Search

Purpose: Pterygium is a common lesion of the ocular surface, and its etiology and pathogenesis are still uncertain. This study aimed to investigate the role of apoptosis and proliferation in pterygium formation and recurrence., Materials and Methods: In this study, p53, Bcl-2, and Ki-67 expression levels were evaluated in primary pterygium ( n  = 35) and recurrent pterygium ( n  = 32) tissue samples and compared with normal conjunctiva ( n  = 30) tissue samples. In addition, recurrent pterygiums were divided into three groups based on recurrence time, and their p53, Bcl-2, and Ki-67 expression levels were compared., Results: The results show that p53, Bcl-2, and Ki-67 expression levels were significantly higher in the pterygium tissue samples as compared to the control group ( p  < 0.001, p  < 0.001, and p  < 0.001, respectively). When primary and recurrent pterygium tissues were compared, bcl-2 expression was higher in recurrent pterygium tissue samples ( p  = 0.003). However, when Ki-67 and p53 expression levels were evaluated, no significant difference was found between primary and recurrent pterygium ( p  = 0.215, p  = 0.321, respectively). Also, p53 and Ki-67 expression were correlated in pterygium tissue samples, and Bcl-2 expression was significantly higher in pterygium that recurrence in the first 6 months after surgery. There was no difference between groups 1, 2, and 3 in terms of p53 and Ki-67 expression., Conclusion: Antiapoptotic mechanisms and proliferation play an important role in the etiopathogenesis of pterygium. Furthermore, Bcl-2 expression may be important in pterygium recurrence.

TP53 mutation is an indicator of poor prognostic in chronic lymphocytic leukemia (CLL). Worse still, CLL patients with TP53 mutation are associated with poor efficacy to current chemotherapeutic, such as Fludarabine. Here, we confirmed that high expression of HDAC1 in CLL patients with TP53 mutation, which is closely related to poor prognosis and drug-resistance. Subsequently, we demonstrated Entinostat (HDAC1 inhibitor) combination with Fludarabine significantly induced apoptosis in TP53 mutations CLL cells. Its mechanism was associated with up-regulation of the pro-apoptotic protein Bax and the down-regulation of HDAC1, HO-1 and BCL-2 proteins. More importantly, we also confirmed that upregulation of HDAC1 could resistant Entinostat-induced apoptosis in TP53 mutations CLL cells by activating the HDAC1/P38/HO-1 pathway. In vivo, we found that Entinostat combination with Fludarabine significantly induced tumor cells apoptosis and prolong survival time in xenograft mouse model. Finally, combining vitro and vivo experiments, we presented the first demonstration that Entinostat combination with Fludarabine had a synergistic effect on the induction of apoptosis in TP53 mutations CLL cells. In conclusion, we provide valuable pre-clinical experimental evidence for the treatment of CLL patients with poor prognosis, especially for TP53 mutations., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Lineage specification of the three germ layers occurs during early embryogenesis and is critical for normal development. The nucleosome remodeling and deacetylase (NuRD) complex is a repressive chromatin modifier that plays a role in lineage commitment. However, the role of chromodomain helicase DNA-binding protein 4 (CHD4), one of the core subunits of the NuRD complex, in neural lineage commitment is poorly understood. Here, we report that the CHD4/NuRD complex plays a critical role in neural differentiation of mouse embryonic stem cells (ESCs). We found that RNAi-mediated Chd4 knockdown suppresses neural differentiation, as did knockdown of methyl-CpG-binding domain protein Mbd3 , another NuRD subunit. Chd4 and Mbd3 knockdowns similarly affected changes in global gene expression during neural differentiation and up-regulated several mesendodermal genes. However, inhibition of mesendodermal genes by knocking out the master regulators of mesendodermal lineages, Brachyury and Eomes, through a CRISPR/Cas9 approach could not restore the impaired neural differentiation caused by the Chd4 knockdown, suggesting that CHD4 controls neural differentiation by not repressing other lineage differentiation processes. Notably, Chd4 knockdown increased the acetylation levels of p53, resulting in increased protein levels of p53. Double knockdown of Chd4 and p53 restored the neural differentiation rate. Furthermore, overexpression of BCL2, a downstream factor of p53, partially rescued the impaired neural differentiation caused by the Chd4 knockdown. Our findings reveal that the CHD4/NuRD complex regulates neural differentiation of ESCs by down-regulating p53., (© 2019 Hirota et al.)

Objective: To investigate the protein expressions of steroid receptor coactivator amplified in breast cancer 1 (AIB1), apoptosis-related protein p53 and B-cell lymphoma-2 (Bcl-2) in epithelial ovarian cancer; to analyze the correlations among the expressions of these three proteins; to explore their correlations with the clinical pathological features and prognosis., Materials and Methods: Immunohistochemistry streptavidin-peroxidase (IHC-SP) method was performed to detect the positive protein expressions of AIB1, Bcl-2, and p53 in the pathological sections of normal ovarian tissues, benign ovarian epithelial tumor, and epithelial ovarian cancer, thereby analyzing the protein expression rates of these genes in different pathological stages, lymphatic metastasis and postoperative recurrent ovarian cancer, and carrying out the correlation analysis of these three proteins., Results: Positive protein expressions of AIB1, Bcl-2, and p53 were identified in the epithelial ovarian cancer tissues, and with an increase in the tumor staging of ovarian cancer, we found that the positive protein expression rate was gradually augmented. Particularly in ovarian cancer with lymphatic metastasis and postoperative recurrence, the positive expression rate was almost 100%. The protein expression of AIB1 was positively correlated with those of the p53 and Bcl-2., Conclusions: We showed that positive expressions of AIB1, Bcl-2, and p53 in ovarian cancer are closely correlated with the pathological staging, metastasis, and recurrence, and the positive protein expression suggests a poor prognosis.

Kinesin spindle protein (KSP) is a microtubule-associated motor protein that is specifically expressed by mitosis cells. It is highly expressed in various types of tumors including hematomalignances and solid tumors. Chemical KSP inhibition has become a novel strategy in the development of anticancer drugs. SB743921 is a selective inhibitor for KSP, which is a mitotic protein essential for cell-cycle progression. Although SB743921 has shown antitumor activities for several types of cancers and entered into clinical trials, its therapeutic effects on breast cancer and mechanisms have not been explored. In this study, we tested the antitumor activity of SB743921 in breast cancer cell lines and partly elucidated its mechanisms. KSP and denticleless E3 ubiquitin-protein ligase homolog (DTL) are overexpressed in breast cancer cells compared with no-cancer tissues. Chemical inhibition of KSP by SB743921 not only reduces proliferation but also induces cell-cycle arrest and leads to apoptosis in breast cancer cells. Treatment of MCF-7 and MDA-MB-231 breast cancer cell lines with SB743921 results in decreased ability of colony formation in culture. SB743921 treatment also causes a KSP accumulation in protein level that is associated with cell arrest. Furthermore, we showed that SB743921 treatment significantly reduces the expression of bcl-2 and cell cycle-related protein DTL, and upregulates p53 and caspase-3 in breast cancer cells. Taken together, these data indicated that SB743921 can be expected to be a novel treatment agent for breast cancers.

