Methods Cell Viability Assay Research Articles

Phillygenin improves diabetic nephropathy by inhibiting inflammation and apoptosis via regulating TLR4/MyD88/NF-κB and PI3K/AKT/GSK3β signaling pathways

BackgroundPhillygenin (PHI), a main bioactive compound found in the fruit of Forsythia suspensa, exhibits antiviral, antioxidant, anti-inflammatory, and antihypertensive activities. However, the molecular mechanisms underlying its effects on diabetic nephropathy (DN) remain unclear. PurposeTo evaluate the therapeutic effects of PHI on DN and elucidate the molecular mechanisms involved. MethodsCell viability assays and RNA-seq analyses were performed to identify potential mechanisms through which PHI regulates HG-induced MPCs. The therapeutic efficacy of PHI was assessed in both DN cells and mouse models. Cytokine levels were measured using ELISA, while the expression levels of key signaling pathways, including TLR4/MyD88/NF-κB and PI3K/AKT/GSK3β along with downstream effectors were analyzed via immunoblotting, immunofluorescence, and immunohistochemical staining. ResultsPHI inhibited inflammatory responses and alleviated apoptosis by reducing the expression levels of IL-6, TNF-α, IL-1β, TLR4, MyD88, NF-κB, and cleaved caspase-3, while enhancing the phosphorylation of PI3K, AKT, GSK3β (Ser9), and pro-caspase-3 in MPCs under HG conditions in vitro. Additionally, in vivo experiments demonstrated that treatment with PHI (50 mg/kg) in db/db mice effectively improved renal function and attenuated kidney injury by reducing the urinary albumin to creatinine ratio (UACR), mitigating podocyte apoptosis, and inhibiting inflammatory via modulation of the TLR4/MyD88/NF-κB and PI3K/AKT/GSK3β signaling pathways. ConclusionPHI inhibits inflammation and apoptosis in vitro and alleviates diabetic kidney injury in db/db mice by interfering TLR4/MyD88/NF-κB and PI3K/AKT/GSK3β signaling pathways. Thus, this study reveals for the first time that PHI is a potential novel therapeutic agent for DN.

Solanum torvum induces ferroptosis to suppress hepatocellular carcinoma

Ethnopharmacological relevanceSolanum torvum Sw. (ST) is used to clear heat toxins, promote blood circulation, and alleviate blood stasis. Therefore, this plant has traditionally been used as an ethnomedicine for common cold, chronic gastritis, and tumors. Aim of the studyThis study aimed to elucidate the mechanism by which ST induces ferroptosis in hepatocellular carcinoma (HCC), the combination effect with lenvatinib, and the impact on lenvatinib-resistant cells. Materials and methodsCell viability assays were performed using different hepatoma cell lines treated with ST. Lipid peroxidation and iron assays were performed using flow cytometry. Molecules involved in the ferroptosis pathway were detected by Western blotting. Finally, a lenvatinib-resistant cell line was established to evaluate the antiproliferative effects of ST. ResultsST ethanol extract inhibited the growth of various hepatoma cell lines. A significant reduction in glutathione peroxidase 4 (GPX4) expression was observed following ST treatment, which was accompanied by increased lipid peroxidation and Fe2+ accumulation. ST induced ferroptosis mainly through heme oxygenase-1 (HO-1) expression. HO-1 knockdown reduced ST-induced lipid peroxidation and reversed GPX4 suppression. Acyl-CoA synthetase long-chain family member 4 (ACSL4) also participated in ST-induced ferroptosis. ST and lenvatinib combination showed an additive effect, and ST retained its potential anti-HCC efficacy in a lenvatinib-resistant cell line. ConclusionThis study demonstrated that the ethanol extract of ST inhibits hepatoma cell growth by inducing ferroptosis. ST displayed an additive effect with lenvatinib in Hep 3B cells and showed remarkable anti-HCC activity in lenvatinib-resistant Hep 3B cells. Collectively, the study shows that ST might have the potential to reduce lenvatinib use in clinical practice and salvage cases of lenvatinib resistance.

More than just a KRAS inhibitor: DCAI abrogates the self-renewal of pancreatic cancer stem cells in vitro

