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PDF file, 51KB, Selected geometrical parameters of the short H���O intermolecular contacts for S-A8.

PDF file, 223KB, Growth inhibition profiles of the NCI60 tumor cell lines by S-A8.

PDF file, 152KB, 1H NMR spectrum of (�)-Boc-A8. The lower panel details the spectrum between 6.4 and 2.4 ppm.

Tumor resistance to antitubulin drugs resulting from P-glycoprotein (Pgp) drug-efflux activity, increased expression of the βIII tubulin isotype, and alterations in the drug-binding sites are major obstacles in cancer therapy. Consequently, novel antitubulin drugs that overcome these challenges are of substantial interest. Here, we study a novel chemotype named furan metotica that localizes to the colchicine-binding site in β-tubulin, inhibits tubulin polymerization, and is not antagonized by Pgp. To elucidate the structure–activity properties of this chiral chemotype, the enantiomers of its most potent member were separated and their absolute configurations determined by X-ray crystallography. Both isomers were active and inhibited all 60 primary cancer cell lines tested at the U.S. National Cancer Institute. They also efficiently killed drug-resistant cancer cells that overexpressed the Pgp drug-efflux pump 106-fold. In vitro, the R-isomer inhibited tubulin polymerization at least 4-fold more potently than the S-isomer, whereas in human cells the difference was 30-fold. Molecular modeling showed that the two isomers bind to β-tubulin in distinct manners: the R-isomer binds in a colchicine-like mode and the S-isomer in a podophyllotoxin-like fashion. In addition, the dynamic binding trajectory and occupancy state of the R-isomer were energetically more favorable then those of the S-isomer, explaining the observed differences in biologic activities. The ability of a racemic drug to assume the binding modes of two prototypical colchicine-site binders represents a novel mechanistic basis for antitubulin activity and paves the way toward a comprehensive design of novel anticancer agents. Mol Cancer Ther; 11(5); 1103–11. ©2012 AACR.

PDF file, 229KB, Growth inhibition profiles of the NCI60 tumor cell lines by R-A8.

Supplementary Tables 1-4, Figures 1-4 from Peloruside- and Laulimalide-Resistant Human Ovarian Carcinoma Cells Have βI-Tubulin Mutations and Altered Expression of βII- and βIII-Tubulin Isotypes

PDF file, 97KB, Legends to the Supplementary Figures.

PDF file, 72KB, Selected Bond Lengths (�) and Torsion Angles (deg) for S-A8.

Supplementary Figure Legends from The Synergistic Combination of the Farnesyl Transferase Inhibitor Lonafarnib and Paclitaxel Enhances Tubulin Acetylation and Requires a Functional Tubulin Deacetylase

Supplementary Figure 2 from The Synergistic Combination of the Farnesyl Transferase Inhibitor Lonafarnib and Paclitaxel Enhances Tubulin Acetylation and Requires a Functional Tubulin Deacetylase

Supplementary Table 1 from The Synergistic Combination of the Farnesyl Transferase Inhibitor Lonafarnib and Paclitaxel Enhances Tubulin Acetylation and Requires a Functional Tubulin Deacetylase

Farnesyl transferase (FT) inhibitors (FTI) are anticancer agents developed to target oncogenic Ras proteins by inhibiting Ras farnesylation. FTIs potently synergize with paclitaxel and other microtubule-stabilizing drugs; however, the mechanistic basis underlying this synergistic interaction remains elusive. Here we show that the FTI lonafarnib affects the microtubule cytoskeleton resulting in microtubule bundle formation, increased microtubule stabilization and acetylation, and suppression of microtubule dynamics. Notably, treatment with the combination of low doses of lonafarnib with paclitaxel markedly enhanced tubulin acetylation (a marker of microtubule stability) as compared with either drug alone. This synergistic effect correlated with FT inhibition and was accompanied by a synergistic increase in mitotic arrest and cell death. Mechanistically, we show that the combination of lonafarnib and paclitaxel inhibits the in vitro deacetylating activity of the only known tubulin deacetylase, histone deacetylase 6 (HDAC6). In addition, the lonafarnib/taxane combination is synergistic only in cells lines expressing the wild-type HDAC6, but not a catalytic-mutant HDAC6, revealing that functional HDAC6 is required for the synergy of lonafarnib with taxanes. Furthermore, tubacin, a specific HDAC6 inhibitor, synergistically enhanced tubulin acetylation in combination with paclitaxel, similar to the combination of lonafarnib and paclitaxel. Taken together, these data suggest a relationship between FT inhibition, HDAC6 function, and cell death, providing insight into the putative molecular basis of the lonafarnib/taxane synergistic antiproliferative combination.