This study was conducted to determine the impact of vitrification on the expression of genes regulating pluripotency and apoptosis in mouse morulae. The morulae were randomly allocated into three groups: (1) untreated (control), (2) exposed to vitrification solution without freezing (toxicity), or (3) vitrified by open-pulled straw method (vitrification). In vitro development was evaluated by morphology and assessed by the blastocyst rate and the blastocyst total cell number. Gene expression in morulae and blastocysts was assessed by quantitative Real Time-PCR (qRT-PCR) and western blot. The results showed that at morulae stage, the POU class 5 homeobox1 (Oct-4) and B-cell lymphoma2 (Bcl2) mRNA levels of vitrification group were significantly lower (P < 0.05) than those of control. Strikingly, the p53 mRNA level was significantly higher in vitrification group. However, the Oct-4, Bcl2 and p53 mRNA levels in mouse blastocysts were not statistically different. Furthermore, western blot results showed that there was no significant difference in Oct-4, Bcl2 and p53 expression at protein level in mouse morulae among three groups. Additionally, the blastocyst rate (96.67%-100.00%) and the average cell number of blastocysts (89.67-92.33) were similar between all groups. The data demonstrate that vitrification transiently changes the mRNA expression of several key genes in mouse morulae regulating early embryo development but does not affect embryo developmental potential in vitro., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Unlabelled: The aim of this study was to describe the clinical features and expression of bcl-2, cyclin D1, p53, and proliferating cell nuclear antigen (PCNA) antibodies in syndromic (nevoid basal cell carcinoma syndrome [NBCCS]) and nonsyndromic patients diagnosed with keratocystic odontogenic tumor (KCOT)., Methods: This descriptive study comprised 5 patients of KCOT associated with NBCCS and 8 patients of nonsyndromic type treated in the Department of Oral Maxillofacial Surgery, Universiti Kebangsaan Malaysia Medical Centre between years 1998 and 2011. The clinical features (site, size, treatment, and recurrence), demographic characteristics, and immunohistochemistry results using antibodies of bcl-2, cyclin D1, p53, and PCNA were examined. The association of the antibody expression and the type of KCOT was analyzed using Fisher exact test., Results: Altogether there were 13 patients, 5 with syndromic KCOT (1 patient met 3 major criteria of NBCCS) and 8 with sporadic KCOT. The age range for syndromic KCT was 11 to 21 years (mean 16.00 years, SD 4.36) and 10 to 54 years (median 24.50 years, interquartile range 19.00) for the nonsyndromic KCOT. Tumor recurrence occurred in 3 patients (7.7%); 1 patient from the syndromic and 2 patients from the nonsyndromic. The most positive expression was observed in PCNA for both the syndromic and nonsyndromic samples and the least positive expression involved the p53., Conclusion: PCNA, bcl-2 protein, and cyclin D1 expressions could be useful in evaluating the proliferative activity of the tumor and the aggressiveness of the clinical presentation; however, the authors would propose for larger sample size research for more definitive results.

Previous studies have shown that GSIV induces apoptotic cell death through upregulation of the pro-apoptotic genes Bax and Bak in Grouper fin cells (GF-1 cells). However, the role of viral genome-encoded protein(s) in this death process remains unknown. In this study, we demonstrated that the Giant seaperch iridovirus (GSIV) genome encoded a serine/threonine kinase (ST kinase) protein, and induced apoptotic cell death via a p53-mediated Bax upregulation approach and a downregulation of Bcl-2 in fish cells. The ST kinase expression profile was identified through Western blot analyses, which indicated that expression started at day 1 h post-infection (PI), increased up to day 3, and then decreased by day 5 PI. This profile indicated the role of ST kinase expression during the early and middle phases of viral replication. We then cloned the ST kinase gene and tested its function in fish cells. The ST kinase was transiently expressed and used to investigate possible novel protein functions. The transient expression of ST kinase in GF-1 cells resulted in apoptotic cell features, as revealed with Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) assays and Hoechst 33258 staining at 24 h (37 %) and 48 h post-transfection (PT) (49 %). Then, through studies on the mechanism of cell death, we found that ST kinase overexpression could upregulate the anti-stress gene p53 and the pro-apoptotic gene Bax at 48 h PT. Interestingly, this upregulation of p53 and Bax also correlated to alterations in the mitochondria function that induced loss of mitochondrial membrane potential (MMP) and activated the initiator caspase-9 and the effector caspase-3 in the downstream. Moreover, when the p53-dependent transcriptional downstream gene was blocked by a specific transcriptional inhibitor, it was found that pifithrin-α not only reduced Bax expression, but also averted cell death in GF-1 cells during the ST kinase overexpression. Taken altogether, these results suggested that aquatic GSIV ST kinase could induce apoptosis via upregulation of p53 and Bax expression, resulting in mitochondrial disruption, which activated a downstream caspases-mediated cell death pathway.

Doxorubicin (DOX) is an efficient drug used in cancer therapy; however, it has severe cardiotoxic side effects. The aim of the present study was to investigate the effects of resveratrol on the adenosine monophosphate-activated protein kinase (AMPK)/P53 pathway in mediating DOX-induced cytotoxicity. H9c2 cells were exposed to 5 µM DOX for 24 h to establish a model of DOX-induced cardiotoxicity. DOX administration amplified P53 and B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) expression and decreased Bcl-2 expression in H9c2 cells. Resveratrol increased the cell viability and decreased the apoptotic rate. In addition, resveratrol markedly increased the phosphorylation of AMPK. Of note, resveratrol protected against DOX-induced increases of P53 and Bax and also prevented the downregulation of Bcl-2 in H9c2 cells. Furthermore, AMPK inhibitor Compound C abolished the protective effects of resveratrol. The results of the present study therefore indicated that resveratrol protected H9c2 cells from DOX-induced apoptosis via the AMPK/P53 pathway.

Many human tumors express high levels of the p53 inhibitor Mdm2, resulting from amplification of the Mdm2 locus or aberrant post-translational regulation of the Mdm2 protein. While the importance of Mdm2 in regulating p53 is clear, Mdm2 also has p53-independent roles. For example, overexpression of Mdm2 results in genomic instability in a p53-independent manner. In addition, Mdm2 has many additional binding partners, some of which, such as the tumor suppressor p73, have also been implicated in genomic instability. In this study, cells and tumors with Mdm2 overexpression and p73 loss exhibit increased genomic instability as compared with either alteration alone and cooperate in development of B-cell lymphomagenesis. Cytogenetic analysis of mouse embryonic fibroblasts and pre-malignant B cells demonstrates that loss of p73 exacerbates the chromosome breaks and fusions observed in Mdm2(Tg) cells. B-cell lymphomas from Mdm2(Tg);p73(+/-) mice retain the remaining p73 allele, exhibit elevated levels of the antiapoptotic protein Bcl2 and thus dampen apoptosis. In summary, Mdm2 overexpression and p73 loss cooperate in genomic instability and tumor development, indicating that the oncogenic function of Mdm2 is a combined effect of inhibiting p53 and p73 functions. Given that p73 is lost or silenced in human B-cell lymphomas, the Mdm2(Tg);p73(+/-) mouse serves as a model for human disease and may provide additional insight into the pathways that contribute to B-cell lymphomagenesis.

