Selenium deficiency exacerbated Bisphenol A-induced intestinal toxicity in chickens: Apoptosis and cell cycle arrest mediated by ROS/P53.

Bisphenol A (BPA) is a phenolic organic synthetic compound that is used as the raw material of polycarbonate plastics, and its safety issues have recently attracted wide attention. Selenium (Se) deficiency has gradually developed into a global disease affecting intestinal function via oxidative stress and apoptosis. However, the toxic effects and potential mechanisms of BPA exposure and Se deficiency in the chicken intestines have not been studied. In this study, BPA exposure and/or Se deficiency models were established in vivo and in vitro to investigate the effects of Se deficiency and BPA on chicken jejunum. The results showed that BPA exposure and/or Se deficiency increased jejunum oxidative stress and DNA damage, activated P53 pathway, led to mitochondrial dysfunction, and induced apoptosis and cell cycle arrest. Using protein-protein molecular docking, we found a strong binding ability between P53 and peroxisome proliferator-activated receptor γ coactivator-1, thereby regulating mitochondrial dysfunctional apoptosis. In addition, we used N-acetyl-L-cysteine and pifithrin-α for in vitro intervention and found that N-acetyl-L-cysteine and pifithrin-α intervention reversed the aforementioned adverse effects. This study clarified the potential mechanism by which Se deficiency exacerbates BPA induced intestinal injury in chickens through reactive oxygen species/P53, which provides a new idea for the study of environmental combined toxicity of Se deficiency, and insights into animal intestinal health from a new perspective.

Identification of neural oscillations and epileptiform changes in human brain organoids.

Brain organoids represent a powerful tool for studying human neurological diseases, particularly those that affect brain growth and structure. However, many diseases manifest with clear evidence of physiological and network abnormality in the absence of anatomical changes, raising the question of whether organoids possess sufficient neural network complexity to model these conditions. Here, we explore the network-level functions of brain organoids using calcium sensor imaging and extracellular recording approaches that together reveal the existence of complex network dynamics reminiscent of intact brain preparations. We demonstrate highly abnormal and epileptiform-like activity in organoids derived from induced pluripotent stem cells from individuals with Rett syndrome, accompanied by transcriptomic differences revealed by single-cell analyses. We also rescue key physiological activities with an unconventional neuroregulatory drug, pifithrin-α. Together, these findings provide an essential foundation for the utilization of brain organoids to study intact and disordered human brain network formation and illustrate their utility in therapeutic discovery.

From Riluzole to Dexpramipexole via Substituted-Benzothiazole Derivatives for Amyotrophic Lateral Sclerosis Disease Treatment: Case Studies.

The 1,3-benzothiazole (BTZ) ring may offer a valid option for scaffold-hopping from indole derivatives. Several BTZs have clinically relevant roles, mainly as CNS medicines and diagnostic agents, with riluzole being one of the most famous examples. Riluzole is currently the only approved drug to treat amyotrophic lateral sclerosis (ALS) but its efficacy is marginal. Several clinical studies have demonstrated only limited improvements in survival, without benefits to motor function in patients with ALS. Despite significant clinical trial efforts to understand the genetic, epigenetic, and molecular pathways linked to ALS pathophysiology, therapeutic translation has remained disappointingly slow, probably due to the complexity and the heterogeneity of this disease. Many other drugs to tackle ALS have been tested for 20 years without any success. Dexpramipexole is a BTZ structural analog of riluzole and was a great hope for the treatment of ALS. In this review, as an interesting case study in the development of a new medicine to treat ALS, we present the strategy of the development of dexpramipexole, which was one of the most promising drugs against ALS.

Spiro-containing derivatives show antiparasitic activity against Trypanosoma brucei through inhibition of the trypanothione reductase enzyme.

