mRNA Expression Levels Of P53 Research Articles

Inhibitory activity of flaxseed oil against CdCl2 induced liver and kidney damage: Histopathology, genotoxicity, and gene expression study

The present work evaluated the effect of flaxseed oil (FO) against toxicity induced by cadmium chloride (CdCl2) in the mouse liver and kidney. Male Swiss albino mice were treated with CdCl2 (4.5 mg/kg, intraperitoneally) with or without FO at three concentrations (4, 8, 12 mL/kg, orally) for two consecutive weeks. To analyze the effects of FO, we used the following techniques: (1) histopathological examination; (2) comet assay; (3) RT-PCR gene expression analysis of tumor necrosis factor (TNF-α) and tumor suppressor protein (p53); and (4) immunohistochemical analysis of caspase-9 protein expression. The gas chromatography-mass spectrometry results showed that FO had a high content of unsaturated fatty acids including, oleic acid, linolenic acid, and linoleic acid. Oral supplementation with FO (12 mL/kg) resulted in a normal histological appearance without alteration in the DNA integrity and gene expression of TNF-α, p53, and caspase-9 in liver and kidney tissues. As expected, CdCl2 remarkably induced loss of histological integrity, increased DNA comet formation, increased TNF-α and p53 mRNA expression levels and increased the immunoreactivity of caspase-9 expression. When FO was given before administration of CdCl2, these histopathological defects were reversed; necrosis, degeneration, inflammatory cell infiltration, hemorrhage, Kupffer cells, and pyknotic cells were all reduced. These histological improvements induced by FO were accompanied by reduced DNA breakage, downregulated mRNA expression of TNF-α and p53, and downregulated immunohistochemical expression of caspase-9 protein. In conclusion, FO and its constituents may act as signaling molecules and modify the expression of genes involved in proinflammatory cytokine production (TNF-α), cell cycle arrest (p53), and apoptosis (caspase-9), thereby improving biological activities and health.

PSVIII-1 Nutrient starvation induces autophagy and alters intestinal barrier function in IPEC-J2 cells

Abstract Autophagy is a cellular process of controlled degradation of damaged organelles and cytoplasmic macromolecules during stress for maintaining homeostasis. Intestinal epithelial cells are barriers against microorganisms, toxins and food antigens. Whether autophagy plays a role in regulating intestinal barrier function is unclear. The objective of this study was to characterize the role of autophagy in starvation-induced alteration of tight junction protein abundance and function in IPEC-J2 cells. Cells were nutrient starved in Krebs-Ringer bicarbonate (KRB) buffer for 0, 0.5, 1, 2, 3, 6, 9 and 12 h. Expression of genes implicated in autophagy regulation (AMPK, MDM2, p53 and DRAM) was determined by RT-PCR. The ratio of protein abundance of microtubule-associated protein light chain 3 (LC3-II/LC3-I) and p62, positive and negative markers of autophagy induction, respectively, was determined by western blotting. Compared with control group (0 h), the relative mRNA expression level of AMPK, MDM2, p53 and DRAM significantly decreased by an average of 52.5% (P < 0.01). Relative to 0 h, the mRNA expression of claudin 1 and claudin 4 significantly increased (108.0%) up to 6 h of starvation and then decreased (31.4%) thereafter (P < 0.01). On the contrary, abundance of claudin 1 and claudin 4 protein was downregulated (49.5%) up to 3 h of starvation and then increased (82.6%) thereafter (P < 0.01). Protein expression of claudin 3 was reduced (P < 0.01) with duration of starvation. There was no change in the protein level of occludin and ZO-1. The ratio of LC3-II/LC3-I significantly increased with duration starvation (P < 0.01), whereas p62 and phospho-AMPK levels decreased up to 6 h of starvation and then increased thereafter (P < 0.01). In summary, autophagy may be implicated in the regulation of tight junction integrity during nutrient starvation in IPEC-J2 cells.

Expression of miR-34a in cataract rats and its related mechanism.