The relevance of heat preconditioning resides in its ability to protect cells from different kinds of injury by induction of heat shock proteins, a process in which the intensity of heat stress (HS) and duration of subsequent recovery are vital. This study evaluates the effects of moderate HS (45 min/43°C) and the time-dependent changes during recovery period of HSP70, Bcl-2 and p53 gene and protein expression in HepG2 cells. We also evaluated the effects of 0.4 mM aspirin (ASA) as a potential pharmacological co-inducer of HSP, both alone and in a combination with HS (ASA + HS). HS alone and ASA + HS caused a major up-regulation of HSP70 mRNA in the first 2 h, while HSP70 protein increased gradually and was especially abundant from 2 h to 24 h. Regarding Bcl-2, all treatments rendered similar results: gene expression was down-regulated in the first 2 h, after which there was protein elevation (12-48 h after HS). mRNA expression of p53 in HS- and (ASA + HS)-cells was down-regulated in the first 12 h. The immediate decrease of p53 protein after HS was followed by a biphasic increase. In conclusion, 0.4 mM ASA + HS does not act as a co-inducer of HSP70 in HepG2 cells, but promotes Bcl-2 protein expression during prolonged treatment. Our suggestion is that hepatic cells are most vulnerable in the first 2-6 h, but may have a high capacity for combating stress 12-24 h after HS. Finally, short-term exposure HS might be a "physiological conditioner" for liver cells to accumulate HSP and Bcl-2 proteins and thus obtain cytoprotection against an additional stress., (© 2015 Wiley Periodicals, Inc.)

Use of the conventional cancer chemotherapy (i.e. vincristine) is limited in tumor cells exhibiting pre-existing or acquired resistance. Here, we found that C6 ceramide (C6) dramatically sensitized vincristine's activity. In vitro, C6 and vincristine coadministration induced substantial necrosis and apoptosis in multiple human cancer cell lines, which were accompanied by a profound AMP-activated protein kinase (AMPK) activation, subsequent p53 activation, mTORC1 inactivation and Bcl-2/HIF-1α downregulation. Such synergistic effects were attenuated by AMPK inactivation through genetic mutation or short hairpin RNA silencing. Coadministration-activated p53 translocated to mitochondria, and formed a complex with cyclophilin-D, leading to mitochondrial permeability transition pore opening and cell necrosis. Disrupting p53-Cyp-D complexation through pharmacological or genetic means reduced costimulation-induced cytotoxicity. In vivo, a liposomal C6 was synthesized, which dramatically enhanced the antiproliferative activity of vincristine on HCT-116 or A2780 xenografts. Together, C6 sensitizes vincristine-induced anticancer activity in vivo and in vitro, involving activating AMPK-p53 signaling., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The current paradigm of metastasis proposes that rare cells within primary tumors acquire metastatic capability via sequential mutations, suggesting that metastases are genetically dissimilar from their primary tumors. This study investigated the changes in the level of expression of a well-defined panel of cell proliferation, differentiation, and apoptosis markers between the primary colorectal cancer (CRC) and the corresponding synchronous lymph node (LN) metastasis from the same patients. DNA flow cytometry and immunostaining of p53, bcl-2, and c-myc were carried out on 36 cases of CRC radical resection specimens with their corresponding LN metastases. There was very low probability that the histological patterns of primary tumors and LN metastases are independent (p < 0.001). Metastatic tumors were significantly more diffusely positive for p53 than the primary tumors (p < 0.001). Conversely, primary tumors were significantly more diffusely positive for c-myc than metastatic tumors (p = 0.011). No significant difference was found between the LNs and the primary tumors in bcl-2 positivity (p = 0.538) and DNA aneuploidy (p = 0.35), with a tendency towards negative bcl-2 and less aneuploidy in LN metastases than primary tumors. In conclusion, LN metastatic colorectal carcinomas have a tendency of being less differentiated, with a higher incidence of diffuse p53 staining, lower incidence of bcl-2 staining, and less aneuploidy in comparison to their primary counterparts suggesting a more aggressive biological behavior, which could indicate the necessity for more aggressive adjuvant therapy.

Osteosarcoma cancer is one of the most lethal malignancies, and there is no effective preventive measure to date. Chamaejasmine is the major ingredient in Stellera chamaejasme L. Except its potent pain-relieving efficacy as reported, chamaejasmine also exerted its anti-tumor activity in several tumor models. Here, we reported that chamaejasmine had a profound anti-proliferative effect on human osteosarcoma cells in a concentration-dependent and time-dependent manner, which was associated with an increase of p21 and bax and a decrease of bcl-2 and consequently increased caspase-3 activity. The main mechanism of anti-tumor potency was mainly attributed to the induction of p53. Chamaejasmine hugely elevated the expression of p53. The results of p53 shRNA experiment further demonstrated that p53 knockdown severely impaired the sensitivity of tested cells to chamaejasmine, implicating the important role of p53 played in chamaejasmine's anti-tumor activity. In conclusion, results showed chamaejasmine induced apoptosis in MG63 cells and could be a candidate drug for osteosarcoma cancer chemoprevention.

Cantharidin, a type of terpenoid, is a chemical compount secreted by the blister beetle or Mylabris phelarata pallas of the Meloidae family. Cantharidin is known to have good antitumor activity. The present study aimed to investigate the anticancer effect of cantharidin and its possible underlying mechanism using tongue squamous cell carcinoma (TSCC) TCA8113 cells. TCA8113 cells were treated with various concentrations of cantharidin, and the cell viability and cytotoxicity were assessed using MTT and LDH assays, respectively. Flow cytometry was conducted to examine cell apoptosis and colorimetric protease assay was performed to analyze caspase-9/3 activities in TCA8113 cells. qPCR and western blot analysis were used to investigate microRNA-214 (miR-214) expression, as well as the expression of p53, Bcl-2 and Bax proteins in TCA8113 cells. miR-214 and anti-miR-214 were transfected with mimics to examine whether miR-214 expression regulated the anticancer effect of cantharidin on TCA8113 cells and p53, Bcl-2 and Bax protein expression. The anticancer effect of cantharidin significantly inhibited cell proliferation and increased cytotoxicity of TSCC Tca8113 cells in a dose- and time-dependent manner. In addition, cantharidin induced cell apoptosis and activated caspase-9/3 activities of TSCC Tca8113 cells. Cantharidin markedly weakened miR-214 expression level, activated p53 protein expression, and suppressed the Bcl-2/Bax signaling pathway in Tca8113 cells. Downregulation of miR-214 increased p53 protein expression and decreased the Bcl-2/Bax signaling pathway of TSCC Tca8113 cells. However, the overexpression of miR-214 reduced the anticancer effect of cantharidin on the proliferation and apoptosis of TSCC Tca8113 cells, inhibited p53 protein expression, and increased the Bcl-2/Bax signaling pathway. The results suggested that cantharidin is a potential anticancer drug that can be used to regulate the proliferation and apoptosis of human TSCC Tca8113 cells. Additionally, its mechanism may partially be associated with the downregulation of miR-214, upregulation of p53 protein expression and suppression of the Bcl-2/Bax signaling pathway.

