Oxygen-independent Manner Research Articles

Photoferroptosis as a potent strategy for neuroblastoma treatment

Photodynamic therapy (PDT) and photothermal therapy (PTT) have been developed to treat tumors with potential of clinical applications due to their high spatiotemporal selectivity and non-invasiveness. Nevertheless, the hypoxia within the tumor microenvironment (TME) limits the efficacy of PDT. PTT has the risk of damaging surrounding normal tissues due to the high temperatures essential for killing tumor cells. Herein, we propose a new tumor treatment strategy based on photo-triggered ferroptosis of tumor cells, which is termed photoferroptosis therapy (PFT). The PFT agent (CuS&AIPH@PEG-PAE@PM) was synthesized by encapsulating a radical generator (2,2’-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, AIPH) and a photothermal agent (copper sulfide, CuS) into an amphiphilic polymer (poly(ethylene glycol)-poly(β-amino ester), PEG-PAE) via self-assembly and a following coating with platelet membrane (PM). Under near-infrared (NIR) irradiation, the PFT agent CuS&AIPH@PEG-PAE@PM generates abundant alkyl radicals (R●) to trigger tumor cell ferroptosis in a moderate temperature and oxygen-independent manner. Meanwhile, the PFT agent also reduces the GSH level and thus suppresses GPX4 expression to promote ferroptosis, which further consolidates the antitumor effect of PFT. The PFT is expected to establish a promising phototherapy strategy against tumors, which has the potential to overcome the limitations of PDT and PTT.

Tween 80 Micelles Loaded with Fe3O4 Nanoparticles and Artemisinin for Combined Oxygen-Independent Ferroptosis Therapy of Cancer.

Artemisinin has an endoperoxide bridge structure, which can be cleaved by ferrous ions to generate various carbonyl radicals in an oxygen-independent manner, highlighting its potential for treating hypoxic tumors. In our study, we fabricated Tween 80 micelles loaded with Fe3O4 nanoparticles and artemisinin for cancer therapy. The synthesized Fe3O4 nanoparticles and drug-loaded micelles have particle sizes of about 5 nm and 80 nm, respectively, both exhibiting excellent dispersibility and stability. After uptake by MCF-7 cells, drug-loaded micelles release Fe2+ and ART into the cytoplasm, effectively inducing the generation of reactive oxygen species (ROS) in hypoxic conditions, thereby enhancing toxicity against cancer cells. In vitro and in vivo studies have demonstrated that ART and Fe3O4 nanoparticles are encapsulated in Tween 80 to form micelles, which effectively prevent premature release during circulation in the body. Although free ART and Fe3O4 nanoparticles can inhibit tumor growth, TW80-Fe3O4-ART micelles demonstrate a more pronounced inhibitory effect, with a tumor suppression rate of up to 85%. A novel strategy based on artemisinin and ferroptosis is thus offered, holding a favorable prospect for hypoxic cancer therapy.

Utilizing Macrophages Missile for Sulfate-Based Nanomedicine Delivery in Lung Cancer Therapy.

Nanomaterial-based drug delivery systems are susceptible to premature drug leakage and systemic toxicity due to lack of specific targeting, and live-cell drug delivery is also prone to be restricted by drug carrier-cell interactions. Here, a method is established to adsorb drug-loaded nanomaterials externally to the live cells, which reduces cytotoxicity caused by drug uptake and improves the bioactivity of the carrier cells and drug release at the lesion site. It was found that polyphenols act like "double-sided tape" to bridge metal-organic framework (MOF) nanoparticles with live macrophages (Mφ), attaching MOFs to the Mφ surface and minimizing intracellular uptake, with no negative effect on cell proliferation. On this basis, a "macrophage missile" with peroxymonosulfate (PMS)-loaded MOF nanoparticles on the cell surface was constructed. As a "propellant", the Mφ, in which bioactivity is preserved, can selectively identify and target tumor cells, precisely bringing nanomedicines to the lesion. MOF nanoparticles are used to load and catalyze PMS, which acts as an exogenous source of reactive oxygen species, showing higher efficacy and lower toxicity in an oxygen-independent manner. The primary study results demonstrate that this innovative combination of biology and nanomaterials remarkably enhances tumor targeting and therapeutic efficacy while reducing systemic side effects. This approach is expected to provide a more effective and safer treatment for lung cancer and holds promise for broader applications in other cancer therapies.

