Albumin-bound paclitaxel augment temozolomide treatment sensitivity of glioblastoma cells by disrupting DNA damage repair and promoting ferroptosis.

BACKGROUND: Temozolomide (TMZ) treatment efficacy in glioblastoma (GBM) patients has been limited by resistance in the clinic. Currently, there are no clinically proven therapeutic options available to restore TMZ treatment sensitivity. Here, we investigated the potential of albumin-bound paclitaxel (ABX), a novel microtubule targeting agent, in sensitizing GBM cells to TMZ and elucidated its underlying molecular mechanism. METHODS: A series of in vivo and in vitro experiments based on two GBM cell lines and two primary GBM cells were designed to evaluate the efficacy of ABX in sensitizing GBM cells to TMZ. Further proteomic analysis and validation experiments were performed to explore the underlying molecular mechanism. Finally, the efficacy and mechanism were validated in GBM patients derived organoids (PDOs) models. RESULTS: ABX exhibited a synergistic inhibitory effect on GBM cells when combined with TMZ in vitro. Combination treatment of TMZ and ABX was highly effective in suppressing GBM progression and significantly prolonged the survival oforthotopic xenograft nude mice, with negligible side effects. Further proteomic analysis and experimental validation demonstrated that the combined treatment of ABX and TMZ can induce sustained DNA damage by disrupting XPC and ERCC1 expression and nuclear localization. Additionally, the combination treatment can enhance ferroptosis through regulating HOXM1 and GPX4 expression. Preclinical drug-sensitivity testing based on GBM PDOs models confirmed that combination therapy was significantly more effective than conventional TMZ monotherapy. CONCLUSION: Our findings suggest that ABX has the potential to enhance TMZ treatment sensitivity in GBM, which provides a promising therapeutic strategy for GBM patients.

Individualized combination therapies based on whole-exome sequencing displayed significant clinical benefits in a glioblastoma patient with secondary osteosarcoma: case report and genetic characterization.

BACKGROUND: The incidence of osteosarcoma as a secondary neoplasm in glioblastoma patient is extremely rare. The genetic characteristic still remains unclear until now. CASE DESCRIPTION: We reported a 47-year-old female patient with multiple intracranial disseminations and infiltrations (splenium of the corpus callosum and lateral ventricular wall) of a rapid progressive glioblastoma underwent occipital craniotomy and total resection of all the enhancing lesions. Whole-exome sequencing and pathological examination revealed glioblastoma, IDH1 wild type, PTEN deficient, TERT mutated, NF1mutated, MGMT unmethylated. After surgery, the patient received combined therapeutic regimen of TTFields (tumor-treating fields) plus pembrolizumab plus temozolomide and TTFields plus everolimus, which displayed significant clinical benefits. During the combined therapeutic course, an extremely rare secondary malignant neoplasm occurred, femur MR and pathological detection of biopsy tissue demonstrated osteosarcoma. The result of whole-exome sequencing revealed 7 germline mutated genes (EPAS1, SETD2, MSH3, BMPR1A, ERCC4, CDH1, AR). Bioinformatic analysis showed the two germline mutations (MSH3 and ERCC4) induced deficiency in the DNA repair machinery, which resulting in the accumulation of mutations and may generate neoantigens contributing to the development of a secondary osteosarcoma in this case. CONCLUSION: Individualized combination therapies based on whole-exome sequencing displayed significant clinical benefits in this case. Germline MSH3 and ERCC4 mutation may induce a secondary osteosarcoma in glioblastoma patients.

Recycling of SLC38A1 to the plasma membrane by DSCR3 promotes acquired temozolomide resistance in glioblastoma.