In recent years it was disclosed, that numerous organotin(IV) derivatives have remarkable cytotoxicity against several types of cancer cells. The property to inhibit cell growth makes these compounds promising for antitumor therapy, as the clinical effectiveness of cisplatin is limited by drug resistance and significant side effects. Tributyltin and triphenyltin are known as endocrine disruptors. Moreover, the compounds exert their toxicity in mammals predominantly through nuclear receptor signaling. Here we present the effects of tributyltin chloride (TBT-Cl) and triphenyltin chloride (TPT-Cl) on cell proliferation, expression of proapoptotic p53, Bax, and antiapoptotic Bcl-2 proteins in human breast cancer MCF-7 cell line. Dose and time dependent (24, 48 and 72 h) cell expositions have demonstrated TBT-Cl as more effective in inhibiting MCF-7 cell proliferation than TPT-Cl. Short time treatment with TBT-Cl displayed marked stimulation of p53 protein expression when compared to TPT-Cl. Both organotin compounds displayed similar mild enhancement of Bax protein expression. The 24h exposition of TPT-Cl induced substantial diminution of Bcl-2 protein expression in comparison with both, untreated cells and TBT-Cl treated cells. Our observations indicate that TBT-Cl and TPT-Cl have different antiproliferative potency and distinct impact on expression of apoptosis marker proteins., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Emerging interest on the interrelationship between the apoptotic and autophagy pathways in the context of cancer chemotherapy is providing exciting discoveries. Complexes formed between molecules from both pathways present potential targets for chemotherapeutics design as disruption of such complexes could alter cell survival. This study demonstrates an important role of Beclin-1 and p53 interaction in cell fate decision of human embryonal carcinoma cells. The findings provide evidence for p53 interaction with Beclin-1 through the BH3 domain of the latter. This interaction facilitated Beclin-1 ubiquitination through lysine 48 linkage, resulting in proteasome-mediated degradation, consequently maintaining a certain constitutive level of Beclin-1. Disruption of Beclin-1-p53 interaction through shRNA-mediated down-regulation of p53 reduced Beclin-1 ubiquitination suggesting requirement of p53 for the process. Reduction of ubiquitination consequently resulted in an increase in Beclin-1 levels with cells showing high autophagic activity. Enforced overexpression of p53 in the p53 down-regulated cells restored ubiquitination of Beclin-1 reducing its level and lowering autophagic activity. The Beclin-1-p53 interaction was also disrupted by exposure to cisplatin-induced stress resulting in higher level of Beclin-1 because of lesser ubiquitination. This higher concentration of Beclin-1 increased autophagy and offered protection to the cells from cisplatin-induced death. Inhibition of autophagy by either pharmacological or genetic means during cisplatin exposure increased apoptotic death in vitro as well as in xenograft tumours grown in vivo confirming the protective nature of autophagy. Therefore, Beclin-1-p53 interaction defines one additional molecular subroutine crucial for cell fate decisions in embryonal carcinoma cells., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Ionizing radiation increases cell mortality in a dose-dependent manner. Increases in DNA double strand breaks, γ-H2AX, p53 phophorylation, and protein levels of p53 and Bax also occur. We investigated the ability of ciprofloxacin (CIP), a widely prescribed antibiotic, to inhibit DNA damage induced by ionizing radiation. Human tumor TK6, NH32 (p53 (-/-) of TK6) cells, and human normal peripheral blood mononuclear cells (PBMCs) were exposed to 2-8 Gy (60)Co-γ-photon radiation. γ-H2AX (an indicator of DNA strand breaks), phosphorylated p53 (responsible for cell-cycle arrest), Bcl-2 (an apoptotic protein, and cell death were measured. Ionizing irradiation increased γ-H2AX amounts in TK6 cells (p53(+/+)) within 1 h in a radiation dose-dependent manner. CIP pretreatment and posttreatment effectively inhibited the increase in γ-H2AX. CIP pretreatment reduced Bcl-2 production but promoted p53 phosphorylation, caspase-3 activation and cell death. In NH32 cells, CIP failed to significantly inhibit the radiation-induced γ-H2AX increase, suggesting that CIP inhibition involves in p53-dependent mechanisms. In normal healthy human PBMCs, CIP failed to block the radiation-induced γ-H2AX increase but effectively increased Bcl-2 production, but blocked the phospho-p53 increase and subsequent cell death. CIP increased Gadd45α, and enhanced p21 protein 24 h postirradiation. Results suggest that CIP exerts its effect in TK6 cells by promoting p53 phosphorylation and inhibiting Bcl-2 production and in PBMCs by inhibiting p53 phosphorylation and increasing Bcl-2 production. Our data are the first to support the view that CIP may be effective to protect normal tissue cells from radiation injury, while enhancing cancer cell death in radiation therapy.

This study was performed to investigate the protective and therapeutic effects of resveratrol (RES) against CdCl2-induced toxicity in rat testes. Seven experimental groups of adult male rats were formulated as follows: A) controls+NS, B) control+vehicle (saline solution of hydroxypropyl cyclodextrin), C) RES treated, D) CdCl2+NS, E) CdCl2+vehicle, F) RES followed by CdCl2 and M) CdCl2 followed by RES. At the end of the protocol, serum levels of FSH, LH and testosterone were measured in all groups, and testicular levels of TBARS and superoxide dismutase (SOD) activity were measured. Epididymal semen analysis was performed, and testicular expression of Bcl-2, p53 and Bax was assessed by RT-PCR. Also, histopathological changes of the testes were examined microscopically. Administration of RES before or after cadmium chloride in rats improved semen parameters including count, motility, daily sperm production and morphology, increased serum concentrations of gonadotropins and testosterone, decreased testicular lipid peroxidation and increased SOD activity. RES not only attenuated cadmium chloride-induced testicular histopathology but was also able to protect against the onset of cadmium chloride testicular toxicity. Cadmium chloride downregulated the anti-apoptotic gene Bcl2 and upregulated the expression of pro-apoptotic genes p53 and Bax. Resveratrol protected against and partially reversed cadmium chloride testicular toxicity via upregulation of Bcl2 and downregulation of p53 and Bax gene expression. The antioxidant activity of RES protects against cadmium chloride testicular toxicity and partially reverses its effect via upregulation of BCl2 and downregulation of p53 and Bax expression.

Aim: To investigate the mechanisms of induction of apoptosis of esophageal cancer cells by esophageal cancer-related gene 2 (ECRG2) in combination with cisplatin (DDP)., Methods: Hoechest staining was performed to analyze the effects of single ECRG2 and ECRG2 in combination with DDP on apoptosis of EC9706 cells. The expression levels of p53 and bcl-2 mRNA and protein were determined by RT-PCR and Western blotting, respectively., Results: The number of apoptotic cells after the treatment with ECRG2 in combination with DDP for 24 hours was more than that after the treatment with single ECRG2. RT-PCR and Western blotting showed that the expression levels of bcl-2 mRNA and protein were both down-regulated, while p53 mRNA and protein were both up-regulated in the cells treated with ECRG2 in combination with DDP compared with those given ECRG2 alone., Conclusion: ECRG2 in combination with DDP can enhance the apoptosis of EC9706 cells, possibly by down-regulating bcl-2 expression and up-regulating p53.

