Yes-mediated phosphorylation of focal adhesion kinase at tyrosine 861 increases metastatic potential of prostate cancer cells

To study the role of FAK signaling complexes in promoting metastatic properties of prostate cancer (PCa) cells, we selected stable, highly migratory variants, termed PC3 Mig-3 and DU145 Mig-3, from two well-characterized PCa cell lines, PC3 and DU145. These variants were not only increased migration and invasion in vitro, but were also more metastatic to lymph nodes following intraprostatic injection into nude mice. Both PC3 Mig-3 and DU145 Mig-3 were specifically increased in phosphorylation of FAK Y861. We therefore examined potential alterations in Src family kinases responsible for FAK phosphorylation and determined only Yes expression was increased. Overexpression of Yes in PC3 parental cells and src−/−fyn−/−yes−/− fibroblasts selectively increased FAK Y861 phosphorylation, and increased migration. Knockdown of Yes in PC3 Mig-3 cells decreased migration and decreased lymph node metastasis following orthotopic implantation of into nude mice. In human specimens, Yes expression was increased in lymph node metastases relative to paired primary tumors from the same patient, and increased pFAK Y861 expression in lymph node metastases correlated with poor prognosis. These results demonstrate a unique role for Yes in phosphorylation of FAK and in promoting PCa metastasis. Therefore, phosphorylated FAK Y861 and increased Yes expression may be predictive markers for PCa metastasis.     

Constitutive activation of pp125fak in newly isolated human breast cancer cell lines

Our laboratory has developed twelve human breast cancer cell lines from primary and metastatic sites. In this report we demonstrate that eight of eight breast cancer cell lines examined exhibit constitutively tyrosine phosphorylated and enzymatically active endogenous pp125fak when grown in monolayer. The activation status of pp125fak in breast cancer cells in monolayer is significantly elevated over that exhibited by normal mammary epithelial cells cultured under the same conditions. Constitutive activation of pp125fak is the only characteristic so far studied that all of these breast cancer cell lines have in common. In contrast to HBC cells, tyrosine phosphorylation of pp125fak in HME cells was low or absent in monolayer culture but was induced to high levels by culturing the cells in Matrigel. Thus tyrosine phosphorylation and activation of pp125fak is a regulated process in normal mammary epithelial cells, but is constitutive in breast cancer cells. Finally, analysis of the ability of normal human mammary epithelial cells and breast cancer cell lines to grow under anchorageindependent conditions indicated that normal human mammary epithelial cells rapidly and uniformly lost viability when not substrateattached, whereas all of the breast cancer cell lines survived for a 3week culture period. Furthermore, a subset of the breast cancer cell lines grew to form large colonies under anchorageindependent conditions. Interestingly, pp125fak activation decreased dramatically in HBC cells cultured for two weeks in suspension, suggesting that activation of this kinase is not necessary for longterm growth under anchorageindependent conditions. These results suggest that constitutive activation of pp125fak results in preferential survival of human breast cancer cells under anchorageindependent conditions but that activation of pp125fak is not the sole mediator of anchorageindependent colony formation.     

Focal adhesion kinase: A prominent determinant in breast cancer initiation,progression and metastasis

Focal adhesion kinase (FAK) is an intracellular non-receptor tyrosine kinase. In addition to its role as a major mediator of signal transduction by integrins, FAK also participates in signaling by a wide range of extracellular stimuli including growth factors, G-protein-coupled receptor agonists, cytokines, and other inflammatory mediators. The link between FAK and breast cancers is strongly suggested by a number of reports showing that FAK gene is amplified and overexpressed in a large fraction of breast cancer specimens. In addition, increased FAK expression and activity frequently correlate with metastatic disease and poor prognosis. Since its discovery in early 1990s, numerous studies have shown a role for FAK in the regulation of cell spreading, adhesion, migration, survival, proliferation, differentiation, and angiogenesis. Many of these studies in cultured cells provided strong evidence to connect FAK expression/activation to the promotion of cancer. Recently, a prominent role of FAK in promoting mammary tumorigenesis, progression and metastasis has been unveiled by different animal models of human breast cancer, including xenograft models in immunodeficient rodents and spontaneous tumor models in transgenic mice that have specific deletion of FAK in the mammary epithelial cells during embryonic or postnatal development. These in vivo studies established FAK as a prominent determinant in mammary cancer initiation, progression and metastasis. Furthermore, a novel function of FAK in maintaining mammary cancer stem/progenitor cells in vivo has been recently reported, which may provide a novel cellular mechanism of FAK in promoting breast cancer initiation and progression. The wealth of knowledge accumulated over almost two decades of research on FAK should help to design potentially novel therapies for breast cancer.     

