Significant Decrease In Cell Survival Research Articles

Transcriptome Analysis Reveals the Important Role of Vitamin B12 in the Response of Natronorubrum daqingense to Salt Stress.

Natronorubrum daqingense JX313T is an extremely halophilic archaea that can grow in a NaCl-saturated environment. The excellent salt tolerance of N. daqingense makes it a high-potential candidate for researching the salt stress mechanisms of halophilic microorganisms from Natronorubrum. In this study, transcriptome analysis revealed that three genes related to the biosynthesis of vitamin B12 were upregulated in response to salt stress. For the wild-type (WT) strain JX313T, the low-salt adaptive mutant LND5, and the vitamin B12 synthesis-deficient strain ΔcobC, the exogenous addition of 10 mg/L of vitamin B12 could maximize their cell survival and biomass in both optimal and salt stress environments. Knockout of cobC resulted in changes in the growth boundary of the strain, as well as a significant decrease in cell survival and biomass, and the inability to synthesize vitamin B12. According to the HPLC analysis, when the external NaCl concentration (w/v) increased from 17.5% (optimal) to 22.5% (5% salt stress), the intracellular accumulation of vitamin B12 in WT increased significantly from (11.54 ± 0.44) mg/L to (15.23 ± 0.20) mg/L. In summary, N. daqingense is capable of absorbing or synthesizing vitamin B12 in response to salt stress, suggesting that vitamin B12 serves as a specific compatible solute effector for N. daqingense during salt stress.

Effect of bisphosphonates and statins on the in vitro radiosensitivity of breast cancer cell lines.

Early-stage breast cancer is usually treated with breast-conserving surgery followed by adjuvant radiation therapy. Acute skin toxicity is a common radiation-induced side effect experienced by many patients. Recently, a combination of bisphosphonates (zoledronic acid) and statins (pravastatin), or ZOPRA, was shown to radio-protect normal tissues by enhancing DNA double-strand breaks (DSB) repair mechanism. However, there are no studies assessing the effect of ZOPRA on cancerous cells. The purpose of this study is to characterize the in vitro effect of the zoledronic acid (ZO), pravastatin (PRA), and ZOPRA treatment on the molecular and cellular radiosensitivity of breast cancer cell lines. Two breast cancer cell lines, MDA MB 231 and MCF-7, were tested. Cells were treated with different concentrations of pravastatin (PRA), zoledronate (ZO), as well as their ZOPRA combination, before irradiation. Anti-γH2AX and anti-pATM immunofluorescence were performed to study DNA DSB repair kinetics. MTT assay was performed to assess cell proliferation and viability, and flow cytometry was performed to analyze the effect of the drugs on the cell cycle distribution. The clonogenic assay was used to assess cell survival. ZO, PRA, and ZOPRA treatments were shown to increase the residual number of γH2AX foci for both cell lines. ZOPRA treatment was also shown to reduce the activity of the ATM kinase in MCF-7. ZOPRA induced a significant decrease in cell survival for both cell lines. Our findings show that pretreatment with ZOPRA can decrease the radioresistance of breast cancer cells at the molecular and cellular levels. The fact that ZOPRA was previously shown to radioprotect normal tissues, makes it a good candidate to become a therapeutic window-widening drug.

Age-related changes in mice behavior and the contribution of lipocalin-2.

Aging causes considerable changes in the nervous system, inducing progressive and long-lasting loss of physiological integrity and synaptic plasticity, leading to impaired brain functioning. These age-related changes quite often culminate in behavioral dysfunctions, such as impaired cognition, which can ultimately result in various forms of neurodegenerative disorders. Still, little is known regarding the effects of aging on behavior. Moreover, the identification of factors involved in regenerative plasticity, in both the young and aged brain, is scarce but crucial from a regenerative point of view and for our understanding on the mechanisms that control the process of normal aging. Recently, we have identified the iron-trafficking protein lipocalin-2 (LCN2) as novel regulator of animal behavior and neuronal plasticity in the young adult brain. On the other hand, others have proposed LCN2 as a biological marker for disease progression in neurodegenerative disorders such as Alzheimer's disease and multiple sclerosis. Still, and even though LCN2 is well accepted as a regulator of neural processes in the healthy and diseased brain, its contribution in the process of normal aging is not known. Here, we performed a broad analysis on the effects of aging in mice behavior, from young adulthood to middle and late ages (2-, 12-, and 18-months of age), and in the absence of LCN2. Significant behavioral differences between aging groups were observed in all the dimensions analyzed and, in mice deficient in LCN2, aging mainly reduced anxiety, while sustained depressive-like behavior observed at younger ages. These behavioral changes imposed by age were further accompanied by a significant decrease in cell survival and neuronal differentiation at the hippocampus. Our results provide insights into the role of LCN2 in the neurobiological processes underlying brain function and behavior attributed to age-related changes.

