At-home Bleaching with a Novel Carbamide Peroxide Polymeric Nanoparticle Gel: A Multicenter Randomized Controlled Trial.

OBJECTIVES: To compare the risk and intensity of tooth sensitivity (TS) as well the effectiveness of at-home bleaching using two carbamide peroxide bleaching gels, specifically a novel polymeric nanoparticle gel (experimental) and a commercial gel (Opalescence PF, Ultradent, South Jordan, Utah, USA), applied at two application times. METHODS: This multicenter, triple-blind, and split-mouth randomized controlled trial was conducted on 80 healthy adults with canine teeth that were shade A2 or darker. The participants all used the experimental and the control gels on one side of a tray, depending on the group to which they were allocated, for 30 or 60 minutes per day over four weeks. The absolute risk and intensity of TS were assessed daily using the five-point Numeric Rating Scale and the 0-10 Visual Analogue Scale. Color change was evaluated with shade guide units (ΔSGU) and a digital spectrophotometer (ΔEab, ΔE00, and ΔWid) at baseline and 30 days postbleaching. The risk and intensity of TS was evaluated by the McNemar and the Wilcoxon signed-rank tests, respectively. Color change (ΔSGU, ΔEab, ΔE00, and ΔWid) was evaluated by Mann-Whitney and paired t-tests (α=0.05). RESULTS: No differences in the risk and intensity of TS were observed based on the bleaching gels used and the times of application (p>0.05). Thirty days after bleaching, there was no significant difference in color change, in terms of the bleaching gels used or the application times (p>0.05). CONCLUSION: The novel carbamide peroxide polymeric nanoparticles gel, when applied for 30 or 60 minutes, produced effective color change and a low rate of tooth sensitivity, as compared to the control group.

Mechanistic validation of a clinical lead stapled peptide that reactivates p53 by dual HDM2 and HDMX targeting.

Hydrocarbon-stapled peptides that display key residues of the p53 transactivation domain have emerged as bona fide clinical candidates for reactivating the tumor suppression function of p53 in cancer by dual targeting of the negative regulators HDM2 and HDMX. A recent study questioned the mechanistic specificity of such stapled peptides based on interrogating their capacity to disrupt p53/HDM2 and p53/HDMX complexes in living cells using a new recombinase enhanced bimolecular luciferase complementation platform (ReBiL). Here, we directly evaluate the cellular uptake, intracellular targeting selectivity and p53-dependent cytotoxicity of the clinical prototype ATSP-7041. We find that under standard serum-containing tissue culture conditions, ATSP-7041 achieves intracellular access without membrane disruption, dose-dependently dissociates both p53/HDM2 and p53/HDMX complexes but not an unrelated protein complex in long-term ReBiL experiments, and is selectively cytotoxic to cancer cells bearing wild-type p53 by inducing a surge in p53 protein level. These studies underscore the importance of a thorough stepwise approach, including consideration of the time-dependence of cellular uptake and intracellular distribution, in evaluating and advancing stapled peptides for clinical translation.

Cell cycle-arrested tumor cells exhibit increased sensitivity towards TRAIL-induced apoptosis.

Resting tumor cells represent a huge challenge during anticancer therapy due to their increased treatment resistance. TNF-related apoptosis-inducing ligand (TRAIL) is a putative future anticancer drug, currently in phases I and II clinical studies. We recently showed that TRAIL is able to target leukemia stem cell surrogates. Here, we tested the ability of TRAIL to target cell cycle-arrested tumor cells. Cell cycle arrest was induced in tumor cell lines and xenografted tumor cells in G0, G1 or G2 using cytotoxic drugs, phase-specific inhibitors or RNA interference against cyclinB and E. Biochemical or molecular arrest at any point of the cell cycle increased TRAIL-induced apoptosis. Accordingly, when cell cycle arrest was disabled by addition of caffeine, the antitumor activity of TRAIL was reduced. Most important for clinical translation, tumor cells from three children with B precursor or T cell acute lymphoblastic leukemia showed increased TRAIL-induced apoptosis upon knockdown of either cyclinB or cyclinE, arresting the cell cycle in G2 or G1, respectively. Taken together and in contrast to most conventional cytotoxic drugs, TRAIL exerts enhanced antitumor activity against cell cycle-arrested tumor cells. Therefore, TRAIL might represent an interesting drug to treat static-tumor disease, for example, during minimal residual disease.

