AIM: To discuss the protection of cistanchis glycosides on ethanol-induced damage in the primary hepatocyte of mice. METHODS: The primary hepatocytes were collected by perfusion in situ method.The cell livability was evaluated by MTT,fluorescence microscopy and flow cytometry were used to evaluate the cell apoptosis and necrosis,immunohistochemistry was used to detect the expression of bcl-2 and c-fos.RESULTS:After the primary hepatocytes were damaged by alcohol,the cell livability decreased,apoptosis and necrosis were obvious,the expression of bcl-2 was decreased while that of c-fos was increased.Cistanchis glycosides could increase the cell livability,ameliorate apoptosis and necrosis,increase the expression of bcl-2,and decrease the expression of c-fos in a dose-dependent manner.CONCLUSION: The protective effects of cistanchis glycosides on ethanol-induced liver damage in the primary hepatocytes is associated with increasing the expression of bcl-2,and decreasing the expression of c-fos;resulting less apoptosis,less necrosis,and increasing the cell livability.
Gastric cancer (GC) is one of the most common cancers worldwide. Tumor microenvironment plays an important role in tumor progression. This study aims to explore the role of cancer-associated fibroblasts (CAFs) in GC and the underlying mechanism.
Abstract The limited number of targetable tumor-specific antigens and the immunosuppressive nature of the microenvironment within solid malignancies represent major barriers to the success of chimeric antigen receptor (CAR)-T cell therapies. Here, using epithelial cell adhesion molecule (EpCAM) as a model antigen, we used alanine scanning of the complementarity-determining region to fine-tune CAR affinity. This allowed us to identify CARs that could spare primary epithelial cells while still effectively targeting EpCAM high tumors. Although affinity-tuned CARs showed suboptimal antitumor activity in vivo, we found that inducible secretion of interleukin-12 (IL-12), under the control of the NFAT promoter, can restore CAR activity to levels close to that of the parental CAR. This strategy was further validated with another affinity-tuned CAR specific for intercellular adhesion molecule-1 (ICAM-1). Only in affinity-tuned CAR-T cells was NFAT activity stringently controlled and restricted to tumors expressing the antigen of interest at high levels. Our study demonstrates the feasibility of specifically gearing CAR-T cells towards recognition of solid tumors by combining inducible IL-12 expression and affinity-tuned CAR.
Oncolytic viruses armed with therapeutic transgenes of interest show great potential in cancer immunotherapy. Here, a novel oncolytic adenovirus carrying a signal regulatory protein‐α (SIRPα)‐IgG1 Fc fusion gene (termed SG635‐SF) was constructed, which could block the CD47 ‘don't eat me’ signal of cancer cells. A strong promoter sequence (CCAU) was chosen to control the expression of the SF fusion protein, and a 5/35 chimeric fiber was utilized to enhance the efficiency of infection. As a result, SG635‐SF was found to specifically proliferate in hTERT‐positive cancer cells and largely increased the abundance of the SF gene. The SF fusion protein was effectively detected, and CD47 was successfully blocked in SK‐OV3 and HO8910 ovarian cancer cells expressing high levels of CD47. Although the ability to induce cell cycle arrest and cell death was comparable to that of the control empty SG635 oncolytic adenovirus in vitro , the antitumor effect of SG635‐SF was significantly superior to that of SG635 in vivo . Furthermore, CD47 was largely blocked and macrophage infiltration distinctly increased in xenograft tissues of SK‐OV3 cells but not in those of CD47‐negative HepG2 cells, indicating that the enhanced antitumor effect of SG635‐SF was CD47‐dependent. Collectively, these findings highlight a potent antitumor effect of SG635‐SF in the treatment of CD47‐positive cancers.
