Anticancer effect of minor phytocannabinoids in preclinical models of multiple myeloma.

Multiple myeloma (MM) is a blood cancer caused by uncontrolled growth of clonal plasmacells. Bone disease is responsible for the severe complications of MM and is caused by myeloma cells infiltrating the bone marrow and inducing osteoclast activation. To date, no treatment for MM is truly curative since patients relapse and become refractory to all drug classes. Cannabinoids are already used as palliative in cancer patients. Furthermore, their proper anticancer effect was demonstrated in many cancer models in vitro, in vivo, and in clinical trials. Anyway, few information was reported on the effect of cannabinoids on MM and no data has been provided on minor phytocannabinoids such as cannabigerol (CBG), cannabichromene (CBC), cannabinol (CBN), and cannabidivarin (CBDV). Scientific literature also reported cannabinoids beneficial effect against bone disease. Here, we examined the cytotoxic activity of CBG, CBC, CBN, and CBDV in vitro in MM cell lines, their effect in modulating MM cells invasion toward bone cells and the bone resorption. Subsequently, according to the in vitro results, we selected CBN for in vivo study in a MM xenograft mice model. Results showed that the phytocannabinoids inhibited MM cell growth and induced necrotic cell death. Moreover, the phytocannabinoids reduced the invasion of MM cells toward osteoblast cells and bone resorption in vitro. Lastly, CBN reduced in vivo tumor mass. Together, our results suggest that CBG, CBC, CBN, and CBDV can be promising anticancer agents for MM.

Cannabigerol Induces Autophagic Cell Death by Inhibiting EGFR-RAS Pathways in Human Pancreatic Ductal Adenocarcinoma Cell Lines.

Pancreatic ductal adenocarcinoma (PDAC) is the most frequent infiltrating type of pancreatic cancer. The poor prognosis associated with this cancer is due to the absence of specific biomarkers, aggressiveness, and treatment resistance. PDAC is a deadly malignancy bearing distinct genetic alterations, the most common being those that result in cancer-causing versions of the KRAS gene. Cannabigerol (CBG) is a non-psychomimetic cannabinoid with anti-inflammatory properties. Regarding the anticancer effect of CBG, up to now, there is only limited evidence in human cancers. To fill this gap, we investigated the effects of CBG on the PDAC cell lines, PANC-1 and MIAPaCa-2. The effect of CBG activity on cell viability, cell death, and EGFR-RAS-associated signaling was investigated. Moreover, the potential synergistic effect of CBG in combination with gemcitabine (GEM) and paclitaxel (PTX) was investigated. MTT was applied to investigate the effect of CBG on PDAC cell line viabilities. Annexin-V and Acridine orange staining, followed by cytofluorimetric analysis and Western blotting, were used to evaluate CBG's effect on cell death. The modulation of EGFR-RAS-associated pathways was determined by Western blot analysis and a Milliplex multiplex assay. Moreover, by employing the MTT data and SynergyFinder Plus software analysis, the effect of the combination of CBG and chemotherapeutic drugs was determined.

Calcium influx, oxidative stress, and apoptosis induced by TRPV1 in chronic myeloid leukemia cells: Synergistic effects with imatinib.

Introduction: Calcium flux is the master second messenger that influences the proliferation-apoptosis balance. The ability of calcium flux alterations to reduce cell growth makes ion channels interesting targets for therapy. Among all, we focused on transient receptor potential vanilloid 1, a ligand-gated cation channel with selectivity for calcium. Its involvement in hematological malignancies is poorly investigated, especially in the field of chronic myeloid leukemia, a malignancy characterized by the accumulation of immature cells. Methods: FACS analysis, Western blot analysis, gene silencing, and cell viability assay were performed to investigate the activation of transient receptor potential vanilloid 1, by N-oleoyl-dopamine, in chronic myeloid leukemia cell lines. Results: We demonstrated that the triggering of transient receptor potential vanilloid 1 inhibits cell growth and promotes apoptosis of chronic myeloid leukemia cells. Its activation induced calcium influx, oxidative stress, ER stress, mitochondria dysfunction, and caspase activation. Interestingly, a synergistic effect exerted by N-oleoyl-dopamine and the standard drug imatinib was found. Conclusion: Overall, our results support that transient receptor potential vanilloid 1 activation could be a promising strategy to enhance conventional therapy and improve the management of chronic myeloid leukemia.

