TMZ@ZIF-8 nanoparticles：preparation，characterization，and in vivo and in vitro anti-tumor studies / 中国药房

OBJECTIVE To prepare zeolite imidazole framework （ZIF）-8 nanoparticles （NPs） loaded with temozolomide （TMZ） （abbreviated as TMZ@ZIF-8 NPs） drug delivery system， thus increasing drug enrichment and anti-glioma effects in lesions. METHODS After preparing ZIF-8 NPs using the room temperature solution reaction method， the impregnation method was used to prepare TMZ@ZIF-8 NPs drug delivery system. Characterization was carried out using transmission electron microscopy， laser particle size， and Fourier transform infrared spectroscopy， and dissolution and anti-tumor activity experiments in vitro and in vivo were conducted. RESULTS TMZ@ZIF-8 NPs were successfully prepared with the particle size of （126.23±7.92） nm， drug loading amount of （28.79±1.26）%， and 72 h cumulative dissolution rate of （72.36±3.62）%. The results of in vitro anti-tumor activity experiments showed that the relative cell survival rate of ZIF-8 NPs remained above 90%； the prepared TMZ@ZIF-8 NPs delivery system exhibited superior inhibition， higher uptake capacity， and better promoting apoptosis effects on the growth and proliferation of C6 cells as compared with the free TMZ. The results of in vivo anti-tumor activity experiments showed that ZIF-8 NPs were not enriched in the brain of rats， and the enrichment effect of TMZ in the brain was not significant， while TMZ@ZIF-8 NPs had a significant enrichment effect in the brain. CONCLUSIONS ZIF-8 NPs can effectively load TMZ， and successfully prepared TMZ@ZIF-8 NPs can improve TMZ uptake ability and anti-glioma effect.

The effects of temozolomide combined with γ-fractional stereotactic radiotherapy on the expression of S100B and exosomal microRNA-330 for non-small cell lung cancer patients with brain metastases / 中国医师进修杂志

Objective:To investigate the effects of temozolomide combined with γ-fractional stereotactic radiotherapy on the expression of S100B and exosomal microRNA-330(miR-330) in the treatment of non-small cell lung cancer (NSCLC) patients with brain metastases.Methods:A total of 82 patients with NSCLC brain metastases from February 2018 to October 2020 were selected prospectively, and they were divided into the control group and the observation group by the random number table method, each with 41 patients. The control group received γ-fractional stereotactic radiotherapy, and the observation group received temozolomide on the basis of the control group. The therapeutic efficacy and prognosis of the two groups were compared, and the levels of serum myelin basic protein (MBP), neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP) levels, liver and kidney function indexes, serum S100B, carcinoembryonic antigen (CEA), exosomal miR-330 were compared between the two groups before and after the treatment. The neurologic function of the patients were evaluated by Mini Mental State Examination (MMSE) and National Institutes Health Stroke Scale (NIHSS).Results:The total remission rate in the observation group was higher than that in the control group: 65.85%(27/41) vs. 34.15%(14/41), there was statistical differences ( χ2 = 8.24, P<0.05), but the disease control rate between the two groups had no significant difference ( P>0.05). After the treatment, the levels of serum MBP, GFAP and NSE in the observation group were lower than those in the control group: (10.13 ± 2.07) μg/L vs. (14.39 ± 2.58) μg/L, (0.57 ± 0.12) μg/L vs. (0.75 ± 0.16) μg/L, (5.09 ± 1.16) μg/L vs. (7.17 ± 1.35) μg/L, there were statistical differences ( P<0.05). The levels alanine aminotransferase, blood urea nitrogen and serum creatinine after treatment between the two groups had no significant differences ( P>0.05). After the treatment, the NIHSS scores in the observation group was lower than that in the control group, MMSE scores was higher than that in the control group: (4.16 ± 0.52) scores vs. (4.73 ± 0.44) scores, (22.07 ± 2.51) scores vs. (20.68 ± 2.19) scores, there were statistical differences ( P<0.05). After treatment, the serum levels of S100B and CEA in the observation group were lower than those in the control group, and the expression of exosomal miR-330 was higher than that in the control group: (62.37 ± 10.54) mg/L vs. (68.05 ± 9.39) mg/L, (12.61 ± 2.05) μg/L vs.(14.08 ± 1.97) μg/L, 0.49 ± 0.12 vs. 0.42 ± 0.05, there were statistical differences ( P<0.05). The median survival time in the observation group was 14.6 months, while that in the control group was 11.50 months. There were no significant differences in the incidence of adverse reactions between the two groups ( P>0.05). Conclusions:Treatment with temozolomide combined with γ-fractional stereotactic radiotherapy for NSCLC patients with brain metastases can improve the therapeutic efficacy, neurological function, inhibit the expression of serum S100B, CEA and exosomal miR-330, and prolong the survival time of patients.

