Pro-apoptotic Proteins P53 Research Articles

Isoquercitrin protects HUVECs against high glucose‑induced apoptosis through regulating p53 proteasomal degradation.

High glucose (HG)-induced endothelial apoptosis serves an important role in the vascular dysfunction associated with diabetes mellitus (DM). It has been reported that isoquercitrin (IQC), a flavonoid glucoside, possesses an anti-DM effect, but the mechanism requires further investigation. The present study investigated the effect of IQC against HG-induced apoptosis in human umbilical vein endothelial cells (HUVECs) and explored its molecular mechanism. HUVECs were treated with 5 or 30 mM glucose for 48 h. Endothelial cell viability was monitored using the Cell Counting Kit-8 assay. Mitochondrial membrane potential was detected by JC-1 staining. Apoptosis was observed by TUNEL staining and flow cytometry. Western blotting was used for the analysis of apoptosis-associated proteins Bax, Bcl-2, cleaved (C)-caspase3, total-caspase3, p53 and phosphorylated p53. Reverse transcription-quantitative PCR was used to analyze the mRNA expression levels of Bax, Bcl-2 and p53. Immunofluorescence staining was utilized to detect the expression levels and distribution of p53 and ubiquitin specific peptidase 10 (USP10) in HUVECs. The results revealed that IQC significantly attenuated HG-induced endothelial apoptosis, as shown by decreased apoptotic cells observed by TUNEL, JC-1 staining and flow cytometry. Moreover, under HG stress, IQC treatment markedly inhibited the increased expression levels of the pro-apoptotic proteins p53, Bax and C-caspase3, and increased the expression levels of the anti-apoptotic protein Bcl-2 in HUVECs. However, the anti-apoptotic effect of IQC against HG was partially blunted by increasing p53 protein levels in vitro. IQC influenced the mRNA expression levels of Bax and Bcl-2 in response to HG, but it did not affect the transcription of p53. Notably, IQC inhibited the HG-induced phosphorylation of p53 at Ser15 and the nuclear transport of USP10, destabilizing p53 and increasing the proteasomal degradation of the p53 protein. The current findings revealed that IQC exerted a protective effect against the HG-induced apoptosis of endothelial cells by regulating the proteasomal degradation of the p53 protein, suggesting that IQC may be used as a novel therapeutic compound to ameliorate DM-induced vascular complications.

Ergothioneine mitigates cisplatin-evoked nephrotoxicity via targeting Nrf2, NF-κB, and apoptotic signaling and inhibiting γ-glutamyl transpeptidase

AimCisplatin is a potent chemotherapeutic agent whose therapeutic application is hindered by the associated nephrotoxicity. Cisplatin-evoked nephrotoxicity has been largely attributed to the induction of oxidative stress and inflammatory responses. The current study aimed at investigating the ability of ergothioneine to mitigate cisplatin-evoked nephrotoxicity and to elucidate the underlining molecular mechanisms. Main methodsWistar rats were treated with a daily dose of ergothioneine (70 mg/kg, po) for fourteen days and a single dose of cisplatin (5 mg/kg, ip) on day ten. On day fifteen, kidneys and blood specimens were collected and subjected to Western blotting, ELISA, histopathological, and spectrophotometric analysis. Key findingsErgothioneine significantly enhanced renal function in cisplatin-treated rats as manifested by increased GFR and decreased serum creatinine and blood urea nitrogen. Ergothioneine effectively reduced the cisplatin-induced oxidative stress and mitigated apoptosis and the histopathological changes. Mechanistically, ergothioneine induced the expression of the antioxidant transcription factor Nrf2 and up-regulated its downstream targets NQO1 and HO-1. Equally important, ergothioneine inhibited γ-glutamyl transpeptidase that plays crucial roles in biotransformation of cisplatin into a toxic metabolite. Additionally, it reduced the pro-apoptotic protein p53 and the inflammatory transcription factor NF-κB along with its downstream pro-inflammatory cytokines TNF-α and IL-1β. SignificanceThe results of the current work shed the light on the ameliorating effect of ergothioneine on cisplatin-evoked nephrotoxicity that is potentially mediated through modulation of Nrf2, p53, and NF-κB signaling and inhibition of γ-glutamyl transpeptidase. This findings support the potential application of ergothioneine in controlling cisplatin-associated nephrotoxicity although clinical investigations are warranted.

