Study On Anti - Tumor Effect Of CXCR4 Antagonist Polypeptide And Toxicology Of Nano Silver

Objective:The chemokine receptor CXCR4 is highly expressed in breast cancer and acute myelocytic leukemia (AML) cells. Its cognate ligand stroma derived factor 1α (SDF-la or CXCL12) is secreted by marrow stromal cells or stromal fibroblasts in tumor microenvironment such as bone marrow, liver, lung, and lymph nodes. CXCR4/CXCL12 axis can directly mediate adhesion, chemotactic and invasive responses through CXCL12-mediated signaling pathways of CXCR4. which results in organ-specific trafficking and metastases of breast cancer. Meanwhile, CXCL12 induces circulating leukemia cells for homing to marrow niches, which provides growth and survival signals for leukemia cells. The axis facilitates leukemia cells beneath marrow stromal cells and leads to formation of minimal residual disease (MRD). which leads to relapse and drug-resistance of leukemia. In addition, CXCL12 in the tumor microenvironment could recruit endothelial progenitors and further allow for tumor angiogenesis. Therefore, disruption of CXCLR4 and CXCL12 interaction is of great significance for inhibiting tumor growth and metastasis and improving the effectiveness of chemotherapy.In the previous studies, we designed a novel peptide E5 according to the structure and sequence characteristics of CXCR4, and proved its inhibitory effect on CXCL12-induced AML cell migration and adhesion. In this study, we try to combine E5 with different chemotherapeutics to investigate its effects both on breast cancer and leukemia model mice and the possible action mechanism. Meanwhile, E5 pharmacokinetic study was performed in healthy mice.Methods:The affinity of E5 to murine breast cancer cell (4T1), murine stromal cell (MS-5) and human umbilical vein endothelial cell (HUVEC) was tested through flow cytometry. Then CCK-8 assay, western blot and Annexin V/PI staining were measured to determine the in vitro cytotoxicity of E5. Meanwhile, the transwell assay and co-cultured condition system were performed to examine the effect of E5 on CXCL12-induced cell migration and adhesion. Thereafter, the mechanism of effect of E5 on CXCR4/CXCL12 axis was conducted by examining the CXCR4 downstream signaling thourgh western blot analysis. CCK-8 assay was used to determine the combination effect of E5 with chemotherapeutics on viability of 4T1 cells co-cultured with or without MS-5. The in vivo combination effect of E5 with chemotherapeutics was evaluated on tumor-bearing mice in breast cancer models, through monitoring the tumor sizes and the survival periods of the mice. As to explore the mechanism of E5 action, the key proteins expressed in tumor tissues, which are associated with downstream signaling pathways of CXCR4 and angiogenesis, were detected by western blot analysis. In a leukemia mice xenograft model, E5-induced AML cells mobilization to peripheral blood was determined through flow cytometry. The effect of E5 combination with chemotherapeutics was investigated through evaluation of leukemia burden in bone marrow, spleen, peripheral blood, liver as well as mice survival by flow cytometry and histological analysis. Pharmacokinetic study of E5 was conducted in the female Balb/c mice after subcutaneous injection of FITC-conjuncted E5.Results:(1) E5 has specific affinity to 4T1 and HUVEC cells which highly express CXCR4. (2) E5 can inhibit 4T1 cell viability and induce apoptosis when the concentration was higher than 25 μM. (3) CXCL12 and MS-5 can enhance the migration rate of both 4T1 and HUVEC cells, which could be inhibited by E5. (4) E5 can interrupt the adhesion of 4T1 cells to stromal cells through abrogating CXCL12-induced Akt and Erk pathway activation. (5) E5 can overcome stromal cell-induced resistance and increases the sensitivity of 4T1 cells to chemotherapeutic drugs (Paclitaxel, Cisplatin, Doxorubicin. or Vincristine). (6) Compared with mono-chemotherapeutics treatment, E5 can enhance inhibitory effect of chemotherapeutic drugs by decreasing tumor burden and prolonging survival period of mice in a breast cancer model. (7) E5 significantly decreases the level of CD31 and phosphorylation of Akt and Erk, indicating E5 could inhibit CXCR4/CXCL12 axis-mediated recruitment of endothelial progenitor for tumor angiogenesis and cell adhesion. (8) E5 significantly mobilizes leukemia cells from stromal microenvironment into peripheral circulation and reduces leukemia burden. (9) Combined with chemotherapeutics. E5 efficiently inhibits infiltration of leukemia cells into bone marrow, spleen and liver, thus prolongs the lifespan of leukemia mice. (10) Liver was the main organ for E5 metabolism after subcutaneous injection of E5, and the concentration of E5 peaked in the blood after 2 hours of administration. The half-life of E5 was about 10 hours in mice.Conclusion:E5 disrupted the interaction of breast cancer cells or AML cells with stromal cells through CXCR4/CXCL12 axis and inhibited cells migration and adhesion activities by abrogating the CXCL12-induced Akt and Erk pathway activation, and resulted in enhancement of tumor cell sensitivity to chemotherapy in vitro and in vivo. Meanwhile, E5 inhibited tumor angiogenesis in breast