Inhibition Effect Of Docetaxel Conjugate Block Copolymers On Prostate Cancer Cell Line PC-3

Prostate cancer is the most prevalent form of cancer for the male, and it is the second leading cause of death in Western countries. In 2008, an estimated 186,320 new cases and 28,660 deaths are expected to occur in the United States alone.Firstline therapies for managing prostate cancer are local treatments such as radical prostatectomy and/or radiotherapy. Following first-line therapy, about 22% of the patients relapse and are treated with hormone therapies including luteinizing hormone-releasing hormone (LHRH) agonists and antiandrogens, constituting second line of therapy. However, acquired resistance to some of these therapies leads to enhanced metastasis. At this stage, median survival rate of prostate cancer patients is about 12 months and necessitates the use of chemotherapeutic agents for effective management of the disease.Chemotherapy has been the main treatment modality in metastatic diseases, which is, however, limited by its intrinsic or acquired multidrug resistance and nonselective toxicity to normal cells. Several chemotherapeutic agents, including docetaxel, are currently available for treatment of prostate cancer.Docetaxel is a semisynthetic product of the European yew Taxus baccata, similar to paclitaxel. At the molecular level, docetaxel disrupts normal processes of microtubule assembly and disassembly. Docetaxel binds tubulin to promote polymerization and prevents depolymerization of microtubules in the absence of guanosine triphosphate.Despite having improved survival efficacy with a docetaxel and prednisone chemotherapy, the improvement is only a progression-free survival of 3 months compared with other chemotherapy regimens available for the treatment of prostate cancer. Furthernore, there was an increase in gastrointestinal (nausea and vomiting) and cardiovascular (thrombosis and pulmonary embolism) toxicities in the docetaxel-treated groups.It is therefore desirable to develop delivery systems that would specifically deliver docetaxel to the tumor site. The advantages of preparing such a targeted-drug delivery system will enhance drug efficacy and reduce drug resistance. This has been a research focus in recent years.Docetaxel-loaded micelles were prepared from poly (ethylene glycol)-b-poly (d, 1-lactide) block copolymers(PEG-PLA).Targeting molecules of luteinizing hormone-releasing hormone (LHRH) was introduced in the PEG terminal, then the LHRH-PEG-PLA block copolymer was synthesized and was used to prepare docetaxel nanoparticles. We expected the nanoparticles could enhance efficiency of docetaxel and minimize its side effects.The advantages of amphipathic block copolymer are listed as follows:â‘ Polylactic acid (PLA) is a synthetic biodegradable polymer. In the aquatic environment, it hydrolyzes into nontoxic hydroxyl-carboxylic acid through ester bond cleavage and then is metabolized into water and carbon dioxide through a citric acid cycle. Polyethylene glycol (PEG) has many advantages, such as good hydrophilicity, flexibility, antiphagocytosis against macrophages, resistance to immunological recognition, non-combination with proteins, and biocompatibility.â‘¡The amphipathic block copolymer can form micelles structures by self-assembly in water with the solvent insoluble polymer block forming the micelles core and solvent soluble block the corona.â‘¢Due to their smaller particle size and modified surface, PEG-PLA block copolymers can avoid being engulfed by phagocytes, hence increase the circulation time in blood. These block copolymers can accumulate in tumor by EPR effect.â‘£micelles'end-group derivative can enable them to actively target special locations for enhancing drug efficacy and decreasing drug toxicity.Using PEG-PLA and LHRH-PEG-PLA as a carrier and modern nanometer micellar targeting technology, we synthesized the PEG-PLA/docetaxel and LHRH-PEG-PLA/ docetaxel. In vitro study, we discussed its anti-tumor function and characteristic. Further, this study had prepared a new docetaxel derivative conjugated to targeting group, LHRH, and tried to determine whether it enhances docetaxel potency in vitro. We have studied the effect and mechanism of PEG-PLA/docetaxel and LHRH-PEG-PLA/docetaxel micelles against PC-3 cell line in order to furnish the theoretical basis for developing and exploiting new drug carrier for anticancer medicine.1.Methods1)After successful Synthesis of PEG-PLA/docetaxel and LHRH-PEG-PLA/docetaxel, they were characterized by 1H-NMR. The size and surface morphology of micelles were evaluated by dynamic light scattering detector and transmission electron microscopy. 