Expression of miR-34a in cataract rats and its related mechanism were investigated. A total of 30 SD rats were selected and divided into three groups: group A: 2-month-old lucent lens, group B: 18-month-old lucent lens, and group C: 18-month-old naturally occurring cataract lens. The lens was taken and measured by LOC III to determine the degree of lens opacity of the three groups of rats. qPCR was used to detect expression of miR-34a and mRNA of SIRT1 and P53. Western blotting was used to detect the protein expression of SIRT1 and P53. Cell apoptosis was detected by flow cytometry. The lens of rats in group C was more turbid than that of groups A and B (P<0.05). The expression levels of miR-34a and P53 mRNA in the rats lens of group C were significantly higher than those in groups A and B, and the expression of SIRT1 mRNA was significantly lower than that of groups A and group B (P<0.05). Expression of miR-34a in group A was significantly higher than that in group B, the mRNA expression of SIRT1 was significantly lower than that in the lucent lens of 18-month-old rats (P<0.05). The expression of SIRT1 protein in group C was significantly lower than that in groups A and group B, while the expression level of P53 protein in group C was significantly higher than that of groups A and B. The expression of SIRT1 protein in group B was significantly higher than that in group A (P<0.05). The apoptosis rate of group C was higher than that of groups A and group B (P<0.05). In conclusion, the upregulation of expression level of miR-34a is related to cataract occurrence in rats, which may be caused by regulation of SIRT1 protein.

Effects of DHA-enriched fish oil on gene expression levels of p53 and NF-κB and PPAR-γ activity in PBMCs of patients with T2DM: A randomized, double-blind, clinical trial

Background and AimsOmega-3 polyunsaturated fatty acids (PUFAs) are natural peroxisome proliferator-activated receptor gamma (PPAR-γ) ligands. Activated PPAR-γ protects the cardiovascular system against atherosclerotic lesion formation and exerts its anti-inflammatory role by suppressing cytokines induced by nuclear factor kappa-B (NF-κB) in endothelial cells (ECs), and it is hypothesized that apoptosis and cell cycle arrest induced by PPAR-γ ligands may be mediated by the p53-dependent pathway. The aim of our study was to investigate the effects of docosahexaenoic acid (DHA)-enriched fish oil supplement on PPAR-γ activity and mRNA expression levels of p53 and NF-κB. Methods and ResultsFifty patients with type 2 diabetes mellitus (T2DM) aged 30–70 years were randomly assigned to receive either 2400 mg/d DHA-rich fish oil or placebo for 8 weeks. Metabolic parameters were assessed at baseline and at the end of the intervention. PPAR-γ activity in the peripheral blood mononuclear cells (PBMCs) was measured using ELISA-based PPAR-γ Transcription Factor Assay Kit, and the gene expression levels of p53 and NF-κB were assessed using real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). On the basis of our finding, 8 weeks of treatment with DHA-rich fish oil increased PPAR-γ activity in PBMCs of subjects with T2DM (p < 0.01) compared to that in placebo (p = 0.4). Between-group comparisons of mean PPAR-γ activity changes showed significant differences (p = 0.03), whereas mRNA expression levels of the p53 and NF-κB genes did not show significant differences between studied groups (p = 0.2 and p = 0.5, respectively). ConclusionOur findings indicated that short-term DHA-rich fish oil supplementation may modulate PPAR-γ activity in PBMCs.

The inhibitory effects of cisplatin-radiation combination treatment on malignant osteosarcoma MG-63 cells and BRCA1-p53 pathways are more efficient than single treatments.

