Nuclear Localization Signal Enhances the Targeting and Therapeutic Efficacy of a Porphyrin-Based Molecular Cargo: A Systemic In Vitro and Ex Vivo Evaluation.

The objective of the present work was to evaluate the potential of a nuclear localization signal (NLS) toward facilitating intracellular delivery and enhancement in the therapeutic efficacy of the molecular cargo. Toward this, an in-house synthesized porphyrin derivative, namely, 5-carboxymethyelene-oxyphenyl-10,15,20-tris(4-methoxyphenyl) porphyrin (UTriMA), was utilized for conjugation with the NLS sequence [PKKKRKV]. The three compounds synthesized during the course of the present work, namely DOTA-Lys-NLS, DOTA-UTriMA-Lys-NLS, and DOTA-Lys-UTriMA, were evaluated for cellular toxicity in cancer cell lines (HT1080), wherein all exhibited minimal dark toxicity. However, during photocytotoxicity studies with DOTA-Lys-UTriMA and DOTA-UTriMA-Lys-NLS conjugates in the same cell line, the latter exhibited significantly higher light-dependent toxicity compared to the former. Furthermore, the photocytotoxicity for DOTA-UTriMA-Lys-NLS in a healthy cell line (WI26VA4) was found to be significantly lower than that observed in the cancer cells. Fluorescence cell imaging studies carried out in HT1080 cancer cells revealed intracellular accumulation for the NLS-conjugated porphyrin (DOTA-UTriMA-Lys-NLS), whereas unconjugated porphyrin (DOTA-Lys-UTriMA) failed to do so. To evaluate the radiotherapeutic effects of the synthesized conjugates, all three compounds were radiolabeled with 177Lu, a well-known therapeutic radionuclide with high radiochemical purity (>95%). During in vitro studies, the [177Lu]Lu-DOTA-UTriMA-Lys-NLS complex exhibited the highest cell binding as well as internalization among the three radiolabeled complexes. Biological distribution studies for the radiolabeled compounds were performed in a fibrosarcoma-bearing small animal model, wherein significantly higher accumulation and prolonged retention of [177Lu]Lu-DOTA-UTriMA-Lys-NLS (9.32 ± 1.27% IA/g at 24 h p.i.) in the tumorous lesion compared to [177Lu]Lu-UTriMA-Lys-DOTA (2.3 ± 0.13% IA/g at 24 h p.i.) and [177Lu]Lu-DOTA-Lys-NLS complexes (0.26 ± 0.17% IA/g at 24 h p.i.) were observed. The results of the biodistribution studies were further corroborated by recording serial SPECT-CT images of fibrosarcoma-bearing Swiss mice administered with [177Lu]Lu-DOTA-UTriMA-Lys-NLS at different time points. Tumor regression studies performed with [177Lu]Lu-DOTA-UTriMA-Lys-NLS in the same animal model with two different doses [250 µCi (9.25 MBq) and 500 µCi (18.5 MBq)] resulted in a significant reduction in tumor mass in the treated group of animals. The above results revealed a definite enhancement in the targeting ability of molecular cargo upon conjugation with NLS and hence indicated that this strategy may be helpful for the preparation of drug-NLS conjugates as multimodal agents.

14-3-3 proteins protect AMPK-phosphorylated ten-eleven translocation-2 (TET2) from PP2A-mediated dephosphorylation.

Ten-eleven translocation-2 (TET2) is a member of the methylcytosine dioxygenase family of enzymes and has been implicated in cancer and aging because of its role as a global epigenetic modifier. TET2 has a large N-terminal domain and a catalytic C-terminal region. Previous reports have demonstrated that the TET2 catalytic domain remains active independently of the N-terminal domain. As such, the function of the N terminus of this large protein remains poorly characterized. Here, using yeast two-hybrid screening, co-immunoprecipitation, and several biochemical assays, we found that several isoforms of the 14-3-3 family of proteins bind TET2. 14-3-3 proteins bound TET2 when it was phosphorylated at Ser-99. In particular, we observed that AMP-activated protein kinase-mediated phosphorylation at Ser-99 promotes TET2 stability and increases global DNA 5-hydroxymethylcytosine levels. The interaction of 14-3-3 proteins with TET2 protected the Ser-99 phosphorylation, and disruption of this interaction both reduced TET2 phosphorylation and decreased TET2 stability. Furthermore, we noted that protein phosphatase 2A can interact with TET2 and dephosphorylate Ser-99. Collectively, these results provide detailed insights into the role of the TET2 N-terminal domain in TET2 regulation. Moreover, they reveal the dynamic nature of TET2 protein regulation that could have therapeutic implications for disease states resulting from reduced TET2 levels or activity.

