2-5-dimethylcelecoxib and Glioblastoma

2,5-Dimethyl-celecoxib (DMC) is a close structural analog of the selective COX-2 inhibitor celecoxib that lacks COX-2-inhibitory function. Thus, DMC is a promising drug for anti-tumor. In this study, we evaluated the efficacy and the molecular basis of DMC in the treatment of human glioblastoma multiforme (GBM). DMC inhibited the growth and proliferation of GBM cell lines (LN229, A172, U251, and U87MG) in a dose-dependent manner (P < 0.001). In GBM cells treated with DMC, detection by flow cytometry showed cell cycle arrest, and proteins involved in cell cycle such as P21 were increased. Compared with control group, Annexin-V/PI-staining in DMC-treatment group was increased, indicating that DMC could induce apoptosis in GBM cells. Also, associated proteins including cleaved caspase 3 and cleaved PARP-1 were increased. It was further explored whether DMC blocked cell cycle and induced apoptosis in GBM cells through CIP2A/PP2A/AKT signaling pathway. After treatment of DMC, the phosphorylation of Akt was reduced while the total Akt level was not affected. DMC suppressed the expression of CIP2A in a time-dependent manner, while the CIP2A overexpression group reversed cell cycle and apoptotic protein expression led by DMC. Finally, in a xenograft model in nude mice using LN229 cells, DMC suppressed tumor growth. These findings proved that DMC could block cell cycle and induce apoptosis in GBM cells by suppressing CIP2A/PP2A/Akt signaling axis, which indicated that DMC could be an effective option for GBM treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; Autoantigens; Brain Neoplasms; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Proliferation; Glioblastoma; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Protein Phosphatase 2; Proto-Oncogene Proteins c-akt; Pyrazoles; Signal Transduction; Sulfonamides

Glioblastoma (GBM) is the deadliest and the most common primary brain tumor in adults. The invasiveness and proliferation of GBM cells can be decreased through the inhibition of Wnt/β-catenin pathway. In this regard, celecoxib is a promising agent, but other COXIBs and 2,5-dimethylcelecoxib (2,5-DMC) await elucidation. Thus, the aim of this study was to analyze the impact of celecoxib, 2,5-DMC, etori-, rofe-, and valdecoxib on GBM cell viability and the activity of Wnt/β-catenin pathway. In addition, the combination of the compounds with temozolomide (TMZ) was also evaluated. Cell cycle distribution and apoptosis, MGMT methylation level, COX-2 and PGE2 EP4 protein levels were also determined in order to better understand the molecular mechanisms exerted by these compounds and to find out which of them can serve best in GBM therapy.. Celecoxib, 2,5-DMC, etori-, rofe- and valdecoxib were evaluated using three commercially available and two patient-derived GBM cell lines. Cell viability was analyzed using MTT assay, whereas alterations in MGMT methylation level were determined using MS-HRM method. The impact of COXIBs, in the presence and absence of TMZ, on Wnt pathway was measured on the basis of the expression of β-catenin target genes. Cell cycle distribution and apoptosis analysis were performed using flow cytometry. COX-2 and PGE2 EP4 receptor expression were evaluated using Western blot analysis.. Wnt/β-catenin pathway was attenuated by COXIBs and 2,5-DMC irrespective of the COX-2 expression profile of the treated cells, their MGMT methylation status, or radio/chemoresistance. Celecoxib and 2,5-DMC were the most cytotoxic. Cell cycle distribution was altered, and apoptosis was induced after the treatment with celecoxib, 2,5-DMC, etori- and valdecoxib in T98G cell line. COXIBs and 2,5-DMC did not influence MGMT methylation status, but inhibited COX-2/PGE2/EP4 pathway.. Not only celecoxib, but also 2,5-DMC, etori-, rofe- and valdecoxib should be further investigated as potential good anti-GBM therapeutics.

Topics: Aged; Antineoplastic Agents, Alkylating; Apoptosis; beta Catenin; Brain Neoplasms; Celecoxib; Cell Cycle; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; DNA Modification Methylases; DNA Repair Enzymes; Dose-Response Relationship, Drug; Etoricoxib; Female; Glioblastoma; Humans; Isoxazoles; Lactones; Male; Methylation; Middle Aged; Neoplasm Proteins; Pyrazoles; Receptors, Prostaglandin E, EP4 Subtype; Sulfonamides; Sulfones; Temozolomide; Tumor Suppressor Proteins; Wnt Signaling Pathway

The proteasome inhibitor bortezomib (Velcade) is known to trigger endoplasmic reticulum (ER) stress via the accumulation of obsolete and damaged proteins. The selective cyclooxygenase-2 (COX-2) inhibitor celecoxib (Celebrex) causes ER stress through a different mechanism (i.e., by causing leakage of calcium from the ER into the cytosol). Each of these two mechanisms has been implicated in the anticancer effects of the respective drug. We therefore investigated whether the combination of these two drugs would lead to further increased ER stress and would enhance their antitumor efficacy. With the use of human glioblastoma cell lines, we show that this is indeed the case. When combined, bortezomib and celecoxib triggered elevated expression of the ER stress markers GRP78/BiP and CHOP/GADD153, caused activation of c-Jun NH(2)-terminal kinase and ER stress-associated caspase-4, and greatly increased apoptotic cell death. Small interfering RNA-mediated knockdown of the protective ER chaperone GRP78/BiP further sensitized the tumor cells to killing by the drug combination. The contribution of celecoxib was independent of the inhibition of COX-2 because a non-coxib analogue of this drug, 2,5-dimethyl-celecoxib (DMC), faithfully and more potently mimicked these combination effects in vitro and in vivo. Taken together, our results show that combining bortezomib with celecoxib or DMC very potently triggers the ER stress response and results in greatly increased glioblastoma cytotoxicity. We propose that this novel drug combination should receive further evaluation as a potentially effective anticancer therapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Boronic Acids; Bortezomib; Celecoxib; Cell Death; Cell Line, Tumor; Cyclooxygenase 2 Inhibitors; Drug Synergism; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Glioblastoma; Heat-Shock Proteins; Humans; Male; Mice; Mice, Nude; Molecular Chaperones; Pyrazines; Pyrazoles; Sulfonamides; Transfection; Xenograft Model Antitumor Assays