| Prostate cancer is one of the most prevalent malignant diseases among menin Western countries, and represents the second leading cause of cancer death,and is becoming more common in China. Localized prostate cancer is typicallytreated with surgery or radiation, and recurrent disease can be controlledtemporarily with androgen ablation. However, almost all prostate carcinomaseventually become hormone refractory and progress rapidly thereafter. Therefore,the development of new molecule-targeted therapies is imperative.Prostate-specific membrane antigen (PSMA) is a type II membraneglycoprotein of 100 kD, with a short intracellular segment, a transmembranedomain, and an extensive extracellular domain. Its expression is independent ofandrogen, increases with disease progression, and becomes the highest inmetastatic, hormone-refractory diseases. In addition, PSMA is abundantlyexpressed in new vessels associated with the tumor but not in normal vessels.Considering these points, the combination of gene therapy and anti-PSMA antibody represents an ideal treatment measure.Caspases are vital elements in transducing apoptotic signals and mediatingapoptosis in mammalian cells. Among the critical cysteine protease familymembers, caspase-3 plays an essential role in the cleavage of diverse cellularproteins. Furin is a cellular endoprotease and has been implicated in theproteolyticy activation of several secreted proteins. Furin-mediated cleavage ofimmunotoxins is both an obligatory and a rate-limiting step for their cytotoxicactivity.ObjectiveObjective:To established a novel fusion gene, consisting of NH2-terminal leadersequence fused with an anti-PSMA antibody (J591), the furin cleavagesequences of diphtheria toxin (Fdt), and the reverse coding sequences of the largeand small subunits of caspase-3 (revcaspase-3) and evaluate the antitumoractivity of immunocasp-3 and immunocasp-3 -secreting lymphocytes in both invitro and in vivo models.Methods:1. To established a novel fusion gene named as immunocasp-3. The pCMVJ591-Fdt-revcaspase-3 plasmid and the empty pCMV vector as a control, weretransfected into Jurkat cells using the Lipofectin reagent. Cells stably expressingthe plasmid were selected with G418. The cells transfected with immunocasp-3are referred to as Jurkat-immunocasp-3. Expression of Jurkat-immunocasp-3was detected by PCR and Western-blot.2. The pCMV-J591-Fdt-revcaspase-3 plasmid was transfected into LNCaPcells (positive for PSMA) and PC-3 cells (negative for PSMA) using theLipofectin reagent.In vitro cell growth was investigated by MTT assay. cellapoptotic index was investigated by flow cytometer and electronic microscope. Transwell filters, which separate the cells but not large molecules, were used forthe cocultivation assay. Tumor cells overexpressing PSMA (LNCaP) and controlcells expressing undetectable PSMA (PC-3) were placed at the bottom chamber,and the transfected Jurkat cells were placed at the top chamber. The ratio ofJurkat: cancer cells was adjusted to 3:1. Viable cells at the bottom chamberswere counted by trypan blue exclusion at the indicated time points aftercocultivation. The percentages of cell killing were determined as follows: 1 - (thenumber of cells cocultured with stably transfected Jurkat/the number of cellscocultured with Jurkat controls)×100%.3. Tumorigenicity assay was performed in nude mice model to assess theantitumor activity of immunocasp-3 in vivo. Tumors were allowed to grow untilthey reached a diameter of 5–7 mm. The mice were then randomly divided intodifferent treatment groups of 5 mice each. One group of mice received 6 doses of10?g of pCMV-J591-Fdt-revcaspase 3 mixed with lipofectamine asintramuscular injections administered in the right posterior limb. Another groupof 5 mice received 3 weekly intravenous injections of 2×106 Jurkat-J591-Fdtrevcaspase3 cells. Control mice were injected with either liposome-mixed emptypCMV vector or unmodified Jurkat cells. Tumor growth was monitored by usinga caliper, with measurement of 2 perpendicular tumor diameters every 3 days,and the volume of the tumor was calculated using the formula: tumor volume =(width)2×length/2. Survival of the mice was also assessed. Cancer and normaltissues were fixed in 10% neutral-buffered formalin, and then processed inparaffin. H&E staining was used for histological verification of tumor growth.To determine if caspase-3 expression was maintained in vivo,immunohistochemistry of the tumors were performed using rabbit anti-humancaspase-3 polyclonal antibody. ResultsResults:1. we successfully established pCMV-J591-Fdt-revcaspase-3 plasmid,referred to immunocasp-3. Vector sequences were confirmed by DNAsequencing. we alse successfully established human CD4+ Jurkat cells overexpressingpCMV-J591-Fdt-revcaspase-3. The expression of the immunocasp-3gene was confirmed by reverse transcription-PCR (RT-PCR) using primersspecific to anti-PSMA antibody fragment (J591) and Fdt-revcaspase 3. Theexpression and secretion of fusion proteins were confirmed by western blotanalysis of cell culture supernatants. As expected, the immunocasp-3-modifiedJurkat cells remained viable and showed growth and proliferation properties thatwere similar to those of unmodified cells, suggesting that the expression of thechimeric protein is associated with low toxicity.2. Fusion proteins were transiently expressed in PSMA-positive LNCaPand PSMA-negative PC-3 cells. Apparent cell death was observed in LNCaPcells but not in PC-3 cells at 48 h after transfection. As observed by the electronicmicroscope, the expression of immunocasp-3 resulted in typical apoptoticchanges?such as shrinkage nuclei, chromatin condensation, plasma membraneblebbing and chromatin breakage fragments. Moreover, Annexin V-FITCstaining performed 48 h after the transfection also revealed that apoptotic cellswere present in 15.3% of the cells transfected with the immunocasp-3 expressionvector, a percentage which was higher than that in the control cells(5.5%).The genetically modified Jurkat cells were then cocultivated in vitro withPSMA-positive LNCaP and PSMA-negative PC-3 cells. The ratio of Jurkat:target cells was adjusted to 3:1, on the basis of pilot experiments designed toidentify the optimal ratio for cell killing. A significant number of LNCaP cells,but not PC-3 cells, were killed by immunocasp-3-secreting Jurkat cells. More than 50% of the cells died within 5 days of coculture, and additional killing ofPSMA-overexpressing tumor cells was seen after longer times.3. We assessed the in vivo antitumor activity of immunocasp-3 in nudemice xenograft models containing PSMA-overexpressing LNCaP cells. Both thevector-lipofectamine-treated group and Jurkat-cell-treated groups showed greaterdecrease in tumor volume and longer survival time than controls. Meanwhile,Jurkat-cell-treated group was more efficient than the vector-lipofectamine groupin reducing tumor size (P < 0.05), suggesting that this strategy of geneadministration yields more effective and sustained antitumor activities in LNCaPxenografts.Immunohistochemical analysis confirmed the presence of caspase-3activity in tumors treated with Jurkat cells expressing J591-Fdt-revcaspase 3, butnot in the normal tissues. The normal tissues displayed normal morphology inHE staining results.This study shows that the immunocasp-3 proteins selectively recognizedand induced apoptotic death in PSMA-overexpressing LNCap cells in vitro andin vivo. Moreover, immunocasp-3 were mainly composed of humanizedantibodies and human endogenous proteins, which rarely aroused the immuneresponse to xenogenic proteins. In addition, apoptotic cells were engulfed bysurrounding cells without causing inflammation, which, together with the abovementionedfetures of ImmunoAIF, contributes to a comparatively low toxicity topatients. Therefore?we suggest that treatment using this gene merits furtherinvestigation and consideration as a molecularly targeted therapeutic measure forprostate cancers. |
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