Disruption of nuclear factor (erythroid-derived-2)-like 2 antioxidant signaling: a mechanism for impaired activation of stem cells and delayed regeneration of skeletal muscle.

Recently we have reported that age-dependent decline in antioxidant levels accelerated apoptosis and skeletal muscle degeneration. Here, we demonstrate genetic ablation of the master cytoprotective transcription factor, nuclear factor (erythroid-derived-2)-like 2 (Nrf2), aggravates cardiotoxin (CTX)-induced tibialis anterior (TA) muscle damage. Disruption of Nrf2 signaling sustained the CTX-induced burden of reactive oxygen species together with compromised expression of antioxidant genes and proteins. Transcript/protein expression of phenotypic markers of muscle differentiation, namely paired box 7 (satellite cell) and early myogenic differentiation and terminal differentiation (myogenin and myosin heavy chain 2) were increased on d 2 and 4 postinjury but later returned to baseline levels on d 8 and 15 in wild-type (WT) mice. In contrast, these responses were persistently augmented in Nrf2-null mice suggesting that regulation of the regeneration-related signaling mechanisms require Nrf2 for normal functioning. Furthermore, Nrf2-null mice displayed slower regeneration marked by dysregulation of embryonic myosin heavy chain temporal expression. Histologic observations illustrated that Nrf2-null mice displayed smaller, immature TA muscle fibers compared with WT counterparts on d 15 after CTX injury. Improvement in TA muscle morphology and gain in muscle mass evident in the WT mice was not noticeable in the Nrf2-null animals. Taken together these data show that the satellite cell activation, proliferation, and differentiation requires a functional Nrf2 system for effective healing following injury.-Shelar, S. B., Narasimhan, M., Shanmugam, G., Litovsky, S. H., Gounder, S. S., Karan, G., Arulvasu, C., Kensler, T. W., Hoidal, J. R., Darley-Usmar, V. M., Rajasekaran, N. S. Disruption of nuclear factor (erythroid-derived-2)-like 2 antioxidant signaling: a mechanism for impaired activation of stem cells and delayed regeneration of skeletal muscle.

Direct microneedle array fabrication off a photomask to deliver collagen through skin.

PURPOSE: To fabricate microneedle arrays directly off a photomask using a simple photolithographical approach and evaluate their potential for delivering collagen. METHODS: A simple photolithographical approach was developed by using photomask consisting of embedded micro-lenses that govern microneedle geometry in a mould free process. Microneedle length was controlled by use of simple glass scaffolds as well as addition of backing layer. The fabricated arrays were tested for their mechanical properties by using a force gauge as well as insertion into human skin with trypan blue staining. Microneedle arrays were then evaluated for the delivery of fluorescent collagen, which was evaluated using a confocal laser scanning microscope. RESULTS: Microneedles with sharp tips ranging between 41.5 ± 8.4 µm and 71.6 ± 13.7 µm as well as of two different lengths of 1336 ± 193 µm and 957 ± 171 µm were fabricated by using the photomasks. The microneedles were robust and resisted fracture forces up to 25 N. They were also shown to penetrate cadaver human skin samples with ease; especially microneedle arrays with shorter length of 957 µm penetrated up to 72% of needles. The needles were shown to enhance permeation of collagen through cadaver rat skin, as compared to passive diffusion of collagen. CONCLUSIONS: A simple and mould free approach of fabricating polymeric microneedle array is proposed. The fabricated microneedle arrays enhance collagen permeation through skin.

