TdT) dUTP Nick-end Labeling Research Articles

The Molecular Effects of a PARP-1/-2 Inhibitor on Proton and Photon Radiotherapy in Head and Neck Cancer Cells

<h3>Purpose/Objective(s)</h3> The responses of human head and neck cancer (HNSCC) cells to photon (XRT) and proton (PRT) radiotherapy can be enhanced by a poly-(ADP-ribose) polymerase (PARP)-1 and PARP-2 inhibitor (PARPi) with undefined underlying mechanisms. We hypothesize that elucidating these mechanisms will facilitate a personalized selection of PARPi in combination with XRT or PRT. Herein, the effects of PARPi on XRT and PRT induced cell death, cell cycle distribution, and related protein expression in HNSCC cells were defined. <h3>Materials/Methods</h3> Human papillomavirus (HPV)-negative HN5, SqCC/Y1, and HPV-positive UPCI-SCC-154, UM-SCC-47 cell lines were tested. Cells were exposed to a 4 Gy PRT or XRT with or without PARPi (1 µM, 1 hour [h] before radiation). Cell cycle distribution, cell necrosis and apoptosis (staining with Annexin-VFITC) were determined using terminal deoxy-nucleotidyltransferase dUTP nickend labeling assay and were analyzed by flow cytometry. Cell mitotic catastrophe (co-staining for cytoplasm with γ- tubulin for nucleus with Dapi), and senescence (assessed using SA-β-gal) were examined. Protein expressions were determined by western blot or by flow cytometry. <h3>Results</h3> PARPi significantly increased XRT and PRT induced cellular senescence at 6 days in the two HPV+ cell lines, with higher percentages of senescence in PRT than XRT cells (<i>P</i> all < 0.05); in the two HPV- cell lines, the PARPi significantly increased the ratio of XRT-induced senescence (<i>P</i> all < 0.01). PARPi caused significantly more cells to undergo mitotic catastrophe after PRT (in 3 cell lines) or XRT (in the two HPV- cell lines), with higher rates of mitotic catastrophe induced by PARPi in PRT vs. XRT group (<i>P</i> all < 0.05). The PARPi led to a trend (non-significant) of increased cell necrosis at 48 h after PRT (in 3 cell lines) or XRT (in 2 cell lines). No effect of PARPi on XRT or PRT induced apoptosis was noted. At 24 h, PARPi significantly prolonged XRT (in two HPV- cell lines) and PRT (in HN5 cells) induced cell cycle arrest at G2/M phase; trends of this prolongation were noted after PRT in SqCC/Y1 and UPCI-SCC-154 cells, and after XRT in UPCI-SCC-154 cells. PARPi caused trends of increasing ratios of specific protein (53BP1 and RAD51 [DNA repair related]; Ki-67 [cell cycle related]) abundant cells at 24 h after PRT (in two HPV- cell lines); and increased cleaved caspase-3 expression after XRT or PRT in four cell lines tested. <h3>Conclusion</h3> In conclusion, PARPi induced higher levels of mitotic catastrophe in combination with PRT versus XRT, promoted XRT and PRT induced senescence, while it led to a trend of increased cell necrosis after PRT and XRT. These upregulating effects of PARPi were cell line dependent. Cell line based delayed DNA damage repair and increase in radiation-induced cell cycle arrest at G2/M phase may have served as parts of the underlying mechanisms. Further studies on personalized therapy of XRT or PRT in combination with PARPi to improve the efficacy of radiotherapy are warranted.

P–124 The beneficial effects of novel quercetin delivery system (loaded on bigel) on male fertility parameters of Non-alcoholic fatty liver model: an in-vivo (rat model) study