Protection against H2O2-evoked toxicity in HT22 hippocampal neuronal cells by geissoschizine methyl ether via inhibiting ERK pathway.

Oxidative stress is considered as an important mechanism underlying the pathology of neurodegenerative disorders. In this study, we utilized an in vitro model where oxidative stress process was evoked by exogenous hydrogen peroxide (H2O2) in HT22 murine hippocampal neurons and evaluated the neuroprotective effects of geissoschizine methyl ether (GME), a naturally occurring alkaloid from the hooks of Uncaria rhynchophylla (Miq.) Jacks. After a 24 h H2O2 (350 μM) insult, a significant decrease in cell survival and a sharp increase in intracellular reactive oxygen species were observed in HT22 cells. Encouragingly, GME (10–200 μM) effectively reversed these abnormal cellular changes induced by H2O2. Moreover, mechanistic studies using Western blot revealed that GME inhibited the increase of phospho-ERK protein expression, but not phospho-p38, caused by H2O2. Molecular docking simulation further revealed a possible binding mode that GME inhibited ERK protein, showing that GME favorably bound to ERK via multiple hydrophobic and hydrogen bond interactions. These findings indicate that GME provide effective neuroprotection via inhibiting ERK pathway and also encourage further ex vivo and in vivo pharmacological investigations of GME in treating oxidative stress-mediated neurological disorders.

PB1857: NRF2 MODULATORS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS: A POSSIBLE NEW THERAPEUTIC APPROACH

Background: Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of resting clonal B lymphocytes in peripheral blood, bone marrow and secondary lymphoid organs. This disease has a notable heterogeneous clinical course, with different prognosis and distinct treatment needs. Oxidative stress (OS) is one of the mechanisms important in CLL development and progression. OS state consists in an unbalance between the production of reactive oxygen species (ROS) and the ability to neutralize them by antioxidant defenses. The nuclear factor erythroid 2–related factor 2, NRF2, is a transcription factor that regulates the expression of several antioxidants and detoxification systems and works as a protective mechanism in normal cells against malignant transformation. However, during the tumorigenic process, NRF2 protects tumor cells from OS effects compromising the therapeutic efficacy. Aims: The study aims to evaluate the therapeutic potential of two NRF2 modulators, Brusatol and ML385, in B cells from CLL patients. Methods: Peripheral blood mononuclear cells (PBMCs) from 13 CLL patients (stage initial, Binet A/B, n=8; advanced stage, Binet C, n=5) were isolated by density gradient using Ficoll-Paque Premium. PBMCs where immediately incubated in absence and presence of Brusatol and ML385. The cytotoxic effect was evaluated 24h and 48h after incubation by fluorometric microculture cytotoxicity assay. After 24h incubation with Brusatol (100nM) and ML385 (100μM) cells were stained with anti-CD5-PerCP, anti-CD19-APC and Annexin V, to assess cell death by flow cytometry. The statistical analysis was carried out in the total CLL patient group and in risk groups. The results were considered statistically significant when p<0.05. Results: CLL patients presented 93,3% (±3.7%) of neoplastic B cells (CD5+/CD19+), 3,7% (±3,0%) of normal B cells (CD5-/CD19+) and 2,7% (±1,2%) of normal T cells (CD5+/CD19-), with no statistical differences regarding staging groups. Brusatol induced a decreased survival rate in CLL cells comparing to ML385, being this effect dependent on the dose and disease stage. Brusatol induced a higher reduction of cell viability in PBMCs from advanced disease patients (Binet C). ML385 did not induced a significant decrease in cell survival, nevertheless its effect is more marked in advanced stage patients at 48h post incubation. IC50 was only achieved at 48h in advanced stage patients and corresponds to 65nM for Brusatol and to 114µM for ML385. NRF2 modulators induced a cytotoxic effect mainly mediated by apoptosis. Cytotoxicity induced by both drugs was more evident in neoplastic B cells, with no significant cytotoxicity in normal B and T cells. In PBMCs incubated with Brusatol an increase of neoplastic B cells in apoptosis about 68% was observed (Brusatol: 91,9% ± 7,1%; Control: 23,7% ± 4,3%), and of 34% in cells incubated with ML385 (ML385: 57,9% ± 10,2%; Control: 23,7% ± 4,3%). No differences of apoptotic cells were observed in advanced stage groups. Summary/Conclusion: Our results showed that neoplastic cells from CLL patients were sensitive to NRF2 modulators, suggesting that these drugs may constitute a new therapeutic approach in CLL patients, particularly in CLL patients in advanced stage. We also demonstrated the low cytotoxic effect of these modulators in normal lymphocytes and its higher efficacy in neoplastic cells from advanced stage patients. Altogether, these results show that these NRF2 modulators have an effect directed to the neoplastic cell and suggest a possible involvement of NRF2 on CLL development and progression. # equally contributing authors.