The poor prognosis of patients with osteosarcoma remains a persistent problem, in particular for patients with unresectable tumors or metastasis. Therefore, combination of radiotherapy and chemotherapy has been considered for patients with metastasis or recurrence, patients unsuitable for surgery and patients refusing surgery. The present study aimed to investigate the effect of the combined treatment with cisplatin and radiation therapy on the biological characteristics of the osteosarcoma cell line MG-63 and the breast cancer 1 (BRCA1)-associated signaling pathways. Cell proliferation was determined using Cell Counting kit-8 assay, and cell apoptosis and cell cycle were assessed by flow cytometry. Cell migration was examined by Transwell assay. The mRNA and protein expression levels of candidate genes, including BRCA1 and p53, were determined by reverse transcription-quantitative PCR and western blotting, respectively. The results demonstrated that combined treatment with radiation and cisplatin significantly inhibited MG-63 cell proliferation compared with radiation or cisplatin treatment alone. Furthermore, radiation, cisplatin or the combined treatment with radiation and cisplatin increased the apoptosis rate of MG-63 cells, which resulted in G2 phase arrest, and significantly decreased the migratory capacity of MG-63 cells. In addition, the apoptosis rate of MG-63 cells following combined radiation and cisplatin treatment was higher compared with the cisplatin group, but lower compared with the radiation group. Furthermore, combined treatment with radiation and cisplatin decreased the mRNA and protein expression levels of BRCA1 and p53. Additionally, combined treatment with radiation and cisplatin had a more potent inhibitory effect on p53 expression than on BRCA1 expression. In addition, combination of radiation and cisplatin had a higher inhibitory effect on Bax protein level and a higher inductive effect on Bcl-2 protein level compared with treatments with radiation and cisplatin alone. The results demonstrated that combined treatment of radiation and cisplatin exhibited superior therapeutic effects on osteosarcoma MG-63 cells compared with radiation or cisplatin treatment alone, which may be mediated by the BRCA1-p53 signaling pathway.

Coenzyme Q10 mitigates ionizing radiation-induced testicular damage in rats through inhibition of oxidative stress and mitochondria-mediated apoptotic cell death

Radiotherapy is a common treatment modality for cancer patients; however, its use is limited by decreasing the probability of fertility in male cancer survivors. Therefore, this study aimed to define the capability of coenzyme Q10 (CoQ10), a potent stimulator of mitochondrial function, in attenuating ionizing radiation (IR)-induced spermatogenesis impairments. Male Sprague Dawley rats were exposed to a single dose of ϒ-rays (10 Gy) and/or treated with CoQ10 (10 mg/kg, orally, for 2 consecutive weeks). IR mediated irregular seminiferous tubules, which were emerged with typical morphological characteristics of apoptosis, and nuclear condensation, while CoQ10 significantly preserved the testicular structure and maintained spermatogenesis, which was displayed by higher levels of serum estradiol and testosterone. CoQ10 remarkably augmented sperm count, motility, and viability while diminished the rate of sperm-defects relatively to their counterparts after IR exposure. CoQ10 modulations in reproductive parameters were underpinned by attenuating IR-induced oxidative stress as evidenced by decreasing lipid peroxidation and increasing the antioxidant enzymes glutathione peroxidase and glutathione-s-transferase activities, and glutathione level. Supporting the involvement of CoQ10 in the anti-apoptotic response, the reduced mRNA expression levels of p53, Puma, and Bax accompanied by the increased Bcl-2 mRNA expression were observed. Subsequently, CoQ10 ameliorated the mitochondria dependent apoptotic pathway through diminishing Bax/Bcl-2 ratio, caspase-3 protein expression, and DNA fragmentation in testes of irradiated rats. Taken together, our findings showed that CoQ10 conserved against IR-induced steroidogenesis disruption through subsiding mitochondria-mediated oxidative stress injury in germinal cells.

Actinomycin V Suppresses Human Non-Small-Cell Lung Carcinoma A549 Cells by Inducing G2/M Phase Arrest and Apoptosis via the p53-Dependent Pathway.