Trypanothione reductase (TR) is a key enzyme that catalyzes the reduction of trypanothione, an antioxidant dithiol that protects Trypanosomatid parasites from oxidative stress induced by mammalian host defense systems. TR is considered an attractive target for the development of novel anti-parasitic agents as it is essential for parasite survival but has no close homologue in humans. We report here the identification of spiro-containing derivatives as inhibitors of TR from Trypanosoma brucei (TbTR), the parasite responsible for Human African Trypanosomiasis. The hit series, identified by high throughput screening, was shown to bind TbTR reversibly and to compete with the trypanothione (TS2) substrate. The prototype compound 1 from this series was also found to impede the growth of Trypanosoma brucei parasites in vitro. The X-ray crystal structure of TbTR in complex with compound 1 solved at 1.98 Å allowed the identification of the hydrophobic pocket where the inhibitor binds, placed close to the catalytic histidine (His 461') and lined by Trp21, Val53, Ile106, Tyr110 and Met113. This new inhibitor is specific for TbTR and no activity was detected against the structurally similar human glutathione reductase (hGR). The central spiro scaffold is known to be suitable for brain active compounds in humans thus representing an attractive starting point for the future treatment of the central nervous system stage of T. brucei infections.

Effects of selenium on the proliferation and apoptosis of sheep spermatogonial stem cells in vitro.

The objective of this study was to investigate effects of selenium (Se) on proliferation and apoptosis of sheep spermatogonial stem cells (SSC) in vitro. The SSC were assigned to five treatment groups (0, 2.0, 4.0, 8.0 and 16.0 µmol/L Se). After treatment with Se for 96 h, cell proliferation and apoptosis were evaluated. The relative abundance of P53 mRNA transcript and protein, cell cycle and apoptosis-related genes were detected using real-time PCR and Western blot quantifications, respectively. The results indicate there were the least cell proliferation rates in the Se16.0 group. Treatments with relatively greater Se concentrations (8.0 and 16.0 µmol/L) resulted in a greater percentage of apoptotic cells, which was consistent with the relative abundances of P53, P21, P27 and pro-apoptosis mRNA transcripts. There were relatively greater ROS concentrations in the control, Se8.0 and Se16.0 groups. Compared with the control group, treatment with the Se concentration of 16.0 µmol/L resulted in an increased abundance of P53, P21, P27 and BAX proteins. Treatment with Pifithrin-α suppressed the increase in abundance of P53 and P21 proteins induced by the relatively greater concentration of Se (16.0 µmol/L), however, did not result in a change in abundances of P27 and BAX proteins. These results indicate the regulatory functions of Se on proliferation and apoptosis of sheep SSC is associated with the P21-mediated P53 signalling pathway. The P27 and BAX proteins have limited functions during the apoptotic process of SSC induced by the relatively greater concentrations of Se.

Pifithrin-Alpha Reduces Methamphetamine Neurotoxicity in Cultured Dopaminergic Neurons.

Methamphetamine (Meth) is a widely abused stimulant. High-dose Meth induces degeneration of dopaminergic neurons through p53-mediated apoptosis. A recent study indicated that treatment with the p53 inhibitor, pifithrin-alpha (PFT-α), antagonized Meth-mediated behavioral deficits in mice. The mechanisms underpinning the protective action of PFT-α against Meth have not been identified, and hence, their investigation is the focus of this study. Primary dopaminergic neuronal cultures were prepared from rat embryonic ventral mesencephalic tissue. High-dose Meth challenge reduced tyrosine hydroxylase immunoreactivity and increased terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling (TUNEL) labeling. PFT-α significantly antagonized these responses. PFT-α also reduced Meth-activated translocation of p53 to the nucleus, an initial step before transcription. Previous studies have indicated that p53 can also activate cell death through transcription-independent pathways. We found that PFT-α attenuated endoplasmic reticulum (ER) stressor thapsigargin (Tg)-mediated loss of dopaminergic neurons. ER stress was further monitored through the release of Gaussia luciferase (GLuc) from SH-SY5Y cells overexpressing GLuc-based Secreted ER Calcium-Modulated Protein (GLuc-SERCaMP). Meth or Tg significantly increased GLuc release in to the media, with PFT-α significantly reducing GLuc release. Additionally, PFT-α significantly attenuated Meth-induced CHOP expression. In conclusion, our data indicate that PFT-α is neuroprotective against Meth-mediated neurodegeneration via transcription-dependent nuclear and -independent cytosolic ER stress pathways.