PURPOSE: Glioblastoma multiforme (GBM) is a primary brain tumor with devastating prognosis. Although the O6-methylguanine-DNA methyltransferase (MGMT) leads to inherent temozolomide (TMZ) resistance, approximately half of GBMs were sufficient to confer acquired TMZ resistance, which express low levels of MGMT. The purpose of this study was to investigate the underlying mechanisms of the acquired TMZ resistance in MGMT-deficient GBM. METHODS: The function of Down syndrome critical region protein 3 (DSCR3) on MGMT-deficient GBM was investigated in vitro and in an orthotopic brain tumor model in mice. Purification of plasma membrane proteins by membrane-cytoplasmic separation and subsequent label free-based quantitative proteomics were used to identified potential protein partners for DSCR3. Immunofluorescence was performed to show the reverse transport of solute carrier family 38 member 1 (SLC38A1) mediated by DSCR3. RESULTS: DSCR3 is upregulated in MGMT-deficient GBM cells during TMZ treatment. Both DSCR3 and SLC38A1 were highly expressed in recurrent GBM patients. Silencing DSCR3 or SLC38A1 expression can increase TMZ sensitivity in MGMT-deficient GBM cells. Combination of proteomics and in vitro experiments show that DSCR3 directly binds internalized SLC38A1 to mediate its sorting into recycling pathway, which maintains the abundance on plasma membrane and enhances uptake of glutamine in MGMT-deficient GBM cells. CONCLUSIONS: DSCR3 is a crucial regulator of acquired TMZ resistance in MGMT-deficient GBM. The DSCR3-dependent recycling of SLC38A1 maintains its abundance on plasma membrane, leading to tumor progression and acquired TMZ resistance in MGMT-deficient GBM.

Quantitative Proteomics Analysis Reveals Nuclear Perturbation in Human Glioma U87 Cells treated with Temozolomide.

Glioblastoma (GBM) is the most malignant and aggressive glioma, which has a very poor prognosis. Temozolomide (TMZ) is still a first-line treatment, but resistance is inevitable even in MGMT-deficient glioblastoma cells. The aims of this study were to comprehend the effect of TMZ on nucleus and the underlying mechanism of acquired TMZ resistance in MGMT-deficient GBM. We show the changes of nuclear proteome in the MGMT-deficient GBM U87 cells treated with TMZ for 1 week. Label-free-based quantitative proteomics were used to investigate nuclear protein abundance change. Subsequently, gene ontology function annotation, KEGG pathway analysis, protein-protein interaction (PPI) network construction analysis of DAPs, and immunofluorescence were applied to validate the quality of proteomics. In total, 457 (455 gene products) significant DAPs were identified, of which 327 were up-regulated and 128 were down-regulated. Bioinformatics analysis uncovered RAD50, MRE11, UBR5, MSH2, MSH6, DDB1, DDB2, RPA1, RBX1, CUL4A, and CUL4B mainly enriched in DNA damage repair related pathway and constituted a protein-protein interaction network. Ribosomal proteins were down-regulated. Cells were in a stress-responsive state, while the entire metabolic level was lowered. SIGNIFICANCE OF THE STUDY: In U87 cell treated with TMZ for 1 week, which resulted in DNA damage, we found various proteins dysregulated in the nucleus. Some proteins related to the DNA damage repair pathway were up-regulated, and there was a strong interaction. We believe this is the potential clues of chemotherapy resistance in tumour cells. These proteins can be used as indicators of tumour resistance screening in the future.

Acquired temozolomide resistance in MGMT-deficient glioblastoma cells is associated with regulation of DNA repair by DHC2.

The acquisition of temozolomide resistance is a major clinical challenge for glioblastoma treatment. Chemoresistance in glioblastoma is largely attributed to repair of temozolomide-induced DNA lesions by O6-methylguanine-DNA methyltransferase (MGMT). However, some MGMT-deficient glioblastomas are still resistant to temozolomide, and the underlying molecular mechanisms remain unclear. We found that DYNC2H1 (DHC2) was expressed more in MGMT-deficient recurrent glioblastoma specimens and its expression strongly correlated to poor progression-free survival in MGMT promotor methylated glioblastoma patients. Furthermore, silencing DHC2, both in vitro and in vivo, enhanced temozolomide-induced DNA damage and significantly improved the efficiency of temozolomide treatment in MGMT-deficient glioblastoma. Using a combination of subcellular proteomics and in vitro analyses, we showed that DHC2 was involved in nuclear localization of the DNA repair proteins, namely XPC and CBX5, and knockdown of either XPC or CBX5 resulted in increased temozolomide-induced DNA damage. In summary, we identified the nuclear transportation of DNA repair proteins by DHC2 as a critical regulator of acquired temozolomide resistance in MGMT-deficient glioblastoma. Our study offers novel insights for improving therapeutic management of MGMT-deficient glioblastoma.

