Induced Cell Growth Inhibition Research Articles

Autophagy suppression enhances DNA damage and cell death upon treatment with PARP inhibitor Niraparib in laryngeal squamous cell carcinoma.

Although poly (ADP-ribose) polymerase (PARP) inhibitors, as anti-tumor drugs targeting the DNA damage response (DDR), have been used for the therapy of various tumors, few researches reported their effect on laryngeal squamous cell carcinoma (LSCC). Here, we first discovered that the PARP-1/2 inhibitor Niraparib could simultaneously induce cell growth inhibition and autophagy in LSCC TU212 and TU686 cells. Niraparib decelerated cell cycle of LSCC by arresting G1 phase and preventing the cells from entering S phase. DNA lesions were also observed upon Niraparib treatment as evidenced by the accumulation of γH2AX and abatement of pRB expression. In addition, autophagy generation was confirmed by the observation of autophagosomes, LC3-positive autophagy-like vacuoles, and obvious conversion of LC3-I to LC3-II. Moreover, blocking autophagy enhanced Niraparib-induced growth inhibition and DNA lesions. Further studies suggested that autophagy suppression could obstruct the activation of checkpoint kinase 1 (Chk1) through elevating proteasomal activity and then impair the capacity of homologous recombination (HR), thereby improving the anti-LSCC efficiency of Niraparib. Collectively, these findings suggested that simultaneous targeting of Niraparib and autophagy might be a promising therapeutic schedule for LSCC in clinic.

MicroRNA-7-5p induces cell growth inhibition, cell cycle arrest and apoptosis by targeting PAK2 in non-small cell lung cancer.

MicroRNAs (miR) are known to be critical regulators in tumor progression. miR‐7‐5p was reported to be involved in several cancers, including glioblastoma, cervical cancer, and melanoma, but its prognostic value and biological function in non‐small‐cell lung cancer (NSCLC) remain unclear. In this study, using quantitative real‐time PCR analysis, we found that miR‐7‐5p was significantly downregulated in NSCLC tissues and cell lines. Lower miR‐7‐5p expression was associated with tumor–node–metastasis stage and tumor size by chi‐squared test. Deceased miR‐7‐5p expression was associated with a worse prognosis in patients with NSCLC using Kaplan–Meier survival analysis and multivariate Cox regression analysis. Experiments in NSCLC cell lines (A549 and H1299) demonstrated that upregulation of miR‐7‐5p significantly suppressed cell proliferation, but induced cell cycle G0/G1 phase arrest and apoptosis using Cell Counting Kit‐8, colony formation, and flow cytometry analysis. Through loss‐of‐function assays, we further demonstrated that downregulation of miR‐7‐5p promoted cell proliferation and cell cycle G1/S transition, but decreased cell apoptosis in SPC‐A1 cells. Furthermore, P21‐activated kinase 2 (PAK2) was predicted and confirmed as a direct target gene of miR‐7‐5p in NSCLC cells by luciferase reporter assay. In addition, we found PAK2 overexpression could partially reverse the effects of miR‐7‐5p on cell proliferation, cell cycle distribution, and apoptosis. We thus concluded that lower expression of miR‐7‐5p was associated with poor prognosis and NSCLC progression by directly targeting PAK2.

Targeting the Warburg effect to overcome lapatinib resistance in esophageal adenocarcinoma