Near-infrared-activated anticancer platinum(IV) complexes directly photooxidize biomolecules in an oxygen-independent manner.

Conventional light-driven cancer therapeutics require oxygen and visible light to indirectly damage biomolecules, limiting their efficacy in deep, hypoxic tumours. Here we report the use of near-infrared-activated small-molecule Pt(IV) photooxidants to directly oxidize intracellular biomolecules in an oxygen-independent manner, achieving controllable and effective elimination of cancer stem cells. These Pt(IV) complexes accumulate in the endoplasmic reticulum and show low toxicity in the dark. Upon irradiation, the resultant metal-enhanced photooxidation effect causes them to robustly photooxidize survival-related biomolecules, induce intense oxidative stress, disrupt intracellular pH (pHi) homeostasis and initiate nonclassical necrosis. In vivo experiments confirm that the lead photooxidant can effectively inhibit tumour growth, suppress metastasis and activate the immune system. Our study validates the concept of metal-enhanced photooxidation and the subsequent chemotherapeutic applications, supporting the development of such localized photooxidants to directly damage intracellular biomolecules and decrease pHi as a strategy for effective metal-based drugs.

Macrophages exosomes-coated Fe3O4 nanoparticles for oxygen independent generation of free radicals and hypoxic cancer therapy

Free radicals, such as H 2 O 2 , O 2 ˉ, •OH, have been extensively applied for cancer therapy. However, their generation is significantly limited due to the hypoxic environment of the tumor. Here, macrophages exosomes coated Fe 3 O 4 nanoparticles loaded with 2,2'-Azobisisoheptonitrile (ABVN) (Fe 3 O 4 -ABVN@Exo NPs) were fabricated to realize photothermally controlled generation of radicals for cancer therapy in an oxygen-independent manner. Upon the irradiation of near infrared light, Fe 3 O 4 generated heat and triggered the release and decomposition of ABVN to produce alkyl radicals, which could damage the DNA, protein, and cause the death of the cells. While the macrophages exosomes could improve the targeting effect and prevent the premature release of ABVN. The nanoparticles indicated a favorable antitumor effect in hypoxic environment, holding a great potential for cancer therapy. A schematic diagram of the fabrication of Fe 3 O 4 -ABVN@Exo NPs and generation of free radicals upon irradiation by a NIR laser. • An initiator, 2,2'-Azobisisoheptonitrile (ABVN), was used as radical generator to be loaded onto Fe 3 O 4 NPs to form Fe 3 O 4 -ABVN NPs for hypoxic cancer therapy in an oxygen-independent manner. • The Fe 3 O 4 -ABVN NPs were encapsulated by exosomes (Fe 3 O 4 -ABVN@Exo NPs) to prevent the premature release and improve the targeting effect. • The free radicals and photothermal effect exhibited synergistic antitumor effect.

DDDR-17. CHEMOTHERAPEUTIC STRESS INDUCES NONCANONICAL HIF1Α SIGNALING UNDER NORMOXIA IN GLIOBLASTOMA DRIVING THERAPEUTIC RESISTANCE

Abstract Glioblastoma (GBM) is an aggressive brain tumor with no cure. Standard treatment includes adjuvant, concomitant temozolomide (TMZ) and radiation, but most patients do not survive more than 15 months. Tumor adaptations to temozolomide (TMZ) contribute to glioblastoma (GBM) chemoresistance and recurrence, and hypoxia inducible factor subunit alpha (HIF1α) and downstream effectors are implicated in attenuated therapeutic responses. We have previously shown that TMZ-dependent hijacking of HIF1α signaling under normoxia is independent from physiological hypoxia in the tumor core. Our results further showed that TMZ promotes HIF1α accumulation enhancing stem-like characteristics of GBM cells driving therapy-resistant populations involved in recurrence. However, noncanonical HIF1α signaling mechanisms are not well understood preventing the success of HIF1α inhibitors in GBM thus far. Here, we investigate how TMZ induces HIF1α in an oxygen-independent manner by characterizing the HIF1a binding profile post-therapy. We cultured patient-derived xenograft (PDX) models of GBM in vitro and found HIF1α expression and hypoxia response element (HRE) activity peak 96h post-therapy using western blot and luciferase assays. Our lab also performed a whole-genome CRISPR-Cas9 knockout screen in GBM cells over 28 days to determine genetic drivers of GBM aggression. HIF1α was found to be particularly enriched in this screen, further supporting the role of chronic hypoxia signaling for GBM proliferation (p-value < 0.05). We show TMZ therapy prevents proteasome-dependent degradation through reduced binding of PHD2 and VHL allowing nuclear translocation of HIF1a through western blot and immunocytochemistry. Thus, we performed immunoprecipitation of HIF1α 96h post-therapy for proteomic characterization to establish how TMZ uniquely regulates HIF1α stabilization in the absence of oxygen tension, and these results will be presented. We will subsequently characterize the regulatory role of TMZ-dependent post-translational modifications to HIF1α. Elucidating the molecular mechanisms of therapy-mediated HIF1α regulation can produce better therapeutic targets and increase TMZ efficacy.

