Temozolomide and radiotherapy in newly diagnosed glioblastoma patients:O^6-methylguanine-DNA methyltransferase (MGMT) promotor methylation status and Ki-67 as biomarkers for survival and response to treatment

Objective:This phase II study aimed at investigating the correlation between O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and protein expression,together with Ki-67 labeling index (LI),to response,time to progression (TTP),and overall survival (OS) in newly diagnosed glioblastoma multiforme (GBM) patients treated with temozolomide (TMZ) concomitant with and adjuvant to radiotherapy (RT).Methods:From June 2005 to August 2008,34 adult patients (18-65 years),PS ≥70,with newly diagnosed GBM received TMZ 75 mg/m2 plus RT up to 60 Gy,followed by TMZ 175 mg/m2 5 days every 4 weeks for 12 doses.MGMT Methylation-specific PCR assay,MGMT protein expression,and Ki-67 expression using immunohistochemistry (IHC) were performed on the tissue blocks.The patients were followed by MRI while MR spectroscopy (MRS) was performed for the stable cases or to confirm progression and accordingly Bevacizumab 10 mg/kg every 2 weeks was added to 7 patients till further progression was proved.Results:31 cases were evaluable,12 (38.7%) had unmethylated MGMT,while 19 (61.3%) were methylated.Seventeen cases (55%) were MGMT immunonegative while 14 cases (45%) were immunopositive.The cut off value of Ki-67 LI in relation to survival was 17%,where 15 were < 17% (48.4%),and 16 were ≥ 17% (51.6%).Response evaluation started after the second dose of the adjuvant TMZ and was repeated every 2 months.The overall disease control rate (ODC) was 74.2%,where 2 patients had complete response (CR),14 had partial response (PR),and 7 had stable disease (SD),while 8 (25.8%) had progressive disease (PD).The ODC was significantly higher among methylated patients and in those with Ki-67 < 17% (P=0.0003).The median overall TTP was 12 months and the median OS was 20 months for all the patients including those who received Bevacizumab for some stable cases or as a salvage treatment in patients with good PS,the MGMT-methylated patients had a higher median TTP of 13 months (range 8 to 18 months,95% CI of 9.36 to 12.9),and OS of 24 months (range 12 to 31 months,95% CI of 16.1 to 21.32),while the unmethylated patients had a median TTP of 6.5 months and a median OS of 12 months,such correlations were highly significant (P=0.0001).MGMT immunoexpression failed to show significant correlation with MGMT promotor methylation or the outcome of the patients.Patients with Ki-67 < 17% had a median TTP of 16 months and median OS of 24 months compared to 7 and 12.5 months respectively for the patients with Ki-67 ≥17%.Significant correlation was found between the ODC,TTP,and OS with age < 52,near total excision,and TMZ doses received ≥ 10.The commonest grade 3 and 4 toxicities was neutropenia recorded in 3 patients (9.67%),thrombocytopenia in 4 patients (12.9%),and one patient with G3 nausea,vomiting,and constipations (3%),all were medically manageable.Conclusion:MGMT promotor methylation status and Ki-67 LI (but not the MGMT protein expression),could serve as prognostic markers for survival,also MGMT could identify the newly diagnosed GBM patients who will have better response to TMZ.

Analysis of clinicopathological and immunohistochemical features of 15 cases with olfactory neuroblastoma

Objective:We studied the clinicopathological and immunohistochemical features of olfactory neuroblastoma(ONB).Methods:The clinic pathological data and immunohistochemical features of 15 cases with ONB were analyzed retrospectively,and the related literatures were reviewed.Results:The tumors were all located in the nasal cavity in 15 cases.According to Kadish's staging system,7 cases were in stage A,5 cases in stage B,and 3 cases in stage C.The morphological features showed small round or ovoid tumor cells divided into pieces or trabeculae by connective tissues which were rich in capillaries.The tumor cells had round or oval nuclei and fine chromatins and lack of cytoplasma.Flexner rosette and Homer-Wright rosette were found in some cases.Acidophilic fibrins were composed of cytoplasmic projection among tumor cells.All cases were positive for NSE,Syn,CgA,1 case was positive for Vimentin,2 cases were positive for S-100,while CKpan,LCA and HMB45 were all negative.Conclusion:ONB is a type of very rare malignant tumors,which could be diagnosed by pathology,and immunohistochemistry is helpful in the diagnosis and differential diagnosis.

Extramedullary skeletal muscle metastasis of glioblastoma:A case report and literature review

Objective The aim of the study was to explore the clinicopathologic, immunophenotypic, and diagnostic features of extramedullary metastases of glioblastoma. Methods One case of extramedullary skeletal muscle metastasis of glioblastoma was studied, including the clinical, histological, and immunohistochemical features. Results A 24-year-old man underwent surgical resection for glioblastoma(WHO grade IV) in the left temporal parietal region followed by radiotherapy and temozolomide therapy. One year and nine months later, he developed an extramedullary skeletal muscle metastasis in L4, and the histology was remarkably different from that of the primary glioblastoma specimen. The immunohistochemical analysis also showed changes. In the metastasis, the small cells were negative for GFAP; weakly positive for S-100; and positive for nestin, NSE, and CD56, with 60% of cells positive for p53 and 40% positive for Ki-67. The giant cells showed strong positivity for GFAP and S-100, and weak expression of p53, Ki-67, nestin, NSE, and CD56. The primary glioblastoma specimen showed strong positivity for GFAP and S-100 and was negative for NSE, nestin, and CD56, with around 25% of the tumor cells positive for p53 and a Ki-67 labeling index of 20%. Conclusion Extraneural metastasis(ENM) is a rare complication of glial tumors and glioma stem cells may be related to the metastasis. Since extraneural metastasis may occur in patients without central nervous symptoms, any unusual signs during the follow-up of patients diagnosed with glioblastoma should not be underestimated.