Abstract Study question Dose quercetin encapsulated in a bigel slow- release delivery system improve male fertility parameters in Non-alcoholic fatty liver (NAFLD) model Summary answer Quercetin in a bigel slow- release delivery system can boost semen parameters in NAFLD rat model What is known already Recent molecular and physiological studies have shown that adverse effects of NAFLD extend far beyond the liver. NAFLD can impair male reproductive function by increasing Reactive Oxidative Stress (ROS) levels, reducing the expression of antioxidant genes and inducing damage in testes immune privilege. Antioxidant therapy and its effectiveness depend on whether the exogenous antioxidant will be readily absorbed to reach high enough that are required to decrease the pathological damages. Quercetin, as an antioxidant, is able to ameliorate oxidative stress. The design of new drug delivery systems using encapsulating antioxidant can boost its durability and effectiveness. Study design, size, duration Bigels were prepared using cottonseed oil/cannabis oil/alginate/ferula gum. Sprague-Dawley rats are housed for 2 weeks, then NAFLD was induced by 58% of dietary calorie as lard and 42 g/L fructose for 16 weeks. The experimental protocol was approved by the ethical committee of Zanjan University of Medical Sciences, Zanjan, Iran. Participants/materials, setting, methods After confirming the NAFLD induction, animals divided into five groups: Control, control NAFLD, received 2 mg/kg Quer loaded on bigels, free bigels, free Quer for 45 days as daily gavage. Semen parameters (count, motility, and morphology), viability (Eosin-nigrosine staining) and serum testosterone levels were analyzed. In addition, histological sections of testicular tissues were investigated by Hematoxylin-Eosin (H&amp;E) staining method. In situ detection of apoptosis was performed using terminal deoxynucleotidyl-transferase dUTP nick end labeling (TUNEL) assay. Main results and the role of chance The sperm count, sperm motility, normal morphology and testosterone level were significantly lower in the NAFLD group than those the controls. Moreover, higher head and tail abnormality percentages were seen in the sperm of these groups. Bigel-Quer significantly improved the serum testosterone level, sperm count, motility, and morphology compared with the NAFLD group. Spermatogenic cells in all stages of differentiation (spermatogonia, primary spermatocytes, early spermatids, late spermatids) are observed and preserved normally in the testicular tubules and lumen filled with mature sperms in the control group. Interestingly, atrophic changes in the testicular tubule architecture with swelling in spermatogonia cells, detachment from tubule membrane, reduced number of mature sperm, and reduced lumen thickness were seen in the NAFLD. In the Quer, bigel and bigel-Quer-treated groups, swelling and vacuolation rate of germ cells decreased. The testicular morphology, and tubule structure were significantly normalized, especially in the bigel-Quer-treated group. Serum testosterone levels significantly increased and reached the healthy control group in the bigel-Quer group. TUNEL-positive cells in testes increased significantly after NAFLD induction. Quantitative analysis showed a significant decrease in testicular TUNEL-positive cells following bigel-Quer treatment, but not in other groups. Limitations, reasons for caution Keeping and daily handling of animals for long-time in animal house for diet-induced NAFLD. NAFLD requires long periods of treatment to get the desired outcome especially in the case of sperm parameters investigation. Wider implications of the findings: The bigel showed synergistic effects with Quer for treating infertility in male rats with NAFLD. Stability and bio-availability of Quer are important aspects that should be considered to justify its supplementation. Empowering antioxidant shield of NAFLD patients by Quer supplementation can improve various damage effects and clinical status of diseases. Trial registration number Not applicable

Differential Response of Müller Cells and Microglia in a Mouse Retinal Detachment Model and Its Implications in Detached and Non-Detached Regions.

Retinal detachment (RD) is a sight-threatening condition, leading to photoreceptor cell death; however, only a few studies provide insight into its effects on the entire retinal region. We examined the spatiotemporal changes in glial responses in a mouse RD model. In electroretinography, a- and b-waves were reduced in a time-dependent manner. Hematoxylin and eosin staining revealed a gradual decrease in the outer nuclear layer throughout the retinal region. Terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay showed that TUNEL-positive photoreceptors increased 5 days after RD and decreased by 14 days. Glial response was evaluated by immunohistochemistry using antibodies against glial fibrillary acidic protein (GFAP, Müller glial marker) and Iba-1 (microglial marker) and osteopontin (OPN, activated microglial marker). GFAP immunoreactivity increased after 7 days in complete RD, and was retained for 14 days. OPN expression increased in microglial cells 3–7 days after RD, and decreased by 14 days in the detached and border regions. Although OPN was not expressed in the intact region, morphologically activated microglial cells were observed. These retinal glial cell responses and photoreceptor degeneration in the border and intact regions suggest that the effects of RD in the border and intact retinal regions need to be understood further.

Ribonucleic acid-binding protein CPSF6 promotes glycolysis and suppresses apoptosis in hepatocellular carcinoma cells by inhibiting the BTG2 expression

Hepatocellular carcinoma (HCC) is currently the sixth most common malignancy and the second major cause of tumor-related deaths in the world. This study aimed to investigate the role of cleavage and polyadenylation factor-6 (CPSF6) and B-cell translocation gene 2 (BTG2) in regulating the glycolysis and apoptosis in HCC cells. The RNA and protein expression of CPSF6 and BTG2 in normal hepatocyte and HCC were, respectively, detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis and Western blot analysis. The viability and apoptosis of transfected Huh-7 cells were, respectively, analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. The expression of apoptosis-related proteins and HK-2 in transfected Huh-7 cells was also detected by Western blot analysis. The levels of glucose and lactate in the culture supernatant of transfected Huh-7 cells were, respectively, detected with the glucose assay kit and lactate assay kit. The interaction of CPSF6 and BTG2 was confirmed by RNA binding protein immunoprecipitation (RIP) assay. As a result, CPSF6 expression was increased while BTG2 expression was decreased in Huh-7 cells. Interference with CPSF6 suppressed the viability and glycolysis, and promoted the apoptosis of Huh-7 cells. Furthermore, CPSF6 interacted with BTG2 and interference with CPSF6 upregulated the BTG2 expression and inhibited the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK)/nuclear factor (NF)-κB pathway. Interference with BTG2 could partially reverse the above cell changes caused by interference with CPSF6. In conclusion, CPSF6 inhibited the BTG2 expression to promote glycolysis and suppress apoptosis in HCC cells by activating AKT/ERK/NF-κB pathway.