To examine the antitumour efficacy and investigate immunological mechanism of combination therapy of IL-2 gene and IL-12 gene transfer with radiation in an immunocompetent murine model that parallel more closely the clinical therapy of head and neck squamous cell carcinoma (HNSCC).Tumors were established in the floor of mouth in C3H/HeJ mice with SCCVII cell line. Lipid-DNA complexed (lipoplexes) by using polycationic liposome-Mediated transduction for HNSCC was transduced in tumor-bearing mouse by direct intratumoral gene transfer. The local tumor was radiated with a dose of 2 Gy in the second day. Tumor sizes were measured before and after the treatment as compared to the different single treatment groups and the controls. After tumors were subculture, the supernatants were collected for IL-2 and IL-12 expression by enzyme-linked immunosorbent assay (ELISA). Natural killer (NK) cell activity and cytotoxic T-lymphocyte (CTL) activity were also assayed by LDH method. CD4+ and CD8+ T-lymphocyte in tumor tissues were examined by immunohistochemistry.HNSCC tumor growth was significantly inhibited following combined IL-2 and IL-12 gene therapy with radiation as compared to IL-2 or IL-12 gene therapy with radiation, single IL-2 or IL-12 gene therapy, radiation alone and the controls. Increased secreted levels of IL-2 and IL-12 protein expression were found in combined and single IL-2 gene or IL-12 gene treated groups. The combination and single gene treated groups produced greater activation of CTL and NK than the controls of all concerned test. The significant CD4+ and CD8+ T lymphocyte infiltration was distributed and the numerous necrosis were seen in tumor tissues after combination therapy.Combined IL-2 gene and IL-12 gene therapy with radiation could significantly inhibited HNSCC tumor growth in the murine model and efficiently induced antitumor immunity of the host.
Abstract Background: Circular RNA plays a crucial role in the occurrence and progression of pancreatic cancer. However, the existence of hsa_circ_0131457 has not been reported yet, necessitating further research. Methods: The expression level of SOX4 in pancreatic cancer cells and tissues was measured using qRT-PCR and immunohistochemistry. The correlation between the expression level of SOX4 in pancreatic cancer tissues and clinicopathological features was analyzed using the Pearson Chi-square test. The survival curve of pancreatic cancer patients was analyzed using the Kaplan-Meier method. The circRNA regulating SOX4 was predicted through bioinformatics and subsequently verified in pancreatic cancer cells and tissues. Bioinformatics was also employed to predict the miRNA and target genes, and a circRNA-miRNA-mRNA regulatory network was constructed. The expression of SFRP2 in pancreatic cancer cells and tissues was measured using qRT-PCR and immunohistochemistry, and the correlation between clinicopathological features and prognosis was analyzed. Lastly, the biological function of SFRP2 was investigated through bioinformatics analysis. Result: The expression of SOX4 was significantly up-regulated in pancreatic cancer cells and tissues. It is also associated with tumor size and T stage, which leads to a poor prognosis. qRT-PCR detection of pancreatic cancer tissue samples from 9 patients revealed a significantly lower expression of hsa_circ_0131457. Further bioinformatics analysis was conducted to establish the hsa_circ_0131457-miR-636-SFRP2 regulation network. Analysis of the target gene SFRP2 demonstrated a significant down-regulation in pancreatic cancer tissues and cells. SFRP2 was found to be negatively correlated with preoperative direct bilirubin, tumor size, T stage, and tumor differentiation, while positively correlated with overall survival time of patients. Conclusion: It is inferred that the hsa_circ_0131457/ miR-636/SFRP2 regulatory mechanism may inhibit the invasion and metastasis of pancreatic cancer.
