An Experiment Study on the Antitumor Activities Induced by Pegylated Liposomal Cytochalasin D
|Course||Biochemistry and Molecular Biology|
|Keywords||Cytochalasin D Pegylated liposome Tumor angiogenesis Apoptosis|
Actin is an evolutionarily conserved protein and one of the major cytoskeletal components in eukaryotic cells, including tumor cells. Actin occurs in two forms, monomeric actin and polymers called microfilaments. Microfilaments are involved in various cellular functions. Recent studies indicated that disruption of microfilaments can cause severe damage to the cell function, even culminating in cell death. Cytochalasin D (CytD) is one of cytochalasins isolated from fungal metabolites that are capable of altering actin polymerization. When CytD is used as a cytotoxic agent in cancer therapy, it will thus cause significant side effects. Many studies have concluded that liposomes are good carriers for the delivery of a variety of drugs, including cytotoxic agents, antibiotics, and antifungal agents. As carriers of anticancer drugs, they have been shown to reduce side effects and promote targeted tumor-cell action. Liposomes preferentially accumulate at tumor sites because they can leak through pores and defects in the capillary endothelium.In the present study, we encapsulate CtyD in the nonaqueous interior of PEG liposomes to prepare a CytD liposome (CytD-PEGL) as a model cytotoxic agent for cancer therapy using a subatmospheric-pressure rotary-evaporator method. Physical and chemical characteristic analysis of the CytD-PEGL by a Marvin nanometer particle size analyzer indicated that the average size, the polydispersity index and Zeta potential of CytD-PEGL were132.6nm,0.175and24.8mV, respectively. Tumor and endothelial cells were treated with various dose of CytD-PEGL and nature isolated CytD (nCytD). Compared with nCytD, cell proliferation in tumor cells treated with CytD-PEGL showed significantly inhibited in a manner similar to that induced by nCytD when detected by MTT assay, and the inhibition of tumor cell proliferation by CytD-PEGL was also shown as time and dose dependent. In vitro detection of cell apoptosis by a TUNEL-based ELISA and direct observation of DNA ladder suggested that both CytD-PEGL and nCytD had the capabilities of inducing significant tumor cell apoptosis also in an equivalent manner. Murine melanoma B16tumor model was established in C57BL/6mice, and the CytD concentrations in the plasma, tumor tissues and major organs were detected by high-performance liquid chromatography for observation of the biodistribution of the CytD-PEGL. Our results showed that CytD-PEGL could be conveniently dissolved in water for i.v. injection, and that it could specifically accumulate in tumor tissues more than nCytD did, indicating a specific characteristic of tumor targeting. The half-time of the CytD-PEGL in the plasma was significantly longer than that of nCytD (4hours versus10minutes). Longer duration of high concentration of CytD was found in the plasma and tumor tissues from the mice treated with CytD-PEGL. For better use of CytD-PEGL, the optimum effective dose of CytD-PEGL was determined in B16melanoma, CT26colorectal carcinoma, and H22hepatoma models, and the results indicated that50μg/kg equivalent of nCytD was the optimum effective dose both for CytD-PEGL and nCytD. Thereafter, the antitumor activities of the CytD-PEGL were also investigated in B16, CT26, and H22murine tumor models by observation of tumor volumes and survival times, the results showed that CytD-PEGL could significantly inhibit tumor growth and prolong survival in a manner similar to or even better than that of cisplatin (DDP, a routine antitumor cytotoxic agent in clinic tumor therapy) did, the tumor volumes in the mice treated with CytD-PEGL were significantly decreased and the survival time significantly longer when compared with the mice treated with CytD and PBS. In addition, when compared with the mice treated with PBS, the mice treated with CytD could also decreased the tumor volumes, but the survival times were not proportionally longer as the mice treated with CytD-PEGL and DDP, suggesting abnormal death in CytD-treated mice. In addition, side effects were specially investigated by observation of the gross measures and the major organs through visual inspection and hematoxylin-eosin staining of the tissue sections. Significant enlargement of live and spleen were found in the mice treated with nCytD, but not in the mice treated with CytD-PEGL and DDP. Compared with the mice treated with nCytD, DDP and PBS, no other adverse consequences were found in the mice treated with CytD-PEGL in gross measures such as weight loss, ruff ling of fur, life span, and behavior. Furthermore, no pathologic changes in the major organs (including liver, lung, kidney, spleens, brain, and heart) were found in the mice CytD-PEGL by microscopic examination. TUNEL-based in situ apoptosis assays showed that CytD-PEGL induced significant tumor cell apoptosis in all three tumor models when comparison with mice treated with nCytD and other control agents. In addition, significant inhibition of tumor angiogenesis was observed in the tumor tissues from the mice treated with CytD-PEGL, but not in the mice treated with nCytD. In addition, we also applied Western blot, Northern blot and realtime quantitative PCR to detect tissue factor (TF) expression in tumor cells in vitro and in tumor tissues in vivo. Significant TF expression only found in the B16melanoma cells and tumor tissues treated with CytD-PEGL, but not in CT26colorectal carcinoma, and H22hepatoma cells and tumor tissues.In summary, our study results indicated that CytD-PEGL could effectively accumulate in tumor tissues, prolong the circulation time of CytD in vivo, effectively inhibit various kinds of tumor growth and prolong the survival time of tumor-bearing mice. The antitumor effects induced by CytD-PEGL were highly related to the induction of tumor cell apoptosis and the inhibition of tumor angiogenesis by CytD-PEGL. Thus, our results indicate that modified forms of CytD, such as CytD-PEGL, may be a potential cytotoxic chemical agent for cancer therapy.