Targeting CAR and Nrf2 improves cyclophosphamide bioactivation while reducing doxorubicin-induced cardiotoxicity in triple-negative breast cancer treatment.

Cyclophosphamide (CPA) and doxorubicin (DOX) are key components of chemotherapy for triple-negative breast cancer (TNBC), although suboptimal outcomes are commonly associated with drug resistance and/or intolerable side effects. Through an approach combining high-throughput screening and chemical modification, we developed CN06 as a dual activator of the constitutive androstane receptor (CAR) and nuclear factor erythroid 2-related factor 2 (Nrf2). CN06 enhances CAR-induced bioactivation of CPA (a prodrug) by provoking hepatic expression of CYP2B6, while repressing DOX-induced cytotoxicity in cardiomyocytes in vitro via stimulating Nrf2-antioxidant signaling. Utilizing a multicellular coculture model incorporating human primary hepatocytes, TNBC cells, and cardiomyocytes, we show that CN06 increased CPA/DOX-mediated TNBC cell death via CAR-dependent CYP2B6 induction and subsequent conversion of CPA to its active metabolite 4-hydroxy-CPA, while protecting against DOX-induced cardiotoxicity by selectively activating Nrf2-antioxidant signaling in cardiomyocytes but not in TNBC cells. Furthermore, CN06 preserves the viability and function of human iPSC-derived cardiomyocytes by modulating antioxidant defenses, decreasing apoptosis, and enhancing the kinetics of contraction and relaxation. Collectively, our findings identify CAR and Nrf2 as potentially novel combined therapeutic targets whereby CN06 holds the potential to improve the efficacy/toxicity ratio of CPA/DOX-containing chemotherapy.

Screening for acute IKr block is insufficient to detect torsades de pointes liability: role of late sodium current.

BACKGROUND: New drugs are routinely screened for IKr blocking properties thought to predict QT prolonging and arrhythmogenic liability. However, recent data suggest that chronic (hours) drug exposure to phosphoinositide 3-kinase inhibitors used in cancer can prolong QT by inhibiting potassium currents and increasing late sodium current (INa-L) in cardiomyocytes. We tested the extent to which IKr blockers with known QT liability generate arrhythmias through this pathway. METHODS AND RESULTS: Acute exposure to dofetilide, an IKr blocker without other recognized electropharmacologic actions, produced no change in ion currents or action potentials in adult mouse cardiomyocytes, which lack IKr. By contrast, 2 to 48 hours of exposure to the drug generated arrhythmogenic afterdepolarizations and ≥15-fold increases in INa-L. Including phosphatidylinositol 3,4,5-trisphosphate, a downstream effector for the phosphoinositide 3-kinase pathway, in the pipette inhibited these effects. INa-L was also increased, and inhibitable by phosphatidylinositol 3,4,5-trisphosphate, with hours of dofetilide exposure in human-induced pluripotent stem cell-derived cardiomyocytes and in Chinese hamster ovary cells transfected with SCN5A, encoding sodium current. Cardiomyocytes from dofetilide-treated mice similarly demonstrated increased INa-L and afterdepolarizations. Other agents with variable IKr-blocking potencies and arrhythmia liability produced a range of effects on INa-L, from marked increases (E-4031, d-sotalol, thioridazine, and erythromycin) to little or no effect (haloperidol, moxifloxacin, and verapamil). CONCLUSIONS: Some but not all drugs designated as arrhythmogenic IKr blockers can generate arrhythmias by augmenting INa-L through the phosphoinositide 3-kinase pathway. These data identify a potential mechanism for individual susceptibility to proarrhythmia and highlight the need for a new paradigm to screen drugs for QT prolonging and arrhythmogenic liability.

Polymeric stent materials dysregulate macrophage and endothelial cell functions: implications for coronary artery stent.

BACKGROUND: Biodegradable polymers have been applied as bulk or coating materials for coronary artery stents. The degradation of polymers, however, could induce endothelial dysfunction and aggravate neointimal formation. Here we use polymeric microparticles to simulate and demonstrate the effects of degraded stent materials on phagocytic activity, cell death and dysfunction of macrophages and endothelial cells. METHODS: Microparticles made of low molecular weight polyesters were incubated with human macrophages and coronary artery endothelial cells (ECs). Microparticle-induced phagocytosis, cytotoxicity, apoptosis, cytokine release and surface marker expression were determined by immunostaining or ELISA. Elastase expression was analyzed by ELISA and the elastase-mediated polymer degradation was assessed by mass spectrometry. RESULTS: We demonstrated that poly(D,L-lactic acid) (PLLA) and polycaprolactone (PCL) microparticles induced cytotoxicity in macrophages and ECs, partially through cell apoptosis. The particle treatment alleviated EC phagocytosis, as opposed to macrophages, but enhanced the expression of vascular cell adhesion molecule (VCAM)-1 along with decreased nitric oxide production, indicating that ECs were activated and lost their capacity to maintain homeostasis. The activation of both cell types induced the release of elastase or elastase-like protease, which further accelerated polymer degradation. CONCLUSIONS: This study revealed that low molecule weight PLLA and PCL microparticles increased cytotoxicity and dysregulated endothelial cell function, which in turn enhanced elastase release and polymer degradation. These indicate that polymer or polymer-coated stents impose a risk of endothelial dysfunction after deployment which can potentially lead to delayed endothelialization, neointimal hyperplasia and late thrombosis.

Effects of increasing carbon nanofiber density in polyurethane composites for inhibiting bladder cancer cell functions.