Development of drugs from natural products has been undergoing a gradual evoluation. Many plant derived compounds have excellent therapeutic potential against various human ailments. They are important sources especially for anticancer agents. A number of promising new agents are in clinical development based on their selective molecular targets in the field of oncology. D-pinitol is a naturally occurring compound derived from soy which has significant pharmacological activitites. Therefore we selected D-pinitol in order to evaluate apoptotic potential in the MCF-7 cell line. Human breast cancer cells were treated with different concentrations of D-pinitol and cytotoxicity was measured by MTT and LDH assays. The mechanism of apoptosis was studied with reference to expression of p53, Bcl-2, Bax and NF-kB proteins. The results revealed that D-pinitol significantly inhibited the proliferation of MCF-7 cells in a concentration-dependent manner, while upregulating the expression of p53, Bax and down regulating Bcl-2 and NF-kB. Thus the results obtained in this study clearly vindicated that D-pinitol induces apotosis in MCF-7 cells through regulation of proteins of pro- and anti-apoptotic cascades.

Both breast cancer resistance protein (BCRP, ABCG2) and apoptosis-related molecules are involved in the development of multidrug resistance (MDR) in cancer cells. However, the association of BCRP with apoptosis-related molecules in the development of MDR is unknown. In this study, we investigated the changes in expression levels of BCRP, Survivin, p53, Bcl-2, Bcl-xL or Bax in cultured MCF-7 and MCF-7/5-FU cells, and explored whether these changes affected the expressions of BCRP. Our data showed that the protein and mRNA expression levels of BCRP, Survivin and Bcl-2 were significantly higher in MCF-7/5-FU cells than in MCF-7 cells, while p53 expression lower in MCF-7/5-FU cells than in MCF-7 cells. Knockdown of Survivin or Bcl-2 in MCF-7/5-FU cells and overexpression of Survivin in MCF-7 cells revealed that Survivin had significant association with BCRP expression. Luciferase reporter gene assays showed that Survivin up-regulated BCRP expression at transcriptional level and this response was mediated through NF-κB(p50) pathway. However, may be due to the physical interaction between p53 and Survivin, p53 directly affected Survivin-regulated BCRP expressions. Interestingly, we found that Survivin would suppress p53 expression. Furthermore, our data revealed that Survivin itself had no apparent effect on NF-κB(p50) and BCRP expression when knockdown of p53 in MCF-7 cells; whereas p53 exerted significant inhibitory action on these when knockdown of Survivin. In conclusion, through down regulation of p53 expression, Survivin attenuates the suppressing effect of p53 on NF-κB(p50) expression and then enhances BCRP expression. This may represent a novel strategy for reversal of BCRP drug transporter activity to modulate MDR in cancer cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aim: The authors have systematically investigated the anticancer potentiality of silver-based nanoparticles (AgNPs) and the mechanism underlying their biological activity in human colon cancer cells., Materials & Methods: Starch-capped AgNPs were synthesized, characterized and their biological activity evaluated through multiple biochemical assays., Results: AgNPs decreased the growth and viability of HCT116 colon cancer cells. AgNP exposure increased apoptosis, as demonstrated by an increase in 4´,6-diamidino-2-phenylindole-stained apoptotic nuclei, BAX/BCL-XL ratio, cleaved poly(ADP-ribose) polymerase, p53, p21 and caspases 3, 8 and 9, and by a decrease in the levels of AKT and NF-κB. The cell population in the G1 phase decreased, and the S-phase population increased after AgNP treatment. AgNPs caused DNA damage and reduced the interaction between p53 and NF-κB. Interestingly, no significant alteration was noted in the levels of p21, BAX/BCL-XL and NF-κB after AgNP treatment in a p53-knockout HCT116 cell line., Conclusion: AgNPs are bona fide anticancer agents that act in a p53-dependent manner. Original submitted 16 March 2012; Revised submitted 25 August 2012; Published online 21 March 2013.

The p53 cancer mutant Y220C is an excellent paradigm for rescuing the function of conformationally unstable p53 mutants because it has a unique surface crevice that can be targeted by small-molecule stabilizers. Here, we have identified a compound, PK7088, which is active in vitro: PK7088 bound to the mutant with a dissociation constant of 140 μM and raised its melting temperature, and we have determined the binding mode of a close structural analogue by X-ray crystallography. We showed that PK7088 is biologically active in cancer cells carrying the Y220C mutant by a battery of tests. PK7088 increased the amount of folded mutant protein with wild-type conformation, as monitored by immunofluorescence, and restored its transcriptional functions. It induced p53-Y220C-dependent growth inhibition, cell-cycle arrest and apoptosis. Most notably, PK7088 increased the expression levels of p21 and the proapoptotic NOXA protein. PK7088 worked synergistically with Nutlin-3 on up-regulating p21 expression, whereas Nutlin-3 on its own had no effect, consistent with its mechanism of action. PK7088 also restored non-transcriptional apoptotic functions of p53 by triggering nuclear export of BAX to the mitochondria. We suggest a set of criteria for assigning activation of p53.

Incidence of hepatocellular carcinoma (HCC) is dramatically increasing and is the third cause of cancer death worldwide. One key approach to control HCC is chemoprevention by naturally occurring agents. This study aims at investigating the antitumor effect of oleanolic acid (OA) and the molecular mechanisms. BALB/c mice were injected subcutaneously with HepG2 cells to establish transplanted tumors. Apoptosis and cell cycle arrest-related markers and signaling cascades were determined by western blot, immunofluorescence, reverse transcriptase-polymerase chain reaction and flow cytometric analysis. OA exhibited inhibitory effect on HCC through induction of apoptosis and cell cycle arrest both in transplanted tumors and in HepG2 cells. OA induced apoptosis through mitochondrial pathway, evidenced by inhibition of Akt/mammalian target of rapamycin pathway, mitochondrial dysfunction, transient increase of adenosine triphosphate, increase of Bax/Bcl-2 ratio, increased release of cytochrome c and activation of caspase/poly (ADP-ribose) polymerase. Activation of mitochondrial apoptotic pathway may be due to reactive oxygen species generated by mitochondrial fatty acid oxidation, resulted from enhancement of lipolysis regulated by cyclic adenosine 3',5'-monophosphate response element-binding protein-hormone-sensitive lipase/peroxisome proliferator-activated receptor γ signaling. OA induced G2/M cell cycle arrest through p21-mediated downregulation of cyclin B1/cdc2. Cyclooxygenase-2 (COX-2) and p53 were involved in OA-exerted effect, and extracellular signal-regulated kinase-p53 signaling played a central role in OA-activated cascades responsible for apoptosis and cell cycle arrest. OA demonstrated significant antitumor activities in HCC in vivo and in vitro models. These data provide new insights into the mechanisms underlying the antitumor effect of OA.