INT6/eIF3e represses E-cadherin expression through HIF2α in lung carcinoma A549 cells.

Hypoxia-inducible factor 2 α (HIF2α), a transcription factor playing a vital role in hypoxia, promotes cancer metastasis. We had previously reported that the cancer-related gene integration site 6/eukaryotic translation initiation factor 3 subunit e (INT6/eIF3e) negatively regulates the protein stability of HIF2α in an oxygen-independent manner. Presently, the downstream targets for INT6/eIF3e-regulated HIF2α are unknown. Given the roles of HIF2α and INT6/eIF3e in epithelial-mesenchymal transition (EMT) that promotes cancer metastasis, we hypothesized that INT6/eIF3e-regulated HIF2α controls EMT. This study shows that INT6/eIF3e knockdown in lung carcinoma A549 cells led to increased expression of HIF2α protein and an EMT-like phenotypic change. The increased HIF2α subsequently repressed the E-cadherin gene. Mechanistically, HIF2α interacts with the twist family bHLH transcription factor 1 (TWIST1) known to regulate EMT process, and binds to the proximal promoter region of E-cadherin, repressing it. Collectively, our work demonstrates that HIF2α, regulated by INT6/eIF3e, represses the E-cadherin gene through TWIST1 to enhance EMT, suggesting a role of the INT6/eIF3e-HIF2α axis in cancer metastasis.