7,8-Dihydroxyflavone suppresses proliferation and induces apoptosis of human osteosarcoma cells.

Recent studies suggest that 7,8-dihydroxyflavone (7,8-DHF) inhibits the development of several tumors. However, its role in osteosarcoma (OS) remains unknown. This study was designed to investigate the effects and underlying mechanisms of 7,8-DHF that may influence OS development. Human OS cell lines (U2OS and 143B) were treated with 7,8-DHF; cell viability and cell migration were assessed by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and wound-healing assay, respectively; and cell death and apoptosis were evaluated by LIVE/DEAD staining and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, respectively. Reactive oxygen species production was measured using 2,7-dichlorodihydrofluorescein diacetate probe. Akt, Bcl-xL/Bcl-2 asociated death promoter (Bad), p38 mitogen-activated protein kinase (MAPK), extracellular regulated protein kinase (ERK), and c-Jun N-terminal kinase (JNK) expression and their respective phosphorylation levels were detected by western blot analysis. We found that 7,8-DHF reduced cell viability in a dose-dependent manner and also promoted apoptosis, inhibited migration, and induced oxidative stress in OS cells. Moreover, 7,8-DHF inhibited Akt, Bad, and p38MAPK, but activated ERK and JNK signals. In summary, our results suggest that 7,8-DHF inhibits OS progression, possibly by regulating Akt/Bad and MAPK signaling. These findings provide new evidence for the pharmacological effects of 7,8-DHF that may improve drug therapy for OS patients.

Melamine exposure from the weaning period causes apoptosis, inflammation, and damage to the blood-brain barrier

The aim of this study to investigate the effects of melamine exposure from the weaning period on the developing brain in rats. Female Wistar albino rats (21 days old, n = 18) were divided into 3 groups, all animals were weighed daily and dose-adjusted. For 3 weeks, 0.1 mL of saline was administered by oral gavage to the control group, 50 mg/kg of melamine to the second group, and 75 mg/kg of melamine to the third group were administered by oral gavage by dissolving in 0.1 mL of saline. On the postnatal 45th day, the rats were sacrificed under anesthesia and brain tissues placed in neutral formalin. After routine tissue processing, brain sections were stained with hematoxylin&eosin(H&E) and Terminal deoxynucleotidyl transferase(TdT) dUTP Nick-End Labeling(TUNEL), IBA-1, Glial Fibrillar Acidic Protein(GFAP), Tumor necrosis factor-α (TNF-α), and SMI-70 antibodies as immunohistochemically. In the results, according to apoptotic index(AI) results, there was a significant increase in the 75 mg/kg and 50 mg/kg melamine groups compared to the control groups (p < 005). There was a significant increase in the number of anti- TNF-α positive neurons and the number of anti-GFAP positive astrocytes in both melamine groups compared to the control group (p < 001). In terms of SMI-71, an increase was found in the 75 mg/kg group compared to other groups (p < 001), while no significant difference was found between the groups in terms of IBA-1 (p > 0.05). It has been observed an increase in dilatation of blood vessels, inflammatory cell infiltration, and endothelial cell degeneration in the 50 mg/kg and 75 mg/kg melamine groups compared to the control group (p < 0.01). there was no statistically significant difference in the body and brain weight between both melamine treatment groups (75 mg/kg and 50 mg/kg) and the control group (p > 005). Melamine exposure (50 mg/kg and 75 mg/kg) from the weaning period causes apoptosis and inflammation in the developing brain, and the disruptions in the blood-brain barrier (BBB) significantly increase exposure to 75 mg/kg.

Retinoic Acid Induced Protein 14 (Rai14) is dispensable for mouse spermatogenesis.

