Hoechst 33342 Research Articles

Quercetin Alleviates Osteoarthritis Progression in Rats by Suppressing Inflammation and Apoptosis via Inhibition of IRAK1/NLRP3 Signaling.

IntroductionQuercetin was recently reported to help protect against osteoarthritis (OA) progression, but the molecular mechanism for that protective affect remains unclear.MethodsHere, OA model rats were intraperitoneally injected with quercetin, and the severity of cartilage damage in the rats was evaluated by H&E, Safranin O, and Toluidine blue, as well as by using the Osteoarthritis Research Society International (OARSI) Scoring System. Additionally, rat chondrocytes were treated with quercetin and then stimulated with IL-1β. The levels of pro-inflammatory cytokines (IL-1β, IL-18, and TNF-α) were detected by ELISA.Cell apoptosis was evaluated by flow cytometry and Hoechst staining. ROS levels were measured using a DCFH-DA probe. Protein expression was evaluated by Western blotting, immunohistochemical staining, and immunofluorescence. ResultsOur data showed that quercetin attenuated the degeneration and erosion of articular cartilage, suppressed inflammation and apoptosis, and downregulated the levels of IRAK1, NLRP3, and caspase-3 expression. In vitro data showed that overexpression of NLRP3 could reverse the suppressive effect of quercetin on IL-1β-induced rat chondrocyte injuries. Importantly, rescue experiments confirmed that quercetin inhibited IL-1β-induced rat chondrocyte injuries in vitro by suppressing the IRAK1/NLRP3 signaling pathway.ConclusionOur study indicated that quercetin inhibits IL-1β-induced inflammation and cartilage degradation by suppressing the IRAK1/NLRP3 signaling pathway.

Abstract LB131: Shortening of mRNA 3′ untranslated region mediates simultaneous overexpression of ABCG2 and CD133 in putative cancer stem cells

Abstract Aim: We investigated the mechanisms driving the concomitant overexpression of ABCG2 and CD133 in cancer stem cells (CSCs) and examined the effect of modulating the underlying machinery on anticancer drug response. Background: Multidrug resistance is a major obstacle to chemotherapy. It is usually associated with increased efflux of anticancer drugs by ATP-binding cassette transporters including ABCG2. ABCG2 is also a key survival factor for CSCs, which lead to recurrence. CSCs have been identified in patient tumor specimens and in cancer cell lines. The CSC hypothesis suggests that anticancer therapies have to target and destroy all residual CSCs in order to produce a durable response. CD133 is a well-characterized stem cell biomarker and it plays crucial role in the self-renewal and tumorigenesis of CSCs. Importantly, ABCG2 and CD133 are co-expressed in CSCs, yet the mechanism(s) driving their concomitant high expression remain elusive. mRNA polyadenylation is a key step regulating the maturation of eukaryotic mRNAs by adding a poly A tail to their 3' ends. Alternative polyadenylation, commonly observed in cancer cells, is a phenomenon responsible for the production of multiple mRNA isoforms bearing different length of 3'-untranslated region (3'UTR). Method: Sub-population of cancer cells from three ABCG2-overexpressing multidrug resistant colon cancer cell lines were sorted by Hoechst dye exclusion or CSC markers (DCLK1+LGR5+BMI1+), respectively, as side population (SP) and putative CSC cells. Expression of ABCG2, CD133, and a few master mRNA polyadenylation trans-acting regulators was compared in SP (or CSC) versus non-SP (or non-CSC) cells. Altered 3'UTR isoforms of ABCG2 and CD133 were examined by 3'RACE assay and 3'UTR isoform specific PCR. The anticancer activity of common chemotherapeutic drugs was examined after genetic modulation of the key mRNA polyadenylation regulators identified. Result: The mRNA stability and expression of both ABCG2 and CD133 were found to be remarkably higher in SP cells (or CSC cells) than in non-SP cells (or non-CSC cells). By 3'RACE assay, shortening of ABCG2 and CD133 3'UTR was evidenced in SP (and CSC) cells but not in non-SP (and non-CSC) cells, thereby increasing mRNA stability and/or translation by escaping the repression from regulatory elements found on long mRNA 3'UTR. Expression of two master mRNA polyadenylation regulators (PABPN1 and CSTF2) was found to be remarkably lower and higher, respectively, in SP (and CSC) than in non-SP (and non-CSC) cells, presumably allowing the preferential utilization of proximal 3'UTR to mediate high expression of ABCG2 and CD133. Genetic silencing of CSTF2 was shown to enhance the anticancer effect of SN-38 and reduce the colony formation capacity of CSC cells. Conclusion: A novel mRNA 3'UTR shortening machinery was shown to mediate the simultaneous overexpression of ABCG2 and CD133 in SP and CSC cells. It may represent useful drug target for circumvention of resistance and eradication of CSCs. Citation Format: Kenneth K.W. To. Shortening of mRNA 3′ untranslated region mediates simultaneous overexpression of ABCG2 and CD133 in putative cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB131.