2)Inhibition effect of PEG-PLA/docetaxel and LHRH-PEG-PLA/docetaxel on prostate cancer cell line PC-3 in vitro were observed by MTT, flow cytometry, morphological study.3)PC-3 cells pretreated with antisense oligonucleotide against LHRH receptor were used for detected LHRH-PEG-PLA/docetaxel efficacy.2.Result1)After successful Synthesis of PEG-PLA/docetaxel and LHRH-PEG-PLA/docetaxel, they were characterized by 1H-NMR.2)The size and surface morphology of micelles were evaluated by dynamic light scattering detector and transmission electron microscopy. Distribution of PEG- PLA/ docetaxel and LHRH-PEG-PLA/docetaxel in solution was unity and symmetry, and their mean diameter was 160 nm.3)There was no significant difference between PEG-PLA/docetaxel and docetaxel on inhibition effect of PC-3 cell line studied by MTT in vitro. Inhibition ratio of both increased with time and concentration. Antiproliferative efficacy in PC-3 cell line exhibited the order LHRH -PEG-PLA/docetaxel> docetaxel, whereas LHRH-PEG-PLA alone had no effect.4)Cell cycle analysis showed that low-dose PEG-PLA/ docetaxel and LHRH-PEG-PLA/docetaxel treatment led to arrest in G2-M phase, whereas high-dose PEG-PLA/docetaxel and LHRH-PEG-PLA/docetaxel treatment led to apoptosis in PC-3 cell line.5)Observed by inverted microscope and Transmission electron microscopy, there was no significant difference between PEG-PLA/docetaxel and docetaxel treatment on PC-3 cell line. So was between LHRH-PEG-PLA/docetaxel and docetaxel treatment.6)Observed by MTT, flow cytometry and morphological study, PEG-PLA/ docetaxel and LHRH-PEG-PLA/docetaxel have no effect on growth inhibition of PC-3 cell line.7)PC-3 cells pretreated with antisense oligonucleotide against LHRH receptor exhibition decreased LHRH-PEG-PLA/docetaxel efficacy, without any change in docetaxel efficacy. Thus, LHRH-PEG-PLA/docetaxel efficacy is likely mediated via LHRH receptor.8)The experimental results indicated that in the anti-tumor process of the LHRH-PEG-PLA/ docetaxel, the Caspase-3 gene expression increased, the Bcl-2 gene expression cut down. The surviving gene expression depended on cell cycle. This decided that low-dose LHRH-PEG-PLA/docetaxel treatment led to arrest in G2-M phase in PC-3 cell line, and little apoptotic cell. 3.conclusion1)PEG-PLA/docetaxel and LHRH-PEG-PLA/docetaxel were synthesized successfully, and both of them could form micelles structures by self-assembly in water.2)PEG-PLA/ docetaxel and LHRH-PEG-PLA/docetaxel have no effect on growth inhibition of PC-3 cell line.3)There was no significant difference between PEG-PLA/docetaxel and docetaxel on inhibition effect of PC-3 cell line.4)It was concluded that LHRH-targeted micelles significantly enhanced the PC-3 cell uptake of encapsulated drug, which also resulted in increased antiproliferative activity of docetaxel.5)Low-dose PEG-PLA/docetaxel and LHRH-PEG-PLA/docetaxel treatment led to arrest in G2-M phase, whereas high-dose PEG-PLA/docetaxel and LHRH-PEG-PLA/docetaxel treatment led to apoptosis in PC-3 cell line.6)The surviving gene expression depended on cell cycle. This was possible mechanism that low-dose LHRH-PEG-PLA/docetaxel treatment led to arrest in G2-M phase in PC-3 cell line, and little apoptotic cell.4.Creative of this research1)PEG-PLA and LHRH-PEG-PLA conjugated with docetaxel by covalent bond, both of them could form "shell-core" structure aqueous soluble micelles.2)PEG-PLA/docetaxel and LHRH-PEG-PLA/docetaxel prepared in this study retained pharmacologic effects of docetaxel on PC-3 cell line.3)LHRH-PEG-PLA/docetaxel micelles significantly enhanced the PC-3 cell uptake of encapsulated drug, and the antitumor efficacy was likely mediated via LHRH receptor.