Near infra-red absorbing Quinolizidine fused curcuminoid-BF2 chelate and its applications in photodynamic therapy using MCF-7 and A549 cells.

Metal-free near-infrared absorbing photosensitizers (PS) have been considered promising candidates for photodynamic therapy. Curcumin, curcuminoid, and its derivatives have therapeutic values due to their anti-inflammatory, antifungal, and antiproliferative properties. Curcuminoid-BF2 chelates have also been studied as cell imaging probes, however, their applications in photodynamic therapy are rare. In this article, we describe the synthesis and therapeutic evaluation of quinolizidine fused curcuminoid-BF2 chelate (Quinolizidine CUR-BF2) containing an acid-sensitive group. This donor-acceptor-donor curcuminoid-BF2 derivative exhibits absorption and emission in the deep red region with an absorption band maximum of ∼647 nm and a weak emission band at approximately 713 nm. It is interesting to note that this derivative has a high molar extinction coefficient (164,655 M-1cm-1). Quinolizidine CUR-BF2 possesses intramolecular charge transfer properties, facilitating the production of singlet oxygen (1O2), which plays a crucial role in cell death. Additionally, Quinolizidine CUR-BF2 can enable the selective release of active ingredients in an acidic medium (pH 5). Furthermore, the nanoaggregates of PS were prepared by encapsulating Quinolizidine CUR-BF2 within Pluronic F127 block co-polymer for better water-dispersibility and enhanced cellular uptake. Dark cytotoxicity of nanoaggregates was found to be negligible, whereas they exhibited significant photoinduced cytotoxicity towards cancer cells (MCF-7 and A549) under irradiation of 635 nm light. Further, the cell death pathway using Quinolizidine CUR-BF2 nanoaggregates as PS is found to occur through apoptosis. Specifically, the present study deals with the successful preparation of Quinolizidine CUR-BF2 nanoaggregates for enhanced water-dispersibility and cellular uptake as well as the efficacy evaluation of developed nanoaggregates for photodynamic therapy.

Smart Magnetic Nanocarriers for Codelivery of Nitric Oxide and Doxorubicin for Enhanced Apoptosis in Cancer Cells.

Extremely short half-life therapeutic molecule nitric oxide (NO) plays significant roles in the functioning of various physiological and pathological processes in the human body, whereas doxorubicin hydrochloride (DOX) is a clinically important anticancer drug widely used in cancer chemotherapy. Thus, the intracellular delivery of these therapeutic molecules is tremendously important to achieve their full potential. Herein, we report a novel approach for the development of highly water-dispersible magnetic nanocarriers for codelivery of NO and DOX. Primarily, bifunctional magnetic nanoparticles enriched with carboxyl and thiol groups were prepared by introducing cysteine onto the surface of citrate-functionalized Fe3O4 nanoparticles. DOX was electrostatically conjugated onto the surface of bifunctional nanoparticles via carboxyl moieties, whereas the thiol group was further nitrosated to provide NO-releasing molecules. The developed magnetic nanocarrier exhibited good aqueous colloidal stability, protein resistance behavior, and high encapsulation efficacy for NO (65.5%) and DOX (85%), as well as sustained release characteristics. Moreover, they showed superior cytotoxicity toward cancer (A549 and MCF-7) cells via apoptosis induction over normal (WI26VA4) cells. Specifically, we have developed magnetic nanocarriers having the capability of dual delivery of NO and DOX, which holds great potential for combinatorial cancer treatment.

Development of a rapid and ultra-sensitive RNA:DNA hybrid immunocapture based biosensor for visual detection of SARS-CoV-2 RNA.