Testing Hybridoma Cells for Mycoplasma Contamination.

Because mycoplasmas are a diverse group of organisms and are difficult to culture, several different strategies for detecting mycoplasma contamination have been developed. To date, no one test is suitable for detecting all of the possible mycoplasmas that may contaminate hybridoma or myeloma cultures. Therefore, it is sensible to consider using several methods. The most commonly used techniques are described here. Mycoplasma screening by growth on microbial medium can be performed on agar plates or in broth culture. Cultures are grown under both aerobic and anaerobic conditions because some common strains of Mycoplasma prefer the lack of oxygen (Mycoplasma buccale, Mycoplasma faucium, M. orale, and M. salivarium). Mycoplasma colonies form a characteristic "fried egg" appearance on agar plates, and this is the diagnostic feature used to confirm mycoplasma contamination. The colonies are small and are most easily seen with an inverted microscope. A quicker method for testing for mycoplasma takes advantage of the DNA-intercalating dye Hoechst 33258. Fixed cells are stained with the dye, and contaminated cultures are detected by the bright, punctate cytoplasmic staining of the Mycoplasma DNA. Finally, commercial kits for the detection of mycoplasmas using colorimetric assays or reporter cells are also described.

Anti-angiogenic, apoptotic and matrix metalloproteinase inhibitory activity of Withania somnifera (ashwagandha) on lung adenocarcinoma cells

IntroductionWithania somnifera belongs to the family Solanaceae, known as Queen of medicinal plants for its enormous use in the medicinal field. Traditionally ashwagandha is used to treat several neurological disorders. This study evaluates the cytotoxic, apoptotic, antiangiogenic and matrix metalloproteinase (MMP) inhibitory activity of W. somnifera on lung adenocarcinoma. MethodologyAqueous and ethanolic extracts were prepared from the roots of the W. somnifera. Qualitative and quantitative phytochemical analyses were performed using the standard protocols. Cytotoxicity was assessed using MTT assay. Further experiments were carried out with IC50 concentration of the extract. Apoptosis and DNA damage were evaluated using AO-EB dual staining, Hoechst staining and Comet assay. Effect of the extract on cell migration was evaluated using scratch assay. Angiogenesis inhibition was evaluated using in ovo CAM assay and angiogenic pathway alterations were evaluated using qRT-PCR and western blotting. Autophagy induction was studied via western blotting. ResultsIn this study, we found antioxidant activity and the presence of certain secondary metabolites in the ethanolic extracts. The extract showed cytotoxic activity on lung adenocarcinoma cells with an IC50 of 99.7 μg/ml. The extract showed significant anti-angiogenic, apoptotic and autophagy induction activity. W. somnifera extract induced significant decrease in the cell migration at lower concentrations indicating the anti-migratory potential. ConclusionOur investigation revealed ethanolic extract of W. somnifera possess significant anti-angiogenic and MMP inhibitory activity and helps in inhibiting the lung adenocarcinoma cells proliferation. Further, our study revealed that the enhanced autophagy induction and apoptotic effects of W. somnifera are responsible for the potential anticancer activity of the extract.

Biocompatible madecassoside encapsulated alginate chitosan nanoparticles, their anti-proliferative activity on C6 glioma cells

Madecassoside (MAD), an ursane type pentacyclic triterpenoid saponin, isolated from Centella asiatica (CA) possesses various pharmacological activities. The aim of this study was to evaluate therapeutic potential of MAD encapsulated alginate chitosan nanoparticles (MACNPs) on C6 glioma cells. Synthesized nanoparticles were characterized by Thermogravimetric analysis (TGA), Differential thermal analysis (DTA), Differential scanning calorimetry (DSC), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). Cell viability and compatibility of both MAD and MACNP were observed on primary astrocytes. Cytotoxicity and anti-proliferative effect of MACNPs were studied on C6 glioma cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5′-ethynyl-2′-deoxyuridine (Edu) assays. MACNPs showed 90% cytotoxicity, and depicted anti-proliferative activity against C6 cells. Annexin V-Alexa flour-488/propidium iodide (PI) staining followed by flow cytometry revealed that MACNPs induced necrosis in C6 glioma cells. Cell death was also observed using acridine orange/ethidium bromide (AO/EtBr) and Hoechst staining. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFDA) assay by flow cytometry showed an increase in reactive oxygen species (ROS) production in MACNP treated C6 glioma cells. MACNPs showed a greater uptake and distribution in C6 glioma cells and inhibited the proliferation of C6 cells via increased intracellular ROS production. Our findings suggest MACNP as a promising drug delivery carrier for the management of glioma and shed new insights to brain malaises.

