Decreased APE-1 by Nitroxoline Enhances Therapeutic Effect in a Temozolomide-resistant Glioblastoma: Correlation with Diffusion Weighted Imaging.
ABSTRACT: Glioblastoma multiforme (GBM) is one of the most aggressive human tumors with poor survival rates. The current standard treatment includes chemotherapy with temozolomide (TMZ), but acquisition of resistance is a persistent clinical problem limiting the successful treatment of GBM. The purpose of our study was to investigate therapeutic effects of nitroxoline (NTX) against TMZ-resistant GBM in vitro and in vivo in TMZ-resistant GBM-bearing mouse model, which was correlated with diffusion-weighted imaging (DWI). For in vitro study, we used TMZ-resistant GBM cell lines and evaluated therapeutic effects of NTX by clonogenic and migration assays. Quantitative RT-PCR was used to investigate the expression level of TMZ-resistant genes after NTX treatment. For in vivo study, we performed 9.4?T MR imaging to obtain T2WI for tumor volume measurement and DWI for assessment of apparent diffusion coefficient (ADC) changes by NTX in TMZ-resistant GBM mice (n?=?8). Moreover, we performed regression analysis for the relationship between ADC and histological findings, which reflects the changes in cellularity and apurinic/apyrimidinic endonuclease-1 (APE-1) expression. We observed the recovery of TMZ-induced morphological changes, a reduced number of colonies and a decreased rate of migration capacity in TMZ-resistant cells after NTX treatment. The expression of APE-1 was significantly decreased in TMZ-resistant cells after NTX treatment compared with those without treatment. In an in vivo study, NTX reduced tumor growth in TMZ-resistant GBM mice (P?=?0.0122). Moreover, ADC was increased in the NTX-treated TMZ-resistant GBM mice compared to the control group (P?=?0.0079), which was prior to a tumor volume decrease. The cellularity and APE-1 expression by histology were negatively correlated with the ADC value, which in turn resulted in longer survival in NTX group. The decreased expression of APE-1 by NTX leads to therapeutic effects and is inversely correlated with ADC in TMZ-resistant GBM. Therefore, NTX is suggested as potential therapeutic candidate against TMZ-resistant GBM.
Project description:Abstract <h4>BACKGROUND</h4> Calculated from a diffusion-weight image (DWI), the apparent diffusion coefficient (ADC) is a quantitative measure that reflects observed net movement of water and correlates to tumor cellularity. We examine the changes in ADC values in patients with LGG treated with dimeric, pan-RAF inhibitor DAY101 (formerly TAK-580/MLN2480). <h4>METHODS</h4> Focusing on ADC change with treatment, we reviewed historical, baseline, and on-treatment brain MRIs for 9 patients enrolled on our institutional, IRB-approved phase 1 trial of DAY101 in children and young adults with radiographically recurrent or progressive LGG harboring MEK/ERK pathway alterations. De-identified DICOM MRI files were independently reviewed. Time points selected included baseline, first follow-up and best response. The pmod molecular image software package was utilized for data processing of ADC estimates. ADC changes were displayed as a histogram with mean values. Results were based upon a single read paradigm. <h4>RESULTS</h4> A shift to lower ADC values for solid components of these tumors was observed, reflecting cellularity and organization; while necrosis correlated with a shift toward higher ADC values. DWI results show reduced ADCs in responding patients, with greater percent change in ADC from baseline associated with deeper responses among treated patients. DWI for treated patients with resultant stable disease showed no significant change in ADC values from baseline while a shift toward higher ADC values was evident in treated, progressing tumors. <h4>CONCLUSION</h4> Preliminary DWI analysis reveals that a reduction in ADC values may correlate with treatment response and a shift toward more normal cellularity in tumors treated with DAY101. This method will be applied to a larger cohort of patients in an ongoing phase 1 trial (NCT03429803) and a planned phase 2 trial.