The Development of reliable and field-compatible detection methods is essential to monitoring and controlling the spread of any global pandemic. We herein report a novel anti-RNA:DNA hybrid (anti-RDH) antibody-based biosensor for visual, colorimetric lateral flow assay, using gold nanoparticles, coupled with transcription-mediated-isothermal-RNA-amplification (TMIRA) for specific and sensitive detection of viral RNA. We have demonstrated its utility for SARS-CoV-2 RNA detection. This technique, which we have named RDH-LFA (anti-RNA:DNA hybrid antibody-based lateral flow assay), exploits anti-RDH antibody for immunocapture of viral RNA hybridized with specific DNA probes in lateral flow assay. This method uses biotinylated-oligonucleotides (DNAB) specific to SARS-CoV-2 RNA (vRNA) to generate a vRNA-DNAB hybrid. The biotin-tagged vRNA-DNAB hybrid molecules bind to streptavidin conjugated with gold nanoparticles. This hybrid complex is trapped by the anti-RDH antibody immobilized on the nitrocellulose membrane resulting in pink color signal leading to visual naked-eye detection in 1 minute. Combining RDH-LFA with isothermal RNA amplification (TMIRA) significantly improves the sensitivity (LOD:10 copies/µl) with a total turnaround time of an hour. More importantly, RDH-LFA coupled with the TMIRA method showed 96.6% sensitivity and 100% specificity for clinical samples when compared to a commercial gold standard reverse-transcription quantitative polymerase-chain-reaction assay. Thus, the present study reports a rapid, sensitive, specific, and simple method for visual detection of viral RNA, which can be used at the point-of-care without requiring sophisticated instrumentation.

Development of Core@Shell γ-Fe2O3@MnxOy@SiO2 Nanoparticles for Hyperthermia, Targeting, and Imaging Applications.

Monodispersed core@shell γ-Fe2O3@MnxOy nanoparticles have been prepared through thermolysis of iron and manganese oleate. Further, these prepared nanoparticles are coated with biocompatible substances such as silica and polyethylene glycol. These particles are highly biocompatible for different cell lines such as normal and cancer cell lines. The nanoparticles are used as hyperthermia agents, and successful hyperthermia treatment in cancer cells is carried out. As compared to γ-Fe2O3@SiO2, γ-Fe2O3@MnxOy@SiO2 shows the enhanced killing of cancer cells through hyperthermia. In order to make them potential candidates for targeting to cancer cells, folic acid (FA) is tagged to the nanoparticles. Fluorescein isothiocyanate (FITC) is also tagged onto these nanoparticles for imaging. The developed γ-Fe2O3@MnxOy@SiO2 nanoparticle can act as a single entity for therapy through AC magnetic field, imaging through FITC and targeting through folic acid simultaneously. This is the first report on this material, which is highly biocompatible for hyperthermia, imaging, and targeting.

Curcumin Encapsulated Casein Nanoparticles: Enhanced Bioavailability and Anticancer Efficacy.

The poor water solubility and bioactivity of drugs can be potentially improved by using suitable nanocarriers. Herein, an economically viable methodology is developed for encapsulation of hydrophobic anticancer agent, curcumin in casein nanoparticles (CasNPs). The successful encapsulation of curcumin was evident from the structural, thermal and spectroscopic analysis of curcumin encapsulated CasNPs (Cur-CasNPs). The CasNPs and Cur-CasNPs samples were lyophilized for their long-term stability and lyophilized powders are found to be stable for more than 6 months at 4-8 °C. From DLS studies, it has been observed that the variation in average size of drug formulations before and after reconstitution were less than 5%. Further, it shows good water-dispersibility, enhanced bioavailability and pH dependent charge conversal feature. Cur-CasNPs showed pH dependent release characteristics with higher at mild acidic environment and enhanced toxicity towards cancer cells (MCF-7) as compared to normal cells (CHO). Moreover, the CasNPs are non-toxic in nature and the developed nanoformulation of drug exhibits substantial cellular internalization and enhanced toxicity towards MCF-7 cells over pure drug, indicating their potential applications.

Immobilization of protein on Fe3O4 nanoparticles for magnetic hyperthermia application.