CircPVT1 Inhibit Apoptosis and Enhance Drug Resistance in Multiple Myeloma

Objective: Circular RNAs (circRNAs) comprise a novel class of widespread non-coding RNAs that could regulate gene expression in eukaryotes. Glucocorticoid resistance is the primary factor of refractory relapse in patients with multiple myeloma (MM). This study was designed to reveal the role of circPVT1 in mediating glucocorticoid resistance in MM through regulating its target genes to inhibit apoptosis of MM cell and promote tumor stem cell survival.Methods and results: We determined with RT-PCR assay that circRNA PVT1 is differentially expressed between glucocorticoid resistant and sensitive MM cells (Figure A), and is closely correlated with prognosis (Figure G and H). We further investigated the role of circPVT1 in MM cell apoptosis and proliferation as well as its downstream signaling pathway mechanism. Silencing circPVT1 enhances sensitivity to glucocorticoid treatment (Figure D), promotes apoptosis and inhibits cell proliferation in resistant MM cell lines and myeloma cells in NOD-SCID mice, resulting up-regulation of the apoptosis pathway as measured by increased Caspase3, PARP and decreased Bcl-2 (Figure E). Measured by CCK-8 cell viability assay, ectopic expression of circPVT1 contributed to inducing proliferation and suppressing apoptosis in sensitive MM cell lines. Using Hoechst Staining, we found that MM stem cell population increases upon ectopic expression and decreases while silencing circPVT1, which revealed circPVT1 could facilitate the proliferation of stem cells to enhance glucocorticoid resistance (Figures F and I).Conclusion: As a downstream of circPVT1, it promotes c-myc nuclear translocation, which intermediated by miRNAs to enhance its targets thus promotes the proliferation of stem cells and inhibits apoptosis. We concluded that circPVT1 may mediate MM glucocorticoid resistance and its downstream signaling pathway may represent a novel target for MM treatment.Figure (A)RT-PCR showing the differential expression of circPVT1 and linear RNA PVT1 between MM.1R and MM.1S cell lines.** P <0.01. Error bars, S.D. (n=3). (B)RT-qPCR results showing the abundance of circPVT1 and PVT1 in MM.1R cells treated with RNase R. The levels of CircPVT1 and PVT1 were normalized to the values measured after mock treatments.** P <0.01. Error bars, S.D. (n=3). (C)The expression of circPVT1 was detected by RT-PCR and was validated by Sanger sequencing. (D)RT-PCR analysis of circPVT1 expression after treatment with siRNA targeting at the circPVT1.** P <0.01. Error bars, S.D. (n=3). (E)Western blot analysis of the levels of PARP, caspase 3, Bcl-2, control of β-actin in MM.1R and MM.1S transfected with circPVT1 siRNA or control siRNA, in absence of dexamethasone 1μM/L for 24 hours. (F)Using Hoechst Staining, analysis of side population of MM.1R transfected with circPVT1 siRNA or control siRNA. (G)RT-PCR showing the differential expression of circPVT1 of GC sensitive and resistant patients. ** P <0.01. Error bars, S.D. (n=28). (H)Kaplan-Meier analysis of the correlation between GC sensitivity and overall survival. (I)Diagram showing c-myc nuclear translocation facilitated by circPVT1, intermediated by miRNAs to enhance the targets of c-myc thus promotes the proliferation of stem cells and inhibits apoptosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Fbxw11 Triggers a Dual Signaling Axis to Exert Proliferation Effects on Lymphocytic Leukemia