Abstract Chimeric antigen receptor (CAR)-T cell therapy has shown robust anti-cancer responses in hematologic malignancies. However, application of this therapeutic approach to solid tumors has been hindered by multiple challenges, one of which is on-target/off-tumor cytotoxicity to normal tissues. Tumor-specific antigens exclusively present on tumor cells are rare. Most CAR-T cells are designed to target tumor associated antigens (TAAs) expressed in high levels on tumor cells. Yet, normal tissues express these antigens as well, albeit at much lower densities.To mitigate the on-target/off-tumor cytotoxicity, we developed a strategy for fine tuning the affinity of CARs to selectively target tumor cells. Epithelial cell adhesion molecule (EpCAM) is highly expressed in epithelial cells and overexpressed in tumor cells in a variety of epithelial carcinomas. High-affinity (nM range) EpCAM-targeting CAR-T cells kill both normal human epithelial cells and EpCAM-high tumor cells in vitro. To develop CAR-T cells specific to EpCAM-high cancers, we identified low-affinity scFv variants by rational design of amino acid substitutions. The affinities of these scFvs were measured by Surface Plasmon Resonance (SPR). CAR-T cells equipped with low-affinity scFvs showed antigen-dependent activation, proliferation and Th1-like cytokine secretion when co-cultured with target cells having varied levels of EpCAM. Importantly, low-affinity CAR-T cells still lysed EpCAM-high tumor cells but spared normal human epithelial cells in vitro. Treatment of MKN28 xenograft mice with low-affinity CAR-T cells resulted in tumor regression and prolonged survival. Moreover, transcriptomic profiling of CAR-T cells revealed significant differences in gene expression levels between high- and low- affinity CARs, particularly as they relate to the functional state of these CAR-T cells, and their resistance to exhaustion. Our results show that rational design of scFv can aid in the identification of CARs that are minimally reactive toward normal tissues while effectively eliminating tumors. Furthermore, affinity-tuned CARs demonstrate better overall fitness and antitumor activity in vivo. This affinity fine-tuning approach shows promise as a general strategy for identifying a therapeutic window for CAR-T cells targeting novel TAAs that may have been overlooked because of basal expression levels in normal tissues. Citation Format: Huan Yang, Janusz Puc, Yanping Yang, Jaclyn E. McCloskey, Yogindra Vedvyas, Hongtao Li, Irene M. Min, Moonsoo M. Jin, Eric v. Hofe. Mitigating on-target off-tumor cytotoxicity of EpCAM CAR-T by affinity tuning [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-381.
To investigate whether RelB-silenced bone marrow-derived dendritic cells (BMDC) pulsed with torpedo acetylcholine receptor (TAChR) immuno-dominant peptide Talpha146~162 can induce tolerance in T cells primed with TAChR.Recombinant lentivirus that produced RelB siRNA and control lentivirus were prepared and used to infect BMDCs. The infected BMDCs were stimulated with LPS,and the resulting cells were designated as DC-siRelB or DC-control, respectively. The mRNA and protein expression of RelB were examined by quantitative real-time PCR and Western blot. Cell surface markers of DC were evaluated by flow cytometry. IL-12 in the supernatant was detected by ELISA. Mice were randomly divided into 6 groups: A1, A2, A3,K1, K2, and K3. On day 0, group A1, A2, and A3 were primed with TAChR in CFA and group K1, K2 and K3 were primed with KLH+CFA. On day 7, group A2 and K2 were injected with Talpha146~162 pulsed DC-siRelB, group A3 and K3 were injected with Talpha146~162 pulsed DC-control, while A1 and K1 group received PBS at the same time. On day 14, lymphocyte proliferative response of the 4 groups were measured.Recombinant lentivirus including RelBshRNA genes was successfully constructed. RelB siRNA knocked down RelB expression in BMDCs obviously. Compared with DC-control, DC-siRelB expressed a significantly lower level of CD80, CD86, and MHC class II on their surface, producing lower level of IL-12. Compared with group A1 and A3, lymphocyte proliferative response to TAChR of A2 group was suppressed significantly (P<0.05). No different lymphocyte proliferative responses to KLH and ConA were seen in group A1, A2 and A3 (P>0.05). No different lymphocyte proliferative responses were seen in group K1, K2 and K3 (P>0.05).Lentiviral-mediated RelB-silenced BMDCs are maturation resistant and can induce antigen-specific tolerance in TAChR primed C57BL/6 mice,which provides a basis for further study of their therapeutic potential in myasthenia gravis.
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