High expression of focal adhesion kinase (p125FAK) in node-negative breast cancer is related to overexpression of HER-2/neu and activated Akt kinase but does not predict outcome

IntroductionFocal adhesion kinase (FAK) regulates multiple cellular processes including growth, differentiation, adhesion, motility and apoptosis. In breast carcinoma, FAK overexpression has been linked to cancer progression but the prognostic relevance remains unknown. In particular, with regard to lymph node-negative breast cancer it is important to identify high-risk patients who would benefit from further adjuvant therapy.MethodsWe analyzed 162 node-negative breast cancer cases to determine the prognostic relevance of FAK expression, and we investigated the relationship of FAK with major associated signaling pathways (HER2, Src, Akt and extracellular regulated kinases) by immunohistochemistry and western blot analysis.ResultsElevated FAK expression did not predict patient outcome, in contrast to tumor grading (P = 0.005), Akt activation (P = 0.0383) and estrogen receptor status (P = 0.0033). Significant positive correlations were observed between elevated FAK expression and HER2 overexpression (P = 0.001), as well as phospho-Src Tyr-215 (P = 0.021) and phospho-Akt (P < 0.001), but not with phospho-ERK1/2 (P = 0.108). Western blot analysis showed a significant correlation of FAK Tyr-861 activation and HER2 overexpression (P = 0.01).ConclusionsImmunohistochemical detection of FAK expression is of no prognostic significance in node-negative breast cancer but provides evidence that HER2 is involved in tumor malignancy and metastatic ability of breast cancer through a novel signaling pathway participating FAK and Src.     

Expression of focal adhesion kinase in small-cell lung carcinoma

BACKGROUND:

The focal adhesion kinase (FAK) is a non‐receptor tyrosine kinase linked to tumor growth, invasion, and metastasis. FAK is overexpressed and associated with prognosis in many cancers, but its prognostic value in small‐cell lung carcinoma (SCLC) is unknown and was the focus of this study.

METHODS:

Total FAK expression was analyzed via immunohistochemistry in tissue microarrays consisting of formalin‐fixed, paraffin‐embedded SCLC specimens from 85 patients. FAK staining scores were tested for correlations with pathological characteristics and clinical outcomes. Phospho‐paxillin was also tested in 35 of the 85 specimens to evaluate whether FAK expression was associated with downstream signaling.

RESULTS:

Specific FAK expression was localized to the cytoplasm of 78/85 (92%) SCLCs. FAK expression was scored low in 11 (13%), moderate in 17 (20%), and high in 50 (59%) SCLCs. FAK staining scores treated as continuous variables did not correlate with SCLC disease stage, response to therapy, recurrence/progression‐free survival, or overall survival. Moreover, total FAK expression did not correlate with phospho‐paxillin Tyr¹¹⁸ expression.

CONCLUSIONS:

Total FAK is strongly expressed in a majority of SCLC tumors. However, the expression evaluated via immunohistochemistry is not a prognostic factor in patients with SCLC. Cancer 2012;. © 2011 American Cancer Society.     

Cytotoxicity, apoptosis induction and anti-metastatic potential of Oroxylum indicum in human breast cancer cells

Despite clinical advances in anticancer therapy, there is still a need for novel anticancer metabolites, with higher efficacy and lesser side effects. Oroxylum indicum (L.) Vent. is a small tree of the Bignoniaceae family which is well known for its food and medicinal properties. In present study, the chemopreventive properties of O. indicum hot and cold non-polar extracts (petroleum ether and chloroform) were investigated with MDA- MB-231 (cancer cells) and WRL-68 (non-tumor cells) by XTT assay. All the extracts, and particularly the petroleum ether hot extract (PHO), exhibited significantly (P<0.05) higher cytotoxicity in MDA-MB-231 when compared to WRL-68 cells. PHO was then tested for apoptosis induction in estrogen receptor (ER)-negative (MDA-MB-231) and ER-positive (MCF-7) breast cancer cells by cellular DNA fragmentation ELISA, where it proved more efficient in the MDA-MB-231 cells. Further, when PHO was tested for anti-metastatic potential in a cell migration inhibition assay, it exhibited beneficial effects. Thus non-polar extracts of O. indicum (especially PHO) can effectively target ER-negative breast cancer cells to induce apoptosis, without harming normal cells by cancer-specific cytotoxicity. Hence, it could be considered as an extract with candidate precursors to possibly harness or alleviate ER-negative breast cancer progression even in advanced stages of malignancy.     