Background: Esophageal adenocarcinoma (EAC) overexpresses HER2. Lapatinib, a dual tyrosine kinase inhibitor blocking HER1 and HER2 pathways failed to improve patient survival. Molecular mechanisms of this lapatinib resistance are still unclear. We therefore explored the role of glycolytic enzyme pyruvate kinase M2 (PKM2), a key regulator of the Warburg effect in the lapatinib resistance mechanism of EAC cells.
 Methods: First we established a lapatinibresistant OE19 (LPR-OE19) cell line from OE19 EAC cells. We then investigated the comparative cell growth inhibition, apoptotic and lactate production effects of HER2 inhibitor lapatinib and PKM2 inhibitor shikonin either alone or in combinations, both in OE19 and LPR-OE19 cells with and without knockdown of PKM2 and HSP40 by siRNAs.
 Results: Lapatinib resistant LPR-OE19 cells showed downregulation of HSP40 both at protein and mRNA levels, but showed upregulation of PKM2 only at the protein level. LPR-OE19 cells showed significantly reduced sensitivity to lapatinib induced cell growth inhibition, apoptosis and decreased cell migration compared to parent OE19 cells. Interestingly, augmented cell growth inhibition, apoptosis and decreased cell migration were observed in LPR-OE19 cells compared to parent OE19 cells when lapatinib was combined with shikonin. Knockdown of PKM2 in LPR-OE19 cells abolished the reduced sensitivity of lapatinib induced cell growth inhibition, and also abolished augmented cell growth inhibition response when lapatinib and shikonin were combined. Contrary to that, knockdown of HSP40 in OE19 cells enhanced PKM2 expression leading to significantly reduced sensitivity to lapatinib induced growth inhibition with augmented growth inhibition when lapatinib and shikonin were combined. Moreover, LPR-OE19 cells showed enhanced lactate production compared to parent OE19. Interestingly, PKM2 and HSP40 co-localize with each other in the cell nucleus suggesting that PKM2 binds to molecular chaperone HSP40.
 Conclusions: These data suggest that targeting HSP40PKM2 interaction could be an innovative therapeutic approach to overcome lapatinib resistance in EAC.

Exenatide induces autophagy and prevents the cell regrowth in HepG2 cells.

The incidence of hepatocellular carcinoma (HCC) keeps rising year by year, and became the second leading cause of cancer-related death. Some studies have found that liraglutide, a GLP-1 analog, may decrease the tumor cells proliferation. Due to this, the aim of this work is to investigate the antiproliferative potential of exenatide, another GLP-1 analog. Cell proliferation was assessed by direct count with Trypan blue dye exclusion. Flow cytometry was used to determinate autophagy and nuclear staining. Morphometric analysis was used to verify senescence and apoptosis. The mechanism that induced cell growth inhibition was analyzed by Western Blot. Treatment with exenatide significantly decreases cell proliferation and increases autophagy, both in relation to control and liraglutide. In addition, mTOR inhibition was greater in cells treated with exenatide. In relation to chronic treatment, exenatide does not allow cellular regrowth by preventing some resistance mechanism that the cells can acquire. These results suggest that exenatide has a potent anti-proliferative activity via mTOR modulation and, among the GLP-1 analogs tested, could be in the future an alternative for HCC treatment.

Abstract 1261: Targeting Wee1 in laryngeal squamous cell carcinoma

Abstract WEE1 is a tyrosine kinase that regulates G2/M cell cycle checkpoint and frequently overexpressed in various tumors. However, the expression and clinical significance of WEE1 in human laryngeal squamous cell carcinoma (LSCC) are still unknown. In this study, we found that WEE1 was highly expressed in LSCC tissues compared with adjacent normal tissues. Importantly, overexpression of WEE1 was correlated with T stages, lymph node metastasis, clinical stages and poor prognosis of LSCC patients. Furthermore, inhibition of WEE1 by MK-1775 induced cell growth inhibition, G2/M cell cycle arrest and apoptosis with the increased intracellular reactive oxygen species (ROS) levels in LSCC cells. Pretreatment with ROS scavenger N-acetyl-L-cysteine could reverse MK-1775-induced ROS accumulation and cell apoptosis in LSCC cells. MK-1775 also inhibited the growth of LSCC xenografts in nude mice. Altogether, these findings suggest that WEE1 is a potential therapeutic target in LSCC, and inhibition of WEE1 is the prospective strategy for LSCC therapy. Note: This abstract was not presented at the meeting. Citation Format: Meng-Ling Yuan, Qi-Wei Jiang, Yang Yang, Jia-Rong Huang, Zhi Shi. Targeting Wee1 in laryngeal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1261.