We report a facile approach for the preparation of protein conjugated glutaric acid functionalized Fe3O4 magnetic nanoparticles (Pro-Glu-MNPs), having improved colloidal stability and heating efficacy. The Pro-Glu-MNPs were prepared by covalent conjugation of BSA protein onto the surface of glutaric acid functionalized Fe3O4 magnetic nanoparticles (Glu-MNPs) obtained through thermal decomposition. XRD and TEM analyses confirmed the formation of crystalline Fe3O4 nanoparticles of average size ~5 nm, whereas the conjugation of BSA protein to them was evident from XPS, FTIR, TGA, DLS and zeta-potential measurements. These Pro-Glu-MNPs showed good colloidal stability in different media (water, phosphate buffer saline, cell culture medium) and exhibited room temperature superparamagnetism with good magnetic field responsivity towards the external magnet. The induction heating studies revealed that the heating efficacy of these Pro-Glu-MNPs was strongly reliant on the particle concentration and their stabilizing media. In addition, they showed enhanced heating efficacy over Glu-MNPs as surface passivation by protein offers colloidal stability to them as well as prevents their aggregation under AC magnetic field. Further, Pro-Glu-MNPs are biocompatible towards normal cells and showed substantial cellular internalization in cancerous cells, suggesting their potential application in hyperthermia therapy.

Effect of structural variation on tumor targeting efficacy of cationically charged porphyrin derivatives: Comparative in-vitro and in-vivo evaluation for possible potential in PET and PDT.

tetracationic (TMPyP) and tricationic porphyrin (TriMPyCOOHP) derivatives were synthesized, characterized and investigated for binding with DNA by Isothermal Titration Calorimetry as well as by UV-Vis spectroscopy in order to study the effect of structural variation on tumor targeting efficacy of cationically charged porphyrin derivatives. Fluorescence cell imaging studies performed in cancer cell lines corroborated the findings of aforementioned studies. Photocytotoxicity experiments in A549 cell lines revealed relatively higher light dependent cytotoxic effects exerted by TMPyP compared to TriMPyCOOHP. In-vivo experiments in tumor bearing animal model revealed relatively longer retention of 68Ga-TMPyP in tumorous lesion compared to that of 68Ga-TriMPyCOOHP. The study reveals that removal of one of the positive charges of the tetracationic porphyrin derivatives significantly reduces their DNA binding ability and cytotoxicity as well as brings changes in the pharmacokinetic pattern and tumor retention in small animal model.

Super Bright Red Upconversion in NaErF4:0.5%Tm@NaYF4:20%Yb Nanoparticles for Anti-counterfeit and Bioimaging Applications.

Nanocrystals having single-band red emission under near-infrared (NIR) excitation through the upconversion process offer great advantages in terms of enhanced cellular imaging in in vitro and in vivo experiments in the biological window (600-900 nm), as a security ink, in photothermal therapy (PTT), in photodynamic therapy (PDT), and so forth but are challenging for materials scientists. In this work, we report for the first time the preparation of a super bright red emitter at 655 nm from monodispersed NaErF4:0.5%Tm@NaYF4:20%Yb nanocrystals (core@active shell). This phosphor exhibits 35 times stronger photoluminescence as compared to NaErF4:0.5%Tm@NaYF4 (core@inactive shell). Here, an Er3+-enriched host matrix works simultaneously as an activator and a sensitizer under NIR excitation. Upconversion red emission at 655 nm arises due to the electronic transition of Er3+ via the involvement of a three-photon absorption (expected to be a two-photon absorption), which has been confirmed via a power-dependent luminescence study. Tm3+ ions incorporated into the core with the active shell act as trapping centers, which promote the red band emission via the back-energy transfer process. Moreover, the active shell containing Yb3+ ions efficiently transfers the energy to the Er3+-enriched core, which suppresses the nonradiative channel rate, and Tm3+ ions act as trapping centers, which reduce the luminescence quenching via reduction of energy migration to the surface of the host lattice. Also, we have shown the potential applications of these nanocrystals: cellular imaging through downconversion and upconversion processes and security ink.

Spontaneous Formation of Cationic Vesicles in Aqueous DDAB-Lecithin Mixtures for Efficient Plasmid DNA Complexation and Gene Transfection.

