Apoptotic Morphological Changes Research Articles

Apoptotic role of marine sponge symbiont Bacillus subtilis NMK17 through the activation of caspase-3 in human breast cancer cell line.

The aim of the present study was to evaluate the diverse potential biological activity of partially purified crude extract (PPCEBS) of marine Bacillus subtilis NMK17 associated with marine sponge Clathria frondifera. Symbionts were isolated from a marine sponge, only the potentialstrain which exhibited apoptosis was sequenced using 16SrRNA and extract of the active strain was subjected to purification using HPLC. The potential pro-apoptotic role of PPCEBS was investigated in MCF-7 human breast cancer cell line for cytotoxicity by MTT assay, which showed dose-dependent cytotoxicity on 24h of exposure. The apoptotic findings demonstrated that PPCEBS significantly induces apoptosis, which was characterised by apoptotic morphological changes. Further, an increased expression of the Caspase 3 and Bax whereas decreased Bcl-2 was confirmed by immunofluorescence and western blotting analysis in MCF-7 cell line, which revealed that PPCEBS has potentapoptosis-inducing property. Added to the desirable apoptotic activity, PPCEBS exhibited excellent antibacterial and antioxidant activities too. The pharmacological effect of the marine sponge-associated bacteria from Gulf of Mannar India needs further attention in discovering new bioactive compounds. Our results suggested that the compounds present in the PPCEBS in marine bacterial B. subtilis NMK17 could be candidates for developing an apoptosis-specific drug with minimal toxicity. This study indicated that marine sponge-associated bacteria could be a good source to find thecytotoxic metabolites which would induce apoptosis and cause cancer cell death. Also, this study explores that marine natural products as a potential source of pharmaceuticals.

ANTICANCER POTENTIAL OF PLANT EXTRACTS FROM RIYADH (SAUDI ARABIA) ON MDA-MB-231 BREAST CANCER CELLS

Background: Medicinal plants have been used in traditional medicine for the treatment of numerous diseases worldwide. There is a dire need for new anticancer agents and plants used in traditional medicine are a particularly useful source. 
 Materials and methods: In this study, extracts of five different plants that grow in the desert of Saudi Arabia were evaluated to assess their cytotoxicity against the MDA-MB-231 breast cancer cell line. Soxhlet extraction was carried out on the leaves and stems using different solvents. The cytotoxicity of these extracts against MDA-MB-231 breast cancer cells was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The apoptotic cellular morphological changes were observed using inverted and fluorescence microscopes. 
 Results: Our results showed that two of the five different medicinal plants (Rumex vesicarius and Malva parviflora) exhibited strong anticancer activity against the breast cancer cells. Specifically, 2 of the 40 extracts (from the five studied plants) showed promising activity. The chloroform extract of the stem of R. vesicarius (RSV CHCL3) exhibited moderate anticancer activity with a half-maximal inhibitory concentration (IC50) of 230 µg/mL while that of the hexane extract of M. parviflora stems (MPS Hex) was 248 µg/mL. Loss of cell integrity, shrinkage of the cytoplasm, and cell detachment were observed in the extract-treated MDA-MB-231 cells.
 Conclusion: R. vesicarius and M. parviflora chloroform and n-hexane stem extracts showed significant cytotoxicity against MDA-MB-231 human breast carcinoma cells.

Alpha pinene modulates UVA-induced oxidative stress, DNA damage and apoptosis in human skin epidermal keratinocytes