Abstract Objectives Growing evidence indicates that pancreatic cancer stem cells (CSCs) contribute to cancer recurrence via chemoresistance, and their growth is sustained by self-renewal. Targeting the self-renewal of pancreatic CSCs is a crucial strategy to eradicate them. Here, we are the first to describe a known KRAS inhibitor, 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), as a novel anti-pancreatic CSC agent that abrogates the self-renewal of pancreatic CSCs. Methods Cell viability assay was used to determine the cytotoxicity of KRAS binders in pancreatic cancer cell lines with either wild-type KRAS (BxPC-3) or clinically relevant KRAS mutations (PANC-1, Capan-2, and MIA PaCa-2). The tumoursphere assay was utilised to investigate the effect of DCAI on the self-renewal of pancreatic CSCs, and its mechanism of action was examined by Western blotting. Results The growth of pancreatic cancer cells remains unaffected by the binding of Benzimidazole (BZIM) to both wild-type and oncogenic KRAS. DCAI and Kobe0065 were equally potent in pancreatic cancer cell lines, except for Capan-2, in which DCAI (GI50=25.8 ± 0.8 µM) was more potent than Kobe0065 (GI50=54.0 ± 1.0 µM). Capan-2 tumourspheres were markedly irresponsive to gemcitabine (IC50>100 µM), while DCAI abrogated the formation of Capan-2 tumourspheres profoundly (IC50=30 µM). Upon treatment with DCAI, CRAF, ERK1, ERK2, and AKT activations were significantly inhibited, and SOX2 expression was greatly reduced in Capan-2 tumourspheres. Conclusions Our present study revealed that DCAI depletes pancreatic CSCs by inhibiting self-renewal via KRAS–CRAF–ERK1/2–SOX2 and KRAS–AKT–SOX2 axes. Our findings suggested that KRAS is a valid therapeutic target in pancreatic CSCs for eradicating cancer recurrence.

FZKA reverses gefitinib resistance by regulating EZH2/Snail/EGFR signaling pathway in lung adenocarcinoma

Ethnopharmacological relevanceFuzheng Kang-Ai (FZKA) decoction is mainly composed of 12 components with different types of herbs. In the last decade, FZKA has been used as an adjuvant treatment for lung cancer in clinical practice. Our previous studies have confirmed that FZKA shows a strong anti-cancer activity, significantly increases the clinical efficacy of gefitinib and reverses gefitinib resistance in non-small cell lung cancer (NSCLC). However, the molecular mechanism still needs to be further elucidated. Aim of the studyThe aim of this study was to investigate the role and mechanism by which FZKA inhibited the cell growth, proliferation and invasion of lung adenocarcinoma(LUAD) and reversed the acquired resistance of gefitinib for the therapy in LUAD. Materials and methodsCell viability assay and EDU assay were used for detecting of cell viability and cell proliferation. Transwell assay was performed to measure cell invasion. Western Blot and qRT-PCR were used for protein and gene expression test. The gene promoter activity was determined by dul-luciferase reporter assay. The in situ expression of protein was measured by cell immunofluorescence. Stabilized cell lines were established for stable overexpression of EZH2. Transient transfection assay was used for gene silence and overexpression. Xenograft tumors and bioluminescent imaging were used for in vivo experiments. ResultsFZKA significantly inhibited the cell viability, proliferation and cell invasion of LUAD, the combination of FZKA and gefitinib had a great synergy on the above processes. Moreover, FZKA significantly decreased EZH2 mRNA and protein expression, FZKA reversed the resistance of gefitinib by down-regulation of EZH2 protein. ERK1/2 kinase mediated the down-regulation of EZH2 reduced by FZKA. In addition, FZKA decreased the expression of Snail and EGFR by decreasing EZH2. Overexpression of Snail and EGFR significantly reversed the effect of FZKA-inhibited cell invasion and cell proliferation. More important, the combination of FZKA and gefitinib enhanced the inhibitory effect on EZH2, Snail and EGFR proteins. Furthermore, the growth inhibition and reversal of gefitinib resistance induced by FZKA were further validated in vivo. Finally, the expression and clinical correlation of EZH2,EGFR and Snail in cancer patients were further validated using bioinformatics analysis. ConclusionsFZKA significantly suppressed tumor progression and reversed gefitinib resistance by regulating the p-ERK1/2-EZH2-Snail/EGFR signaling pathway in LUAD.

Plumbagin modulates RANKL-induced osteoclast and osteoblast differentiation

BackgroundBone remodeling is a balanced, dynamic function of two counteractive processes: osteoblasts-mediated bone formation and osteoclasts-dependent bone resorption. An imbalance between these two processes causes bone diseases. Herein, the effects and molecular modus operandi of plumbagin- a bioactive phytometabolite from Plumbago sp. have been investigated on osteoblast and osteoclast differentiation. Material and MethodsCell viability assay and Alizarin red-S staining assessed the plumbagin effects on pre-osteoblastic MC3T3-E1 cell viability and differentiation. Alkaline phosphatase activity, the osteoblast-specific genes, and protein expression were investigated by colorimetry, quantitative-PCR and immunoblotting, respectively. TRAP assay and osteoclast-specific proteins' expression assessed plumbagin's effect on osteoclastogenesis. During plumbagin-driven osteoclastogenesis, we used paired-end RNA-sequencing data to identify the key up-/down-regulated DEGs, validation by qPCR, and bioinformatics-based pathway analysis. ResultsPlumbagin at a very low dose (0.25 µM) significantly promoted osteoblast differentiation, as evident from alkaline phosphatase, matrix mineralization, and enhanced expression of osteoblast-specific markers, including Runx2 and its target genes. The reduced TRAP activity, osteoclast-specific protein expression, and RNA sequencing suggested RANKL-induced osteoclastogenesis inhibition by plumbagin at 2 µM.. ConclusionPlumbagin significantly enhances osteoblast differentiation by enhanced matrix mineralization and osteoblast-specific gene expression but suppresses the RANKL-induced osteoclastogenesis by regulating key osteoclast-specific DEGs and protein expression. Plumbagin can thus significantly influence bone biology and affect bone diseases.

