藤黄酸联合顺铂对人骨肉瘤细胞作用的实验研究

[目的]本实验主要研究藤黄酸(gambogic acid,GA)在骨肉瘤中的抗肿瘤作用,检验藤黄酸与传统的骨肉瘤化疗药物顺铂(cisplatin,CDDP)联用是否具有协同作用,探讨其作用的机制。[方法]采用CCK-8法测定不同浓度藤黄酸、顺铂单用及联合应用对人骨肉瘤细胞MG-63的增殖抑制作用,PI染色流式细胞术检测细胞周期变化,Annexin V-FITC/PI双染色检测细胞凋亡变化。[结果]藤黄酸对MG-63具有明显生长抑制作用,且具有浓度依赖性,IC 25为0.52μg/ml,藤黄酸诱导MG-63细胞产生G2/M期细胞周期阻滞和凋亡,而顺铂则诱导产生G0/G1期细胞周期阻滞和凋亡。藤黄酸与顺铂联用时增殖抑制及诱导凋亡作用比单用时明显增强。[结论]藤黄酸通过诱导MG-63细胞周期阻滞和凋亡产生抗人骨肉瘤细胞增殖作用,藤黄酸与顺铂联用具有协同作用。     

Anticancer effects of zoledronic acid against human osteosarcoma cells.

Based on neoadjuvant chemotherapy, the prognosis of osteosarcoma patients has improved dramatically. However, due to therapy resistance in patient subgroups, the development of new treatment strategies is still of utmost importance. The aim of our study was to test the effects of the nitrogen-containing bisphosphonate zoledronic acid (ZOL) on osteosarcoma cell lines (N = 9). Exposure to ZOL at low micromolar concentrations induced a dose- and time-dependent block of DNA synthesis and cell cycle progression followed by microfilament breakdown and apoptosis induction. The ZOL-induced cell cycle accumulation in S phase was accompanied by significant changes in the expression of cyclins and cyclin-dependent kinase inhibitors with a prominent loss of cyclin E and D1. ZOL not only inhibited growth but also migration of osteosarcoma cells. The mevalonate pathway intermediary geranyl-geraniol (GGOH) but not farnesol (FOH) significantly inhibited the anticancer effects of ZOL against osteosarcoma cells. Correspondingly, ZOL sensitivity correlated with the blockade of protein geranylgeranylation indicated by unprenylated Rap1. Overexpression of even high levels of P-glycoprotein, as frequently present in therapy-resistant osteosarcomas, did not impair the anticancer activity of ZOL. Summarizing, our data suggest that ZOL, which selectively accumulates in the bone, represents a promising agent to improve osteosarcoma therapy.     

Metastatic tumor cells detection and anti‐metastatic potential with vesicular stomatitis virus in immunocompetent murine model of osteosarcoma

Sarcomas are associated with a high incidence of lung metastasis, which leads to a high‐risk of cancer death. This study was performed to explore the pre‐clinical theranostic potential of a novel fully functional recombinant vesicular stomatitis virus carrying imaging gene Katushka (rVSV‐K), as virotherapy and circulating tumor cells (CTCs) detection in the syngeneic mouse model of osteosarcoma with spontaneous pulmonary metastases. Recombinant VSV‐K was generated and evaluated in vitro on human and murine osteosarcoma cells. Spontaneous osteosarcoma metastases were established in immune‐competent mice by implanting subcutaneously syngeneic osteosarcoma LM8 cells. The vector was injected into the tumor‐bearing mice via jugular vein either once or repeatedly. To assess effectiveness, primary tumor growth and development of lung metastasis as well as survival were evaluated. We found that rVSV‐K efficiently replicated in and killed all osteosarcoma cell lines in time‐dependent manners. Both single or repeated systemic injections of the virus did not inhibit the growth of the primary tumor, but the repeated administration could effectively suppress the development of lung metastases and was likely responsible for the observed increase in survival. Furthermore, we demonstrated, for the first time, that CTCs in blood samples from syngeneic osteosarcoma‐bearing mice were successfully detected by utilizing rVSV‐K ex vivo. Our results show that repeated systemic injections of rVSV‐K are an effective anti‐metastatic agent against osteosarcoma in immune‐competent mice and this virus to be a useful tool for detection of osteosarcoma CTCs, suggesting that further development of future viral‐based theranostic approach in patients with osteosarcoma is warranted. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2562–2569, 2018.