Dysregulation of signal conduction will break the balance of normal hematopoiesis and cause various blood diseases. Ubiquitin-proteasome system (UPS) takes part in both physiological and pathological processes by post-translational regulating intracellular signal transduction pathways. Fbxw11 as a constituent of the SCF (Skp1-Cul1-F box) E3 ubiquitin ligase complex, regulates various signal transduction pathways and its pathological roles in tumorigenesis have been suggested. However the role of Fbxw11 in the development of leukemia and the underlying mechanisms remain largely unknown.To assess the clinical relevance of Fbxw11, the expression of Fbxw11 in AL patients was also studied by real-time PCR. The expression of Fbxw11 was higher in newly diagnosed ALL (p=0.0304) and relapsed ALL patients (p=0.0389) than that in healthy donors. However, results from a follow-up study demonstrated that the expression of Fbxw11 was dramatically decreased after chemotherapy achieving CR (p=0.0202). These results were conformed in the BloodSpot database.To study in vitro effects of Fbxw11 on leukemia cells, the lymphocytic leukemia cell line, L1210, was stably transfected with Fbxw11 transcript variants and defined as L1210-control, L1210-Fbxw11a, L1210-Fbxw11c and L1210-Fbxw11d, respectively. Increased percentage of S/G2/M phase cells was detected in L1210 cells with high level Fbxw11 by PI stainingand Ki-67/hoechst33342 staining. Fbxw11 significantly promoted the growth of L1210 cells. The proliferation effects of Fbxw11 on the leukemia cells were also tested in vivo . Tumor growth was significantly higher in groups with high level Fbxw11 variants. Significantly higher rate of Brdu positive cells were detected by confocal microscopy in slides formed by L1210 cells expressing high level Fbxw11 variants than control slides .To further investigate the pro-proliferation mechanism by Fbxw11 transcript variants, Agilent Sureprint G3 Mouse Gene Expression Microarray was performed after total RNA was isolated from control L1210 cells and those expressing high level Fbxw11 transcript variants. Categories of NF-κB and Wnt/β-catenin signaling pathway has high enrichment score in KEGG and IPA respectively. Categories of regulation of cell proliferation and positive regulation of T cell proliferation have high enrichment score in GO biological process. Cell cycle regulator, CyclinD1 and CyclinD3, which were regulated by multiple transcription factors including NF-κB and β-catenin, were significantly up-regulated in L1210 cells expressing high level Fbxw11 transcript variants at protein level. The expression of other NF-κB-regulated genes and β-catenin/TCF-regulated genes were also up-regulated in L1210 cells with high level Fbxw11 transcript variants, which was consistent with the array data. Activation of both NF-κB and β-catenin/TCF signaling pathways was detected in 293T cells expressing high level Fbxw11 transcript variants by dual luciferase reporter system. Furthermore, combination of low dose inhibitors for NF-κB and β-catenin/TCF pathways (ICG-001 and JSH-23) completely diminished pro-proliferation effects of Fbxw11 though the use of low dose either inhibitor had little effect.Taken together, Fbxw11 was up-regulated in ALL patients. High level expression of Fbxw11 stimulated the proliferation of L1210 lymphocytic leukemia cells in vitro and promoted tumor formation in vivo through cell cycle regulation. Most importantly, activation of both NF-κB and β-catenin/TCF signaling pathways were involved in this process. This work highlights dual signaling activation and illustrates the mechanism of Fbxw11 on the development of lymphocytic leukemia and implys possible approaches for leukemia therapy.The work was supported by the Grants 81300376, 81370634, 81570153, from the National Natural Science Foundation of China (NSFC); 14JCQNJC10600 and 17 JCZDJC35000 from the Tianjin Science and Technology Programs DisclosuresNo relevant conflicts of interest to declare.

Potential application of two cobalt (III) Schiff base complexes in cancer chemotherapy: Leads from a study using breast and lung cancer cells

Cobalt (III) Schiff base complexes are of attraction in the context of their potential application in cancer therapy. The aim of this study has been to find the mechanism of action of cobalt (III) Schiff base complexes 1 and 2, the synthesis and characterization of which have already been reported, in inhibiting growth of human breast cancer cell MCF-7 and lung cancer cell A549. The already proclaimed anti-proliferative effect of the cobalt complexes was ascertained using MTT cytotoxicity assay. More assays such as Acridine orange & Ethidium bromide staining, AnnexinV-Cy3 staining, Hoechst staining, comet assay, and Reactive Oxygen Species (ROS) assay- all supported the cytotoxic property of the complexes. Moreover, the expression levels of mRNA of pro- and antiapoptotic genes also supported the effectiveness of cobalt complexes by modifying the ratio of Bax: Bcl-2. In addition, the cobalt complexes induced apoptosis in MCF- 7 and A549 cells through modulation of pro-apoptotic, anti-apoptotic, and ROS modulatory gene expressions. The present study validates the scientific evidence for antiproliferative efficacy of cobalt complexes against MCF-7 and A549 cells. Thus, this study takes cobalt complexes 1 and 2 to a step higher towards their use as anticancer agents.

Malformin-A1 (MA1) Sensitizes Chemoresistant Ovarian Cancer Cells to Cisplatin-Induced Apoptosis.

High-grade epithelial ovarian cancer is a fatal disease in women frequently associated with drug resistance and poor outcomes. We previously demonstrated that a marine-derived compound MalforminA1 (MA1) was cytotoxic for the breast cancer cell line MCF-7. In this study, we aimed to examine the effect of MA1 on human ovarian cancer cells. The potential cytotoxicity of MA1was tested on cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780CP) ovarian cancer cell lines using AlamarBlue assay, Hoechst dye, flow cytometry, Western blot, and RT-qPCR. MA1 had higher cytotoxic activity on A2780S (IC50 = 0.23 µM) and A2780CP (IC50 = 0.34 µM) cell lines when compared to cisplatin (IC50 = 31.4 µM and 76.9 µM, respectively). Flow cytometry analysis confirmed the cytotoxic effect of MA1. The synergistic effect of the two drugs was obvious, since only 13% of A2780S and 7% of A2780CP cells remained alive after 24 h of treatment with both MA1 and cisplatin. Moreover, we examined the expression of bcl2, p53, caspase3/9 genes at RNA and protein levels using RT-qPCR and Western blot, respectively, to figure out the cell death mechanism induced by MA1. A significant down-regulation in bcl2 and p53 genes was observed in treated cells compared to non-treated cells (p < 0.05), suggesting that MA1 may not follow the canonical pathway to induce apoptosis in ovarian cancer cell lines. MalforminA1 showed promising anticancer activity by inducing cytotoxicity in cisplatin-sensitive and cisplatin-resistant cancer cell lines. Interestingly, a synergistic effect was observed when MA1 was combined with cisplatin, leading to it overcoming its resistance to cisplatin.

