Objective To explore the effect of pyropheophorbide-a methyl ester-mediated photodynamic therapy (MPPa-PDT) on the apoptosis in human osteosarcoma cell line MG63 and the underlying mechanism. Methods Human osteosarcoma MG63 cells in logarithmic growth phase were divided into 4 groups: blank control group (control group), the MPPa treatment group (MPPa group), the light irradiation group (LED group), and MPPa-PDT treatment group (MPPa-PDT group). MPPa-PDT group and MPPa group were incubated with MPPa (0.75?μmol/ L) for 20 hours in dark condition; control group and LED group were incubated with equal volume of fresh medium for 20 hours in the same condition. After washing with PBS and replacement with fresh culture medium, LED group and MPPa-PDT group cells were exposed to light (4.8 J/cm2) for 120 seconds. After light exposure, all groups were cultured in dark condition again. Then cellular morphology changes were observed by an inverted phase contrast microscopy, endoplasmic reticulum morphology changes were observed by transmission electron microscopy, cellular apoptosis was detected by Hoechst33258 nuclear staining, cell apoptotic rate and the levels of Ca in cells were analyzed by flow cytometry, the expression of p-PERK, C/EBP homologous protein (CHOP), cleaved-Caspase-12 were assayed by Western blot. Results In MPPa-PDT group, the retracted and round cells were observed; Hoechst33258 nuclear staining showed nuclear condensation, fragmentation, and other typical apoptotic morphological changes; the cell apoptotic rate (48.76%±3.54%) was significantly higher than that of control group (5.04%±0.41%), MPPa group (5.33%±0.38%), and LED group (6.48%±0.46%) (P < 0.05); the levels of Ca2+ in cells (485.29±58.77) was also significantly higher than that of control group (97.24±4.77), MPPa group (97.95±6.30), and LED group (101.17±5.26) (P < 0.05); swelling endoplasmic reticulum was observed under transmission electron microscope; the expressions of p-PERK, CHOP, and cleaved-Caspase-12 gradually increased at 1, 3, and 6 hours after treatment respectively, which were significantly higher than those of the other groups (P < 0.05). There was no typical apoptotic morphological changes and endoplasmic reticulum morphological changes in control group, MPPa group, and LED group, and there was no significant difference in the above indexes among 3 groups (P > 0.05). Conclusion MPPa-PDT can significantly induce apoptosis in MG63 cells. The endoplasmic reticulum stress pathway is involved in the MPPa-PDT induced apoptosis.
As the most common primary malignant bone tumor in children and adolescents, osteosarcoma has the characteristics of high malignancy, easy metastasis and poor prognosis. The recurrence, metastasis and multi-drug resistance of osteosarcoma are the main problems that limit the therapeutic effect and survival rate of osteosarcoma. Among them, lung metastasis is often the main target organ for distant metastasis of osteosarcoma. In recent years, people have paid attention to the signaling pathway of the occurrence and development of osteosarcoma and made in-depth studies on its mechanism. A variety of relevant signaling pathways have been constantly clarified. At present, there is still a lack of systematic and multi-directional exploration and summary on the signaling pathway related to the pulmonary metastasis of osteosarcoma. This paper explores the new direction of targeted therapy for osteosarcoma by elucidating the relationship between the signaling pathway associated with osteosarcoma and the pulmonary metastasis of osteosarcoma.
Objective To investigate the possibility of using extendible distal femoral replacements in the treatment of osteosarcoma in growing individuals. Methods From December 1999 to March 2003, 3 cases (2 were typeⅡB, 1 was type ⅡA) with osteosarcoma were treated byextendible distal femoral replacements. Of the 3 cases, 2 underwent prosthesis extention operation, 1 was not operated. Results After the removal of tumor, the extremities of 2 patients were shortened by 4 to 5 cm within 2 to 3 years. After the lengthening procedure, the affected extremities were of equal length to the unaffected extremities and no drag symptoms of blood vessel and nerves were observed. Follow-up was done for 2 months to 3 years. There was no aseptic loosening. The function of joints was fairly good. Conclusion Extendible distal femoral replacements is an easy, convenient, and effective way to treat osteosarcoma.
Abstract：Pulmonary metastasectomy is an important curative option for patients with osteogenic and softtissue sarcoma spread to the lungs. Complete surgical removal of pulmonary metastases can improve survival and is recommended under certain criteria. Specific issues that require consideration when planning pulmonary metastasectomy include: preoperative assessment of the operation index and contraindications, choice of surgical strategies, pulmonary parenchymal preservation, and the role of lymphadenectomy. With the development of iconography and chemotherapy, the emergence of targeted drugs, and the innovation of radiotherapy, the concept of the diagnosis and treatment for pulmonary metastases from osteogenic and softtissue sarcoma is also undergoing great changes.
Objective To review the research progress of the treatment of osteosarcoma, and to thoroughly understand its current state of research and prospect so as to lay a sol id foundation for the cl inical treatment. Methods The cl inical and experimental research l iteratures about treatment of osteosarcoma were extensively reviewed and analyzed. Results The present treatment of osteosarcoma is still need to comprehensive therapy which combine chemotherapy and surgical treatment. There are some progresses in gene therapy and molecular targeting therapy which can improve survival rate. Furthermore, well-designed studies and cl inical trials are needed to evaluate the potential therapeutic impact before they are used in cl inical. Conclusion Advancement in chemotherapeutic regimens has improved survival and l imb-sparing surgery in the treatment of osteosarcoma, but the progress of gene therapy and molecular targeting therapy gives new hope for osteosarcoma patients.