Project description:<h4>Purpose</h4>Glioblastoma (GBM) is one of the deadliest cancers with no cure. While conventional MRI has been widely adopted to examine GBM clinically, accurate neuroimaging assessment of tumor histopathology for improved diagnosis, surgical planning, and treatment evaluation remains an unmet need in the clinical management of GBMs.<h4>Experimental design</h4>We employ a novel diffusion histology imaging (DHI) approach, combining diffusion basis spectrum imaging (DBSI) and machine learning, to detect, differentiate, and quantify areas of high cellularity, tumor necrosis, and tumor infiltration in GBM.<h4>Results</h4>Gadolinium-enhanced T1-weighted or hyperintense fluid-attenuated inversion recovery failed to reflect the morphologic complexity underlying tumor in patients with GBM. Contrary to the conventional wisdom that apparent diffusion coefficient (ADC) negatively correlates with increased tumor cellularity, we demonstrate disagreement between ADC and histologically confirmed tumor cellularity in GBM specimens, whereas DBSI-derived restricted isotropic diffusion fraction positively correlated with tumor cellularity in the same specimens. By incorporating DBSI metrics as classifiers for a supervised machine learning algorithm, we accurately predicted high tumor cellularity, tumor necrosis, and tumor infiltration with 87.5%, 89.0%, and 93.4% accuracy, respectively.<h4>Conclusions</h4>Our results suggest that DHI could serve as a favorable alternative to current neuroimaging techniques in guiding biopsy or surgery as well as monitoring therapeutic response in the treatment of GBM.
Project description:Background:Evaluate the utility of diffusion-weighted imaging (DWI) for the assessment of local recurrence of glioblastoma (GBM) on imaging performed 24 h following MRI-guided laser interstitial thermal therapy (LITT). We hypothesize that microscopic peritumoral infiltration correlates with early subtle variations on DWI images and apparent diffusion coefficient (ADC) maps. Methods:Of 64 patients with GBM treated with LITT, 39 had MRI scans within 24 h after undergoing LITT. Patterns on DWI images and ADC maps 24 h following LITT were correlated with areas of future GBM recurrence identified through coregistration of subsequent MRI examinations. In the areas of suspected recurrence within the periphery of post-LITT lesions, signal intensity values on ADC maps were recorded and compared with the remaining peritumoral ring. Results:Thirty-nine patients with GBM met the inclusion criteria. For predicting recurrent GBM, areas of decreased DWI signal and increased signal on ADC maps within the expected peritumoral ring of restricted diffusion identified 24 h following LITT showed 86.1% sensitivity, 75.2% specificity, and high correlation (r = 0.53) with future areas of GBM recurrence (P < .01). Areas of future recurrence demonstrated a 37% increase in the ADC value (P < .001), compared with findings in the surrounding treated peritumoral region. A significantly greater area under the receiver operating characteristics curve was determined for ADC values (P < .01). Conclusions:DWI obtained 24 h following LITT can help predict the location of GBM recurrence months before the development of abnormal enhancement. This may alter future treatment planning, perhaps suggesting areas that may be targeted for additional therapy.
Project description:Glioblastomas (GBM) often acquire resistance against temozolomide (TMZ) after continuous treatment and recur as TMZ-resistant GBM (TMZ-R-GBM). Lomustine (CCNU) and nimustine (ACNU), which were previously used as standard therapeutic agents against GBM before TMZ, have occasionally been used for the salvage therapy of TMZ-R-GBM; however, their efficacy has not yet been thoroughly examined. Therefore, we investigated the antitumor effects of CCNU and ACNU against TMZ-R-GBM. As a model of TMZ-R-GBM, TMZ resistant clones of human GBM cell lines (U87, U251MG, and U343MG) were established (TMZ-R-cells) by the culture of each GBM cells under continuous TMZ treatment, and the antitumor effects of TMZ, CCNU, or ACNU against these cells were analyzed in vitro and in vivo. As a result, although growth arrest and apoptosis were triggered in all TMZ-R-cells after the administration of each drug, the antitumor effects of TMZ against TMZ-R-cells were significantly reduced compared to those of parental cells, whereas CCNU and ACNU demonstrated efficient antitumor effects on TMZ-R-cells as well as parental cells. It was also demonstrated that TMZ resistance of TMZ-R-cells was regulated at the initiation of DNA damage response. Furthermore, survival in mice was significantly prolonged by systemic treatment with CCNU or ACNU but not TMZ after implantation of TMZ-R-cells. These findings suggest that CCNU or ACNU may serve as a therapeutic agent in salvage treatment against TMZ-R-GBM.