Intravitreal injection of EV11, a novel aryl ketone amide, inhibits choroidal neovascularization via AKT/ERK1/2 pathway

PurposeChoroidal neovascularization (CNV) is the major cause of irreversible vision loss associated with age-related macular disease (AMD). The currently clinical chemical therapeutic strategies are of high cost and facing supply chain shortage. In our study, we aim to investigate EV11, a novel derivative from Sorafenib, as a new approach to inhibit the formation of CNV. MethodsCell viability assay, wound healing assay, transwell assay and tube formation assay were applied to explore the effects of EV11 on human vascular endothelial cells (HUVECs). Western blotting analysis was performed to investigate the pathways when EV11 acts on HUVECs. Laser-induced CNV in mice and intravitreal injection of EV11 were applied to find out the efficacy of the drug in vivo. Histological examination and electroretinogram (ERG) evaluated the retinal morphology and visual function after drug application. ResultsEV11 influenced the HUVECs cell viability as the concentration increasing after 24 hour incubation. It influenced HUVECs through suppressing AKT and ERK1/2 pathway. EV11 reduced CNV area with the optimal concentration of 200uM in mice eyes and compared with Bevacizumab, it had the same effect. The retinal thickness around the optic in each group was not influenced. The amplitudes of the a- and b-waves on scotopic and photopic ERG were not reduced after intravitreal injection. ConclusionThe present study indicated that EV11 affected the proliferation, migration and tube formation of HUVECs, inhibited the area of neovascular of laser induced choroidal neovascularization in mice eyes with no toxicity. EV11 could block the AKT/ERK1/2 signaling pathway in effects of HUVECs. This study unveiled a novel perspective drug EV11 to be a potential candidate for neovascularization.

The natural compound from Garcinia bracteata mainly induces GSDME-mediated pyroptosis in esophageal cancer cells

BackgroundPyroptosis, an inflammatory form of programmed cell death (PCD), is reported to play important roles in the treatment of tumors. In our previous studies, we found that neobractatin (NBT), a caged prenylxanthone isolated from edible fruits of Garcinia bracteata C. Y. Wu ex Y. H. Li, showed anticancer effects against different cancer cells. However, the effect of NBT on pyroptosis is not well understood. PurposeThis study aims to investigate whether and how GSDME-mediated pyroptosis contributes to NBT-induced antitumor effects in esophageal cancer (EC) cells. MethodsCell viability assay and colony formation assay were used to determine the anticancer effects of NBT in esophageal cancer cells. Lactate dehydrogenase (LDH) release assay and microscopy imaging were used to detect the main characteristic of pyroptosis. CRISPR–Cas9 knockout and siRNA knockdown were performed to verify the roles of GSDME and caspase-3 in NBT-induced pyroptosis. Flow cytometry was used to measure the reactive oxygen species (ROS) level and cell apoptosis. The changes of related protein level were detected by Western blot. Furthermore, animal experiments were used to verify the in vivo effect of NBT. ResultsThe results showed that NBT reduced the viability of EC cells mainly through GSDME-mediated pyroptosis. Morphologically, NBT induced cell swelling and formed large bubbles emerging from plasma membrane in wild type EC cells. Furthermore, NBT induced the cleavage of GSDME by activating caspase-3 in EC cells. On the other hand, caspase-3 activated by NBT also induced apoptosis especially at high dosage. Knocking down GSDME switched NBT-induced cell death from mainly pyroptosis to apoptosis in vivo and in vitro. Mechanistic studies indicated that NBT led to accumulation of ROS, which then regulated the phosphorylation of both JNK and MEK/ERK. In the absence of ROS or caspase-3, NBT-induced pyroptosis and apoptosis were completely reversed. Moreover, NBT showed a significant antitumor effect in both the KYSE150 and GSDME knockout KYSE150-/- xenograft models by inducing pyroptosis and apoptosis, respectively. ConclusionOur results indicated that natural compound NBT could induce GSDME-mediated pyroptosis and apoptosis in esophageal cancer cells, making it a potential therapeutic drug in clinical treatment.