L-Arginine Inhibited Inflammatory Response and Oxidative Stress Induced by Lipopolysaccharide via Arginase-1 Signaling in IPEC-J2 Cells

This study aimed to explore the effect of L-arginine on lipopolysaccharide (LPS)-induced inflammatory response and oxidative stress in IPEC-2 cells. We found that the expression of toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), cluster of differentiation 14 (CD14), nuclear factor-kappaBp65 (NF-κBp65), chemokine-8 (IL-8), tumor necrosis factor (TNF-α) and chemokine-6 (IL-6) mRNA were significantly increased by LPS. Exposure to LPS induced oxidative stress as reactive oxygen species (ROS) and malonaldehyde (MDA) production were increased while glutathione peroxidase (GSH-Px) were decreased in LPS-treated cells compared to those in the control. LPS administration also effectively induced cell growth inhibition through induction of G0/G1 cell cycle arrest. However, compared with the LPS group, cells co-treatment with L-arginine effectively increased cell viability and promoted the cell cycle into the S phase; L-arginine exhibited an anti-inflammatory effect in alleviating inflammation induced by LPS by reducing the abundance of TLR4, MyD88, CD14, NF-κBp65, and IL-8 transcripts. Cells treated with LPS+L-arginine significantly enhanced the content of GSH-Px, while they decreased the production of ROS and MDA compared with the LPS group. Furthermore, L-arginine increased the activity of arginase-1 (Arg-1), while Arg-1 inhibitor abolished the protection of arginine against LPS-induced inflammation and oxidative stress. Taken together, these results suggested that L-arginine exerted its anti-inflammatory and antioxidant effects to protect IPEC-J2 cells from inflammatory response and oxidative stress challenged by LPS at least partly via the Arg-1 signaling pathway.

BZW2 gene knockdown induces cell growth inhibition, G1 arrest and apoptosis in muscle-invasive bladder cancers: A microarray pathway analysis.

Bladder cancer is among the most common cancers all over the world. The function of basic leucine zipper and W2 domains 2 (BZW2) in tumour progression has been reported. However, the biological function of BZW2 in muscle‐invasive bladder cancers (MIBCs) remains to be determined. The aim of the present study was to reveal the expression and roles of BZW2 in human MIBCs and to explore the molecular mechanisms underlying these functions. Clinically, BZW2 expression was higher in MIBC tissues than the adjacent non‐tumour tissues. Knocking down BZW2 using shRNA inhibited cell proliferation and G1/S cell cycle progression in vitro, and induced apoptosis in both 5637 and T24 cells. Moreover, in vivo studies with mice xenograft models confirmed the anti‐proliferative effects of BZW2‐knockdown, providing a future therapeutic target. We also performed biochemical microarray analysis to identify the potential signalling pathways, disease states and functions which could be affected by suppressing BZW2 in MIBC cells. Collectively, our findings suggest BZW2 has an oncogenic role in MIBCs and serves as a promising target for molecular diagnosis and gene therapy.

CDK7 inhibitor suppresses tumor progression through blocking the cell cycle at the G2/M phase and inhibiting transcriptional activity in cervical cancer.

BackgroundThe disordered cell cycle and dysregulated expression of numerous oncogenes involved in tumor-relevant processes are highly related to the tumorigenesis of cervical cancer. Cyclin-dependent kinase 7 (CDK7) constitutes the indispensable catalytic subunit of CDK-activating kinase (CAK), which is required for both cell cycle transition and transcriptional regulation. However, research regarding the antitumor effects of CDK7 inhibition in cervical cancer remains unclear.PurposeOur study aims to explore the antineoplastic effects of the CDK7 inhibitor THZ1 in cervical cancer cells and to find a potential agent for cervical cancer treatment.MethodsThe CRISPR-Cas9 system was used to knock down CDK7. The Cell Counting Kit-8 (CCK-8) assay was used to detect the cell viability after CDK7 depletion and THZ1 treatment. Western blot was employed to detect protein expression. The expression levels of mRNA were assayed through qRT-PCR. Flow cytometry analysis was used to assay the apoptotic cells and cell cycle distribution. Gene expression microarray analysis was used to identify the differential expression of the genes. Subcutaneous xenograft mouse model was performed to test the antineoplastic effects of THZ1 in vivo.ResultsWe revealed that the genetic depletion of CDK7 using the CRISPR-Cas9 system exhibited great cell growth inhibition in cervical cancer cell lines, consistent with the effects of CDK7 blocking using THZ1. Cervical cancer cells were highly sensitive to THZ1 treatment, and a low concentration of THZ1 could induce substantial cell apoptosis. THZ1 specifically perturbed the phosphorylation of cell cycle regulator CDK1 and decreased the expression of cyclin B1, leading to a cell cycle blockage at the G2/M phase and inducing cell growth inhibition. The gene expression microarray analysis showed that massive oncogene transcripts, especially those associated with tumorigenesis, were preferential suppressed after THZ1 treatment. The qRT-PCR confirmed that several essential oncogenes in tumorigenesis (c-MYC, hTERT, RAD51, and BCL-2) and HPV viral oncogenes (E6 and E7) were preferentially repressed by THZ1. Moreover, THZ1 exhibited substantial antineoplastic effects against cervical cancer in vivo without inducing obvious side effects.ConclusionThese findings indicated that the CDK7 inhibitor THZ1 is a potential option in cervical cancer treatment owing to its ability to inhibit cell cycle progression and transcriptional activity.