Abstract 3003: The effects of autophagy inhibition on HNSCC sensitivity to CTX

Abstract Background: Cetuximab (CTX) is a monoclonal antibody commonly used in the treatment of head and neck squamous cell carcinomas (HNSCCs) that acts by targeting the epidermal growth factor receptor (EGFR). Addition of CTX to standard multi-agent chemotherapy only slightly improves the response rate from 20% to 36%, highlighting the urgent need for improved treatment. Autophagy is a naturally occurring mechanism activated within cells to protect them from harsh conditions or stress but can also provide cancerous cells with protection against further treatment attempts. Here, we examine the efficacy of combining CTX with the autophagy inhibitor, SAR405, as well as investigating the specific mechanism of CTX-induced autophagy. Methods: To measure autophagy, we utilized the Promega LC3 reporter assay, Western blotting for LC3, immunofluorescence for LC3, and an acridine orange assay. We ran our assays using the HNSCC cell lines A253 (HPV-), UM-SCC1 (HPV+), and its CTX-resistant derivative, UM-SCC1-C5 (HPV+). Clonogenic assays were run to measure colony formation, cell counting kit 8 (CCK8) assays to determine cell survival, and flow cytometry to investigate cell cycle status. Results: In this study, we first were able to demonstrate that CTX can activate pro-survival autophagy. CTX-treated cells had higher LC3 flux and more LC3 puncta than control cells as assessed by western blot and immunofluorescence staining, respectively. CTX-resistant cells demonstrated higher basal LC3 flux compared to the CTX-sensitive parental cells. We also discovered that the use of the autophagy inhibitor, SAR405, could help improve tumor response when combined with CTX. The addition of SAR405 to CTX treatment led to a decrease in colony formation and a decrease in cell survival, overcoming resistance to CTX. Interestingly, we discovered that SAR405 alone led to cell cycle arrest in the G1 phase but did not lead to a statistically significant decrease in cell survival compared to the control, showing that SAR405 is able to arrest cell growth but is not cytotoxic. Finally, knockdown of EGFR or LAPTM4B prevented CTX-induced activation of autophagy. Conclusion: Through our work, we have been able to demonstrate that CTX is able to activate autophagy, and that this initiation of autophagy plays a cytoprotective role in HNSCC cells. The addition of the autophagy inhibitor SAR405 improved response to CTX treatment. The promising results obtained thus far open the door to future studies investigating the efficacy of combining CTX with more specific autophagy inhibitors. Citation Format: Samantha T. Bradley, Yong-Syu Lee, Justin H. Skiba, Jaimee Eckers, Adam D. Swick, Rong Hu, Kwang P. Nickel, Zafer Gurel, Pippa F. Cosper, Randall J. Kimple. The effects of autophagy inhibition on HNSCC sensitivity to CTX [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3003.

Knockdown of &lt;i&gt;FLT4&lt;/i&gt;, &lt;i&gt;Nup98&lt;/i&gt;, and &lt;i&gt;Nup205&lt;/i&gt; cellular genes as a suppressor for the viral activity of Influenza A/WSN/33 (H1N1) in A549 cell culture

Objectives. To evaluate the effect of cellular genes FLT4, Nup98, and Nup205 on the reproduction of the influenza A virus in A549 human lung cancer cell line.Methods. The work was carried out using the equipment of the center for collective use of the I.I. Mechnikov Research Institute of Vaccines and Sera (Russia). The virus-containing fluid was collected within three days from the moment of transfection and infection and the intensity of viral reproduction was assessed by viral titration and hemagglutination reaction. The viral RNA concentration was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR). To calculate statistically significant differences between groups, the nonparametric Mann–Whitney test was used.Results. In cells treated with small interfering RNAs (siRNAs) targeted at FLT4, Nup98, and Nup205 genes, a significant decrease in their expression and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) was observed at a multiplicity of infection (MOI) = 0.1. Additionally, it was found that a decrease in the expression of target genes using siRNA does not lead to a significant decrease in cell survival. The viral titer in cells treated with siRNA FLT4.2, Nup98.1, and Nup205 on the first day was lower by an average of 1.0 lg, and on the second and third days, by 2.2–2.3 lg, compared to cells treated with nonspecific siRNA. During real-time RT-PCR, a significant decrease in the concentration of viral RNA was observed with siRNA Nup98.1 (up to 190 times) and Nup205 (up to 30 times) on the first day, 26 and 29 times on the second day, and 6 and 30 times on the third day, respectively. For FLT4.2 siRNA, the number of viral RNA copies decreased by 23, 18, and 16 times on the first, second, and third days. Similar results were obtained when determining the hemagglutinating activity of the virus. The hemagglutinating activity on the third day most strongly decreased in cells treated with siRNA Nup205 and FLT4.2 (16 times). In cells treated with siRNA FLT4.1, Nup98.1, and Nup98.2, hemagglutinating activity decreased by 8 times.Conclusions. In the present study, three cellular genes (FLT4, Nup98, and Nup205) were identified—the decrease in the expression of which effectively suppresses viral reproduction— and the original siRNA sequences were obtained. The results obtained are important for creating therapeutic and prophylactic medication, whose action is based on the RNA interference mechanism.