Transcriptome analysis upon potassium usnate exposure reveals ATF3-induced apoptosis in human gastric and colon cancer cells

BackgroundPotassium usnate (KU), a water-soluble form of usnic acid, shows anticancer activity. However, the underlying mechanisms have not been fully elucidated. PurposeWe aimed to identify the pathways involved in anticancer effects of KU in human gastric cancer (GC) and colorectal cancer (CRC) cells using RNA-sequencing (RNA-seq) based transcriptome analysis. Study DesignWe analyzed the cytotoxic effects of KU to identify the common molecular events in GC and CRC cells upon KU exposure using unbiased approaches. MethodsCell viability assays and western blot experiments were used to examine apoptotic changes, cell cycle arrest, and endoplasmic reticulum (ER) stress-induced cellular responses in KU-treated cells. Total RNA from KU-treated human GC and CRC cells was prepared for RNA-seq analysis. Gene ontology term and gene set enrichment analyses were used to identify the key mediators of the cytotoxic effects of KU. The expression of ER stress-induced apoptotic markers was evaluated using quantitative reverse-transcription PCR and western blot analysis. Chromatin immunoprecipitation assays for ATF3 and H3K27ac, and ATF3 knockdown were employed to verify the underlying molecular mechanisms. The inhibitory effect of KU on tumor growth in vivo was validated with metastatic tumor nodule formations in a mouse liver model. ResultsKU exerted cytotoxicity in human GC and CRC cells through the activation of the ER stress-induced apoptotic pathway. KU stimulated ATF3 expression, an important mediator of molecular events of apoptosis. ATF3 binds to the promoter region of ATF3, CHOP, GADD34, GADD45A, DR5, and PUMA genes and subsequently promoted apoptotic events. Knockdown of ATF3 significantly reduced the expression of ATF3 target genes and the cytotoxic effects of KU. The intraperitoneal injection of KU induced ATF3 and the apoptosis of implanted colon cancer cells, resulting in reduced metastatic tumor growth in the mouse livers. ConclusionKU exerts cytotoxic effects in human GC and CRC cells by triggering ER stress-induced apoptosis via an ATF3 dependent pathway.

14,15β-dihydroxyklaineanone inhibits HepG2 cell proliferation and migration through p38MAPK pathway

Eurycoma longifolia Jack (Simaroubaceae) is commonly distributed in the Southeast Asia and Indo China, which has been shown to possess antianxiety, antibacterial, anticancer, antifungal, anti-inflammatory, antimalarial and antioxidant biological activities. 14,15β-dihydroxyklaineanone is a diterpene isolated from E.longifolia Jack, which is cytotoxic against human lung cancer and human breast cancer cell lines. However, the effects and underlying mechanisms of 14,15β-dihydroxyklaineanone on hepatocellular carcinoma remain unknown. Cell viability assay and colony formation assay were used to measure HepG2 cell proliferation. Flow cytometry was used to analyse cell cycle and apoptosis. Wound-healing assay and transwell assay were used to observe cells migration. RNA sequencing and the enrichment of differentially expressed genes (DEGs) in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to find and determine underlying pathways. We found that 14,15β-dihydroxyklaineanone inhibited the growth and migration of HepG2 cells but did not induce cell apoptosis. 14,15β-dihydroxyklaineanone induced S cell cycle arrest by downregulating the expression levels of cyclin A, p-CDK2, cyclin B1, p21, E2F-1 and PCNA. In addition, RNA sequencing showed that 14,15β-dihydroxyklaineanone regulated MAPK pathway by increasing the expression levels of phosphor-p38. Downregulating of p38 via both p38 inhibitor (SB203580) and p38-siRNA could antagonize the inhibition of cell proliferation and migration andreverse the changes in p-p38, E-cadherin, N-cadherin and PCNA expression induced by 14,15β-dihydroxyklaineanone treatment. 14,15β-dihydroxyklaineanone inhibited cell proliferation and migration through regulating p38 MAPK pathway in HCC cells.

Anti‐tumor activity of the ethanolic extract of Micromeria fruticosa on human breast and colon cancer cells