Hyperhomocysteinemia (HHcy) is a risk factor for cardiovascular disease and has a strong correlation with heart failure. However, the effects of HHcy on cardiac tissue remain less well understood. To elucidate the role of p53-dependent apoptosis in HHcy-induced cardiac injury, we fed ApoE(-/-) mice with high methionine diet to establish HHcy model. Serum Hcy, cardiac enzymes, and lipids were measured. The protein levels of Noxa, DNMT1, caspases-3/9, and p53 were determined by enzyme-linked immunosorbent assay. Bcl-2 and Bax proteins were detected by immunohistochemistry staining. S-adenosyl methionine and S-adenosyl homocysteine concentrations were determined by high-performance liquid chromatography. The mRNA levels of p53 and DNMT1 were analyzed by real-time polymerase chain reaction (PCR) and the methylation levels of p53 were analyzed by nested methylation-specific-PCR. Our data showed that the concentrations of serum Hcy and lipids were increased in Meth group compared with the N-control group, which indicated that the model was established successfully. The expression levels of p53 and Noxa were increased in Meth group, while the methylation status of p53 was hypomethylation. The activities of caspase-3/9 were increased in Meth group compared with the N-control group. In addition, immunohistochemistry staining showed that the expression of Bax was significantly increased in Meth and Meth-F group compared with the N-control group. In summary, HHcy induces cardiac injury by up-regulation of p53-dependent pro-apoptotic related genes Noxa and Bax, while p53 DNA hypomethylation is a key molecular mechanism in pathological process induced by HHcy.

Aims: The aim of this study was to investigate the anti proliferative activity of 3-deoxyanthocyanin extracted from red sorghum bran on human breast cancer cell line MCF 7. The confirmatory tests were carried out in vitro through the expression studies of p(53) and (bcl) 2 genes in MCF 7 cells., Method: The 3-deoxyanthocyanins were isolated from red sorghum bran and cytotoxic studies were performed in MCF 7 cell line by MTT assay. The mRNA expression levels of p(53) and (bcl) 2 genes were performed using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in MCF 7 cells., Key Findings: On cytotoxic studies, the present data indicates sorghum anthocyanins, which showed 84.09% of inhibition in the proliferation of MCF 7 cells, and the CTC(50) value was 300 μg/ml. The sorghum 3-deoxyanthocyanins induced apoptosis in MCF 7 was mediated by stimulation of the p(53) gene and down regulation of the (bcl) 2 gene., Significance: The significance of our work was the anthocyanin isolated from red sorghum bran inhibits the proliferation of human breast cancer cell line., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In a prospective multicenter phase II trial of radioembolization with yttrium-90 ((90)Y-RE) in chemorefractory liver-dominant metastatic colorectal cancer (mCRC), we showed that median survival was 12.6 months (95% CI 7.0-18.3) with 48% of 50 patients achieving disease control. In this extension retrospective study, we analyzed whether a panel of biomarkers, known to be associated to an adverse clinical outcome, underwent variations in CRC liver metastases pre and post (90)Y-RE.Of the 50 patients included in the study, 29 pre-(90)Y-RE therapy and 15 post-(90)Y-RE had liver biopsy specimens available. In these series we investigated survivin, p53, Bcl-2 and Ki-67 expression pre- and post-(90)Y-RE by immuhistochemistry (IHC). Our findings evidenced a decrease of survivin (77% vs 33%), p53 (93% vs 73%), Bcl-2 (37% vs 26%) expression as well as of Ki-67 proliferation index (62.5% vs 40%) on liver biopsies collected post-(90)Y-RE as compared to pre-(90)Y-RE. In the subset of 13 matched liver metastases we further confirmed the reduction of survivin (92.3% vs 53.8%; p = 0.06), p53 (100% vs 69.2%; p = 0.05) and Bcl-2 (69.2% vs 53.8%; p = 0.05) expression post-(90)Y-RE. This biomarker modulation was accompanied by morphological changes as steatohepatitis, hepatocyte necrosis, collagen deposition, proliferating and/or bile duct ectasia, focal sinusoidal dilatation and fibrosis.Although our analysis was conducted in a very limited number cases, these changes appear strictly related to the response to (90)Y-RE therapy and may deserve further investigation on a larger series of patients.

Objective: The aim of this study was to determine by immunohistochemistry the presence and significance of p53 and bcl-2 proteins in oral lichen planus (OLP) and oral squamous cell carcinoma (OSCC)., Study Design: We used 21 cases diagnosed as OLP 16 diagnosed as OSCC and four normal gingival biopsies taken from healthy patients were used as controls. Slides were processed for immunohistochemistry using anti-p53 and anti-bcl-2 monoclonal antibodies., Results: We found p53 immunoexpression in 71.4% OLP cases and 68.7% OSCC cases, with no immunoexpression in control cases. Bcl-2 was negative for all OLP and OSCC cases, and mild positivity was observed in normal tissue. We found significant correlation among p53 expression and OSCC malignancy., Conclusions: Our results suggest that TP53 system mainly promotes a hyperproliferative state by cell cycle arrest of the OLP epithelial cells for repairing damaged DNA nor apoptosis and that anti-apoptotic action of bcl-2 is not important in this disease.

Background: Nowadays it remains a controversial issue whether a correlation exists between the apoptosis rate of tumor tissue and the prognosis of the patients. The aim of the study is to investigate the relationships of apoptotic genes and apoptotic index of osteosarcoma tissue to prognosis of the patients, meanwhile to explore the valid prognostic biomarkers of osteosarcoma that will enhance efficacy of clinical treatments for osteosarcoma., Methods: In our studies, the immunohistochemical ABC and terminal DNA breakpoints in situ 3-hydroxy end labeling (TUNEL) techniques were used to detect the expressions of p53, c-MYC, BCL-2 and apoptotic index in 56 osteosarcoma specimens. The relationships between apoptotic genes expression and apoptotic index in osteosarcoma tissue and their correlations with pathologic classification and prognosis of osteosarcoma cases were analyzed., Results: We found that the expressions of p53, c-MYC, and BCL-2 were negatively correlated with apoptotic index of osteosarcoma tissue, were not correlated with pathological types of osteosarcoma, and were closely related to prognosis of the patients. Moreover, apoptotic index of osteosarcoma tissue was positively correlated with the long term survival of the patients., Conclusion: We concluded that the expressions of p53, c-MYC, BCL-2 protein and apoptotic index could be used as potential biomarks for predicting the progression and prognosis of osteosarcoma, and for optimizing clinical treatments., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Unlabelled: This study aim was to identify the relations between tumor suppressor genes (p53, bcl-2, PTEN), nuclear proliferative antigen Ki-67 and epidemiologic, morphologic and histologic patient related factors, in colorectal cancer., Materials and Methods: Twenty-two prospectively collected colorectal cancer resection specimens were histologically prepared, using standard paraffin-embedded and Hematoxylin-Eosin staining method; for immunohistochemical study, the Streptavidin-Biotin (sABC)/Horseradish Peroxidase (HRP) method was used., Results: P53 was positive in 86.36% of cases, more intense (>50%) in rectal cancer and in women 59.16 ± 9.49-year-old; the G1/2 adenocarcinoma was dominant. Bcl-2 was positive in 18.18% of the cases, in distal colorectal cancer, only in men, 63.5 ± 13.2-year-old. PTEN was positive in 95.45% of the cases; intense positivity was recorded in 12 men and six women, 61.47 ± 11.67-year-old, in rectal topography. Ki-67 was positive in 86.36% of the cases, more intense in four men and four women, 63.45 ± 12.22-year-old, in proximal and advanced colorectal cancers (pT3N1/2 - 62.5%)., Conclusions: Tumor suppressor genes mutations are often present in colorectal cancer; the intensity of the expression of these mutations varies, which could explain the different prognosis for these patients.