AimsThis study aims to evaluate the protective effect of alpha pinene (AP), an essential oil monoterpene, against ultraviolet-A (UVA; 320–400 nm) induced cellular damages in human skin epidermal keratinocytes (HaCaT cells). Materials and methodsIn this study, HaCaT cells were subjected to single UVA-irradiation (10 J/cm2) in the presence and absence of AP (30 μM) then different cellular end points were analyzed. The protective effect of AP against UVA-induced cytotoxicity was evaluated by MTT-based metabolic assay. Generation of reactive oxygen species (ROS), alteration of mitochondrial membrane potential (MMP), DNA single- and double strand breaks (SSBs and DSBs) and apoptotic morphological changes during different treatment conditions were measured by fluorescence microscopy and spectrofluorometry. Modulatory role of AP against UVA-mediated inflammatory markers expression, nucleotide excision repair (NER) proteins and apoptotic markers expression during AP and/or UVA treatment were studied by western blot. Key findingsPretreatment with AP prevented UVA-induced cytotoxicity, generation of ROS, lipid peroxidation and DNA stand breaks probably through its antioxidant property. AP also inhibited UVA-induced inflammatory mediators such as NF-κB, TNF-α and IL-6 expression in HaCaT cells. Further, AP modulates NER proteins via activation of p53 and p21 thereby subsequently prevent the formation of UVA-induced cyclobutane pyrimidine dimers (CPDs). We also noticed that AP inhibits apoptotic cell death by preventing UVA-induced loss of mitochondrial membrane potential through modulating Bax/Bcl-2 expression in HaCaT cells. SignificanceThe present findings suggest that AP prevent UVA-induced oxidative stress, inflammation, DNA damages and apoptosis in human skin cells.

Sodium fluoride causes oxidative stress and apoptosis in cementoblasts

ObjectivesToothpaste with fluoride concentration up to 5000 ppm are recommended to the patients who are susceptible to root caries; however, the effects of fluoride on cementoblasts have received less attention. MethodsThe OCCM-30 cells were exposed to 0,0.5, 5, 10 mM NaF respectively. A TUNEL (TdT-mediated dUTP-biotin nick end labeling) assay kit was used to detect the DNA fragmentation. Hoechst staining was used to determine changes of nuclear morphology. Real-time quantitative RT-PCR and Western blotting were performed to investigate the mRNA and protein expression of caspase-3,-8,-9, cleaved Poly (ADP-ribose) polymerase (PARP) and Fas-ligand (Fas-L), a ligand of death receptor. CA-DCF-DA [5 (6)-Carboxy-2′,7′-dichlorofluorescein diacetate] was used to measure the generation of reactive oxygen species (ROS) in OCCM-30 cells after the NaF stimulation. ResultsThe results showed apoptotic morphological changes and DNA fragmentation in OCCM-30 cells exposed to high concentration of NaF. 10 mM NaF induced the expression of cleaved caspase-3,-8,-9 and cleaved Poly (ADP-ribose) polymerase (PARP). The mRNA expression of the Fas-L was also increased in cells exposed to 5 mM NaF. Furthermore, 10 mM NaF stimulation resulted in a significant generation of ROS in the OCCM-30 cells. ConclusionsOur research demonstrated that apoptosis is activated by NaF in OCCM-30 cells through both of the extrinsic death receptor-dependent and oxidative stress-related intrinsic apoptotic pathway. Clinical significanceMore consideration should be given about the fluoride concentration and the frequency of dental products when used to prevent the root caries for patients with gingival recession.

Phytochemical analysis and antioxidant and anticancer activities of mastic gum resin from Pistacia atlantica subspecies kurdica.

BackgroundThe mastic gum resin has been used in traditional Kurdish medicine for treating various disorders such as topical wound and gastric ulcer. The study designed to evaluate the total polyphenol and flavonoid content, free radical scavenging activity, and anticancer effects of mastic gum resin derived from Pistacia atlantica subspecies kurdica.Materials and methodsFolin -Ciocalteau and the aluminum chloride colorimetric assays were used to determine the total phenol and flavonoid contents in the mastic gum resin respectively. Whereas, DPPH and ABTS+ assays were used to determine the antioxidant activities of mastic gum resin. Regarding anticancer activities, the MTT assay was used to study the effect of mastic gum resin on the proliferation of various cancer cells and the morphological changes were identified after Acridine Orange/Propidium Iodide staining. Flow cytometry was applied to determine the influence of mastic gum resin on the apoptosis rate by Annexin V double staining and to investigate the influence on cell cycle progression. Caspase colorimetric assay was used to estimate the hallmark enzyme of apoptosis, and finally RNA were obtained from COLO205 cells and analyzed by qRT-PCR analyses.ResultsThe MTT results showed that the mastic gum resin at concentrations from 0.01 to 100 μM induced death of cancer cells in a dose and time-dependent manner. The mastic gum resin suppressed proliferation of human cancer cells with 72 h IC50 value of 15.34 ± 0.21, 11.52 ± 0.18, 8.11 ± 0.23 and 5.2 ± 0.8 μg/mL for bile duct cancer (cholangiocarcinoma) (KMBC), pancreatic carcinoma (PANC-1), gastric adenocarcinoma (CRL-1739), and colonic adenocarcinoma (COLO205) cells, respectively. Normal human colon fibroblast (CCD-18Co) cells were not adversely affected by resin treatment. Flow cytometry showed that the mastic gum resin significantly (P<0.05) arrested COLO205 cell proliferation at the G2/M phase of cell cycle. The resin caused apoptotic morphological changes in COLO205 cells. The apoptotic effect to mastic gum resin was via the mitochondrial as shown by the up-regulation of Bax, down-regulation of Bcl-2 genes, and activation of caspase-9 and -3 activities.ConclusionIt was confirmed that the antiproliferative efficacy of the resin is positively correlated with its polyphenolic contents, suggesting a causal link related to exudate content of phenolic acid and flavonoids. The results revealed that the mastic gum resin has potential to be developed as an anticancer and antioxidant product due to its high content of polyphenol compounds.

