The research advances of CAR-T cell therapy in solid tumor / 医学研究生学报

Chimeric antigen receptor T-cell immunotherapy (CAR-T) has gradually become a new therapeutic strategy for malignant tumor following more than 30 years of development. CAR-T therapy has achieved remarkable success in treating refractory hematological malignancies but less encouraging in solid tumors. There are many difficulties and challenges in the treatment of solid tumors such as low infiltration, immunosuppressive microenvironment, off-target effect. To overcome the limitation of CAR-T cells in the application of solid tumors, many new strategies were explored. Here, we will give a brief review on the recent progress about improvement approaches to improve the effectiveness of CAR-T cells in the treatment of solid tumors.

Advances in research on the function and disease of desmoglein 2 / 医学研究生学报

Desmoglein (DSG) is an important constituent of desmosome, a single transmembrane glycoprotein that binds to desmocollin (DSC), regulates intercellular adhesion and transmits intracellular and extracelluar signals. Studies have shown that DSG2, one subtype of the DSG, is abnormally expressed in tumor cells and has certain value for targeted treatment and prognosis. This paper reviews the structure, biological functions and related diseases of DSG2 protein.

Reconsidering CAR-T cell therapy for solid tumors / 医学研究生学报

Chimeric antigen receptor T cells (CAR-T) have achieved remarkable therapeutic effects in the treatment of malignant hematological tumors, but there are still some problems including poor efficacy and possible toxicity risks in the treatment of solid tumors. This paper analyzes the reasons for the poor efficacy of CAR-T cells in the treatment of solid tumors, including tumor heterogeneity, difficulty in homing of CAR-T cells, immunosuppressive microenvironment, off-target effect,T cell exhaustion and so on, and proposes the strategies and future trends in improving the efficacy of CAR-T in the treatment of solid tumors.

MetFab-DOX reduces doxorubicin-resistance of HepG2 / DOX human hepatocellular carcinoma cells / 医学研究生学报

Objective MetFab-DOX can inhibit the proliferation of hepatocellular carcinoma HepG2 cells, but few researches have been conducted on the effect of MetFab-DOX on doxorubicin-resistant HepG2 cells. This study aimed to constructed doxorubicin-resistant HepG2 cell lines and explored the effect of MetFab-DOX on their drug resistance.Methods Using high-dose intermittent induction, we constructed the doxorubicin-resistant hepatocellular carcinoma cell model HepG2/DOX and divided the cells into a blank control, a DOX (5 μg/mL), and an MetFab-DOX group (containing 5 μg/mL doxorubicin). After treatment, we detected the effects of MetFab-DOX on the proliferation, apoptosis, internalization and biological function of the HepG2/DOX cells by CCK8 assay, FCM, cell immunofluorescence, wound healing assay and Transwell invasion assay, respectively. We also established a tumor-bearing model in the nude mouse and examined the effects of MetFab-DOX on the volume and morphology of the tumor.Results The drug resistance index of the HepG2/DOX cells treated with DOX and MetFab-DOX was markedly reduced, with statistically significant difference between the HepG2 and HepG2/DOX cells (P<0.05). After 24 hours of treatment, the cell apoptosis rate was remarkably higher in the MetFab-DOX than in the DOX group (19.87% vs 8.09%, P<0.05), and so was it at 48 hours (41.27% vs 16.15%, P<0.01). The internalization in the cells showed no statistically significant difference between the MetFab-DOX and DOX groups at 30 minutes, while the fluorescence intensity of doxorubicin was markedly higher in the former than in the latter group at 60 and 120 minutes. The cell scratch healing rate was lower in the MetFab-DOX than in the DOX and blank control groups at 24 hours (14.46% vs 16.80% and 19.88%, P<0.05), but higher in the former than in the latter two groups at 48 hours (22.60% vs 36.96% and 56.43%, P<0.01). The number of the membrane-penetrating cells per visual field was significantly decreased in the MetFab-DOX and DOX groups as compared with that in the blank control (646.18 and 880.51 vs 1043.52, P<0.05), and even lower in the MetFab-DOX than in the DOX group (P<0.05). After 40 days of treatment, the tumor inhibition rate was remarkably higher in the MetFab-DOX than in the DOX group (64% vs 35.27%, P<0.05). In the blank control group, the transplanted tumor cells were irregularly arranged and proliferative tumors varied in volume and constituted a larger proportion. The proliferation of the cells was slightly reduced in the DOX group as compared with that in the control. In the MetFab-DOX group, the tumor cells showed a significant shrinkage and a decreased number.Conclusion MetFab-DOX can effectively reduce the doxorubicin-resistance of hepatocellular carcinoma, and the underlying mechanism may be associated with its abilities of increasing the accumulation in drug-induced cells and inducing cell apoptosis.