Micromeria fruticosa (L.) Druce subsp. Serpyllifolia (Lamiaceae) is a perennial, aromatic shrub that usually distributed in the Mediterranean regions. The aerial parts of the plant are widely used to treat many diseases, including abdominal pains, diarrhea, colds, eye infections, heart disorders and wounds. A few reports have confirmed various therapeutic potentialities of its extract, such as anti‐inflammatory, gastroprotective, analgesic activities, antiobesity, and antidiabetic activities.AimTo investigate the mechanistic pathway for the antitumor activity of the ethanolic extract of M. fruticosa on two different cancer cell lines, namely human breast adenocarcinoma (MCF‐7), human colorectal cancer (HCT‐116) cell lines.MethodsCell viability assay, DNA profile with PI staining, Annexin‐V/PI apoptosis assay, microarray analysis, qRT‐PCR were used to assess the effect of M. fruticosa on cytotoxicity, cell cycle proliferation, apoptosis, and genes expression profile in treated cell lines.ResultsM. fruticosa is found to exhibit potential and effective cytotoxic activity against MCF‐7 and HCT‐116 cancer cell lines by attenuating cell proliferation and inducing cell cycle arrest. Microarray analysis revealed a significant reduction in cdk1, cyclin B1 and survivin levels upon M. fruticosa treatment. The qRT‐PCR confirmed the downregulation of cdk1, cyclin B1 that correlates with the observed G2 arrest in both cell lines. Moreover, results showed that the decreased levels of the cell death inhibitor, survivin is correlated to the promoted cell death in the treated cell lines.ConclusionThe findings suggest promising antiproliferative potential Micromeria fruticosa in breast and colon cancers. Further studies are needed to characterize the potential active constituent.Support or Funding InformationThis research was funded by University of Sharjah Seed Grant no.1701090113‐P and UOS‐Boehringer Ingelheim award.

β-Elemene inhibits the metastasis of multidrug-resistant gastric cancer cells through miR-1323/Cbl-b/EGFR pathway

Backgroundβ-Elemene is a natural agent extracted from the traditional Chinese herbal medicine Curcuma wenyujin that is a promising novel plant-derived drug with broad-spectrum anticancer activity. Our previous study identified an enhanced capacity for metastasis in multidrug resistant (MDR) gastric cancer and breast cancer cells. However, the anti-metastatic effects of β-Elemene on MDR cancer cells remain unknown. PurposeIn this study, we posit the hypothesis that β-elemene possesses antimetastatic effects on MDR cancer cells. MethodsCell viability assay was used to assess the resistance of SGC7901/ADR cells and the cytotoxic effects of β-Elemene. Wound healing, transwell assay and lung metastatic mice model were used to the anti-metastasis effects of β-Elemene. MicroRNA microarray analysis was used to explore potential regulated miRNAs. Luciferase reporter assay was used to identify the direct target. Human MMP antibody array, western blot, immunoprecipitation, qRT-PCR analyses and immunohistochemistry were conducted to investigate the underlying anti-metastasis mechanism of β-Elemene. ResultsIn this study, we found that β-Elemene significantly inhibited the metastatic capacity of MDR gastric cells in vivo and in vitro. Mechanistically, we found that β-Elemene regulated MMP-2/9 expression and reversed epithelial-mesenchymal transition. Further studies showed that β-Elemene upregulated Cbl-b expression, resulting in inhibition of the EGFR-ERK/AKT pathways, which regulate MMP-2/9. Additionally, we confirmed that β-Elemene upregulated Cbl-b by inhibiting miR-1323 expression. Finally, we found that numbers of metastatic tumor nodules were significantly decreased in the lungs of nude mice after β-Elemene treatment. ConclusionOur results suggested that β-Elemene inhibits the metastasis of MDR gastric cancer cells by modulating the miR-1323/Cbl-b/EGFR signaling axis.

Withaferin A triggers G2/M arrest and intrinsic apoptosis in glioblastoma cells via ATF4-ATF3-CHOP axis.

ObjectiveWithaferin A (WA) is a bioactive compound with a remarkable anti‐cancer effect derived from Withania somnifera, commonly known as ashwagandha. However, the anti‐cancer mechanisms of WA in glioblastoma multiforme (GBM) are still unclear.Materials and MethodsCell viability assays and xenografted nude mice were used to evaluate the effects of WA, along with flow cytometry to detect apoptosis and cell cycle of GBM. RNA‐seq analysis, Western blotting, immunofluorescence staining, qRT‐PCR and siRNA gene silencing were carried out to determine the signalling pathways affected by WA.ResultsWithaferin A significantly inhibited the growth of GBM in vitro and in vivo and triggered the intrinsic apoptosis of GBM cells by up‐regulating expression of Bim and Bad. WA arrested GBM cells at the G2/M phase of the cell cycle through dephosphorylating Thr161 of CDK1 by activating p53‐independent p21 up‐regulation. Knockdown of p21 restored cell cycle progression and cell viability by down‐regulating the expression of Bad rather than Bim. We demonstrated that endoplasmic reticulum (ER) stress induced by WA through the ATF4‐ATF3‐CHOP axis, initiated apoptosis and G2/M arrest in GBM cells.ConclusionWe revealed a novel pathway that elucidated WA activation of apoptosis and G2/M arrest in GBM cells through the ATF4‐ATF3‐CHOP axis. This discovery is important for optimization of WA‐based regimens for prevention and/or treatment of GBM.