Actinomycin V, extracted and separated from marine-derived actinomycete Streptomyces sp., as the superior potential replacement of actinomycin D (which showed defect for its hepatotoxicity) has revealed an ideal effect in the suppression of migration and invasion in human breast cancer cells as referred to in our previous study. In this study, the involvement of p53 in the cell cycle arrest and pro-apoptotic action of actinomycin V was investigated in human non-small-cell lung carcinoma A549 cells. Results from the 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide assay showed that cytotoxic activity of actinomycin V on A549 cells (with wild-type p53) was stronger than the NCI-H1299 cells (p53-deficient). Actinomycin V upregulated both of the protein and mRNA expression levels of p53, p21Waf1/Cip1 and Bax in A549 cells. For this situation, actinomycin V decreased the M-phase related proteins (Cdc2, Cdc25A and Cyclin B1) expression, arrested cells in G2/M phase and subsequently triggered apoptosis by mediating the Bcl-2 family proteins’ expression (Bax and Bcl-2). Furthermore, the effects of cell cycle arrest and apoptosis in A549 cells which were induced by actinomycin V could be reversed by the pifithrin-α, a specific inhibitor of p53 transcriptional activity. Collectively, our results suggest that actinomycin V causes up-regulation of p53 by which the growth of A549 cells is suppressed for cell cycle arrest and apoptosis.

Correlation of APOBEC3G Polymorphism with Human Papillomavirus (HPV) Persistent Infection and Progression of Cervical Lesions.

BackgroundWe studied the effect of APOBEC3G on persistent human papillomavirus (HPV) infection and the correlation between APOBEC3G polymorphism and HPV persistent infection and cervical disease progression in Uygur women in China.Material/MethodsFrom January 2015 to December 2017, we enrolled 529 Uygur ethnic group patients with HPV infection. SIHA cells were transfected with APOBEC3G. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were used to detect mRNA and protein expression levels of APOBEC3G and HPV E6 and p53. Exon 3 of APOBEC3G was sequenced by first-generation sequencing.ResultsThe mRNA and protein expression levels of APOBEC3G in the cervical cancer group were significantly higher than in the cervical intraepithelial neoplasia (CIN) group (p<0.05). The mRNA and protein expression levels of APOBEC3G in the CIN group were significantly higher than in the non-cervical lesions group (p<0.05). The mRNA and protein expression levels of HPV E6 in SIHA cells transfected with APOBEC3G were significantly lower than in the control group and the no-load group (p<0.05), and the mRNA and protein expression levels of p53 were significantly higher than in the control group and the no-load group (p<0.05). There was a polymorphic locus rs5757465 on exon 3 of APOBEC3G in Uygur women, and this rare CC type was a risk factor for cervical lesions and cervical cancer (OR=3.714, 95%CI: 1.916–7.202, p<0.05).ConclusionsAPOBEC3G is involved in continuous HPV infection, cervical prelesions, and the development of cervical cancer, and the rare genotype (CC) of APOBEC3G may be one of the factors causing cervical lesions in Uygur women who have HPV infection.

Research update for articles published in EJCI in 2017.

Our findings showed that p53 mRNA expression levels are upregulated in epicardial adipose tissue (EAT) from patients with heart failure (HF). This upregulation was also found in myocardium [2]. Our group have described its upregulation in EAT by sympathetic system. This article is protected by copyright. All rights reserved.

Matrine derivate MASM protects murine MC3T3-E1 osteoblastic cells against dexamethasone-induced apoptosis via the regulation of USP14/p53

Matrine derivative MASM (M19) is a quinolizine alkaloid with diverse pharmacological effects including preventing postmenopausal osteoporosis. In the current study, we observed that pretreatment with M19 inhibited cell apoptosis of murine osteoblastic MC3T3-E1 and primary osteoblasts induced by 1 μM dexamethasone in a dose-dependent manner. Our study also showed that pretreatment with M19 significantly prevented the upregulation of p53 that is caused by dexamethasone but had no effect on the p53 mRNA expression levels. Further immunoprecipitation (IP) experiments showed that M19 treatment increases the ubiquitination of p53 in dexamethasone-treated MC3T3-E1 cells. The expression of USP14, a deubiquitinating enzyme, increased with dexamethasone and decreased with M19 pretreatment. Co-IP experiments demonstrated the interaction between USP14 and p53, and the induced effect of a USP14 inhibitor (IU1) on p53 ubiquitination, which indicated that USP14 is a potential deubiquitinase for p53. Furthermore, pretreatment with IU1 or a p53 inhibitor (PFT-α) partially blocked dexamethasone-induced apoptosis of MC3T3-E1 cells. The overexpression of USP14 or p53 reversed the antiapoptotic effect of M19 in dexamethasone-treated MC3T3-E1 cells. In addition, PFT-α treatment remarkably blocked the effects of USP14 overexpression. In summary, our findings suggest that M19 exerts protective effects on dexamethasone-treated MC3T3-E1 cells by regulating USP14/p53.

