Upregulation Of Apoptotic Genes Research Articles

Pituitary tumor‑transforming gene 1 regulates the senescence and apoptosis of oral squamous cell carcinoma in a p21‑dependent DNA damage response manner.

Pituitary tumor‑transforming gene 1 (PTTG1), also known as securin, is a proto‑oncogene involved in the development of various cancers by promoting cell proliferation and mobility. However, its underlying biological mechanisms in oral squamous cell carcinoma (OSCC) progression remain unclear. in the present study, it was sought to elucidate the role of PTTG1 as an oncogene in OSCC progression and was attempted to unravel the underlying mechanism and impact of PTTG1 expression on cell cycle, cell death, and cellular senescence. The effect of double strand break on PTTG1 expression was investigated in OSCC growth. To identify the role of PTTG1 in OSCC growth, the cell viability and senescence was analyzed by EdU and senescence‑associated beta‑galactosidase (SA‑β‑gal) assay, respectively. To verify the DNA damage‑induced senescence of PTTG1, the chromosomal damage in OSCC was analyzed in vitro. Finally, the effect of PTTG1 on tumor growth and gene expression related to cell viability and DNA damaged‑induced senescence was investigated in vivo. PTTG1 expression was compared between OSCC and healthy patient samples (n=32) using reverse transcription‑quantitative PCR and immunohistochemistry; and it was found that PTTG1 expression was upregulated in OSCC. Small interfering RNA‑mediated knockdown of PTTG1 in two OSCC cell lines revealed that PTTG1 downregulation significantly inhibited cell proliferation and arrested the cell cycle pathway as evidenced by changes in checkpoint genes (such as cyclin D1, E and B1). PTTG1 knockdown also increased apoptosis, as evidenced by the upregulation of apoptotic genes [such as cleaved (c‑) Caspase‑7 and c‑poly (ADP‑ribose) polymerase]. Moreover, PTTG1 downregulation promoted cellular senescence, as shown by western blotting and SA‑β‑gal staining. Finally, senescence‑induced DNA damage was observed in OSCC cells, which accelerates genomic instability, through chromosomal damage analysis. Taken together, the present findings suggested that PTTG1 acts as a proto‑oncogene; regulates cell proliferation, cell cycle, cellular senescence and DNA damage in OSCC; and may serve as a novel diagnostic biomarker and potential therapeutic target for OSCC.

Vitamin A ameliorates valproic acid-induced autism-like symptoms in developing zebrafish larvae by attenuating oxidative stress and apoptosis

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social deficits and repetitive/stereotyped behaviors. Prenatal exposure to valproic acid (VPA) has been reported to induce ASD-like symptoms in human and rodents. However, the etiology and pathogenesis of ASD have not been well elucidated. This study aimed to explore the mechanisms underlying VPA-induced ASD-like behaviors using zebrafish model and investigated whether vitamin A could prevent VPA-induced neurotoxicity. Here, zebrafish embryos were exposed to 0, 25 and 50 μM VPA from 4 to 96 h post fertilization (hpf) and the neurotoxicity was assessed. Our results showed that VPA affected the normal development of zebrafish larvae and induced ASD-like behaviors, including reduced locomotor activity, decreased distance near conspecifics, impaired social interaction and repetitive swimming behaviors. Exposure to VPA decreased the GFP signal in transgenic HuC:egfp zebrafish according to the negative effect of VPA on the expression of neurodevelopmental genes. In addition, VPA enhanced oxidative stress by promoting the production of reactive oxygen species (ROS) and hydrogen peroxide (H2O2) and inhibiting the activity of superoxide dismutase, then triggered apoptosis by upregulation of apoptotic genes. These adverse outcomes were mitigated by vitamin A, suggesting that vitamin A rescued VPA-induced ASD-like symptoms by inhibiting oxidative stress and apoptosis. Overall, this study identified vitamin A as a promising strategy for future therapeutic regulator of VPA-induced ASD-like behaviors.