FAK和VEGF在胃癌中的表达及其相互关系

目的检测粘着斑激酶(FAK)和血管内皮生长因子(VEGF)在胃癌中的表达及其与侵袭和转移的关系,并探讨二者的相关性.方法采用免疫组织化学sABC法,观察86例胃癌及30例非胃癌组织中FAK和VEGF的表达情况.结果FAK和VEGF在胃癌中的阳性率分别为80%和59%.在非胃癌组织中的阳性表达率分别为10%和13%.FAK和VEGF在侵及浆膜层胃癌病例中的表达明显高于未侵及浆膜层者,二者之间有显著差异性(P＜0.05);有淋巴结转移组与无淋巴结转移组比较有明显差异(P＜0.05),FAK与VEGF阳性表达呈正相关(P＜0.01).结论FAK、VEGF在胃癌的侵袭和转移中起重要作用,二者在胃癌中表达升高可以作预测胃癌侵袭和转移的指标.     

Mitoxantrone,vincristine, and 5-fluorouracil with leucovorin modulation as induction chemotherapy prior to high-dose intensification in metastatic breast cancer

Summary We treated 39 women with newly diagnosed stage IV breast cancer with a new regimen of mitoxantrone 18 mg/m² on days 1, 29, 57, vincristine 1.4 mg/m² (maximum 2.0 mg) on days 1, 8, 15, 22, 29, 36, 43, 50, and 5-fluorouracil 375 mg/m² on days 15–20, 43–47, 71–75 with leucovorin modulation 500 mg/m² before each 5FU infusion (MVF). This regimen was utilized as an initial cytoreductive or induction program for these patients prior to high-dose intensification with autologous stem cell rescue. Ten patients (25%) obtained a clinical complete response and six patients (15%) obtained a partial response for an overall response rate of 40%. In addition, 10 patients had evaluable disease that was improved or stable (primarily bone and/or bone marrow metastases) after MVF induction. Thus, 26 patients (65%) were eligible for high-dose intensification with autologous stem cell rescue after MVF induction. Toxicity was primarily a mild mucositis and more commonly peripheral neuropathy. MVF therapy is an active treatment program for metastatic breast cancer but the neurotoxicity makes it difficult to recommend for widespread use.     

Specific cytoplasmic alpha-fetoprotein binding protein in MCF-7 human breast cancer cells and primary breast cancer tissue

Summary Direct evidence was obtained for the existence of a specific high affinity alpha-fetoprotein (AFP)-binding protein in the cytosol of both MCF-7 human breast cancer cultured cells and primary breast cancer tissue from postmenopausal women using a nitrocellulose blotting assay. Scatchard analysis of the binding data for MCF-7 cells at 37° C revealed the presence of a single class of AFP binding sites with an apparent K_d of 4.5 × 10^–8 M, and 75,000 binding sites per cell. All 9 primary breast cancer cytosols obtained from postmenopausal women also contained measureable levels of this specific AFP-binding protein. The number of AFP molecules specifically bound varied considerably between patients and ranged from 29–250 fmol per mg cytosol protein. Levels of AFP-binding protein levels and estrogen receptor measured in these same breast cancer cytosols showed a positive statistical correlation (r = 0.85). Taken together, the present evidence for the existence of a specific cytoplasmic AFP-binding protein in MCF-7 cells and previously reported evidence forde novo synthesis of free immunoreactive and bound nonimmunoreactive forms of cytoplasmic AFP by MCF-7 cells is consistent with the conclusion that most of the endogenous AFP synthesized in breast cancer cells is rapidly bound to specific cytoplasmic AFP-receptors, and that binding of AFP to these receptors masks its immunoreactivity. The association of AFP synthesis with rapidly growing fetal liver and adult regenerating liver, germ-cell tumors, immature uterus, and breast cancer cells suggests that a positive correlation exists between cytoplasmic AFP-receptor levels and the proliferative capacity of malignant breast tumors, and therefore such measurements may provide useful therapeutic and/or prognostic information in individual patients.     