A feasible strategy of fabricating hybrid drugs encapsulated polymeric nanoparticles for the treatment of gastric cancer therapy

Chemotherapy of hybrid-drugs has become one of the widely used therapies for the prevention of cancers with promising clinical results. This study aimed to create compostable polymeric nanoparticles (PPNPs) for the codelivery of capsaicin (CAPS) and biotin (BT) and to examine the anticancer activity of the drug delivery mechanism (CAPS/BT@PPNPs) against in vitro and in vivo gastric cancer cells. Because of the insufficiency of double antitumor drugs BT and CAPS compressed in polymer-based bio-degradable nanoparticles, the encapsulation of BT and CAPS antitumor drugs co-loaded with polyethylene glycol and polylactidecoglycolide nanoparticles (PPNPs) is effective. Transmission electron microscopy (TEM) was utilized to investigate the structure of BT@PPNPs, CAPS@PPNPs, and BT/CAPS@PPNPs, as well as their shape and size. Human gastric carcinoma cell lines, such as SGC-791 and NCI-N87, were also induced to apoptosis in vitro by BT/CAPS@PPNPs. Morphologies and cell death were observed using bio-chemical staining methods, AO/EB (acridine orange/ethidium bromide) and Hoechst. Flow cytometry in dual staining was also used to confirm the mechanistic investigations of apoptosis. Overall, this dual drug delivery strategy suggests that BT/CAPS@PPNPs could be used as a new method to increase the efficacy of gastric therapeutics.

Dihydromyricetin promotes apoptosis, suppresses proliferation and tumor necrosis factor-α-mediated nuclear factor kappa-B activation in nasopharyngeal carcinoma CNE-2 cell.

To investigate the efficacy of dihydromyricetin (DMY) on nasopharyngeal carcinoma (NPC) cell proliferation, apoptosis and to reveal the underlying mechanism in vitro experiments. The CNE-2 cell line was treated with different concentrations of DMY and the effects of DMY on cell viability and proliferation were evaluated using cell counting kit-8 (CCK-8) assay and plate colony formation assay. Cellular apoptosis was detected by flow cytometry following Annexin V fluorescein isothiocyanate/propidine iodide staining. Nuclei morphology was observed under a fluorescence microscope following Hoechst 333258 staining. The expression of phosphorylated inhibitor of nuclear factor kappa-B kinase subunit beta (p-IKKβ), phosphorylated inhibitor of nuclear factor kappa-B kinase subunit alpha (p-IKKα), inhibitor of nuclear factor kappa-B alpha (IκB-α), nuclear factor kappa-B (NF-κB)/p65 was examined by Western blot analysis and the nuclear translocation of NF-κB/p65 was observed using a confocal laser scanning microscopy. DMY inhibited the proliferative capability and colony formation of NPC CNE-2 cells. Meanwhile, DMY induced apoptosis of CNE-2 cells in a dose and time-dependent manner via upregulating B-cell lymphoma-2 associated X, but downregulating B-cell lymphoma-2 and pro-caspase-3. Importantly, we found that DMY suppressed tumor necrosis factor alpha (TNF-α)-mediated NF-κB activation via inhibiting p-IKKβ, p-IKKα and blocking NF-κB subunit p65. Our experiments demonstrated that DMY had significant antiproliferative and apoptosisinducing effects on CNE-2 cells. Additionally, DMY promoted inactivation of p-IKKβ, p-IKKα, and blocked the nuclear translocation of NF-κB subunit p65. These results suggest that DMY may be an important therapeutic approach for NPC.