Cellular Cytotoxicity and Oxidative Potential of Recurrent Molds of the Genus Aspergillus Series Versicolores.

Molds are ubiquitous biological pollutants in bioaerosols. Among these molds, the genus Aspergillus is found in the majority of indoor air samples, and includes several species with pathogenic and toxigenic properties. Aspergillus species in the series Versicolores remain little known despite recurrence in bioaerosols. In order to investigate their toxicity, we studied 22 isolates of clinical and environmental origin, corresponding to seven different species of the series Versicolores. Spore suspensions and ethyl acetate extracts prepared from fungal isolates were subjected to oxidative potential measurement using the dithiothreitol (DTT) test and cell survival measurement. The DTT tests showed that all species of the series Versicolores had an oxidative potential, either by their spores (especially for Aspergillus jensenii) or by the extracts (especially from Aspergillus amoenus). Measurements of cell survival of A549 and HaCaT cell lines showed that only the spore suspension containing 105 spores/mL of Aspergillus jensenii caused a significant decrease in survival after 72 h of exposure. The same tests performed with mixtures of 105 spores/mL showed a potentiation of the cytotoxic effect, with a significant decrease in cell survival for mixtures containing spores of two species (on A549 cells, p = 0.05 and HaCaT cells, p = 0.001) or three different species (on HaCaT cells, p = 0.05). Cell survival assays after 72 h of exposure to the fungal extracts showed that Aspergillus puulaauensis extract was the most cytotoxic (IC50 < 25 µg/mL), while Aspergillus fructus caused no significant decrease in cell survival.

IFNγ reduces viability in human conjunctival goblet cells in vitro

AbstractPurposeConjunctival goblets cells play an important role in the homeostasis of the ocular surface and are known to be affected by inflammatory cytokines in diseases of the ocular surface. An understanding of the role of interleukin (IL)‐4, IL‐13, IL‐17, and interferon (IFN)γ in conjunctival goblet cell survival and function is necessary to explore the pathogenesis of ocular surface diseases. Previous studies have shown that IFNγ reduces the survival of the goblet cells, whereas IL‐13, IL‐4 and IL‐17A induce proliferation in mouse and rat models (1). The purpose of this study was to investigate the effect of IL‐4, IL‐13, IL‐17A and IFNγ on human goblet cell survival assessed by LDH analysis.MethodsHuman goblet cell cultures were grown from cultured donor conjunctiva. The goblet cells were seeded into wells and stimulated with IL‐4, IL‐13, IL‐17A and IFNγ with 100 ng/mL for 48 hours, respectively. Cell viability was evaluated by LDH assays.ResultsStimulation with IFNγ showed significant decrease in goblet cell viability with LDH assay (p&lt;0.01). Stimulation with IL‐4, IL‐13 and IL‐17A showed no significant decrease in cell survival compared to control. Significant differences in cell survival were observed between stimulation with IFNγ and IL‐4 (p&lt;0.001) and IL‐13 (p&lt;0.05), respectively.ConclusionsOur study indicates that IFNγ reduces human conjunctival goblet cell survival in contrast to the Th2‐associated cytokines IL‐4 and IL‐13.ReferenceGarcía‐Posadas L, Contreras‐Ruiz L, Soriano‐Romaní L, Dartt DA, Diebold Y. Conjunctival Goblet Cell Function: Effect of Contact Lens Wear and Cytokines. Eye Contact Lens 2016;42(2): 83–90.Conflict of InterestThis work was supported by a research grant from LEO Pharma.

MiR-495 regulates cell proliferation and apoptosis in H2O2 stimulated rat spinal cord neurons through targeting signal transducer and activator of transcription 3 (STAT3).