Synergistic sensitization of hUMSCs-derived supernatant combined with temozolomide in different glioma cell lines / 中国病理生理杂志

AIM:To explore the synergistic sensitization effect of human umbilical cord mesenchymal stem cell culture supernatant(hUMSC-CM)combined with temozolomide(TMZ)on various glioma cell lines,and to elucidate the underlying mechanisms.METHODS:The hUMSC-CM was harvested using two different serum deprivation tech-niques at 24 and 48 h,and was converted into freeze-dried powder,which was then given to rat malignant glioma cell line RG-2,human astrocytoma cell line U251 and human glioblastoma cell line LN-428 at 5 concentrations(0,1,3,6 and 9 g/L).The effectiveness and sensitivity of hUMSC-CM for inhibiting growth of glioma cells at 24,48 and 72 h were as-sessed using CCK-8 assay.Hematoxylin-eosin(HE)staining combined with CCK-8 assay was employed to evaluate the chemotherapy sensitivity of glioma cells after 48 h of treatment with TMZ at 6 concentrations(0,25,50,100,200 and 400 μmol/L).Two concentrations(3 and 9 g/L)of hUMSC-CM and 3 concentrations(50,100 and 200 μmol/L)of TMZ were chosen for concurrent treatment of glioma cells to assess the proliferation and pathological alterations.TUNEL staining was utilized to detect apoptosis.Flow cytometry was utilized to analyze cell cycle modifications.The expression alterations of apoptosis-inducing proteins,cleaved caspase-3,cleaved caspase-8 and cleaved PARP1,as well as autophagy-inducing proteins beclin-1 and LC3,were examined using Western blot to investigate the synergistic sensitization mechanism of hUMSC-CM combined with TMZ in vitro.RESULTS:The susceptibility of glioma cell lines to hUMSC-CM and TMZ varied,with RG-2 showing the highest sensitivity,followed by U251,and then LN-428.The inhibitory effect of hUMSC-CM(3 and 9 g/L)and TMZ(50,100 and 200 μmol/L)combined treatment on glioma cells was significantly greater than that that of single-agent treatments(P＜0.05),demonstrating a dose-and concentration-dependent enhancement.Notably,the combination of 9 g/L hUMSC-CM(C9)with 50 μmol/L TMZ(T50)effectively suppressed glioma cell growth.CCK-8 as-say indicated a significant reduction of cell viability in C9+T50 group compared with either C9 or T50 alone(P＜0.05).HE staining and TUNEL staining revealed pronounced morphological changes and significant apoptotic features in glioma cells treated with C9+T50.Flow cytometric analysis confirmed that C9+T50 induced cell cycle arrest in glioma cells.Fur-thermore,compared with control group,the levels of cleaved caspase-3,cleaved caspase-8,cleaved PARP1,beclin-1,and LC3-Ⅱ/LC3-Ⅰ were significantly elevated in the C9+T50-treated glioma cells(P＜0.01).CONCLUSION:(1)The concomitant administration of hUMSC-CM and TMZ exerts a broad inhibitory effect on glioma cells,with a synergistic sen-sitization observed across different cell lines.(2)The enhancement of glioma cell sensitivity to TMZ by hUMSC-CM may be attributed to the modulation of caspase-8/caspase-3/PARP1 signaling pathway and the induction of both apoptosis and autophagy in glioma cells.

Signal mining and analysis of temozolomide adverse events based on FAERS datadase / 中国药师

Objective To mine temozolomide-related adverse drug event(ADE)signals in the real world and to provide a reference for the safe clinical use of temozolomide.Methods The U.S.Food and Drug Administration Adverse Event Reporting System(FAERS)was used to collect the ADE reporting data of temozolomide in the FAERS database from January 1,2004 to December 31,2022.The signal mining was performed using the report ratio method and Bayesian confidence interval progressive neural network method to analyze the occurrence of ADE.Results In the database,there were 24 725 ADE reports with temozolomide as the primary suspected drug,and a total of 300 ADE signals were identified,involving 23 system organ categories,and the top 5 were blood and lymphatic system diseases,systemic diseases and various reactions of administration sites,various examinations,various neurological diseases,various injuries,poisoning and procedural complications etc.the most frequently reported ADE signals included thrombocytopenia,low platelet count,neutral granulocytopenia,pancytopenia,convulsive attacks,and febrile neutropenia.42 new suspected adverse reactions were discovered,which were not recorded in the instructions,such as pseudomonas skin infection,herpetic meningoencephalitis,hypoglossal nerve paralysis,porokeratosis,etc.Conclusion The common adverse reactions of temozolomide in the real world are generally consistent with the instructions,but some new suspicious adverse reactions have been discovered.During clinical drug use,special attention should be paid to these new adverse reactions,and it is recommended to monitor patients'adverse reactions and take appropriate measures in a timely manner.