PD01-01 VHL-MEDIATED UBIQUITINATION OF THE KINASE MPS1 REGULATES THE MITOTIC CHECKPOINT IN CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 May 2022PD01-01 VHL-MEDIATED UBIQUITINATION OF THE KINASE MPS1 REGULATES THE MITOTIC CHECKPOINT IN CLEAR CELL RENAL CELL CARCINOMA Mark Woodford, Sarah Backe, Rebecca Sager, Oleg Shapiro, Imad Nsouli, Laura Wengert, Gennady Bratslavsky, Dimitra Bourboulia, and Mehdi Mollapour Mark WoodfordMark Woodford More articles by this author , Sarah BackeSarah Backe More articles by this author , Rebecca SagerRebecca Sager More articles by this author , Oleg ShapiroOleg Shapiro More articles by this author , Imad NsouliImad Nsouli More articles by this author , Laura WengertLaura Wengert More articles by this author , Gennady BratslavskyGennady Bratslavsky More articles by this author , Dimitra BourbouliaDimitra Bourboulia More articles by this author , and Mehdi MollapourMehdi Mollapour More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002516.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Abnormal chromosome segregation during mitosis causes aneuploidy, a hallmark of cancers associated with high risk for tumorigenesis, which is normally regulated by the mitotic checkpoint. Mps1 kinase activity is essential for spindle checkpoint signaling. Mps1 is over-expressed in clear cell renal cell carcinoma (ccRCC) and requires the molecular chaperone Hsp90 for its activity. Mps1 phosphorylates Hsp90, regulating chaperone function of numerous oncogenic client proteins, including Mps1, and conferring tumor selectivity of Hsp90 inhibitors in ccRCC. The most frequent alteration leading to ccRCC is loss of von Hippel Lindau (VHL), the recognition subunit of an E3-ubiquitin ligase complex that targets proteins for degradation. As Mps1 is over-expressed in ccRCC, the objective of the current study was to determine whether Mps1 gets targeted for degradation by VHL and whether this regulates the mitotic checkpoint. METHODS: VHL-mediated ubiquitination of Mps1 was examined in vitro and VHL-dependent degradation of Mps1 was examined in ccRCC cell lines by Western blot. Site-directed mutagenesis was utilized to determine Mps1 ubiquitination sites and the effect of increased Mps1 stability on mitotic checkpoint was determined using flow cytometry. RESULTS: Mps1 kinase is ubiquitinated by a VHL containing E3-ubiquitin ligase complex and Mps1 activity is essential for its ubiquitination. Re-expression of VHL in VHL-null ccRCC cell lines leads to proteasomal degradation of Mps1. VHL degrades Mps1 in an oxygen independent manner by ubiquitination of Mps1-K86, K827, and K848. Mps1 ubiquitination regulates cell cycle progression via exit from the mitotic checkpoint. CONCLUSIONS: Mps1 is targeted for degradation by the tumor suppressor VHL in a hypoxia-independent manner and Mps1 is over-expressed in VHL-null ccRCC. VHL-mediated ubiquitination of Mps1 regulates mitotic checkpoint progression. Mps1 stability additionally mediates Hsp90 post-translational modification and tumor selectivity of Hsp90 inhibitors. Source of Funding: This work was also supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number, R01GM139932 (D.B.), R35GM139584 (M.M.), and R01GM124256 (M.M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the Urology Care Foundation-American Urological Association (M.M.) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e30 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Mark Woodford More articles by this author Sarah Backe More articles by this author Rebecca Sager More articles by this author Oleg Shapiro More articles by this author Imad Nsouli More articles by this author Laura Wengert More articles by this author Gennady Bratslavsky More articles by this author Dimitra Bourboulia More articles by this author Mehdi Mollapour More articles by this author Expand All Advertisement PDF DownloadLoading ...

Oxygen-Independent Sulfate Radical for Stimuli-Responsive Tumor Nanotherapy.

Variant modalities are quested and merged into the tumor nanotherapy by leveraging the excitation from external or intratumoral incentives. However, the ubiquitous hypoxia and the insufficient content of hydrogen peroxide (H2O2) in tumor microenvironments inevitably hinder the effective production of reactive oxygen species (ROS). To radically extricate from the shackles, peroxymonosulfate (PMS: HSO5 −)‐loaded hollow mesoporous copper sulfide (CuS) nanoparticles (NPs) are prepared as the distinct ROS donors for sulfate radical (•SO4 −)‐mediated and stimuli‐responsive tumor nanotherapy in an oxygen‐independent manner. In this therapeutic modality, the second near‐infrared laser irradiation, together with the released copper ions as well as the heat produced by CuS after illumination, work together to activate PMS thus triply ensuring the copious production of •SO4 −. Different from conventional ROS, the emergence of •SO4 −, possessing a longer half‐life and more rapid reaction, is independent of the oxygen (O2) and H2O2 content within the tumor. In addition, this engineered nanosystem also exerts the function of photoacoustic imaging and skin restoration on the corresponding animal models. This study reveals the enormous potential of sulfate radical in oncotherapy and broadens pave for exploring the application of multifunctional and stimuli‐responsive nanosystems in biomedicine.

Oxygen-independent Free Radicals Induced by Photothermal Effect of Fe3O4 for Hypoxic Cancer Therapy

Cancer treatments based on reactive oxygen species (ROS) have emerged as effective clinical methods. However, the generation of ROS is significantly suppressed owing to the hypoxic tumor microenvironment. ROS generated in an oxygen-independent manner provides a novel strategy for cancer therapy. Here, we use an initiator as therapeutic drug to realize light controlled generation of free radicals. The initiator was loaded onto Fe3O4 nanoparticles. Upon the irradiation of near infrared light, the temperature of the nanoparticles increased, inducing the decomposition of the initiator to produce alkyl radicals. This therapeutic system showed remarkable damage to cells in hypoxic environment.

Clostridioides difficile strain-dependent and strain-independent adaptations to a microaerobic environment.