Supplementary Figure 1 from The Synergistic Combination of the Farnesyl Transferase Inhibitor Lonafarnib and Paclitaxel Enhances Tubulin Acetylation and Requires a Functional Tubulin Deacetylase

We synthesized a new inhibitor of tubulin polymerization, the pyrrole (1-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrrol-3-yl)(3,4,5-trimethoxy-phenyl)methanone 6 (RS6077). Compound 6 inhibited the growth of multiple cancer cell lines, with IC

We synthesized new aroyl diheterocyclic pyrrole (ARDHEP)

2-Difluoromethoxyestratriene derivatives were designed to improve potency and in vivo stability of the drug candidate 2-methoxyestradiol (2ME2). Compound evaluation in vitro against the proliferation of MCF-7 and MDA MB-231 breast cancer cells, as inhibitors of tubulin polymerisation and also steroid sulfatase (STS) both in cell lysates and in whole cells, showed promising activities. In antiproliferative assays 2-difluoromethoxyestradiol was less potent than 2ME2, but its sulfamates were often more potent than the corresponding nonfluorinated analogues. The fluorinated bis-sulfamate is a promising anti-proliferative agent in MCF-7 cells (GI50 0.28 µM) vs the known 2- methoxyestradiol-3,17-O,O-bissulfamate (STX140, GI50 0.52 µM), confirming the utility of our approach. Compounds were also evaluated in the NCI 60-cell line panel and the fluorinated bissulfamate displayed very good overall activities with a sub-micromolar average GI50. It was a very potent STS inhibitor in whole JEG-3 cells (IC50 3.7 nM) similar to STX140 (4.2 nM) and additionally interferes with tubulin assembly in vitro and colchicine binding to tubulin. An Xray study of 2-difluoromethoxy-3-benzyloxyestra-1,3,5(10)-trien-17- one examined conformational aspects of the fluorinated substituent. The known related derivative 2-difluoromethyl-3-sulfamoyloxyestrone was evaluated for STS inhibition in whole JEG-3 cells and showed an excellent IC50 of 55 pM.

Objective: Several anti-tubulin agents were introduced for the cancer treatment so far. Despite successes in the treatment of cancer, these agents cause toxic side effects, including peripheral neuropathy. Comparing anti-tubulin agents, indibulin seemed to cause minimal peripheral neuropathy, but its poor aqueous solubility and other potential clinical problems have led to its remaining in a preclinical stage. Methods: Herein, indibulin analogues were synthesized and evaluated for their in vitro anti-cancer activity using MTT assay (on the MCF-7, T47-D, MDA-MB231 and NIH-3T3 cell lines), annexin V/PI staining assay, cell cycle analysis, anti-tubulin assay and caspase 3/7 activation assay. Results: One of the compounds, 4a, showed good anti-proliferative activity against MCF-7 cells (IC50: 7.5 μM) and low toxicity on a normal cell line (IC50 > 100 μM). All of the tested compounds showed lower cytotoxicity on normal cell line in comparison to reference compound, indibulin. In the annexin V/PI staining assay, induction of apoptosis in the MCF-7 cell line was observed. Cell cycle analysis illustrated an increasing proportion of cells in the sub-G-1 phase, consistent with an increasing proportion of apoptotic cells. No increase in G2/M cells was observed, consistent with the absence of anti-tubulin activity. A caspase 3/7 assay protocol showed that apoptosis induction by more potent compounds was due to activation of caspase 3. Conclusion: Newly synthesized compounds exerted acceptable anticancer activity and further investigation of current scaffold would be beneficial.