The atopic dermatitis-like lesion and the associated MRSA infection and barrier dysfunction can be alleviated by 2,4-dimethoxy-6-methylbenzene-1,3-diol from Antrodia camphorata.

BACKGROUND: Atopic dermatitis (AD) is an inflammatory skin disease with an associated barrier dysfunction and Staphylococcus aureus infection. The mainstay steroid and calcineurin inhibitor therapy shows some adverse effects. 2,4-Dimethoxy-6-methylbenzene-1,3-diol (DMD) is a benzenoid isolated from Antrodia camphorata. OBJECTIVE: We investigated the inhibitory effect of DMD on methicillin-resistant S. aureus (MRSA), the chemokine production in stimulated keratinocytes, and the AD-like lesion found in ovalbumin (OVA)-sensitized mice. METHODS: The antimicrobial effect and cutaneous barrier function were evaluated using an in vitro culture model and an in vivo mouse model of AD-like skin. RESULTS: DMD exhibited a comparative minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against MRSA with nalidixic acid, a conventional antibiotic. The MIC and MBC for DMD was 78.1 and 156.3 µg/ml, respectively. DMD also showed the ability to eliminate the clinical bacteria isolates with resistance to methicillin and vancomycin. The DNA polymerase and gyrase inhibition evoked by DMD for bacterial lethality was proposed. In the activated keratinocytes, DMD stopped the upregulation of chemokines (CCL5 and CCL17) and increased the expression of differentiation proteins (filaggrin, involucrin, and integrin ß-1). Topical application of DMD facilely penetrated into the skin, with AD-like skin displaying 2.5-fold greater permeation than healthy skin. The in vivo assessment using the mouse model with OVA sensitization and MRSA inoculation revealed a reduction of transepidermal water loss (TEWL) and bacterial burden by DMD by about 2- and 100-fold, respectively. Differentiation proteins were also restored after topical DMD delivery. CONCLUSION: Our data demonstrated an advanced concept of AD treatment by combined barrier repair and bacterial eradication with a sole agent for ameliorating the overall complications.

Phytochemical Analysis of Anti-Inflammatory and Antioxidant Effects of Mahonia aquifolium Flower and Fruit Extracts.

Oxidative stress and inflammation are interlinked processes. The aim of the study was to perform a phytochemical analysis and to evaluate the antioxidant and anti-inflammatory activities of ethanolic Mahonia aquifolium flower (MF), green fruit (MGF), and ripe fruit (MRF) extracts. Plant extract chemical composition was evaluated by HLPC. A DPPH test was used for the in vitro antioxidant activity. The in vivo antioxidant effects and the anti-inflammatory potential were tested on a rat turpentine oil-induced inflammation, by measuring serum nitric oxide (NOx) and TNF-alpha, total oxidative status (TOS), total antioxidant reactivity (TAR), oxidative stress index (OSI), 3-nitrothyrosine (3NT), malondialdehyde (MDA), and total thiols (SH). Extracts were administrated orally in three dilutions (100%, 50%, and 25%) for seven days prior to inflammation. The effects were compared to diclofenac. The HPLC polyphenol and alkaloid analysis revealed chlorogenic acid as the most abundant compound. All extracts had a good in vitro antioxidant activity, decreased NOx, TOS, and 3NT, and increased SH. TNF-alpha was reduced, and TAR increased only by MF and MGF. MDA was not influenced. Our findings suggest that M. aquifolium has anti-inflammatory and antioxidant effects that support the use in primary prevention of the inflammatory processes.

