Elevated ratio of MMP2/MMP9 activity is associated with poor response to chemotherapy in osteosarcoma

Background: Matrix metalloproteinases (MMPs) are crucially involved in the regulation of multiple stages of cancer progression. Elevated MMP levels have been associated with the development of metastases and poor prognosis in several types of cancer. However, the role of MMPs in osteosarcoma and their prognostic value is still unclear. Available data are conflicting, most likely due to different technical approaches. We hypothesized that in contrast to total mRNA or protein levels frequently analyzed in previous studies the enzymatic activities of MMPs and their inhibitors the tissue inhibitors of matrix metalloproteinases (TIMPs) are closer related to their biological functions. We therefore aimed to evaluate the reliability of different zymography techniques for the quantification of MMP and TIMP activities in osteosarcoma biopsies in order to investigate their distribution, possible regulation and prognostic value. Methods: All analyses were done using cryo-conserved osteosarcoma pretreatment biopsies (n = 18). Gene and protein expression of MMPs and TIMPs were analyzed by RT-qPCR and western blot analysis, respectively. Overall MMP activity was analyzed by in situ zymography, individual MMP activities were analyzed by gelatin zymography. Reverse zymography was used to detect and quantify TIMP activities. Results: Strong overall MMP activities could be detected in osteosarcoma pretreatment biopsies with MMP2 and MMP9 as predominant active MMPs. In contrast to total RNA or protein expression MMP2 and MMP9 activities showed significant quantitative differences between good and poor responders. While MMP9 activity was high in the good responder group and significantly decreased in the poor responder group, MMP2 activity showed a reverse distribution. Likewise, significant differences were detected concerning the activity of TIMPs resulting in a negative correlation of TIMP1 activity with MMP2 activity (p = 0.044) and negative correlations of TIMP2 and TIMP3 with MMP9 activity (p = 0.007 and p = 0.006). Conclusion: In contrast to mRNA or protein levels MMP and TIMP activities showed significant differences between the analyzed good and poor responder groups. A shift from MMP9 to predominant MMP2 activity is associated with poor response to chemotherapy suggesting that the ratio of MMP2/MMP9 activity might be a valuable and easily accessible marker to predict the response to chemotherapy in osteosarcoma

Overexpression of Inosine 5′-Monophosphate Dehydrogenase Type II Mediates Chemoresistance to Human Osteosarcoma Cells

overexpression in osteosarcoma patients with poor response to chemotherapy. The aim of this study was to provide evidence for direct involvement of IMPDH2 in the development of chemoresistance..IMPDH2 is directly involved in the development of chemoresistance in osteosarcoma cells, suggesting that targeting of IMPDH2 by RNAi or more effective pharmacological inhibitors in combination with chemotherapy might be a promising means of overcoming chemoresistance in osteosarcomas with high IMPDH2 expression

Optimization of the chicken chorioallantoic membrane assay as reliable in vivo model for the analysis of osteosarcoma.

Survival rates of osteosarcoma patients could not be significantly improved by conventional chemotherapeutic treatment regimens since the introduction of high-dose chemotherapy 35 years ago. Therefore, there is a strong clinical need for new therapeutic targets and personalized treatment strategies, requiring reliable in vivo model systems for the identification and testing of potential new treatment approaches. Conventional in vivo rodent experiments face ethical issues, are time consuming and costly, being of particular relevance in orphan diseases like osteosarcoma. An attractive alternative to such animal experiments is the chicken chorioallantoic membrane (CAM) assay. The CAM is a highly vascularized, non-innervated extra-embryonic membrane that is perfectly suited for the engraftment of tumor cells. However, only few reports are available for osteosarcoma and reported data are inconsistent. Therefore, the aim of this study was the adaptation and optimization of the CAM assay for its application in osteosarcoma research. Tumor take rates and volumes of osteosarcoma that developed on the CAM were analyzed after modification of several experimental parameters, including egg windowing, CAM pretreatment, inoculation technique and many more. Eight osteosarcoma cell lines were investigated. Our optimized OS-CAM-assay was finally validated against a rat animal xenograft model. Using the cell line MNNG HOS as reference we could improve the tumor take rates from 51% to 94%, the viability of the embryos from initially 40% to >80% and achieved a threefold increase of the tumor volumes. We were able to generate solid tumors from all eight osteosarcoma cell lines used in this study and could reproduce results that were obtained using an osteosarcoma rat animal model. The CAM assay can bridge the gap between in vitro cell culture and in vivo animal experiments. As reliable in vivo model for osteosarcoma research the optimized CAM assay may speed up preclinical data collection and simplifies research on potential new agents towards personalized treatment strategies. Further, in accordance with Russell's and Burch's "Principles of Humane Experimental Technique" the reasonable use of this model provides a refinement by minimizing pain and suffering of animals and supports a considerable reduction and/or replacement of animal experiments