Abstract 160: Novel strategy targeting breast cancer stem cells CD44+/CD24-low responsible for breast cancer progression

Abstract The present study has been designed to isolate the highly tumorigenic sub-population of cells from the breast cancer cell line (T47-D). The effect of Manuka honey (MH), Frankincense essential oil (F), Metformin (M) and combined treatment on eradicating breast cancer stem cells (BCSCs) that are responsible for cancer progression was investigated. The isolated (BCSCs) were subjected to different concentrations of (MH), (F), (M) and combined treatment to visualize their effects on BCSCs viability, morphological changes, caspase-3 activity, apoptosis induction, clonogenic survival, β-catenin, ALDH6A1, Notch-1, Notch-4 and Nonog protein expression, using western blot analysis and protein localization by immunocytochemical technique. Inhibited viability of isolated breast cancer stem cells, which showed apoptotic morphological changes were observed beside increased caspase-3 activity and inhibited clonogenic survival compared to control. At the protein level, our data showed that (MH), (F), (M) and combined synergistic treatment decreased the expression of β-catenin levels, ALDH6A1 and Notch-1 compared to control. In addition, (MH) showed no effect on Notch-4 expression, but significantly decreased the expression level of Nanog. In contrast, (F) treatment enhanced the protein expression of Nanog level in a dose dependent manner compared to control. Furthermore, (MH), (F), (M), and combined treatment showed conspicuously decrease in the nuclear and cytoplasmic level of β-catenin and ALDH6A1 of CD44+/CD24-low (BCSCs) compared to control. Taken together, natural compounds of Manuka honey and Frankincense essential oils and their combination with Metformin have been emerged as a potential strategy to target and eradicate (BCSCs). Citation Format: Fatma A. Abouelnazar, Ahmed S. Sultan. Novel strategy targeting breast cancer stem cells CD44+/CD24-low responsible for breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 160.

A new niclosamide derivatives-B17 can inhibit urological cancers growth through apoptosis-related pathway.

The incidence and mortality rate of urological cancers is increasing yearly. Niclosamide has been repurposed as an anti‐cancer drug in recent years. Synthesized derivative of niclosamide was testified for its anti‐cancer activity in urological cancers. MTT assay was used to measure the cytotoxicity effect of niclosamide and its derivatives in urological cancer cell lines. Migratory ability was monitored by scratch migration assay. Apoptosis and cell cycle changes were analyzed by annexin V and PI staining. The apoptosis‐related signal proteins were evaluated by western blotting. T24 had the best drug sensitivity with the lowest IC 50 in niclosamide and B17 treatment than DU145 and Caki‐1 cells. After niclosamide and B17 treatment, the mitotic cells were decreased, but apoptotic bodies and morphology changes were not prominent in T24, Caki‐1, and DU145 cells. The migratory ability was inhibited in niclosamide treatment than control group on Caki‐1 cells and niclosamide and B17 treatment than control group on DU145 cells. Early apoptosis cells were increased after niclosamide and B17 treatment than control group without cell cycle changes in T24, Caki‐1, and DU145 cells. Programmed cell death was activated majorly through PAPR and bcl‐2 in T24 and caspase‐3 in Caki‐1 cells, respectively. Niclosamide and B17 derivative had good ability in inhibition proliferation and migratory ability in T24, Caki‐1, and DU145 cells without prominent morphology and apoptotic body changes. UCC cells are more sensitive to niclosamide and B17 treatment. Early apoptosis was induced after niclosamide and B17 treatment through different mechanisms in T24, Caki‐1, and DU145 cells.