Chromosomal abnormalities in human breast cancer

This review emphasizes cytogenetic changes and DNA analyses by Southern blot in primary breast tumors, rather than metastases, established cell lines, and pleural effusions. The data suggests that the most frequently altered chromosomes and chromosome regions are 1p, 1q, 2q, 3p, 5, 6q, 8p, 8q, 11p, 11q, 12, 13q, 14q, 16, 17p, and 17q. Changes on 8q, 11p, 11q, 13q, and 17q appear to be associated with either progression of the disease or poor prognosis. Alterations on 1p and 3p may represent early events in the development of breast cancer.     

Potential role of p53 on metallothionein induction in human epithelial breast cancer cells

The expression and induction of metallothionein has been associated with protection against oxidative stress and apoptosis. This study examines the effect of tumour suppressor protein p53 on metallothionein expression following CdCl2 treatment in eight human epithelial breast cancer cell lines differing in p53 and oestrogen-receptor status. Cells were treated with 10 microM CdCl2 for 24 h and metallothionein protein levels were measured by cadmium binding assay. MCF7 cells which are p53-positive (p53+) and oestrogen-receptor-positive showed a large induction in metallothionein synthesis by 10.79+/-1.36-fold. Other breast cancer cell lines which are p53-negative (p53-) and oestrogen-receptor-negative or weakly oestrogen-receptor-positive showed a small induction ranging from 1.40+/-0.10 to 3.65+/-0.30-fold. RT-PCR analysis showed an induction of metallothionein mRNA in MCF7 cells by about 1.61+/-0.08-fold, while in HCC1806 cells (p53-, oestrogen-receptor-negative) by 1.11+/-0.13-fold, and in MDA-MB-231 (p53-, oestrogen-receptor-negative) by 1.25+/-0.06-fold. Metallothionein localisation was determined by immunohistochemical staining. Prior to metal treatment, metallothionein was localised mainly in the cytoplasm of MCF7 and MDA-MB-231 cells. After treatment with 10 microM CdCl2 for 24 h, MCF7 cells showed intense nuclear and cytoplasmic staining for metallothionein, while MDA-MB-231 cells showed staining in the cytoplasm with weak nuclear staining. Apoptosis induced by 10-40 microM CdCl2 at time points between 4 and 48 h was examined with TUNEL assay. In MCF7 cells, apoptosis increased with higher concentrations of CdCl2, it peaked at 6-8 h and appeared again at 48 h for all concentrations of CdCl2 tested. In MDA-MB-231 cells, apoptosis remained at low levels for 10-40 microM CdCl2 at all time points. Studies on cadmium uptake showed similar uptake and accumulation of cadmium at 8 and 24 h in all the cell lines. The data demonstrate that treatment of epithelial breast cancer cells with 10 microM CdCl2 for 24 h caused a greater induction of metallothionein protein and mRNA expression in p53+ and oestrogen-receptor-positive cells as compared to p53- and oestrogen-receptor-negative or weakly oestrogen-receptor-positive cells. This effect may be associated with the occurrence of apoptosis and suggests a role for p53 and oestrogen-receptor on the expression and induction of metallothionein in epithelial cells.     

p53-Dependent repression of focal adhesion kinase in response to estradiol in breast cancer cell-lines

Mutations in the TP53 suppressor gene are frequent in breast cancers. These mutations are associated with poor prognosis, thought to be due to proliferative advantage and poor response to chemotherapy associated with loss of p53 function. The focal adhesion kinase (FAK/PTK2), a tyrosine kinase, is over-expressed in a variety of human tumors including breast cancers. FAK is a critical regulator of adhesion and motility and its over-expression is associated with increased metastatic potential. Recently, FAK promoter has been shown to contain p53 responsive elements and to be down-regulated by DNA-damage in a p53-dependent manner. Here, we have used five estrogen-dependent breast cancer cells lines with different p53 status, including an isogenic model, to show that FAK expression was regulated in a p53-dependent manner in response to estradiol. FAK protein and mRNA expression were down-regulated by estradiol in wild-type but not mutant p53 cells. Moreover, silencing wild-type p53 increased FAK expression, while over expressing p53 repressed FAK expression. ChIP experiment showed that p53 bound to FAK promoter in the presence of estradiol in p53 wild-type but not in mutant p53 cells, suggesting a direct role of p53 in down regulating FAK mRNA expression. FAK mRNA expression was also found to correlate with TP53 mutation status in a series of breast tumors. Finally, loss of FAK down-regulation in p53 mutant cells was correlated with increased proliferation and invasion upon estradiol stimulation, while FAK silencing reduced invasion. These results suggest that p53 is an important down regulator of FAK and that loss of p53 function in breast cancer may contribute to the metastatic potential of estrogen-responsive tumors through uncontrolled FAK expression upon estrogens stimulation.     