Dichloromethane fraction of Moringa oleifera leaf methanolic extract selectively inhibits breast cancer cells (MCF7) by induction of apoptosis via upregulation of Bax, p53 and caspase 8 expressions.

Moringa oleifera is a well-known medicinal plant which has anti-cancer and other biological activities. This research aims to determine the cytotoxic and apoptotic effect of M. oleifera leave extract on the breast cancer (MCF7) cells. The extracts were prepared using hexane, dichloromethane, chloroform and n-butanol by fractionating the crude 80% methanol extract of the plant leaves. The cytotoxic effect of the extracts on MCF7 cells were determined using CellTiter 96® AQueous One Solution Cell Proliferation (MTS) assay. The apoptosis study was conducted using Annexin V-FITC analysis and confirmed by Western blotting using selected proteins, which are p53, Bax, cytochrome c and caspase 8. Our results showed that the dichloromethane (DF-CME-MOL) extract was selectively cytotoxic to MCF7 cells (5μg/mL) without significantly inhibiting the non-cancerous breast (MCF 10A) cells. It had the highest selectivity index (SI) value of 9.5 among the tested extracts. It also induced early apoptosis and increased the expressions of pro-apoptotic proteins Bax, caspase 8 and p53 in MCF7 cells. Gas chromatography-mass spectrometry analysis (GC-MS) analysis showed that the major compounds found in DF-CME-MOL were benzeneacetonitrile, 4-hydroxy- and benzeneacetic acid, 4-hydroxy-, methyl ester among others that were detected. Thus, DF-CME-MOL extract was found to inhibit the proliferation of MCF7 cells by apoptosis induction, which is likely due to the activities of the detected phytochemical compounds of the extract.

Molecular insights into a mechanism of resveratrol action using hybrid computational docking/CoMFA and machine learning approach

A phytoalexin, Resveratrol remains a legendary anticancer drug candidate in the archives of scientific literature. Although earlier wet-lab experiments rendering its multiple biological targets, for example, epidermal growth factors, Pro-apoptotic protein p53, sirtuins, and first apoptosis signal (Fas) receptor, Mouse double minute 2 (MDM2) ubiquitin-protein ligase, Estrogen receptor, Quinone reductase, etc. However, notwithstanding some notable successes, identification of an appropriate Resveratrol target(s) has remained a major challenge using physical methods, and hereby limiting its translation into an effective therapeutic(s). Thus, computational insights are much needed to establish proof-of-concept towards potential Resveratrol target(s) with minimum error rate, narrow down the search space, and to assess a more accurate Resveratrol signaling pathway/mechanism at the starting point. Herein, a brute-force technique combining computational receptor-, ligand-based virtual screening, and classification-based machine learning, reveals the precise mechanism of Resveratrol action. Overall, MDM2 ubiquitin-protein ligase (4OGN.pdb) and co-crystallized quinone reductases 2 (4QOH.pdb) were found two suitable drug targets in the case of Resveratrol derivatives. Indeed, carotenoid cleaving oxygenase together with later twos gave gigantic momentum in guiding the rational drug design of Resveratrol derivatives. These molecular modeling insights would be useful for Resveratrol lead optimization into a more precise science. Communicated by Ramaswamy H. Sarma

MiR-489-3p modulates cell proliferation and apoptosis in cerulein-induced acute pancreatitis by targeting X-linked inhibitor of apoptosis protein

IntroductionThis study aims to explore the effect and mechanism of miR�489-3p on the proliferation and apoptosis of pancreatic acinar cells in acute pancreatitis (AP).Material and methodsX-linked inhibitor of apoptosis protein (XIAP) and miR-489-3p expression in serum of AP patients, pancreatic AR42J cells, and rat AP tissues were measured using quantitative reverse transcription poly�merase chain reaction and western blot. The effect of miR-489-3p on pro�liferation and apoptosis was determined by MTT assay and flow cytometry. The relationship between XIAP and miR-489-3p was verified using luciferase assay RNA immunoprecipitation assay. Histological changes in rat pancreatic tissues were observed via haematoxylin and eosin staining.ResultsMeasurement of miR-489-3p and XIAP expressions in AP patients revealed a negative correlation between miR-489-3p and XIAP. Increased miR-489-3p expression in AP patients indicated a poor prognosis. Cerulein was used to induce AP in AR42J cells and standard deviation rats. Exper�iments in cells and AP rat models showed that miR-489-3p can increase cell apoptosis and inhibit cell proliferation by regulating XIAP, as shown by elevated expressions of pro-apoptotic proteins (p53 and Bax) and decreased expression of proliferation indicator (Ki-67) after transfection of miR-489- 3p mimics. Meanwhile, knockdown of miR-489-3p abrogated the inhibitory effects of miR-489-3p on cell proliferation and the promotion on cell apop�tosis. Luciferase assay and RNA immunoprecipitation assay confirmed that XIAP can directly bind miR-489-3p.ConclusionsWe concluded that miR-489-3p modulates cell proliferation and apoptosis in AP by targeting XIAP. Given that high expression of miR�489-3p in AP indicated poor prognosis, it raises the possibility that miR�489-3p might be a novel and valuable therapeutic target and a prognosis indicator for AP.