BackgroundRetinoic Acid Induced Protein 14 (Rai14) is an evolutionarily conserved gene that is highly expressed in the testis. Previous experiments have reported that small interfering RNA (siRNA)-mediated gene knockdown (KD) of Rai14 in rat testis disrupted spermatid polarity and transport. Of note, a gene knockout (KO) model is considered the “gold standard” for in vivo assessment of crucial gene functions. Herein, we used CRISPR/Cas9-based gene editing to investigate the in vivo role of Rai14 in mouse testis.MethodsSperm concentration and motility were assayed using a computer-assisted sperm analysis (CASA) system. Histological and immunofluorescence (IF) staining and transmission electron microscopy (TEM) were used to visualize the effects of Rai14 KO in the testes and epididymides. Terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) was used to determine apoptotic cells. Gene transcript levels were calculated by real-time quantitative PCR.ResultsRai14 KO in mice depicted normal fertility and complete spermatogenesis, which is in sharp contrast with the results reported previously in a Rai14 KD rat model. Sperm parameters and cellular apoptosis did not appear to differ between wild-type (WT) and KO group. Mechanistically, in contrast to the well-known role of Rai14 in modulating the dynamics of F-actin at the ectoplasmic specialization (ES) junction in the testis, morphological changes of ES junction exhibited no differences between Rai14 KO and WT testes. Moreover, the F-actin surrounded at the ES junction was also comparable between the two groups.ConclusionIn summary, our study demonstrates that Rai14 is dispensable for mouse spermatogenesis and fertility. Although the results of this study were negative, the phenotypic information obtained herein provide an enhanced understanding of the role of Rai14 in the testis, and researchers may refer to these results to avoid conducting redundant experiments.

MiR-182 inhibits oxidative stress and epithelial cell apoptosis in lens of cataract rats through PI3K/Akt signaling pathway.

The aim of this study was to investigate the influences of micro ribonucleic acid (miR)-182 on oxidative stress and epithelial cell apoptosis in the lens of cataract rats through the phosphatidylinositol 3-hydroxy kinase/protein kinase B (PI3K/Akt) pathway. A total of 36 Sprague-Dawley rats were randomly assigned into three groups, including normal group (n=12), model group (n=12), and miR-182 mimics group (n=12). Rats in normal group were first normally fed. After establishing the cataract model, rats in model group were intraperitoneally injected with normal saline. Meanwhile, rats in miR-182 mimics group were intraperitoneally injected with miR-182 mimics. At 7 d after operation, materials were sampled. The expressions of B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax) were detected via immunofluorescence. The protein expressions of PI3K and Akt were detected using Western blotting. Moreover, the expression level of miR-182 was measured via qPCR. Cell apoptosis was evaluated using terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL). In addition, the content of superoxide dismutase (SOD) and malondialdehyde (MDA) was determined using enzyme-linked immunosorbent assay (ELISA). Compared with normal group, both model group and miR-182 mimics group exhibited significantly up-regulated expression level of Bax and down-regulated expression of Bcl-2 (p<0.05). MiR-182 mimics group had markedly lower expression level of Bax and higher expression level of Bcl-2 than model group (p<0.05). Western blotting results demonstrated that the protein expression levels of PI3K and Akt in model and miR-182 mimics groups were considerably higher than those in normal group (p<0.05). Meanwhile, their protein expression levels in miR-182 mimics group were significantly higher than those in model group (p<0.05). In comparison with normal group, the expression level of miR-182 was markedly up-regulated in both model group and miR-182 mimics group (p<0.05). Moreover, its expression level in miR-182 mimics group was considerably higher than that in model group (p<0.05). TUNEL-positive cells increased significantly in both model group and miR-182 mimics group when compared with normal group (p<0.05). However, they were remarkably reduced in miR-182 mimics group when compared with model group (p<0.05). Compared with normal group, model and miR-182 groups exhibited substantially decreased SOD content and increased MDA content (p<0.05). MiR-182 inhibits oxidative stress and epithelial cell apoptosis in the lens of cataract rats by activating the PI3K/Akt signaling pathway.

Increased FOXM1 Expression by Cisplatin Inhibits Paclitaxel-Related Apoptosis in Cisplatin-Resistant Human Oral Squamous Cell Carcinoma (OSCC) Cell Lines.

Cisplatin and paclitaxel are commonly used to treat oral cancer, but their use is often limited because of acquired drug resistance. Here, we tested the effects of combined cisplatin and paclitaxel on three parental (YD-8, YD-9, and YD-38) and three cisplatin-resistant (YD-8/CIS, YD-9/CIS, and YD-38/CIS) oral squamous cell carcinoma (OSCC) cell lines using cell proliferation assays and combination index analysis. We detected forkhead box protein M1 (FOXM1) mRNA and protein expression via real-time qPCR and Western blot assays. Cell death of the cisplatin-resistant cell lines in response to these drugs with or without a FOXM1 inhibitor (forkhead domain inhibitory compound 6) was then measured by propidium iodide staining and TdT dUTP nick end labeling (TUNEL) assays. In all six OSCC cell lines, cell growth was more inhibited by paclitaxel alone than combination therapy. Cisplatin-induced overexpression of FOXM1 showed the same trend only in cisplatin-resistant cell lines, indicating that it was associated with inhibition of paclitaxel-related apoptosis. In summary, these results suggest that, in three cisplatin-resistant cell lines, the combination of cisplatin and paclitaxel had an antagonistic effect, likely because cisplatin blocks paclitaxel-induced apoptosis. Cisplatin-induced FOXM1 overexpression may explain the failure of this combination.