BackgroundMicroRNA-495 (miR-495) is a post-translational modulator that performs several functions, and it is involved in several disease states. On the other hand, the physiological functions of miR-495 in H2O2 stimulated mouse spinal cord neuronal dysfunction have not yet been fully understood.MethodsIn this study, we speculated that miR-495 may regulate the expression of STAT3 in the processes of neuronal proliferation and apoptosis following spinal cord injury (SCI). Cell viability was assessed with methyl thiazolyl tetrazolium (MTT) assay. Caspase-3 activity was assayed with ELISA. Cellular apoptotic changes were measured with TUNEL assay. Intracellular ROS production was determined by measuring uptake of dichlorodihydrofluorescein diacetate (DCFH-DA; PCR was used to assay the mRNA expression of STAT3 gene bearing predicted targeting positions for miR-495, while qRT-PCR was used to measure miR-495 mRNA.ResultsThe results demonstrated that treatment of SCNs with H2O2 led to a significant decrease in cell survival, while it enhanced apoptosis. The H2O2 treatment induced cell membrane dysfunction, and increased ROS levels and DNA damage. Interestingly, the expression of miR-495 was markedly suppressed when SCNs were exposed to H2O2. However, miR-495 overexpression reversed H2O2-induced cytotoxicity and apoptosis in SCNs. Moreover, H2O2 exposure elevated protein and mRNA concentrations of STAT3 in SCNs. Bioinformatics analysis showed likely binding domains of miR-495 in the 3'-untranslated regions of STAT3 in SCNs. MiR-495 loss-of-function and gain-of-function significantly up-regulated and down-regulated both STAT3 mRNA and protein expressions, respectively, in SCNs.ConclusionsmiR-495 overexpression inhibited H2O2-induced SCN dysfunction. This mechanism was mediated through the down-regulation of STAT3 expression.

Targeting Aurora A Kinase (AAK) in Platinum-Resistant High Grade Serous Ovarian Cancer.

Aurora A kinase (AAK) involved in G2-M transition is functionally involved in centrosome maturation and maintaining an active spindle assembly checkpoint. We tested the hypothesis that in platinum-taxane resistant high grade serous ovarian cancer (HGSOC) inhibition of AAK involved in G2-M transition would enhance the anti-tumor activity of cisplatin (CP) or paclitaxel (PT). Using HGSOC cell lines from platinum-taxane refractory patients that do not harbor BRCA1/2 mutations, we tested the anti-tumor activity of CP, or PT alone or in combination with the AAK inhibitor alisertib (AL). Treatment with CP for 3 h or PT for 6 h followed sequentially by AL for 48 h led to a significant decrease in cell survival (p < 0.001) compared to treatment with either drug alone in HGSOC cells but not in immortalized normal human ovarian surface epithelium or normal human fallopian tube secretory epithelium cells. The treatment with CP or PT followed by AL also led to a significant increase in reactive oxygen species (p < 0.05), apoptosis (p < 0.001) and accumulation of cells in G2/M that was accompanied by a modest increase in expression of AAK. Downregulation of AAK, but not aurora B kinase, with targeted siRNAs also significantly enhanced apoptosis by CP or PT, suggesting that AL specifically targeted AAK. In summary, in HGSOC without BRCA1/2 mutations, CP, or PT resistance can potentially be circumvented by sequential treatment with AL that inhibits AAK involved in G2-M transition.

Effects of Gadolinium MRI Contrast Agents on DNA Damage and Cell Survival when Used in Combination with Radiation.

Gadolinium is a commonly used contrast agent for magnetic resonance imaging (MRI). The goal of this work was to determine how MRI contrast agents affect radiosensitivity for tumour cells. Using a 225kVp X-ray cabinet source, immunofluorescence and clonogenic assays were performed on six cancer cell lines: lung (H460), pancreas (MiaPaCa2), prostate (DU145), breast (MCF7), brain (U87) and liver (HEPG2). Dotarem® contrast agent, at concentrations of 0.2, 2 and 20 mM, was used to determine its effect on DNA damage and cell survival. Measurements were performed using inductively coupled plasma mass spectrometry (ICP-MS) to determine the amount of gadolinium taken up by each cell line for each concentration. A statistically significant increase in DNA damage was seen for all cell lines at a dose of 1 Gy for concentrations of 2 and 20 mM, at 1 h postirradiation. At 24 h postirradiation, most of the DNA damage had been repaired, with approximately 90% repair for almost all doses of radiation and concentrations of Dotarem. Clonogenic results showed no statistically significant decrease in cell survival for any cell line or concentration. Uptake measurements showed cell line-specific variations in uptake, with MCF7 and HEPG2 cells having a high percentage uptake compared to other cell lines, with 151.4 ± 0.3 × 10-15 g and 194.8 ± 0.4 × 10-15 g per cell, respectively, at 2 mM Dotarem concentration. In this work, a variability in gadolinium uptake was observed between cell lines. A significant increase was seen in initial levels of DNA damage after 1 Gy irradiation for all six cancer cell lines; however, no significant decrease in cell survival was seen with the clonogenic assay. The observation of high levels of repair suggest that while initial levels of DNA damage are increased, this damage is almost entirely repaired within 24 h, and does not affect the ability of cells to survive and produce colonies.