Evaluation of Neuroendocrine and Immunological Parameters in Animals Following Chronic Combined Exposure to Xenobiotics

Background: A high prevalence of chromium and benzene compounds in the environment associated with motor vehicle and industrial operations arouses interest in the study of these xenobiotics in a long-term experiment. The objective of this work was to analyze effects of a chronic combined exposure to chromium and benzene on the hypothalamic-pituitary-adrenocortical (HPA) and immune systems of male Wistar rats. Materials and methods: Eighty male Wistar rats were administered potassium dichromate and benzene with drinking water in doses equaling one maximum permissible concentration (MPC) during 135 days. The hypothalamus, pituitary gland, adrenal glands, thymus, and spleen were then studied using morphometric, histological, and electron microscopy methods. The streptavidin-biotin peroxidase method was used to determine the expression of pro-apoptotic protein p53 and anti-apoptotic protein bcl2. We also measured the body, thymus and spleen weights of animals, nucleated cell counts in the thymus, spleen, and bone marrow and evaluated the cellular composition of the spleen and bone marrow as well as spontaneous and concanavalin A-induced secretion of interferon gamma (IFN-γ), IL-4, IL-6, and IL-10 cytokines by splenocytes. Results: We established an adverse effect of the exposure on the HPA function expressed in the activation of its secretory activity, blocking the release of hypothalamic neuropeptides at the level of the neurohypophysis and leading to ultrastructural damage to the neurosecretory cells of the hypothalamus, pituitary adenocytes and adrenal cortical cells, as well as in an increase in the programmed death of thymocytes. We also observed a decrease in the thymus weight and thymocyte counts and a complex of structural and functional changes indicating the status of its accidental involution in the exposed rats. The revealed decrease in the splenocyte count in the experimental group was accompanied by an increase in the size of the white spleen pulp. An increase in the induced production of the main immunoregulatory cytokines IFN-γ and IL-4 by splenocytes was found. Conclusion: Our findings can be used to analyze impairments of the HPA and immune systems in workers with a chronic combined exposure to benzene and chromium compounds in the occupational setting.

Expression profile analysis of a new testis-specifically expressed gene C17ORF64 and its association with cell apoptosis in MCF-7 cells.

With the increasing incidence of male infertility, identification and investigation the functions of new genes related to spermatogenesis are effective avenues to elucidate the decline of testicular function. In this study, a new gene, C17ORF64 (chromosome 17 open reading frame 64), was identified from mouse testes and its potential function was studied.RT-PCR and qRT-PCR assay showed that C17ORF64 mRNA was expressed exclusively in mouse testes and up-regulated from the 3-week old to 6-month old testes during postpartum development, which is consistent with C17ORF64 protein expression profile by western blotting analysis. Immunohistochemical analysis revealed that C17ORF64 protein was mainly localized in the cytoplasm of spermatogonia and spermatocytes, which is verified by GFP- labeled C17ORF64 gene expressed in GC-1 cells. C17ORF64 overexpression not only promoted cell apoptosis in MCF-7 cells, but also significantly decreased cell viability via MTT assay. Flow cytometric assay showed that C17ORF64 overexpression could inhibit cell cycle progression by arresting G1/S transition. Western blot and qRT-PCR analysis revealed that C17ORF64 overexpression inhibited the expression of anti-apoptotic protein bcl-2 and increased the expressions of pro-apoptotic protein caspase-3, caspase-8, caspase-9, Bax, P21 and P53. Taken together, our results confirmed C17ORF64 testis-specific expression pattern and, for the first time, demonstrated that C17ORF64 could inhibit cell viability and accelerate apoptosis in MCF-7 cells through caspase-3 regulatory pathways.