Apoptosis of germ cells in the normal testis of the Japanese quail (Coturnix coturnix japonica)

It has been established that excess germ cells in normal and in pathological conditions are removed from testicular tissue by the mechanism of apoptosis. Studies on germ cell apoptosis in avian species are grossly lacking, and there are only a few reports on induced germ cell degenerations in the testis tissue of birds. This study was designed to investigate the process of apoptosis of germ cells in the Japanese quail (Coturnix coturnix japonica). Germ cell degenerations were investigated in birds of all age groups, namely pre-pubertal, pubertal, adult, and aged. Apoptosis of germ cells in the quails, as shown by hematoxylin & eosin (H&E), TdT dUTP Nick End Labeling (TUNEL) assay and electron microscopy, was similar to that observed in previous studies of germ cells and somatic cells of mammalian species. The observed morphological features of these apoptotic cells ranged from irregular plasma and nuclear membranes in the early stage of apoptosis to rupture of the nuclear membrane, condensation of nuclear material, as well as fragments of apoptotic bodies, in later stages of apoptosis. In the TUNEL-positive cell counts, there was a significant difference between the mean cell counts for the four age groups (P < 0.05). Post hoc analysis revealed a highly significant difference in the aged group relative to the pubertal and adult age groups, while the cell counts of the pre-pubertal group were significantly higher than those of the pubertal group. However, there was no significant difference between cell counts of the pre-pubertal and the adult, and between the pre-pubertal and the aged groups.

Fraxetin inhibits interleukin-1β-induced apoptosis, inflammation, and matrix degradation in chondrocytes and protects rat cartilage in vivo

Osteoarthritis (OA) is a disease characterized by degeneration of the joint complex due to cartilage destruction. Fraxetin, a widely used and studied coumarin compound extracted from a traditional Chinese herb (Qin Pi), has shown anti-inflammatory and antioxidant properties, but its effects on OA have not been studied. In the present study, western blotting, immunofluorescence, and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) were used to evaluate the effects of fraxetin on IL-1β-induced apoptotic activity, inflammatory responses, and catabolism in rat chondrocytes. The results showed that fraxetin prevented IL-1β-induced apoptosis of chondrocytes and inhibited inflammatory mediator release by regulating the Toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)/nuclear factor (NF)-κB pathway in chondrocytes. Additionally, fraxetin suppressed the upregulation of matrix metalloproteinase 13 (MMP13) and degradation of collagen II in the extracellular matrix (ECM). Moreover, the effects of fraxetin in vivo were assessed in a monosodium iodoacetate (MIA)-induced rat model of OA using hematoxylin and eosin (H&E) and Safranin O-fast green staining and magnetic resonance imaging (MRI). The results showed that fraxetin protected the cartilage against destruction. In conclusion, fraxetin could be a potential therapeutic for OA.

Detection of Embryonic Suspensor Cell Death by Whole-Mount TUNEL Assay in Tobacco.

Embryonic suspensor in angiosperms is a short-lived structure that connects the embryo to surrounding maternal tissues, which is necessary for early embryogenesis. Timely degeneration via programed cell death is the most distinct feature of the suspensor during embryogenesis. Therefore, the molecular mechanism regulating suspensor cell death is worth in-depth study for embryonic development. However, this process can hardly be detected using conventional methods since early embryos are deeply embedded in the seed coats and inaccessible through traditional tissue section. Hence, it is necessary to develop a reliable protocol for terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) analysis using limited living early embryos. Here, we provide a detailed protocol for the whole-mount detection of suspensor cell death using a TUNEL system in tobacco. This method is especially useful for the direct and rapid detection of the spatial-temporal characters of programed cell death during embryogenesis, as well as for the diminishment of the artifacts during material treatment by traditional methods.

Long non-coding RNA LINC00858 aggravates the oncogenic phenotypes of ovarian cancer cells through miR-134-5p/RAD18 signaling.