Effects of Apatinib combined with Temozolomide on levels of sPD-1 and sPD-L1 in patients with drug-resistant recurrent glioblastoma

Abstract Objective This study aimed to explore the effects of Apatinib combined with Temozolomide (TMZ) on the levels of Soluble PD-1 (sPD-1) and Soluble Programmed Death-1 Ligand (sPD-L1) in patients with drug-resistant recurrent Glioblastoma (GB). Study design A total of 69 patients with recurrent GB from September 2020 to March 2022 were recruited and assigned to the control group (n = 34) and observation group (n = 35) according to different treatment options after tumor recurrence. The control group was treated with TMZ, and the observation group was treated with Apatinib combined with TMZ. Levels of sPD-1 and spd-l1, clinical efficacy, survival time and adverse reactions were observed and compared between the two groups. Results General data including gender, age, body mass index, and combined diseases indicated no statistical significance between groups (p > 0.05). Before the intervention, sPD-1 and sPD-L1 levels were not significantly different in the two groups (p > 0.05). After interventions, levels of PD-1 and sPD-L1 levels decreased significantly (p < 0.05). The objective remission rate and clinical benefit rate of the observation group were higher and overall survival and progression-free survival were longer than those of the control group (p < 0.05). No significant difference was observed in major adverse reactions among patients (p > 0.05). Conclusions Apatinib combined with TMZ is safe and effective in the treatment of recurrent GB. The combined application of the two can reduce the levels of sPD-1 and sPD-L1, which has important clinical application value.

Temozolomide alleviates breast carcinoma via the inhibition of EGFR/ERK/ MMP-1 pathway with induction of apoptotic events

Purpose: To evaluate the chemotherapeutic activity of temozolomide counter to mammary carcinoma. Methods: In-vitro anticancer activity has been conducted on MCF7 cells, and mammary carcinoma has been induced in Wistar rats by introduction of 7, 12-Dimethylbenz(a)anthracene (DMBA), which was sustained for 24 weeks. Histopathology, immunohistochemistry, cell proliferation study and apoptosis assay via TUNEL method was conducted to evaluate an antineoplastic activity of temozolomide in rat breast tissue. Results: IC50 value of temozolomide in MCF7 cell has been obtained as 103 µM, which demonstrated an initiation of apoptosis. The temozolomide treatment facilitated cell cycle arrest in G2/M and S phase dose dependently. The treatment with temozolomide suggested decrease of the hyperplastic abrasions and renovation of the typical histological features of mammary tissue. Moreover, temozolomide therapy caused the downregulation of epidermal growth factor receptor, extracellular signal-regulated kinase, and metalloproteinase-1 expression and upstream of p53 and caspase-3 proliferation to indicate an initiation of apoptotic events. Conclusions: The occurrence of mammary carcinoma has been significantly decreased by activation of apoptotic pathway and abrogation of cellular propagation that allowable for developing a suitable mechanistic pathway of temozolomide in order to facilitate chemotherapeutic approach.

Inhibition effect of temozolomide on proliferation of human glioma cells by autophagy-induced pyroptosis / 中华内分泌外科杂志

Objective:To investigate the effect of temozolomide on autophagy of human glioma cells, and the inhibition of autophagy induced pyrocytosis on the proliferation of human glioma cells.Methods:2-64 μ mol/L of temozolomide was used to treat glioma U251 cells cultured in vitro. MTT assay was used to detect cell viability, MDC staining was used to detect autophagic vesicles in cells, cloning assay was used to detect cell proliferation, RT qPCR was used to detect the expression level of pyroptosis related mRNA in cells, Western blot was used to detect the expression of autophagy related proteins and pyroptosis related proteins in cells, and the relationship between autophagy and pyroptosis was detected by adding autophagy inhibitors.Results:Temozolamide could induce autophagy of human glioma cells, and significantly induce tumor cells to pyroptosis, thereby inhibiting the proliferation of tumor cells; RT qPCR results showed that caspase-1, GSDMD, IL-1 after temozolomide administration compared with the normal group β, the mRNA expression levels of IL-18 and NLRP3 increased significantly; Western blot results showed that Cleaved-caspase-1, Cleaved-N-terminalGSDMD, IL-1 β、IL-18 and NLRP3 protein were up-regulated; The incidence of pyroptosis decreased after the addition of autophagy inhibitors.Conclusion:Temozolamide can induce autophagy of human glioma cells, and then lead to pyroptosis, which plays an inhibitory role in proliferation.

A robust luminescent assay for screening alkyladenine DNA glycosylase inhibitors to overcome DNA repair and temozolomide drug resistance / 药物分析学报

Temozolomide(TMZ)is an anticancer agent used to treat glioblastoma,typically following radiation therapy and/or surgical resection.However,despite its effectiveness,at least 50％of patients do not respond to TMZ,which is associated with repair and/or tolerance of TMZ-induced DNA lesions.Studies have demonstrated that alkyladenine DNA glycosylase(AAG),an enzyme that triggers the base excision repair(BER)pathway by excising TMZ-induced N3-methyladenine(3meA)and N7-methylguanine le-sions,is overexpressed in glioblastoma tissues compared to normal tissues.Therefore,it is essential to develop a rapid and efficient screening method for AAG inhibitors to overcome TMZ resistance in glio-blastomas.Herein,we report a robust time-resolved photoluminescence platform for identifying AAG inhibitors with improved sensitivity compared to conventional steady-state spectroscopic methods.As a proof-of-concept,this assay was used to screen 1440 food and drug administration-approved drugs against AAG,resulting in the repurposing of sunitinib as a potential AAG inhibitor.Sunitinib restored glioblastoma(GBM)cancer cell sensitivity to TMZ,inhibited GBM cell proliferation and stem cell char-acteristics,and induced GBM cell cycle arrest.Overall,this strategy offers a new method for the rapid identification of small-molecule inhibitors of BER enzyme activities that can prevent false negatives due to a fluorescent background.