Identification of Key Candidate Proteins and Pathways Associated with Temozolomide Resistance in Glioblastoma Based on Subcellular Proteomics and Bioinformatical Analysis.

TMZ resistance remains one of the main reasons why treatment of glioblastoma (GBM) fails. In order to investigate the underlying proteins and pathways associated with TMZ resistance, we conducted a cytoplasmic proteome research of U87 cells treated with TMZ for 1 week, followed by differentially expressed proteins (DEPs) screening, KEGG pathway analysis, protein-protein interaction (PPI) network construction, and validation of key candidate proteins in TCGA dataset. A total of 161 DEPs including 65 upregulated proteins and 96 downregulated proteins were identified. Upregulated DEPs were mainly related to regulation in actin cytoskeleton, focal adhesion, and phagosome and PI3K-AKT signaling pathways which were consistent with our previous studies. Further, the most significant module consisted of 28 downregulated proteins that were filtered from the PPI network, and 9 proteins (DHX9, HNRNPR, RPL3, HNRNPA3, SF1, DDX5, EIF5B, BTF3, and RPL8) among them were identified as the key candidate proteins, which were significantly associated with prognosis of GBM patients and mainly involved in ribosome and spliceosome pathway. Taking the above into consideration, we firstly identified candidate proteins and pathways associated with TMZ resistance in GBM using proteomics and bioinformatic analysis, and these proteins could be potential biomarkers for prevention or prediction of TMZ resistance in the future.

The Impact of MMP-2 and Its Specific Inhibitor TIMP-2 Expression on the WHO Grade and Prognosis of Gliomas in Chinese Population: a Meta-Analysis.

So far, the prognostic value of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of matrix metalloproteinase 2 (TIMP-2) expressions in patients with gliomas has been widely reported, especially in China. But, the results were inconsistent. Thus, we conducted a meta-analysis to determine the correlation of MMP-2 and TIMP-2 expressions with the prognosis of patients with gliomas. Identical search strategies were used to search relevant literature in electronic databases updated to May 1, 2015, and odds ratios (ORs) with 95 % confidence intervals (95 % CIs) were estimated. Funnel plots and Egger's tests were conducted for the evaluation of publication bias, and heterogeneity and sensitivity were also analyzed. Finally, a total of 25 studies involving 1572 patients were included in the meta-analysis. Coincidentally, all these studies were conducted in Chinese population. It was found that MMP-2 expression was significantly associated with high-WHO grade gliomas (n = 24, OR = 6.54, CI = 4.98-8.60; I 2 = 0 %, P = 0.911) and poor overall survival (OS), while it did not correlate to age (n = 2, OR = 0.78, CI = 0.35-1.74; I 2 = 0 %, P = 0.621) and gender (n = 2, OR = 1.15, CI = 0.51-2.62; I 2 = 0 %, P = 0.995). Moreover, the results of the pooled analysis indicated that there was no association between TIMP-2 expression and the WHO grade of gliomas (n = 7, OR = 1.02, 95 % CI = 0.68-1.54; I 2 = 71.4 %, P = 0.002), but the ratio of MMP-2 and TIMP-2 (MMP-2/TIMP-2) rose with the increase of the WHO grade of gliomas. In conclusion, there was no correlation between TIMP-2 expression and the WHO grade of gliomas, while MMP-2 expression was potently associated with high-WHO grade of gliomas.

Akt and ß-catenin contribute to TMZ resistance and EMT of MGMT negative malignant glioma cell line.