ObjectiveTo investigate the feasibility and effectiveness of proximal tibial hemiprosthesis replacement in the first stage and prosthesis revision in the second stage in reducing the risk of length discrepancy of limbs in children with proximal tibial osteosarcoma.MethodsBetween 2009 and 2013, 3 children with conventional osteosarcoma at the proximal tibia (stage ⅡB) were treated. There were 2 boys and 1 girl. They were 12, 13, and 13 years old, respectively. After 4 courses of preoperative chemotherapy, the proximal tumor segmental resection and proximal tibial hemiprosthesis replacement were performed. Then the patients underwent prosthetic revision in the second stage when they were 20, 17, and 17 years old, respectively.ResultsAll patients successfully completed two stages of operations. The length discrepancy of lower limb after the second stage operation were 19, 7, and 21 mm, respectively. Three patients were followed up 13, 3, and 27 months after the second stage operation, and the lower extremities functions were satisfactory. The Musculoskeletal Tumor Society (MSTS) score was 26, 27, and 25, respectively.ConclusionThe proximal tibial hemiprosthesis replacement in the first stage combined with prosthesis revision in the second stage for treating the proximal tibia osteosarcoma in children can keep the distal femur growth ability, reduce the length discreapancy of lower limb, and obtain satisfactory stability and good function.
Objective To design, construct and select the optimal repl ication-defective recombinant adenovirus mediated short hairpin RNA (shRNA) which is transduced into human osteosarcoma cells to silence c-myc gene expression, and to construct the recombinant adenovirus vector expressing c-myc-shRNA and determine its viral titer. Methods Three pairs of complementary single-stranded ol igonucleotides (ss ol igos) were designed and synthesized, and then they were annealed to create a double-stranded ol igonucleotide (ds ol igos).The ds ol igos were cloned into pENTR/U6 vector to produce the shuttle plasmid pENTR/U6-shRNA, which was transduced into osteosarcoma cells by l iposome after sequencing. The plasmid with good silence effect was chosen by RT-PCR to perform the LR recombination reaction to the adenovirus backbone plasmid. The expression clone was transfected into HEK293A cells to produce repl ication-incompetent recombinant adenovirus mediated shRNA against c-myc whose cytopathic effect was observed and viral titer was determined by the viral particle (VP) method and 50% tissue culture infective dose (TCID50). Results Ds ol igos, which was verified by electrophoresis, was cloned into pENTR/U6 vector to produce pENTR/U6-shRNA shuttle plasmid, which was confirmed to be corrected by sequencing. The optimal plasmid with good silence effect was chosen by RT-PCR from the three pairs of double-stranded ol igonucleotide. By Pac I enzyme, the l inearrization repl ication-defective recombinant adenovirus mediated shRNA was constructed to perform the LR recombination reaction to the adenovirus backbone plasmid. The cytopathic effect and vacuole phenomenon of adenovirus mediated shRNA appeared at 3 days and became obvious at 6 days. The adenovirus virus titer in the first generation was 5.23 × 109 VP/mL, and reached 2.26 × 1012 VP/mL via 3-4 generations’ ampl ification. The viral titer was 10-3.8/0.1 mL determined by VP method and TCID50. Conclusion The recombinant adenovirus mediated shRNA c-myc is constructed in vitro through RNA interference technology.
ObjectiveTo investigate the effects of cinobufagin on the apoptosis in U-2OS osteosarcomas cells (U-2OS cells) and explore its potential mechanism. MethodsThe cytostatic effects of cinobufagin (10, 20, 50, 100, 200, and 400 nmol/L) on U-2OS cells were evaluated by MTT assay at 24, 48, and 72 hours after culture; simple U-2OS cells served as control group. The impact of cinobufagin (100 nmol/L) on the apoptosis in U-2OS cells was determined by flow cytometry at 48 hours after culture, which were treated with cinobufagin (experimental group) or with cinobufagin plus Z-VAD-FMK (control group), and simple U-2OS cells served as blank control group. The Caspase-3 activity was measured by Caspase-3 activity assay kit at 48 hours after culture, which were treated with cinobufagin (20, 50, and 100 nmol/L), and simple U-2OS cells served as control group.The expression of apoptosis signal pathway related proteins in U-2OS cells treated with cinobufagin were detected by Western blot at 48 hours after culture, which were treated with cinobufagin (20, 50, and 100 nmol/L), and simple U-2OS cells served as control group. ResultsThe results of MTT assay showed that cinobufagin inhibited the proliferation of U-2OS cells in a dose- and time-dependent manners. At each time point, the growth rate of U-2OS cells was significantly reduced with the increasing cinobufagin concentration, and as time prolonged, the growth rate of U-2OS cells behaved the same way in the same group. There were significant differences among different time points and groups (P<0.05). The apoptotic rate of experimental group (46.87%±11.23%) was significantly higher than that of the control group (2.34%±0.98%) and blank control group (1.04%±0.25%) (P<0.05). The Caspase-3 activity in 20, 50, and 100 nmol/L groups were 1.14±0.32, 1.31±0.41, and 1.92±0.54, respectively, which were significantly higher than that in control group (P<0.05). Compared with 20 and 50 nmol/L groups, 100 nmol/L group significantly increased the Caspase-3 activity in U-2OS cells (P<0.05). Compared with the control group, the expressions of cleaved Caspase-3, cleaved Caspase-9, and Bax were obviously up-regulated; the Bcl-2 expression was down-regulated; and the ratio of Bax/Bcl-2 was increased in different cinobufagin-treated groups (P<0.05). The same tendency was seen in different cinobufagin-treated goups, showing significant differences among groups (P<0.05). ConclusionCinobufagin can inhibite the proliferation of U-2OS cells, and induce cell apoptosis. The potential mechanism of cinobufagin-induced apoptosis may be related to the mitochondria-mediated pathway.