Design, synthesis, in vitro and in silico evaluation of a new series of oxadiazole-based anticancer agents as potential Akt and FAK inhibitors

In the current work, new 1,3,4-oxadiazole derivatives were synthesized and investigated for their cytotoxic effects on A549 human lung adenocarcinoma, C6 rat glioma and NIH/3T3 mouse embryonic fibroblast cell lines. Compounds 2, 6 and 9 were found to be the most potent anticancer agents against A549 and C6 cell lines and therefore their effects on apoptosis, caspase-3 activation, Akt, FAK, mitochondrial membrane potential and ultrastructural morphological changes were evaluated. N-(5-Nitrothiazol-2-yl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)methyl]-1,3,4-oxadiazol-2-yl]thio]acetamide (9) increased early and late apoptotic cell population in A549 and C6 cells more than cisplatin and caused more mitochondrial membrane depolarization in both cell lines than cisplatin. On the other hand, N-(6-methoxybenzothiazol-2-yl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)methyl]-1,3,4-oxadiazol-2-yl]thio]acetamide (6) caused higher caspase-3 activation than cisplatin in both cell lines. Compound 6 showed significant Akt inhibitory activity in both cell lines. Moreover, compound 6 significantly inhibited FAK (Phospho-Tyr397) activity in C6 cell line. Molecular docking simulations demonstrated that compound 6 fitted into the active sites of Akt and FAK with high affinity and substrate-specific interactions. Furthermore, compounds 2, 6 and 9 caused apoptotic morphological changes in both cell lines obtained from micrographs by transmission electron microscopy. A computational study for the prediction of ADME properties of all compounds was also performed. These compounds did not violate Lipinski's rule, making them potential orally bioavailable anticancer agents.

Diethyl Blechnic, a Novel Natural Product Isolated from Salvia miltiorrhiza Bunge, Inhibits Doxorubicin-Induced Apoptosis by Inhibiting ROS and Activating JNK1/2.

Doxorubicin (DOX) is a widely used antineoplastic agent in clinics. However, its clinical application is largely limited by its cardiotoxicity. Diethyl blechnic (DB) is a novel compound isolated from Salvia miltiorrhiza Bunge. Here, we study the effect of DB on DOX-induced cardiotoxicity and its underlying mechanisms. Cellular viability was tested by 3-[-4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and protein level was evaluated by Western blotting. 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining was performed to determine the mitochondrial membrane potential (MMP). Hoechst 33342 staining and TUNEL staining was performed to test the apoptosis. Reactive oxygen species (ROS) generation was investigated by using flow cytometry. DB significantly inhibited DOX-induced apoptosis in H9c2 cells and primary cultured cardiomyocytes. Moreover, DB decreased cell apoptotic morphological changes and reversed the mitochondrial membrane potential induced by DOX. Meanwhile, pre-treatment with DB increased the expression levels of B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra-large (Bcl-xl), and survivin and reduced the expression levels of Bcl-2-associated X protein (Bax), p-p53, cytochrome c (cyt c), and cleaved-caspase 3, 7, 8, 9 in the protein levels in DOX-treated H9c2 cells. Furthermore, DB suppressed ROS generation. The DB-mediated protective effects were accompanied by increased c-Jun N-terminal kinase1/2 (JNK1/2) expression. In addition, SP600125, the inhibitor of JNK1/2, abolished the protective effect of DB. We concluded that DB protected cardiomyocytes against DOX-induced cytotoxicity by inhibiting ROS and activating the JNK1/2 pathway. Therefore, DB is a promising candidate as a cardioprotective agent against DOX-induced cardiotoxicity.