Upregulation of apoptotic genes and downregulation of target genes of Sonic Hedgehog signaling pathway in DAOY medulloblastoma cell line treated with arsenic trioxide

Sonic hedgehog (SHH) medulloblastoma etiology is associated with the SHH molecular pathway activation at different levels. We investigated the effect of arsenic trioxide as a downstream-level inhibitor of the SHH signaling pathway on morphology, cytotoxicity, migration, and SHH-related and apoptotic gene expression of DAOY cells. Cells were treated at various arsenic trioxide (ATO)concentrations (1, 2, 3, 5, and 10 μM) for different times (24 and 48 hr). Following treatments, the morphology of the cells was investigated at ×20 and ×40 magnification by an inverted microscope. Then, cytotoxicity was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays. Cell migration was analyzed through the wound-healing assay. Furthermore, the expression of SHH-related (GLI1, GLI2, SMO, and MYCN) and apoptotic genes (BAX, BCL2, and TP53) was assessed by real-time quantitative polymerase chain reaction (qPCR). Finally, GLI1, SMO, and MYCN markers were analyzed through immunocytochemistry. Data were analyzed by SPSS (version 16) and P≤0.05 was considered significant. Morphological changes were seen at 3 and 2 μM in 24 and 48 hr of treatment, respectively. The MTT assay showed a dose-dependent cytotoxicity indicating an IC50 value of 3.39±0.35 and 2.05±0.64 μM in 24 and 48hr treatment, respectively. In addition, the trypan blue assay showed higher IC50 values of 4.29±0.25 and 3.92±0.22 μM in 24 and 48 hr treatment, respectively. The wound-healing assay indicated a dose-dependent reduction of cell migration speed showing a 50% reduction at 2.89±0.26 μM. Significant downregulation of GLI1 and GLI2, as well as the upregulation of BAX, BAX/BCL2 ratio, and TP53 were evident. Significant increases in GLI1 and MYCN markers were also evident in immunocytochemistry. ATO, as a downstream effective inhibitor of the SHH pathway, substantially leads to cell death, cell migration inhibition, apoptosis upregulation, and downregulation of SHH target genes in DAOY medulloblastoma. Since ATO is a toxic chemotherapeutic agent, it must be used at low concentrations (2 μM) in order not to damage healthy cells.

Curcumin protects against fenvalerate-induced neurotoxicity in zebrafish (Danio rerio) larvae through inhibition of oxidative stress

Fenvalerate (FEN), a typical type II pyrethroid pesticide, is widely used in agriculture. FEN has been detected in the environment and human body. However, the neurotoxicity of FEN has not been well elucidated. This study aimed to explore the mechanisms underlying FEN-induced neurotoxicity using the zebrafish (Danio rerio) model. We also investigated whether curcumin (CUR), a polyphenol antioxidant that exhibits neuroprotective properties, can prevent FEN-induced neurotoxicity. Here, zebrafish embryos were exposed to 0, 3.5, 7 and 14 μg/L of FEN from 4 to 96 h post fertilization (hpf) and neurotoxicity was assessed. Our results showed that FEN decreased the survival rate, heart rate, body length and spontaneous movement, and increased malformation rate. FEN caused neurobehavioral alterations, including decreased swimming distance and velocity, movement time and clockwise rotation times. FEN also suppressed neurogenesis in transgenic HuC:egfp zebrafish, reduced cholinesterase activity and downregulated the expression of neurodevelopment related genes (elavl3, gfap, gap43 and mbp). In addition, FEN enhanced oxidative stress via excessive reactive oxygen species and antioxidant enzyme inhibition, then triggered apoptosis by upregulation of apoptotic genes (p53, bcl-2, bax and caspase 3). These adverse outcomes were alleviated by CUR, indicating that CUR mitigated FEN-induced neurotoxicity by inhibiting oxidative stress. Overall, this study revealed that CUR ameliorated FEN-induced neurotoxicity via its antioxidant, indicating a promising protection of CUR against environmental pollutant-induced developmental anomalies.

Nitrogen and copper-doped saffron-based carbon dots: Synthesis, characterization, and cytotoxic effects on human colorectal cancer cells

AimDoped carbon dots (CDs) have attracted tremendous attention in cancer therapy. We aimed to synthesize copper, nitrogen-doped carbon dots (Cu, N-CDs) from saffron and investigated their effects on HCT-116 and HT-29 colorectal cancer (CRC) cells. Main methodsCDs were synthesized by hydrothermal method and characterized by transmission electron microscopy (TEM), energy-dispersive X-ray (EDX), Fourier transform infrared (FT-IR) spectroscopy, ultraviolet-visible (UV–Vis) absorption spectroscopy, and fluorescence spectroscopy. HCT-116 and HT-29 cells were incubated with saffron, N-CDs, and Cu, N-CDs for 24 and 48 h for cell viability. Cellular uptake and intracellular reactive oxygen species (ROS) were evaluated by immunofluorescence microscopy. Oil Red O staining was used to monitor lipid accumulation. Apoptosis was evaluated using acridine orange/propidium iodide (AO/PI) staining and quantitative real-time polymerase chain reaction (Q-PCR) assay. The expression of miRNA-182 and miRNA-21 was measured by Q-PCR, while the generation of nitric oxide (NO) and lysyl oxidase (LOX) activity was calculated by colorimetric methods. Key findingsCDs were successfully prepared and characterized. Cell viability decreased in the treated cells dose- and time-dependently. HCT-116 and HT-29 cells uptook Cu, N-CDs with a high level of ROS generation. The Oil Red O staining showed lipid accumulation. Concomitant with an up-regulation of apoptotic genes (p < 0.05), AO/PI staining showed increased apoptosis in the treated cells. In comparison to control cells, NO generation, and miRNA-182 and miRNA-21 expression significantly changed in the Cu, N-CDs treated cells (p < 0.05). SignificanceThe results indicated that Cu, N-CDs could inhibit CRC cells through the induction of ROS generation and apoptosis.

