| Osteosarcoma is the most common primary malignant tumor of bone for adolescent or children, and have the distinct characteristic of early metastasis,80%of patients have already had pulmonary metastasis when the clinical diagnosis of osteosarcoma is made. In recent decades, remarkable advance in the treatment of osteosarcoma has been made. Though nowadays nearly80%of patients can have their invalid extremities preserved after treatment and the5-year survival rate has been elevated from approximately20%to nearly80%, still more than50%of the osteosarcoma patients would die as a result of relapse and metastasis. There are still many difficulties and suspects in the treatment of osteosarcoma, so it’s very necessary to explore the more effective and less toxic treatments. In recent10years, gene therapy, immunotherapy and molecular targeted therapy of tumor have become a promising strategy for the treatment of osteosarcoma. The exploring of new effective treatment is now a significant direction in osteosarcoma research. Herein, we describe experiments conducted with a fusion gene, immunocasp-6, which was generated by fusing a HER2-specific single-chain Ab, a single-chain Pseudomonas exotoxin A and an active caspase-6which can directly cleave lamin A leading to nucleus damage inducing programmed cell death. IntroductionThe human epidermal growth factor receptor-2(HER2), a member of the epithelial growth factor receptor family, transduces cell signaling and plays key roles in cell differentiation, adhesion, and motility. Sufficient evidence has suggested that patients with HER2-overexpressing tumors exhibit a reduced response to conventional treatments. The HER2protein is reportedly overexpressed in several human malignant tumor, including human breast and ovarian cancer, salivary gland adenocarcinoma, gastric cancer and osteosarcoma. Since it is overexpressed in tumor cells but is not detected in normal cells, HER2is an ideal target molecular for cancer gene therapy to exploit differences at the molecular level between normal and malignant cells.Caspases are vital elements in transferring apoptotic signals and executing apoptosis in mammalian cells. Caspase-6is one of effective caspases during the cell apoptotic program. Activation of caspase-6induces apoptosis by cleaving lamin A and other substrates. Unlike its wild-type zymogen counterpart, active caspase-6constructed with subunits in reverse order, is capable of autocatalytic processing in vitro independent of apoptotic signals, and can induce apoptosis of tumor cells, which thereby makes it an attractive candidate for gene therapy.Pseudomonas exotoxin A (PEA) is a single toxin molecule comprising of three large structure domain (Ⅰ Ⅱ, Ⅲ), Domain Ⅱ of PEA is responsible for the molecules to target cells, Domain Ⅱ of PEA is responsible for efficiently transferring the cellular toxicity domain to the cytoplasm, Domain Ⅱ of PEA is responsible for inducing cell dead. Domain Ⅱ of PEA has been reported to efficiently transfer the cellular toxicity domain to the cytoplasm. By replacing the cellular toxicity domain of PEA with active caspase-6, we sought to translocate the caspase into tumor cells in which it would induce tumor cell apoptosis. As a well-recognized Ab, e23sFv, derived from a mouse mAb against human HER2, has been confirmed to bind the extracellular domain of HER2protein with high affinity and to be internalized by endocytosis. The highly specific antibody to antigen suggests that e23sFV of the immunocasp-6fusion gene can specifically recognize and bind to HER2overexpreing osteosarcom, but not cells with undetectable HER2. After immunocasp-6fusion gene was internalized into cells by the domain II of PEA, caspase-6can recongnize and cut cell substrate and carry out pro-apoptosis function, consequently we constructed the immunocasp-6fusion gene can specifically and efficiently suppress the HER2overexpressing tumors. Even though this novel immunocasp-6has been proven to be effective in inducing apop-tosis in the HER2-overexpressing human breast tumor cell line, SKBR-3, their effects on human osteosarcoma is still unclear. Thus the purpose of the present study is to extend our immunocasp-6strategy to the treatment of HER2-overexpressing malignant tumor, and to verify that the immunocasp-6can specifically and efficiently suppress the HER2-overexpressing osteosarcoma in vitro as well as