New metal complexes derived from diacetylmonoxime-n(4)antipyrinylthiosemicarbazone: Synthesis, characterization and evaluation of antitumor activity against Ehrlich solid tumors induced in mice

The present study aimed to synthesize new metal complexes of diacetylmonoxime-N(4)antipyrinylthiosemicarbazone ligand and evaluate their antitumor activity. New complexes with ferric, cobalt, nickel and copper ions were prepared. Elemental, 1H Nuclear magnetic resonance, Mass spectroscopy, Electron paramagnetic resonance, Fourier Transform InfraredSpectroscopy, Ultraviolet–visible and thermal gravimetricanalysis were used to characterize the obtained complexes 1–11. An in vivo tumor model was established to investigate the effect of the naked ligand and its metal complexes 2, 5 and 8. Ehrlich ascites carcinoma solid tumor was induced in mice through subcutaneous inoculation of Ehrlich ascites carcinoma cells. The volumes of the formed solid tumors, the alanine transaminase, aspartate transaminase, albumin concentration in the serum, as well as the levels of Ki67 and p53 proteins in tumor and liver tissues were detected. All the tested complexes, especially complex 5, possessed proliferative inhibition manifested as the reduction of the tumor volume, Alanine aminotransferase & Aspartate aminotransferase activity, and the level of the Ki67 protein. Additionally, they restored the albumin concentration to normal levels as well increased the level of pro-apoptotic p53 protein. In conclusion, the antitumor activity of the newly synthesized metal complexes against Ehrlich ascites carcinoma solid tumors was proved to be mediated by the inhibition of Ki67 and induction of p53 proteins.

Пифитрин-альфа тормозит дифференцировку вновь образованных клеток субгранулярной зоны зубчатой извилины у крыс линии Крушинского–Молодкиной при аудиогенном киндлинге

One of the main goal of modern neurobiology is search for approaches to prevent structural violations in the brain. In particular, damage to the hippocampus after epileptic activity. Epilepsy leads to an increase in proliferation in the hippocampal neurogenic niche - subgranular cells layer of dental gyrus. In recent years, there is a prevalent idea that new-born cells contribute to epileptogenesis to a greater extent than prevent neurodegenerative damage. We suggested that proapoptotic protein p53 can be one of possible therapeutic targets for epilepsy and neurodegenerative consequences of this disease.In present work, we used The Krushinsky - Molodkina (KM) inbred rat strain. This strain is genetically prone to audiogenic seizures (AGS). Audiogenic kindling induced epileptiform activity in the limbic system and the cortex. It has been shown that in initial stages of epileptogenesis 4 AGS lead to increase in proliferation, aberrant migration in hilus and acceleration of differentiation of newborn cells. We did not show any disturbance in apoptosis nor autophagy in early development of limbic epilepsy. Pifitrin-α (chemical inhibitor of p53) did not lead to changes in apoptosis and autophagy but caused increase in proliferation and migration of newborn cells in granular cell layer and hilus of dental gyrus. However, in a week after the last seizure the number of differentiated cells decreased despite a significant increase in the number of newborn cells in the group with inactivation of p53 compared to the vehicle control group. Decreased number of differentiated cells tells that pifitrin -α may be used as a potential therapeutic agent for reduction in neurodegeneration which is associated with epilepsy.

Chemopreventive effect of dieckol against 7,12-dimethylbenz(a)anthracene induced skin carcinogenesis model by modulatory influence on biochemical and antioxidant biomarkers.