Efficacy of intensity modulated radiotherapy with sequential chemotherapy in the treatment of high-grade glioma and analysis of influential factors / 中国基层医药

Objective:To investigate the efficacy of intensity-modulated radiotherapy with sequential chemotherapy in the treatment of high-grade glioma and analyze the influential factors.Methods:A total of 56 patients with high-grade glioma who received treatment in Yantai Municipal Laiyang Central Hospital from January 2014 to January 2016 were retrospectively analyzed. All patients underwent three-dimensional conformal radiotherapy or enhanced radiotherapy. The use of bevacizumab, pathological grade, and preoperative and postoperative Karnofsky Performance Status scores in all patients were recorded. Cox and other proportional risk regression models were used to analyze the predictors of patient mortality and receiver operating characteristic (ROC) curve analysis was performed.Results:All patients were followed up to April 2022. Follow-up results showed that the median survival time of patients receiving concurrent chemotherapy with temozolomide and adjuvant chemotherapy with temozolomide was 11.6 months. Univariate analysis showed that pathological grade, Karnofsky Performance Status scores, and the degree of tumor resection were correlated with the prognosis of patients ( P = 0.022, 0.049, 0.022). Multivariate analysis showed that the degree of tumor resection and pathological grade were the independent influential factors of prognosis ( P = 0.010, 0.010). Survival curve analysis revealed that the median survival time of patients subjected to total tumor resection was 12.6 months and that of patients subjected to partial tumor resection was 4.8 months. The median survival time of patients subjected to total tumor resection was longer than that of patients subjected to partial tumor resection. The median survival time of patients with WHO grade Ⅲ tumors was 25.2 months, and it was 6.3 months for patients with WHO grade Ⅳ tumors. The median survival time of patients with WHO grade Ⅲ tumors was longer than that of patients with WHO grade Ⅳ tumors. The receiver operating characteristic curve analysis results showed that the area under the receiver operating characteristic curve plotted for using WHO classification of tumors in the neurological system and surgical methods to predict the death of patients with high-grade glioma was 0.783 and 0.814, respectively. WHO tumor grade and surgical methods for prediction of prognosis of high-grade glioma had high accuracy. Conclusion:Low pathological grade and total resection are independent protective factors for the prognosis of patients with high-grade glioma.

Apoptosis-promoting effect of temozolomide loaded nanoparticles combined with low intensity focused ultrasound on human glioma cells / 中华超声影像学杂志

Objective:To prepare liquid-gas phase modified nanoparticles (TMZ/PFP/PLGA NPs) of perfluoropentane (PFP) and temozolomide (TMZ) encapsulated by polylactic-glycolic acid copolymer (PLGA), combined with low intensity focused ultrasound (LIFU) irradiation, and to investigate its ultrasound imaging ability and intervention effect on human glioma cells in vitro.Methods:TMZ/PFP/PLGA NPs were prepared by compound emulsion method. The basic physical and chemical properties and drug loading ability of TMZ/PFP/PLGA NPs were detected. CCK-8 assay was used to detect the cytotoxicity of nanoparticles in vitro and the effect of synergistic intervention with LIFU on the survival rate of glioma cells. The expression levels of apoptosis related proteins Bcl-2, Bax and caspase-3 were detected by Western blot.Results:Under transmission electron microscope, TMZ/PFP/PLGA NPs showed a circular core-shell structure with regular morphology, particle size was (137.9±63.31)nm, encapsulation efficiency of TMZ was (83.01±5.57)%, drug loading was (3.19±0.22)%. The survival rate of U251 cells was still above 70% after 24 hours of co-incubation with nanoparticles. Under the synergistic effect of LIFU irradiation, the apoptosis of U251 cells was accelerated and the survival rate of U251 cells was significantly decreased. The results of Western blot showed that the synergic intervention could significantly down-regulate the expression of apoptosis related protein Bcl-2, and significantly up-regulate the expression of Bax protein and caspase-3 protein (all P<0.05). Conclusions:TMZ/PFP/PLGA NPs have good basic physical and chemical properties. TMZ/PFP/PLGA NPs have low cytotoxicity in vitro while efficiently loading chemotherapeutic drug timozolomide. Synergistic intervention under LIFU irradiation can significantly accelerate the apoptosis of U251 glioma cells, which has a good application prospect.