Growth Inhibition and Apoptosis of Ehrlich Ascites Carcinoma Cells by Methanol Extract from the Calyx of Hibiscus Sabdariffa Linn

Objectives: This study focused on the antioxidant and anticancer activities of methanolic extract of Hibiscus sabdariffa calyx. Methods: In vitro methods were used to determine the different types of phytochemical content and antioxidant activity of methanolic extract of Hibiscus sabdariffa calyx as well as its cytotoxic effect against Ehrlich ascites carcinoma cells. In vivo, the antineoplastic effect of methanolic extract of Hibiscus sabdariffa calyx against Ehrlich ascites carcinoma was also evaluated by determining the viable tumor cell count, survival time, body weight gain, and hematological profi les of experimental mice along with observing morphological changes of Ehrlich ascites carcinoma cells by fl uorescence microscopy and determining the expression of p53 gene in Ehrlich ascites carcinoma cell assayed by reverse transcription-polymerase chain reaction. Chemical composition of methanolic extract of Hibiscus sabdariffa calyx was analyzed by gas chromatography–mass spectrometry. Results: Flavonoid rich methanolic extract of Hibiscus sabdariffa calyx had showed remarkable antioxidant activity and in in vitro assay, methanolic extract of Hibiscus sabdariffa calyx had potent inhibitory effect against Ehrlich ascites carcinoma cells with a half maximal inhibitory concentration of 28.16 µg/mL. Apoptotic morphological changes and increased expression of p53 gene in Ehrlich ascites carcinoma cells derived from methanolic extract of Hibiscus sabdariffa calyx-treated (5 and 10 mg/kg/day) mice were also observed. Moreover, chemical constituents identifi ed by gas chromatography–mass spectrometry analyses were likely related to this activity. Conclusion: Methanolic extract of Hibiscus sabdariffa calyx has promising antioxidant and antineoplastic potential and can be considered as a probable new source for antitumor agent.

Cadmium-induced apoptosis in neuronal cells is mediated by Fas/FasL-mediated mitochondrial apoptotic signaling pathway

Cadmium (Cd) is a toxic metal capable of damaging brain. Studies have demonstrated that Cd can induce apoptosis in neuronal cells. The CD95/APO-1 (Fas)/Fas Ligand (FasL) signaling pathway is one of the primary apoptosis pathways, but the role and regulatory mechanism of this pathway in neuronal cells remain unclear. Here, we demonstrated the underlying mechanism of the Fas/FasL system involving the mitochondrial apoptotic pathway in neuronal cells. Primary rat cerebral cortical neurons and PC12 cells were exposed to Cd, which significantly activated expression of Fas, FasL, Fas-associated death domain (FADD) and cleaved caspase-8. However, expression of cleaved caspase-8 decreased at 20 µM Cd in primary cerebral cortical neurons. Importantly, Cd-induced apoptotic morphological changes and increase in the apoptosis rate were partially blocked by Z-IETD-FMK, which is a specific inhibitor of caspase-8. Cd-mediated increase of apoptosis rate was inhibited by anti-FasL antibody. Furthermore, our data revealed that Z-IETD-FMK also blocked increase of truncated BH3 interacting domain death agonist (tBID)/BID, decrease of the B-cell lymphoma 2 (Bcl-2)/Bcl-2 associate X protein (Bax) ratio and mitochondrial membrane potential (MMP), release of cytochrome c, as well as cleavage of caspase-9/3 and poly (ADP-ribose) polymerase (PARP) induced by Cd. Taken together, our results demonstrate that the Fas/FasL-mediated mitochondrial apoptotic pathway plays an important role in Cd-induced neuronal apoptosis.

Novel pregnenolone derivatives modulate apoptosis via Bcl-2 family genes in hepatocellular carcinoma in vitro