Clostridioides difficile (formerly Clostridium difficile ) colonizes the gastrointestinal tract following disruption of the microbiota and can initiate a spectrum of clinical manifestations ranging from asymptomatic to life-threatening colitis. Following antibiotic treatment, luminal oxygen concentrations increase, exposing gut microbes to potentially toxic reactive oxygen species. Though typically regarded as a strict anaerobe, C. difficile can grow at low oxygen concentrations. How this bacterium adapts to a microaerobic environment and whether those responses to oxygen are conserved amongst strains is not entirely understood. Here, two C. difficile strains (630 and CD196) were cultured in 1.5% oxygen and the transcriptional response to long-term oxygen exposure was evaluated via RNA-sequencing. During growth in a microaerobic environment, several genes predicted to protect against oxidative stress were upregulated, including those for rubrerythrins and rubredoxins. Transcription of genes involved in metal homeostasis was also positively correlated with increased oxygen levels and these genes were amongst the most differentially transcribed. To directly compare the transcriptional landscape between C. difficile strains, a ‘consensus-genome’ was generated. On the basis of the identified conserved genes, basal transcriptional differences as well as variations in the response to oxygen were evaluated. While several responses were similar between the strains, there were significant differences in the abundance of transcripts involved in amino acid and carbohydrate metabolism. Furthermore, intracellular metal concentrations significantly varied both in an oxygen-dependent and oxygen-independent manner. Overall, these results indicate that C. difficile adapts to grow in a low oxygen environment through transcriptional changes, though the specific strategy employed varies between strains.

Characterisation of the redox centers of ethylbenzene dehydrogenase

Ethylbenzene dehydrogenase (EbDH), the initial enzyme of anaerobic ethylbenzene degradation from the beta-proteobacterium Aromatoleumaromaticum, is a soluble periplasmic molybdenum enzyme consisting of three subunits. It contains a Mo-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD) cofactor and an 4Fe–4S cluster (FS0) in the α-subunit, three 4Fe–4S clusters (FS1 to FS3) and a 3Fe–4S cluster (FS4) in the β-subunit and a heme b cofactor in the γ-subunit. Ethylbenzene is hydroxylated by a water molecule in an oxygen-independent manner at the Mo-bis-MGD cofactor, which is reduced from the MoVI to the MoIV state in two subsequent one-electron steps. The electrons are then transferred via the Fe–S clusters to the heme b cofactor. In this report, we determine the midpoint redox potentials of the Mo-bis-MGD cofactor and FS1–FS4 by EPR spectroscopy, and that of the heme b cofactor by electrochemically induced redox difference spectroscopy. We obtained relatively high values of > 250 mV both for the MoVI–MoV redox couple and the heme b cofactor, whereas FS2 is only reduced at a very low redox potential, causing magnetic coupling with the neighboring FS1 and FS3. We compare the results with the data on related enzymes and interpret their significance for the function of EbDH.Graphical abstract

Emerging Designs of Aggregation-Induced Emission Agents for Enhanced Phototherapy Applications

Emerging Designs of Aggregation-Induced Emission Agents for Enhanced Phototherapy Applications

Intrinsic nucleus-targeted ultra-small metal\u2013organic framework for the type I sonodynamic treatment of orthotopic pancreatic carcinoma

BackgroundSonodynamic therapy (SDT) strategies exhibit a high tissue penetration depth and can achieve therapeutic efficacy by facilitating the intertumoral release of reactive oxygen species (ROS) with a short lifespan and limited diffusion capabilities. The majority of SDT systems developed to date are of the highly O2-dependent type II variety, limiting their therapeutic utility in pancreatic cancer and other hypoxic solid tumor types.ResultsHerein, a nucleus-targeted ultra-small Ti-tetrakis(4-carboxyphenyl)porphyrin (TCPP) metal–organic framework (MOF) platform was synthesized and shown to be an effective mediator of SDT. This MOF was capable of generating large quantities of ROS in an oxygen-independent manner in response to low-intensity ultrasound (US) irradiation (0.5 W cm−2), thereby facilitating both type I and type II SDT. This approach thus holds great promise for the treatment of highly hypoxic orthotopic pancreatic carcinoma solid tumors. This Ti-TCPP MOF was able to induce in vitro cellular apoptosis by directly destroying DNA and inducing S phase cell cycle arrest following US irradiation. The prolonged circulation, high intratumoral accumulation, and nucleus-targeting attributes of these MOF preparations significantly also served to significantly inhibit orthotopic pancreatic tumor growth and prolong the survival of tumor-bearing mice following Ti-TCPP + US treatment. Moreover, this Ti-TCPP MOF was almost completely cleared from mice within 7 days of treatment, and no apparent treatment-associated toxicity was observed.ConclusionThe nucleus-targeted ultra-small Ti-TCPP MOF developed herein represents an effective approach to the enhanced SDT treatment of tumors in response to low-intensity US irradiation.Graphic abstract