     

Inhibition of matrix metalloproteinase-14 in osteosarcoma cells by clodronate

BACKGROUND: Bisphosphonates reduce the bone metastasis formation and angiogenesis but the exact molecular mechanisms involved are unclear. Progelatinase A (proMMP-2; 78 KDa) is activated up during the tumor spread and metastasis by a cell surface-associated matrix metalloproteinase (membrane-type matrix metalloproteinase [MT1-MMP] or MMP-14). MATERIAL AND METHODS: We evaluated the effects of a bisphosphonate (clodronate) on MT1-MMP mRNA expression and protein production, catalytic activity and proteolytic activation of proMMP-2 by cultured human MG-63 osteosarcoma cells. RESULTS: Clodronate, at therapeutically attainable noncytotoxic concentrations, dose-dependently inhibited phorbol myristic acetate (PMA)-induced proteolytic activation of proMMP-2 by human MG-63 osteosarcoma cells. Clodronate also downregulated the PMA-induced expression of MT1-MMP mRNA and protein production in human MG-63 osteosarcoma cells, as evidenced by Northern analysis and fluorescent immunohistochemistry. Furthermore, clodronate inhibited directly and dose-dependently MT1-MMP activity, and the MT1-MMP inhibition by clodronate was reduced in the presence of an increased (5 mM) Ca(2+) concentrations when compared to physiological (1 mM) Ca(2+) concentrations. CONCLUSION: We conclude that (1) the extracellular/cell-associated mechanism of bisphosphonate involves inhibition of MT1-MMP catalytic activity eventually by chelation, and that (2) intracellular mechanism involves downregulation of induced MT1-MMP mRNA and protein expression. The inhibition and downregulation of MT1-MMP by clodronate can be related to their ability to reduce MG-63 osteosarcoma cell invasion and spread. These findings may, at least in part, explain at molecular level the antitumor and antibone resorption activities of clodronate observed in clinical studies.     

Role of integrin‐linked kinase in osteosarcoma progression

Although integrin‐linked kinase (ILK) has been suggested to play a role in the tumorigenesis of a number of human epithelial carcinomas, little is known of its role in musculoskeletal sarcoma. The authors studied ILK expression by immunohistochemistry using osteosarcoma prechemotherapy specimens from 56 patients, and investigated the prognostic implications of the findings obtained. It was found that ILK overexpression was significantly correlated with the presence of distant metastasis (p = 0.008) and that it was an independent prognostic factor for both poor overall survival and poor event‐free survival (p = 0.015 and 0.010, respectively). During a transfection experiment conducted by transfecting osteosarcoma cells with ILK siRNA, VEGF concentrations were measured using an ELISA kit, and then compared with those of untransfected controls to evaluate its angiogenic effects. In addition, apoptotic percentages were measured by Annexin‐V flow cytometry, and invasive properties were evaluated by measuring the numbers of non‐migrating cells in a Boyden chamber. It was found that ILK downregulation significantly decreased angiogenesis, increased apoptosis, and decreased invasiveness of osteosarcoma cells. These results show that ILK is a promising prognostic factor in osteosarcoma and a novel potential therapeutic target for the treatment of osteosarcoma. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1668–1675, 2013     

Eradication of osteosarcoma by fluorescence‐guided surgery with tumor labeling by a killer‐reporter adenovirus

