Radiosensitivity Of Lovo Cell Enhanced By Immunoliposomal Docetaxel

BacgroundDocetaxel is a semisynthetic taxane that affects polymerized tubulin both topromote microtubule formation and to inhibit its disassembly. Docetaxel is a phase-specific agent that may offer the potential for enhancement of radiosensitization. As the poor targeted, the toxities to the normal tissues limit the extents of application. Liposomes can enhance the therapeutic expontent as the carries of druges, but it has no targeted. Colorectal cancer is one of the most common malignant diseases in the world. In the treatment of colorectal cancer, local recurrence is a major problem. To improve overall survival and reduce local recurrence rate after surgery, additional radiotherapy was given either preoperatively or postoperatively.In order to enhance its beneficial radiosensitizing capability, docetaxel was incorporated into liposomes with antibody conjugate for the targeted delivery to specifical cancer cells. Combination sterical stabilization with targetability, docetaxel immunoliposomes were prepared. In this study, we attempt to investigate the radiosensitization of colon carcinomas targeting with docetaxel liposome conjugated to antibody specific for carcinoembryonic antigen (CEA). The aim of this study is selective delivery of docetaxel and enhancing the radiation sensitivity on human colon cancer cell. In order to increase radiotherapy effects of local tumor and to improve both the quality of life and overall survival, radiosensitizer should be targeted to deliver into the tumor and limited to normal tissue. This workshopconsidered a new approach by use targeted radiosensitizer to improve radiotherapy. MethodsPreparation of immunoliposomal docetaxel: A new PEG-PE derivative that had been endgroup-functionalized with cyanuric chloride was first synthesized. Anti-carcinoembryonic antigen (CEA) monoclonal antibody can simply be coupled to this membrane anchor in mild basic conditions (pH 8.8) without the need for previous antibody derivatizations. The entrapment efficiency and the quality of prepared docetaxel immunoliposomes were evaluated. In vitro cell association of docetaxel immunoliposomes binding was determined. The function and activity of the coupling anti-CEA monoclonal antibody was also analyzed.Enhancement of radiosensitivity by immunoliposomal docetaxel in human colon adenocarcinoma LoVo cell: The optimal combination of immunoliposomal docetaxel and radiation in vitro was obtained by the cytotoxicity evaluation of immunoliposomal docetaxel and a single radiation treatment in human colon adenocarcinoma cell lines.The effect of immunoliposomal docetaxel in combination with radiation on the expression of apoptosis-related proteins and EGFR : The expression of apoptosis-associated proteins p53, Bcl-2 and Bax were detected by flow cytometry in LoVo cells. Fas -. FasL and survivin proteins were detected by immunohistochemical staining and were quantified using semi-quantitative analysis by image analysis system. Epidermal growth factor receptor (EGFR) was detected with Western blot. Carcinoembryonic antigen expressed in cell cultures was assessed by Sandwhich ELISA. ResultsThe mean particle size of docetaxel immunoliposomes was 213±17.4 nm. Encapsulation efficiency of docetaxel immunoliposomes was 97.6 %. Using flow cytometry to analyze the targetability of docetaxel immunoliposomes show a higher degree of in vitro cell binding compared to conventional docetaxel liposomes. Enzyme-linked immunosorbent assay (EILSA) indicated that the immunoreactivity ofantibodies on liposome was retained completely. A stable and selective targeted agent has been achieved by biophysical evaluation of docetaxel immunoliposomes. The MTT assay indicated the cellular growth inhibitory rates were dose-response and time- response. HT29 cells shown a strong cytotoxicity to immunoliposomal docetaxel compared with LoVo cells. Both cell lines showed a dose-response to a single radiation shot. The combined treatment LoVo cells were tested by clonogenic assay. 2nM, lOOnM immunoliposomal docetaxel enhanced radiosensitivity ( SERDo. SERoq were 1.70, 1.78 and 1.47, 1.63 respectivly), but 2nM liposomal docetaxel was less radiosensitive ( SERdo, SERoq werel.02, 1.03). Flow cytometric analysis showed the concomitant effect of 2nM docetaxel with 2Gy radiation exposure, significantly increased the percentage of G2-M cells and induced apoptosis (P O.01), by comparison with non-targeted docetaxel. TUNEL staining cells revealed a condensed TUNEL-positive nucleus and reflected apoptotic cell death. Immunoliposomal docetaxel induced the numbers of TUNEL positive nuclei increased (43.6%), in contrast to liposomal docetaxel (17.7 %). Targeted docetaxel also increased the DNA ladder formation in the combined treatment LoVo cells. Morphological evidence of apoptotic cells could be distinguished under the fluorescence microscope after staining with Hochest 33342. Many nuclear fragments were observed in LoVo cells with docetaxel radiosensitization The Immunoliposomal docetaxel treated LoVo cells showed Bax increased, but no significant changes in the expression of p53 and bcl-2 proteins were observed. By the effect of docetaxel radiosensitization, FasN FasL in LoVo cells were expressed higher than LoVo cells with non-targeted docetaxel treatment (p<0.01). Combined with radiation, docetaxel could inhibit the expression of survivin. EGFR was down-regulated by Immunoliposomal docetaxel. After exposures of docetaxel and radiation , carcinoembryonic antigen was increased in the cell cultures. ConclusionA stable and selective targeted agent has been achieved by biophysical evaluation of docetaxel immunoliposomes. A stable and selective targeted agent has beenachieved by biophysical evaluation of docetaxel immunoliposomes. The immunoliposomal docetaxel is able to enhance the radiosensitivity in LoVo cell. The different pathways involved in promotion of apoptosis undergo docetaxel radiosensitization.