Project description:Glioblastoma multiforme (GBM) is one of the most aggressive cancers. Despite recent advances in multimodal therapies, high-grade glioma remains fatal. Temozolomide (TMZ) is an alkylating agent used worldwide for the clinical treatment of GBM; however, the innate and acquired resistance of GBM limits its application. Here, we found that TMZ inhibited the proliferation and induced the G2/M arrest of GBM cells. Therefore, we performed microarrays to identify the cell cycle- and apoptosis-related genes affected by TMZ. Notably, GADD45A was found to be up-regulated by TMZ in both cell cycle and apoptosis arrays. Furthermore, GADD45A knockdown (GADD45A<sup>kd</sup>) enhanced the cell growth arrest and cell death induced by TMZ, even in natural (T98) and adapted (TR-U373) TMZ-resistant cells. Interestingly, GADD45A<sup>kd</sup> decreased the expression of O<sup>6</sup>-methylguanine-DNA methyltransferase (MGMT) in TMZ-resistant cells (T98 and TR-U373). In MGMT-deficient/TMZ-sensitive cells (U87 and U373), GADD45A<sup>kd</sup> decreased TMZ-induced TP53 expression. Thus, in this study, we investigated the genes influenced by TMZ that were important in GBM therapy, and revealed that GADD45A plays a protective role against TMZ treatment which may through TP53-dependent and MGMT-dependent pathway in TMZ-sensitive and TMZ-resistant GBM, respectively. This protective role of GADD45A against TMZ treatment may provide a new therapeutic strategy for GBM treatment.
Project description:Glioblastoma (GBM) is an aggressive and incurable tumor of the brain with limited treatment options. Current first-line standard of care is the DNA alkylating agent temozolomide (TMZ), but this treatment strategy adds only ~4 months to median survival due to the rapid development of resistance. While some mechanisms of TMZ resistance have been identified, they are not fully understood. There are few effective strategies to manage therapy resistant GBM, and we lack diverse preclinical models of acquired TMZ resistance in which to test therapeutic strategies on TMZ resistant GBM. In this study, we create and characterize two new GBM cell lines resistant to TMZ in vitro, based on the 8MGBA and 42MGBA cell lines. Analysis of the TMZ resistant (TMZres) variants in conjunction with their parental, sensitive cell lines shows that acquisition of TMZ resistance is accompanied by broad phenotypic changes, including increased proliferation, migration, chromosomal aberrations, and secretion of cytosolic lipids. Importantly, each TMZ resistant model captures a different facet of the "go" (8MGBA-TMZres) or "grow" (42MGBA-TMZres) hypothesis of GBM behavior. These in vitro model systems will be important additions to the available tools for investigators seeking to define molecular mechanisms of acquired TMZ resistance.
Project description:<b>Background: </b>The ability to treat glioblastoma (GBM) using the chemotherapeutic agent temozolomide (TMZ) has been hampered by the development of therapeutic resistance. In this study, we assessed the ability of the isoquinoline alkaloid berberine to alter GBM TMZ resistance using two different TMZ-resistant cell lines to mimic a physiologically relevant GBM experimental system.<br><br><b>Methods: </b>By treating these resistant cell lines with berberine followed by TMZ, we were able to assess the chemosensitivity of these cells and their parental strains, based on their performance in the MTT and colony formation assays, as well as on the degree of detectable apoptosis that was detected in the strains. Furthermore, we used Western blotting to assess autophagic responses in these cell lines, and we extended this work into a xenograft mouse model to assess the in vivo efficacy of berberine.<br><br><b>Results: </b>Through these experiments, our findings indicated that berberine enhanced autophagy and apoptosis in TMZ-resistant cells upon TMZ treatment in a manner that was linked with ERK1/2 signaling. Similarly, when used in vivo, berberine increased GBM sensitivity to TMZ through ERK1/2 signaling pathways.<br><br><b>Conclusions: </b>These findings demonstrate that berberine is an effective method of increasing the sensitization of GBM cells to TMZ treatment in a manner that is dependent upon the ERK1/2-mediated induction of autophagy, thus making berberine a potentially viable therapeutic agent for GBM treatment.