Ovarian cancer is a common gynecological cancer. Herein, we focused on the function and probable mechanisms of LINC00858 in ovarian cancer. Real-time quantitative polymerase chain reaction (RT-qPCR) was employed for detecting the expression of LINC00858, miR-134-5p and RAD18 E3 ubiquitin protein ligase (RAD18). Cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) and apoptosis were detected by cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), transwell, terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) and western bolt experiments, as appropriate. Interplays between LINC00858, miR-134-5p and RAD18 were detected by RNA immunoprecipitation (RIP), RNA pull down and luciferase reporter assays. LINC00858 were up-regulated in ovarian cancer tissues and cells, and its expression was elevated in advanced samples compared to early ones. Knocking down LINC00858 inhibited cell proliferation, motility and EMT, but accelerated cell apoptosis in ovarian cancer. Moreover, could be sponged by LINC00858 sponged miR-134-5p to enhance RAD18 expression in ovarian cancer. Also, silenced RAD18 could also restrain oncogenic behaviors of ovarian cancer cells. Rescue experiments showed that overexpressing RAD18 reversed the effects caused by knocking down LINC00858 on cellular processes. LINC00858 sequestered miR-134-5p to elevate RAD18 expression, resulting in aggravated development of ovarian cancer. This might provide promising targets for treating patients with ovarian cancer.

Hydroxytyrosol enhances cisplatin-induced ototoxicity: Possible relation to the alteration in the activity of JNK and AIF pathways

Hydroxytyrosol (HT), a polyphenol widely contained as an ester in olive fruits and olive leaves, exhibits a broad spectrum of effectiveness. The present study was designed to investigate the effect of HT alone as well as in the combination with cisplatin on the House Ear Institute-Organ of Corti 1 cells (HEI-OC1) and C57BL/6 cochlear hair cells in vitro. The cell viability was measured by cell counting kit-8 (CCK8) assay. The levels of reactive oxygen species were evaluated by Dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining. The expression of phosphorylated Jun N-terminal kinase (p-JNK) and cleaved-caspase 3 was assessed by Western blotting. The apoptosis was detected by terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) staining. The distribution of apoptosis inducing factor (AIF) was determined by immunofluorescent staining. HT alleviated the levels of reactive oxygen species in both untreated state and after cisplatin stimulus. However, HT at concentration of 100 μM decreased the cell viability of HEI-OC1 from 100 ± 17.38% in control group to 50.17 ± 1.89% and increased the expression of p-JNK and c-caspase 3 from 0.62 ± 0.10, 0.20 ± 0.050 in the control group to 1.24 ± 0.18, 0.85 ± 0.18 in the group treated with 30 μM cisplatin, as well as to 1.64 ± 0.14, 1.44 ± 0.12 in the group with 30 μM cisplatin +100 μM HT, respectively. Meanwhile, HT triggered AIF transferring to nuclei and, also, led to cochlear HCs arranging disorderly and missing. Moreover, HT elevated the expression of p-JNK and c-caspase 3 from 1.00 ± 0.27, 1.00 ± 0.26 in the control group to 2.23 ± 0.24, 22.87 ± 3.80 in the group with 30 μM cisplatin, and to 2.75 ± 0.23, 31.56 ± 3.86 in the group with 30 μM cisplatin+100 μM HT correspondingly. Taken together, data from this work reveal that HT itself possesses toxic effect on HCs mainly thorough AIF-dependent apoptosis, while, it aggravates the ototoxicity-caused by cisplatin via both JNK and AIF pathways related apoptosis. Findings from this work offer clear evidence that that HT might not be recommended to utilize for preventing cisplatin-induced ototoxicity.

Effects of MC-LR on histological structure and cell apoptosis in the kidney of grass carp (Ctenopharyngodon idella)

Microcystin-LR (MC-LR) is a well-known hepatotoxin; however, increasing evidence suggests that it might induce kidney injury. Grass carp (Ctenopharyngodon idella) is one of the most important farmed species and may be affected by MC-LR releasing into waterbody during cyanobacterial bloom. Here, this present study aimed to explore the nephrotoxicity of grass carp by MC-LR. The grass carp received a single intraperitoneal injection of different doses of MC-LR (0, 25, 75, and 100 μg/kg body weight (BW)), and the kidneys were isolated at 24 and 96 h post-injection (hpi). Histopathological examination revealed kidney lesions, with severe hemorrhage, necrosis of the interstitium, and dilation of Bowman's capsule in the 75 and 100 μg MC-LR/kg BW groups. Under transmission electron microscopy, a larger number of swelling and vacuolated degeneration of mitochondria were observed; moreover, apoptotic features, such as condensed chromatin and shrinkage of cells, were observed in the 75 and 100 μg MC-LR/kg BW groups at 96 hpi. MC-LR significantly upregulated the number of apoptotic cells in the 75 and 100 μg/kg BW groups at 96 hpi as indicated by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay (P < 0. 05). The results of quantitative assays showed that the mRNA expression of Bax, caspase-9, and caspase-3 in grass carp kidney were significantly increased at 96 hpi in the 75 and 100 μg MC-LR/kg BW groups compared with that in the control group, but Bcl-2 mRNA expression was significantly decreased in all the treatment groups at 24 and 96 hpi. Taken together, these results indicated that MC-LR damaged the kidney structure and resulted in renal apoptosis which may occur via the mitochondrial pathway.

