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    • CD CD belong to the costimulatory adhesion

    2021-10-16

    CD80/CD86 belong to the costimulatory adhesion molecule family, which can activate T Hyper Assembly Cloning receptor by the costimulatory pathway and have been used as indicators of DC maturation in numerous studies. In addition, MHC-II, as the principal component of antigen presentation, was significantly elevated after antigen loading. Therefore, CD80, CD86 and MHC-II molecules are considered markers of DC maturation (Hunter et al., 2007). IL-12 is one of the most important parameters of DC maturation (Athiemorales et al., 2004). In our research, CD40 × HER2ScDb could significantly upregulate the expression of CD80/CD86/MHC-II compared with that of the NS group and Ag group, and the IL-12 expression of the Ag + CD40 × HER2ScDb group also increased remarkably, which was significantly different from that of the NS group and Ag group. However, there was no significant difference between the Ag + CD40 × HER2ScDb group and Ag + TNF-α group (P > 0.05). Our results were consistent with the research of Mangsbo (Mangsbo et al., 2015). Comparable growth inhibitory effects were observed for the Ag + CD40 × HER2 ScDb and Ag + TNF-α groups in 4T1 cell lines, which indicated that CD40 × HER2ScDb could restrain the proliferation of 4T1 by stimulating tumor-specific immunoreactions. The in vivo murine model studies revealed the remarkable inhibitory effects on T6-17 cells in the CD40 × HER2 ScDb and CD40 mAb groups. Moreover, HE staining demonstrated that CTLs could be assembled in tumor tissues after CD40 × HER2 ScDb was administered. Caspase-3 is a critical protease for activation in the early stage of apoptosis and is the final apoptotic executioner (Liu et al., 1997). In many studies (He et al., 2005; Jerome et al., 2003), Caspase-3 has been used as an indicator of CTL function. In this study, IHC staining of Caspase-3 demonstrated that Caspase-3 was abundantly expressed in the T6-17 cytoplasm after CD40 × HER2 ScDb or CD40 mAb treatment, which indicated that CD40 × HER2 ScDb could induce the apoptosis of 4T1 cells by stimulating CTLs. HER2 is overexpressed in a wide range of human malignancies, and its expression level is correlated with poor clinical outcome in patients with several cancers, such as breast cancer, gastric cancer and prostate cancer (Roh and Pippin, 2000; Zhang et al., 2009; Liu et al., 2005). Immunofluorescence experiments confirmed that CD40 × HER2 ScDb had a strong affinity for HER2 expressed on the surface of T6-17 cells. The cytotoxicity assays indicated that CD40 × HER2 ScDb could remarkably inhibit the proliferation of T6-17 cells. Akt is one of the major downstream effectors of PI3K and is phosphorylated after PI3K receives an upstream activation signal. Akt phosphorylation regulates cell proliferation, differentiation, apoptosis and migration by activating or inhibiting its downstream target proteins. Therefore, phosphorylation of Akt is an important indicator of HER2 expression, which affects tumor cell proliferation and metastasis (Shoman et al., 2005). Extracellular signal regulated kinase (ERK) is an important member of the mitogen activated protein kinase (MAPK) family, which plays a vital role in regulating cell proliferation, differentiation, and apoptosis (Eblen et al., 2002). HER2 could activate ERK via MAPK. Activated ERK regulates the function of downstream target genes by phosphorylating transcription factors, cytoskeleton-associated proteins, and other substrates (Eblen et al., 2002). Therefore, the phosphorylation level of ERK can reflect the activation level of the MAPK/ERK signaling pathway. In this study, the expression levels of phosphorylated Akt and ERK in T6-17 cells were detected under different interventions. Western blotting was performed to determine the HER2-targeting mechanism of CD40 × HER2 ScDb. Our experiments showed that CD40 × HER2 ScDb could significantly inhibit the phosphorylation of ERK and Akt compared with that of the NS and CD40 ScFv groups. Therefore, we speculated that CD40 × HER2 ScDb could inhibit the proliferation of tumor cells and promote their apoptosis by inhibiting the PI3K/AKT and MAPK/ERK1/2 signaling pathways. In the present study, we evaluated the function of CD40 × HER2 ScDb in vivo by constructing a T6-17 tumor model in BALB/c-nude mice. By comparing the differences in the tumor growth curves of each group, we found that the CD40 × HER2 ScDb group and the trastuzumab group had inhibited tumor proliferation, and the difference was significant compared with that of the NS group (P < 0.05). No significant difference was found between the CD40 × HER2 ScDb group and the trastuzumab group. HE staining of tissue slices indicated that after CD40 × HER2 ScDb was administered, T6-17 cells lost their normal morphology, the chromatin aggregated and the nucleus volume decreased. In some fields, only the blurred nuclear outline or nuclear debris could be found, which indicated that CD40 × HER2 ScDb could induce the apoptosis of T6-17 in vivo by targeting HER2 and blocking the downstream signal pathway.