A series of pregnenolone derivatives were synthesized and assessed for anti-cancer activity against hepatocellular carcinoma cell line (HepG2). The synthesized hetero-steroids (compounds 3, 4, 5, 6, 7, 8a and 8b) were evaluated for their cytotoxic activities using MTT (3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay. Apoptotic activity was assessed using dual acridine orange/ethidium bromide staining method and DNA fragmentation assay. Pro-apoptotic genes (Bax and Bak) and anti-apoptotic genes (Bcl-2 and Bcl-xL) were analyzed using quantitative real time PCR. The results revealed that compounds 4 and 6 displayed cytotoxic activity (IC50s, 36.97 ± 2.18 and 18.46 ± 0.64 μM, respectively), while compounds 5 and 7 exhibited weak cytotoxic activity (IC50s, 93.87 ± 8.30 μM and 93.48 ± 4.14 μM, respectively). All synthesized heterocyclic pregnenolone derivatives induced apoptosis through DNA fragmentation. Compounds 4 and 6 increased early and late apoptotic cell percentages while compounds 3, 5, 7 and 8b increased either early or late apoptotic cell percentage. Moreover, compounds 3, 6 and 8b up-regulated the expression level of Bak gene. On the other hand, compounds 4, 5, 7 and 8a down-regulated the Bcl-2 expression level, besides, compounds 5, 7 and 8a down-regulated the Bcl-xL expression level. Compounds 5, 7, 8a and 8b increased the Bak/Bcl-xL ratio, besides, compound 8a raised the Bax/Bcl-xL ratio whereas compound 5 elevated Bax/Bcl-2 and Bak/Bcl-2 ratios. The present work introduced novel pro-apoptotic pregnenolone derivatives that acted against HepG2 cells through DNA fragmentation, apoptotic morphological changes and were able to increase the pro-apoptotic/anti-apoptotic ratios of Bcl-2 family genes. This study particularly revealed that the cytotoxic compound 4 is the most promising pro-apoptotic compound among other synthesized derivatives where it induced apoptosis (late and early) through the down-regulation of Bcl-2 gene expression level.

Nuclear apoptotic volume decrease in individual cells: Confocal microscopy imaging and kinetic modeling

The dynamics of nuclear morphology changes during apoptosis remains poorly investigated and understood. Using 3D time-lapse confocal microscopy we performed a study of early-stage apoptotic nuclear morphological changes induced by etoposide in single living HepG2 cells. These observations provide a definitive evidence that nuclear apoptotic volume decrease (AVD) is occurring simultaneously with peripheral chromatin condensation (so called “apoptotic ring”). In order to describe quantitatively the dynamics of nuclear morphological changes in the early stage of apoptosis we suggest a general molecular kinetic model, which fits well the obtained experimental data in our study. Results of this work may clarify molecular mechanisms of nuclear morphology changes during apoptosis.

Evaluation of the Cytotoxic and Autophagic Effects of Atorvastatin on MCF-7 Breast Cancer Cells

Background:Recently, cytotoxic effects of statins on breast cancer cells have been reported. However, the mechanism of anti-proliferative effects is currently unknown. Autophagy is non-apoptotic programmed cell death, which is characterized by degradation of cytoplasmic components and as having a role in cancer pathogenesis.Aims:To investigate the anti-proliferative effects of atorvastatin on MCF-7 human breast adenocarcinoma cells with respect to both autophagy and apoptosis.Study Design:Cell culture study.Methods:Cell viability was analyzed using WST-1 cell proliferation assay. Apoptosis was determined by the TUNEL method, whereas autophagy was assessed by Beclin-1 and LC3B immunofluorescence staining. Ultrastructural analysis of cells was performed by electron microscopy.Results:Atorvastatin reduced MCF-7 cell proliferation in a dose- and time-dependent manner inducing TUNEL-, Beclin-1-, and LC3B-positive cells. Moreover, ultrastructural analysis showed apoptotic, autophagic, and necrotic morphological changes in treatment groups. A statistically significant increase in the apoptotic index was detected with higher concentrations of atorvastatin at 24 h and 48 h (p<0.05).Conclusion:The anti-proliferative effects of atorvastatin on breast cancer cells is mediated by the induction of both apoptosis and autophagy which shows statins as a potential treatment option for breast cancer.

Resveratrol inhibits the growth of gastric cancer via the Wnt/β-catenin pathway.

The inhibitory effect of resveratrol on the growth of gastric cancer cells through downregulation of the Wnt/β-catenin signaling pathway were studied. First we determined the effective concentration of resveratrol on the growth and proliferation of MGC-803 gastric cancer cells. Methylthiazolyl tetrazolium assay showed that resveratrol significantly inhibited the proliferation of MGC-803 cells in a dose-dependent manner. Resveratrol induced apoptotic morphological changes in MGC-803 cells. Reverse transcription-polymerase chain reaction and western blot analysis showed that resveratrol downregulated the expression of three important components of the Wnt signaling pathway, β-catenin, c-myc, and cyclin D1, at the mRNA and protein levels. Overall, resveratrol inhibits the growth of MGC-803 cells by inhibiting the Wnt signaling pathway. This study provides a new idea and direction for the antitumor mechanism of resveratrol.