In a previous study, we developed fluorescence‐guided surgery (FGS) for osteosarcoma using an orthotopic model with 143B human osteosarcoma cells expressing red fluorescent protein (RFP) implanted into the intramedullary cavity of the tibia in nude mice. The FGS‐treated mice had a significantly higher disease‐free survival (DFS) rate than the bright‐light surgery (BLS). However, although FGS significantly reduced the recurrence of the primary tumor, it did not reduce lung metastasis. In the present study, we utilized the OBP‐401 telomerase‐dependent killer‐reporter adenovirus, carrying green fluorescent protein (GFP), to label human osteosarcoma in situ in orthotopic mouse models. OBP‐401‐illuminated human osteosarcoma cell lines, 143B and MNNG/HOS cells in vitro and in vivo. OBP‐401 tumor illumination enabled effective FGS of the 143B‐derived orthotopic mouse model of human osteosarcoma model as well as FGS eradication of residual cancer cells after BLS. OBP‐401‐assisted FGS significantly inhibited local recurrence and lung metastasis after surgery, thereby prolonging DFS and overall survival (OS), achieving a very important improvement of therapeutic outcomes over our previously reported FGS study. These therapeutic benefits of FGS were demonstrated using a clinically‐viable methodology of direct labeling of human osteosarcoma in situ with the OBP‐401 killer‐reporter adenovirus in contrast with previous reports, which used genetically engineered labeled cells or antibody‐based fluorescent labels for FGS. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:836–844, 2016.     

PEDF‐derived synthetic peptides exhibit antitumor activity in an orthotopic model of human osteosarcoma

Pigment epithelium‐derived factor (PEDF) is one of the most potent inhibitors of angiogenesis, and has recently been demonstrated to have an important multifunctional role in tumor growth, invasion, and metastasis. However, relatively little is known of mechanisms through which PEDF exerts its antitumor activity. Therefore, with the aim of identifying potential functional epitopes specifically against osteosarcoma, we evaluated the bioactivity of four 25‐mer synthetic PEDF‐derived peptides (termed StVOrth‐1, ‐2 ‐3, and ‐4) against a human osteosarcoma cell line, SaOS‐2. We found that StVOrth‐2 (residues 78–102) predominantly inhibited tumor cell proliferation, while StVOrth‐3 (residues 90–114) markedly increased cellular adhesion to collagen type‐1, with StVOrth‐4 (residues 387–411) demonstrating most significant inhibition of Matrigel invasion. Furthermore, we show that StVOrth‐1 (residues 40–64), ‐2 and ‐3 induce osteoblastic differentiation, evidenced by increased mineralized nodule formation. Interestingly, although no peptide inhibited angiogenesis in the tube formation assay, StVOrth‐3 and ‐4 markedly suppressed VEGF expression. We further tested the activity of StVOrth‐2 and StVOrth‐3 in vivo, in an orthotopic model of osteosarcoma and found that both peptides significantly inhibited primary tumor growth and the development of pulmonary metastases. Together these results provide greater insight into the potential mechanisms through which PEDF exerts its antitumor function. Furthermore, this raises the possibility of developing short PEDF fragments as lead compounds for the treatment of osteosarcoma. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1671–1680, 2007     

Targeting Cdk11 in osteosarcoma cells using the CRISPR‐cas9 system

Osteosarcoma is the most common type primary malignant tumor of bone. Patients with regional osteosarcoma are routinely treated with surgery and chemotherapy. In addition, many patients with metastatic or recurrent osteosarcoma show poor prognosis with current chemotherapy agents. Therefore, it is important to improve the general condition and the overall survival rate of patients with osteosarcoma by identifying novel therapeutic strategies. Recent studies have revealed that CDK11 is essential in osteosarcoma cell growth and survival by inhibiting CDK11 mRNA expression with RNAi. Here, we apply the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐Cas9 system, a robust and highly efficient novel genome editing tool, to determine the effect of targeting endogenous CDK11 gene at the DNA level in osteosarcoma cell lines. We show that CDK11 can be efficiently silenced by CRISPR‐Cas9. Inhibition of CDK11 is associated with decreased cell proliferation and viability, and induces cell death in osteosarcoma cell lines KHOS and U‐2OS. Furthermore, the migration and invasion activities are also markedly reduced by CDK11 knockout. These results demonstrate that CRISPR‐Cas9 system is a useful tool for the modification of endogenous CDK11 gene expression, and CRISPR‐Cas9 targeted CDK11 knockout may be a promising therapeutic regimen for the treatment of osteosarcoma. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:199–207, 2015.