Electronic Band-Engineered Nanomaterials for Biosafety and Biomedical Application

ConspectusInteraction between nanomaterials (NMs) and biological systems can be beneficial for biological functions but also can present hazards to humans. Nanotoxicology and nanomedicine, as two subdisciplines of nanotechnology, share the same goal of making safer NMs for biomedical application. NMs with unique electronic properties have been widely used for biomedical applications, such as bacterial inactivation, wound healing, tumor therapy, and Alzheimer’s disease therapy. Meanwhile, the biosafety of NMs has become a hot topic, and development of effective “safe-by-design” strategies will be beneficial for the wide applications of NMs in the biomedical field. However, it is currently hard to establish a property–activity relationship between NMs and their biosafety and biomedical applications, especially for electronic band structure including conduction band energy (Ec), valence band energy (Ev), Fermi energy (Ef), and bandgap energy (Eg). Eg determines the suitable lights used to excite NMs, and Ec and Ev determine the redox abilities of photoinduced electrons and holes, while Ef dominates the charge transfer process within NMs. Therefore, through modulating the electronic band structure of NMs, not only can the biosafety of NMs be elevated, but also the photoelectronic performance can be improved, providing a profound understanding to the design of functional NMs for the biomedical application with excellent biocompatibility.In this account, we focus on our recent progress in electronic band structure-modulated NMs for biosafety and biomedical application. First, we investigate the toxicities of NMs with different Ec levels and establish safe-by-design strategies to make safer NMs through modulating their electronic properties, such as tuning Ec values of NMs out of the biological redox potential range and tuning the Ef edge far away from the Ev edge. Second, we propose that deep level defect, resonance energy transfer, and narrow band gap intensely correlate with the photothermal performance of NMs and rationally designed heterostructures can significantly improve the photothermal conversion efficacies of these NMs. Third, we introduce a series of NMs with unique heterostructure to promote photoinduced electron–hole spatial separation and improve photodynamic performance for antibacterial and anticancer applications. Among these heterostructures, the thermally retractable heterostructure can create a favorable microenvironment for photodynamic therapy; Z-scheme heterostructure can simultaneously produce oxygen and reactive oxygen species for photodynamic therapy against hypoxic tumor; plasmon–pyroelectric heterostructure can thermally generate reactive oxygen species in an oxygen-independent manner for hypoxic tumor therapy. Furthermore, we describe the photooxidation and antioxidant abilities of NMs for treating Alzheimer’s disease through inhibiting amyloid-β self-assembly and scarifying reactive oxygen species. Finally, we propose the challenges and perspectives of electronic band structure-modulated biomedical application of NMs. We expect that this field will attract increasing research interest and create new opportunities for biosafety and biomedical studies of NMs.

Hypoxia-Adapted Sono-chemodynamic Treatment of Orthotopic Pancreatic Carcinoma Using Copper Metal-Organic Frameworks Loaded with an Ultrasound-Induced Free Radical Initiator.