60S acidic ribosomal protein P1 (RPLP1) is elevated in human endometriotic tissue and in a murine model of endometriosis and is essential for endometriotic epithelial cell survival in vitro.

Endometriosis is a female disease which is defined as the presence of ectopic endometrial tissue and is dependent on estrogen for its survival in these ectopic locations. Expression of the ribosomal protein large P1 (RPLP1) is associated with cell proliferation and invasion in several pathologies, but a role in the pathophysiology of endometriosis has not been explored. In this study, we aimed to evaluate the expression and function of RPLP1 with respect to endometriosis pathophysiology. RPLP1 protein was localised by immunohistochemistry (IHC) in eutopic and ectopic tissue from 28 subjects with confirmed endometriosis and from 20 women without signs or symptoms of the disease, while transcript levels were evaluated by qRT-PCR in 77 endometriotic lesions and 55 matched eutopic endometrial biopsies, and protein expression was evaluated using western blotting in 20 of these matched samples. To evaluate the mechanism for enhanced lesion expression of RPLP1, an experimental murine model of endometriosis was used and RPLP1 expression was localized using IHC. In vitro studies using an endometriosis cell line coupled with shRNA knockdown was used to demonstrate its role in cell survival. Expression of RPLP1 mRNA and protein were significantly higher in ectopic lesion tissue compared to paired eutopic endometrium and immunohistochemical localisation revealed predominant localisation to epithelial cells. This pattern of lesion RPLP1 was recapitulated in mice with experimentally induced endometriosis. Stable knockdown of RPLP1 protein resulted in a significant decrease in cell survival in vitro. These studies reveal that RPLP1 is associated with cell proliferation and/or survival and may play a role in the pathophysiology of endometriosis.

67P - PARP inhibition enhances cisplatin sensitivity in cervical cancer by modulating β-catenin signaling

BackgroundCisplatin (CDDP) is used in the treatment of locally advanced disease (FIGO stage IIB-IVA) as well recurrent/metastatic cervical cancer. However toxic side effects and acquired resistance limits its efficacy. Enhanced DNA repair is one of the mechanisms responsible for acquired cisplatin resistance. Poly(ADP-ribose) polymerase (PARP) inhibitors have been approved for treating BRCA mutated cancers like breast and ovary cancer, however, little is known about the therapeutic efficacy and mechanism of action of PARP inhibitors in non-BRCA cancer like cervical cancer, either as a single agent or in combination with cisplatin.Table: 67PCombined effect of PJ34 and CDDP on cisplatin sensitivity in cervical cancer cell linesTable: 67PCancer cell parameterOnly CDDP (10μM)Only PJ34 (10μM)CDDP(10μM) + PJ34(10μM)p value/fold differenceClonogenic formation fraction#0.23±0.08 (H) 0.31± 0.06 (S)0.67±0.09 (H) 0.65±0.01 (S)0.13±0.05 (H) 0.15±0.07 (S)* (H) * (S)Fold migration0.71±0.06 (H) 0.75±0.03 (S)0.89±0.07 (H) 0.99±0.08 (S)0.54±0.05 (H) 0.34±0.02 (S)** (H) * (S)Relative invasion43.4±2.25 (H) 47.9±7.07 (S)66.6±5.94 (H) 70.5±5.74 (S)15.8±2.81 (H) 23.4± 4.13 (S)** (H) ** (S)IC50 value##8.25μM (H) 10.8μM (S)31.5μM (H) 33.0μM (S)3.6μM (H) 3.4μM (S)2.29 fold 3.18 fold MethodsEffect of PARP-1 inhibition (PJ34/PARP-1siRNA) was determined in vitro on cell viability, apoptosis, cell cycle progression, proliferation, invasion and metastasis, clonogenecity and β-catenin signaling in cervical cancer cell lines HeLa and SiHa. ResultsCombination of CDDP with PJ34 or PARP-1 siRNA significantly reduced the cell proliferation and induced cell cycle arrest and apoptosis. Also, a significant decrease in cell survival as well as cell invasion and migration was observed as compared with either CDDP/PJ34 alone. The enhanced CDDP sensitivity by PARP-1 inhibition was determined to be due to inhibition of β-catenin signaling as shown by a decrease in MMP-2 activity, MMP-9, c-myc and cyclin-D1 expression upon treatment with PJ34 and PARP-1 siRNA. ConclusionsOur data provides experimental evidence on the contribution of PARP-1 inhibition in enhancing the cytotoxicity of CDDP in cervical cancer cells. We also present novel findings on the suppression of β-catenin and its downstream signaling components by PARP-1 inhibitor.#: 5μM of CDDP was used both alone and in combination with PJ34; ##: A gradient of CDDP from 0-15μM was used to evaluate the combined effect of PJ34 and CDDP on IC50 value of CDDP. p: *<0.05, **<0.01. (H): HeLa; (S): SiHa. Legal entity responsible for the studyThe authors. FundingAll India Institute of Medical Sciences. DisclosureAll authors have declared no conflicts of interest.