Cationic liposomes have become an attractive tool to deliver genes and interfering RNA into cells. Herein, we report the application of spontaneously formed cationic vesicles in mixtures of lecithin and cationic amphiphiles for efficient transfection of plasmid DNA and siRNA into cells. The average hydrodynamic diameter of the phospholipid vesicles was modulated by changing the ratio of dihexadecyldimethylammonium bromide (DDAB) to phospholipid in the vesicles. The vesicles were characterized by dynamic light scattering, ζ potential, and small-angle X-ray scattering. Depending on the ratio of DDAB to phospholipid, the average size of the vesicles can be varied in the range of 150-300 nm with a ζ potential of +40 mV. The ability of these cationic vesicles to form lipoplexes upon binding with pDNA is demonstrated by ζ potential, isothermal titration calorimetry, gel retardation, and DNase I digestion assay. The enthalpy of binding between pDNA and cationic liposome was found to be -5.7 (±0.8) kJ/mol. The cellular uptake studies of lipoplexes observed by fluorescence microscopy confirmed good transfection efficiency of DDAB liposomes in MCF-7 and HeLa cells. The fluorescent imaging analysis showed effective gene delivery and expression of green fluorescent protein. In addition, the formulation has demonstrated an ability to deliver small interfering RNA (siBRD4) for efficient gene silencing as seen by a significant decrease in BRD4 protein level in siBRD4-treated cells. Comparison of the transfection efficiency of different formulations suggests that DDAB-rich mixed phospholipid vesicles with size <200 nm are better than large size vesicles for improved endocytosis and gene expression.

Identification of Nrf2-responsive microRNA networks as putative mediators of myocardial reductive stress.

Although recent advances in the treatment of acute coronary heart disease have reduced mortality rates, few therapeutic strategies exist to mitigate the progressive loss of cardiac function that manifests as heart failure. Nuclear factor, erythroid 2 like 2 (Nfe2l2, Nrf2) is a transcriptional regulator that is known to confer transient myocardial cytoprotection following acute ischemic insult; however, its sustained activation paradoxically causes a reductive environment characterized by excessive antioxidant activity. We previously identified a subset of 16 microRNAs (miRNA) significantly diminished in Nrf2-ablated (Nrf2-/-) mouse hearts, leading to the hypothesis that increasing levels of Nrf2 activation augments miRNA induction and post-transcriptional dysregulation. Here, we report the identification of distinct miRNA signatures (i.e. "reductomiRs") associated with Nrf2 overexpression in a cardiac-specific and constitutively active Nrf2 transgenic (caNrf2-Tg) mice expressing low (TgL) and high (TgH) levels. We also found several Nrf2 dose-responsive miRNAs harboring proximal antioxidant response elements (AREs), implicating these "reductomiRs" as putative meditators of Nrf2-dependent post-transcriptional regulation. Analysis of mRNA-sequencing identified a complex network of miRNAs and effector mRNAs encoding known pathological hallmarks of cardiac stress-response. Altogether, these data support Nrf2 as a putative regulator of cardiac miRNA expression and provide novel candidates for future mechanistic investigation to understand the relationship between myocardial reductive stress and cardiac pathophysiology.

Reductive stress impairs myogenic differentiation.

Myo-satellite cells regenerate and differentiate into skeletal muscle (SM) after acute or chronic injury. Changes in the redox milieu towards the oxidative arm at the wound site are known to compromise SM regeneration. Recently, we reported that abrogation of Nrf2/antioxidant signaling promotes oxidative stress and impairs SM regeneration in C57/Bl6 mice. Here, we investigated whether the activation of intracellular Nrf2 signaling favors antioxidant transcription and promotes myoblast differentiation. Satellite cell-like C2C12 myoblasts were treated with sulforaphane (SF; 1.0 & 5.0 µM) to activate Nrf2/antioxidant signaling during proliferation and differentiation (i.e. formation of myotubes/myofibers). SF-mediated Nrf2 activation resulted in increased expression of Nrf2-antioxidants (e.g. GCLC and G6PD) and augmented the production of reduced glutathione (GSH) leading to a reductive redox state. Surprisingly, this resulted in significant inhibition of myoblast differentiation, as observed from morphological changes and reduced expression of MyoD, Pax7, and Myh2, due to reductive stress (RS). Furthermore, supplementation of N-acetyl-cysteine (NAC) or GSH-ester or genetic knock-down of Keap1 (using siRNA) also resulted in RS-driven inhibition of differentiation. Interestingly, withdrawing Nrf2 activation rescued differentiation potential and formation of myotubes/myofibers from C2C12 myoblasts. Thus, abrogation of physiological ROS signaling through over-activation of Nrf2 (i.e. RS) and developing RS hampers differentiation of muscle satellite cells.