Marcadores tumorales CA125, He4 e índice ROMA en cáncer de ovario / Tumor markers CA125, He4, ROME index in ovarian cancer / Marcadores tumorais CA125, He4 e índice ROMA no câncer de ovário

El cáncer de ovario es un problema de salud pública para el cual no se cuenta con métodos de tamizaje estandarizados, no obstante, los marcadores Ca125, He4 y el índice de Roma tiene un gran valor en el diagnóstico y pronóstico de esta patología. Objetivo. Analizar el comportamiento de los marcadores tumorales Ca125 y He4 e índice de Roma en la predicción de malignidad en pacientes con masas ováricas. Materiales y Métodos. Se tomaron los resultados de laboratorio de 112 mujeres atendidas en el Hospital General Ambato de los valores séricos de Ca125, He4 y su correspondiente cálculo del índice de Roma. Se los dividió en el grupo pre y postmenopáusico, maligno y benigno. Resultados. El análisis de los resultados definió la relación de Ca 125 y He4 con el diagnóstico de cáncer de ovario con un nivel de confianza del 95% y valor de p<0,05. La probabilidad de diferenciar cáncer de ovario de procesos benigno para Ca125, He4 e índice de Roma fue del 93,33%, 84,4 y 99,7, respectivamente. Conclusiones. El mejor predictor de malignidad es el índice de Roma. Se encontraron valores séricos elevados de He4 mayores para pacientes postmenopáusicas. Se requieren más estudios que avalen un método de tamizaje estandarizado para el cáncer de ovario.

Ovarian cancer is a public health problem for which there are no standardized screening methods; however, Ca125, He4 and Rome index markers are of great value in the diagnosis and prognosis of this pathology. Objective. To analyze the behavior of tumor markers Ca125 and He4 and Rome index in the prediction of malignancy in patients with ovarian masses. Materials and Methods. The laboratory results of 112 women attended at Hospital General Ambato were taken for serum Ca125, He4 and their corresponding calculation of the Rome index. They were divided into premenopausal and postmenopausal, malignant and benign groups. Results. Analysis of the results defined the relationship of Ca 125 and He4 with the diagnosis of ovarian cancer with a confidence level of 95% and value of p<0.05. The probability of differentiating ovarian cancer from benign processes for Ca125, He4 and Rome index was 93.33%, 84.4 and 99.7, respectively. Conclusions. The best predictor of malignancy is the Rome index. Elevated serum He4 values were found to be higher for postmenopausal patients. Further studies are needed to support a standardized screening method for ovarian cancer.

O câncer do ovário é um problema de saúde pública para o qual não existem métodos de triagem padronizados. No entanto, os marcadores índice Ca125, He4 e Roma são de grande valor no diagnóstico e prognóstico desta patologia. Objetivo. Analisar o comportamento dos marcadores tumorais Ca125 e He4 e o índice de Roma na previsão de malignidade em pacientes com massas ovarianas. Materiais e métodos. Os resultados laboratoriais de 112 mulheres tratadas no Hospital Geral Ambato foram tomados para o soro Ca125, He4 e seu correspondente cálculo do índice de Roma. Eles foram divididos em grupos pré e pós-menopausa, malignos e benignos. Resultados. A análise dos resultados definiu a associação de Ca125 e He4 com o diagnóstico de câncer de ovário a um nível de confiança de 95% e valor de p<0,05. A probabilidade de diferenciar o câncer de ovário dos processos benignos para Ca125, He4 e índice de Roma foi de 93,33%, 84,4 e 99,7, respectivamente. Conclusões. O melhor preditor de malignidade é o índice de Roma. Os valores elevados de soro He4 foram considerados mais altos para pacientes na pós-menopausa. São necessários mais estudos para apoiar um método padronizado de triagem para o câncer de ovário.

Mild overexpression of a novel transcription factor Grainy head like-2 (Grhl2) elicits hybrid phenotype in human astrocytoma cells

The Grainy head like-2 (Grhl2) transcription factor plays a major role in embryonic and cancer development. The role of Grhl2 has been intensively studied in various cancers but not for brain cancer. Hence, in this study, we provide a preliminary understanding on the role of Grhl2 that regulate the transition of astrocytoma cells. The human A172 astrocytoma cell line, a mesenchymal cell characterized by mild overexpression of Grhl2 transcription factor, was used in this study. At first, the Grhl2 stably overexpressing A172 clones into three types i.e., Grhl2+ (mild), Grhl2++ (moderate) and Grhl2+++ (high) based on mRNA and protein expression levels of Grhl2 were characterized. Phenotypic characteristics of vector and Grhl2+ cells were observed using phase contrast microscopy. Quantitative PCR (qPCR), Western blot and immunofluorescence were used to detect the level of mesenchymal markers (N-cadherin/vimentin) and also epithelial markers (E-cadherin/ ?- catenin) in vector and Grhl2+ cells. The migration and invasion characteristics of vector and Grhl2+ cells were determined by scratch assay and Boyden chamber assay. Further, the Grhl2+ cells were characterized to determine the effect of temozolomide chemotherapy drug which were widely used in treating brain cancer. As expected, in phase contrast image, we observed the mesenchymal characteristic of A172 cells becomes hybrid phenotype i.e., mixture of mesenchymal (spindle-like fibroblast morphology) and epithelial (cobblestone like appearance) cells upon Grhl2 mild expression (Grhl2+) when compared to vector cells. Further, we found that there was a significant upregulation of E-cadherin at both mRNA and protein levels in Grhl2+ cells when compared to vector cells. There was a significant upregulation of ?-catenin, N-cadherin and vimentin at mRNA levels, but there was no significant upregulation at the protein levels in Grhl2+ cells compared to the vector cells. The migration and invasion were diminished in Grhl2+ cells when compared to the vector control cells. We observed that the Grhl2+ were sensitive to the temozolomide compared to the vector cells. This infers that the Grhl2+ cells were unable to attain complete transition of mesenchymal to epithelial state, and hence we categorized the Grhl2+ cells as hybrid phenotype. The results provide a better understanding of the largely unknown function of Grhl2 in human astrocytoma cells as tumor progression or suppression.