Novel compounds containing polar and nonpolar substitutions on the prop-2-en-1-on linker of the trans-indol-3-ylacrylamide scaffold were designed by modeling within the colchicine site of tubulin and then synthesized to determine the role of such substitutions on tubulin polymerization. We first determined the in vitro antiproliferation activities of the compounds against cancer cell lines with a particular focus on hepatocellular carcinoma. The results indicated that five of the compounds showed moderate antitumor activities. When the tubulin polymerization inhibitory effect of these compounds was evaluated, compound 13 was determined to be a tubulin polymerization inhibitor. Furthermore, cell cycle analysis for compound 13 resulted in G2/M-phase arrest in Huh7 cells. The results indicated that polar substitutions on the indole acrylamide scaffold enhance potency against tubulin polymerization. However, the substitution-related bioactivity shifting was not observed on the cancer cell lines since the inhibition mechanisms of the compounds may vary. (C) 2022 Elsevier B.V. All rights reserved.

Lymphomas are among the ten most common cancers, and, although progress has been achieved in increasing survival, there is still an unmet need for more effective therapeutic approaches, including better options for patients with refractory tumors that initially respond but then relapse. The lack of effective alternative treatment options highlights the need to develop new therapeutic strategies capable of improving survival prospects for lymphoma patients. Herein, we describe the identification and exploration of the SAR of a series of [1,2]oxazolo[5,4-e]isoindoles as potent small molecules that bind to the colchicine site of tubulin and that have promise for the treatment of refractory lymphomas. Exploration of the chemical space of this class of compounds at the pyrrole moiety and at the [1,2]oxazole ring highlighted two compounds bearing a 3,5-dimethoxybenzyl and a 3,4,5-trimethoxybenzyl group as potent candidates and showed that structural modifications at the isoxazole moiety are generally not favorable for activity. The two best candidates showed efficacy against different lymphoma histotypes and displayed 88 and 80% inhibition of colchicine binding fitting well into the colchicine pocket, as demonstrated by X-ray crystallography T2R-TTL–complexes, docking and thermodynamic analysis of the tubulin-colchicine complex structure. These results were confirmed by transcriptome data, thus indicating [1,2]oxazolo[5,4-e]isoindoles are promising candidates as antitubulin agents for the treatment of refractory lymphomas.

Organosulfur compounds show cytotoxic potential towards many tumor cell lines. Disulfides and thiosulfonates act through apoptotic processes, inducing proteins associated with apoptosis, endoplasmic reticulum stress, and the unfolded protein response. Three p-substituted symmetric diaryl disulfides and three diaryl thiosulfonates were synthesized and analyzed for inhibition of tubulin polymerization and for human cancer cell cytotoxic activity against seven tumor cell lines and a non-tumor cell line. S-(4-methoxyphenyl)-4-methoxybenzenesulfonothioate (6) exhibited inhibition of tubulin polymerization and showed the best antiproliferative potential, especially against the 786-0 cell line, being six times more selective as compared with the non-tumor cell line. In addition, compound 6 was able to activate caspase-3 after 24 and 48 h treatments of the 786-0 cell line and induced cell-cycle arrest in the G2/M stage at the highest concentration evaluated at 24 and 48 h. Compound 6 was able to cause complete inhibition of proliferation, inducing the death of 786-0 cells, by increasing the number of cells at G2/M and greater activation of caspase-3.

A promising design paradigm for small-molecule inhibitors of tubulin polymerization that bind to the colchicine site draws structural inspiration from the natural products colchicine and combretastatin A-4 (CA4). Our previous studies with benzocycloalkenyl and heteroaromatic ring systems yielded promising inhibitors with dihydronaphthalene and benzosuberene analogues featuring phenolic (KGP03 and KGP18) and aniline (KGP05 and KGP156) congeners emerging as lead agents. These molecules demonstrated dual mechanism of action, functioning as both potent vascular disrupting agents (VDAs) and as highly cytotoxic anticancer agents. A further series of analogues was designed to extend functional group diversity and investigate regioisomeric tolerance. Ten new molecules were effective inhibitors of tubulin polymerization (IC(50) < 5 µM) with seven of these exhibiting highly potent activity comparable to CA4, KGP18, and KGP03. For one of the most effective agents, dose-dependent vascular shutdown was demonstrated using dynamic bioluminescence imaging in a human prostate tumor xenograft growing in a rat.