Cell death mechanisms of the anti-cancer drug etoposide on human cardiomyocytes isolated from pluripotent stem cells.

Etoposide (ETP) and anthracyclines are applied for wide anti-cancer treatments. However, the ETP-induced cardiotoxicity remains to be a major safety issue and the underlying cardiotoxic mechanisms are not well understood. This study is aiming to unravel the cardiotoxicity profile of ETP in comparison to anthracyclines using physiologically relevant human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). Using xCELLigence real-time cell analyser (RTCA), we found that single high dose of ETP induces irreversible increase in hPSC-CMs beating rate and decrease in beating amplitude. We also identified 58 deregulated genes consisting of 33 upregulated and 25 downregulated genes in hPSC-CMs after ETP treatment. Gene ontology (GO) and pathway analysis showed that most upregulated genes are enriched in GO categories like positive regulation of apoptotic process, regulation of cell death, and mitochondria organization, whereas most downregulated genes were enriched in GO categories like cytoskeletal organization, muscle contraction, and Ca2+ ion homeostasis. Moreover, we also found upregulation in 5 miRNAs (has-miR-486-3p, has-miR-34c-5p, has-miR-4423-3p, has-miR-182-5p, and has-miR-139-5p) which play role in muscle contraction, arginine and proline metabolism, and hypertrophic cardiomyopathy (HCM). Immunostaining and transmission electron microscopy also confirmed the cytoskeletal and mitochondrial damage in hPSC-CMs treated with ETP, as well as noticeable alterations in intracellular calcium handling and mitochondrial membrane potential were also observed. The apoptosis inhibitor, Pifithrin-α, found to protect hPSC-CMs from ETP-induced cardiotoxicity, whereas hPSC-CMs treated with ferroptosis inhibitor, Liproxstatin-1, showed significant recovery in hPSC-CMs functional properties like beating rate and amplitude after ETP treatment. We suggest that the damage to mitochondria is a major contributing factor involved in ETP-induced cardiotoxicity and the activation of the p53-mediated ferroptosis pathway by ETP is likely the critical pathway in ETP-induced cardiotoxicity. We also conclude that the genomic biomarkers identified in this study will significantly contribute to develop and predict potential cardiotoxic effects of novel anti-cancer drugs in vitro.

Intermittent low dose irradiation enhances the effectiveness of radio- and chemo-therapy for human colorectal adenocarcinoma cell line HT-29.

Low dose irradiation (LDIR) induces hormesis and adaptive response in organism and mammalian cell lines. Notably, LDIR generates distinct biological effects in cancer cells from normal cells, e.g., it may affect the growth of cancer cells via the activation of certain cell signaling pathway, which does not exist in normal cells. Therefore, LDIR is considered as a promising assistant method of clinical cancer therapy. In this study, we chose human colorectal adenocarcinoma cell line HT-29 as the experimental model, and investigated the differential biological effects between 250 mGy single dose LDIR and 250 mGy intermittent LDIR pretreatments in high dose irradiation (HDIR) radiotherapy and 5-fluorouracil (5-FU) based chemotherapy. Through the cell growth assays, we observed that 250 mGy intermittent LDIR pretreatment significantly increased the killing effect of both radiotherapy and chemotherapy. Western blotting results showed that intermittent LDIR pretreatment apparently activated the ATM/p53 (ataxia telangiectasia mutated, ATM) pathway in radiotherapy; it also activated ERK and p38MAPK pathways in chemotherapy. When we used chemical inhibitors to block the ATM/p53 or p38MAPK pathways, the intermittent LDIR induced cell growth inhibitions were reversed. However, blockage of ERK pathway could not affect the cell growth inhibiton in chemotherapy. Taken together, our findings evaluated the intermittent LDIR as a potential valuable method that can enhance the effectiveness of radiotherapy and chemotherapy, especially in the radio- or chemo-resistant tumor types.