Luteolin: a flavonoid with a multifaceted anticancer potential.

Therapeutic effect of phytochemicals has been emphasized in the traditional medicine owing to the presence of bioactive molecules, such as polyphenols. Luteolin is a flavone belonging to the flavonoid class of polyphenolic phytochemicals with healing effect on hypertension, inflammatory disorders, and cancer due to its action as pro-oxidants and antioxidants. The anticancer profile of luteolin is of interest due to the toxic effect of contemporary chemotherapy paradigm, leading to the pressing need for the development and identification of physiologically benevolent anticancer agents and molecules. Luteolin exerts anticancer activity by downregulation of key regulatory pathways associated with oncogenesis, in addition to the induction of oxidative stress, cell cycle arrest, upregulation of apoptotic genes, and inhibition of cell proliferation and angiogenesis in cancer cells. In this review, we discuss about the anticancer profile of luteolin.

Establishment of effective and safe recipient preparation for germ-cell transplantation with intra-testicular busulfan treatment in pre-pubertal Barbari goats

The busulfan, an alkylating agent, suppresses endogenous spermatogenesis in recipient testes. However, considering a wide variation in the effects of busulfan among animal species, its dosage and route of infusion need optimization to prepare effective and safe recipients. Thus, the current study aimed to create a suitable recipient goat model for germ cell (Gc) transplantation through a single intra-testicular (i.t.) busulfan infusion under ultrasonographic (USG) guidance. As observed through the infusion of trypan blue under USG guidance into mediastinum testis (MT) of pre-pubertal Barbari bucks, 3–5 mL of trypan blue solution could fill almost 80% of seminiferous tubules. Thereafter, in Experiment-1, the effect of different busulfan doses (mg/kg) i.e. 0 [negative control, Group (Gr) 1; 0 mg/kg-MT], 1 (Gr 2; 1 mg/kg-MT), 2 (Gr 3; 2 mg/kg-MT), and 3 (Gr 4; 3 mg/kg-MT) were studied. Further, in Experiment-2, sterilizing effects of busulfan infusion through two different routes [MT or cavum vaginale (CV)] were compared. Following i.t. busulfan treatment, no adverse physiological effects or body weight loss were detected. The histological analyses demonstrate a dose-dependent depletion of Gc with almost complete loss of Gc and spermatogenic activities in Gr 3 and 4, and extensive fibrosis in Gr 4. A considerable suppression of spermatogenesis marked with devoid of endogenous spermatogonial population and absence of significant (P > 0.05) effect on key hematological variables were observed in 2 mg/kg-MT Gr. These findings coupled with the results of significant (P < 0.05) down-regulation of marker genes of undifferentiated spermatogonia (THY-1 and PLZF), Gc pluripotency (UCHL-1, OCT-4, and DDX-4), and adhesion (E-cadherin and β-integrin); up-regulation of apoptotic genes (ID - 4 and BCL-6), and unchanged expression of Sertoli cell marker (vimentin), confirmed the safe and efficient depletion of endogenous Gc in 2 mg/kg-MT Gr. Furthermore, the effect of busulfan infusion on scrotal-testicular biometry, endocrine variables (plasma cortisol and testosterone), and Gc removal was more evident when busulfan was infused into MT than into CV. Overall, the results demonstrated that 2.0 mg/kg is an optimal single dose of busulfan when infused into the MT under USG guidance for the preparation of pre-pubertal recipient bucks. Overall, this study provides a basis to prepare suitable recipients through providing an available niche for efficient Gc transplantation in goats.