PKCα is required for Akt-mTORC1 activation in non-small cell lung carcinoma (NSCLC) with EGFR mutation.

Mutational activation of the epidermal growth factor receptor (EGFR) is a major player in the pathogenesis of non-small cell lung cancer (NSCLC). NSCLC patients with constitutively active EGFR mutations (mEGFR) eventually develop drug resistance against EGFR tyrosine-kinase inhibitors (TKIs); therefore, better understandings of key components of mEGFR signaling are required. Here, we initially observed aberrantly high expression of protein kinase Cα (PKCα) in lung adenocarcinomas, especially those with mEGFR, and proceeded to examine the role of PKCα in the regulation of the signaling pathways downstream of mutant EGFR (mtEGFR). The results showed that NSCLC cell lines with constitutively active EGFR mutations tend to have very or moderately high PKCα levels. Furthermore, PKCα was constitutively activated in HCC827 and H4006 cells which have an EGFR deletion mutation in exon 19. Interestingly, mtEGFR was not required for the induction of PKCα at protein and message levels, suggesting that the increased levels of PKCα are due to independent selection. Whereas, mtEGFR activity was required for robust activation of PKCα. Loss of functions studies revealed that the NSCLC cells rely heavily on PKCα for the activation of the mTORC1 signaling pathway. Unexpectedly, the results demonstrated that PKCα was required for activation of Akt upstream of mTOR but only in cells with the mtEGFR and with the increased expression of PKCα. Functionally, inhibition of PKCα in HCC827 led to caspase-3-dependent apoptosis and a significant decrease in cell survival in response to cellular stress induced by serum starvation. In summary, the results identified important roles of PKCα in regulating mTORC1 activity in lung cancer cells, whereby a primary switching occurs from PKCα-independent to PKCα-dependent signaling in the presence of mEGFR. The results present PKCα as a potential synergistic target of personalized treatment for NSCLC with constitutively active mutant forms of EGFR and constitutively active PKCα.

Correction: The Molecular Mechanisms of the Antibacterial Effect of Picosecond Laser Generated Silver Nanoparticles and Their Toxicity to Human Cells.

[This corrects the article DOI: 10.1371/journal.pone.0160078.].

Effects of mobile phone prolonged radiation on kidney cells; an in-vitro study

Introduction: Mobile phones have been shown to have hazardous effects on the human renal system, reproduction and development, central nervous system and immune function. Objectives: This study aimed to investigate whether the mobile phone prolonged radiofrequency (RF) radiation could affect healthy kidney cells in an in-vitro study. Materials and Methods: Human kidney healthy cells in three groups were placed into three batches. The first and second groups (exposed groups) were exposed to a mobile phone simulator for 1 and 2 h/d, respectively. The irradiation was performed for 8 consecutive days. For the third group (control group), no radiation was applied to the samples and they completed the assay cycle under identical conditions. Cells’ survival was evaluated using MTT [3-(4,5-dimethylthiazol–2- yl)-2,5-diphenyltetrazolium bromide] assay. Statistical significance of the differences between control and exposed groups were assessed using the repeated measure ANOVA method (P<0.05). Results: Results showed that radiation exposure from mobile phones simulator decreased the kidney cell survival in the exposed groups (up to 40%). ANOVA test revealed that a significant decrease in cell survival in the exposed group compared to the control group (P=0.014). No significant differences between the irradiated groups were found (P>0.05). Conclusion: Mobile phone exposure affects kidney cells survival during an in vitro condition. To study the effects of mobile phone radiation on kidney, further in vivo studies on mammalians are needed.

The association between p53 protein phosphorylation at serine 15, serine 20 and sensitivity of cells isolated from patients with ovarian cancer and cell lines to chemotherapy in in vitro study.