Inhibition of connective tissue growth factor overexpression decreases growth of hepatocellular carcinoma cells in vitro and in vivo / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>We have previously found that connective tissue growth factor (CTGF) is highly expressed in a rat model of liver cancer. Here, we examined expression of CTGF in human hepatocellular carcinoma (HCC) cells and its effect on cell growth.</p><p><b>METHODS</b>Real-time PCR was used to observe expression of CTGF in human HCC cell lines HepG2, SMMC-7721, MHCC-97H and LO2. siRNA for the CTGF gene was designed, synthesized and cloned into a Plk0.1-GFP-SP6 vector to construct a lentivirus-mediated shRNA/CTGF. CTGF mRNA and protein expression in HepG2 cells treated by CTGF-specific shRNA was evaluated by real-time PCR and Western blotting. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was utilized to evaluate the growth effect, and a colony formation assay was used for observing clonogenic growth. In vivo, tumor cell proliferation was evaluated in a nude mouse model of xenotransplantation. Statistical significance was determined by t test for comparison between two groups, or analysis of variance (ANOVA) for multiple groups.</p><p><b>RESULTS</b>Immunohistochemical staining of CTGF was seen in 35 of 40 HCC samples (87.5%). CTGF was overexpressed 5-fold in 20 HCC tissues, compared with surrounding non-tumor liver tissue. CTGF mRNA level was 5 - 8-fold higher in HepG2, SMMC-7721 and MHCC-97H than in LO2 cells. This indicated that the inhibition rate of cell growth was 43% after knockdown of CTGF expression (P < 0.05). Soft agar colony formation assay showed that siRNA mediated knockdown of CTGF inhibited colony formation in soft agar of HepG2 cells (P < 0.05). The volume of tumors from CTGF-shRNA-expressing cells only accounted for 35% of the tumors from the scrambled control-infected HepG2 cells (P < 0.05).</p><p><b>CONCLUSIONS</b>CTGF was overexpressed in human HCC cells and downregulation of CTGF inhibited HCC growth in vitro and in vivo. Knockdown of CTGF may be a potential therapeutic strategy for treatment of HCC.</p>

Screening and identification of human anti-c-Met Fab from a phage antibody library / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To screen anti-c-Met Fab from a phage antibody library and identify its binding activity.</p><p><b>METHODS</b>The expression of c-Met of HCC lines was identified by Western blot and immunofluorescence. Antibodies against c-Met were screened with immobilized antigen. After five rounds of panning, 30 randomly selected clones were identified by phage ELISA to select specific clones with high affinity. The positive clones were selected for Fab soluble expression in TOP10F and the binding activities were analysed in HCC lines.</p><p><b>RESULTS</b>c-Met expressed in HCC membrane was confirmed by Western blot and immunofluorescence. A Fab fragment named AM2-26 with fine activity to c-Met was selected. AM2-26 binding specificity was confirmed by IP, FACS and immunofluorescence.</p><p><b>CONCLUSION</b>The anti-c-Met Fab binding to c-Met in HCC provides a promising candidate for the biotherapy of hepatoma.</p>

Expression of MRP1/CD_9 in cervical squamous cell carcinoma tissue and its clinical significance / 肿瘤研究与临床

Objective To investigate the clinical significance in MRP1/CD_9 expression in cervical squamous cancer tissues and normal cervical tissues.Methods The expression of MRP1/CD_9 were assayed by SABC immunohistochemical methods in 53 cases of cervical cancer tissues and 13 cases of normal cervical tissues.Results Positive expression of MRP1/CD_9 was detected in 13 normal cervical tissue.MRP1/D_9 ex- pression is down-regulated in cervical carcinoma(P