Inhibition effect of oxyepiberberine isolated from Coptis chinensis franch. On non-small cell lung cancer based on a network pharmacology approach and experimental validation

Ethnopharmacological relevanceAs an important Chinese herb, Coptis chinensis Franch. (Huanglian, HL) has a long history of usage for clearing heat, eliminating dampness, purging fire and detoxification in Traditional Chinese Medicine (TCM). HL, also called goldthread, was frequently used for the treatment of typhoid, tuberculosis, epidemic cerebrospinal meningitis, pertussis, and other lung-related diseases. Modern research has shown that HL and its main compounds also have anti-tumor effects. However, studies have not reported whether its main compounds inhibit Non-small cell lung cancer (NSCLC) development and progression. ObjectiveThis study aimed to find out the potential targets and mechanisms of Oxyepiberberine (OPB) isolated from HL in the treatment of NSCLC, using network pharmacology and biological experimental. MethodsSilica gel chromatography column was used to isolate OPB from HL, and the structure of OPB was elucidated using different spectroscopic analysis methods, including 1H-nuclear magnetic resonance (NMR), 13C-NMR and electrospray ionization mass spectrometry (ESI/MS). MTT assay was performed to determine cell proliferation of OPB on A549, H1975 and BEAS-2B cells. Then, the potential targets, pathways and hub genes of OPB for treating NSCLC were screened out through network pharmacology. Based on the results of network pharmacology, core targets of OPB for treating NSCLC were docking with OPB via molecular docking. Wound healing, plate clone, Hoechst staining, and western blot assay were used to verify the function of OPB in treatment of NSCLC. ResultsOPB was isolated from the HL, its molecular formula was identified as C20H17NO5. Through MTT, OPB significantly inhibited the proliferation of H1975 cells and A549 cells, and A549 was chosen as the test cancer cell. Through network pharmacology, 22 potential targets, 156 related-pathways, and 6 hub genes were screened out. The results of molecular docking showed that SRC, BRAF, and MMP9 were the core targets of OPB against NSCLC. Through biological experimental, it was found that OPB inhibited growth and migration of A549 cells. In addition, OPB induced apoptosis in A549 cells. Through western blot assay, the expressions of Src, ERK1/2 and other four proteins were down-regulated, which suggested that OPB inhibited the proliferation of lung cancer cells by down-regulating SRC-FAK-RAS-RAF-MEK-ERK pathway, so as to achieve the anti-NSCLC effect. ConclusionOur study demonstrated that anti-NSCLC effect of OPB through network and experiments, which provided a theoretical basis for the clinical antitumor of OPB, and provided a foundation for further study of OPB.

자색밀(아리흑) 추출물의 기능성 성분 및 생리활성

This study aimed to identify the phytochemical compounds in purple wheat bran ‘Arriheuk’ extract, with water, 50% ethanol and 70% ethanol and determine its physiological activities. Six types of flavonoids were identified in ‘Arriheuk’ extract using a HPLC analysis. The highest total polyphenol content was observed in the 70% ethanol extract, while the highest total flavonoid content was obtained both from the water and the 70% ethanol extracts of ‘Arriheuk’. 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity was significantly high in the 48-h water extract and the 12-h 70% ethanol extract, while the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging activity was not significantly different between the two extracts. Cell viability, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, was high for cells treated with 70% ethanol extract. The apoptosis protective effect, confirmed by Hoechst staining, showed that the morphological features were significantly reduced in cells treated with the 70% ethanol extract. Oil-Red-O staining was used to confirm the protective effect of ‘Arriheuk’ extracts on the intracellular lipid accumulation in cells, and the results showed the best inhibitory effect was observed at the 70% ethanol extract. However, the effect decreased in a concentration dependent manner. These results may improve the usage of ‘Arriheuk’ as a food material in the future.

EMBR-23. KIF11 DEPENDENCY ON P53 MUTATIONAL STATUS IN MEDULLOBLASTOMA

IntroductionKIF11, a mitotic kinesin, is a component responsible for assembly and maintenance of mitotic spindle during mitosis. Tumor cells can upregulate KIF11. Inhibition of KIF11 results monopolar spindle formation, resulting in monoastral mitosis in cells. This activates the spindle assembly checkpoint, cells are arrested and prevented from entering cell cycle, resulting in cell death via apoptosis or necrosis, cell division with aneuploidy or mitotic slippage without division into tetraploid G1 phase. MethodsWe hypothesized that the effect of KIF11 inhibition on medulloblastoma (MB) is dependent of its p53 mutational status. ResultsOur findings on Hoechst staining demonstrated a small molecule inhibitor of KIF11 which induced apoptosis in p53-wildtype MB cells at 48h (p<0.0001), was able to trigger mitotic catastrophe (p = 0.0010) in p53-mutant MB cells at 24h and subsequent necrosis (p=0.0039) at 48h. KIF11 inhibitor exerted anti-proliferative effects on five MB cell lines at nanomolar concentration range, independent of its p53 mutational status. Cells treated with KIF11 inhibitor were arrested in G2/M phase. Apoptosis was observed on Annexin V flow cytometry 24h after treatment, followed by necrosis after 48h in p53-wildtype cells. In contrast, treated p53-mutant cells underwent necrosis at 24h. Differences in cell death mechanisms upon KIF11 inhibition was confirmed on immunoblotting by upregulated p53 expression and presence of cleaved-PARP and DNA-damage marker in p53-wildtype cells, indicative of apoptosis. While inhibition of KIF11 and increased p53 expression were observed only after 48h, cleaved-PARP expression was detected as early as 24h in p53-wildtype, suggesting KIF11-independent, cleaved-PARP-mediated cell death at 24h. In contrast, treated p53-mutant cells showed decreased p53 expression and absence of cleaved-PARP and DNA-damage marker after 24h. ConclusionsOur results suggest that when mitotic arrest is induced, p53-mutant MB cells undergo mitotic catastrophe and necrosis while p53-wildtype MB cells predominantly undergo apoptosis.