Role of Clock Genes and Circadian Rhythm in Renal Cell Carcinoma: Recent Evidence and Therapeutic Consequences.

Circadian rhythm regulates cellular differentiation and physiology and shapes the immune response. Altered expression of clock genes might lead to the onset of common malignant cancers, including Renal Cell Carcinoma (RCC). Data from Cancer Genome Atlas (TCGA) indicate that clock genes PER1-3, CRY2, CLOCK, NR1D2 and RORα are overexpressed in RCC tissues and correlate with patients' prognosis. The expression of clock genes could finely tune transcription factor activity in RCC and is associated with the extent of immune cell infiltration. The clock system interacts with hypoxia-induced factor-1α (HIF-1α) and regulates the circadian oscillation of mammalian target of rapamycin (mTOR) activity thereby conditioning the antitumor effect of mTOR inhibitors. The stimulation of natural killer (NK) cell activity exerted by the administration of interferon-α, a cornerstone of the first era of immunotherapy for RCC, relevantly varies according to circadian dosing time. Recent evidence demonstrated that time-of-day infusion directly affects the efficacy of immune checkpoint inhibitors in cancer patients. Compounds targeting the circadian clock have been identified and their role in the era of immunotherapy deserves to be further investigated. In this review, we aimed at addressing the impact of clock genes on the natural history of kidney cancer and their potential therapeutic implications.

TRPML2 Mucolipin Channels Drive the Response of Glioma Stem Cells to Temozolomide and Affect the Overall Survival in Glioblastoma Patients.

The survival of patients with glioblastoma (GBM) is poor. The main cause is the presence of glioma stem cells (GSCs), exceptionally resistant to temozolomide (TMZ) treatment. This last may be related to the heterogeneous expression of ion channels, among them TRPML2. Its mRNA expression was evaluated in two different neural stem cell (NS/PC) lines and sixteen GBM stem-like cells by qRT-PCR. The response to TMZ was evaluated in undifferentiated or differentiated GSCs, and in TRPML2-induced or silenced GSCs. The relationship between TRPML2 expression and responsiveness to TMZ treatment was evaluated by MTT assay showing that increased TRPML2 mRNA levels are associated with resistance to TMZ. This research was deepened by qRT-PCR and western blot analysis. PI3K/AKT and JAK/STAT pathways as well as ABC and SLC drug transporters were involved. Finally, the relationship between TRPML2 expression and overall survival (OS) and progression-free survival (PFS) in patient-derived GSCs was evaluated by Kaplan-Meier analysis. The expression of TRPML2 mRNA correlates with worse OS and PFS in GBM patients. Thus, the expression of TRPML2 in GSCs influences the responsiveness to TMZ in vitro and affects OS and PFS in GBM patients.

Highly Potent and Selective Dopamine D4 Receptor Antagonists Potentially Useful for the Treatment of Glioblastoma.

To better understand the role of dopamine D4 receptor (D4R) in glioblastoma (GBM), in the present paper, new ligands endowed with high affinity and selectivity for D4R were discovered starting from the brain penetrant and D4R selective lead compound 1-(3-(4-phenylpiperazin-1-yl)propyl)-3,4-dihydroquinolin-2(1H)-one (6). In particular, the D4R antagonist 24, showing the highest affinity and selectivity over D2R and D3R within the series (D2/D4 = 8318, D3/D4 = 3715), and the biased ligand 29, partially activating D4R Gi-/Go-protein and blocking ß-arrestin recruitment, emerged as the most interesting compounds. These compounds, evaluated for their GBM antitumor activity, induced a decreased viability of GBM cell lines and primary GBM stem cells (GSC#83), with the maximal efficacy being reached at a concentration of 10 µM. Interestingly, the treatment with both compounds 24 and 29 induced an increased effect in reducing the cell viability with respect to temozolomide, which is the first-choice chemotherapeutic drug in GBM.

Coexpression of TRPML1 and TRPML2 Mucolipin Channels Affects the Survival of Glioblastoma Patients.