Methylation-associated miR193b silencing activates master drivers of aggressive prostate cancer.

240 Background: Epigenetic changes in DNA methylation, histone modifications and miRNA expression have major roles in prostate cancer (PC) initiation and progression. Previous screening for epigenetically regulated miRNAs in PC revealed hypermethylation of the miR-193b promoter, but no related functional studies in PC have been reported. Methods: Methylation was measured by methylation-specific PCR. Quantitative RT-PCR was used to validate the regulation of potential miR-193b targets. Luciferase reporter assays were applied to detect the regulation of miR-193b-targets. The promoter methylation status was modified by DNMT and HDAC inhibitors (5-azacytidine and mocetinostat). Results: miR-193b level is inversely correlated with the degree of its promoter’s methylation in the TCGA cohort. Distinct miR-193b promoter methylation patterns were detected in multiple normal and PC cell lines. Overexpression of miR-193b in PC cells with low miR193b level induced cell growth inhibition, apoptosis, or inhibition of invasion. Previously reported top 150-downregulated genes by miR-193b in liposarcoma showed a high correlation with PC progression in multiple PC cohorts. We further identified FOXM1 and RRM2 as the direct targets of miR-193b. Knockdown of FOXM1 and RRM2 in PC cells phenocopied miR-193b with respect to inhibition of invasion and induction of apoptosis. Furthermore, combination treatment with DNMT and HDAC inhibitors released the methylation-regulated silencing of miR-193b in PC cells, resulting the inhibition of FOXM1 and RRM2 expression. Conclusions: We revealed the tumor suppressive function of miR-193b in PC. Overexpression of miR-193b in PC cells with low miR193b level induced cell growth inhibition, apoptosis, or inhibition of invasion. A gene set identified in liposarcoma cells by overexpression of miR-193b showed a high correlation with PC progression in multiple PC cohorts. FOXM1 and RRM2 may be the key targets of miR-193b in PC. Our findings suggest that methylation-silencing miR-193b in PC may release the inhibition of some key oncogenes to contribute PC progression, which could provide a possible therapeutic mechanism for PC therapy.

Honokiol alleviates sepsis-induced acute kidney injury in mice by targeting the miR-218-5p/heme oxygenase-1 signaling pathway

BackgroundHonokiol is a low-molecular-weight natural product and has been reported to exhibit anti-inflammatory activity.ObjectivesOur study aimed to investigate the influence of honokiol on sepsis-induced acute kidney injury (AKI) in a mouse model.Material and methodsA cecal ligation and puncture (CLP) surgical operation was performed to establish a sepsis-induced acute kidney injury model in mice. Renal histomorphological analysis was performed with periodic acid-Schiff (PAS) staining. The levels of inflammatory markers in serum were measured by ELISA assay. The mRNA and protein levels were assayed by RT-qPCR and western blotting, respectively. Annexin V-FITC/PI staining was used to evaluate glomerular mesangial cell (GMC) apoptosis.ResultsThe results revealed that honokiol significantly increased the survival rate in mice undergoing a CLP operation. Inflammatory cytokines, such as TNF-α, IL-6 and IL-1β, were significantly inhibited in honokiol-treated septic mice compared with the CLP group. In addition, honokiol showed the ability to reverse CLP-induced AKI in septic mice. Furthermore, heme oxygenase-1 (HO-1) expression levels were significantly up-regulated and miR-218-5p was markedly down-regulated in honokiol-treated septic mice as compared to CLP-operated mice. Bioinformatics and experimental measurements showed that HO-1 was a direct target of miR-218-5p. In vitro experiments showed that both honokiol and miR-218-5p inhibitors blocked lipopolysaccharide (LPS)-induced cell growth inhibition and GMC apoptosis by increasing the expression of HO-1.ConclusionsHonokiol ameliorated AKI in septic mice and LPS-induced GMC dysfunction, and the underlying mechanism was mediated, at least partially, through the regulation of miR-218-5p/HO-1 signaling.

