An evaluation of the interaction of pixantrone with formaldehyde-releasing drugs in cancer cells.

PURPOSE: Pixantrone is a synthetic aza-anthracenedione currently used in the treatment of non-Hodgkin's lymphoma. The drug is firmly established as a poison of the nuclear enzyme topoisomerase II, however, pixantrone can also generate covalent drug-DNA adducts following activation by formaldehyde. While pixantrone-DNA adducts form proficiently in vitro, little evidence is presently at hand to indicate their existence within cells. The molecular nature of these lesions within cancer cells exposed to pixantrone and formaldehyde-releasing prodrugs was characterized along with the cellular responses to their formation. METHODS: In vitro crosslinking assays, [14C] scintillation counting analyses and alkaline comet assays were applied to characterize pixantrone-DNA adducts. Flow cytometry, cell growth inhibition and clonogenic assays were used to measure cancer cell kill and survival. RESULTS: Pixantrone-DNA adducts were not detectable in MCF-7 breast cancer cells exposed to [14C] pixantrone (10-40 µM) alone, however the addition of the formaldehyde-releasing prodrug AN9 yielded readily measurable levels of the lesion at ~ 1 adduct per 10 kb of genomic DNA. Co-administration with AN9 completely reversed topoisomerase II-associated DNA damage induction by pixantrone yet potentiated cell kill by the drug, suggesting that pixantrone-DNA adducts may promote a topoisomerase II-independent mechanism of cell death. Pixantrone-DNA adduct-forming treatments generally conferred mild synergism in multiple cell lines in various cell death and clonogenic assays, while pixantrone analogues either incapable or relatively defective in forming DNA adducts demonstrated antagonism when combined with AN9. CONCLUSIONS: The features unique to pixantrone-DNA adducts may be leveraged to enhance cancer cell kill and may be used to guide the design of pixantrone analogues that generate adducts with more favorable anticancer properties.

Effect of Benzo[a]Pyrene on Spindle Misorientation and Fidelity of Chromosome Segregation in Lung Epithelial BEAS-2B Cells.

Benzo[a]pyrene (B[a]P) is an environmental carcinogen found in tobacco smoke. It leads to high levels of DNA adducts in the lungs of cigarette smokers contributing to genomic instability. Alterations in the mitotic spindle apparatus play a major role in the generation of genomic instability through promoting chromosome mis-segregation and aneuploidy. To date, the effect of B[a]P exposure on altering the mitotic apparatus in normal lung epithelial cells remains unknown. In our study, BEAS-2B human bronchial epithelial cells were exposed to B[a]P and spindle dynamics were evaluated. Confocal imaging showed that B[a]P exposure significantly alters spindles misorientation, leading to chromosome mis-segregations in the form of chromosome lags and bridges. In addition, centrosome duplication and premature centriole disengagement were induced leading to misaligned and multipolar spindle formation. Comparative genomic analysis of mitotic spindle associated genes, revealed downregulation of AurA-Plk1-AurB signaling cascade by B[a]P. In addition, we analyzed the status of p53 and its downstream p21 in B[a]P-treated cells and showed a suppression of p53-p21 axis. When the extent of DNA damage associated with induced mitotic abnormalities was investigated using γ-H2AX, a significant increase and persistence in DNA damage was observed. Overall, our findings show that B[a]P potently induces mitotic abnormalities, DNA damage, and genetic instability.

OLA1 regulates protein synthesis and integrated stress response by inhibiting eIF2 ternary complex formation.

Translation is a fundamental cellular process, and its dysregulation can contribute to human diseases such as cancer. During translation initiation the eukaryotic initiation factor 2 (eIF2) forms a ternary complex (TC) with GTP and the initiator methionyl-tRNA (tRNAi), mediating ribosomal recruitment of tRNAi. Limiting TC availability is a central mechanism for triggering the integrated stress response (ISR), which suppresses global translation in response to various cellular stresses, but induces specific proteins such as ATF4. This study shows that OLA1, a member of the ancient Obg family of GTPases, is an eIF2-regulatory protein that inhibits protein synthesis and promotes ISR by binding eIF2, hydrolyzing GTP, and interfering with TC formation. OLA1 thus represents a novel mechanism of translational control affecting de novo TC formation, different from the traditional model in which phosphorylation of eIF2α blocks the regeneration of TC. Depletion of OLA1 caused a hypoactive ISR and greater survival in stressed cells. In vivo, OLA1-knockdown rendered cancer cells deficient in ISR and the downstream proapoptotic effector, CHOP, promoting tumor growth and metastasis. Our work suggests that OLA1 is a novel translational GTPase and plays a suppressive role in translation and cell survival, as well as cancer growth and progression.

