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Temozolomide (TMZ) is an alkylating agent used for the treatment of glioblastoma. The late autophagy inhibitor chloroquine (CQ) inhibits glioblastoma tumors in a p53-independent and p53-dependent manner. We addressed a possible beneficial effect of combination treatment with TMZ and CQ by examining the molecular and cellular mechanism of co-treatment. Combination treatment of U87 cell (wild type p53) with TMZ and CQ synergistically reduced cell proliferation and enhanced apoptosis, with increased sub-G 1 hypodiploid cells and caspase activation. This effect was abolished by a pan-caspase inhibitor, Z-VAD-FMK. TMZ induced autophagy, and the addition of CQ further increased autophagic vacuoles. Inhibition of early stages of autophagy by Beclin 1 knockdown and 3-methyladenine pretreatment prevented the enhanced effect of the combination treatment. The combination treatment also upregulated p53 and phospho-p53 levels, whereas p53 knockdown or overexpression of mutant p53 abolished the combination effect. In contrast, combination therapy had no enhanced effect on U373 cell (mutant p53) proliferation and apoptosis within 3 d, although TMZ induced autophagy and co-treatment with CQ increased autophagic vacuole accumulation. However, long term combination treatment for 9–10 d effectively decreased clonal and cellular growth with increased G 2 –M arrest. This effect was also abolished by Beclin 1 knockdown. Our data support the beneficial effect of combination treatment with TMZ and CQ in glioma via differential autophagy-associated mechanisms, depending on p53 status.

DNA lesion-induced centrosomal abnormalities during the replication phase are relatively unknown. Here, we report that RNAi-mediated depletion of RRM1 induces cell-cycle arrest at the replication phase, along with severe DNA damage and centrosomal amplification. Interestingly, CHK1 depletion synergistically increased RRM1-depletion-induced centrosomal amplification. In response to hydroxyurea, CHK1 was delocalized from the centrosome by RRM1 depletion. Moreover, CDK1, which functions in centrosome separation and is inhibited by CHK1, was found to be essential for RRMI1-depletion-induced centrosomal amplification. Thus, we herein demonstrate that RRM1 preserves chromosomal stability via the CHK1- and CDK1-dependent stabilization of the centrosomal integrity at the replication stage.

The present study was performed to investigate the possible combined use of the Cox-2 inhibitor niflumic acid and the PPARγ ligand ciglitazone and to elucidate the mechanisms underlying enhanced apoptosis by this combination treatment in human lung cancer cells. Combined niflumic acid–ciglitazone treatment synergistically induced apoptotic cell death, activated caspase-9, caspase-3, and induced caspase-3-mediated PARP cleavage. The combination treatment also triggered apoptosis through caspase-8/Bid/Bax activation, and the inhibition of caspase-8 suppressed caspase-8/Bid activation, caspase-3-mediated PARP cleavage, and concomitant apoptosis. In addition, combined niflumic acid–ciglitazone treatment significantly induced ER stress responses, and suppression of CHOP expression significantly attenuated the combined niflumic acid–ciglitazone treatment-induced activation of caspase-8 and caspase-3, and the subsequent apoptotic cell death, indicating a role of ER stress in caspase-8 activation and apoptosis. Interestingly, the pro-apoptotic effects of combined niflumic acid–ciglitazone treatment were realized through Cox-2- and PPARγ-independent mechanisms. Taken together, these results suggest that sequential ER stress and caspase-8 activation are critical in combined niflumic acid–ciglitazone treatment-induced apoptosis in human lung cancer cells.

The kringle domain of urokinase-type plasminogen activator (UK1) has anti-angiogenic and anti-tumor effects. Celecoxib, an inhibitor of cyclooxygenase type 2, also suppresses angiogenesis and tumor growth. To look for potential additive effects in their activities, we examined the anti-angiogenic and anti-tumor effects of the combination of UK1 and celecoxib for malignant gliomas. In vitro , the combination of UK1 and celecoxib enhanced inhibition of proliferation, migration, and tube formation of endothelial cells, although showing no enhancement of inhibition of U87 cell growth. However, in vivo models, combination treatment of intracerebral U87 malignant glioma xenografts in nude mice with UK1 (10 mg/kg/day) and celecoxib (10 mg/kg/day) at lower doses resulted in even more potent inhibition of tumor growth than each monotherapy (by 81% compared to untreated tumors), with drastic decrease of the expression of angiogenesis-related factors and increase of apoptosis in the tumor tissues. Interestingly, UK1 inhibited VEGF or bFGF-induced phosphorylation of ERK1/2 in ECs, whereas celecoxib showed no such effects. However, celecoxib inhibited U87 cell growth and directly suppressed their VEGF production. Therefore, our data suggest that combined use at low doses of UK1 and celecoxib with different anti-angiogenic mechanisms provides a desirable strategy for anti-glioma therapy.