Skin cancer is the commonly found type, which contributes to 40% of whole cancer incidences worldwide. Dieckol is an active compound occurs in the marine algae with many biological benefits. In this exploration, we intended to investigate the therapeutic potency of dieckol against the 7,12-dimethylbenz(a)anthracene (DMBA)-triggered skin carcinogenesis in mice. The skin cancer was stimulated to the animals via injecting the 25 μg of DMBA in 100 μL of acetone in shaved dorsal portion along with the 30 mg/kg of dieckol supplementation for 25 week. The antioxidant enzymes and phase-I and -II detoxifying enzymes in the test animals were inspected via standard protocols. Pro-inflammatory markers (IL-6, IL-1β, and TNF-α) level was examined via ELISA kits and the expression of inflammatory molecular markers like p-NF-ƙB, IƙBα and p-IƙBα were studied through western blotting. The expression status of pro- and anti-apoptotic proteins (p53, Bax, Bcl-2, caspase-3, caspase-9, COX-2, TGF-β1) was investigated via real-time polymerase chain reaction (RT-PCR). Our results revealed that the 30 mg/kg of dieckol supplementation noticeably regained the body and liver weight and also diminished the tumor incidence in the DMBA-incited animals. Dieckol treatment exhibited an enhanced antioxidants (SOD, CAT, GPx, and GSH) and reduced phase-I enzymes Cyt-p450 and Cyt-b5 in the DMBA-induced animals. Dieckol also diminished the pro-inflammatory modulators like IL-6, IL-1β and TNF-α. Western blotting result evidenced that the dieckol was inhibited the IƙB/NF-ƙB signaling pathway. RT-PCR study proved the enhanced expression of pro-apoptotic protein (p53, Bax, caspase-3 and -9) in the dieckol treated animals. Histological study also confirmed the therapeutic benefits of Dieckol. Altogether with these findings, it was clear that the dieckol has appreciably allayed the DMBA activated skin tumorigenesis in the mice and it could be a promising agent to treat the human skin cancer in future.

Novel 1,3,4-oxadiazole Targets STAT3 Signaling to Induce Antitumor Effect in Lung Cancer.

Lung cancer is the leading type of malignancy in terms of occurrence and mortality in the global context. STAT3 is an oncogenic transcription factor that is persistently activated in many types of human malignancies, including lung cancer. In the present report, new oxadiazole conjugated indazoles were synthesized and examined for their anticancer potential in a panel of cancer cell lines. Among the new compounds, 2-(3-(6-chloro-5-methylpyridin-3-yl)phenyl)-5-(1-methyl-1H-indazol-3-yl)-1,3,4-oxadiazole (CHK9) showed consistently good cytotoxicity towards lung cancer cells with IC50 values ranging between 4.8–5.1 µM. The proapoptotic effect of CHK9 was further demonstrated by Annexin-FITC staining and TUNEL assay. In addition, the effect of CHK9 on the activation of STAT3 in lung cancer cells was examined. CHK9 reduced the phosphorylation of STAT3Y705 in a dose-dependent manner. CHK9 had no effect on the activation and expression of JAK2 and STAT5. It also reduced the STAT3-dependent luciferase reporter gene expression. CHK9 increased the expression of proapoptotic (p53 and Bax) proteins and decreased the expression of the antiapoptotic (Bcl-2, Bcl-xL, BID, and ICAM-1) proteins. CHK9 displayed a significant reduction in the number of tumor nodules in the in vivo lung cancer model with suppression of STAT3 activation in tumor tissues. CHK9 did not show substantial toxicity in the normal murine model. Overall, CHK9 inhibits the growth of lung cancer cells and tumors by interfering with the STAT3 signaling pathway.

Palmatine induces G2/M phase arrest and mitochondrial-associated pathway apoptosis in colon cancer cells by targeting AURKA

Studies have shown that palmatine (PAL) has anti-cancer effects. However, the activity and potential mechanisms of PAL against colorectal cancer remain elusive. The results showed that PAL significantly inhibited the proliferation of colon cancer cells in vitro and in vivo without significant effect on non-tumorigenic colon cells. Target prediction and clinical sample database analysis suggested that PAL may contribute to colon cancer cells phase arrest and apoptosis by targeting aurora kinase A (AURKA). Inhibition and overexpression of AURKA proved that PAL induces G2/M phase arrest and apoptosis in colon cancer cells by targeting AURKA. Moreover, PAL promoted intracellular Reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (ΔΨm). PAL reduced the levels of AURKA, Bcl-xl and Bcl2 proteins, and promoted the expression of pro-apoptotic proteins P53, P73, Caspase3 and Caspase9, as well as the increase of cytochrome c (cyt. c) in cell lysates in vitro and in vivo. Together, our study confirmed that PAL induced G2/M phase arrest and mitochondrial-associated pathway apoptosis in colon cancer cells by targeting AURKA. PAL may provide a novel solution for the treatment of colon cancer by serving as a new AURKA inhibitor.

Treatment of Brain Edema by Wogonoside Is Associated with Inhibition of Neuronal Apoptosis and SIRT1 Activation in Rats.