Re(η6‐C6H5‐benzimidazole)2]+ and Derivatives as Dye Mimics; Synthesis, UV Absorption Studies and DFT Calculations

AbstractFunctionalizations of highly oxidation and hydrolysis stable mono‐cationic rhenium bis‐arene complexes [Re(η6‐C6H6)2]+ are of great interest. We directly built up structural features of the well‐known Hoechst Dye on the coordinated arenes as a model for prospective DNA minor groove binding studies. Extensions of the aromatic bis‐arene unit with functionalized and derivatized benzimidazole moieties resulted in a deep orange colour of the complexes, showing UV/Vis absorption spectra with multiple transition maxima. These have been assigned with support of DFT calculations to gain information about their charge transfer natures. The different transitions of the complexes, which are either intra‐ligand, ligand‐to‐ligand or metal‐to‐ligand charge transitions, were additionally compared and discussed with the spectra of the corresponding free ligands.

Targeting Jab1 using hesperidin (dietary phytocompound) for inducing apoptosis in HeLa cervical cancer cells.

Plant flavonoids have been emerged as a potent anticancerous agent by exhibiting significant growth inhibitory potential and apoptotic induction in several carcinomas via targeting several oncoproteins. However, inverse association of hesperidin with Jab1 oncoprotein in cervical cancer has rarely been reported. Thus, we have intended our research study towards establishing this unexplored inverse correlation of hesperidin with Jab1 which could further prevent cervical cancer progression. Our research findings clearly demonstrated that hesperidin treatment resulted in Jab1 gene down-regulation and p27 up-regulation in a dose-dependent manner in HeLa cancer cells. These gene modulations might occur via excessive reactive oxygen species (ROS) generation and caspase-3 activation which further resulted in apoptotic induction. Increase in apoptotic cells was confirmed through Hoechst staining and cell cycle analysis. Thus, these results strongly suggested that Jab1 is a potent therapeutic target of hesperidin to suppress cell growth and trigger apoptosis in HeLa cells. PRACTICAL APPLICATIONS: Dietary flavonoids play a crucial role in the management of numerous malignancies via targeting several mutated oncogenes. Our study strongly exhibited that hesperidin treatment suppressed the HeLa cancer cell proliferation via increased ROS generation and reduced Jab1 mRNA expression. Thus, the inference of Jab1-mediated intracellular signals by hesperidin might be a novel approach to control cervical cancer.

Tanshinone IIA sensitizes TRAIL-induced apoptosis in glioblastoma through inducing the expression of death receptors (and suppressing STAT3 activation)

ObjectiveThis work was designed to explore whether the combination of Tanshinone IIA (T-IIA) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has a direct anti-cancer effect in glioblastoma (GBM) and the possible mechanisms. MethodsGBM cells (U-87 and U-251 MG) were treated with T-IIA or/and TRAIL, or the expression of death receptors (DRs), DR4 and DR5, was suppressed in GBM cells. The activity of GBM cells was determined by MTT, and the apoptosis was assessed by Hoechst33342 staining and flow cytometry. The expression levels of cleaved caspase-3/8/9, phosphorylated (p)-STAT3 as well as DR4 and DR5 in GBM cells were assessed by Western blotting. A nude mouse xenograft model was constructed to evaluate the effects of T-IIA and TRAIL cotreatment on tumor growth and apoptosis in vivo. ResultsAfter T-IIA treatment, GBM cells resumed the sensitivity to TRAIL-induced apoptosis dependent on inhibition of p-STAT3 and activation of DR4, DR5 and caspases. DR4 or/and DR5 knockdown significantly abated the co-effect of T-IIA and TRAIL on GBM cell apoptosis and proliferation. Furthermore, T-IIA and TRAIL cotreatment markedly inhibited the growth of transplanted tumor and activated U87 cell apoptosis in nude mice. ConclusionT-IIA increases TRAIL-induced apoptosis by downregulating STAT3 and upregulating DR4 and DR5, indicating T-IIA therapy as a novel treatment strategy for TRAIL-resistant GBM.

Hirsutanol A inhibits T-acute lymphocytic leukemia Jurkat cell viability through cell cycle arrest and p53-dependent induction of apoptosis.