     

化疗药上调DR4、DR5表达体外逆转人骨肉瘤对TRAIL的耐受

目的研究肿瘤坏死因子相关凋亡诱导配体（TRAIL）与化疗药对人骨肉瘤的联合作用及其受体（TRAILR）在人骨肉瘤中的表达间的关系。方法应用细胞计数、AO／EB染色、流式细胞术等方法比较检测阿霉素、顺铂、甲氨喋呤、紫杉醇单独作用和与TRAIL联合作用于MG-63及骨肉瘤组织的细胞毒效应；应用原位杂交、Western Blot方法检测人骨肉瘤细胞系MG-63及新鲜骨肉瘤组织用药前后TRAILR的表达。结果阿霉素、顺铂和紫杉醇增强死亡受体的表达，且与TRAIL联合作用诱导人骨肉瘤细胞凋亡的效率显著强于单用。结论阿霉素、顺铂、紫杉醇通过上调DR4、DR5表达逆转人骨肉瘤对TRAIL的耐受。     

骨肉瘤抑癌基因表达及其增殖活性的临床意义

目的 检测ｐ１６,ｐ５３基因蛋白及增殖细胞核抗原在骨肉瘤中的存在情况,探讨其与骨肉瘤发生,发展和预后的关系。方法 采用改良的免疫组化,ＡＢＣ法,对３２例骨肉瘤手术标本和１５例正常骨组织标本进行ｐ１６,ｐ５３基因蛋白,ＰＣＮＡ的检测。结果 ｐ１６,ｐ５３基因蛋白,ＰＣＮＡ的阳性表达分别为３７．５％,７５．０％,１００％,焉正常对照差异有显著性;     

Evaluation of P‐glycoprotein (Pgp) expression in human osteosarcoma by high‐throughput tissue microarray

Survival of osteosarcoma patients is currently limited by the development of metastases and multidrug resistance (MDR). A well‐established cause of MDR involves overexpression of P‐glycoprotein (Pgp) in tumor cells. However, some discrepancies still exist as to the clinical significance of Pgp in osteosarcoma. We sought to elucidate further whether the Pgp expression correlated with clinical behavior in a series of patients with osteosarcoma via high‐throughput tissue microarray (TMA) analysis. Immunohistochemical analysis of Pgp expression in a TMA of 114 specimens with a retrospective review of 70 osteosarcoma patients admitted to the Massachusetts General Hospital (MGH) was performed. High Pgp expression was correlated with metastasis development and poor response to pre‐operative chemotherapy in osteosarcoma patients. Eighteen of the fifty‐seven patients initially admitted with primary osteosarcoma showed high Pgp expression. Among these 18 patients with high Pgp expression, 13 of 18 (72%) patients eventually developed metastases. There was no significant clinical relevance between Pgp expression and osteosarcoma survival. These results support that high expression of Pgp is important, but cannot be assigned as, an individual predictor in the development of human osteosarcoma. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1606–1612, 2016.     

mTOR signaling plays a critical role in the defects observed in muscle‐derived stem/progenitor cells isolated from a murine model of accelerated aging

Mice expressing reduced levels of ERCC1‐XPF (Ercc1 ^−/Δ mice) demonstrate premature onset of age‐related changes due to decreased repair of DNA damage. Muscle‐derived stem/progenitor cells (MDSPCs) isolated from Ercc1 ^−/Δ mice have an impaired capacity for cell differentiation. The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth in response to nutrient, hormone, and oxygen levels. Inhibition of the mTOR pathway extends the lifespan of several species. Here, we examined the role of mTOR in regulating the MDSPC dysfunction that occurs with accelerated aging. We show that mTOR signaling pathways are activated in Ercc1 ^−/Δ MDSPCs compared with wild‐type (WT) MDSPCs. Additionally, inhibiting mTOR with rapamycin promoted autophagy and improved the myogenic differentiation capacity of the Ercc1 ^−/Δ MDSPCs. The percent of apoptotic and senescent cells in Ercc1 ^−/Δ MDSPC cultures was decreased upon mTOR inhibition. These results establish that mTOR signaling contributes to stem cell dysfunction and cell fate decisions in response to endogenous DNA damage. Therefore, mTOR represents a potential therapeutic target for improving defective, aged stem cells. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1375–1382, 2017.