76P - Proton-sensitizing effect of small molecule inhibitor of P300 histone acetyltransferase C646 in human pancreatic cancer cells

BackgroundHistone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Recent studies have demonstrated that several chromatin-regulating proteins can modulate cellular responses to other cytotoxic modalities including ionizing radiation and chemotherapeutic drugs. Even though histone acetylation is one of the key mechanisms of epigenetic regulation, relatively little is known about the cancer therapeutic potential of HAT inhibitors. This is in stark contrast to the well-studied effects of HDAC inhibitors. In this study, we investigated the proton beam-sensitizing effect of C646, a selective small molecule inhibitor of p300 histone acetyltransferase in human pancreatic cancer cells. MethodsCell viability assay (CCK-8; Dojindo), Clonogenic survival assay, Cell cycle analysis (PI staining). Annexin V-FITC and PI staining, γ-H2AX foci, Western blot, BxPC-3 Xenograft tumor model, TUNEL assay. ResultsAsPC-1, BxPC-3 and Mia-paca2 cells exhibited increase in radiosensitivity when exposed to C646 at all doses of proton beam tested. C646 pre-treatment induce led to increase of sub-G1 population and abolishment of G2/M arrest. Flow cytometry analysis with annexin V staining and Western blot analysis showed that The pre-treatment with C646 significantly enhanced proton-induced apoptotic cell death through the down-regulation of anti-apoptotic molecules. Our in vivo results demonstrate synergistic effects of combination therapy with C646 and proton beam. In BxPC-3 xenograft tumor model, C646 pre-treatment increased proton-induced tumor growth inhibition and apoptotic cell death in tumor tissues. In addition, the combination treatment of C646 with proton beam increased DNA damage and decreased activation of DNA repair pathway compared with proton alone. ConclusionsOur results suggest that the C646 may increase proton- induced apoptosis through the modulation of various pro- and anti- apoptotic molecules in human pancreatic cancer cells. These results provide proof of principle that the inhibition of histone acetylation by HATis can be exploited to develop new proton-sensitizer. Legal entity responsible for the studyThe authors. FundingNational Research Foundation of Korea (NRF). DisclosureAll authors have declared no conflicts of interest.

Taxol-Resistant Gene 1 (Txr1) Mediates Oxaliplatin Resistance by Inducing Autophagy in Human Nasopharyngeal Carcinoma Cells.

BackgroundOxaliplatin (L-OHP) is an important chemotherapy regimen for nasopharyngeal carcinoma (NPC), but can fail due to drug resistance. In this study, the role of Txr1 (taxol-resistant gene 1) in oxaliplatin resistance was investigated.Material/MethodsCell viability assay was carried out using the CellTiter-Glo Luminescent Cell Viability Assay Kit. CNE1 and CNE2 cells were cultured continuously with gradually increasing concentrations of L-OHP for 6 months to establish drug-resistant cell lines. Autophagy was detected by electron microscopy. Txr1 expression in NPC cells was detected via Western blotting and real-time quantitative PCR (qRT-PCR).ResultsIn L-OHP-resistant CNE1/L-OHP and CNE2/L-OHP cells, mRNA and protein expression of Txr1 increased compared to the parental cells, and downregulation of Txr1 re-sensitized drug-resistant cells to L-OHP. Moreover, we found that Txr1-mediated L-OHP resistance was associated with increased autophagy. Txr1-overexpression cells developed L-OHP resistance and a high level of autophagy. Inhibiting autophagy using 2 different methods – inhibition of autophagy-related gene expression and autophagy inhibitor – attenuated L-OHP resistance of NPC cells.ConclusionsWe conclude that the detection of Txr1 might become a good indicator to evaluate the treatment and prognosis of nasopharyngeal carcinoma. Our data suggest that further investigation of Txr1 in the setting of L-OHP resistance is warranted.

Micro-RNA-181a suppresses progestin-promoted breast cancer cell growth

BackgroundRecent investigations have indicated that hormone therapy may increase the risk of breast cancer (BC), and the addition of synthetic progestins may play a critical role in this. Several studies have pointed out the important role of progesterone receptor membrane component 1 (PGRMC1) in the development of BC, especially with hormone therapy using progestins. Although the deregulation of microRNA-181a (miR-181a) is often associated with human BC, the effect of miR-181a on PGRMC1 expression during hormone therapy has not been investigated. MethodsCell viability assay and apoptosis assay were performed to investigate the pro-BC effect of progestin (norethisterone, NET) and anti-BC effect of miR-181a on MCF-7 cells. Quantitative RT-PCR and Western blot analysis were used to evaluate gene expressions in the NET-treated MCF-7 cells. ResultsNET dose-dependently increased BC cell viability and this effect was accompanied by increased expression of PGRMC1. Overexpression of miR-181a strongly reduced the cell viability of MCF-7 cells, mainly through increased apoptosis, which was evidenced by substantially increased gene expression of pro-apoptosis factors such as BAX and CASPASE 9 in miR-181a overexpressed cells. Importantly, miR-181a abrogated NET-stimulated cell viability and PGRMC1 expression. ConclusionsWe provide evidence that miR-181a promotes MCF-7 cell apoptosis. Moreover, miR-181a suppressed NET-provoked cell viability and PGRMC1 expression in MCF-7 cells. These data may suggest a therapeutic strategy of using miR-181a to reduce BC risk in progestin hormone replacement therapy.