Among brain cancers, glioblastoma (GBM) is the most malignant glioma with an extremely poor prognosis. It is characterized by high cell heterogeneity, which can be linked to its high malignancy. We have previously demonstrated that TRPML1 channels affect the OS of GBM patients. Herein, by RT-PCR, FACS and Western blot, we demonstrated that TRPML1 and TRPML2 channels are differently expressed in GBM patients and cell lines. Moreover, these channels partially colocalized in ER and lysosomal compartments in GBM cell lines, as evaluated by confocal analysis. Interestingly, the silencing of TRPML1 or TRPML2 by RNA interference results in the decrease in the other receptor at protein level. Moreover, the double knockdown of TRPML1 and TRPML2 leads to increased GBM cell survival with respect to single-channel-silenced cells, and improves migration and invasion ability of U251 cells. Finally, the Kaplan-Meier survival analysis demonstrated that patients with high TRPML2 expression in absence of TRPML1 expression strongly correlates with short OS, whereas high TRPML1 associated with low TRPML2 mRNA expression correlates with longer OS in GBM patients. The worst OS in GBM patients is associated with the loss of both TRPML1 and TRPML2 channels.

The Prognostic Value of the Circulating Tumor Cell-Based Four mRNA Scoring System: A New Non-Invasive Setting for the Management of Bladder Cancer.

Bladder cancer (BC) is one of the most expensive lifetime cancers to treat because of the high recurrence rate, repeated surgeries, and long-term cystoscopy monitoring and treatment. The lack of an accurate classification system predicting the risk of recurrence or progression leads to the search for new biomarkers and strategies. Our pilot study aimed to identify a prognostic gene signature in circulating tumor cells (CTCs) isolated by ScreenCell devices from muscle invasive and non-muscle invasive BC patients. Through the PubMed database and Cancer Genome Atlas dataset, a panel of 15 genes modulated in BC with respect to normal tissues was selected. Their expression was evaluated in CTCs and thanks to the univariate and multivariate Cox regression analysis, EGFR, TRPM4, TWIST1, and ZEB1 were recognized as prognostic biomarkers. Thereafter, by using the risk score model, we demonstrated that this 4-gene signature significantly grouped patients into high- and low-risk in terms of recurrence free survival (HR = 2.704, 95% CI = 1.010−7.313, Log-rank p < 0.050). Overall, we identified a new prognostic signature that directly impacted the prediction of recurrence, improving the choice of the best treatment for BC patients.

Unveiling the Molecular Mechanisms Driving the Capsaicin-Induced Immunomodulatory Effects on PD-L1 Expression in Bladder and Renal Cancer Cell Lines.

The blockade of the PD-L1/PD-1 immune checkpoint has promising efficacy in cancer treatment. However, few patients with bladder cancer (BC) or renal cell carcinoma (RCC) respond to this approach. Thus, it is important to implement a strategy to stimulate the immune anti-tumor response. In this scenario, our study evaluated the effects of a low capsaicin (CPS) dose in BC and RCC cell lines. Western blot, qRT-PCR and confocal microscopy were used to assess PD-L1 mRNA and protein expression. Alterations to the cellular oxidative status and changes to the antioxidant NME4 levels, mRNA modulation of cytokines, growth factors, transcriptional factors and oncogene, and the activation of Stat1/Stat3 pathways were examined using Western blot, cytofluorimetry and qRT-PCR profiling assays. In BC, CPS triggers an altered stress oxidative-mediated DNA double-strand break response and increases the PD-L1 expression. On the contrary, in RCC, CPS, by stimulating an efficient DNA damage repair response, thus triggering protein carbonylation, reduces the PD-L1 expression. Overall, our results show that CPS mediates a multi-faceted approach. In modulating PD-L1 expression, there is a rationale for CPS exploitation as a stimulus that increases BC cells' response to immunotherapy or as an immune adjuvant to improve the efficacy of the conventional therapy in RCC patients.

Evening Primrose Oil Improves Chemotherapeutic Effects in Human Pancreatic Ductal Adenocarcinoma Cell Lines-A Preclinical Study.

Evening Primrose oil (EPO), obtained from the seeds of Evening Primrose (Oenothera L.), is largely used as a dietary supplement, especially after cancer diagnosis. Human pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease correlated with poor clinical prognosis and a very low response rate to common chemotherapy. The aim of this work was to study the potential ability of EPO to improve the effects of chemotherapeutic drugs in PANC-1 and MIAPaCa-2 cell lines. Cytotoxicity, cell death, reactive oxygen species (ROS) production and EPO anticancer activity associated with the main chemotherapeutic drugs commonly used in therapy were investigated. Results showed that EPO reduced PDAC cell viability and increased paclitaxel efficacy. This evidence suggests that EPO may be used as a potential supplement to increase chemotherapeutic efficacy in PDAC therapy.