Glioblastoma is one of the most lethal cancers in central nervous system, and some individual cells that cannot be isolated for surgical resection and also show treatment-resistance induce poor prognosis. Hence, in order to research these cells, we treated temozolomide (TMZ)-sensitive U87MG cells with 400µM TMZ in culture media for over 6months and established TMZ-resistant cell line designated as U87/TR. We detected the MGMT status through pyrosequencing and western blotting, and we also assessed the proliferation, migration, EMT-like changes and possible activated signaling pathways in U87/TR cells. Our results demonstrated that U87/TR was MGMT negative, which indicated that MGMT made no contribution for TMZ-resistance of U87/TR. And U87/TR cells displayed cell cycle arrest, higher capacity for migration and EMT-like changes including both phenotype and characteristic proteins. We also revealed that both ß-catenin and the phosphorylation level of Akt and PRAS40 were increased in U87/TR, while we did not observe the phosphorylation of mTOR in U87/TR. It indicated that activation of Akt and Wnt/ß-catenin pathways may be response for the chemo-resistance and increased invasion of U87/TR cells, and the phosphorylation of PRAS40 and inactivated mTOR may be related to cell cycle arrest in U87/TR cells.

[RNA interference of PC4 and SFRS1 interacting protein 1 inhibits invasion and migration of U87 glioma cells].

OBJECTIVE: To investigate the effect of small interfering RNA (siRNA)-mediated silencing of PC4 and SFRS1 interacting protein 1 (PSIP1) on invasion and migration of human glioma U87 cells. METHODS: Chemically synthesized siRNA targeting PSIP1 gene was transfected into U87 cells via lipofectamine, and the gene silencing effect was determined using real-time PCR. The changes in the invasion and migration abilities of the transfected cells were assessed with Transwell assay and wound healing assay, respectively. Western blotting was used to analyze the expression of N-cadherin, ß-catenin and the transcription factor Slug. RESULTS: The mRNA and protein level of PSIP1 was significantly reduced in U87 cells after transfection with PSIP1 siRNA (P<0.0001). PSIP1 knockdown in U87 cells resulted in significant suppression of cell invasion and migration abilities (P<0.01) and also reduced N-cadherin, ß-catenin and Slug expressions. CONCLUSION: s Silencing of PSIP1 impairs the invasion and migration abilities of glioma cells and lowers the expressions of N-cadherin, ß-catenin and Slug, suggesting that PSIP1 may regulate Slug by classical Wnt/ß-catenin signaling pathway to modulate epithelial-mesenchymal transition and promote the invasion and migration of glioma cells.

The usefulness of xuefu zhuyu tang for patients with angina pectoris: a meta-analysis and systematic review.

Objective. To evaluate the efficacy of Xuefu Zhuyu Tang (XFZYT) for treating angina pectoris (AP). Methods. Six databases were searched (up to December, 2013). Eligible randomized controlled trials (RCTs) evaluating the efficiency of XFZYT plus traditional antianginal medications (TAMs) compared with TAMs alone in patients with AP were included. The outcomes were relief of anginal symptoms (RAS) and improvement of electrocardiogram (ECG) and blood high-density lipoprotein cholesterol (HDL-C) level. Result. Finally 14 RCTs were included. There were evidences that XFZYT combined with TAMs was more effective in improving RAS (RR = 1.29; 95% CI = [1.20, 1.38]), ECG (RR = 1.37; 95% CI = [1.22, 1.54]), and blood HDL-C level (MD = 0.29 mmol/L; 95% CI = [0.23, 0.35]) compared with TAMs alone. Our meta-analysis also showed the pooled number needed to treat (NNT) of the group with stable angina pectoris (SAP) was smaller in improving RAS (4.2 versus 5.7) and ECG (3.1 versus 5.5) compared with the group with both SAP and unstable angina pectoris (UAP). Conclusion. Combination therapy with XFZYT and TAMs is more effective in treating AP compared with TAMs alone. And XFZYT may be a more suitable choice for the treatment of SAP. However, the findings should be interpreted with caution due to the mediocre methodological quality of the included RCTs.