An In Vitro Study on the Cytotoxicity and Genotoxicity of Silver Sulfide Quantum Dots Coated with Meso-2,3-dimercaptosuccinic Acid.

Silver sulfide (Ag2S) quantum dots (QDs) are highly promising nanomaterials in bioimaging systems due to their high activities for both imaging and drug/gene delivery. There is insufficient research on the toxicity of Ag2S QDs coated with meso-2,3-dimercaptosuccinic acid (DMSA). In this study, we aimed to determine the cytotoxicity of Ag2S QDs coated with DMSA in Chinese hamster lung fibroblast (V79) cells over a wide range of concentrations (5-2000 μg/mL). Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake (NRU) assays. The genotoxic and apoptotic effects of DMSA/Ag2S QDs were also assessed by comet assay and real-time polymerase chain reaction technique, respectively. Cell viability was 54.0±4.8% and 65.7±4.1% at the highest dose (2000 μg/mL) of Ag2S QDs using the MTT and NRU assays, respectively. Although cell viability decreased above 400 μg/mL (MTT assay) and 800 μg/mL (NRU assay), DNA damage was not induced by DMSA/Ag2S QDs at the studied concentrations. The mRNA expression levels of p53, caspase-3, caspase-9, Bax, Bcl-2, and survivin genes were altered in the cells exposed to 500 and 1000 μg/mL DMSA/Ag2S QDs. The cytotoxic effects of DMSA/Ag2S QDs may occur at high doses through the apoptotic pathways. However, DMSA/Ag2S QDs appear to be biocompatible at low doses, making them well suited for cell labeling applications.

The effect of resveratrol supplementation on the expression levels of factors associated with cellular senescence and sCD163/sTWEAK ratio in patients with type 2 diabetes mellitus: study protocol for a double-blind controlled randomised clinical trial

IntroductionOver the past decades, the number of people with type 2 diabetes (T2D) has increased globally. One of the major complications in these patients is cardiovascular disease; it seems that...

Difference in pathogenicity of 2 strains of avian leukosis virus subgroup J in broiler chicken

Avian leukosis virus subgroup J has been found to infect many types of chickens with various genetic backgrounds. The ALV-J strain NX0101, which was isolated from broiler breeders in 2001, mainly induces the formation of myeloid cell tumors. However, strain HN10PY01, which was recently isolated from laying hens, mainly induces the formation of myeloid cell tumors and hemangioma. In order to determine the difference in pathogenicity of the 2 strains in broiler chickens, 2 groups of chicken embryos were infected with NA0101 and HN10PY01 separately. A comparison was made of the mortality, oncogenicity, body weights, indexes for immune organs, levels of ALV group-specific antigen p27, and mRNA expression levels of the tumor-related gene, p53, in ALV-J-infected birds and immune organs of theses chickens in response to Newcastle Disease Virus (NDV) and avian influenza virus subtype H9 (AIV-H9) vaccination. The results indicated that strain NX0101 was highly pathogenic in broiler chickens and led to a 30% mortality rate and 45% oncogenicity, compared with the HN10PY01-infected birds. Weight of chickens was also significantly lower after 15 wk (P < 0.05). In addition, the mRNA expression levels of tumor-related p53 in medulla, liver, and lung in broilers infected with strain NX0101 were significantly higher than those infected with strain HN10PY01 (P < 0.05). These results indicated that strain NX0101 had a higher replication ability in broiler chickens. The findings of this study will contribute to further elucidating the mechanisms underlying host susceptibility and tumor classification in ALV-J-infected chickens.