cancer mice probably by blocking CXCR4/CXCL12 axis-mediated recruitment of endothelial progenitor. On the basis of these data, E5 plays an important role in inhibitory effect of tumorigenesis and could be a potential therapeutic agent to improve the clinic benefits of current therapies for breast cancer and leukemia.Objective:The silver nanoparticles (AgNPs) have been widely explored for being used as a powerful antibacterial agent in the household products, electronics, food packages, and medical devices due to its much stronger antimicrobial performance than conventional silver compounds. With the increase in applications, exposure to AgNPs-containing products has become a serious concern for its impacts upon the health, environment, and safety. AgNPs have been found to induce cytotoxicity and genotoxicity in vitro, such as inhibit cell growth, enhance cell membrane permeability, induce intracellular oxidative stress reaction and secretion of inflammatory factors and cytokines, change cell conjunction and cause DNA damage. In addition, many studies have shown that AgNPs could enter model animals through inhalation, oral or intravenous exposure, and distribute to most major target organs, particularly to the liver, lungs, kidneys, and spleen. As AgNPs can dissolve Ag ions constantly, it is hard to distinguish toxicological characteristics of AgNPs and Ag ions. Thus, the precise differences in the mechanisms of toxicity between AgNPs and Ag ions remain elusive. Intravenous administration of AgNPs to mice could simulate the case when medical personnel or patient wounds were exposed to AgNPs. Therefore, the aim of the present study was to assess the potential toxicity of AgNPs when intravenously exposed to mice and compare its impacts with that of Ag ions.Methods:AgNPs (10,75, and 110 nm) used in this study were coated with citrate. TEM observation and DLS analysis were performed to examine the physical and chemical properties in different mediums. Then the viability and apoptosis of primary human umbilical vein endothelial cells (HUVEC) were tested in the presence of AgNPs or silver nitrate (AgNO3) through CCK-8 assay and Hoechst/PI staining. Meanwhile, the cell uptake of AgNPs was determined by ICP-MS analysis and TEM observation. The effects of AgNPs and AgNO3 on intercellular conjunction, cell cytoskeleton and intracellular ROS were examined by confocal microscopy analysis and the flow cytometer after VE-cadherin staining, phalloidin staining, and 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) assay, respectively. The in vivo location of AgNPs and toxicity of AgNPs and AgNO3 were evaluated following single or multiple intravenous administrations to mice, using TEM observation, histological analysis of tissue sections, routine clinical parameters of serum analysis and Elisa assay of the complement components C3.Results:(1) TEM observations show that the three kinds of AgNPs are homogeneously spherical, and the average diameter for AgNP-10, AgNP-75 and AgNP-110 was determined as 11 ± 1 nm,76 ± 6 nm and 107 ± 8 nm; The surface of AgNPs are negatively charged, and they were equally well dispersed in both water and serum-containing medium. The average hydrodynamic diameter for AgNP-10, AgNP-75 and AgNP-110 was 9.8 ±3.2 nm,72.0 ± 0.9 nm and 99.3 ± 1.7 nm respectively in water. (2) AgNPs could be taken up by vascular endothelial cells through endocytosis and located in autolysosomes and vesicles, while AgNO3 was taken up little. (3) AgNPs inhibited cell viability in concentration and size dependent manners. However, cells exposed to AgNO3 showed a steeper decrease in viability as Ag concentrations were increased. (4) AgNPs incubation induced the elevation of intracellular ROS in concentration-dependent and down-regulation of VE-cadherin between the endothelial cells and affected the cytoskeleton actin reorganization. The cytotoxicity of AgNPs could be rescued by antioxidant N-acetylcysteine (NAC). In contrast, AgNO3 caused direct cell necrosis at higher concentrations and without ROS induction at lower concentration. (5) TEM observations showed that AgNPs located nearby microvascular structures among hepatic cells. Inflammatory infiltrates were detected in the vessel walls of liver, kidney, and lung when mono- or multi-dose intravenous injections of AgNPs to mice, and the surrounding inflammatory infiltrates mainly consisted of monocytes and neutrophils. The decrease of complement components C3 showed the activation of the complement system induced by AgNPs. Intravenous exposure of AgNPs could induce moderate inflammation in the liver of mice. However, intravenous administration of AgNO3 did not cause appreciable peripheral inflammation in liver, kidney and lung.Conclusion:AgNPs could be taken up by vascular endothelial cells and induce intracellular ROS elevation, which was closely related to disruption the integrity of endothelial layer. The AgNPs-induced leakiness of endothelial cells could attract immune cells in liver, kidney and lung tissues and mediate the common peripheral inflammation through intravenous exposure. In contrary, AgNO3 was taken up little, which may involve another mechanism of cytotoxicity independent of elevating intracellular ROS.