HP1a-mediated heterochromatin formation inhibits high dietary sugar-induced tumor progression

High dietary sugar (HDS) is a modern dietary concern that involves excessive consumption of carbohydrates and added sugars, and increases the risk of metabolic disorders and associated cancers. However, epigenetic mechanisms by which HDS induces tumor progression remain unclear. Here, we investigate the role of heterochromatin, an important yet poorly understood part of the epigenome, in HDS-induced tumor progression of Drosophila Ras/Src and Ras/scrib tumor systems. We found that increased heterochromatin formation with overexpression of heterochromatin protein 1a (HP1a), specifically in tumor cells, not only decreases HDS-induced tumor growth/burden but also drastically improves survival of Drosophila with HDS and Ras/Src or Ras/scrib tumors. Moreover, HDS reduces heterochromatin levels in tumor cells. Mechanistically, we demonstrated that increased heterochromatin formation decreases wingless (wg) and Hippo (Hpo) signaling, thereby promoting apoptosis, via inhibition of Yorkie (Yki) nuclear accumulation and upregulation of apoptotic genes, and reduces DNA damage in tumor cells under HDS. Taken together, our work identified a novel epigenetic mechanism by which HP1a-mediated heterochromatin formation suppresses HDS-induced tumor progression likely by decreasing wingless and Hippo signaling, increasing apoptosis, and maintaining genome stability. Our model explains that the molecular, cellular, and organismal aspects of HDS-aggravated tumor progression are dependent on heterochromatin formation, and highlights heterochromatin as a therapeutic target for cancers associated with HDS-induced metabolic disorders.

Fetal hypoxia and apoptosis following maternal porcine reproductive and respiratory syndrome virus (PRRSV) infection

BackgroundMechanisms of fetal death following maternal PRRSV2 infection remain uncharacterized, although hypoxia from umbilical cord lesions and/or placental detachment due to apoptosis are hypothesized. We performed two experiments examining hypoxia and apoptosis in PRRSV-infected and non-infected, third-trimester fetuses to elucidate possible associations with fetal death. Fetuses were selected based on four phenotypic infection groups: fetuses from non-challenged control gilts (CTRL); low viral load fetuses (LVL; Exp 1) or uninfected fetuses (UNINF; Exp 2) from inoculated gilts; viable high viral load fetuses (HVL-VIA); and HVL meconium-stained fetuses (HVL-MEC).ResultsIn experiment 1, paraffin embedded fetal tissues collected 21 days post maternal infection (DPI) were examined for DNA fragmentation associated with apoptosis. Positively stained foci were larger and more numerous (P < 0.05) in heart, liver, and thymus of HVL-VIA and HVL-MEC compared to CTRL and LVL fetuses. In experiment 2, group differences in gene expression within the hypoxia (HIF1a, IDO1, VEGFa, LDHA, NOS2, NOX1) and apoptosis (CASP3, CASP7, CASP8, CASP9, RIPK1, RIPK3) pathways were assessed by RT-qPCR in fetal tissues collected at 12 DPI. High viral load fetuses showed differential expression relative to the CTRL and UNINF (P < 0.05 for all). Brain tissue from HVL-VIA and HVL-MEC fetuses presented increased expression of CASP7, CASP8, RIPK3, HIF1a and IDO1. Fetal heart showed increased expression of CASP8, HIF1a, IDO and NOX1 and a decrease in NOS2 expression in infected groups. CASP7, CASP9, RIPK1 and RIPK3 were only increased in the heart of HVL-VIA while VEGFa was only increased for HVL-MEC fetuses. Thymus from HVL-MEC had decreased expression of CASP9 and there was increased IDO1 in all infected fetuses.ConclusionsThere is strong evidence of apoptosis occurring in the heart, liver and thymus of highly viral load fetuses at 21 DPI. Furthermore, there was clear upregulation of apoptotic genes in the heart of high viral load infected fetuses and less prominent upregulation in the brain of PRRSV-infected fetuses, whereas thymus appears to be spared at 12 DPI. There was no strong evidence of hypoxia at 12 DPI in brain and thymus but some indication of hypoxia occurring in fetal heart.