The study included a total of 17 ameloblastomas diagnosed in a range of 15 years. Clinical data processing was followed by histopathological and immunohistochemical analysis, following the expression of p53, Bcl-2 and Ki67 in the tumor epithelial compartments. Lesions predominated in the age range 20-40 years, to male gender, being located mainly in the mandible (88.2%). Typical follicular ameloblastoma was present in 70.6% of analyzed tumors. P53 and Bcl-2 immunoexpression was identified especially in the peripheral cells, the number of marked cells being over 50%, while the percentage of positive cells from the stellate reticulum was below 10%. The Ki67 positivity index was below 10% in both compartments. Squamous and granular cells reveal no stain for the investigated markers. Peripheral columnar cells are active compartment of ameloblastomas being involved in the processes of proliferation-apoptosis.

Aim: Clinically electroacupuncture (EA) is proved an effective therapy for vascular dementia (VD), but their mechanisms remain uncertain. The aim of this study was to determine whether EA protects pyramidal cells from apoptosis in hippocampus of a VD rat model by inhibiting the expression of p53 and Noxa., Methods: One month after a VD animal model was established by bilateral occlusion of common carotid arteries, EA treatment was given at "Baihui" (DU20), "Dazhui" (DU14), and "Shenshu" (BL23). The learning and memory ability was assessed by Morris water maze. Neuronal apoptosis in hippocampus was evaluated with hematoxylin-eosin (HE) staining, and the expression of p53 and Noxa was analyzed by confocal laser scanning microscope with immunofluorescence staining., Results: Expressions of p53 and Noxa in the EA group and sham-operated group were less than in the VD model group (P < 0.01), and the expression of p53 was positively correlated to expression of Noxa in hippocampus of VD rats (r = 0.918, P < 0.01). EA treatment could reduce the amount of apoptotic neurons in hippocampal CA1 area of rats with VD. The average latency in the Morris water maze test was significantly shorter, and escape strategies improved from edge and random searches to more linear swim pathway in the EA group compared with the VD model group (P < 0.01)., Conclusions: The increasing expressions of p53 and Noxa play important roles in the pathogenesis of VD. EA improves learning and memory ability and protects pyramidal cells from apoptosis by blocking expression of p53 and Noxa in the hippocampal CA1 region of VD rats. These results suggest a novel mechanism of EA treatment to VD., (© 2010 Blackwell Publishing Ltd.)

Background: Although alterations in the mechanisms of apoptosis are an integral part of the tumor phenotype, their precise role in endometrial carcinoma is still obscure. The aim was to determine whether Bcl-2 plays a similar biological role in endometrial cancer as in breast cancer, endometrial cancer being also a hormone-dependent tumor., Materials and Methods: The expression of the apoptosis-related Bcl-2 and p53 genes, together with Ki67, E-cadherin, c-erb-B2 and estrogen and progesterone receptors were studied in 136 formalin-fixed, paraffin-embedded endometrial carcinoma samples by means of immunohistochemistry., Results: Bcl-2 expression correlated directly and significantly with E-cadherin (r=0.22, p=0.011) estrogen receptor (r=0.18, p=0.04) and progesterone receptor expression (r=0.30, p=0.0006), and inversely with surgical stage (r=-0.20, p=0.024). Mutant p53 expression was directly and significantly associated with increasing patient age (r=0.25, p=0.007), tumor grade (r=0.37, p<0.001), Ki67 (r=0.47, p<0.0001), c-erb-B2 expression (r=0.21, p=0.012) and with E-cadherin expression (r=0.19, p=0.026)., Conclusion: Bcl-2 and p53 are independently and significantly co-expressed with E-cadherin in endometrial carcinoma. Furthermore, the expression of Bcl-2 is also significantly associated with the expression of both progesterone and estrogen receptors, in that order, suggesting that, analogously to breast cancer, apoptosis is hormonally regulated to some degree also in endometrial cancer.

Nearly half of human cancers harbor p53 mutations, which can promote cancerous growth, metastasis, and resistance to therapy. The gain of function of mutant p53 is partly mediated by its ability to form a complex with NF-Y or p63/p73. Here, we demonstrate that TopBP1 mediates these activities in cancer, and we provide both in vitro and in vivo evidence to support its role. We show that TopBP1 interacts with p53 hot spot mutants and NF-YA and promotes mutant p53 and p300 recruitment to NF-Y target gene promoters. TopBP1 also facilitates mutant p53 interaction with and inhibition of the transcriptional activities of p63/p73. Depletion of TopBP1 in mutant p53 cancer cells leads to downregulation of NF-Y target genes cyclin A and Cdk1 and upregulation of p63/p73 target genes such as Bax and Noxa. Mutant p53-mediated resistance to chemotherapeutic agents depends on TopBP1. The growth-promoting activity of mutant p53 in a xenograft model also requires TopBP1. Thus, TopBP1 mediates mutant p53 gain of function in cancer. Since TopBP1 is often overexpressed in cancer cells and is recruited to cooperate with mutant p53 for tumor progression, TopBP1/mutant p53 interaction may be a new therapeutic target in cancer.

The aim of the study was to assess the microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression in ovarian serous carcinoma and to examine their relation with apoptosis.Paraffin-embedded specimens of 41 cases of ovarian serous carcinomas were evaluated by immunohistochemistry for VEGF, p53, and bcl-2 expression. MVD was assessed with CD31 staining. We investigated the association of tumor angiogenesis (MVD and VEGF) with clinicopathologic factors, p53 overexpression, and bcl-2 expression.There was a significant correlation between high MVD and suboptimal debulking and advanced stage disease. A significant negative correlation was expressed between bcl-2 and VEGF expression. In univariate analysis, only stage had a significant impact on disease-free survival.The results of this study suggest that higher degree of angiogenesis is associated with suboptimal debulking and advanced-stage disease. Expression of VEGF had negative association with VEGF expression.