Enhanced anti-tumour effects of Vinca alkaloids given separately from cytostatic therapies.

BACKGROUND AND PURPOSE: In polychemotherapy protocols, that is for treatment of neuroblastoma and Ewing sarcoma, Vinca alkaloids and cell cycle-arresting drugs are usually administered on the same day. Here we studied whether this combination enables the optimal antitumour effects of Vinca alkaloids to be manifested. EXPERIMENTAL APPROACH: Vinca alkaloids were tested in a preclinical mouse model in vivo and in vitro in combination with cell cycle-arresting drugs. Signalling pathways were characterized using RNA interference. KEY RESULTS: In vitro, knockdown of cyclins significantly inhibited vincristine-induced cell death indicating, in accordance with previous findings, Vinca alkaloids require active cell cycling and M-phase transition for induction of cell death. In contrast, anthracyclines, irradiation and dexamethasone arrested the cell cycle and acted like cytostatic drugs. The combination of Vinca alkaloids with cytostatic therapeutics resulted in diminished cell death in 31 of 36 (86%) tumour cell lines. In a preclinical tumour model, anthracyclines significantly inhibited the antitumour effect of Vinca alkaloids in vivo. Antitumour effects of Vinca alkaloids in the presence of cytostatic drugs were restored by caffeine, which maintained active cell cycling, or by knockdown of p53, which prevented drug-induced cell cycle arrest. Therapeutically most important, optimal antitumour effects were obtained in vivo upon separating the application of Vinca alkaloids from cytostatic therapeutics. CONCLUSION AND IMPLICATIONS: Clinical trials are required to prove whether Vinca alkaloids act more efficiently in cancer patients if they are applied uncoupled from cytostatic therapies. On a conceptual level, our data suggest the implementation of polychemotherapy protocols based on molecular mechanisms of drug-drug interactions. LINKED ARTICLE: This article is commented on by Solary, pp 1555-1557 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12101.

NOXA as critical mediator for drug combinations in polychemotherapy.

During polychemotherapy, cytotoxic drugs are given in combinations to enhance their anti-tumor effectiveness. For most drug combinations, underlying signaling mechanisms responsible for positive drug-drug interactions remain elusive. Here, we prove a decisive role for the Bcl-2 family member NOXA to mediate cell death by certain drug combinations, even if drugs were combined which acted independently from NOXA, when given alone. In proof-of-principle studies, betulinic acid, doxorubicin and vincristine induced cell death in a p53- and NOXA-independent pathway involving mitochondrial pore formation, release of cytochrome c and caspase activation. In contrast, when betulinic acid was combined with either doxorubicine or vincristine, cell death signaling changed considerably; the drug combinations clearly depended on both p53 and NOXA. Similarly and of high clinical relevance, in patient-derived childhood acute leukemia samples the drug combinations, but not the single drugs depended on p53 and NOXA, as shown by RNA interference studies in patient-derived cells. Our data emphasize that NOXA represents an important target molecule for combinations of drugs that alone do not target NOXA. NOXA might have a special role in regulating apoptosis sensitivity in the complex interplay of polychemotherapy. Deciphering the differences in signaling of single drugs and drug combinations might enable designing highly effective novel polychemotherapy regimens.

An immunohistochemical investigation into the presence and localization of cytochrome P-450 isozymes in organogenesis-stage rat conceptal tissue.