in vivo.Materials and methodsPlasmid and DNA construct Recombinant immunocasp-6was generated by sequential fusion of the genes of a signal peptide (Met-Lys-His-Leu-Trp-Phe-Phe-Leu-Leu-Leu-Val-Ala-Ala-Pro-Arg-Trp-Val-Leu-Ser-) consisting of a single chain HER2antibody (e23sFv), a Pseudomonas exotoxin A (PEA) translocation domain (from aa253to412) and an active caspase-6. The immunocasp-6was cloned into a pCMV plasmid, namely pCMV-immunocasp-6.Cell culture. Human osteosarcoma cell line SOSP-9607-E10, with relatively high metastatic potential, was derived from a17-year-old male patient who had been diag-nosed of tibial osteosarcoma and underwent osteotomy and established from these cells by continuous in vitro cultivation for over120transfer generations in one year. SOSP-9607-E10cells were maintained in DMEM or RPMI-1640(Invitrogen) supplemented with10%fetal bovine serum (FBS) and4mmol/1L-glutamine.Detection of HER2antigen on the surface of osteosarcoma SOSP-9607-E10cells. Osteosarcoma SOSP-9607-E10cells were stained with antibodies recognizing HER2as the primary antibodies and tested by indirect immunofluorescence staining and flow cytometry detection according to the standard procedure when they growed to a certain number. Osteosarcoma tissue of82patients came from bone cancer research and tested by the PLA and tested by immunohistochemistry assay according to the standard procedure of the Vectastain Elite ABC kit per the manufacturer’s instructions prior to digital photography under an Olympus Eclipse E600microscope with a Spot RT slider camera and imaging software.Cell transient transfection. At24h before transfection, cells were seeded in12or96-well plates at1x105or5x103cells per well. pCMV-immunocasp-6or pCMV vector alone,1μg, encapsulated by2μl Lipofectamine2000(Invitrogen) were mixed, incubated for20min at room temperature to form DNA-liposome mixture. Then the mixture was administered to cells and incubated in a humidified incubator at37℃with5%CO2for6h, then the medium was removed and cells were resuspended in complete medium.Cell viability assay. Viability of the transiently transfected cells was tested by using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT) assay. Briefly, after the cells adhered to the low lamber of96-well plates, the cells were divided randomly into three groups, namely the mock group, the control group and the immunocasp-6group, and transfected with pCMV-immunocasp-6or pCMV vector. Thereafter, the cells were cultured in96-well plates for12to96h, then incubated with20μl of1.5mg/ml MTT for4h. After that, the cells were treated with150μl DMSO. OD at A490nm were determined using the Sunrise microplate reader (Tecan). Each assay was performed in triplicate on at least three independent occasions.Flow cytometry assay for detection of apoptosis. SOSP-9607-E10cells were seeded at a density of1x105cells per well on slides in12-well Costar transwell plates, and transfected with pCMV-immunocasp-6or pCMV vector when cell number was at a density of3x105cells per well. After48h of transfection, cells in the lower chamber were collected, stained with Annexin V-FITC/PI following standard procedures and finally analyzed by FCM.Immunofluorescence. After transfection SOSP-9607-E10cells were incubated in complete medium for an additional48h. Then, the cells in the lower chamber were fixed in paraformaldehyde solution (4%in phosphate buffered saline (PBS), pH7.4), permeabilized with PBS containing0.1%Triton X-100, and blocked with2%normal rabbit serum. Then the cells were stained with antibodies recognizing caspase-6(C20,1:200; Santa Cruz Biotechnologies) as the primary antibodies, with biotin-linked anti-goat IgG (1:100; Santa Cruz Biotechnologies) and FITC-linked anti-rabbit IgG (1:100; Sigma) as the secondary antibodies. The staining was examined using a fluorescence microscope (Japanese Olympus Co.).Electronic microscopy assay. SOSP-9607-E10cells were harvested48h after transfection, and then fixed in2.5%glutaraldehyde, dehydrated and embedded to observe morphologic change with a transmission electron microscope.Immunohistochemistry assay. The transfected cells were cultured on coverslips as mention above, and then fixed with a freshly prepared paraformaldehyde solution for30min at room temperature, and permeabilized with0.1%Triton X-100for15min on ice.The sample use goat anti-human Caspase-6as the first antibody (C20,1:200; Santa Cruz Biotechnologies), followed by biotin-linked anti-rabbit IgG (1:100; Santa Cruz Biotechnology) as the secondary antibody and then processed with the Vectastain Elite ABC kit per the manufacturer’s instructions prior to digital photography under an Olympus Eclipse E600microscope with a Spot RT slider camera and imaging software.Antitumor activity of immunocasp-6in vivo. Six-to eight-week-old BALB/c athymic mice were purchased from National Rodent Laboratory Animal Resources, Shanghai Branch (Shanghai, China), and were cared and used in compliance with institutional guidelines. The mice were inoculated s.c. with2x106SOSP-9607-E10cells. Tumors were allowed to grow until they reached a diameter of5-7mm (day0). The mice were then randomly divided into different two groups, namely the immunocasp-6group and vector group.The mice bearing SOSP-9607-E10tumors were subjected to liposome-mediated immunocasp-6or vector treatments. pCMV-immunocasp-6or pCMV vector alone,10μg, encapsulated by20μl Lipofectamine2000was administered i.m. to mice. Nine mice were utilized for each treatment, each mouse was administered every3days for a total of five times. All the BALB/c athymic mice were killed two weeks later.The volume of the tumor, the body weight of the mice and the net weight of the tumor were observed and analyzed by statistics.HE (hematoxylin-eosin) staining. Xenograft tumors were fixed in paraformaldehyde solution and embedded in paraffin after treatment, paraffin-embedded tissue sections were dewaxed, hydrated. HE (hematoxylin-eosin) staining was performed in accordance with the manufacture’s instructions. Hematoxylin was used to counterstain the sections.TUNEL staining. TUNEL staining was performed on paraffin sections, using the TdT-FragELTM DNA Fragmentation Detection kit (Calbiochem) in accordance with the manufacture’s instructions. Hematoxylin was used to counterstain the sections.Immunohistochemistry assay. Xenograft tumors and muscle tissues were fixed in paraformaldehyde solution and embedded in paraffin after treatment, paraffin-embedded tissue sections were dewaxed, hydrated, and incubated in0.3%methanol-H2O2for20min to remove endogenous peroxidase. Next, they were dried and blocked for1h with the appropriate serum in a humidified chamber. Primary antibody was added overnight at4℃. The sample use goat anti-human Caspase-6as the first antibody (C20,1:200; Santa Cruz Biotechnologies), followed by biotin-linked anti-rabbit IgG (1:100; Santa Cruz Biotechnology) as the secondary antibody and then processed with the Vectastain Elite ABC kit per the manufacturer’s instructions prior to digital photography under an Olympus Eclipse E600microscope with a Spot RT slider camera and imaging software.Assessment of immunocasp-6effects on human osteosarcoma lung metastasis. Eighteen athymic, six-to eight-week-old Balb/c mice were inoculated with2x105SOSP-9607-E10cells into thighbone marrow. The mice were then divided randomly into two groups, nine mice in each group, for i.m. liposome-mediated immunocasp-6or vector treatments as indicated above. The treatment was performed once every3days for two weeks, then once a week thereafter for five weeks. The number of the neonatal tumors in lungs were counted, mouse survival times were recorded and the neonatal mass was tested by H&E staining.Statistical analyses. The data are expressed as the mean±SD. Statistical analyses were performed with the SPSS13.0software package for Windows (SPSS, Chicago, IL). Cell viability assay were analyzed by the analysis of covariance (ANCOVA, dunnett T3) method. The volume of the tumor, the body weight of the mice and the net weight of the tumor were analyzed using independent-samples t-test (the data of the volume of the tumor and the body weight of the mice were obtained from the data before treatment subtracted by the data after treatment). The survival rates were analyzed using the Kaplan-Meier method, and comparisons among treatment groups were obtained using the log-rank test. Statistical significance was based on a value of P<0.05.ResultsImmunocasp-6effectively and specifically suppresses the growth of osteosarcoma SOSP-9607-E10cells in vitro. Xu et al. have found the immunocasp-6fusion gene can specifical bind to HER2-overexpressing breast tumor cells, internalizes, under-goes autoprocessing between PEA Arg279and Gly280, and induces cell apoptosis, but