A3 Adenosine Receptor Antagonists with Nucleoside Structures and Their Anticancer Activity.

The overexpression of the A3 adenosine receptor (AR) in a number of cancer cell types makes it an attractive target for tumor diagnosis and therapy. Hence, in the search for new A3AR ligands, a series of novel 2,N6-disubstituted adenosines (Ados) was synthesized and tested in radioligand binding and functional assays at ARs. Derivatives bearing a 2-phenethylamino group in the N6-position were found to exert higher A3AR affinity and selectivity than the corresponding N6-(2,2-diphenylethyl) analogues. 2-Chloro-N6-phenylethylAdo (15) was found to be a potent full A3AR agonist with a Ki of 0.024 nM and an EC50 of 14 nM, in a cAMP accumulation assay. Unlike 15, the other ligands behaved as A3AR antagonists, which concentration-dependently reduced cell growth and exerted cytostatic activity on the prostate cancer cell line PC3, showing comparable and even more pronounced effects with respect to the ones elicited by the reference full agonist Cl-IB-MECA. In particular, the N6-(2,2-diphenylethyl)-2-phenylethynylAdo (12: GI50 = 14 µM, TGI = 29 µM, and LC50 = 59 µM) showed the highest activity proving to be a potential antitumor agent. The cytostatic effect of both A3AR agonist (Cl-IB-MECA) and antagonists (12 and other newly synthesized compounds) confirm previous observations according to which, in addition to the involvement of A3ARs, other cellular mechanisms are responsible for the anticancer effects of these ligands.

The Mucolipin TRPML2 Channel Enhances the Sensitivity of Multiple Myeloma Cell Lines to Ibrutinib and/or Bortezomib Treatment.

Multiple myeloma (MM) is a haematological B cell malignancy characterised by clonal proliferation of plasma cells and their accumulation in the bone marrow. The aim of the present study is the evaluation of biological effects of Ibrutinib in human MM cell lines alone or in combination with different doses of Bortezomib. In addition, the relationship between the expression of TRPML2 channels and chemosensitivity of different MM cell lines to Ibrutinib administered alone or in combination with Bortezomib has been evaluated. By RT-PCR and Western blot analysis, we found that the Ibrutinib-resistant U266 cells showed lower TRPML2 expression, whereas higher TRPML2 mRNA and protein levels were evidenced in RPMI cells. Moreover, TRPML2 gene silencing in RPMI cells markedly reverted the effects induced by Ibrutinib alone or in combination with Bortezomib suggesting that the sensitivity to Ibrutinib is TRPML2 mediated. In conclusion, this study suggests that the expression of TRPML2 in MM cells increases the sensitivity to Ibrutinib treatment, suggesting for a potential stratification of Ibrutinib sensitivity of MM patients on the basis of the TRPML2 expression. Furthermore, studies in vitro and in vivo should still be necessary to completely address the molecular mechanisms and the potential role of TRPML2 channels in therapy and prognosis of MM patients.

Functional In Vitro Assessment of VEGFA/NOTCH2 Signaling Pathway and pRB Proteasomal Degradation and the Clinical Relevance of Mucolipin TRPML2 Overexpression in Glioblastoma Patients.

Glioblastoma (GBM) is the most malignant glioma with an extremely poor prognosis. It is characterized by high vascularization and its growth depends on the formation of new blood vessels. We have previously demonstrated that TRPML2 mucolipin channel expression increases with the glioma pathological grade. Herein by ddPCR and Western blot we found that the silencing of TRPML2 inhibits expression of the VEGFA/Notch2 angiogenic pathway. Moreover, the VEGFA/Notch2 expression increased in T98 and U251 cells stimulated with the TRPML2 agonist, ML2-SA1, or by enforced-TRPML2 levels. In addition, changes in TRPML2 expression or ML2-SA1-induced stimulation, affected Notch2 activation and VEGFA release. An increased invasion capability, associated with a reduced VEGF/VEGFR2 expression and increased vimentin and CD44 epithelial-mesenchymal transition markers in siTRPML2, but not in enforced-TRPML2 or ML2-SA1-stimulated glioma cells, was demonstrated. Furthermore, an increased sensitivity to Doxorubicin cytotoxicity was demonstrated in siTRPML2, whereas ML2-SA1-treated GBM cells were more resistant. The role of proteasome in Cathepsin B-dependent and -independent pRB degradation in siTRPML2 compared with siGLO cells was studied. Finally, through Kaplan-Meier analysis, we found that high TRPML2 mRNA expression strongly correlates with short survival in GBM patients, supporting TRPML2 as a negative prognostic factor in GBM patients.