Anti-tumor activity and mechanisms of IDO1 inhibitor in combined treatment with temozolomide on human glioma cell lines / 药学学报

We analyzed the anticancer effect and mechanism of the novel indoleamine 2,‍3-dioxygenase 1 (IDO1) inhibitor NLG-919 combined with temozolomide (TMZ) on human glioma cell lines. The anti-tumor activity of NLG-919 and temozolomide after single and combined treatments was detected by MTT assay. Colony formation assay, invasion assay and migration assays were used to detect the effects of NLG-919 and temozolomide alone or in combination on proliferation, invasion and migration of human glioma cells. A flow cytometry assay was used to detect cell apoptosis, cell cycle arrest, reactive oxygen species (ROS) production and mitochondrial membrane potential damage (JC-1). An immunofluorescence assay was used to detect the expression level of IDO1 and HPLC was used to detect the expression level of L-kynurenine (Kyn) to explore the anti-tumor mechanism of NLG-919 and temozolomide. The results show that NLG-919 had a weak in vitro inhibitory effect compared to that of temozolomide. The IC50 of NLG-919 on U251 cells and U87 after 72 h was 26.9 and 30.7 μmol·L-1, respectively. However, when NLG-919 was used in combination with temozolomide, its anti-glioma activity was significantly increased. Compared with the single treatment, the combination treatment had a potent ability to inhibit proliferation, invasion and migration of glioma cells. Combination treatment improved the capacity of temozolomide to induce cell cycle arrest and inhibit the growth of glioma cells. NLG-919 significantly down-regulated the expression and activity of IDO1 in glioma cells, and the inhibitory effect was improved after combination with temozolomide, and effectively blocked the production of Kyn through the metabolism of L-tryptophan (Trp). In conclusion, the IDO1 inhibitor NLG-919 and temozolomide showed synergistic effects in the anticancer therapy of human glioma cell lines.

Clinical efficacy of temozolomide combined with X-ray stereotactic radiotherapy in the treatment of recurrent glioma / 中国医师杂志

Objective:To investigate the curative efficacy of X-ray stereotactic radiotherapy combined with temozolomide in the treatment of recurrent glioma.Methods:48 patients with recurrent glioma treated in Mianyang Central Hospital from January 2018 to January 2019 were retrospectively selected, including 24 patients treated with stereotactic radiotherapy as the control group and 24 patients treated with temozolomide combined with stereotactic radiotherapy as the observation group. The treatment effect, inflammatory factor level, incidence of adverse events and survival rate were compared between the two groups.Results:The complete remission rate and total effective rate in the observation group were significantly higher than those in the control group (66.7% vs 37.5%, 87.5% vs 62.5%) (all P<0.05). There were no significant differences in serum levels of hepatocyte growth factor (HGF), tumor necrosis factor-α (TNF-α) and interleukin-17 (IL-17) between the two groups before treatment (all P>0.05). After treatment, the serum levels of HGF, TNF-α and IL-17 in observation group was significantly lower than those in control group (all P<0.05). The incidence of adverse events in the observation group was significantly lower than that in the control group ( P<0.05). During follow-up of 6, 12 and 18 months, the survival rate of patients in the observation group was significantly higher than that in the control group, with statistically significant difference (all P<0.05). Conclusions:The combination of X-ray stereotactic radiotherapy with temozolomide in the treatment of recurrent glioma shows better clinical outcome and extended survival rate. To conclude, this combined treatment is recommended in further clinical promotion.

Inhibition of Ciliogenesis Enhances the Cellular Sensitivity to Temozolomide and Ionizing Radiation in Human Glioblastoma Cells / 生物医学与环境科学（英文）

Objective@#To investigate the function of primary cilia in regulating the cellular response to temozolomide (TMZ) and ionizing radiation (IR) in glioblastoma (GBM).@*Methods@#GBM cells were treated with TMZ or X-ray/carbon ion. The primary cilia were examined by immunostaining with Arl13b and γ-tubulin, and the cellular resistance ability was measured by cell viability assay or survival fraction assay. Combining with cilia ablation by IFT88 depletion or chloral hydrate and induction by lithium chloride, the autophagy was measured by acridine orange staining assay. The DNA damage repair ability was estimated by the kinetic curve of γH2AX foci, and the DNA-dependent protein kinase (DNA-PK) activation was detected by immunostaining assay.@*Results@#Primary cilia were frequently preserved in GBM, and the induction of ciliogenesis decreased cell proliferation. TMZ and IR promoted ciliogenesis in dose- and time-dependent manners, and the suppression of ciliogenesis significantly enhanced the cellular sensitivity to TMZ and IR. The inhibition of ciliogenesis elevated the lethal effects of TMZ and IR via the impairment of autophagy and DNA damage repair. The interference of ciliogenesis reduced DNA-PK activation, and the knockdown of DNA-PK led to cilium formation and elongation.@*Conclusion@#Primary cilia play a vital role in regulating the cellular sensitivity to TMZ and IR in GBM cells through mediating autophagy and DNA damage repair.