To investigate the presence and localization of a variety of xenobiotic biotransforming isozymes of the cytochrome P-450 superfamily in the organogenesis-stage rat conceptal tissue, pregnant female rats were dosed with one of two inducing agents, 3-methylcholanthrene [3MC, 40 mg/kg body weight, ip, day 7 post coitum (pc)] and phenobarbital (PB, 40 mg/kg, ip, days 5, 6, 7 and 8 pc), or with their vehicles (3MC, olive oil; PB, 0.9% NaCl) as controls. The conceptuses were allowed to grow either in vivo, or in vitro, using the whole embryo culture system, from days 9.5 to 11.5 pc. The embryos and isolated visceral yolk sacs were submitted to immunohistochemical investigation using light microscopy. The livers of the dams served as positive controls. Polyclonal and monoclonal antibodies raised against a variety of cytochrome P-450 isozymes were used in the alkaline phosphatase-anti-alkaline phosphatase enzyme immune complex method. All pre-induced dam livers showed positive staining with all polyclonal and monoclonal antibodies tested. The presence of P450IA1 was detected in the visceral yolk sac of both ex vivo and cultured conceptuses, preinduced in utero with 3MC, with the appropriate polyclonal antibodies but not with the monoclonal antibodies. P450IIB1/2 was detected in the visceral yolk sac of both ex vivo and cultured conceptuses, pre-induced in utero by phenobarbital, with the appropriate polyclonal antibodies, but not with the monoclonal antibodies. No staining was seen in any embryo proper, with any vehicle-treated conceptal tissue, or with antibodies raised against P-450s IIE1, IIIA or IVA. Our results support the hypothesis that the organogenesis-stage rat conceptus contains, in the visceral yolk sac, a 3MC-inducible P-450 isozyme similar, but not identical, to adult IA1. They also provide evidence that a PB-inducible isozyme similar, but not identical, to adult IIB1/2, is present in the visceral yolk sac at this stage of conceptus development.

Genetically engineered V79 Chinese hamster cells for stable expression of human cytochrome P450IA2.

V79 Chinese hamster cells were genetically engineered for stable expression of human P450IA2. Full length cDNA, encoding human P450IA2, was inserted into an SV40 early promoter containing eukaryotic expression vector and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to the neomycin derivative G418) into V79 Chinese hamster cells. The recombinant expression vector was introduced into two different V79 sublines, one expressing an endogenous acetyltransferase (V79-NH), the other not (V79-MZ). The presence of human cytochrome CYP1A2 cDNA in the G418 resistant V79 cell clones was confirmed by Southern blotting. The transcription of the cDNA into mRNA was detected by Northern blotting and the translation into an authentic cytochrome P450IA2 protein was shown by Western blotting. The enzymatic activity in these cells was determined by the cytochrome P450IA2-dependent methoxy-, ethoxy-, benzoxy-, and pentoxyresorufin dealkylation activity.

Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17 beta-estradiol and 2-aminofluorene in V79 Chinese hamster cells.

In continuation of our work toward the establishment of a working cell bank for metabolic and toxicological studies, V79 Chinese hamster cells were genetically engineered for stable expression of rat cytochrome P450IA2. Full-length cDNA encoding rat P450IA2 was obtained by searching a cDNA library made from Aroclor 1254-induced rat liver mRNA and by joining a small 5'-end fragment to a fragment containing the rest of the cDNA. The sequence of the cDNA was confirmed by DNA sequencing and comparison to a previously published cDNA sequence. The reconstructed full-length cDNA was inserted into a simian virus 40 early promoter-containing eukaryotic expression vector and cotransferred with the neomycin phosphotransferase gene as a selective marker into V79 cells by the calcium/phosphate-coprecipitation technique. G418-resistant V79 cell clones were checked for chromosomal integration of the cDNA by Southern blotting, for expression of authentic mRNA and protein by northern and western blotting, and for P450IA2-specific enzymatic activities such as hydroxylation of 17 beta-estradiol and 2-aminofluorene.