Elucidation of anti-proliferative and anti-angiogenic potential ofnovel imidazo[2,1-b][1,3,4]thiadiazole derivatives in Ehrlich ascites tumor model

Introduction and Aim:Synthesis of antineoplastic drugs is challenging and shrouded with possibilities of multidrug resistance and numerous side effects. Imidazo[2,1-b][1,3,4]thiadiazole moieties exhibit tremendous scope as novel anti-cancer molecules. In the present study, we synthesize and characterize a series of 5(a-e) Imidazo[2,l-b][1, 3,4]thiadiazole derivatives andevaluate them for antiproliferative properties using in vivo, in vitro , andin silicoapproach. Materials and Methods:The in vivostudies conducted using murine Ehrlich Ascites Cancer (EAC) cell model establishes that treatment with 5c reduces the tumorigenesisby promoting apoptosis inEAC-bearing mice. The cells retrieved from the control andtreatment arms of EAC cell bearing mice were used for nuclear and Giemsa staining, DNA fragmentation, RT-PCR and chorioallantoic membrane evaluations. Results:5cinduces apoptosis, plasma membrane degradation, up-regulation of apoptotic genes and anti-angiogenic characteristics. In vitro evaluation of 5cusing,various cancer cell linesagainst normal fibroblast 3T3 L1 cells confirm 5c sensitivity to MCF-7 with IC50value of 8?M. 5c exudes marked reduction in cell viability, dual nuclear staining,and long-term colony formationassays in thesecells. Conclusion:5c inhibits the growth and proliferation of cancer cells. The results of our molecular docking predictions further substantiate our claim. This study is valuable as 5c exhibitsapromising approach for the treatment of cancer and itsanti-proliferative potential can be exploited for designing novel anticancer drugs in the near future. Keywords:Imidazo[2,1-b][1,3,4]thiadiazole derivatives;NMRcytotoxicity, EAC cell;apoptosis;anti-angiogenesis.

Apoptosis induction and proliferation inhibition by silibinin encapsulated in nanoparticles in MIA PaCa-2 cancer cells and deregulation of some miRNAs.

Objective(s):Silibinin, as an herbal compound, has anti-cancer activity. Because of low solubility of silibinin in water and body fluids, it was encapsulated in polymersome nanoparticles and its effects were evaluated on pancreatic cancer cells and cancer stem cells.Materials and Methods:MIA PaCa-2 pancreatic cancer cells were treated with different doses of silibinin encapsulated in polymersome nanoparticles (SPNs). Stemness of MIA PaCa-2 cells was evaluated by hanging drop technique and CD133, CD24, and CD44 staining. The effects of SPNs on cell cycle, apoptosis and the expression of several genes and miRNAs were investigated. Results:IC50 of SPNs was determined to be 40 µg/ml after 24 hr. Our analysis showed that >98% of MIA PaCa-2 cells expressed three stem cell markers. FACS analysis showed a decrease in these markers in SPNs-treated cells. PI/AnnexinV staining revealed that 40 µg/ml and 50 µg/ml of SPNs increased apoptosis up to ~40% and >80% of treated cells, respectively. Upregulation of miR-34a, miR-126, and miR-let7b and downregulation of miR-155, miR-222 and miR-21 was observed in SPNs-treated cells. In addition, downregulation of some genes involved in proliferation or migration such as AKT3, MASPINE, and SERPINEA12, and upregulation of apoptotic genes were observed in treated cells.Conclusion:Our results suggested that SPNs induced apoptosis and inhibited migration and proliferation in pancreatic cells and cancer stem cells through suppression of some onco-miRs and induction of some tumor suppressive miRs, as well as their targets.

Molecular docking, anti-proliferative activity and induction of apoptosis in human liver cancer cells treated with androstane derivatives: Implication of PI3K/AKT/mTOR pathway

Worldwide, cancer is still an area with high unmet medical need. Lead optimization efforts towards structure-based drug design were employed to discover newly synthesized hetero-steroid derivatives with promising anticancer effects against hepatocellular carcinoma (HCC). The aim of our study is to evaluate the anti-proliferative activity and the mechanism, a dual PI3K/mTOR inhibitor, and mechanism of action of a series of heterocylic androstane derivatives as anti-HCC agent. The cytotoxic effects of different heterocylic androstanes and 5FU as single agents, were assessed against both HepG2 cells and Non-malignant MDCK cell line to assess the toxicity. Then the underlying mechanism of compound 4 as most promising compound was evaluated using molecular docking, MTT assay, cell cycle analysis, DNA fragmentation, and real-time PCR. The results of MTT assay showed potential cytotoxic effect for compound 4 and 5 against liver cancer cell line with IC50 value 39.81 and 57.54 μM, respectively. Inhibition of the PI3K/AKT/mTOR pathway was achieved by compound 4, which was documented by molecular docking and augmented by gene expression analysis. Detailed mechanism revealed that compound 4 induced cell cycle arrest, DNA fragmentation, and induction of apoptosis by inhibition of anti-apoptotic genes, and upregulation of apoptotic genes. Our results shed a light on aminopyrazoloandrostane derivative 4 as an inhibitor of the PI3K/AKT/mTOR pathway, which might be acting as promising anti-liver cancer agent. Our data support further investigation of agents targeting the PI3K/AKT/mTOR.