BACKGROUND: During recent years, a number of anti-tubulin agents were introduced for treatment of diverse types of cancer. Despite their potential in the treatment of cancer, drug resistance and adverse toxicity, such as peripheral neuropathy, are some of the negative effects of anti-tubulin agents. Among anti-tubulin agents, indibulin was found to cause minimal peripheral neuropathy. Thus far, however, indibulin has not entered clinical usage, caused in part by its poor aqueous solubility and other developmental problems in preclinical evaluation. OBJECTIVES: With respect to need for finding potent and safe anticancer agents, in our current research work, we synthesized several indibulin-related diarylpyrrole derivatives and investigated their anti-cancer activity. METHODS: Cell cultur studies were perfomred using the MTT cell viability assay on the breast cancer cell lines MCF-7, T47-D, and MDA-MB231 and also NIH-3 T3 cells as representative of a normal cell line. The activity of some of the synthesized compounds for tubulin interaction was studied using colchicine binding and tubulin polymerization assays. The annexin V-FITC/PI method and flow cytometric analysis were used for studying apoptosis induction and cell cycle distribution. RESULTS AND CONCLUSION: Two of the synthesized compounds, 4f and 4 g, showed high activity on the MDA-MB231 cell line (IC(50) = 11.82 and 13.33 μM, (respectively) and low toxicity on the normal fibroblast cells (IC(50) > 100 μM). All of the tested compounds were more potent on T47-D cancer cells and less toxic on NIH-3 T3 normal cells in comparison to reference compound, indibulin. The tubulin polymerization inhibition assay and [(3)H]colchicine binding assay showed that the main mechanism of cell death by the potent synthesized compounds was not related to an interaction with tubulin. In the annexin V/PI staining assay, the induction of apoptosis in the MCF-7 and MDA-MB231 cell lines was observed. Cell cycle analysis illustrated an increased percentage of sub-G-1 cells in the MDA-MB231 cell line as a further indication of cell death through induction of apoptosis. [Figure: see text]

A tritiated derivative of the sponge-derived natural product spongistatin 1 was prepared, and its interactions with tubulin were examined. [

Many clinically used agents active in cancer chemotherapy exert their activity through the induction of cell death (apoptosis) by targeting microtubules, altering protein function or inhibiting DNA synthesis. The benzo[b]thiophene scaffold holds a pivotal place as a pharmacophore for the development of anticancer agents, and, in addition, this scaffold has many pharmacological activities. We have developed a flexible method for the construction of a new series of 2-aryl-3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophenes as potent antiproliferative agents, giving access to a wide range of substitution patterns at the 2-position of the 6-methoxybenzo[b]thiophene common intermediate. In the present study, all the synthesized compounds retained the 3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophene moiety, and the structure-activity relationship was examined by modification of the aryl group at its 2-position with electron-withdrawing (F) or electron-releasing (alkyl and alkoxy) groups. We found that small substituents, such as fluorine or methyl, could be placed in the para-position of the 2-phenyl ring, and these modifications only slightly reduced antiproliferative activity relative to the unsubstituted 2-phenyl analogue. Compounds 3a and 3b, bearing the phenyl and para-fluorophenyl at the 2-position of the 6-methoxybenzo[b]thiophene nucleus, respectively, exhibited the greatest antiproliferative activity among the tested compounds. The treatment of both Caco2 (not metastatic) and HCT-116 (metastatic) colon carcinoma cells with 3a or 3b triggered a significant induction of apoptosis as demonstrated by the increased expression of cleaved-poly(ADP-ribose) polymerase (PARP), receptor-interacting protein (RIP) and caspase-3 proteins. The same effect was not observed with non-transformed colon 841 CoN cells. A potential additional effect during mitosis for 3a in metastatic cells and for 3b in non-metastatic cells was also observed.

A new biological scaffold was produced by replacing the 6π-electron phenyl ring-B of a natural flavone skeleton with a 10π-electron benzothiophene (BT). Since aromatic rings are important for ligand protein interactions, this expansion of the π-electron system of ring-B might change the bioactivity profile. One of the resulting novel natural product-inspired compounds, 2-(benzo[b]thiophen-3-yl)-5-hydroxy-7-isopropoxy-6-methoxyflavone (6), effectively arrested the cell cycle at the G2/M phase and displayed significant antiproliferative effects with IC50 values of 0.05–0.08 μM against multiple human tumor cell lines, including a multidrug resistant line. A structure-activity relationship study revealed that a 10π-electron system with high aromaticity, juxtaposed 4-oxo and 5-hydroxy groups, and 7-alkoxy groups were important for potent antimitotic activity. Interestingly, two BT-flavonols (3-hydroxyflavone), 16 and 20, with 3-hydroxy and 5-alkoxy groups, induced distinct biological profiles affecting the cell cycle at the G1/S phase by inhibition of DNA replication through an interaction with topoisomerase I.