The association between p53 protein phosphorylated at serine 15 (Ser15), serine 20 (Ser20) and ovarian tumor cell sensitivity after chemotherapy was analyzed in order to define the influence of p53 activation on tumor cell sensitivity to chemotherapy. The study was performed on ovarian cancer cell line (OvBH-1), colon adenocarcinoma metastasis to ovary (SW626) and on cells isolated from ascitic fluids from patients with ovarian cancer: with (p53+) or without (p53-) p53 nuclear protein accumulation. p53 protein, Ser15, Ser20, Bax, Noxa and PgP protein expression was evaluated by means of immunocytochemical staining before and after chemotherapy. Cell viability after treatment was estimated using MTT assay. Cell lines and tumor cells p53+, p53- revealed a significant decrease in cell survival after camptothecin, paclitaxel, cisplatin treatment, compared to the control group (p < 0.01). In p53+ group, the expression of Ser20 significantly increased after camptothecin and paclitaxel (p < 0.05). Ser15, Ser20, Bax, Noxa expression correlated with MTT and depended on p53+, p53- tumor cell and the drug used (p < 0.05). Expression of Bax and Noxa were dependent on the type of tumor cells and drug used. The correlation between Ser15, Ser20 and Bax, Noxa expression was found in cell lines and tumor cells (p < 0.05). Our study suggests that the relation between Ser15 or Ser20 and tumor cell viability might reflect their role in tumor sensitivity on chemotherapy in dependent p53 protein status. Revealed association between p53 protein phosphorylated at Ser15, Ser20 and Bax, Noxa protein expression determined the apoptotic activity of tumor cells.

Bovine herpes virus type 4 alters TNF-α and IL-8 profiles and impairs the survival of bovine endometrial epithelial cells

Bovine herpes virus type 4 (BoHV-4) can be transmitted by contaminated semen to cows at the time of breeding and may cause uterine disease. The aim of this study was to characterize the susceptibility of bovine endometrial epithelial cells (bEEC) to BoHV-4 by using an in vitro model. When bEEC were challenged with different multiplicity of infection (MOI; from 0.001 to 10) of BoHV-4 for 6days, a significant decrease in cell survival with increasing MOI was observed. The bEEC were subsequently challenged with BoHV-4 MOI 0.1 for 7days. During the first 4days, numbers increased in a similar way in controls and infected group (p<0.01 when compared to Day 0). After Day 4, numbers of live cells in infected samples decreased when compared to controls and were lower than control at Day 7 (p<0.01). From titration and qPCR, increasing number of viral particles was observed from Day 1, and reached a plateau at Day 5. Concentrations of IL-8 increased with time and were higher in supernatants from infected cells than in controls (p<0.0001). TNF-α concentrations presented similar profile as cell survival ones. In conclusion, the survival of bEEC was strongly impaired by BoHV-4 infection in a time and dose dependent manner and supernatant cytokine profiles were altered. This information supports BoHV-4 implication in clinical cases of uterine diseases and the existence of a risk of BoHV-4 transmission from infected males through animal breeding.

Sodium perbarate and benzalkonium chloride induce DNA damage in Chang conjunctival epithelial cells

Content and objective: To investigate and compare the toxic effects of benzalkonium chloride (BAC) and new type oxidative preservative sodium perborate (NaBO3) on DNA damage, reactive oxygen species (ROS), and cell survival in immortalized human Chang conjunctival cells.Materials and methods: Cells were exposed to BAC and NaBO3 in concentrations of 0.00001–0.001% for 30 min. Cell viability was measured by the MTT test. Alkaline comet assay was used to detect DNA damage. Mitochondrial membrane potential (MMP), cell apoptosis, and ROS production were detected by flow cytometry analysis.Results: Significant changes in the relative cell survival rate in cells were observed after exposure to 0.0005–0.001% BAC for 30 min (p < 0.001). DNA damage and intracellular ROS were observed in a dose-dependent manner with BAC exposure (p < 0.001). However, 0.001% BAC induced less ROS than 0.0005% BAC. A decrease in MMP was also recorded. NaBO3 did not induce the decrease in cell survival and MMP in low concentration but could induce DNA damage and ROS generation in a 0.001% concentration (p < 0.001).Discussion and conclusions: BAC can induce DNA damage in human conjunctival epithelial cells; this effect may be related to oxidative stress. Although NaBO3 did not induce a significant decrease in cell survival and MMP, DNA damage and ROS generation were still detected in high concentration. New type oxidative preservative has less toxicity than the old type, but it still has the tendency of producing genotoxic changes in an in vitro test system.