MP64-08 A NOVEL BIGUANIDE DERIVATIVE DRUG, IM176, INHIBITS PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2018MP64-08 A NOVEL BIGUANIDE DERIVATIVE DRUG, IM176, INHIBITS PROSTATE CANCER Choung-Soo KIM, Wonseok Choi, YOONRIM KIM, and Bong-Min Kim Choung-Soo KIMChoung-Soo KIM More articles by this author , Wonseok ChoiWonseok Choi More articles by this author , YOONRIM KIMYOONRIM KIM More articles by this author , and Bong-Min KimBong-Min Kim More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2053AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Biguanides were originally developed for the treatment of hyperglycemia and type 2 diabetes mellitus. Recently, the biguanides metformin and phenformin have been shown to exert potential anticancer effects in prostate cancer. We evaluated the anti-cancer efficacy and mechanism of IM176, a novel biguanide derivate drug on prostate cancer using prostate cancer cell lines and patient-derived castration-resistant prostate cancer cell lines. METHODS Cell viability assay, annexin V-FITC apoptosis detection, microscopy with immunofluorescence staining, real-time quantitative reverse transcription-polymerase chain reaction, and western blotting were conducted. Efficacy of IM176 was also evaluated using 2 cell lines derived from castration-resistant prostate cancer patient. RESULTS IM176 dose-dependently inhibited cell viability in all prostate cancer cell lines at lowest IC50 concentrations (LNCaP: 18.5μM, 22Rv1: 36.8 μM) compared to those of metformin, and phenformin. IM176-mediated AMPK activation caused mTOR inhibition, and a decrement in the phosphorylation of p70S6K1 and S6. IM176 inhibited the expression of AR, AR-splice variant 7 (AR-V7) and prostate-specific antigen in LNCaP and 22Rv1. IM176 induced apoptosis with increased levels of cleavage of caspase-3, and annexin V-positive / PI-positive, respectively. Moreover, IM176 inhibited cell viability at lowest IC50 concentrations in 2 cell lines derived from castration-resistant prostate cancer patient. We evaluate the relationship between AMPK-mTOR pathway and AR signaling pathway by blocking each pathway separately. After AR knockdown, phosphorylation of AMPK was significantly increased. However, AR and AR-V7 were not increased after treating AMPK inhibitor, Compound C. CONCLUSIONS IM176 showed comparable anti-tumor effects via AMPK-mTOR pathway and AR signaling pathway with the lowest IC50 compared to other biguanide derivative drugs in prostate cancer cell lines, including patient-derived castration resistant prostate cancer cell line and may be a novel anti-cancer drug for the treatment of prostate cancer. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e853 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Choung-Soo KIM More articles by this author Wonseok Choi More articles by this author YOONRIM KIM More articles by this author Bong-Min Kim More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Antiviral effect of 25-hydroxycholesterol against porcine reproductive and respiratory syndrome virus in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that causes economically huge losses to the pig industry worldwide. Current control of PRRSV infection remains inadequate although various means have been implemented. Thus, investigating novel antiviral therapeutics to combat PRRSV infection is essential. In the present study, the antiviral effect in vitro of 25-hydroxycholesterol (25HC) against PRRSV was investigated. Cell viability assay was performed to examine the impact of 25HC on the cell viability. Indirect immunofluorescence assay and virus titration were utilized to evaluate the levels of PRRSV growth. Viral attachment assay, penetration assay and release assay were conducted to investigate the antiviral mechanism of 25HC against PRRSV. Real-time RT-PCR assay was used to analyse the effect of 25HC on the genome synthesis of PRRSV. We demonstrated that the growth of PRRSV was significantly inhibited in 25HC-pretreated cells and PRRSV-infected cells by 25HC. Moreover, 25HC could impair the attachment and entry of PRRSV in vitro, but not affect viral genome synthesis and virion release. Our findings clearly indicate that 25HC can exert antiviral effect against PRRSV infection in vitro, suggesting that 25HC might be a novel potential agent to control PRRSV infection.