Project description:Glioblastoma (GBM) is the most common and lethal tumor of the central nervous system with highly infiltrative and resistant to chemotherapy. Temozolomide (TMZ) is widely used as the first-line treatment for the therapy of GBM. However, a considerable percentage inherent or acquired resistance in GBM accounts for many treatment failures of the TMZ chemotherapy. Therefore, a deeper understanding of the molecular characteristics underlying TMZ resistance and the identification of novel therapeutic target is urgent. Here, we show that MALAT1 was significantly upregulated in TMZ-resistant GBM cells. On the other hand, MALAT1 knockdown reduces TMZ resistance of GBM cells both in vitro and in vivo by inhibiting cell proliferation and promoting apoptosis. We also show that miR-101 overexpression reduced TMZ resistance of GBM cells and played an antagonistic role compared with MALAT1. Importantly, we demonstrate that MALAT1 promoted the chemoresistance through suppressing miR-101 signaling pathway via directly binding it in GBM cells. In conclusion, our study indicates that knockdown of MALAT1 reverses chemoresistance to TMZ via promoting miR-101 regulatory network in GBM and thus offers a novel prognostic marker and potential target for GBM TMZ-based chemotherapy.
Project description:Glioblastoma multiforme (GBM) is the most common malignant tumor of the central nervous system. Temozolomide (TMZ)-based adjuvant treatment has improved overall survival, but clinical outcomes remain poor; TMZ resistance is one of the main reasons for this. Here, we report a new phosphatidylinositide 3-kinase inhibitor, XH30; this study aimed to assess the antitumor activity of this compound against TMZ-resistant GBM. XH30 inhibited cell proliferation in TMZ-resistant GBM cells (U251/TMZ and T98G) and induced cell cycle arrest in the G1 phase. In an orthotopic mouse model, XH30 suppressed TMZ-resistant tumor growth. XH30 was also shown to enhance TMZ cytotoxicity both in vitro and in vivo. Mechanistically, the synergistic effect of XH30 may be attributed to its repression of the key transcription factor GLI1 via the noncanonical hedgehog signaling pathway. XH30 reversed sonic hedgehog-triggered GLI1 activation and decreased GLI1 activation by insulin-like growth factor 1 via the noncanonical hedgehog signaling pathway. These results indicate that XH30 may represent a novel therapeutic option for TMZ-resistant GBM.
Project description:Acquired chemoresistance is a major limiting factor in the clinical treatment of glioblastoma (GBM). However, the mechanism by which GBM acquires therapeutic resistance remains unclear. Here, we aimed to investigate whether METTL3-mediated N6-methyladenosine (m<sup>6</sup>A) modification contributes to the temozolomide (TMZ) resistance in GBM. We demonstrated that METTL3 METTL3-mediated m<sup>6</sup>A modification were significantly elevated in TMZ-resistant GBM cells. Functionally, METTL3 overexpression impaired the TMZ-sensitivity of GBM cells. In contrast, METTL3 silencing or DAA-mediated total methylation inhibition improved the sensitivity of TMZ-resistant GBM cells to TMZ <i>in vitro</i> and <i>in vivo</i>. Furthermore, we found that two critical DNA repair genes (MGMT and APNG) were m<sup>6</sup>A-modified by METTL3, whereas inhibited by METTL3 silencing or DAA-mediated total methylation inhibition, which is crucial for METTL3-improved TMZ resistance in GBM cells. Collectively, METTL3 acts as a critical promoter of TMZ resistance in glioma and extends the current understanding of m<sup>6</sup>A related signaling, thereby providing new insights into the field of glioma treatment.