The effects of cannabidiol via TRPV2 channel in chronic myeloid leukemia cells and its combination with imatinib.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by accumulation of immature cells in bone marrow and peripheral blood. Although successful results were obtained with tyrosine kinase inhibitors, several patients showed resistance. For this reason, the identification of new strategies and therapeutic biomarkers represents an attractive goal. The role of transient receptor potential (TRP) ion channels as possible drug targets has been elucidated in different types of cancer. Among natural compounds known to activate TRPs, cannabidiol (CBD) displays anticancer properties. By using FACS analysis, confocal microscopy, gene silencing, and cell growth assay, we demonstrated that CBD, through TRPV2, inhibits cell proliferation and cell cycle in CML cells. It promoted mitochondria dysfunction and mitophagy as shown by mitochondrial mass reduction and up-regulation of several mitophagy markers. These effects were associated with changes in the expression of octamer-binding transcription factor 4 and PU.1 markers regulated during cellular differentiation. Interestingly, a synergistic effect by combining CBD with the standard drug imatinib was found and imatinib-resistant cells remain susceptible to CBD effects. Therefore, the targeting of TRPV2 by using CBD, through the activation of mitophagy and the reduction in stemness, could be a promising strategy to enhance conventional therapy and improve the prognosis of CML patients.

Correlation between High PD-L1 and EMT/Invasive Genes Expression and Reduced Recurrence-Free Survival in Blood-Circulating Tumor Cells from Patients with Non-Muscle-Invasive Bladder Cancer.

BACKGROUND: PD-L1 represents a crucial immune checkpoint molecule in the tumor microenvironment, identified as a key target for cancer immunotherapy. A correlation between PD-L1 and EMT-related genes expression in various human cancers has been suggested. METHODS: By ScreenCell filtration, digital droplet PCR and confocal microscopy analysis, we aimed to investigate the expression of PD-L1 and EMT/invasive genes (TWIST1, ZEB1, VIMENTIN, TIMP2) in circulating tumor cells (CTCs) collected from the blood of non-muscle-invasive bladder cancer (NMIBC) patients, assessing the prognostic value of these biomarkers in the disease. Welchs' test and Mann-Whitney U test, correlation index, Kaplan-Meier, Univariate and Multivariate Cox hazard proportional analysis were used. RESULTS: Higher PD-L1, TIMP2 and VIM mRNA levels were found in pT1 compared to pTa NMIBC. As evaluated by Kaplan-Meier and Univariate and Multivariate Cox analysis, enhancement of PD-L1, TWIST1 and TIMP2 expression reduces the recurrent free survival in NMIBC patients. CONCLUSIONS: High PD-L1, TWIST1 and TIMP2 mRNAs mark the recurrent-NMIBC patients and by reducing the RFS represent negative prognostic biomarkers in these patients.

Mechanosensation and Mechanotransduction in Natural Killer Cells.

Natural killer (NK) cells are a main subset of innate lymphocytes that contribute to host immune protection against viruses and tumors by mediating target cell killing and secreting a wide array of cytokines. Their functions are finely regulated by a balance between activating and inhibitory receptors and involve also adhesive interactions. Mechanotransduction is the process in which physical forces sensed by mechanosensors are translated into chemical signaling. Herein, we report findings on the involvement of this mechanism that is mainly mediated by actin cytoskeleton, in the regulation of NK cell adhesion, migration, tissue infiltration and functions. Actin represents the structural basis for NK cell immunological synapse (NKIS) and polarization of secretory apparatus. NK-target cell interaction involves the formation of both uropods and membrane nanotubes that allow target cell interaction over long distances. Actin retrograde flow (ARF) regulates NK cell signaling and controls the equilibrium between activation versus inhibition. Activating NKIS is associated with rapid lamellipodial ARF, whereas lower centripetal actin flow is present during inhibitory NKIS where ß actin can associate with the tyrosine phosphatase SHP-1. Overall, a better knowledge of mechanotransduction might represent a future challenge: Realization of nanomaterials tailored for NK cells, would be important to translate in vitro studies in in vivo new immunotherapeutic approaches.