Effects and mechanisms of resveratrol on arsenic trioxide-induced cytotoxicity in H9c2 cells

Objective To investigate the protective effects and mechanisms of resveratrol on arsenic trioxide (As2O3) -induced cytotoxicity in H9c2 cells. Methods Cell viability assay, lactate dehydrogenase (LDH) release, caspase-3 activity assay, reactive oxygen species (ROS) generation and cellular calcium levels were measured in H9c2 cells when cells single exposure to As2O3 and combinational treated with As2O3 and resveratrol. Results Pretreating H9c2 cells with resveratrol (40, 50, 90, 100 μg/ml) reduced the extensive cell death induced by As2O3(P 0.05). Treatment with resveratrol (100 μg/ml) had no effect on intracellular calcium concentration (P>0.05). Conclusions Resveratrol can ameliorate As2O3-induced cytotoxicity in H9c2 cells in vitro. Its mechanism probably related to reducing the ROS generation. Key words: Resveratrol; Arsenic trioxide; Cytotoxicity; Mechanisim

Actein Inhibits Cell Proliferation and Migration in Human Osteosarcoma.

BackgroundOsteosarcoma is one of the most common malignant bone cancers worldwide. Although the traditional chemotherapies have made some progression in the past decades, the mortality of osteosarcoma in children and adolescent is very high. Herein, the role of actein in osteosarcoma was explored.Material/MethodsCell viability assay was performed in osteosarcoma cell lines 143B and U2OS. Colony formation analysis was included when cells were treated with different doses of actin. Cell cycle assay was conducted to further examine the role of actein. Cell apoptotic rate and the relative activities of caspase-3, caspase-8, and caspase-9 were detected in 143B and U2OS osteosarcoma cells. Moreover, transwell assays were used to explore the effects of actein on cell metastasis.ResultsActein significantly inhibited osteosarcoma cell viability in a time- and dose-dependent manner. Actein also dramatically suppressed the colony formation ability in osteosarcoma143B and U2OS cells. It was revealed that osteosarcoma cells were arrested in G0/G1 phase in the cell cycle progression and induced to apoptosis by administration of actein. The activities of pro-apoptotic factors such as caspase-3 and caspase-9 were significantly increased by actein. Furthermore, administration of actein decreased cell migrated and invasive abilities in both 143B and U2OS cell lines.ConclusionsActein inhibits tumor growth by inducing cell apoptosis in osteosarcoma. The inhibitive roles of actein in cell proliferation, migration and invasion suggest that actein may serve as a potential therapeutic agent in the treatment of osteosarcoma.

The natural secolignan peperomin E induces apoptosis of human gastric carcinoma cells via the mitochondrial and PI3K/Akt signaling pathways in vitro and in vivo

BackgroundPeperomin E (PepE) is a type of secolignan that is a major component of the plant Peperomia dindygulensis. It has been shown to exert anticancer effects in various cancer cell lines; however, the effects of PepE on human gastric cancer remain unexplored. PurposeThe aim of this study was to investigate the effectiveness of PepE as a treatment of gastric cancer and to identify the underlying mechanisms of its anticancer activity. Study designThe efficacy of PepE was examined using human gastric carcinoma SGC-7901, BGC-823, MKN-45 cell lines and normal gastric epithelial GES-1 cell line as an in vitro model and SGC-7901 xenograft mice as an in vivo model. MethodsCell viability assays were used to examine the anticancer effect of 0–204.8µM concentrations of PepE in vitro. Additionally, flow cytometry and western blotting were used to elucidate the mechanism with a particular focus on apoptosis. SGC-7901 cells were injected into BALB/c mice, which were then treated with 5 or 15mg/kg/day dose of PepE. The in vivo activity of PepE was investigated by measuring tumors and conducting immunohistochemistry experiments. The safety of PepE was investigated by measuring blood biochemical parameters and conducting histopathological analysis. Taxol was used throughout as a positive control. ResultsThe results showed that PepE exhibited antiproliferative effects against gastric cancer cells and induced their apoptosis in a dose dependent manner with lower toxicity against normal gastric epithelial cells. Mechanistic evaluations indicated that PepE induced apoptosis by reducing the mitochondrial membrane potential (MTP), inducing cytochrome C release from mitochondria, reducing the ratio of Bcl-2/Bax and Bcl-xl/Bad, increasing activation of caspase-3, and decreasing the levels of PI3K and pAkt. The apoptotic effect of PepE on SGC-7901 cells was partially blocked by an Akt activator SC79. PepE potently inhibited in vivo tumor growth with no obvious toxicity following subcutaneous inoculation of SGC-7901 cells in nude mice. ConclusionsThese findings indicate that PepE can inhibit cell proliferation and induce apoptosis of gastric cancer cells through mitochondrial and PI3K/Akt signaling pathways with relative safety and may be a novel effective chemotherapeutic agent against gastric cancer.