Biosynthesis of palladium nanoparticles using Poplar leaf extract and its application in Suzuki coupling reaction.

A green route for the synthesis of palladium (Pd) nanoparticles (Pd NPs) employing Poplar leaf extract as a reducing and capping agent is described. The as-prepared Pd NPs are spherical with a face centred cubic structure, a particle distribution of 2.2-6.8 nm and an average particle size of 4.2 nm. The application of this catalyst toward homogeneous Suzuki coupling reactions was investigated. The Pd NPs afforded a yield of 98.86% in the Suzuki coupling reaction of 4-bromotoluene with phenylboronic acid using 0.01 mmol% of the catalyst at 60°C for 30 min under an air atmosphere.

Effect of potential role of p53 on embryo development arrest induced by H2O2 in mouse.

During mammalian embryo development in vitro, mechanism of embryonic development arrest caused by oxidative stress has not been clear so far. The tumor suppressor protein p53 controls cell cycle and programmed cell death by regulating relevant signal pathway. Recent researches revealed that the concentration and distribution of p53 are closely related with reactive oxygen species (ROS). The main objective of this experiment was to explore the role of p53 on embryonic development arrest caused by oxidative stress. Results showed that embryo arrest at two-four-cell stage was significantly increased in the presence of 50 µM H2O2 (39.01 ± 2.74 vs. 77.20 ± 5.34%, p < 0.05). Supplementation of N-acetyl-L-cysteine (NAC) obviously reduced the ratio of development arrest (39.01 ± 2.74 vs. 71.18 ± 5.34%, p < 0.05), which was accompanied by an increase in ROS level, and H2O2 treatment sharply increased messenger RNA (mRNA) expression and protein levels of p53 and p53-ser15. Further increased transcription of GADD45a and p21, a downstream of p53, has an especially significant effect on the mRNA expression of GADD45a. However, expressions of cdc2 were reduced by H2O2. In addition, using Pifithrin-α (PFT-α), the suppresser of p53, the result showed that GADD45a and p21 were significantly downregulated, but the cell cycle gene cdc2 was significantly upregulated, while the protein level of p53 and p53-ser15 was significantly decreased. Taken together, these results demonstrate that ROS could activate p53 and regulate p53 target genes to influence early embryo development in in vitro culture.

Preclinical effects of honokiol on treating glioblastoma multiforme via G1 phase arrest and cell apoptosis.

BACKGROUND: Our previous study showed that honokiol, a bioactive polyphenol, can traverse the blood-brain barrier and kills neuroblastoma cells. PURPOSE: In this study, we further evaluated the preclinical effects of honokiol on development of malignant glioma and the possible mechanisms. METHODS: Effects of honokiol on viability, caspase activities, apoptosis, and cell cycle arrest in human glioma U87 MG or U373MG cells were assayed. As to the mechanisms, levels of inactive or phosphorylated (p) p53, p21, CDK6, CDK4, cyclin D1, and E2F1 were immunodetected. Pifithrin-α (PFN-α), a p53 inhibitor, was pretreated into the cells. Finally, our in vitro findings were confirmed using intracranial nude mice implanted with U87 MG cells. RESULTS: Exposure of human U87 MG glioma cells to honokiol decreased the cell viability. In parallel, honokiol induced activations of caspase-8, -9, and -3, apoptosis, and G1 cell cycle arrest. Treatment of U87 MG cells with honokiol increased p53 phosphorylation and p21 levels. Honokiol provoked signal-transducing downregulation of CDK6, CDK4, cyclin D1, phosphorylated (p)RB, and E2F1. Pretreatment of U87 MG cells with PFN-α significantly reversed honokiol-induced p53 phosphorylation and p21 augmentation. Honokiol-induced alterations in levels of CDK6, CDK4, cyclin D1, p-RB, and E2F1 were attenuated by PFN-α. Furthermore, honokiol could induce apoptotic insults to human U373MG glioma cells. In our in vivo model, administration of honokiol prolonged the survival rate of nude mice implanted with U87 MG cells and induced caspase-3 activation and chronological changes in p53, p21, CDK6, CDK4, cyclin D1, p-RB, and E2F1. CONCLUSIONS: Honokiol can repress human glioma growth by inducing apoptosis and cell cycle arrest in tumor cells though activating a p53/cyclin D1/CDK6/CDK4/E2F1-dependent pathway. Our results suggest the potential of honokiol in therapies for human malignant gliomas.