Ru(II)-Based Amino Acid Complexes Show Promise for Leukemia Treatment: Cytotoxicity and Some Light on their Mechanism of Action.

Ruthenium is attracting considerable interest as the basis for new compounds to treat diseases, and studies have shown that complexes with different structures have significant antineoplastic and antimetastatic potential against several types of tumors, including tumors resistant to cisplatin drugs. We examined the cytotoxic, genotoxic, and pro-apoptotic activities of six ruthenium complexes containing amino acid with general formulation [Ru(AA)(bipy)(dppb)]PF6, where AA = amino acid (alanine, glycine, leucine, lysine, methionine, or tryptophan); bipy = 2,2´-bipyridine; and dppb = [1,4-bis(diphenylphosphine)butane], against A549 (lung carcinoma) and K562 (chronic myelogenous leukemia) cancer cells. The results show that the ruthenium complexes tested were able to induce cytotoxicity in A549 and K562 cancer cells. Complex 1 containing alanine inhibited the cell viability of A549 and K562 tumor cells by inducing apoptosis, as evidenced by an increased number of Annexin V-positive cells and the induction of DNA damage and cell cycle arrest. Complex 1 was able to induce caspase-mediated apoptosis in K562 cells through the mitochondrial dysfunction, the upregulation of apoptotic genes, and the downregulation of Bcl2 anti-apoptotic gene. Besides being cytotoxic to K562 and A549 cells, ruthenium complex containing alanine shows low cytotoxicity and genotoxicity against non-tumor cells. These results suggest that the ruthenium (II) complex is a potential safe and efficient antineoplastic candidate for leukemia treatment.

Abstract B26: Novel lncRNA regulates PAX3/FOXO1 expression and is essential for RMS tumorigenesis

Abstract Rhabdomyosarcoma (RMS) is a pediatric cancer of the developing skeletal muscle, and the mechanisms that drive this tumor are not completely understood. Despite the genetic alterations discovered in this disease, therapeutics targeting these mutations have not improved patient survival over the past 25 years. Since more than 60% of patients with metastatic disease do not survive, this creates an urgency to identify new vulnerabilities to exploit for therapeutic development. To do this we performed a comprehensive evaluation of the epigenomic landscape across 16 RMS orthotopic patient-derived xenograft tumors. We identified 41 super enhancers unique to RMS tumors. Within these, we discovered 9 novel lncRNAs of which 3 have potential cis-regulatory activity. We hypothesize that these lncRNAs are required for RMS tumorigenesis. Indeed, from preliminary knockdown analysis of one of these lncRNAs, lnc19_31, we observed rapid cell death in RMS cell lines and concomitant upregulation of apoptotic genes based on RNA-seq, such as TP53. Remarkably, we also observed more than 70 cancer consensus genes that were up- or downregulated, including genes that are frequently mutated in RMS patients such as BCOR and FGFR4. Within these we were excited to discover the loss of PAX3/FOX1 translocation, as this is a major driver of the alveolar subtype and often associated with poor prognosis. Taken together, we hypothesize that lncRNA deregulation is required for expression of key oncogenes and tumor suppressor and essential for RMS cell survival and tumorigenesis. These data will shed insight into a new underlying mechanism that drives this disease. These exciting findings present potential tumor vulnerabilities and, thus, new opportunities for future therapeutic discoveries that would otherwise be beyond our reach. Citation Format: Xiang Chen, Mireya Herrera-Herrera, Justina McEvoy. Novel lncRNA regulates PAX3/FOXO1 expression and is essential for RMS tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B26.

P-183 - Protection of kidney function with human antioxidation protein α1-microglobulin (A1M) in a mouse 177Lu-DOTATATE radiation therapy model

Human α1-microglobulin (A1M) is an antioxidant with reductase and radical-binding properties. A1M has been shown to protect cells and tissues against oxidative stress-related injury in vitro and in vivo. Here, we have studied the in vivo protection effect of A1M against irradiation-induced tissue damage in a mouse peptide receptor radionuclide therapy (PRRT) model. In PRRT, which is in clinical use today, infused radiolabeled peptides induce tumor killing but can also cause injury of healthy tissue, particularly the kidneys where peptides are retained. Thus, we studied the short and long-term effects of the radiolabeled peptide 177Lu-DOTATATE (DOTA), with or without co-injections of A1M, in nude BALB/c mice. Both DOTA and A1M were localized preferentially to kidneys and displayed simultaneous biodistribution in renal tubules. The DOTA-injections resulted in double-stranded DNA-breaks and up-regulation of apoptotic genes in the short-term mice, and radiation-related deaths, decreased renal function and disturbed renal histology in the long-term mice. All of these findings were counteracted with A1M. This suggests that co-administration of A1M during PRRT can increase tolerability and improve tumor control by allowing higher activity or more frequent doses.