Electrostatically bound lanreotide peptide - gold nanoparticle conjugates for enhanced uptake in SSTR2-positive cancer cells.

Lanreotide peptide (LP) has high affinity to somatostatin receptors like SSTR2 and is commonly used in the treatment of neuro-endocrine tumors. The main objective of this study is to target gold nanoparticles (AuNPs) towards SSTR2-positive cancer cells using lanreotide peptide (LP) as the targeting agent for enhanced tumor uptake and antitumor activity. pH mediated changes in the surface potential of LP and AuNP is used to prepare electrostatically bound AuNP-LP complexes. AuNP-LP complex formation was demonstrated by UV-Visible spectroscopy, surface potential, dynamic light scattering (DLS), small angle X-ray scattering and HR-TEM. Confocal microscopy and flow cytometric studies show that AuNP-LP complex has higher cellular uptake in SSTR2 expressed cancer cells (MCF-7 and AR42J) than in CHO cells. The enhanced cellular uptake of LP coated AuNPs lead to ~1.5 to 2-fold GSH depletion and enhanced ROS generation in MCF-7 cells. The preferential cytotoxicity of the AuNP-LP complex towards MCF-7 and AR42J cells, as revealed by MTT assay, is consistent with the increased cellular uptake. Our studies demonstrate that LP coated AuNP can be used as an effective platform to selectively target SSTR2 positive cancer cells for combination therapy approaches involving gold nanoparticles.

PRDM16 suppresses HIF-targeted gene expression in kidney cancer.

Analysis of transcriptomic data demonstrates extensive epigenetic gene silencing of the transcription factor PRDM16 in renal cancer. We show that restoration of PRDM16 in RCC cells suppresses in vivo tumor growth. RNaseq analysis reveals that PRDM16 imparts a predominantly repressive effect on the RCC transcriptome including suppression of the gene encoding semaphorin 5B (SEMA5B). SEMA5B is a HIF target gene highly expressed in RCC that promotes in vivo tumor growth. Functional studies demonstrate that PRDM16's repressive properties, mediated by physical interaction with the transcriptional corepressors C-terminal binding proteins (CtBP1/2), are required for suppression of both SEMA5B expression and in vivo tumor growth. Finally, we show that reconstitution of RCC cells with a PRDM16 mutant unable to bind CtBPs nullifies PRDM16's effects on both SEMA5B repression and tumor growth suppression. Collectively, our data uncover a novel epigenetic basis by which HIF target gene expression is amplified in kidney cancer and a new mechanism by which PRDM16 exerts its tumor suppressive effects.

Integrative Epigenetic and Gene Expression Analysis of Renal Tumor Progression to Metastasis.

The Cancer Genome Atlas (TCGA) and other large-scale genomic data pipelines have been integral to the current understanding of the molecular events underlying renal cell carcinoma (RCC). These data networks have focused mostly on primary RCC, which often demonstrates indolent behavior. However, metastatic disease is the major cause of mortality associated with RCC and data sets examining metastatic tumors are sparse. Therefore, a more comprehensive analysis of gene expression and DNA methylome profiling of metastatic RCC in addition to primary RCC and normal kidney was performed. Integrative analysis of the methylome and transcriptome identified over 30 RCC-specific genes whose mRNA expression inversely correlated with promoter methylation, including several known targets of hypoxia inducible factors. Notably, genes encoding several metabolism-related proteins were identified as differentially regulated via methylation including hexokinase 2, aldolase C, stearoyl-CoA desaturase, and estrogen-related receptor-γ (ESRRG), which has a known role in the regulation of nuclear-encoded mitochondrial metabolism genes. Several gene expression changes could portend prognosis in the TCGA cohort. Mechanistically, ESRRG loss occurs via DNA methylation and histone repressive silencing mediated by the polycomb repressor complex 2. Restoration of ESRRG in RCC lines suppresses migratory and invasive phenotypes independently of its canonical role in mitochondrial metabolism. IMPLICATIONS: Collectively, these data provide significant insight into the biology of aggressive RCC and demonstrate a novel role for DNA methylation in the promotion of HIF signaling and invasive phenotypes in renal cancer.