BackgroundBrain edema and neuronal apoptosis are closely associated with loss of neurological function and death in rats with subarachnoid hemorrhage (SAH). The present study investigated the effect of wogonoside on brain edema induced by SAH in rats and studied the mechanism involved.Material/MethodsThe rats were intra-gastrically administered 10, 20, 50, 100, 150 and 200 mg/kg doses of wogonoside 24 h prior to SAH induction. Western blotting was used to assess levels of pro-apoptotic protein, SIRT1, ZO-1, and p53 protein expression. Apoptotic nuclei were detected using immunofluorescence and TUNEL staining.ResultsWogonoside treatment significantly suppressed edema formation in SAH-induced rats. Pre-treatment with wogonoside exhibited an inhibitory effect on SAH-induced extravascular Evans blue staining in rats. The expression of ZO-1, Occludin, and Claudin-5 proteins was increased by wogonoside in the SAH-induced rats. The inhibitory effect of SAH was completely reversed in the rats treated with the 200 mg/kg dose of wogonoside. The expression of SIRT1 protein was upregulated, and p53 and AC-p53 were downregulated by wogonoside in SAH rats. Wogonoside treatment significantly reduced SAH-mediated promotion of Bax, Puma, Noxa, Bid, and cleaved Caspase-3 expression. In the SAH-induced rats, pre-treatment with wogonoside reduced the TUNEL-positive cell count.ConclusionsThe present study demonstrated that wogonoside prevents brain edema development and apoptosis of neurons in rats by promoting SIRT1 expression and suppression of p53 activation. Therefore, wogonoside has therapeutic potential for the treatment of edema and needs to be investigated further to completely define the mechanism involved.

Исследование роли нерецепторной тирозинкиназы c-Abl в регуляции р53-зависимой активации ERK1/2-зависимого каскада

Protein p53 is a transcription factor that induces aging, cell cycle arrest and apoptosis in response to cell stress. Published findings suggest that proapoptotic protein p53 is involved not only in apoptosis, but also in the regulation of neural differentiation along with the activation of ERK1/2 signaling cascade. However, the nature and the mechanisms of p53 effects on ERK1/2 are not completely understood. Here, we examine the intracellular mechanisms underlying p53-dependent activation of ERK1/2. The study was performed on undifferentiated PC12 cells. For short-term activation of TrkA receptors the cells were incubated with NGF. Our results demonstrate that p53 activation with Nutlin-3 results in the increase of TrkA/cRaf/ERK cascade and c-Abl activity. c-Abl inhibition under p53 activation had no effect on TrkA receptors, but was associated with enhanced activity of cRaf and ERK1/2. At the same time, combined application of Nutlin-3 with c-Abl and TrkA inhibitors evoked considerable decrease of ERK1/2 activity. Our results suggest that p53 activates ERK1/2 cascade either in complex with c-Abl or in c-Abl-independent manner by modulating the c-Raf or upstream targets.

Features of Bcl2 and p53 proteins synthesis in pancreatic islets of normotensive and hypertensive rats with streptozotocin-induced diabetes

In our previous studies it was established that the remodeling of pancreatic islets with decreasing β-cell population density is formed in hypertensive SHR rats. The imbalance between the synthesis of proapoptotic and antiapoptotic factors may be one of the possible causes of disturbed formation of the endocrinocyte population in the pancreas. The aim of the research was to study the parameters of Bcl2 and p53 proteins synthesis in pancreatic islets in normotensive and hypertensive rats in the streptozotocin-induced diabetes mellitus development. Materials and methods . The study was performed on 30 normotensive male Wistar rats (systolic BP=105.0 ±1.1 mm Hg) and 25 hypertensive SHR rats (systolic BP=155.7 ±0.9 mm Hg) with fasting normoglycemia (4.73 ± 0.10 mmol/l). Bcl2 and p53 proteins were detected in histological pancreas sections by immunofluorescence method. The relative area of Bcl2- and p53-immunopositive material, concentration of proteins in endocrinocytes, their content in the islets and apoptosis index p53/Bcl2 were analyzed in pancreatic islands. Results. The area of relative immunofluorescence to the Bcl2 protein was 2 times less, and the protein content was 3 times lower in pancreatic islets of hypertensive rats (SHR) compared with normotensive Wistar rats. At the same time, no statistical differences in the area of the immunopositive material to the p53 protein and its content in the islets between the experimental groups were revealed. The development of streptozotocin-induced diabetes in Wistar rats was accompanied by approximately 2-fold decrease in the Bcl2 protein expression in pancreatic islets, a significant increase in the specific content of p53 protein and a 3.8-fold increase in the apoptosis index of p53/Bcl2. In pancreatic islets of SHR rats, diabetes mellitus development was accompanied by 2-fold increase in the specific content of the proapoptotic protein p53 without the reduction of the antiapoptotic protein Bcl2 synthesis. At the same time, the p53/Bcl2 apoptosis index in SHR rats remained statistically higher than in Wistar rats. Conclusions. Endocrine cells of pancreatic islets of SHR rats are characterized by the prevalence of proapoptotic protein p53 expression as compared with Wistar line normotensive rats. The development of streptozotocin diabetes in Wistar rats leads to a significant decrease in the number of endocrinocytes synthesizing the antiapoptotic protein Bcl2. At the same time, an increase in the synthesis of the proapoptotic protein p53 in endocrinocytes in diabetes is observed both in normotensive and in hypertensive rats.