High doses of S-methylcysteine cause hypoxia-induced cardiomyocyte apoptosis accompanied by engulfment of mitochondaria by nucleus

Despite its important role as a medicinal plant, some studies reported a toxic effect for garlic (Allium sativum) when given in higher doses. Herein, we investigated the possible cardiotoxic effects of high doses of S-methylcysteine (SMC), a water soluble organosulfur compound present in garlic. Rats were orally administered SMC at a low dose (50mg), high dose (150mg) and very high dose (300mg)/kg body weight, or saline (control) for 10days. High and very high doses of SMC resulted in a significant increase in serum cardiac injury biomarkers [aspartate transaminase (AST), lactate dehydrogenase (LDH), creatine kinase (CK) and cardiac troponin T (cTnT)], as well as oxidative stress marker nitric oxide (NO) concentration in heart and a significant decrease in cardiac superoxide dismutase (SOD) activity. Moreover, ultrastructure findings in myocardium of rats treated by high and very high doses showed inter-bundle vacuolation, loss of myofibrils, and centripetal movement of mitochondria towards nucleus. The mitochondria were partially surrounded by nuclear membrane at high dose SMC, and completely engulfed by nucleus at very high dose. This centripetal movement of mitochondria accompanied by cardiomyocytes hypoxia-induced apoptosis as evident by increasing TUNEL positive cells as well as upregulation of apoptotic genes (caspase3 and Bax), hypoxia inducible factor 1 alpha (HIF1α), dynein light chain 1 (DYNLL1) and downregulation of the anti-apoptotic marker, Bcl2. We conclude that high and very high doses of SMC cause hypoxia induced cardiomyocyte apoptosis accompanied by engulfment of mitochondria by nucleus.

Aluminum doping tunes band gap energy level as well as oxidative stress-mediated cytotoxicity of ZnO nanoparticles in MCF-7 cells.

We investigated whether Aluminum (Al) doping tunes band gap energy level as well as selective cytotoxicity of ZnO nanoparticles in human breast cancer cells (MCF-7). Pure and Al-doped ZnO nanoparticles were prepared by a simple sol-gel method. Characterization study confirmed the formation of single phase of AlxZn1-xO nanocrystals with the size range of 33–55 nm. Al-doping increased the band gap energy of ZnO nanoparticles (from 3.51 eV for pure to 3.87 eV for Al-doped ZnO). Al-doping also enhanced the cytotoxicity and oxidative stress response of ZnO nanoparticles in MCF-7 cells. The IC50 for undoped ZnO nanoparticles was 44 μg/ml while for the Al-doped ZnO counterparts was 31 μg/ml. Up-regulation of apoptotic genes (e.g. p53, bax/bcl2 ratio, caspase-3 & caspase-9) along with loss of mitochondrial membrane potential suggested that Al-doped ZnO nanoparticles induced apoptosis in MCF-7 cells through mitochondrial pathway. Importantly, Al-doping did not change the benign nature of ZnO nanoparticles towards normal cells suggesting that Al-doping improves the selective cytotoxicity of ZnO nanoparticles toward MCF-7 cells without affecting the normal cells. Our results indicated a novel approach through which the inherent selective cytotoxicity of ZnO nanoparticles against cancer cells can be further improved.

A combination of high dose rate (10X FFF/2400 MU/min/10 MV X-rays) and total low dose (0.5 Gy) induces a higher rate of apoptosis in melanoma cells in vitro and superior preservation of normal melanocytes.