Biochemical and Epigenetic Insights into L-2-Hydroxyglutarate, a Potential Therapeutic Target in Renal Cancer.

PURPOSE: Elevation of L-2-hydroxylgutarate (L-2-HG) in renal cell carcinoma (RCC) is due in part to reduced expression of L-2-HG dehydrogenase (L2HGDH). However, the contribution of L-2-HG to renal carcinogenesis and insight into the biochemistry and targets of this small molecule remains to be elucidated. EXPERIMENTAL DESIGN: Genetic and pharmacologic approaches to modulate L-2-HG levels were assessed for effects on in vitro and in vivo phenotypes. Metabolomics was used to dissect the biochemical mechanisms that promote L-2-HG accumulation in RCC cells. Transcriptomic analysis was utilized to identify relevant targets of L-2-HG. Finally, bioinformatic and metabolomic analyses were used to assess the L-2-HG/L2HGDH axis as a function of patient outcome and cancer progression. RESULTS: L2HGDH suppresses both in vitro cell migration and in vivo tumor growth and these effects are mediated by L2HGDH's catalytic activity. Biochemical studies indicate that glutamine is the predominant carbon source for L-2-HG via the activity of malate dehydrogenase 2 (MDH2). Inhibition of the glutamine-MDH2 axis suppresses in vitro phenotypes in an L-2-HG-dependent manner. Moreover, in vivo growth of RCC cells with basal elevation of L-2-HG is suppressed by glutaminase inhibition. Transcriptomic and functional analyses demonstrate that the histone demethylase KDM6A is a target of L-2-HG in RCC. Finally, increased L-2-HG levels, L2HGDH copy loss, and lower L2HGDH expression are associated with tumor progression and/or worsened prognosis in patients with RCC. CONCLUSIONS: Collectively, our studies provide biochemical and mechanistic insight into the biology of this small molecule and provide new opportunities for treating L-2-HG-driven kidney cancers.

Thioredoxin-dependent regulation of AIF-mediated DNA damage.

The thioredoxin (Trx) system is one major redox system in mammalian cells. One of its component, Trx, is involved in redox homeostasis and many cellular biological processes through participating in disulfide reduction, S-nitrosylation/S-denitrosylation reactions and protein-protein interactions. In this study, we report the identification of a novel interaction between cytosolic/nuclear Trx1 and apoptosis inducing factor (AIF), and the redox sensitivity and biological significance of the Trx-AIF interaction was characterized. Cytosolic Trx1 but not mitochondrial Trx2 was observed to interact with AIF under physiological conditions and Trx1's active site cysteines were crucial for the interaction. Under oxidative stress conditions, Trx-AIF interaction was disrupted. When the treated cells were allowed to recover from oxidative stress by means of removal of the oxidants, interaction between Trx1 and AIF was re-established time-dependently, which underpins the biological relevance of a Trx-dependent redox regulation of AIF-mediated cell death. Indeed, in times of oxidative stress, nuclear translocation of AIF was found to occur concurrently with perturbations to the Trx-AIF interaction. Once localized in the nucleus, reduced Trx1 hindered the interaction between AIF and DNA, thereby bringing about an attenuation of AIF-mediated DNA damage. In conclusion, characterization of the Trx-AIF interaction has led to an understanding of the effect of reduced Trx1 on possibly regulating AIF-dependent cell death through impeding AIF-mediated DNA damage. Importantly, identification of the novel interaction between Trx1 and AIF has provided opportunities to design and develop therapeutically relevant strategies that either promote or prevent this protein-protein interaction for the treatment of different disease states.