Acute lymphocytic leukemia (ALL) is a type of childhood leukemia with the highest incidence; T-acute lymphocytic leukemia (T-ALL) is far more difficult to treat than B-acute lymphocytic leukemia (B-ALL) and has a poor long-term prognosis. Therefore, there is an urgent requirement to develop effective drugs for the treatment of T-ALL. Hirsutanol A is a natural sesquiterpenoid compound. The aim of the present study was to evaluate the in vitro anticancer activity of hirsutanol A against T-acute lymphocytic leukemia Jurkat cells and investigate the mechanism of action. A Cell Counting Kit-8 assay demonstrated that hirsutanol A inhibited the viability of Jurkat cells in a dose- and time-dependent manner. In addition, hirsutanol A induced cell cycle arrest at the G2 phase as determined via flow cytometry. Furthermore, Hoechst staining, Annexin V-FITC/propidium iodide double staining, mitochondrial membrane potential detection using JC-1 and western blot analysis of apoptotic proteins indicated that the inhibitory effect of hirsutanol A on Jurkat cells was associated with the induction of apoptosis. Of note, hirsutanol A induced the expression of the tumor suppressor p53, whereas simultaneous treatment with pifithrin-α, an inhibitor of p53, significantly reduced Jurkat cell apoptosis induced by hirsutanol A. In summary, the present study suggested that hirsutanol A inhibited Jurkat cell viability through induction of cell cycle arrest and p53-dependent initiation of apoptosis, thus hirsutanol may serve as a promising compound for the treatment of T-ALL.

Neuropeptide Y prevents nucleus pulposus cells from cell apoptosis and IL‑1β‑induced extracellular matrix degradation

ABSTRACT Intervertebral disc degeneration (IDD) is characterized by excessive inflammatory reaction, and neuropeptide Y (NPY) was reported to have anti-inflammatory effect. However, the effect of NPY on NP cells has not been investigated up to date. This study aimed to clarify the role of NPY on the process of IDD. Fourteen fresh human lumbar intervertebral discs were harvested, and degeneration-related proteins were examined. Pfirrmann grading system was used to evaluate IDD. Rat nucleus pulposus (NP) cells were used to investigate the effect of NPY on the proliferation, apoptosis, and extracellular matrix (ECM) in NP cell induced by IL-1βin vitro. The expression levels of NPY and its receptors (type 1 receptor, Y1R, and type 2 receptor, Y2R) were detected via immunohistochemical analysis, western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and proliferation were explored using cell counting kit-8 assay, western blot, and immunofluorescence analysis. Cell apoptosis was investigated by Hoechst staining, JC-1 Staining, annexin V-FITC/PI double staining, and western blot. The secretion of NPY from NP cells was determined via enzyme-linked immunosorbent assay (ELISA). The expression of anabolic and catabolic gene was analyzed by qRT-PCR, western blot, immunofluorescence analysis, and ELISA. The expression of Y2R was significantly increased in both human degenerative intervertebral discs and IL-1β-induced NP cells. Although no positive results for NPY indicated by western blot both in vivo and in vitro, ELISA results demonstrated that the secretion of NPY from NP cells was increased by low-concentration IL-1β, but was decreased when the concentration of IL-1β was 30 ng/ml and above. In addition, NPY could promote NP cells proliferation and protect NP cells against IL‑1β‑induced apoptosis via suppressing mitochondrial-mediated apoptosis pathway. What’s more, NPY can suppress the expression of catabolic gene and ameliorate IL-1β- induced matrix degeneration in NP cells. In conclusion, NPY could promote NP cell proliferation and alleviate IL‑1β‑induced cell apoptosis via mitochondrial pathway. In addition, NPY can suppress the expression of ECM‑catabolic proteinases and ameliorate IL-1β- induced ECM degeneration in vitro.

3D Bioprinting of Model Tissues That Mimic the Tumor Microenvironment.

The tumor microenvironment (TME) influences cancer progression. Therefore, engineered TME models are being developed for fundamental research and anti-cancer drug screening. This paper reports the biofabrication of 3D-printed avascular structures that recapitulate several features of the TME. The tumor is represented by a hydrogel droplet uniformly loaded with breast cancer cells (106 cells/mL); it is embedded in the same type of hydrogel containing primary cells—tumor-associated fibroblasts isolated from the peritumoral environment and peripheral blood mononuclear cells. Hoechst staining of cryosectioned tissue constructs demonstrated that cells remodeled the hydrogel and remained viable for weeks. Histological sections revealed heterotypic aggregates of malignant and peritumoral cells; moreover, the constituent cells proliferated in vitro. To investigate the interactions responsible for the experimentally observed cellular rearrangements, we built lattice models of the bioprinted constructs and simulated their evolution using Metropolis Monte Carlo methods. Although unable to replicate the complexity of the TME, the approach presented here enables the self-assembly and co-culture of several cell types of the TME. Further studies will evaluate whether the bioprinted constructs can evolve in vivo in animal models. If they become connected to the host vasculature, they may turn into a fully organized TME.