Purification, characterization and anti-tumor activities of polysaccharides from Ecklonia kurome obtained by three different extraction methods

To investigate and compare the effects of different extraction methods on the structure and anti-tumor activity of Ecklonia kurome polysaccharides (EP), three techniques, namely hot water extraction (HW), ultrasonic-assisted extraction (UA) and enzyme-assisted extraction (EA), were used to extract EP, and three crude EPs were purified by DEAE-cellulose and gel filtration chromatography. The significant antitumor active components in each method were screened by MTT assay and named as HW-EP5, UA-EP4 and EA-EP3, respectively. The molecular weight, FT-IR assay and NMR showed that HW-EP5, UA-EP4 and EA-EP3 were pyran polysaccharides with a molecular weight of 14,466, 15,922 and 16,947 Da, respectively. HW-EP5 contained the most monosaccharides and the highest content of sulfate and uronic acid. HW-EP5 had an even and smooth sheet-like appearance, while UA-EP4 and EA-EP3 exhibited irregular and rough fragments. All three polysaccharides can inhibit the migration of human breast cancer cells (MCF-7) and promote its apoptosis. All three polysaccharides promoted caspase activity during apoptosis. HW-EP5 and UA-EP4 up-regulated the expression of proapoptotic proteins Bax and p53, while EA-EP3 only up-regulated the expression of p53. These experimental results indicate that Ecklonia kurome polysaccharides, especially HW-EP5, have great potential as a natural medicine for the treatment of breast cancer.

Cerium Aminoclay-A Potential Hybrid Biomaterial for Anticancer Therapy.

In this study, novel biomedical properties of Ce-aminoclay (CeAC) were investigated through in vitro and in vivo assays. CeAC (≥500 μg/mL) can selectively kill cancer cells (A549, Huh-1, AGS, C33A, HCT116, and MCF-7 cells) while leaving most normal cells unharmed (WI-38 and CCD-18Co cells). Notably, it displayed a high contrast of simultaneous imaging in HeLa cells by blue photoluminescence without any fluorescence dye. Its anticancer mechanism has been fully demonstrated through apoptosis assays; herein CeAC induced high-level apoptosis (16%), which promoted the expression of proapoptotic proteins (Bax, p53, and caspase 9) in tumor cells. Besides, its biological behavior was determined through antitumor effects using intravenous and intratumoral administration routes in mice implanted with HCT116 cells. During a 40 day trial, the tumor volume and tumor weight were reduced by a maximum of 92.24 and 86.11%, respectively. The results indicate that CeAC exhibits high bioavailability and therapeutic potential based on its unique characteristics, including high antioxidant capacity and electrostatic interaction between its amino functional groups and the mucosal surface of cells. In summary, it is suggested that CeAC, with its high bioimaging contrast, can be a promising anticancer agent for future biomedical applications.

Synergistic Effect of Epigenetic Inhibitors Decitabine and Suberoylanilide Hydroxamic Acid on Colorectal Cancer In vitro.