MCC1019, a selective inhibitor of the Polo-box domain of Polo-like kinase 1 as novel, potent anticancer candidate

Polo-like kinase (PLK1) has been identified as a potential target for cancer treatment. Although a number of small molecules have been investigated as PLK1 inhibitors, many of which showed limited selectivity. PLK1 harbors a regulatory domain, the Polo box domain (PBD), which has a key regulatory function for kinase activity and substrate recognition. We report on 3-bromomethyl-benzofuran-2-carboxylic acid ethyl ester (designated: MCC1019) as selective PLK1 inhibitor targeting PLK1 PBD. Cytotoxicity and fluorescence polarization-based screening were applied to a library of 1162 drug-like compounds to identify potential inhibitors of PLK1 PBD. The activity of compound MC1019 against the PLK1 PBD was confirmed using fluorescence polarization and microscale thermophoresis. This compound exerted specificity towards PLK1 over PLK2 and PLK3. MCC1019 showed cytotoxic activity in a panel of different cancer cell lines. Mechanistic investigations in A549 lung adenocarcinoma cells revealed that MCC1019 induced cell growth inhibition through inactivation of AKT signaling pathway, it also induced prolonged mitotic arrest—a phenomenon known as mitotic catastrophe, which is followed by immediate cell death via apoptosis and necroptosis. MCC1019 significantly inhibited tumor growth in vivo in a murine lung cancer model without affecting body weight or vital organ size, and reduced the growth of metastatic lesions in the lung. We propose MCC1019 as promising anti-cancer drug candidate.

Celastrol Inhibits the Growth of Ovarian Cancer Cells in vitro and in vivo

Celastrol is a natural triterpene isolated from the Chinese plant Thunder God Vine with potent antitumor activity. However, the effect of celastrol on the growth of ovarian cancer cells in vitro and in vivo is still unclear. In this study, we found that celastrol induced cell growth inhibition, cell cycle arrest in G2/M phase and apoptosis with the increased intracellular reactive oxygen species (ROS) accumulation in ovarian cancer cells. Pretreatment with ROS scavenger N-acetyl-cysteine totally blocked the apoptosis induced by celastrol. Additionally, celastrol inhibited the growth of ovarian cancer xenografts in nude mice. Altogether, these findings suggest celastrol is a potential therapeutic agent for treating ovarian cancer.

Brosimone I, an isoprenoid-substituted flavonoid, induces cell cycle G1 phase arrest and apoptosis through ROS-dependent endoplasmic reticulum stress in HCT116 human colon cancer cells.

Brosimone I is an isoprenoid-substituted flavonoid from Artocarpus heterophyllus. Here, we reported for the first time that brosimone I induced cell cycle G1 phase arrest and apoptosis in HCT116 human colon cancer cells. Brosimone I treatment increased the cytosolic Ca2+ level, and subsequently activated the CaMKKβ-AMPK pathway. STO-609, a CaMKKβ inhibitor, and compound C, an AMPK-specific inhibitor, attenuated brosimone I-induced loss of cell viability in HCT116 cells. Furthermore, brosimone I enhanced ER stress. Salubrinal, an ER stress inhibitor, reduced brosimone I-induced cell growth inhibition. In addition, brosimone I was found to increase ROS generation and the inhibition of ROS formation by NAC, a ROS inhibitor, attenuated brosimone I-induced cell death, cytosolic Ca2+ increase, and ER stress markers. Collectively, our findings reveal that brosimone I induces cell cycle G1 phase arrest and apoptosis via the induction of ROS-mediated increased cytosolic Ca2+, ER stress, and the activation of the CaMKKβ-AMPK signaling pathway.

Anticancer activity of britannin through the downregulation of cyclin D1 and CDK4 in human breast cancer cells.