Screening and identification of ligand-protein interactions using functionalized heat shock protein 90-fluorescent mesoporous silica-indium phosphide/zinc sulfide quantum dot nanocomposites

Currently, nanosphere-based ligand fishing cannot be accomplished with imaging processing, although this step is important for real-time identification. Herein, a ligand fishing technique combined with real-time imaging is presented for the identification of ligands for heat shock protein 90α (Hsp 90α) from a complex matrix, Alisma plantago-aquatica Linn. crude extract, using Hsp 90α-functionalized mesoporous silica nanoparticle (MSN)-InP/ZnS quantum dot (QD) nanocomposites as a support material. Twenty ligands for Hsp 90α were screened, and their structures were identified by mass spectrometry. The activities of the ligands were verified by real-time imaging of cells apoptotic morphological changes. Quantitative analysis showed that Alisma plantago-aquatica Linn contained 8.19μg/g Alisol F, which regarded as one typical component of Alisma plantago-aquatica Linn, and the extraction ratio of Alisol F was 76.2%. The precision for five replicate measurements was 7.0% (RSD). The prepared nanocomposites were also used to screen proteins from a mixture of cellular extracts, and five proteins from HeLa cells were identified as potential client proteins of Hsp 90α.

Investigation of antioxidant and anticancer potential of fucoidan from Sargassum polycystum

The present study was aimed to evaluate the antioxidant and anticancer potential of fucoidan isolated from Sargassum polycystum. The isolated fucoidan was successfully purified by DEAE cellulose-ion exchange chromatography and dialysis. Totally four active fractions (F1–F4) were collected and explored its chemical constitution by calorimetric assays. Among them, fraction 2 (F2) showed the higher yield percentage, fucose and sulphate content. Further, monosaccharide composition, structural and functional properties of the F2 was analyzed by HPLC, FTIR and NMR. F2 shows highest DPPH radical scavenging activity (55.94 ± 0.69%), reducing power (0.33 absorbance rate), hydrogen peroxide scavenging activity (71.76 ± 2.14%) and nitric oxide radical scavenging activity (51.81 ± 1.04%) at 1000 μg/ml. The cell viability of MCF-7 and HCT-15 cell lines was proportionate to the concentration of F2 with an estimated IC50 was 20 and 50 μg/ml respectively. The fluorescence and confocal laser scanning microscopic analysis demonstrated the apoptotic morphological changes and cell mediated death in F2 treated cancer cells. Higher amount of LDH release was found in the F2 treated cancer cells than the control group. Thus, the present finding proved that the isolated F2 encompasses significant antioxidant and anticancer property.

Homoisopogon a from Ophiopogon japonicus induces apoptosis in A549 – a non small cell lung cancer cell line

Lung cancer is the leading cause of death among Vietnamese people and can be divided into two major groups: small cell lung cancer (SCLC), and non-small cell lung cancer (NSCLC). It is estimated that 85% of patients was diagnosed as NSCLC. Therapy drugs that targeted lung cancer tumors with the epidermal growth factor receptor (EGFR) may initially provide benefit, but over time tumors can develop resistance. The recent discovery of EGFR-tyrosine kinase inhibitors (EGFR-TKIs) has provided a new target for the theurapeutic agents in non-small cell lung cancer treatment. In our study, homoisopogon A, a new compound isolated from the tubers of Ophiopogon japonicus (Mach mon, in Vietnamese) effectively showed the cytotoxic activity to the EGFR and TKI-resistant NSCLC cell lines including human lung carcinoma A549, human lung adenocarcinoma NCI-H1975 and human lung adenocarcinoma NCI-H1650. The IC50 values of homoisopogon A against A549, NCI-H1975 and NCI-H1650 were determined as 6.26, 19.51, and 24.66 µM, respectively. Preliminary study on the mechanism of action using flow cytometry analysis was performed. Homoisopogon A treatment of A549 cells at concentration of 25 μM generated apoptosis in 27.5% of cells (7% early apoptosis and 20.5% late apoptosis) after 24h of treatment. The effect increased significantly at the concentration of 50 μM, the homoisoflavanone generated apoptosis in 83.8% of cells (23.5% early apoptosis and 60.3% late apoptosis). Treatment with homoisopogon A for 48h also resulted in typical apoptotic morphological changes in A549 cells under microscopic observation. The results strongly suggested that homoisopogon A induces apoptosis in EGFR and TKI-resistant-A549 cells, thus resulting in the cytotoxicity.