Colorectal Cancer (CRC) is a common cause of oncological deaths worldwide. Alterations of the epigenetic landscape constitute a well-documented hallmark of CRC phenotype. The accumulation of aberrant DNA methylation and histone acetylation plays a major role in altering gene activity and driving tumor onset, progression and metastasis. In this study, we evaluated the effect of Suberoylanilide Hydroxamic Acid (SAHA), a panhistone deacetylase inhibitor, and Decitabine (DAC), a DNA methyltransferase inhibitor, either alone or in combination, on Caco-2 human colon cancer cell line in vitro. Our results showed that SAHA and DAC, separately, significantly decreased cell proliferation, induced apoptosis and cell cycle arrest of Caco-2 cell line. On the other hand, the sequential treatment of Caco-2 cells, first with DAC and then with SAHA, induced a synergistic anti-tumor effect with a significant enhancement of growth inhibition and apoptosis induction in Caco-2 cell line as compared to cells treated with either drug alone. Furthermore, the combination therapy upregulates protein expression levels of pro-apoptotic proteins Bax, p53 and cytochrome c, downregulates the expression of antiapoptotic Bcl-2 protein and increases the cleavage of procaspases 8 and 9; this suggests that the combination activates apoptosis via both the intrinsic and extrinsic pathways. Mechanistically, we demonstrated that the synergistic anti-neoplastic activity of combined SAHA and DAC involves an effect on PI3K/AKT and Wnt/β-catenin signaling. In conclusion, our results provide evidence for the profound anti-tumorigenic effect of sequentially combined SAHA and DAC in the CRC cell line and offer new insights into the corresponding underlined molecular mechanism.

Pereskia bleo Leaves Extract Induces Cell Death via Cell Cycle Arrest and Apoptosis in Cervical Cancer Cells HeLa

Introduction: Pereskia bleo is a leafy and edible plant, locally known as “Pokok Jarum Tujuh Bilah” which has anticancer properties. This study purposed to determine the cytotoxic effects of P. bleo leaves extracts on several well-known cancer cells and elucidate its underlying mechanism in inducing cell death.Methods: Cytotoxic activity on selected cell lines was determined using MTT assay. Mechanism of cell death was investigated through cell cycle and Annexin V assay. Expression of apoptotic proteins was measured by flow cytometry method.Results: Ethyl acetate extract of P. bleo leaves (PBEA) appeared to have the strongest IC50 value (14.37 ± 8.40 μg/ml) and most active against HeLa cells was further studied for apoptosis. The cell cycle investigation by flow cytometry evidenced the increment of PBEA treated HeLa cells in G0/G1 phase and apoptotic event was detected in Annexin V assay. Analysis of apoptotic protein showed pro-apoptotic proteins (Bax, p53 and caspase 3) were triggered where as anti-apoptotic protein Bcl-2 was suppressed in treated HeLa cells.Conclusions: Our findings demonstrated that PBEA treatment induced cell death in HeLa cells by p53-mediated mechanism through arresting cell cycle at G0/G1 phase and mitochondrial-mediated pathway with involvement of pro-apoptotic proteins, anti-apoptotic protein, and caspase 3.

Silica Nanoparticles Provoke Cell Death Independent of p53 and BAX in Human Colon Cancer Cells.

Several in vitro studies have suggested that silica nanoparticles (NPs) might induce adverse effects in gut cells. Here, we used the human colon cancer epithelial cell line HCT116 to study the potential cytotoxic effects of ingested silica NPs in the presence or absence of serum. Furthermore, we evaluated different physico-chemical parameters important for the assessment of nanoparticle safety, including primary particle size (12, 70, 200, and 500 nm) and surface modification (–NH2 and –COOH). Silica NPs triggered cytotoxicity, as evidenced by reduced metabolism and enhanced membrane leakage. Automated microscopy revealed that the silica NPs promoted apoptosis and necrosis proportional to the administered specific surface area dose. Cytotoxicity of silica NPs was suppressed by increasing amount of serum and surface modification. Furthermore, inhibition of caspases partially prevented silica NP-induced cytotoxicity. In order to investigate the role of specific cell death pathways in more detail, we used isogenic derivatives of HCT116 cells which lack the pro-apoptotic proteins p53 or BAX. In contrast to the anticancer drug cisplatin, silica NPs induced cell death independent of the p53–BAX axis. In conclusion, silica NPs initiated cell death in colon cancer cells dependent on the specific surface area and presence of serum. Further studies in vivo are warranted to address potential cytotoxic actions in the gut epithelium. The unintended toxicity of silica NPs as observed here could also be beneficial. As loss of p53 in colon cancer cells contributes to resistance against anticancer drugs, and thus to reoccurrence of colon cancer, targeted delivery of silica NPs could be envisioned to also deplete p53 deficient tumor cells.