Plumbagin Enhanced Inhibitory Effect of Temozolomide on Migration and Invasion of Human Glioma U251 Cells Through E-cadherin / 肿瘤防治研究

Objective To investigate the inhibitory effect and mechanism of temozolomide on migration and invasion of U251 human glioma cells enhanced by plumbagin. Methods CCK-8 method was used to study the effects of plumbagin, temozolomide and plumbagin+temozolomide on the proliferation of glioma U251 cells. Cell scratch test was used to detect the migration of U251 cells in the control (DMSO), plumbagin, temozolomide and plumbagin+temozolomide groups for 48h. Transwell assay was used to detect the effect of the combination therapy on the invasion of U251 cells. Western blot was used to detect the relative expression levels of E-cadherin in three groups. Results CCK-8 showed that the proliferation inhibition rate of U251 cells treated with plumbagin (1.25 μmol/L) combined with temozolomide (200 μmol/L) for 48h was 75.69%, significantly higher than that treated with plumbagin alone (P=0.012) or temozolomide alone (P=0.034). Cell scratch assay showed that the combination of plumbagin and temozolomide could significantly enhance the inhibition effect of temozolomide on the migration of U251 cells (P=0.023). Transwell assay showed that the invasion ability of U251 cells was significantly decreased after the combination therapy (P < 0.05). The protein expression of E-cadherin in the combination group was significantly higher than those in plumbagin or temozolomide groups (P < 0.05). Conclusion Plumbagin combined with temozolomide can inhibit the migration and invasion of glioma cells and enhance the sensitivity of glioma cells to temozolomide. And the effect is achieved by the protein expression of E-cadherin.

Establishment of a mouse model bearing orthotopic temozolomide-resistant glioma / 南方医科大学学报

OBJECTIVE@#To establish a mouse model bearing orthotopic temozolomide (TMZ)-resistant glioma that mimics the development of drug resistance in gliomas @*METHODS@#Seventy-eight adult C57BL/6 mice were randomly divided into 6 groups (@*RESULTS@#The mouse models bearing TMZresistant glioma was successfully established. The cells from the high-dose induced group showed a significantly higher colony-forming rate than those from the high-dose control group (@*CONCLUSIONS@#Progressive increase of TMZ doses in mice bearing orthotopic gliomas can effectively induce TMZ resistance of the gliomas.

Effect of lenalidomide combined with temozolomide on drug resistance and O6-methylguanine-DNA methyltransferase gene epigenetic modification of U251/TR cells / 中华神经医学杂志

Objective:To investigate the effect of lenalidomide (LEN) combined with temozolomide (TMZ) on proliferation, invasion, drug resistance and O6-methylguanine-DNA methyltransferase ( MGMT) gene epigenetic modification of TMZ-resistant human glioblastoma cell line U251/TR. Methods:A TMZ-resistant human glioma cell line, U251/TR, was successfully established by stepwise exposure of U251 parental cells to TMZ. U251/TR cells were divided into dimethyl sulfoxide (DMSO) group, LEN group, TMZ group and LEN+TMZ group (DMSO group: without any drug intervention; LEN group, TMZ group, and LEN+TMZ group were pretreated with 100 μmol/L LEN, 200 μmol/L TMZ, 100 μmol/L LEN+200 μmol/L TMZ, respectively; the drugs were administered once every 24 h). The proliferation rate of these cells in each group was detected by sulfonylrhodamine B colorimetric assay at different time points (24, 48, 72, and 96 h after treatment). At 96 h after treatment, the invasion and migration abilities of cells in each group were detected by Transwell assay; the proliferation cycle of cells in each group was detected by flow cytometry; Western blotting, immunohistochemical staining and immunofluorescence staining were used to detect the MGMT protein expression, and the MGMT mRNA expression in cells of each group was detected by reverse transcription-PCR; methylation specific PCR was used to detect the MGMT gene promoter methylation in each group of cells. Results:The cell proliferation rate of LEN+TMZ group was significantly decreased as compared with TMZ, LEN, and DMSO groups at 24, 48, 72 and 96 h after treatment ( P<0.05). At 96 h after treatment, LEN+TMZ group had significantly decreased number of transmembrane cells, and significantly increased ratio of cells at G0/G1 phase as compared with the other 3 groups ( P<0.05); the MGMT protein and mRNA expression levels in TMZ group and LEN+TMZ group were significantly lower than those in LEN group and DMSO group ( P<0.05); and the number of cells with strong or moderate MGMT expression in TMZ group and LEN+TMZ group was obviously less than that in LEN group and DMSO group, and the MGMT fluorescence intensity in TMZ group and LEN+TMZ group (+) was obviously lower than that in LEN group (+++) and DMSO group (+++). The MGMT gene promoter was unmethylated in all groups. Conclusion:LEN alone does not obviously inhibit the proliferation and invasion of U251/TR cells; but LEN combined with TMZ could inhibit the proliferation and invasion of U251/TR cells and co-reverse the drug resistance of U251/TR cells, whose mechanism is not related to the changes of MGMT gene promoter methylation.