Influence of the IMPDH inhibitor mycophenolic acid (MPA) on cell proliferation of different Saos-2 cell lines.

<p><b>A:</b> MPA was added to the culture medium at the indicated concentrations and cell proliferation was determined using WST-1 assay at 0, 24, 48, and 72 h. <b>B:</b> Saos-2 wild-type cells were treated with the indicated concentrations of MPA for 48 h with or without the addition of 10 µM guanine. Cell proliferation was analyzed by WST-1 assay at 450 nm. Analyses were performed in triplicate and the results are presented as mean ± SD.</p

Chemosensitivity of Saos-2 cdsIMPDH2 cells cotransfected with an <i>IMPDH2-</i>specific shRNA construct.

<p><b>A:</b> Stable Saos-2 cdsIMPDH2 cells were cotransfected with three different shRNA constructs directed against <i>IMPDH2</i>. Stable cell lines were generated, and <i>IMPDH2</i> mRNA expression was analyzed by quantitative PCR and compared to that in Saos-2 wild-type, Saos-2 pCMS, and Saos-2 cdsIMPDH2 cells. <b>B:</b> Western blot analysis of IMPDH2 expression in Saos2 cdsIMPDH2 cells cotransfected with an IMPDH2 specific shRNA construct. <b>C:</b> Analysis of chemosensitivity of Saos-2 wild-type, Saos-2 pCMS, Saos-2 cdsIMPDH2, and Saos-2 cdsIMPDH2 cells stable cotransfected with shRNA construct specific for <i>IMPDH2</i>. Cells were treated with cisplatin at the indicated concentrations for 72 h before cell viability was analyzed by propidiumiodide staining. Analyses were performed in triplicate and the results are presented as mean ± SD. (** <i>p</i><0.01).</p

Chemoresistance in <i>IMPDH2</i>-overexpressing Saos-2 cells.

<p><b>A:</b> Western blot analysis of IMPDH2 expression in Saos-2 wild-type cells, Saos-2 cells transfected with the empty vector (Saos-2 pCMS), Saos-2 cells overexpressing <i>IMPDH2</i> (Saos-2 cdsIMPDH2), and Saos-2 cells transfected with a shRNA construct directed against <i>IMPDH2</i> (Saos-2 shIMPDH2). <b>B:</b> Cell viability of different Saos-2 cell lines after treatment with cisplatin at the indicated concentrations for 72 h. Analyses were performed in triplicate and the results are presented as mean ± SD (** <i>p</i><0.01 compared to Saos-2 wild-type cells). <b>C:</b> Cell viability of different Saos-2 cell lines after treatment with methotrexate (MTX) at the indicated concentrations for 96 h. Analyses were performed in triplicate and the results are presented as mean ± SD (** <i>p</i><0.01 compared to Saos-2 wild-type cells).</p

Chemosensitivity of Saos-2 wild-type, Saos-2 pCMS, Saos-2 shIMPDH2, and Saos-2 cdsIMPDH2 cells treated with the IMPDH inhibitor mycophenolic acid (MPA) at the indicated concentrations for 72 h with or without cisplatin (2.5 µg/ml).

<p>Analyses were performed in triplicate and the results are presented as mean ± SD.</p