4-Fluorobenzaldehyde limonene-based thiosemicarbazone induces apoptosis in PC-3 human prostate cancer cells

AimsThis study evaluated parameters of toxicity and antiproliferative effects of (+)-N(1)-4-fluorobenzaldehyde-N(4)-{1-methyl-1-[(1R)-4-methylcyclohexene-3-il]-ethyl}-thiossemicarbazone (4-FTSC) in PC-3 adenocarcinoma prostate cells. Main methodsCytotoxicity of 4-FTSC in PC-3 cells was evaluated using MTT assay. Morphology examination of PC-3 cells treated with 4-FTSC was also performed as well as the cell death mechanisms induced were investigated using flow cytometry. Parameters of toxicity of 4-FTSC was conducted by the investigation of its potential myelotoxicity and lymphotoxicity, hemolytic activity and acute oral toxicity profile. Key findings4-FTSC showed promising cytotoxic effects against PC-3 cells (IC50 = 18.46 μM). It also triggered apoptotic morphological changes, phosphatidylserine externalization and a significant increase of DNA fragmentation in PC-3 cells. Moreover, 4-FTSC did not show changes in the PC-3 cell cycle with levels of p21, p27, NFĸB and cyclin D1 similar to those found in both control and treated cells. 4-FTSC also promoted an increase of p53 levels associated with mitochondrial impairment through loss of ∆Ψm and ROS overproduction. 4-FTSC-induced cell death mechanism in PC-3 cells involved activation of caspase-3/-7 through apoptosis intrinsic pathway via caspase-9. Regarding toxicological profile, 4-FTSC showed in vitro lymphotoxicity, although with low cytotoxicity for bone marrow progenitors and no hemolytic potential. Moreover, it was classified as GHS category 5 (LD50 > 2000–5000 mg/Kg), suggesting it has low acute oral systemic toxicity. Significance4-FTSC seems to be a promising candidate to be used as a clinical tool in prostate cancer treatment. Further studies are required to better clarify its toxicopharmacological effects found in this compound.

Down-regulation of STAT3 induces the apoptosis and G1 cell cycle arrest in esophageal carcinoma ECA109 cells

BackgroundSignal transducer and activator of transcription 3 (STAT3) is persistently activated in a wide variety of epithelial cancers. Aberrant activity of STAT3 correlates with tumor growth, invasion and metastasis, which makes it a potential therapeutic target of cancer. To explore the biological role of STAT3 in esophageal cancer, we used small hairpin RNA to knockdown the expression of the STAT3 gene in the esophageal carcinoma ECA109 cell line and the cell apoptosis, cell cycle and cell migration were investigated.MethodsThe cell apoptosis was tested using DNA ladder, mitochondrial membrane potential assay, TUNEL assay, annexin V-PI staining. Cell cycle phases were estimated using flow cytometry analysis. The mRNA and proteins related to apoptosis and cell cycle were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. And cell migration was investigated by in vitro Transwell assay. The data were analyzed with two-sample Student’s t test and ANOVA followed by the LSD post hoc test.ResultsOur results showed that knockdown of STAT3 in ECA109 cells induced noticeable apoptotic morphological changes like cell shrinkage, apoptotic vacuoles, membrane blebbing time-dependently. In addition, DNA ladder, TUNEL assay, Annexin V-PI staining and declined level of cleaved Caspase-3 indicated that down-regulation of STAT3 could induce apoptosis in ECA109 cells. Flow cytometry analysis displayed the induction of G1-phase cell cycle arrest of ECA109 cells by STAT3 decreasing, consistent with the descend of c-Myc and cyclin D1 in protein levels. Furthermore, STAT3 knockdown suppressed the expression of matrix metalloproteinases-9, sushi domain containing 2 and urokinase plasminogen activator in ECA109 cells and inhibited cell migration ability.ConclusionsKnockdown of STAT3 could induce the apoptosis and G1 cell cycle arrest in esophageal carcinoma ECA109 cells, and inhibit the migration ability of cells as well.