非小细胞肺癌组织中FAK和pY397FAK的表达

目的:探讨粘着斑激酶(focaladh sionkinase,FAK)和磷酸化FAK(phosph FAK,pY397FAK)在非小细胞肺癌(no smallcelllungcancer,NSCLC)和正常支气管 黏膜上皮中的表达,及FAK在NSCLC发生 浸润和转移中的作用。方法:应用免疫组织 化学方法检测FAK和pY397FAK在60例 NSCLC和19例正常支气管黏膜上皮中的表 达,并与临床病理指标和Ki 67表达进行联 合分析。结果:FAK和pY397FAK在NSCL 中表达为83.3%(50/60)和55.0%(33 60),高于正常支气管黏膜上皮[57.9%(11 19)和21.1%(4/19)],差异有统计学意义 P=0.030、P=0.010。FAK和pY397FAK表 达升高与TNM分期及淋巴结转移有关,在 Ⅲ期和淋巴结转移组的阳性率高于Ⅰ+Ⅱ 期和无淋巴结转移组,差异有统计学意义, 均<0.05。FAK的强阳性表达率在低分化 组中(73.1%,19/26)显著高于高 中分化组 (47.1%,16/34),P=0.043。结论:FAK和 pY397FAK过表达在NSCLC发生和进展中发 挥重要作用。FAK和pY397FAK可能作为判 断NSCLC生物学行为的指标和治疗的新靶 点。     

p21活化激酶4在乳腺癌细胞中的表达及定位

目的:研究p21激活激酶4（PAK4）在人乳腺癌细胞和组织中的表达和定位。方法:蛋白印迹法检测PAK4在人乳腺癌细胞系中蛋白和基因的表达,激光共聚焦扫描显微镜检测内源PAK4在MCF-7乳腺癌细胞中的定位。结果:PAK4在人乳腺肿瘤细胞系中蛋白和基因表达水平增高,激光共聚焦扫描显微镜观察发现PAK4主要定位于MCF-7细胞质中。结论:PAK4在人乳腺癌细胞系中高表达,主要定位于乳腺癌组织细胞质内,可能是人乳腺癌重要的调节因子。     

Concomitant intensive chemoradiotherapy induction in non-metastatic inflammatory breast cancer: long-term follow-up

The aim of this study was to evaluate with a long follow-up the efficacy of concomitant chemoradiotherapy in non-metastatic inflammatory breast cancer (IBC) and to evaluate the breast conservation rate. Between 1990 and 2000, 66 non-metastatic patients with IBC were treated with chemotherapy and concomitant irradiation. The induction chemotherapy consisted of epirubicine, cyclophosphamide and vindesine, in association with split-course bi-fractionated irradiation to a total dose of 65 Gy with concomitant cisplatin and 5-fluorouracil. Maintenance chemotherapy consisted of high-dose methotrexate and six cycles of epirubicine, cyclophosphamide and fluorouracil. Hormonal treatment was given if indicated. Mastectomy was not systemic. Among 65 evaluable patients, 57 (87.6%) achieved a complete clinical response and had a breast conservation. Only six loco regional relapses were noted in six patients with a delay of 20 months and with concomitant metastatic dissemination in four cases. Median disease-free survival (DFS) was 28 months. Median overall survival (OS) was 63 months and median follow-up was 55.5 months. Induction chemotherapy and concomitant irradiation is feasible in patients with IBC, permitting a breast conservation with a high rate of local control with an OS comparable to that of the best recent series.     