Baicalin Suppresses Bilirubin-Induced Apoptosis and Inflammation by Regulating p38 Mitogen-Activated Protein Kinases (MAPK) Signaling in Neonatal Neurons.

BackgroundHyperbilirubinemia is associated with central nervous system damage in preterm neonates due to the neurotoxicity of bilirubin. This study explored the possible mechanisms of bilirubin’s neurotoxicity, and the protective effect of baicalin (BAI) was also investigated.Material/MethodsIsolated neonatal rat hippocampal neurons were exposed to free bilirubin (Bf). BAI was used to treat these neurons. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cell viability. Terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assay was used to detect apoptosis. Contents of inflammatory cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Protein expression and phosphorylation levels were assessed by Western blotting. Nuclear translocation was observed by immunofluorescent staining.ResultsBf incubation significantly induced apoptosis and decreased viabilities of neurons. The phosphorylation levels of MAP kinase kinase (MKK)3, MKK6, p38 mitogen-activated protein kinases (MAPK), nuclear translocation level of p65, and the expression levels of cleaved caspase3 and tumor necrosis factor (TNF)α were found to be dramatically higher in Bf-incubated neurons. BAI pre-treatment, however, increased cell viability by reducing cell apoptosis. BAI pre-treatment also reduced phosphorylation levels of MKK3, MKK6, p38 MAPK, and nuclear translocation level of p65, as well as the expression levels of cleaved caspase3 and TNFα, in Bf-incubated neurons.ConclusionsBAI suppressed bilirubin-induced neuron apoptosis and inflammation by deactivating p38 MAPK signaling.

N-acetyl-l-cysteine mediated regulation of DNA fragmentation, an apoptotic event, against methoxychlor toxicity in the granulosa cells of ovarian antral follicles

Methoxychlor (MXC), an organo-chlorine insecticide, is a reproductive toxicant in females, causing apoptosis-mediated follicular atresia. To elucidate the potentials of Methoxychlor as a geno-toxicant, granulosa cells of healthy antral follicles, exposed to MXC and antioxidant, N-acetyl-l-cysteine, were studied by the terminal deoxynucleotidyltransferase-dUTP nick end-labelling and single-cell gel electrophoresis (comet) assays.MXC caused DNA fragmentation, as revealed by the increased incidence of dark brown condensed TUNEL positive cells in contrast with lightly brown TUNEL negative cells with maximum TUNEL positive cells were observed in 100 μg/mL MXC treated groups. Quantitatively, maximum geno-toxicity was exhibited at highest MXC treatment with percent tail DNA as 17.87 ± 0.85, 41.16 ± 3.94, and 47.73 ± 3.71 in comparison with control (0.65 ± 0.03, 2.91 ± 0.27, and 7.16 ± 1.39) after 24, 48 and 72 h exposure duration, respectively. MXC treated groups exhibited Type 1-Type 3 comets as compared to Type 0 comets in control groups. Supplementation of NAC led to significant (p < 0.05) decline in geno-toxicity in MXC treated groups with maximum amelioration observed at 5 and 10 mM.Consequently, increased DNA damage attributed to the granulosa cells apoptosis in response to Methoxychlor exposure was significantly combated by NAC supplementation, preventing the geno-toxicity induced cyto-toxicity in GCs.

The protective effect of betanin and copper on spinal cord ischemia–reperfusion injury

Context: Both copper and betanin have been implicated as having significant bioactivity against ischemic damage in a variety of experimental and clinical settings. The aim of this study is to investigate whether betanin and copper have any protective effect on spinal cord in an ischemia–reperfusion (I/R) model in rats. Design: Spraque-Dawley rats were used in four groups: Sham group (n = 7), control group (laparotomy and cross-clamping of aorta, n = 7), betanin treatment group (dosage of 100 mg/kg of betanin administered intraperitoneally (i.p.) 60 min before laparotomy, n = 7), copper sulfate treatment group (administered copper sulfate i.p. at a dose of 0.1 mg/kg/day for 7 days before laparotomy, n = 7). Malondialdehyde (MDA), glutathione (GSH) levels, myeloperoxidase (MPO) and superoxide dismutase (SOD) activity were measured. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was also performed to evaluate apoptosis. Setting: Kafkas University, Faculty of Medicine, Kars, Turkey. Results: I/R injury was successfully demonstrated with the surgical model. Betanin and copper treatment significantly decreased MDA levels, MPO activity and the number of apoptotic cells in the spinal cord. Betanin and copper treatment significantly increased GSH levels. Copper treatment significantly increased SOD activity, whereas betanin was not as effective. Apoptotic cells were significantly decreased in both treatment groups. Conclusion: I/R injury of the spinal cord can be successfully demonstrated by aortic clamping in this surgical model. Betanin/Copper sulphate has ameliorative effects against operative I/R injury. Low toxicity of those agents makes them ideal targets for clinical research for this purpose.