Both apoptotic induction and cell cycle blockade in cancer cells are effective strategies to eliminate cancer cells. Many conventional cancer drugs that induce apoptosis and inhibit cell cycle progression have been reported as potential therapeutics for various types of cancer. Britannin is a natural sesquiterpene lactone that its profound anticancer properties were revealed in our previous study. In this study, we evaluated the effects of britannin on the cell cycle distribution and also cell cycle-related proteins. Analysis of cell cycle distribution was carried out using flow cytometer. The effects of britannin on cyclin D1 and CDK4 expression were evaluated using the Western blot. The obtained results show that britannin at the low concentrations induces cell growth inhibition mainly through G1-phase arrest while it seems that apoptosis contributes to cell growth inhibitory effect of high doses of britannin. Reduction of cyclin D1 and CDK4 protein levels were also observed after treating cancer cells with britannin. The obtained results reveal that britannin can inhibit MCF-7 and MDA-MB-468 breast cancer cells proliferation through arresting cell cycle progression through cyclin D1/CDK4-mediated pathway.

Selective Pharmacologic Inhibition of Paroxysmal Nocturnal Hemoglobinuria Clones

Paroxysmal nocturnal hemoglobinuria (PNH) is usually associated with reduced bone marrow (BM) capacity caused by acquired idiopathic aplastic anemia (AA). PIGA mutations lead to a partial or total deficiency of glycosylphosphatidyl-inositol (GPI) anchor proteins (AP). AA is characteristically accompanied by the presence of often tiny GPI-AP deficient clones, which in a significant proportion of patients (10-15%), irrespective of the initial success of immunosuppressive therapy, will evolve to produce manifest hemolytic PNH. Indeed in our cohort of BM failure patients (n=319), 41% of AA patients had a PNH clone present (0.02-20% of granulocytes) (AA/PNH), 14% of patients had primary PNH (primary PNH), and 8% had a history of PNH post AA (secondary PNH).To date, drug development for PNH has focused on designing supportive therapies to prevent transfusions due to hemolysis or thrombotic complications. In addition to the current FDA approved C5 inhibitor eculizumab, new, more convenient and effective complement blockers are under development. Apart from hematopoietic stem cell (HSC) transplantation, no direct strategies targeting basic pathophysiologic mechanisms of PNH have been ventured to prevent evolution of PNH clones and cure the disease. In early AA/PNH syndrome, the PIGA mutant HSCs are rare and unlikely contribute to significant blood cell production. While in later stages of manifest hemolytic PNH, hematopoiesis relies most frequently on mutant HSCs and thus elimination of these cells would result in AA.We hypothesized that if a selective inhibitor of GPI-AP-deficient [GPI-AP (-)] cells can be developed, it could be used primarily in AA/PNH patients with a small clone size. The hope would be to prevent both later expansion of GPI-AP d(-) cells and development of manifest PNH.To discover compounds acting selectively against GPI-AP (-) cells, we subjected wild type (WT) and GPI-AP (-) cell lines (K562, TF-1) to a high-throughput screen using a platform of 3000 bio-active molecules to identify hits and chemical compounds capable of selectively eliminating GPI-AP (-) cells. Our robotic screen yielded several top hits including GR -89696 fumarate, D-cycloserine and CGS-15943. Dose-response experiments confirmed CGS-15943 as a candidate growth inhibitor of GPI-AP (-) cells. CGS-15943 is an adenosine receptor antagonist and non-phosphodiesterase inhibitor which has previously been shown to inhibit cancer cell growth via PI3K/Akt pathway.Low range dose CGS-15943 (1uM) induced cell growth inhibition in K562 and TF-1 GPI-AP (-) cells by 4.7 fold and 3.2 fold, respectively. No cell growth arrest was observed in K562 WT and TF-1 WT cells, as the percentage of alive cells was >95% upon drug treatment. Mixed competition assays were conducted in vitro using equal ratios of K562 and TF-1 WT and GPI-AP (-) cells exposed to CGS-15943 (1uM). Six days after culture, flow cytometric analysis of CD59 surface expression revealed that CGS-15943 allowed for preferential survival of WT cells (84.7 % K562, 96.3% TF-1) vs. GPI-AP (-) cells (15.3% K562, 3.7% TF-1). CGS-15943 induced an increase in the % of AnnexinV+/PI- and AnnexinV+/PI+ in TF-1 GPI-AP (-) cells (12.04% and 44.82, respectively). Similar results were obtained in K562 GPI-AP (-) cells (15.84% and 21.08%). Mononuclear cells of a PNH patient were stimulated with CD3/28 beads in presence of CGS-15943. Flow cytometric analysis indicates a dose dependent growth inhibition effect on GPI-AP (-) lymphocytes after 3 days of culture. Previous reported observations from our group identified that the survival differences between GPI-AP (-) and WT cells largely depend on active PI3K signaling pathway. Our pilot investigation of CGS-15943 - indicates that CGS-15943 induces an decrease in the protein expression of the PI3K isoform - p110γ - exclusively in GPI-AP (-) cells possibly suggesting that CGS-15943 inhibits the catalytic subunit of- p110γ.In sum, we describe that the small molecule compound CGS-15943 selectively eliminates GPI-AP (-) cells in vitro, in both cell lines and in primary PNH cells most likely interfering with the PI3K/AKT survival pathway. DisclosuresMaciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.