The herbal compound geniposide rescues formaldehyde-induced apoptosis in N2a neuroblastoma cells

The herbal medicine Tong Luo Jiu Nao （TLJN） contains geniposide （GP） and ginsenoside Rgl at a molar ratio of i0：1. Rgl is the major component of another herbal medicine, panax notoginseng saponin （PNS）. TLJN has been shown to strengthen brain function in humans, and in animals it improves learning and memory. We have previously shown that TLJN reduces amyloi- dogenic processing in Alzheimer＇s disease （AD） mouse models. Together this suggests TLJN may be a potential treatment for patients with dementia. Because chronic damage of the central nervous system by formaldehyde （FA） has been presented as a risk factor for age-associated cognitive dysfunction, in the present study we investigated the protective effect of both TLJN and GP in neuron-like cells exposed to FA. FA-exposed murine N2a neuroblastoma cells were incubated with TLJN, its main in- gredient GP, as well as PNS, to measure cell viability and morphology, the rate of apoptosis and expression of genes encoding Akt, FOXO3, Bcl2 and p53. The CCK-8 assay, cytoskeletal staining and flow cytometry were used to test cell viability, mor- phology and apoptosis, respectively. Fluorescent quantitative real-time PCR （qRT-PCR） was used to monitor changes in gene expression, and HPLC to determine the rate of FA clearance. Treatment of N2a cells with 0.09 mmol L-1 FA for 24 h signifi- cantly reduced cell viability, changed cell morphology and promoted apoptosis. Both TLJN and GP conferred neuroprotection to FA-treated N2a cells, whereas PNS, which had to be used at lower concentrations because of its toxicity, did not. Our data demonstrate that TLJN can rescue neuronal damage caused by FA and that its main ingredient, GP, has a major role in this ef- ficacy. This presents purified GP as a drug or lead compound for the treatment of AD.

Visualizing the microtubule-associated protein tau in the nucleus

Although tau is mainly known as an axonal microtubule-associated protein,many studies indicate that it is not restricted to this subcellular compartment.Assessing tau’s subcellular distribution,however,is not trivial as is evident from transgenic mouse studies.When human tau is over-expressed,it can be immunohistochemically localized to axons and the somatodendritic domain,modeling what is found in neurodegenerative diseases such as Alzheimer’s disease.Yet,in wild-type mice,despite its abundance,tau is difficult to visualize even in the axon.It is even more challenging to detect this protein in the nucleus,where tau has been proposed to protect DNA from damage.To establish a framework for future studies into tau’s nuclear functions,we compared several methods to visualize endogenous nuclear tau in cell lines and mouse brain.While depending on the fixation and permeabilization protocol,we were able to detect nuclear tau in SH-SY5Y human neuroblastoma cells,we failed to do so in N2a murine neuroblastoma cells.As a second method we used subcellular fractionation of mouse tissue and found that in the nucleus tau is mainly present in a hypophosphorylated form.When either full-length or truncated human tau was expressed,both accumulated in the cytoplasm,but were also found in the nuclear fraction.Because subcellular fractionation methods have their limitations,we finally isolated nuclei to probe for nuclear tau and found that the nuclei were free of cytoplasmic contamination.Together our analysis identifies several protocols for detecting tau in the nucleus where it is found in a less phosphorylated form.

Toxicity of graphene oxide and multi-walled carbon nanotubes against human cells and zebrafish

Graphene possesses unique physical and chemical properties, which have inspired a wide range of potential biomedical applications. However, little is known about the adverse effects of graphene on the human body and ecological environment. The purpose of our work is to make assessment on the toxicity of graphene oxide (GO) against human cell line (human bone marrow neuroblastoma cell line and human epithelial carcinoma cell line) and zebrafish (Danio rerio) by comparing the toxic effects of GO with its sister, multi-walled carbon nanotubes (MWNTs). The results show that GO has a moderate toxicity to organisms since it can induce minor (about 20%) cell growth inhibition and slight hatching delay of zebrafish embryos at a dosage of 50 mg/L, but did not result in significant increase of apoptosis in embryo, while MWNTs exhibit acute toxicity leading to a strong inhibition of cell proliferation and serious morphological defects in developing embryos even at relatively low concentration of 25 mg/L. The distinctive toxicity of GO and MWNTs should be ascribed to the different models of interaction between nanomaterials and organisms, which arises from the different geometric structures of nanomaterials. Collectively, our work suggests that GO does actual toxicity to organisms posing potential environmental risks and the result is also shedding light on the geometrical structure-dependent toxicity of graphitic nanomaterials.

Transcriptome profile of human neuroblastoma cells in the hypomagnetic field

Research has shown that the hypomagnetic field(HMF)can affect embryo development,cell proliferation,learning and memory,and in vitro tubulin assembly.In the present study,we aimed to elucidate the molecular mechanism by which the HMF exerts its effect,by comparing the transcriptome profiles of human neuroblastoma cells exposed to either the HMF or the geomagnetic field.A total of 2464 differentially expressed genes(DEGs)were identified,216 of which were up-regulated and2248 of which were down-regulated after exposure to the HMF.These DEGs were found to be significantly clustered into several key processes,namely macromolecule localization,protein transport,RNA processing,and brain function.Seventeen DEGs were verified by real-time quantitative PCR,and the expression levels of nine of these DEGs were measured every 6 h.Most notably,MAPK1 and CRY2,showed significant up-and down-regulation,respectively,during the first 6 h of HMF exposure,which suggests involvement of the MAPK pathway and cryptochrome in the early bio-HMF response.Our results provide insights into the molecular mechanisms underlying the observed biological effects of the HMF.