A class I histone deacetylase inhibitor, entinostat, enhances lapatinib efficacy in HER2-overexpressing breast cancer cells through FOXO3-mediated Bim1 expression.

Although there are effective HER2-targeted agents, novel combination strategies in HER2-overexpressing breast cancers are needed for patients whose tumors develop drug resistance. To develop new therapeutic strategy, we investigated the combinational effect of entinostat, an oral isoform-selective histone deacetylase type I inhibitor, and lapatinib, a HER2/EGFR dual tyrosine kinase inhibitor, in HER2+ breast cancer cells. We assessed the combinational synergistic effect and its mechanism by CellTiter Blue assay, flow cytometry, anchorage-independent growth, quantitative real-time PCR, small interfering RNA, Western blotting, and mammary fat pad xenograft mouse models. We found that compared with entinostat or lapatinib alone, the two drugs in combination synergistically inhibited proliferation (P < 0.001), reduced in vitro colony formation (P < 0.05), and resulted in significant in vivo tumor shrinkage or growth inhibition in two xenograft mouse models (BT474 and SUM190, P < 0.001). The synergistic anti-tumor activity of the entinostat/lapatinib combination was due to downregulation of phosphorylated Akt, which activated transcriptional activity of FOXO3, resulting in induction of Bim1 (a BH3 domain-containing pro-apoptotic protein). Furthermore, entinostat sensitized trastuzumab/lapatinib-resistance-HER2-overexpressing cells to the trastuzumab/lapatinib combination and enhanced the anti-proliferation effect compare with single or double combination treatment. This study provides evidence that entinostat has enhanced anti-tumor effect in combination with HER2-targeted reagent, lapatinib, and resulting in induction of apoptosis by FOXO3-mediated Bim1 expression. Our finding justifies for conducting a clinical trial of combinational treatment with entinostat, lapatinib, and trastuzumab in patients with HER2-overexpressing breast cancer resistant to trastuzumab-based treatment.

New anthracenedione derivatives with improved biological activity by virtue of stable drug-DNA adduct formation.

Mitoxantrone is an anticancer agent that acts as a topoisomerase II poison, however, it can also be activated by formaldehyde to form DNA adducts. Pixantrone, a 2-aza-anthracenedione with terminal primary amino groups in its side chains, forms formaldehyde-mediated adducts with DNA more efficiently than mitoxantrone. Molecular modeling studies indicated that extension of the "linker" region of anthracenedione side arms would allow the terminal primary amino greater flexibility and thus access to the guanine residues on the opposite DNA strand. New derivatives based on the pixantrone and mitoxantrone backbones were synthesized, and these incorporated primary amino groups as well as extended side chains. The stability of DNA adducts increased with increasing side chain length of the derivatives. A mitoxantrone derivative bearing extended side chains (7) formed the most stable adducts with ∼100-fold enhanced stability compared to mitoxantrone. This finding is of great interest because long-lived drug-DNA adducts are expected to perturb DNA-dependent functions at all stages of the cell cycle.

Pixantrone can be activated by formaldehyde to generate a potent DNA adduct forming agent.

Mitoxantrone is an anti-cancer agent used in the treatment of breast and prostate cancers. It is classified as a topoisomerase II poison, however can also be activated by formaldehyde to generate drug-DNA adducts. Despite identification of this novel form of mitoxantrone-DNA interaction, excessively high, biologically irrelevant drug concentrations are necessary to generate adducts. A search for mitoxantrone analogues that could potentially undergo this reaction with DNA more efficiently identified Pixantrone as an ideal candidate. An in vitro crosslinking assay demonstrated that Pixantrone is efficiently activated by formaldehyde to generate covalent drug-DNA adducts capable of stabilizing double-stranded DNA in denaturing conditions. Pixantrone-DNA adduct formation is both concentration and time dependent and the reaction exhibits an absolute requirement for formaldehyde. In a direct comparison with mitoxantrone-DNA adduct formation, Pixantrone exhibited a 10- to 100-fold greater propensity to generate adducts at equimolar formaldehyde and drug concentrations. Pixantrone-DNA adducts are thermally and temporally labile, yet they exhibit a greater thermal midpoint temperature and an extended half-life at 37 degrees C when compared to mitoxantrone-DNA adducts. Unlike mitoxantrone, this enhanced stability, coupled with a greater propensity to form covalent drug-DNA adducts, may endow formaldehyde-activated Pixantrone with the attributes required for Pixantrone-DNA adducts to be biologically active.