FFAR1-and FFAR4-dependent activation of Hippo pathway mediates DHA-induced apoptosis of androgen-independent prostate cancer cells

Evidence indicates that diets enriched in Docosahexaenoic acid (DHA), a 22:6 n-3 polyunsaturated fatty acid, reduces the risk of prostate cancer, but the biochemical mechanisms are unclear. The Hippo pathway has been well established as a tumor suppressor pathway and is involved in many diverse biologic processes including cell growth, cell death, and organ size control in organisms. Here we showed that DHA induces cell growth inhibition and apoptosis of human androgen-independent prostate cancer cells dependent on the Hippo pathway. DHA inactivates YAP by promoting phosphorylation in androgen-independent prostate cancer cell lines, accompanied by increased YAP cytoplasm translocation. We also observed that DHA-induced YAP phosphorylation was reversed by both the LATS1 and MST1 siRNAs. Further experiments showed that the mechanism of DHA-induced YAP phosphorylation associated with FFAR1 and FFAR4. Down-regulation of FFAR1 and FFAR4 resulted in reduced YAP phosphorylation and reversed DHA-induced YAP phosphorylation. In addition, DHA-induced YAP phosphorylation was abolished by dominant negative Gαs and PKA inhibitor H-89. Overall, these findings define a mechanism by which FFAR1-and FFAR4-dependent activation of Hippo pathway mediates DHA-induced apoptosis of androgen-independent prostate cancer cells, thus providing a promising therapeutic target for prostate cancer.

How to Ward Off Retinal Toxicity of Perfluorooctane and Other Perfluorocarbon Liquids?

Reactive and underfluorinated impurities are acknowledged as a source of cytotoxicity of perfluorocarbon liquids (PFCLs) used as blood substitutes. To determine whether this is also a relevant factor in retinal toxicity, we analyzed eight PFO batches associated with adverse ocular events. (A) The amount of reactive and underflurinated impurities was analyzed by fluoride-selective potentiometry and expressed as H-value. (B) Cytotoxicity of these batches was determined by an ISO 10993-5-compliant extractive test and compared to published data generated with a direct-contact method. (C) A toxic PFO batch (061014) was purified to remove reactive and underfluorinated impurities. (A) and (B) -measurements were repeated after that. (D) The dose dependence of the H-value and cytotoxicity was determined in a dilution experiment. (A) The batches revealed H-values ranging from 1.400 ppm to 4.500 ppm. (B) All batches induced cell growth inhibition; seven must be classified as cytotoxic. Findings from ISO-conform extractive and direct-contact methods showed no difference. (C) After all reactive and underfluorinated impurities in batch 061014 were removed, the H-value dropped to <10 ppm and cytotoxicity disappeared. (D) Cytotoxicity increases gradually as the H-value rises. The clinical relevance of the H-value as a safety parameter for PFO endotamponades could be proven. The H-value is a measure for reactive and underfluorinated impurities that cause toxicity of PFCLs and should be incorporated in each endotamponade specification with a limit of 10 ppm to prove the effectiveness of the ultra-purification required and ensure a safe product. Despite the fact that an (ISO)-standard literally is a "standard" only, which cannot cover all imaginable possibilities, the incorporation of the H-value determination into the relevant ISO standard has been initiated. If a thorough risk assessment results in risks that cannot be detected and/or managed by the effective standard, additional investigations have to be performed.

Inhibition of WEE1 Suppresses the Tumor Growth in Laryngeal Squamous Cell Carcinoma.