Induction of apoptosis by L-NMMA, via FKHRL1/ROCK pathway in human gastric cancer cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the apoptosis-inducing effect of endogenous nitric oxide (NO) suppression in gastric cancer cells and its mechanisms.</p><p><b>METHODS</b>Apoptosis of gastric cancer cells was detected by flow cytometry. Expression of phosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1 in gastric cancer cells was analyzed using Western blotting. Immunofluorescence assay was performed to localize the intracellular phosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1. Transfection of FKHRL1-HA wild type and mutant FKHRL1-HA T32A constructs was performed by lipofectamine plus reagent. NO generation was determined by Griess reaction.</p><p><b>RESULTS</b>Gastric cancer cells were significantly apoptotic after treatment with N(G)-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), compared with the control (P<0.01). The apoptosis of gastric cancer cells induced by L-NMMA was dose-dependent and time-independent. However, the Z-DEVD-fmk, a caspase-3, 6, 7, 8, 10 inhibitor, did not prevent the apoptosis. The immunofluorescence assays showed that FKHRL1 protein was strongly expressed in the nucleu and p-FKHRL1 thr-32 protein was strongly expressed in the cytoplasm of SGC-7901 cells when endogenous nitric oxide generation was blocked by L-NMMA, but no change in FKHRL1 ser-253 phosphorylation. Nevertheless, ROCK protein was strongly expressed in p-FKHRL1 thr-32-positive SGC-7901 cells. The wortmannin, an inhibitor of phosphoinositol-3-OH kinase (PI3K), did not block the phosphorylated FKHRL1 thr-32 protein induced by L-NMMA. However, Y-27632, a specific inhibitor of the protein kinase ROCK, significantly blocked apoptosis induced by phosphorylated FKHRL1 thr-32 (P < 0.01), which was mediated by L-NMMA. A significant decrease in NO generation (P < 0.01) and a significant increase in apoptosis (P < 0.01) were observed when FKHRL1-HA wild-type cells were transfected, which caused increased FKHRL1 thr-32 phosphorylation.</p><p><b>CONCLUSIONS</b>L-NMMA triggers gastric carcinoma cell apoptosis, possibly by promoting FKHRL1 thr-32 phosphorylation and initiating signal of FKHRL1 to ROCK kinase. This apoptotic signaling process is PI3K/Akt as well as caspase-3 independent.</p>

Nanoparticles as a vaccine adjuvant of anti-idiotypic antibody against schistosomiasis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The development of new adjuvants for human use has been the focus of attention. This study's aim is to explore the possibility of using nanoparticle Ca nanoparticles (CA) as a vaccine adjuvant of anti-idiotypic antibody NP30 against schistosomiasis and its protective mechanisms.</p><p><b>METHODS</b>Nanoparticle CA-NP30 conjugate (CA-NP30) was fabricated. BALB/c mice were immunized actively with CA-NP30 to evaluate its effects of protective immunity on mice. The serum levels of specific IgG, IgG1 and IgG2a antibodies against NP30 and the concentrations of IFN-gamma and IL-4 in supernatant of splenocytes were determined via ELISA.</p><p><b>RESULTS</b>Nanoparticle CA could enhance significantly the protective immunity of NP30 against infection of Schistosoma japonicum and the worm reduction rose from 36.0% (NP30 alone) to 52.6%. The serum levels of specific IgG, IgG1 and IgG2a antibodies against NP30 increased remarkably, as compared with those of the group immunized with NP30 alone. The concentration of IFN-gamma in supernatant of splenocyte was drastically elevated [the groups immunized with CA-NP30 and NP30 alone were (493.80 +/- 400.74) pg/ml and (39.03 +/- 39.58) pg/ml, respectively], but the concentration of IL-4 showed no significant difference from that of NP30 alone [(27.94 +/- 9.84) pg/ml vs (27.28 +/- 14.44) pg/ml].</p><p><b>CONCLUSIONS</b>Nanoparticle CA could act as a vaccine adjuvant of anti-idiotypic antibody NP30 against schistosomiasis. The mechanism could be that CA-NP30 enhances humoral and cellular immune responses in mice.</p>