Gambogic Acid Inhibits Malignant Melanoma Cell Proliferation Through Mitochondrial p66shc/ROS-p53/Bax-Mediated Apoptosis.

BACKGROUND/AIMS: Malignant melanoma has high metastatic potential, is highly resistant to chemotherapy, and has a poor survival rate. Gambogic acid (GA), a polyprenylated xanthone extracted from a traditional Chinese medicinal herb, has been proven to exhibit antitumor activity. The present study aimed to investigate the signaling pathways that mediated GA-induced inhibition of human malignant skin melanoma proliferation. METHODS: The study was conducted using A375 cells and the corresponding tumor transplanted in nude mice. RESULTS: Incubation of A375 cells with 1-10 µg/ml GA decreased cell viability and increased apoptosis. GA concentration-dependently increased p66shc expression and intracellular ROS levels. GA also decreased the oxygen consumption rate and the mitochondrial membrane potential (MMP) in A375 cells. Experimental inhibition of p66shc by siRNA suppressed GA-induced increase of ROS, decrease of oxygen consumption rate, MMP and cell viability, whilst suppressing GA-induced increase of apoptosis. GA concentration-dependently upregulated p53 and Bax expression in A375 cells. GA also increased p53-TA-luciferase activity and p53-binding to Bax promoter, which was inhibited by Sip53. Experimental inhibition of p53 with Sip53 blocked GA-induced decrease of the oxygen consumption rate and cell viability, and blocked the increase of apoptosis. In tumor-bearing nude mice, GA notably inhibited tumor growth, and this action was suppressed by N-acetylcysteine (NAC), a potent antioxidant, and by PFT-α, a p53 inhibitor. In A375 tumors transplanted in nude mice, GA increased both p66shc and p53 expression. NAC and PFT-α treatment did not significantly affect p66shc expression in tumors grown in mice treated with GA. In contrast, both NAC and PFT-α treatment inhibited GA-induced p53 expression in mouse tumors. CONCLUSION: Results provided novel preclinical insights into the chemotherapeutic use of GA by highlighting the importance of p66shc/ROS-p53/Bax pathways in the antitumor effect of GA in malignant melanoma.

3-H-[1,2]Dithiole as a New Anti-Trypanosoma cruzi Chemotype: Biological and Mechanism of Action Studies.

The current pharmacological Chagas disease treatments, using Nifurtimox or Benznidazole, show limited therapeutic results and are associated with potential side effects, like mutagenicity. Using random screening we have identified new chemotypes that were able to inhibit relevant targets of the Trypanosoma cruzi. We found 3H-[1,2]dithioles with the ability to inhibit Trypanosoma cruzi triosephosphate isomerase (TcTIM). Herein, we studied the structural modifications of this chemotype to analyze the influence of volume, lipophilicity and electronic properties in the anti-T. cruzi activity. Their selectivity to parasites vs. mammalian cells was also examined. To get insights into a possible mechanism of action, the inhibition of the enzymatic activity of TcTIM and cruzipain, using the isolated enzymes, and the inhibition of membrane sterol biosynthesis and excreted metabolites, using the whole parasite, were achieved. We found that this structural framework is interesting for the generation of innovative drugs for the treatment of Chagas disease.

Comparison of the essential oil content and composition of the spathe, buds and pollen of Phoenix dactylifera.