Heterocyclic scaffolds are widely utilized for drug design by taking into account the molecular structure of therapeutic targets that are related to a broad spectrum of ailments, including tumors. Such compounds display various covalent and non-covalent interactions with the specific residues of the target proteins while causing their inhibition. There is a substantial number of heterocyclic compounds approved for cancer treatment, and these compounds function by interacting with different therapeutic targets involved in tumorogenesis. In this review, we trace and emphasize the privileged heterocyclic pharmacophores that have immense potency against several essential chemotherapeutic tumor targets: microtubules, kinases and carbonic anhydrases. Potent compounds currently undergoing pre-clinical and clinical studies have also been assessed for ascertaining the effective class of chemical scaffolds that have significant therapeutic potential against multiple malignancies. In addition, we also describe briefly the role of heterocyclic compounds in various chemotherapy regimens. The optimized molecular hybridization of delineated motifs may result in the discovery of more active anticancer therapeutics and circumvent the development of resistance by specific targets in the future.

Three different series of cis-restricted analogues of combretastatin A-4 (CA-4), corresponding to thirty-nine molecules that contained a pyrrole nucleus interposed between the two aryl rings, were prepared by a palladium-mediated coupling approach and evaluated for their antiproliferative activity against six human cancer cell lines. In the two series of 1,2-diaryl pyrrole derivatives, results suggested that the presence of the 3′,4′,5′-trimethoxyphenyl moiety at the N-1 position of the pyrrole ring was more favorable for antiproliferative activity. In the series of 3,4-diarylpyrrole analogues, three compounds (11i-k) exhibited maximal antiproliferative activity, showing excellent antiproliferative activity against the CA-4 resistant HT-29 cells. Inhibition of tubulin polymerization of selected 1,2 pyrrole derivatives (9a, 9c, 9o and 10a) was similar to that observed with CA-4, while the isomeric 3,4-pyrrole analogues 11i-k were generally from 1.5- to 2-fold more active than CA-4. Compounds 11j and 11k were the only compounds that showed activity as inhibitors of colchicine binding comparable to that CA-4. Compound 11j had biological properties consistent with its intracellular target being tubulin. This compound was able to block the cell cycle in metaphase and to induce significant apoptosis at a concentration of 25 nM, following the mitochondrial pathway, with low toxicity for normal cells. More importantly, compound 11j exerted activity in vivo superior to that of CA-4P, being able to significantly reduce tumor growth in a syngeneic murine tumor model even at the lower dose tested (5.0 mg/kg).

Long-term survivors of glioblastoma multiforme (GBM) are at high risk of developing second primary neoplasms, including leukemia. For these patients, the use of classic tyrosine kinase inhibitors (TKIs), such as imatinib mesylate, is strongly discouraged, since this treatment causes a tremendous increase of tumor and stem cell migration and invasion. We aimed to develop agents useful for the treatment of patients with GBM and chronic myeloid leukemia (CML) using an alternative mechanism of action from the TKIs, specifically based on the inhibition of tubulin polymerization. Compounds 7 and 25, as planned, not only inhibited tubulin polymerization, but also inhibited the proliferation of both GMB and CML cells, including those expressing the T315I mutation, at nanomolar concentrations. In in vivo experiments in BALB/cnu/nu mice injected subcutaneously with U87MG cells, in vivo, 7 significantly inhibited GBM cancer cell proliferation, in vivo tumorigenesis, and tumor growth, tumorigenesis and angiogenesis. Compound 7 was found to block human topoisomerase II (hTopoII) selectively and completely, at a concentration of 100 μM.