The efficacy of sonodynamic therapy (SDT) is largely dependent upon oxygen availability to generate deleterious reactive oxygen species, and as such, hypoxic microenvironments greatly constrain the efficacy of SDT. Development of free radical generators that are not dependent on oxygen and related combination treatment strategies thus have the potential to enhance the antitumor potential of SDT. Combined treatment strategies are expected to improve the efficacy of sonodynamic antitumor therapy. As metal-organic framework (MOF) platforms are highly amenable to integration with other therapeutic approaches, we herein report the development of tumor microenvironment (TME)-responsive nanoparticles constructed by embedding the azo initiator 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (AIPH) into hypoxia-triggered copper metal-organic framework (Cu-MOF) nanovectors to achieve synergistic sono-chemodynamic therapy in an orthotopic murine pancreatic carcinoma model system. When exposed to hypoxic conditions within the TME, this Cu-MOF structure underwent degradation, leading to the release of Cu2+ and AIPH. Cu2+ was then able to deplete local glutathione stores, resulting in the reduction of Cu2+ to Cu+, which then reacts with endogenous H2O2 in a Fenton-like reaction to yield cytotoxic hydroxyl radicals (•OH) for chemodynamic therapy. When exposed to ultrasound irradiation, AIPH further degraded in an oxygen-independent manner to yield nitrogen bubbles and alkyl radicals, the former of which enhanced the ability of these nanoparticles to penetrate deeply into the tumor. The resultant radicals induced substantial DNA damage and apoptotic cell death within target tumors under different levels of oxygen availability. As such, this hypoxic TME-responsive synergistic sono-chemodynamic approach offers an ideal means of achieving oxygen-independent free radical generation and enhanced treatment efficacy.

Effects of LET on oxygen-dependent and-independent generation of hydrogen peroxide in water irradiated by carbon-ion beams

Linear energy transfer (LET) dependence of yields of O2-dependent and O2-independent hydrogen peroxide (H2O2) in water irradiated by ionizing radiation was investigated. The radiation-induced hydroxyl radical (•OH) generation in an aqueous solution was reported to occur in two different localization densities, the milli-molar (relatively sparse) and/or molar (markedly-dense) levels. In the milli-molar-level •OH generation atmosphere, •OH generated at a molecular distance of ∼7 nm are likely unable to interact. However, in the molar-level •OH generation atmosphere, several •OH were generated with a molecular distance of 1 nm or less, and two •OH can react to directly make H2O2. An aliquot of ultra-pure water was irradiated by 290-MeV/nucleon carbon-ion beams at the Heavy-Ion Medical Accelerator in Chiba (HIMAC, NIRS/QST, Chiba, Japan). Irradiation experiments were performed under aerobic or hypoxic (<0.5% oxygen) conditions, and several LET conditions (13, 20, 40, 60, 80, or >100 keV/μm). H2O2 generation in irradiated samples was estimated by three methods. The amount of H2O2 generated per dose was estimated and compared. O2-independent H2O2 generation, i.e. H2O2 generation under hypoxic conditions, increased with increasing LET. On the other hand, the O2-dependent H2O2 generation, i.e. subtraction of H2O2 generation under hypoxic conditions from H2O2 generation under aerobic conditions, decreased with increasing LET. This suggests that the markedly-dense •OH generation is positively correlated with LET. High-LET beams generate H2O2 in an oxygen-independent manner.

Tablet-like TiO2/C nanocomposites for repeated type I sonodynamic therapy of pancreatic cancer

Sonodynamic therapy (SDT) represents a viable approach to overcoming the limited ability of photodynamic therapy to penetrate biological barriers. However, pancreatic tumors contain a hypoxic microenvironment that limits the efficacy of oxygen-dependent type II SDT, complicating efforts to develop reliable, stable, and hypoxia-tolerant sonosensitizer. Herein, a tablet-like TiO2/C nanocomposite with a metal-organic-framework (MOF)-derived carbon structure was designed and found to be hypoxia-tolerant and stable in response to repeated ultrasound irradiation, enabling the TiO2/C-mediated generation of large quantities of reactive oxygen species (ROS) and thereby achieving efficacious type I SDT. Importantly, this nanocomposite continued to generate ROS in response to repeated ultrasound irradiation, and was able to induce tumor cell apoptosis via SDT-induced DNA damage in vitro and in vivo. This TiO2/C nanocomposite also exhibited good biocompatibility and did not induce any apparent toxicity in vitro and in vivo. Together, these data highlight TiO2/C as a valuable nanocomposite capable of facilitating repeated type I SDT, making it a promising tool for the treatment of hypoxic solid pancreatic tumors. Statement of significanceIn this research, a tablet-like TiO2/C nanocomposite with a metal-organic-framework (MOF)-derived carbon structure was designed, which exhibited great stability upon repeated ultrasound irradiation, hypoxic-tolerant ability and good biocompatibility. After ultrasound irradiation, TiO2/C could efficiently generate reactive oxygen species in an oxygen-independent manner, which overcame the limitation of pure TiO2 nanoparticles. Therefore, it was applied to repeated type I sonodynamic therapy of hypoxic pancreatic tumor.