P53 mediates PEDF‑induced autophagy in human umbilical vein endothelial cells through sestrin2 signaling

Autophagy is a conserved catabolic process by which cytoplasmic components are delivered into lysosomes for degradation. Pigment epithelium-derived factor (PEDF) has been reported to be associated with autophagy and can induce p53 expression; however, the mechanism relating PEDF with autophagy in endothelial cells remains poorly understood. The present study aimed to investigate the association between the PEDF-p53-sestrin pathway and autophagy in human umbilical vein endothelial cells (HUVECs). PEDF-induced autophagy was examined by fluorescence microscopy and western blot analysis. p53 small interfering (si)RNA and sestrin2 siRNA were constructed and transfected into HUVECs prior to PEDF treatment. The protein expression levels of microtubule-associated protein light chain 3 (LC3) I, LC3 II and p62 were evaluated by western blot analysis, and the mRNA expression levels of p53 and sestrin2 were determined using reverse transcription-quantitative polymerase chain reaction analysis. The regulation of mechanistic target of rapamycin (mTOR) was reflected by p70S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) protein expression levels, as determined by western blot analysis. PEDF could induce HUVEC autophagy by sequentially inducing p53 and sestrin2 expression, as observed by fluorescence microscopy and western blot analysis. Conversely, the induction of sestrin2 by PEDF was eliminated by p53 siRNA. In addition, p53 siRNA and sestrin2 siRNA could attenuate PEDF-induced HUVEC autophagy. Inhibition of mTOR may be the mechanism responsible for PEDF-induced autophagy; as p70S6K and 4E-BP1 phosphorylation levels were significantly upregulated in p53 siRNA-treated and sestrin2 siRNA-treated groups. The findings of the present study indicated that PEDF may trigger autophagy in HUVECs by inducing p53 and sestrin2 expression, and inhibiting mTOR expression; these findings may contribute to the improved understanding of diseases, including cancer and atherosclerosis.

Regulation of p53 in the red-eared slider (Trachemys scripta elegans) in response to salinity stress

The freshwater red-eared slider (Trachemys scripta elegans) is found not only in freshwater but also in coastal saline habitats. Hyperosmotic salinity can induce cell damage. p53, regarded as the guardian of the genome, is very important and versatile in response to the change of environment. In this study, the role of p53 in T. s. elegans under environmental salinity change will be explored. The results indicated that amino acid sequence of p53 showed high similarity to p53 of other species. In addition, the expression of p53 showed differences in various tissues under normal condition. Under salinity stress, the mRNA levels of p53 in the liver increased significantly at 48 h with 15‰ group (15 practical salinity units-exposed group). In the heart, p53 mRNA levels increased at 6 h in 5‰ (5 practical salinity units) and 15‰ groups. Furthermore, the changes of p21 mRNA expression levels in liver and heart were similar to p53, while cyclin D1, cyclin-dependent kinase4 (CDK4) and cyclin-dependent kinase6 (CDK6) showed opposite changes to p53. Moreover, Bax and caspase 3 mRNA expression levels were similar to p53, respectively, while Bcl-2 showed opposite changes. The positive cells of apoptosis were found in the liver of 15‰ at 48 h and 30 d of chronic stress. Taken together, these results indicated that the T. s. elegans may protect itself by regulating cell cycle progression and apoptosis of damaged cells under salinity stress, which played an important role for T. s. elegans in salinity adaptation.

Correlation of p16 and nm23-H1 expression levels with incidence and prognosis of soft tissue sarcoma.