The aim of this study was to determine the apoptotic effects, toxicity, and radiosensitization of total low dose irradiation delivered at a high dose rate in vitro to melanoma cells, normal human epidermal melanocytes (HEM), or normal human dermal fibroblasts (HDF) and to study the effect of mitochondrial inhibition in combination with radiation to enhance apoptosis in melanoma cells. Cells irradiated using 10X flattening filter-free (FFF) 10 MV X-rays at a dose rate of 400 or 2400 MU/min and a total dose of 0.25–8 Gy were analyzed by cell/colony counting, MitoTracker, MTT, and DNA-damage assays, as well as by quantitative real-time reverse transcriptase PCR in the presence or absence of mitochondrial respiration inhibitors. A dose rate of 2400 MU/min killed on average five-fold more melanoma cells than a dose rate 400 MU/min at a total dose of 0.5 Gy and preserved 80% survival of HEM and 90% survival of HDF. Increased apoptosis at the 2400 MU/min dose rate is mediated by greater DNA damage, reduced cell proliferation, upregulation of apoptotic genes, and downregulation of cell cycle genes. HEM and HDF were relatively unharmed at 2400 MU/min. Radiation induced upregulation of mitochondrial respiration in both normal and cancer cells, and blocking the respiration with inhibitors enhanced apoptosis only in melanoma cells. A high dose rate with a low total dose (2400 MU/min, 0.5 Gy/10X FFF 10 MV X-rays) enhances radiosensitivity of melanoma cells while reducing radiotoxicity toward HEM and HDF. Selective cytotoxicity of melanoma cells is increased by blocking mitochondrial respiration.

Anti-proliferation Effects of Isorhamnetin on Lung Cancer Cells in Vitro and in Vivo

Isorhamnetin (Iso), a novel and essential monomer derived from total flavones of Hippophae rhamnoides that has long been used as a traditional Chinese medicine for angina pectoris and acute myocardial infarction, has also shown a spectrum of antitumor activity. However, little is known about the mechanisms of action Iso on cancer cells. To investigate the effects of Iso on A549 lung cancer cells and underlying mechanisms. A549 cells were treated with 10~320 μg/ml Iso. Their morphological and cellular characteristics were assessed by light and electronic microscopy. Growth inhibition was analyzed by MTT, clonogenic and growth curve assays. Apoptotic characteristics of cells were determined by flow cytometry (FCM), DNA fragmentation, single cell gel electrophoresis (comet) assay, immunocytochemistry and terminal deoxynucleotidyl transferase nick end labeling (TUNEL) . Tumor models were setup by transplanting Lewis lung carcinoma cells into C57BL/6 mice, and the weights and sizes of tumors were measured. Iso markedly inhibited the growth of A549 cells with induction of apoptotic changes. Iso at 20 μg/ml, could induce A549 cell apoptosis, up-regulate the expression of apoptosis genes Bax, Caspase-3 and P53, and down-regulate the expression of Bcl-2, cyclinD1 and PCNA protein. The tumors in tumor-bearing mice treated with Iso were significantly smaller than in the control group. The results of apoptosis-related genes, PCNA, cyclinD1 and other protein expression levels of transplanted Lewis cells were the same as those of A549 cells in vitro. Iso, a natural single compound isolated from total flavones, has antiproliferative activity against lung cancer in vitro and in vivo. Its mechanisms of action may involve apoptosis of cells induced by down-regulation of oncogenes and up-regulation of apoptotic genes.

Pharmacokinetics and pharmacodynamics of phenethyl isothiocyanate: implications in breast cancer prevention.

Phenethyl isothiocyanate (PEITC)-a naturally occurring isothiocyanate in cruciferous vegetables-has been extensively studied as a chemopreventive agent in several preclinical species and in humans. Pharmacokinetic features of unchanged PEITC are (I) linear and first-order absorption, (II) high protein binding and capacity-limited tissue distribution, and (III) reversible metabolism and capacity-limited hepatic elimination. Membrane transport of PEITC is mediated by BCRP, multidrug resistance-associated protein (MRP) 1, and MRP2 transporters belonging to the ATP-binding-cassette (ABC) family. PEITC is metabolized by glutathione S-transferase (GST) in the liver, with the glutathione conjugate of PEITC undergoing further conversion to mercapturic acid by N-acetyl transferase in rats and humans. PEITC modulates the activity and expression of numerous phase I and phase II drug-metabolizing enzymes and can inhibit the metabolism of procarcinogens to form carcinogens and increase carcinogen elimination. In recent years, several in vitro and in vivo studies have elucidated molecular mechanisms underlying the pharmacodynamics of PEITC in breast cancer that include cancer cell apoptosis by upregulation of apoptotic genes, cell cycle arrest at G2/M phase by generation of reactive oxygen species and depletion of intracellular glutathione, downregulation of the estrogen receptor, decrease in sensitivity to estrogen, and inhibition of tumor metastasis. Inhibition of angiogenesis is one of the recently reported mechanisms of breast cancer prevention by PEITC. Complex pharmacokinetics and pharmacodynamics of PEITC necessitate a systems-biology approach in parallel with PK/PD modeling to develop PEITC as a therapeutic agent for treating cancers.