Growth-related oncogene produced in human breast cancer cells and regulated by Syk protein-tyrosine kinase

Syk, a nonreceptor type of protein tyrosine kinase widely expressed in hematopoietic cells, is a candidate suppressor gene in human breast cancer. Reduced expression of Syk protein is correlated with poor prognosis, while its overexpression can reduce the malignant phenotype of breast cancer cells. The mechanism of action of Syk remains unclear. In this study, we utilized low Syk-expressing, highly invasive MDA-MB-231 and high Syk-expressing, less invasive MCF-7 breast cancer cells to investigate the possibility that part of the functional effects of Syk are mediated by cytokines known to play roles in cell migration, invasion or metastasis. Using protein array technology, we determined that MDA-MB-231 cells secrete a number of cytokines known to regulate cellular growth and motility. One such cytokine, growth-related oncogene (GRO), has previously not been described in breast cancer. Of the compounds detected in the culture supernatant of MDA-MB-231, GRO was the only one that was significantly altered by modulation of Syk expression; overexpression of Syk caused a marked reduction in secreted levels of GRO. Conversely, downregulation of the relatively high levels of Syk produced in MCF-7 cells upregulated GRO secretion. At the mRNA level, overexpression of Syk in MDA-MB-231 differentially regulated the 3 GRO isotypes such that message levels of GROalpha and gamma were downregulated while that of GRObeta was not affected. Matrigel invasion assays demonstrated a link between Syk expression and resulting GRO activity in mediating the invasive potential of MDA-MB-231 cells. In summary, our findings provide evidence that human breast cancer cells express and secrete GRO and implicate this cytokine as an essential mediator of the antiinvasive properties of Syk tyrosine kinase.     

Expression of pp32 gene family members in breast cancer

The pp32 gene family consists of at least three closely related members, pp32, pp32r1 and pp32r2. In spite of a high degree of identity at the nucleotide level, pp32 functionally behaves as a tumor suppressor where as pp32r1 and pp32r2 are pro-oncogenic. The purpose of this pilot study was to determine pp32-related expression and whether alternative gene use among the pp32 family members occurred in human breast cancer. As a first step, in situ hybridization with a riboprobe capable of hybridizing with all the three members showed abundant pp32-related mRNA in benign ducts and acini and in infiltrating ductal carcinomas. A total of 100/102 cases were positive. Further, a detailed molecular analysis by RT-PCR, cloning, and sequencing was performed in five frozen infiltrating breast carcinomas and matched benign breast tissues. Oncogenic pp32r1 (5/5) and pp32r2 (3/5) expression was observed in carcinomas where as benign breast tissues expressed pp32. 4/5 carcinomas continued to express pp32 but one was devoid of pp32 expression. These results suggest that alternative expression of pp32 family members may be common in human breast cancer and the analysis of the profile of pp32-related expression might be helpful in understanding the role of these genes in breast cancer pathogenesis.     

A novel low-molecular weight inhibitor of focal adhesion kinase, TAE226, inhibits glioma growth

Glioblastomas are highly lethal cancers that resist current therapies. Novel therapies under development target molecular mechanisms that promote glioblastoma growth. In glioblastoma patient specimens, the non-receptor tyrosine kinase focal adhesion kinase (FAK) is overexpressed. Upon growth factor receptor stimulation or integrin engagement, FAK is activated by phosphorylation on critical tyrosine residues. Activated FAK initiates a signal transduction cascade which promotes glioma growth and invasion by increasing cellular adhesion, migration, invasion, and proliferation. We find that human glioma cell lines express different levels of total FAK protein and activating phosphorylation of tyrosine residues Tyr397, Tyr861, and Tyr925. As all glioma cell lines examined expressed phosphorylated FAK, we examined the efficacy of a novel low-molecular weight inhibitor of FAK, TAE226, against human glioma cell lines. TAE226 inhibited the phosphorylation of FAK as well as the downstream effectors AKT, extracellular signal-related kinase, and S6 ribosomal protein in multiple glioma cell lines. TAE226 induced a concentration-dependent decrease in cellular proliferation with an associated G(2) cell cycle arrest in every cell line and an increase in apoptosis in a cell-line-specific manner. TAE226 also decreased glioma cell adhesion, migration, and invasion through an artificial extracellular matrix. Together, these data demonstrate the potential benefit of TAE226 for glioma therapy.