not in cells with undetectable HER2. To investigate whether the same cytotoxic effect could be achieved in osteosarcoma cells, we first tested the cell viability of the transiently transfected cells by MTT assay, The finding discovered the transient expression of the immunocasp-6led to an apparent delay in cell viability. In other words, tumors cells in the immunocasp-6treatment group have weaker viability than those in the mock or control group (P=0.013, P=0.007, respectively), while there is no significantly difference between the mock group and control group (P=0.989). The Annexin V-FITC staining48h after the transfection revealed that the percentages of apoptotic cells in the immunocasp-6group were31.4%, while only6%in the vector group. When tumor cell growth was stunted, the morphological change of transfected cells were tested. Immunofluorescence test discovered that transiently transfected cells had enriched or chipped nuclear. Transmission electron microscopy presented typical apoptotic changes in cells, including chromatin condensation and its margination at the nuclear periphery, cellular shrinkage and blebbing, and formation of so-called apoptotic bodies. Furthermore, immunohistochemistry staining with anti-caspase-6antibody revealed that most of the cells were caspase-6positive, suggesting that caspase-6was effective in inducing tumor cell apoptosis.Immunocasp-6transduction-induced HER2-overexpressing osteosarcoma cell death in subcutaneously transplanted nude mice. As showed in Table Ⅱ, the growth of the tumor in the liposome-mediated pCMV-immunocasp-6treatment group was significantly slower than that of the pCMV vector group (t=3.177, P=0.001), the body weight of the mice in the immunocasp-6group was significantly heavier than that of the vector group (t=-5.153, P=0.0002), and the net weight of the tumor in the immunocasp-6group was significantly less than that of the vector group (t=5.407,P=0.0006). Then the osteosarcoma tissues were collected and H&E (hematoxylin and eosin) stain was performed. The staining discovered tumor tissues were in poor condition in which most of tumor tissue was dead. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) staining, demonstrated that most of the tumor tissue in the immunocasp-6group were in the state of apoptosis. Thereafter, immunohistochemistry analysis confirmed the presence of caspase-6in tumor tissues in the immunocasp-6group, but not in those treated with vector and muscle tissues in either immunocasp-6group or vector group. These findings suggest that immunocasp-6can specifically and efficiently induce tumor tissue apoptosis.Inhibitory effect of immunocasp-6on lung metastasis of HER2-overexpressing osteosarcoma in vivo. The neonatal mass were diagnosed as osteosarcoma by H&E staining. The findings disovered that the number of neonatal tumors in lungs in the immunocasp-6group was significantly less than that in the vector group. Furthermore, the mice in the immunocasp-6group survived longer than that in the vector group. SOSP-9607-E10tumors were more likely to metastasize to the lung in the absence of immunocasp-6treatment, suggesting that immunocasp-6can prevent or slow osteosarcoma metastasis.ConclusionOur study has proved that immunocasp-6can specifically recognize and bind to HER2overexpreing osteosarcoma SOSP-9607-E10cells, after internalized into cells they can recongnize and cut cell substrate and carry out pro-apoptosis function,while they have no damage to HER2-low(or no)expressing tissues. In a word, in our constructed fusion gene, e23sfv still can selectively recognize and bind to HER2antigen, and recombinant Caspase-6still have high pro-apoptosis activity.Different from usually built immunotoxin molecular, caspase-6is the end of the common effect of protease in different cell apoptosis pathway and is direct practitioners of cell apoptosis,therefore, the fusion gene we built will more effectively promote the HER2positive tumor cell apoptosis and won’t damage to normal cells. Furthermore, the immunocasp-6fusion gene generated by fusing a humanized HER2-specific single-chain Ab, a single-chain Pseudomonas exotoxin A and an active caspase-6has low immunogenicity and can be helpful for repeated usage, Which can provide a basis for a sustainable, efficient, safe and special gene therapy to HER2overexpress tumors. |
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