WEE1 is a tyrosine kinase that regulates G2/M cell cycle checkpoint and frequently overexpressed in various tumors. However, the expression and clinical significance of WEE1 in human laryngeal squamous cell carcinoma (LSCC) are still unknown. In this study, we found that WEE1 was highly expressed in LSCC tissues compared with adjacent normal tissues. Importantly, overexpression of WEE1 was correlated with T stages, lymph node metastasis, clinical stages and poor prognosis of LSCC patients. Furthermore, inhibition of WEE1 by MK-1775 induced cell growth inhibition, cell cycle arrest and apoptosis with the increased intracellular reactive oxygen species (ROS) levels in LSCC cells. Pretreatment with ROS scavenger N-acetyl-L-cysteine could reverse MK-1775-induced ROS accumulation and cell apoptosis in LSCC cells. MK-1775 also inhibited the growth of LSCC xenografts in nude mice. Altogether, these findings suggest that WEE1 is a potential therapeutic target in LSCC, and inhibition of WEE1 is the prospective strategy for LSCC therapy.

Combination of pristimerin and paclitaxel additively induces autophagy in human breast cancer cells via ERK1/2 regulation.

Pristimerin, a quinonemethide triterpenoid, has demonstrated anticancer activity against a number of types of cancer, including breast cancer. However, its mechanism of action remains unclear. The present study investigated the autophagy-induced anticancer efficacy of pristimerin on MDA-MB-231 human breast cancer cells. Pristimerin inhibited the growth of these cells in a concentration-dependent manner. Treatment with pristimerin dose-dependently induced an increase of light chain 3B (LC3-II), whereas autophagy inhibitor 3-methyladenine (3-MA) inhibited pristimerin-induced LC3-II accumulation and cytotoxic effects. Autophagy was also activated by paclitaxel as observed by an elevated LC3-II level. Although 24 µM paclitaxel induced autophagy without cytotoxicity, combined with pristimerin it additively induced cell growth inhibition and autophagy induction. Autophagy induction was measured with an autophagy detection kit and LC3-II levels were monitored by western blot analysis. Treatment with 3-MA inhibited LC3-II accumulation and cell death induced by a combination of paclitaxel and pristimerin. Pristimerin and paclitaxel inhibited extracellular signal-regulated kinase (ERK)1/2/p90RSK signaling, consistent with autophagy indicators, namely p62 degradation and beclin 1 expression. In addition, ERK activator ceramide C6 treatment suppressed the LC3-II levels induced by a combination of paclitaxel and pristimerin. These results suggested that exposure to pristimerin induced autophagic cell death, whereas a combination treatment of pristimerin and paclitaxel resulted in an additive effect on ERK-dependent autophagic cell death.

Intra‑tumoral treatment with oxygen‑ozone in glioblastoma: A systematic literature search and results of a case series

Despite progress in surgery and radiochemotherapy, the prognosis of glioblastoma (GB) remains poor. GB cells exhibit a preference for hypoxia to maintain their tumor-forming capacity. Treatment strategies utilizing oxygen (O2) or ozone (O3) and generating reactive oxygen species induce cell growth inhibition and apoptosis. The anti-tumorigenic properties of O2-O3 are accompanied by a key role in regulating immunogenicity. The present study reported a case series of an intra-tumoral O2-O3 application in recurrent GB. Following surgery in combination with standard radiochemotherapy, O2-O3 (5 ml at 40 µg/ml) was applied every four weeks into the tumor vicinity. The patients received a median of 27 (range, 3–44) O2-O3 applications. In addition, a systematic literature search was performed in order to evaluate the role of O3 in the treatment of malignancies. The median overall survival rate was 40 (range, 16–53) months. The median survival rate following the first recurrence or the initiation of the O2-O3 treatment, respectively, was 34 (range, 12–53) months. In one patient, a local infection and in another, hemorrhage occurred, necessitating in both the temporary removal of the reservoir. The data from the present study support the potential benefit of an intra-tumoral O2-O3 application in recurrent GB. The scientific literature revealed by the bibliographic search suggests that O3 may be considered a viable adjuvant therapy in oncological patients. The present study may serve as a starting point for further observational and clinical studies elucidating the cellular and systemic effects of O2 and/or O3 and demonstrating their efficacy and safety in larger patient samples.