development of a series of promising molecular inhibitors for use in clinical oncology and targeted therapy [16]. Suppression of EGFR expression by siRNA or inhibition of EGFR kinase activity by an agent, such as gefitinib, has already proved to be of clinical benefit in the treatment of certain cancer cell types [17–19]. Yao et al. [20] revealed that mitochondrial location of EGFR was independent of receptor endocytosis and that gefitinib decreased cell viability as well as the quantity of mitochondrial EGFR. Previously, we have shown that treatment of glioma cells with human umbilical cord blood stem cells (hUCBSC) inhibited the expression of XIAP, FAK and cyclin D1 with simultaneous upregulation of PTEN [21–24]. Based on our previous results, we hypothesized that hUCBSC could efficiently inhibit EGFR expression as well as its translocation to mitochondria. Hence, in the present study, we investigated the mitochondrial localization of EGFR expression in human GBM from several GBM patient– derived specimens and validated similar phenomenon in standard glioma cell lines. In addition, we demonstrate that EGFR translocation to mitochondria and its co-localization with FAK is highly inhibited after treatment with hUCBSC. Results EGFR co-localizes with FAK in clinical samples Since GBM-patient derived specimens reproduce the genotypic and phenotypic characteristics of GBM more faithfully than standard glioma cell lines [25,26], we first examined EGFR status in GBM-patient derived specimens. Using an immunohistochem- istry-based strategy, we identified six GBM-patient derived specimens with high expression of EGFR as compared to normal brain specimen (Fig. 1A). The mRNA expression levels of EGFR corresponded to the immunohistochemistry results (Fig. 1B). However, compared to the expression of EGFR, we did not observe much expression of FAK in GBM-patient derived samples. On the contrary, expression of c-Src was higher in samples where EGFR expression is low (for e.g., hGBM9, hGBM13) (Fig. 1C). Next, we checked the protein levels of these molecules. EGFR protein expression levels almost correspond to the levels observed with immunohistochemistry and mRNA expression (Fig. 1D). Contrary to this, pEGFR levels can be seen in only hGBM2 and hGBM6 (Fig. 1E). On the other hand, FAK expression is seen in all the samples with faint expression in hGBM8. Higher expression of p-Fak was observed in hGBM6 and hGBM9 samples with faint expression in remaining clinical samples. c-Src expression was seen in all GBM samples with faint expression in hGBM8. Phospho-c-Src expression was observed only in hGBM2, hGBM3, hGBM9 and hGBM13 samples with very faint expression in hGBM6. Once we confirmed the presence of EGFR and FAK in GBM-patient derived samples, we checked for the co-localization of active forms of FAK and EGFR in mitochondria. In all clinical samples, co-localization of pEGFR with pFAK is clearly established in the mitochondria (Fig. 2). Having confirmed the expression of EGFR in clinical samples and its association with FAK, we evaluated the significance of hUCBSC treatment in glioma cells and xenografts in regards to EGFR status. hUCBSC treatment inhibits EGFR Once we confirmed the expression status of EGFR and FAK in clinical samples, we evaluated the expression levels of EGFR, FAK and other molecules in human glioma cell lines both in vitro and in vivo with reference to the treatment effect of hUCBSC in these glioma cells. Expression of EGFR is highly downregulated in hUCBSC-treated U87, U251 and 5310 glioma cells as compared Inhibition of EGFR Localization to Mitochondria to their controls (Fig. 3A). Similarly, hUCBSC significantly reduced EGFR expression in U251 and 5310 in vivo xenografts (Fig. 3B). Further, we checked the expression of EGFR, FAK and c-Src in glioma cells lines and their co-cultures with hUCBSC. We observed that in co-cultures of glioma cells with hUCBSC, the mRNA levels of EGFR, FAK and c-Src are downregulated (Fig. 3C, 3D). This is associated with the downregulation of these molecules at protein levels also (Fig. 3G). We observed that EGFR and FAK are more significantly downregulated compared to c-Src (Fig. 3H). To confirm these results, we checked the expression of these molecules in nude mice brain xenografts. Similar to the in vitro results, in hUCBSC-treated brain tissues also, mRNAs of EGFR, FAK and c-Src were downregulated (Figs. 3E, 3F). This ultimately resulted in the lower expression of these molecules at protein levels (Figs. 3I, 3J). These results prove that hUCBSC downregulate EGFR, FAK and c-Src at both transcriptional and translational stages in both in vitro and in vivo conditions. Since EGFR plays a vital role in the proliferation of glioma cells, we tested the expression of Ki67 in U87, U251 and 5310 glioma cells. These cells express high levels of the ubiquitous proliferation marker Ki67 (Fig. 4A); treatment with hUCBSC lowered the expression of Ki67 indicating EGFR-mediated proliferation of glioma cells is inhibited by hUCBSC treatments. Further, we checked the co-localization of EGFR and FAK in glioma cells. As expected pEGFR co-localized with pFAK in glioma cells and this co-localization was completely inhibited by hUCBSC treatment (Fig. 4B). To confirm these results, we treated U251, U87 and 5310 cells with external supply of EGF and observed higher expression of EGFR and FAK as compared to control glioma cells (Fig. 4C). In another experiment, we treated glioma cells with EGF initially and then co-cultured these cells with hUCBSC for 72 hours. Even though glioma cells were supplied with exogenous EGF, hUCBSC were highly efficient in downregulating both EGFR and FAK in these treatments (Fig. 4D). To further substantiate these results, we performed cell proliferation assays based on BrdU incorporation. In all three glioma cell lines of the present study, hUCBSC inhibited cell proliferation by more than 80% (Fig. 5A). The inhibition of cell proliferation was more pronounced in hUCBSC-treated 5310 cell lines. In another experiment, we observed that exogenous supply of EGF increased cell proliferation of glioma cells by about 10% (Fig. 5B). However, hUCBSC were able to inhibit proliferation of exogenous EGF- supplied cells to ,80% than control glioma cells. Further to compare and evaluate the efficiency of hUCBSC with that of Temozolomide (TMZ), we performed combination treatments of hUCBSC and TMZ on glioma cells. TMZ alone inhibited the proliferation of glioma cells to ,40% (Fig. 5C). However, combination treatments of hUCBSC and TMZ at different time intervals showed profound effect on the cell proliferation of glioma cells compared to single TMZ treatments. These experiments confirm the efficacy of hUCBSC against EGF-treated glioma cells and the effect of hUCBSC in inhibiting glioma cell proliferation. hUCBSC inhibits translocation of phospho-EGFR to mitochondria of glioma cells EGFR activation stimulates many complex intracellular signal- ing pathways. Apart from this activation, EGFR translocates to mitochondria and co-localizes with FAK to further activate mitochondria-mediated signaling in glioma cells. Since we observed that hUCBSC treatments inhibited EGFR expression in glioma cells, we decided to further evaluate translocation of the active form of EGFR to mitochondria in glioma cells. In all these cell lines, translocation of activated EGFR to mitochondria was PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31884