dependent manner and reached the peak at 72 h( P <0.05 or P <0.01). Conclusion: Insertion of luciferase didn’t significantly change the inhibitory effect and inhibitory mode ofATV onA549-luc cells.ATV exerted its in vitro inhibitory effect onA549-luc and A549 cells by inducing cell apoptosis.

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Over-expression of miR-488-5p decreases proliferation and migration ability of cervical cancer C33Acells through TEM8

ZHANG Pei ; YUE Qingfen ; HOU Qingxia ; HAN Jingfeng

Chinese Journal of Cancer Biotherapy.2018;25(12):1270-1275. doi:10.3872/j.issn.1007-385x.2018.12.010

Objective: To observe the expression of miR-488-5p in cervical cancer tissues and to explore its effect on the proliferation and migration of cervical cancer C33Acells. Methods: 12 pairs of cervical cancer tissues and corresponding paracancer tissues from patients, who underwent total hysterectomy at the Luoyang Central Hospital of Zhengzhou University from March 2017 to September 2017, were collected for this study; and the expression of miR-488-5p was detected by fluorescence quantitative and real-time polymerase chain reaction (qRT-PCR). Lipofectamine 3000 was used to transfect miR-488-5p (experiment group) and miR-NC (control group) into cervical cancer C33Acells. Cell cycle distribution was detected by Flow cytometry. Cell proliferation was assessed by CCK8 assay and Transwell assay was used to detect cell migration. Bioinformatics software was used to predict the possible target genes of miR-488-5p, and luciferase activity assay was used to verify the binding of miR-488-5p to target genes. The expressions of tumor endothelial marker 8 (TEM8) and downstream EGFR signaling pathway related proteins in two groups were detected by qRT-PCR and Western blotting. Results: The relative expression level of miR-488-5p in cervical cancer tissues (1.33±0.20) was significantly lower than that in paracancer tissues (3.68±0.45) (P<0.01). The relative expression level of miR-488-5p in the experimental group (25.23±3.11) was significantly higher than that in the control group (1.02±0.10) (P<0.01). The percentage of C33A cells at G0/G1 phase in experimental group (53.39±2.48)% was significantly higher than that in control group (39.57±1.21)% (P<0.01). When the culture time extended to 96 h and 120 h, the proliferation ability of C33Acells in experimental group was significantly lower than that in control group (P<0.05), and the number of migrated cells in the experimental group (117.90±18.86) was significantly less than that in the control group (295.10±19.33) (P <0.01). Luciferase activity assay confirmed that miR-488-5p could directly bind with TEM8 and inhibit its expression (P<0.01). The relative expression of TEM8 mRNAin experimental group (0.42±0.06) was significantly lower than that in control group (1.00 ± 0.06) (P<0.01).After transfection with miR-488-5p for 48h, the protein expressions of TEM8, p-EGFR, p-ERK and pAKT were significantly lower than those in control group (P <0.01). Conclusion: The expression of miR-488-5p in cervical cancer tissues was decreased. Over-expression of miR-488-5p could inhibit the cell cycle progression of cervical cancer cells and reduce the proliferation and migration of cervical cancer cells. The mechanism may be related to the interference of TEM8 gene expression.

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Expression of HOPX in cervical cancer tissues and blood serum and its correlation to CEAand CA125

HUANG Lifeng ; LIU Jingli ; WANG Baishi ; ZHANG Lixia ; YANG Zhen

Chinese Journal of Cancer Biotherapy.2018;25(12):1276-1281. doi:10.3872/j.issn.1007-385x.2018.12.011

Objective: To investigate the expression of HOPX gene in cervical cancer tissues and blood serum as well as its effect on cervical cancer HeLa cells, and to analyze its correlation to tumor maker CEAand CA125. Methods: 50 pairs of cervical cancer tissues and para-cancerous tissues as well as the peripheral blood samples from patients with cervical cancer, who were treated at Tianjin Binhai People’s Hospital and Tianjin Wuqing People’s Hospital from June 2015 to December 2017, were collected for this study; in addition, 50 samples of blood serum from healthy people were used as control. Real-time quantitative PCR (qRT-PCR) and immumohistochemical staining (IHC) were used to detect mRNA and protein expressions of HOPX in tissue and serum samples, NCBI-GEO data base and TCGA data base were used to collect the information on HOPX gene and patients’prognosis, and the correlation between HOPX expression and patients’prognosis was analyzed. Vectors over-expressing HOPX or control vectors were transfected into HeLa cells; MTT assay and colony formation assay were used to examine the proliferation ability of HeLa cells, Tranwell assay was used to detect the migration and invasion of HeLa cells, and Western blotting was used to detect the expression of EMT-related proteins. Results: Both sample examination and data base information showed that the expression level of HOPX was down-regulated in tissue and serum samples of cervical cancer patients and was positively related with the survival of patients (r=0.736， P<0.05); while it’s expression was negatively related to the level of CEAand CA125 in cervical cancer tissues and serum (r=-0.678, P<0.05). HOPX over-expression inhibited cell proliferation, migration and invasion, promoted the expression of E-cadherin but inhibited the expression of Vimentin and ICAM1 (all P<0.05 or P<0.001). Conclusion: HOPX is low expressed in cervical cancer tissues and blood samples, and negatively correlated with CEA and CA125, but positively correlated with the survival of patients. Thus, combination of HOPX and CEA/CA125 may improve the early diagnosis rate of cervical cancer and provide a new strategy for precision treatment of cervical cancer in future.

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