Several medicinal applications have been reported for different components of date palm. The inflorescence of the male date tree is composed of spathe that surrounds many buds containing pollen. In this study, a comparison between the content and composition of the essential oils obtained from these three components of an inflorescence was made. After obtaining each oil using hydro-distillation method, the oil yield was measured as the weight ratio of the oil to the distilled sample (w/w %) and the chemical composition of the oil was determined by gas chromatography-mass spectrometry analysis. It was observed that the pollen possessed the most oil content (1.47%) composed of 68.04% oleic acid, whereas the content of this fatty acid in the spathe and bud oils was found to be less than 0.05% and 5.65%, respectively. Spathe oil was dominated by 3,4-dimethoxytoluene (52.90%) while the main constituent of the bud oil was trans-caryophyllene (44.20%).

Effect of smear layer deproteinizing on resin-dentine interface with self-etch adhesive.

OBJECTIVES: This study aimed to investigate deproteinizing effect of sodium-hypochlorite (NaOCl) and mild acidic hypochlorous-acid (HOCl) pretreatment on smear layer-covered dentine and to evaluate their effects on morphological characteristics of resin-dentine interface with self-etch adhesive. METHODS: Human coronal-dentine discs with standardized smear layer were pretreated with 6% NaOCl or 50ppm HOCl for 15s or 30s. Their deproteinizing effects at the treated smear layer-covered dentine surfaces were determined by the measurement of amide:phosphate ratio using ATR-FTIR analysis. In addition, using TEM, micromorphological alterations of hybridized complex and nanoleakage expression were evaluated at the interface of a self-etch adhesive (Clearfil SE Bond) to the pretreated dentine surface with or without subsequent application of a reducing agent (p-Toluenesulfinic acid salt; Accel(®)). RESULTS: Both pretreatments of NaOCl and HOCl significantly reduced the amide:phosphate ratio as compared with the no-pretreated group (p<0.05), coincident with the elimination of the hybridized smear layer on their bonded interfaces. Nanoleakage within the hybrid layer was found in the no-pretreated and NaOCl-pretreated groups, whereas the subsequent reducing agent application changed the reticular nanoleakage to spotted type. HOCl-pretreated groups showed less nanoleakage expression in a spotted pattern, regardless of reducing agent application. CONCLUSIONS: NaOCl and HOCl solutions could remove the organic component on the smear layer-covered dentine, which could eliminate the hybridized smear layer created by self-etch adhesive, leading to the reduction of nanoleakage expression within hybrid layer. CLINICAL SIGNIFICANCE: Smear layer deproteinizing could modify dentine surface, giving an appropriate substrate for bonding to self-etch adhesive system.

Oridonin induces apoptosis in SW1990 pancreatic cancer cells via p53- and caspase-dependent induction of p38 MAPK.

Oridonin, an active component isolated from Rabdosia rubescens, has been reported to exhibit antitumor effects. In the present study, we evaluated the antitumor activity and the mechanisms of action of oridonin in pancreatic cancer. Oridonin treatment significantly induced apoptotic cell death in SW1990 pancreatic cancer cells in a dose-dependent manner. Additionally, cell apoptosis was markedly inhibited by PFT α (pifithrin α), a p53-specific inhibitor, which was applied to evaluate the function of p53, showing that p53 was responsible for the cytotoxity of oridonin. Moreover, oridonin increased the expression of p-p53 with a concomitant increase in p21 in the SW1990 cells. Following treatment with mitogen-activated protein kinase (MAPK) inhibitors, PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and SB203580 (p38 inhibitor), the cytotoxity of oridonin was not influenced by JNK (SP600125) and ERK (PD98059), but these effects were opposite to the cytotoxity of oridonin observed with SP203580 treatment. These findings confirmed that orodonin-induced apoptosis was p38-dependent, but JNK- and ERK-independent. Furthermore, the activation of the p38 kinase promoted the activation of p53 and its downstream target p21, and further caused caspase-9 and -3 activation, as demonstrated by evidence showing that the p38 inhibitor SB203580 not only blocked the phosphorylation of p38 but also reduced the activation of p53, p21 and caspase-9 and -3. Collectively, these results suggest that p53-dependent and caspase-dependent induction of p38 MAPK directly participates in apoptosis induced by oridonin.