MiR-26a inhibits myocardial cell apoptosis in rats with acute myocardial infarction through GSK-3β pathway.

To study the influence of micro ribonucleic acid (miR)-26a on myocardial cell apoptosis in rats with acute myocardial infarction (AMI) through the glycogen synthase kinase 3 beta (GSK-3β) pathway. A total of 36 Sprague-Dawley rats were randomly divided into sham group (n=12), model group (n=12), and miR-26a mimics group (n=12). Only the heart was exposed, and normal saline was intraperitoneally injected postoperatively in sham group, and the model of AMI was prepared in model group. Besides, after modeling, miR-26a mimics were injected into the left ventricle in miR-26a mimics group. At 48 h after operation, sampling was performed. Then, the expressions of B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax), as well as the protein expression of phosphorylated GSK-3β (p-GSK-3β) were detected via immunohistochemistry and Western blotting, respectively. Moreover, the expression level of miR-26a was measured via quantitative polymerase chain reaction (qPCR), and cell apoptosis was evaluated using terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. Compared with those in sham group, the expression level of Bax was substantially raised, but that of Bcl-2 was notably lowered in model group and miR-26 mimics group (p<0.05), and miR-26 mimics group had a markedly lower expression level of Bax and a remarkably higher expression level of Bcl-2 than model group (p<0.05). According to Western blotting results, the protein expression level of p-GSK-3β in model and miR-26a mimics groups was considerably higher than that in sham group (p<0.05), and miR-26a mimics group exhibited a notably higher protein expression level of p-GSK-3β than model group (p<0.05). In comparison with that in sham group, the expression level of miR-26a rose markedly in both model group and miR-26a mimics group (p<0.05), and its expression level in miR-26a mimics group was dramatically higher than that in model group (p<0.05). Additionally, the TUNEL-positive cells were considerably increased in both model group and miR-26a mimics group in comparison with that in sham group (p<0.05), and miR-26a mimics group had markedly fewer TUNEL-positive cells than model group (p<0.05). MiR-26a activates the GSK-3β signaling pathway to inhibit myocardial cell apoptosis after AMI.

Molecular Imaging of Extracellular Tumor pH to Reveal Effects of Locoregional Therapy on Liver Cancer Microenvironment.

To establish magnetic resonance (MR)-based molecular imaging paradigms for the noninvasive monitoring of extracellular pH (pHe) as a functional surrogate biomarker for metabolic changes induced by locoregional therapy of liver cancer. Thirty-two VX2 tumor-bearing New Zealand white rabbits underwent longitudinal imaging on clinical 3T-MRI and CT scanners before and up to 2 weeks after complete conventional transarterial chemoembolization (cTACE) using ethiodized oil (lipiodol) and doxorubicin. MR-spectroscopic imaging (MRSI) was employed for pHe mapping. Multiparametric MRI and CT were performed to quantify tumor enhancement, diffusion, and lipiodol coverage of the tumor posttherapy. In addition, incomplete cTACE with reduced chemoembolic doses was applied to mimic undertreatment and exploit pHe mapping to detect viable tumor residuals. Imaging findings were correlated with histopathologic markers indicative of metabolic state (HIF-1α, GLUT-1, and LAMP-2) and viability (proliferating cell nuclear antigen and terminal deoxynucleotidyl-transferase dUTP nick-end labeling). Untreated VX2 tumors demonstrated a significantly lower pHe (6.80 ± 0.09) than liver parenchyma (7.19 ± 0.03, P < 0.001). Upregulation of HIF-1α, GLUT-1, and LAMP-2 confirmed a hyperglycolytic tumor phenotype and acidosis. A gradual tumor pHe increase toward normalization similar to parenchyma was revealed within 2 weeks after complete cTACE, which correlated with decreasing detectability of metabolic markers. In contrast, pHe mapping after incomplete cTACE indicated both acidic viable residuals and increased tumor pHe of treated regions. Multimodal imaging revealed durable tumor devascularization immediately after complete cTACE, gradually increasing necrosis, and sustained lipiodol coverage of the tumor. MRSI-based pHe mapping can serve as a longitudinal monitoring tool for viable tumors. As most liver tumors are hyperglycolytic creating microenvironmental acidosis, therapy-induced normalization of tumor pHe may be used as a functional biomarker for positive therapeutic outcome.