and colony formation assay showed that EGR1 over-expression had no effect on osteosarcoma cell growth. Our findings suggest that the expression of EGR does not have general effects on growth and instead exerts regulatory effects that appear to be cell- type-specific. We showed up-regulation of EGR1 following cisplatin, methotrexate, etoposide, or doxorubicin treatment, each of which exerts cytotoxic effects by different pharmacological mechanisms. EGR1 can be rapidly induced by many stimuli, including growth factors, cytokines, ultraviolet light, anti-tumour agents, and various stresses [8,10,24, Cao, 1992 #88,25,28–35]. The distinct types of stress caused by anti-tumor drugs might promote up-regulation of EGR1, although anti-tumor drugs exert different pathways. Further, we examined which signaling pathway promotes EGR1 expression following anti-tumor agent treatment. We treated osteosarcoma cell lines with anti-tumor agent and some specific inhibitors including ERK inhibitor, HIF1-a inhibitor, JAK2 inhibitor, LY294002, and others but we were unable to inhibit EGR1 expression effectively. Further examination for regulation mechanisms of EGR1 expression is needed. The principle mode of action of doxorubicin, an anthracycline antibiotic, appears to be its ability to cross-link DNA and RNA, thereby affecting DNA and RNA synthesis [36,37]. However, recent studies have demonstrated that genotoxic (i.e., DNA damaging) agents, including many important cancer chemother- apy drugs, can have significant and selective effects on the expression of certain inducible genes [38]. It has also been demonstrated that noncytotoxic doses of the DNA cross-linking cancer chemotherapy drugs MMC, cisplatin, and carboplatin were effective at significantly altering the expression of the MDR1 gene coding for the multidrug resistance protein P- glycoprotein [37]. We were therefore interested in whether chemotherapy agents might similarly alter the expression of inducible invasion-related genes, and thereby potentially alter tumor invasiveness, and found that anti-tumour agents increased the expression of EGR1, and EGR1 decreased that of uPA and uPAR. The uPA system is thought to play roles in several different processes important to tumor progression including angiogenesis, tumor growth, and metastasis [39]. Expression of uPA and uPAR frequently indicates a poor prognosis, and is in some cases predictive of invasion and metastasis. uPAR is also thought to play roles in the growth and metastasis of human osteosarcoma [40–44]. We showed that forced expression of EGR1 inhibited expression of uPA and uPAR. In addition, EGR1 decreased the activity of uPA. These findings suggest that up-regulation of EGR1 following chemotherapy inhibits osteosarcoma migration via uPA system. Many signaling pathways activate transcription factors that act on the uPAR promoter, driving uPAR expression in cancer [45]. uPAR transcription is controlled by ERK through activator protein 1 transcription factors [46]. Hypoxia-inducible factor 1a drive uPAR expression through a hypoxia responsive element in the uPAR promoter [47]. Nuclear factor-kB also activates uPAR expression [48]. Thus, multiple signaling inputs can up-regulate uPAR transcription in tumors. We could not detect the pathways that promote down-regulation of uPA/uPAR. Further examination for regulation mechanisms of uPA/uPAR system is needed. Recently, many molecular target drugs have been developed [49–52]. In addition, several Notch signal inhibitors have been tested as molecular target drugs [53–55]. We previously reported that activation of Notch signaling promotes the progression of human osteosarcoma [56]. We examined the EGR1 expression by c-secretase inhibitor, a pharmacological agent known to effectively Chemotherapy Prevents OS Invasion by EGR1 block Notch activation. EGR1 was up-regulated by c-secretase inhibitor in human osteosarcoma cell lines (data not shown). These findings suggest that EGR1 expression will also be up-regulated by molecular target drugs. In summary, anti-tumor agents increased the expression of EGR1, and EGR1 decreased osteosarcoma invasion. Our findings suggest that even though chemotherapy could not prevent osteosarcoma growth in chemotherapy poor responders, chemo- therapy prevents osteosarcoma cell migration into blood vessel by down-regulation of urokinase plasminogen activation via up- regulation of EGR1 during chemotherapy periods. Supporting Information Figure S1 Anti-tumor agent treatment increased the expression of EGR1. Following 48 h or 5 days drug treatments, total RNA extracted from osteosarcoma cell lines were analyzed by real-time PCR. Following 48 h treatment, cisplatin, metho- trexate, etoposide or doxorubicin increased EGR1 expression in 143B cell and Saos-2 cells. Following 5 days treatment, cisplatin increased EGR1 expression in 143B cell. Following 5 days treatment, etoposide or doxorubicin increased EGR1 expression in Saos-2 cells. The comparative Ct (DDCt) method was used to determine fold change in expression using GAPDH or ACTB. Experiments were performed in triplicate with similar results [error bars represent mean (SD)]. (TIF) Figure S2 Chemotherapy increased EGR1 expression. Total RNA extracted from osteosarcoma patients’ biopsy specimens and excised tumors following chemotherapy were used for real-time PCR. Real-time PCR revealed that 3 of 3 excised specimens of osteosarcoma increased EGR1 expression 7.87- to 1.73-fold (A). One day after 4 mg doxorubicin treatment, RNA was extracted from tumor in nude mice xenograft models. Real- time PCR revealed that low dose chemotherapy increased EGR1 expression in vivo (B) (P,0.05). The comparative Ct (DDCt) method was used to determine fold change in expression. These experiments were performed in triplicate with similar results [error bars represent mean (SD)]. (TIF) Figure S3 Forced expression of EGR1 does not affect osteosarcoma cell growth in vitro. We transfected control vector or EGR1 expression vector, and examined osteosarcoma cell growth. MTT assay revealed that growth of viable 143B, Saos- 2, and HOS cells over 8 days was not affected by forced expression of EGR1 (A). These experiments were performed in triplicate with similar results [error bars represent mean (SD)]. Colony formation assay revealed that forced expression of EGR1 did not affect the number of colonies in soft agar (B). These experiments were performed in triplicate with similar results [error bars represent mean (SD)]. (TIF) Figure S4 Forced expression of EGR1 decreased the expression of uPA and uPAR. Cell lysate were prepared from control vector or EGR1 expression vector stably transfected cells. ELISA assay showed that forced expression of EGR1 decreased the expression of uPA and uPAR proteins in 143B (P,0.05) (A). The expression of uPA and uPAR decreased in Saos-2 and HOS (P,0.05) (B, C). These experiments were in triplicate with similar results [error bars represent mean (SD)]. (TIF) PLoS ONE | www.plosone.org 10 January 2011 | Volume 6 | Issue 1 | e16234