The design, synthesis, and biological evaluation of a series novel N1‑methyl pyrazolo[4,3-d]pyrimidines as inhibitors of tubulin polymerization and colchicine binding were described here. Synthesis of target compounds involved alkylation of the pyrazolo scaffold, which afforded two regioisomers. These were separated, characterized and identified with (1)H NMR and NOESY spectroscopy. All compounds, except 10, inhibited [(3)H] colchicine binding to tubulin, and the potent inhibition was similar to that obtained with CA-4. Compounds 9 and 11–13 strongly inhibited the polymerization of tubulin, with IC(50) values of 0.45, 0.42, 0.49 and 0.42 μM, respectively. Compounds 14–16 inhibited the polymerization of tubulin with IC(50s) near ~1 μM. Compounds 9 12, 13 and 16 inhibited MCF-7 breast cancer cell lines and circumvented βIII-tubulin mediated cancer cell resistance to taxanes and other MTAs, and compounds 9–17 circumvented Pgp-mediated drug resistance. In the standard NCI testing protocol, compound 9 exhibited excellent potency with low to sub nanomolar GI(50) values (≤10 nM) against most tumor cell lines, including several multidrug resistant phenotypes. Compound 9 was significantly (P

BackgroundGlioma is sensitive to microtubule-targeting agents (MTAs), but most MTAs do not cross the blood brain barrier (BBB). To address this limitation, we developed the new chemical entity, ST-401, a brain-penetrant MTA.MethodsSynthesis of ST-401. Measures of MT assembly and dynamics. Cell proliferation and viability of patient-derived (PD) glioma in culture. Measure of tumor microtube (TM) parameters using immunofluorescence analysis and machine learning-based workflow. Pharmacokinetics (PK) and experimental toxicity in mice. In vivo antitumor activity in the RCAS/tv-a PDGFB-driven glioma (PDGFB-glioma) mouse model.ResultsWe discovered that ST-401 disrupts microtubule (MT) function through gentle and reverisible reduction in MT assembly that triggers mitotic delay and cell death in interphase. ST-401 inhibits the formation of TMs, MT-rich structures that connect glioma to a network that promotes resistance to DNA damage. PK analysis of ST-401 in mice shows brain penetration reaching antitumor concentrations, and in vivo testing of ST-401 in a xenograft flank tumor mouse model demonstrates significant antitumor activity and no over toxicity in mice. In the PDGFB-glioma mouse model, ST-401 enhances the therapeutic efficacies of temozolomide (TMZ) and radiation therapy (RT).ConclusionOur study identifies hallmarks of glioma tumorigenesis that are sensitive to MTAs and reports ST-401 as a promising chemical scaffold to develop brain-penetrant MTAs.

Most cancer therapeutics developed to date are excluded from the brain and are therefore ineffective in treating glioma, the most devastating type of brain cancer in adults. Microtubule targeting agents (MTAs) are indispensable medicines to treat a wide range of solid and hematopoietic tumors, and evidence suggests that glioma is sensitive to MTAs; but most MTAs do not cross the blood-brain-barrier. To address this limitation, we developed a brain-penetrant MTA, ST-401, that inhibited the growth of human glioma in culture at nanomolar concentrations. ST-401 bound to the colchicine site, inhibited tubulin assembly and reversibly reduced microtubule (MT) dynamics. Testing of ST-401 on the NCI 60 cancer cell panel indicated that its anti-tumor activity does not correlate with any of the compounds tested thus far through this platform but showed weak correlations for two MTA that work through distinct mechanisms: taxol (p=0.46) and vinblastine (p=0.34). Thus, ST-401 may kill cancer cells through a novel mechanism related to disruption of MT function. We discovered that ST-401 killed patient-derived (PD) glioma isolates in both mitosis and interphase, and inhibited the formation of tumor microtubes, MT-rich structures that connects glioma cells to a network that is resistant to standard therapies. Pharmacokinetic analysis of ST-401 in mice shows brain penetration reaching antitumor concentrations, and in vivo testing of ST-401 in a xenograft model demonstrated significant antitumor activity. In an immunocompetent mouse model of platelet-derived growth factor B-driven glioma, ST-401 significantly enhanced the therapeutic efficacies of standard care therapies temozolomide and radiation therapy. Our study identified novel aspects of glioma tumorigenesis that exhibit enhanced sensitivity to MTAs and shows that the brain-penetrant MTA, ST-401, represents a promising chemical scaffold to develop therapeutics for the treatment of patients diagnosed with glioma.