Comparative transcriptomics reveals PrrAB-mediated control of metabolic, respiration, energy-generating, and dormancy pathways in Mycobacterium smegmatis

BackgroundMycobacterium smegmatis is a saprophytic bacterium frequently used as a genetic surrogate to study pathogenic Mycobacterium tuberculosis. The PrrAB two-component genetic regulatory system is essential in M. tuberculosis and represents an attractive therapeutic target. In this study, transcriptomic analysis (RNA-seq) of an M. smegmatis ΔprrAB mutant was used to define the PrrAB regulon and provide insights into the essential nature of PrrAB in M. tuberculosis.ResultsRNA-seq differential expression analysis of M. smegmatis wild-type (WT), ΔprrAB mutant, and complementation strains revealed that during in vitro exponential growth, PrrAB regulates 167 genes (q < 0.05), 57% of which are induced in the WT background. Gene ontology and cluster of orthologous groups analyses showed that PrrAB regulates genes participating in ion homeostasis, redox balance, metabolism, and energy production. PrrAB induced transcription of dosR (devR), a response regulator gene that promotes latent infection in M. tuberculosis and 21 of the 25 M. smegmatis DosRS regulon homologues. Compared to the WT and complementation strains, the ΔprrAB mutant exhibited an exaggerated delayed growth phenotype upon exposure to potassium cyanide and respiratory inhibition. Gene expression profiling correlated with these growth deficiency results, revealing that PrrAB induces transcription of the high-affinity cytochrome bd oxidase genes under both aerobic and hypoxic conditions. ATP synthesis was ~ 64% lower in the ΔprrAB mutant relative to the WT strain, further demonstrating that PrrAB regulates energy production.ConclusionsThe M. smegmatis PrrAB two-component system regulates respiratory and oxidative phosphorylation pathways, potentially to provide tolerance against the dynamic environmental conditions experienced in its natural ecological niche. PrrAB positively regulates ATP levels during exponential growth, presumably through transcriptional activation of both terminal respiratory branches (cytochrome c bc1-aa3 and cytochrome bd oxidases), despite transcriptional repression of ATP synthase genes. Additionally, PrrAB positively regulates expression of the dormancy-associated dosR response regulator genes in an oxygen-independent manner, which may serve to fine-tune sensory perception of environmental stimuli associated with metabolic repression.

Elevated FGF23 and disordered renal mineral handling with reduced bone mineralization in chronically erythropoietin over-expressing transgenic mice

Fibroblast Growth Factor 23 (FGF23) is a phosphaturic factor causing increased renal phosphate excretion as well as suppression of 1,25 (OH)2-vitamin D3. Highly elevated FGF23 can promote development of rickets and osteomalacia. We and others previously reported that acute application of erythropoietin (EPO) stimulates FGF23 production. Considering that EPO is clinically used as chronic treatment against anemia, we used here the Tg6 mouse model that constitutively overexpresses human EPO in an oxygen-independent manner, to examine the consequences of long-term EPO therapy on mineral and bone metabolism. Six to eight weeks old female Tg6 mice showed elevated intact and C-terminal fragment of FGF23 but normal plasma levels of PTH, calcitriol, calcium and phosphate. Renal function showed moderate alterations with higher urea and creatinine clearance and mild albuminuria. Renal phosphate excretion was normal whereas mild hypercalciuria was found. Renal expression of the key proteins TRPV5 and calbindin D28k involved in active calcium reabsorption was reduced in Tg6 mice. Plasma levels of the bone turnover marker osteocalcin were comparable between groups. However, urinary excretion of deoxypyridinoline (DPD) was lower in Tg6 mice. MicroCT analysis showed reduced total, cortical, and trabecular bone mineral density in femora from Tg6 mice. Our data reveal that chronic elevation of EPO is associated with high FGF23 levels and disturbed mineral homeostasis resulting in reduced bone mineral density. These observations imply the need to study the impact of therapeutically applied EPO on bone mineralization in patients, especially those suffering from chronic kidney disease.