ERK Phosphorylation Regulates the Aml1/Runx1 Splice Variants and the TRP Channels Expression during the Differentiation of Glioma Stem Cell Lines.

The identification of cancer stem cells in brain tumors paved the way for new therapeutic approaches. Recently, a role for the transcriptional factor Runx1/Aml1 and the downstream ion channel genes in brain cancer development and progression has been suggested. This study aimed to explore the expression and the role of Runx1/Aml1, its Aml1b and Aml1c splice variants and the downstream TRPA1 and TRPV1 ion channels in undifferentiated and day-14 differentiated neural stem cells (NSCs and D-NSCs) and glioblastoma stem cells (GSCs and D-GSCs) lines with different proneural (PN) or mesenchymal (MES) phenotype. Gene and protein expression were evaluated by qRT-PCR, cytofluorimetric, western blot and confocal microscopy analyses. Moreover, by western blot, we observed that ERK phosphorylation enhances the Aml1b and Aml1c protein expression during glioma differentiation. Furthermore, the agonists of TRPA1 and TRPV1 channels stimulated apoptosis/necrosis in GSCs and D-GSCs as evaluated by Annexin V and PI staining and cytofluorimetric analysis. Finally, by qRT-PCR, the modulation of Wnt/ß catenin, FGF, and TGFß/SMAD signaling pathways in PN- and MES-GSCs was reported. Overall, our results provide new evidence regarding Runx1/Aml1 isoform overexpression and modulation in TRP channel expression during gliomagenesis, thus offering new directions for glioblastoma therapy.

Transient Receptor Potential (TRP) Channels in Haematological Malignancies: An Update.

Transient receptor potential (TRP) channels are improving their importance in different cancers, becoming suitable as promising candidates for precision medicine. Their important contribution in calcium trafficking inside and outside cells is coming to light from many papers published so far. Encouraging results on the correlation between TRP and overall survival (OS) and progression-free survival (PFS) in cancer patients are available, and there are as many promising data from in vitro studies. For what concerns haematological malignancy, the role of TRPs is still not elucidated, and data regarding TRP channel expression have demonstrated great variability throughout blood cancer so far. Thus, the aim of this review is to highlight the most recent findings on TRP channels in leukaemia and lymphoma, demonstrating their important contribution in the perspective of personalised therapies.

Knock-Down of Mucolipin 1 Channel Promotes Tumor Progression and Invasion in Human Glioblastoma Cell Lines.

Among cancers that affect the central nervous system, glioblastoma is the most common. Given the negative prognostic significance of transient receptor potential mucolipin 1 (TRPML1) channel reduction in patients with glioblastoma, as discussed in previous publications, the aim of the current study was to investigate the biological advantage of TRPML1 loss for glioma cells. Human glioblastoma primary cancer cells (FSL and FCL) and glioblastoma cell lines (T98 and U251) were used for that purpose. TRPML1 silencing in T98 cells induces defective autophagy, nitric oxide (NO) production, and cathepsin B-dependent apoptosis in the first 48 h and then apoptotic-resistant cells proliferate with a high growth rate with respect to control cells. In U251 cells, knock-down of TRPML1 stimulates NO generation and protein oxidation, arrests cell cycle at G2/M phase, and induces autophagy leading to cathepsin B-dependent senescence. Finally, in both cell lines, the long-term effects of TRPML1 silencing promote survival and invasion capacity with respect to control cells. Silencing of TRPML1 also affects the phenotype of glioblastoma primary cells. FSL cells show increased proliferation ability, while FCL cells enter into senescence associated with an increased invasion ability. In conclusion, although the molecular heterogeneity among different glioblastoma cell lines mirrors the intercellular heterogeneity in cancer cells, our data support TRPML1 downregulation as a negative prognostic factor in glioblastoma.