Preliminary exploration of relevant mechanisms and effects of LncRNA LY6E-DT on temozolomide resistance in high grade glioma cell U251 / 中华内分泌外科杂志

Objective:To investigate the effect of long non-coding RNA (LncRNA) LY6E-DT on temozolomide (TMZ) resistance in high grade glioma cells (HGG) cells and its mechanism.Methods:Bioinformatics screened the LncRNA correlated to the expression of O6-methylguanine-DNA methyltransferase (MGMT) in HGG tissue, and excavated potentially related microRNA (miRNA) . HGG cell U251 was cultured and randomly divided into control group, resistance group and interference group. Resistance group and interference group were treated with 0.2-16.0 μg·ml -1 TMZ gradient incremental induction, and isosmotic PBS was used to treat control group cells. Control group and resistance group were transfected with control vector, while the interference group was transfected with shRNA interference vector targeting LY6E-DT by lentivirus. The semi-inhibitory concentration (IC 50) of TMZ in each group was detected by CCK-8 experiment, the expression levels of LY6E-DT, miR-125a-5p and MGMT mRNA in each group were detected by real-time fluorescent quantitative PCR (RT-qPCR) , and the expression levels of MGMT protein were detected by Western blot. The effect of miR-125a-5p on MGMT and LY6E-DT was detected by luciferase reporter gene method. Results:LY6E-DT expression was positively correlated with MGMT (Pearson correlation coefficient = 0.32, P<0.001) , and it was found that LY6E-DT expression was significantly lower in HGG tissues, but it was not conducive to the prognosis of HGG patients; In the control group, resistance group and interference group, the expressions of LY6E-DT were 1.000±0.047, 3.704±0.402 and 0.743±0.064; the expressions of miR-125a-5p were 1.000±0.049, 0.351±0.031 and 0.934±0.050; the expressions of MGMT mRNA were 1.000±0.017, 5.205±0.462 and 3.183±0.667; the expressions of MGMT protein are 0.108±0.012, 0.891±0.063 and 0.375±0.038; TMZ IC 50 are 6.79±0.71, 30.52±3.69 and 15.64±2.25 μg·ml -1. Compared with the control group, LY6E-DT, MGMT and TMZ IC50 in the resistance group were significantly higher, while LY6E-DT expression in the interference group was significantly lower, MGMT and TMZ IC 50 were significantly higher ( P<0.05) ; Compared with the resistance group, the expression of LY6E-DT, MGMT mRNA and protein and TMZ IC 50 decreased significantly in the interference group ( P<0.05) . miR-125a-5p significantly inhibited luciferase expression of MGMT 3'UTR ( P<0.01) , while LY6E-DT significantly restored the expression level ( P<0.01) . Conclusion:LY6E-DT can promote MGMT expression and TMZ resistance in HGG cells, which is related to the inhibition of miR-125a-5p expression.

Downregulation of hsa_circ_0000936 sensitizes resistant glioma cells to temozolomide by sponging miR-1294

Glioma is one of the most aggressive forms of brain tumor and is hallmarked by high rate of mortality,metastasis and drug resistance. Herein, we explore the role of circular RNA (circRNA) hsa_circ_0000936 inthe resistance of glioma cells to temozolomide (TMZ). In this study, Relative changes in gene expression levelswere compared using qRT-PCR. The role of hsa_circ_0000936 was characterized by cell count kit -8 assay andflow cytometry. Luciferase reporter assay was carried out for target validation.We found that hsa_circ_0000936was upregulated in glioma tissues as compared to their adjacent normal tissues. Increased expression ofhsa_circ_0000936 was found in the glioma tissues of patients showing resistance to TMZ compared with thatof patients showing sensitivity to TMZ. The upregulation of hsa_circ_0000936 was also confirmed in TMZresistant glioma cells. miR-1294 was downregulated in TMZ-resistant glioma cells and identified as a directtarget of hsa_circ_0000936. Downregulation of hsa_circ_0000936 increased the sensitivity of TMZ-resistantglioma cells towards TMZ. Moreover, restoration of miR-1294 could abrogate the promoting effect ofhsa_circ_0000936 on TMZ resistance in TMZ-resistant glioma cells. In conclusion, downregulation ofhsa_circ_0000936 sensitizes TMZ-resistant glioma cells to TMZ by sponging miR-1294, suggesting thathsa_circ_0000936 may be a potential target for overcoming the resistance of glioma cells to TMZ.