The anti-cancer activities of Vernonia amygdalina extract in human breast cancer cell lines are mediated through caspase-dependent and p53-independent pathways.

Breast cancer is currently the leading cause of cancer-related deaths among women globally. Notably, medicinal plant extracts may be a potential source for treatments of breast cancer. Vernonia amygdalina (VA) is a woody shrub reported to have not only diverse therapeutic effects but also anti-cancer properties. However, current research about the mechanisms of the anti-cancer potential of VA has been limited. This study aimed to investigate the mechanisms of action of VA that underlie its anti-cancer effects in human breast cancer cell lines (MCF-7 and MDA-MB-231 cells). Results from MTT assay revealed that VA inhibits the proliferation of MCF-7 and MDA-MB-231, in a time- and dose-dependent manner. The underlying mechanism of this growth inhibition involved the stimulation of cell-type specific G1/S phase cell cycle arrest in only MCF-7 cells, and not in MDA-MB-231 cells. While the growth arrest was associated with increased levels of p53 and p21, and a concomitant decrease in the levels of cyclin D1 and cyclin E, it was shown that VA causes cell cycle arrest through a p53-independent pathway as tested by the wild type p53 inhibitor, pifithrin-α. Furthermore, this study revealed that VA induces apoptosis in the two cell lines, as indicated by the increase in Annexin V-positive cells and sub-G1 population, and that this VA-induced apoptosis occurred through both extrinsic and intrinsic apoptotic pathways. The apoptosis in MCF-7 cells was also likely to be caspase-dependent and not p53 transcriptional-dependent. Given that approximately 70% of diagnosed breast cancers express ER-α, a crucial finding was that VA inhibits the expression of ER-α and its downstream player, Akt, highlighting the potential clinical significance of VA. Moreover, VA exhibits synergism when combined with doxorubicin, suggesting that it can complement current chemotherapy. Overall, this study demonstrates the potential applications of VA as an anti-cancer drug for breast cancer treatment.

The novel p53-dependent metastatic and apoptotic pathway induced by vitexin in human oral cancer OC2 cells.

Vitexin, identified as apigenin-8-C-D-glucopyranoside, a natural flavonoid compound found in certain herbs such as hawthorn herb, has been reported to exhibit anti-oxidative, anti-inflammatory, anti-metastatic and antitumor properties. The aim of this study was to investigate the possible existence of p53-dependent pathway underlying vitexin-induced metastasis and apoptosis in human oral cancer cells, OC2 cells. Vitexin decreased cell viability significantly. Meanwhile, the expression of tumor suppressor p53 and a small group of its downstream genes, p21(WAF1) and Bax, were upregulated. The p53 inhibitor pifithrin-α (PFT-α) knockdown of the signaling of p53 led vitexin to lose its antitumor effect and inhibited the expression of p53 downstream genes, p2(WAF1) and Bax. Vitexin had anti-metastatic potential accompanied with increasing plasminogen activator inhibitor 1 (PAI-1) accumulation and decreasing matrix metalloproteinase-2 expression. Our present study evidenced, by using p53 inhibitor PFT-α, PAI-1 and peroxisome proliferator-activated receptor γ are downstream genes of p53 in vitexin-induced signaling. MAPK inhibitor PD98059 decreased the OC2 cells viability significantly. The expression of p53 and its downstream genes p21(WAF1) and Bax were enhanced by blocking the activation of p42/p44 MAPK in response to treatment with vitexin. Moreover, p42/p44 MAPK played a negative role in p53-dependent metastasis and apoptosis. We give evidence for the first time that the novel p53-dependent metastatic and apoptotic pathway induced by vitexin in human oral cancer OC2 cells.