The expression levels of p16 and nm23-H1 genes in soft tissue sarcoma (STS) were evaluated to investigate correlation of the expression levels with the incidence and prognosis of STS. Tumor tissues and para-carcinoma normal tissues were collected from 64 STS patients. The messenger ribonucleic acid (mRNA) expression levels of p16 and nm23-H1 in the tissues were detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the protein expression levels of p16 and nm23-H1 in tissues were detected using immunohistochemistry. Spearman's correlation analysis was used for the correlation between expression levels of p16 and nm23-H1 in STS tissues and the correlation between p16 and nm23-H1 mRNA and protein expression. Moreover, the correlation of p16 and nm23-H1 expression levels in tumor tissues with pathological parameters and prognosis of STS patients were analyzed combined with clinical data. Results of RT-qPCR showed that mRNA expression levels of p16 and nm23-H1 in tumor tissues of STS patients were significantly lower than those in para-carcinoma normal tissues (P<0.01). Results of immunohistochemistry showed that the positive expression rates of p16 and nm23-H1 in tumor tissues of STS patients (43.75 and 39.06% respectively) were significantly lower than those in para-carcinoma normal tissues (85.93 and 89.06% respectively). The expression of p16 and nm23-H1 mRNA was positively correlated with protein expression levels. There was a positive correlation between the expression levels of p16 and nm23-H1 in tumor tissues of STS patients. The negative expression of p16 in tumor tissues of STS patients correlated with tumor size, tumor metastasis and clinical staging, and the negative expression of nm23-H1 correlated with tumor metastasis and clinical staging. The overall 5-year survival rate of patients was 54.68%, and the prognosis of patients with positive expression levels of p16 and nm23-H1 was better. Univariate survival analyses revealed that p16 and nm23-H1 were influencing factors of the overall survival rate of STS patients. p16 and nm23-H1 expression in STS is low, and their expression levels are closely related to the pathological parameters and prognosis of STS patients, so they can serve as reference indexes for prognosis estimation of STS.

Identification of a novel regulatory mechanism involved in inhibition of transcription of suvivin mRNA in breast cancer cells via p21cip–mediated regulation

Purpose: To evaluate the effect of p21Cip1 on survivin transcription levels in breast carcinoma, and to investigate the potential mechanisms.Methods: Epirubicin, a p21Cip1 activator, was used to treat MCF7 cells. Under the action of normal biological functions of p53, pEGFP-C2-p21 was transfected into MCF7 cells by lipofectamine and positive clones were screened out with G418. The expression levels of p21cip1, p53 and survivin mRNA were quantitated by real-time fluorescent polymerase chain reaction (RQ-PCR). MTT assay was utilized to measure cellular viability and proliferation after transfection. Flow cytometry was employed to determine the cell cycle. Hoechst 33342 staining was carried out to assess cell apoptosis. Lastly, several transcription factor sites located at the promoter region of survivin gene, such as, sp1 site, E2F site and p300/CBP, were measured by p21 overexpression using RT-PCR.Results: Following epirubicin treatment, within 24 h, the expression levels of endogenous p21cip1 and p53 were up-regulated, whereas that of survivin was down-regulated. After transfection treatment, p21 inhibited the proliferation of MCF7 cells on days 3 and 4, and MCF7 cells overexpressed p21 mRNA, whereas the level of survivin mRNA in MCF7-p21 groups was markedly down-regulated relative to control group, but overexpression of p21 was not sufficient to cause changes in p53 gene expression. The overexpressed p21 resulted in G1/G0 phase arrest based on cell cycle analysis, but apoptosis was not induced. In addition, co-transcription factors E2F-1, sp1 and p300/CBP mRNA levels decreased significantly compared with normal p21 expression groups.Conclusion: P21cip1 may down-regulate the expression of survivin gene partially by inhibiting the expression level of HAT.Keywords: Cyclin-dependent kinase inhibitor 1, Phosphoprotein p53, Survivin, Breast carcinoma, G1/G0 phase arrest, Epirubicin, Lipofectamine

Expression of P53, BAX, and BCL-2 in human malignant melanoma and squamous cell carcinoma cells after tea tree oil treatment in vitro.