Polyurethane (PU) is a versatile elastomer that is commonly used in biomedical applications. In turn, materials derived from nanotechnology, specifically carbon nanofibers (CNFs), have received increasing attention for their potential use in biomedical applications. Recent studies have shown that the dispersion of CNFs in PU significantly enhances composite nanoscale surface roughness, tensile properties, and thermal stability. Although there have been studies concerning normal primary cell functions on such nanocomposites, there have been few studies detailing cancer cell responses. Since many patients who require bladder transplants have suffered from bladder cancer, the ideal bladder prosthetic material should not only promote normal primary human urothelial cell (HUC) function, but also inhibit the return of bladder cancerous cell activity. This study examined the correlation between transitional (UMUC) and squamous (or SCaBER) urothelial carcinoma cells and HUC on PU:CNF nanocomposites of varying PU and CNF weight ratios (from pure PU to 4:1 [PU:CNF volume ratios], 2:1, 1:1, 1:2, and 1:4 composites to pure CNF). Composites were characterized for mechanical properties, wettability, surface roughness, and chemical composition by atomic force microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, and goniometry. The adhesion and proliferation of UMUC and SCaBER cancer cells were assessed by MTS assays. Cellular responses were further quantified by measuring the amounts of nuclear mitotic protein 22 (NMP-22), vascular endothelial growth factor (VEGF), and tumor necrosis factor alpha. Results demonstrated that both UMUC and SCaBER cell proliferation rates decreased over time on substrates with increased CNF in PU. In addition, with the exception of VEGF from UMUC (which was the same across all materials), composites containing the most CNF activated cancer cells (UMUC and SCaBER) the least, as shown by their decreased expression of NMP-22, tumor necrosis factor alpha, and VEGF. Moreover, the adhesion of HUC increased on composites containing more CNF than PU. Overall levels of NMP-22 were significantly lower in HUC than in cancerous UMUC and SCaBER cells on PU:CNF composites. Thus, this study provided a novel nanocomposite consisting of CNF and PU that should be further studied for inhibiting the return of cancerous bladder tissue and for promoting normal non-cancerous bladder tissue formation.

Decreased lung carcinoma cell density on select polymer nanometer surface features for lung replacement therapies.

Poly(lactic-co-glycolic) acid (PLGA) has been widely used as a biomaterial in regenerative medicine because of its biocompatibility and biodegradability properties. Previous studies have shown that cells (such as bladder smooth muscle cells, chondrocytes, and osteoblasts) respond differently to nanostructured PLGA surfaces compared with nanosmooth surfaces. The purpose of the present in vitro research was to prepare PLGA films with various nanometer surface features and determine whether lung cancer epithelial cells respond differently to such topographies. To create nanosurface features on PLGA, different sized (190 nm, 300 nm, 400 nm, and 530 nm diameter) polystyrene beads were used to cast polydimethylsiloxane (PDMS) molds which were used as templates to create nanofeatured PLGA films. Atomic force microscopy (AFM) images and root mean square roughness (RMS) values indicated that the intended spherical surface nanotopographies on PLGA with RMS values of 2.23, 5.03, 5.42, and 36.90 nm were formed by employing 190, 300, 400, and 530 nm beads. A solution evaporation method was also utilized to modify PLGA surface features by using 8 wt% (to obtain an AFM RMS value of 0.62 nm) and 4 wt% (to obtain an AFM RMS value of 2.23 nm) PLGA in chloroform solutions. Most importantly, lung cancer epithelial cells adhered less on the PLGA surfaces with RMS values of 0.62, 2.23, and 5.42 nm after four hours of culture compared with any other PLGA surface created here. After three days, PLGA surfaces with an RMS value of 0.62 nm had much lower cell density than any other sample. In this manner, PLGA with specific nanometer surface features may inhibit lung cancer cell density which may provide an important biomaterial for the treatment of lung cancer (from drug delivery to regenerative medicine).

The role of polymer nanosurface roughness and submicron pores in improving bladder urothelial cell density and inhibiting calcium oxalate stone formation.

Synthetic polymers have been proposed for replacing resected cancerous bladder tissue. However, conventional (or nanosmooth) polymers used in such applications (such as poly(ether) urethane (PU) and poly-lactic-co-glycolic acid (PLGA)) often fail clinically due to poor bladder tissue regeneration, low cytocompatibility properties, and excessive calcium stone formation. For the successful reconstruction of bladder tissue, polymer surfaces should be modified to combat these common problems. Along these lines, implementing nanoscale surface features that mimic the natural roughness of bladder tissue on polymer surfaces can promote appropriate cell growth, accelerate bladder tissue regeneration and inhibit bladder calcium stone formation. To test this hypothesis, in this study, the cytocompatibility properties of both a non-biodegradable polymer (PU) and a biodegradable polymer (PLGA) were investigated after etching in chemicals (HNO(3) and NaOH, respectively) to create nanoscale surface features. After chemical etching, PU possessed submicron sized pores and numerous nanometer surface features while PLGA possessed few pores and large amounts of nanometer surface roughness. Results from this study strongly supported the assertion that nanometer scale surface roughness produced on PU and PLGA promoted the density of urothelial cells (cells that line the interior of the bladder), with the greatest urothelial cell densities observed on nanorough PLGA. In addition, compared to respective conventional polymers, the results provided evidence that nanorough PU and PLGA inhibited calcium oxalate stone formation; submicron pored nanorough PU inhibited calcium oxalate stone formation the most. Thus, results from the present study suggest the importance of nanometer topographical cues for designing better materials for bladder tissue engineering applications.