Tea tree oil (TTO) is an essential oil obtained by steam distillation from the leaves of Melaleuca alternifolia (Myrtaceae). This oil has traditionally been used for the treatment of various skin infections. The present study aimed to investigate the cytotoxic effects of TTO against two representative types of human skin cancer, namely malignant melanoma (A-375) and squamous cell carcinoma (HEp-2).To outline the basic molecular mechanism involved in apoptosis induction in A-375 and HEp-2 cell lines, Annexin V/PI staining for apoptosis detection, cell cycle analysis were monitored using flow cytometry and mRNA expression levels of the apoptosis-regulatory genes P53, BAX, and BCL-2 were determined by real-time PCR and western blot after treatment with TTO. Results showed that TTO exhibited a strong cytotoxicity towards A-375 and HEp-2 cell lines, with IC50 values of 0.038% (v/v) and 0.024% (v/v) respectively. This cytotoxicity resulted from TTO induced apoptosis in both A-375 and HEp-2 cell lines as evidenced by morphological features of apoptosis and Annexin V/PI staining results in addition to the activation of caspase-3/7 and -9, upregulation of pro-apoptotic genes (P53 and BAX) and downregulation of the anti-apoptotic gene BCL-2. Additionally, cell cycle analysis showed that TTO caused cell cycle arrest mainly at G2/M phase. Taken together, the results of this study reveal that TTO is an effective apoptosis inducer in A-375 and HEp-2 cancer cell lines, indicating that it could be a promising chemopreventive candidate to be used in topical formulations against melanoma and squamous cell cancers; however, further in vivo studies may be warranted.

Influence of microgravity-induced intervertebral disc degeneration of rats on expression levels of p53/p16 and proinflammatory factors.

Association of expression levels of tumor suppressor proteins p53 and p16 and inflammatory factors in simulated weightlessness with the degree of lumbar disc degeneration of rats was investigated. Magnetic resonance imaging (MRI) examination was performed for rats in control group and experimental group, and the intervertebral disc of rats in both groups was detected and analyzed using hematoxylin and eosin (H&E) histopathological staining. The mRNA expression levels of proinflammatory factors, interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were detected, and p53 and p16 mRNA and protein expression levels were detected. MRI results showed significant intervertebral disc injury in experimental group. Results of H&E staining revealed that the intervertebral disc injury in experimental group was more serious with obvious signs of degeneration than that in control group. The mRNA expression levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in rats in experimental group were significantly increased compared with those in control group, indicating that the degree of lumbar disc degeneration of rats in simulated weightlessness is closely related to the inflammatory factors. RT-PCR and western blotting proved that both p53 and p16 mRNA and protein expression levels in experimental group were obviously increased. Results of t-test manifested that there were statistically significant differences in p53 and p16 expression levels between control group and experimental group (P<0.01). The abnormal expression levels of p53 and p16 genes have close association with the degree of lumbar disc degeneration of rats in simulated weightlessness, and the lumbar disc degeneration is also closely related to the increased expression levels of inflammatory factors.

Antitumor and anti-inflammatory effects of oligosaccharides from Cistanche deserticola extract on spinal cord injury

In the present study, the pharmacological effects of oligosaccharides from Cistanche deserticola extract on inflammation, oxidative stress, and apoptosis in male albino rats with spinal cord injury were investigated. Lipid peroxidation, GSH, catalase, superoxide dismutase, acetylcholine esterase, GPx, ROS, and nitric acid were significantly altered in the rats with spinal cord injury. The mRNA expression levels of IL-6, TNF-α, cyclooxygenase-2, iNOS, p53, caspase-3, bax, and pro-NGF were reduced by >20% following extract supplementation. Protein expression levels of caspase-3 and pro-NGF were also reduced by >20%. The number of p53 positive cells was 1, 79, 54, 33, and 19 in groups GI–GV, respectively, and the corresponding numbers of caspase-3 positive cells were 2, 87, 51, 23, and 14. Based on the present results, the use of oligosaccharides from Cistanche deserticola extract was effective against inflammation, oxidative stress, and apoptosis in spinal cord injury male albino rats.