The objective was to investigate the upstream apoptotic mechanisms that were triggered by a styrylpyrone derivative, goniothalamin (GTN), in tumor protein p53 (TP53)-positive and -negative hepatocellular carcinoma (HCC)-derived cells. Effects of GTN were evaluated by the flow cytometry, alkaline comet assay, immunocytochemistry, small-hairpin RNA interference, mitochondria/cytosol fractionation, quantitative reverse transcription-polymerase chain reaction, immunoblotting analysis and caspase 3 activity assays in two HCC-derived cell lines. Results indicated that GTN triggered phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, also known as NOXA)-mediated apoptosis via TP53-dependent and -independent pathways. In TP53-positive SK-Hep1 cells, GTN furthermore induced TP53 transcription-dependent and -independent apoptosis. After GTN treatment, accumulation of reactive oxygen species, formation of DNA double-strand breaks, transactivation of TP53 and/or PMAIP1 gene, translocation of TP53 and/or PMAIP1 proteins to mitochondria, release of cytochrome c from mitochondria, cleavage of caspases and induction of apoptosis in both cell lines were sustained. GTN might represent a novel class of anticancer drug that induces apoptosis in HCC-derived cells through PMAIP1 transactivation regardless of the status of TP53 gene., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Glioblastoma is the most malignant form of adult brain tumor and is associated with a dismal prognosis. Emerging data suggest that Notch signaling participates principally in the formation and malignant progression of glioblastoma. Resveratrol is a terpenoid that exhibits broad pro-apoptotic activity in various types of cancers, including glioblastoma. However, the effects of resveratrol on Notch signaling in glioblastomas have not yet been fully elucidated. We demonstrated that resveratrol strongly suppressed cell growth and induced apoptosis in A172 and T98G glioblastoma cells, which have low active Notch-1 expression and a heterozygous p53 mutation. Our results suggest that resveratrol significantly activates intracellular Notch-1 and restores wild-type p53 expression in a time-dependent manner. Significant de-phosphorylation of Akt, increased Bax expression, decreased Bcl-2 expression and cleavage of caspase-3 were also observed in resveratrol-induced apoptosis in glioblastoma cells. Moreover, simultaneous treatment with resveratrol and a Notch-1 inhibitor (MRK-003) partially attenuated the apoptosis and completely blocked the activation of Notch-1 and the increase in wild-type p53. This suggests that restoration of wild-type p53 expression depends on Notch-1 activation. In addition, the de-phosphorylation of Akt, increased expression of Bax and cleavage of caspase-3 were not fully reversed by MRK-003 treatment, suggesting that p53 restoration is not the only mechanism underlying resveratrol-induced apoptosis. Taken together, we confirmed the anti-proliferative and pro-apoptotic effects of resveratrol on glioblastoma cells and revealed Notch-1 activation-dependent restoration of p53 as an important causative mechanism.

Boldenone is an anabolic steroid developed for veterinary use. Recently, it is used by bodybuilders in both off-season and pre-contest, where it is well known for increasing vascularity while preparing for a bodybuilding contest. So, the present study was designed to investigate the possible effect of using growth promoter boldenone undecylenate on the rabbit liver tissue. Thirty-two adult New Zealand rabbits were divided into four groups (8 animals each). Control group includes animals that injected intramuscularly with olive oil and dissected after 3 weeks. The experimental groups include animals that receive one, two and three intramuscular injections of 5 mg/kg body weight boldenone, respectively. The animals were dissected after 3, 6 and 9 weeks respectively, where the interval of each dose of boldenon was 3 weeks. Small pieces of the liver tissues were sent for the histopathological examination. Apoptotic p53 and antiapoptotic Bc1-2 proteins were localized immunohistochemically. Histological observations of the liver tissue showed that the sinusoidal congestion was the most prominent feature that extended from the centrilobular to the periportal regions. Hepatocellular vacuolation in the centrilobular region was also detected. Liver immunohistochemical observation showed a significant increase of the apoptotic protein p53 and a significant decrease in the antiapoptotic Bc1-2 proteins. The highest frequency of p53 positive cells was observed in the liver sections of three dose of boldenone injections, while the lowest in control group, also the highest frequency of Bcl-2 positive cells was observed in the liver sections of control group while the lowest in three dose of boldenone injections. The present results investigate that people should be careful if they want to use such steroids to enhance their strength and endurance.

αβ and γδ lineage T cells are thought to arise from a common CD4(-)CD8(-) progenitor in the thymus. However, the molecular pathways controlling fate selection and maturation of these two lineages remain poorly understood. We demonstrated recently that a ubiquitously expressed ribosomal protein, Rpl22, is selectively required for the development of αβ lineage T cells. Germline ablation of Rpl22 impairs development of αβ lineage, but not γδ lineage, T cells through activation of a p53-dependent checkpoint. In this study, we investigate the downstream effectors used by p53 to impair T cell development. We found that many p53 targets were induced in Rpl22(-/-) thymocytes, including miR-34a, PUMA, p21(waf), Bax, and Noxa. Notably, the proapoptotic factor Bim, while not a direct p53 target, was also strongly induced in Rpl22(-/-) T cells. Gain-of-function analysis indicated that overexpression of miR-34a caused a developmental arrest reminiscent of that induced by p53 in Rpl22-deficient T cells; however, only a few p53 targets alleviated developmental arrest when individually ablated by gene targeting or knockdown. Co-elimination of PUMA and Bim resulted in a nearly complete restoration of development of Rpl22(-/-) thymocytes, indicating that p53-mediated arrest is enforced principally through effects on cell survival. Surprisingly, co-elimination of the primary p53 regulators of cell cycle arrest (p21(waf)) and apoptosis (PUMA) actually abrogated the partial rescue caused by loss of PUMA alone, suggesting that the G1 checkpoint protein p21(waf) facilitates thymocyte development in some contexts.

Aims: Solamargine, a steroidal glycoalkaloid isolated from S. incanum, has been shown to induce apoptosis in several cancer cell lines. In this study, the involvement of p53 in the pro-apoptotic action of solamargine was investigated in human osteosarcoma U2OS cells., Main Methods: The cytotoxicity of solamargine was evaluated by MTT assay. Solamargine-induced apoptosis was evidenced by chromatin condensation, formation of apoptotic bodies and exposure of phosphatidylserine on the extracellular surface as revealed by DAPI nuclear staining, transmission electron microscopy and flow cytometry, respectively. mRNA expressions of p53 and Bax were investigated using real time-PCR. Western blot was used to examine the changes in the expression levels of p53, Bax, Bcl-2, caspase-3, caspase-9 and cytochrome c. Subcellular localization of p53 was verified by immunofluorescence staining and cell fractionation., Key Findings: Solamargine substantially reduced cell viability and induced apoptosis in osteosarcoma U2OS cells. In this connection, solamargine increased the mRNA and protein expressions of p53 and Bax (a pro-apoptotic protein downstream to p53). The expression of Bcl-2 (an anti-apoptotic protein) was also reduced. Furthermore, solamargine induced mitochondrial translocation of p53, loss of mitochondrial membrane potential, cytochrome c release and activation of caspase-9 and -3. p53-specific transcriptional inhibitor pifithrin-α or mitochondrial translocation inhibitor pifithrin-μ partially reversed solamargine-induced apoptosis., Significance: Solamargine activates the mitochondria-mediated apoptotic pathway in U2OS cells via both p53 transcription-dependent and -independent mechanisms. This compound may merit further investigation as a potential therapeutic agent for the treatment of cancer., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background/aims: Coumarins are natural compounds found in many plants that possess medical value by itself and its modified derivatives., Method: Six novel coumarin derivatives were synthesized and examined for their potential anticancer cytotoxicity., Result: Among the 6 derivatives, 3,5-dimethyl-(7)H-furo[3,2-g]chromen-7-one (DMFC) presented the strongest cytotoxicity against human hepatoma HepG2 cells in vitro with an IC(50) value of 8.46 ± 0.28 μM in a 48-hour treatment. Further experiments revealed that DMFC induced apoptosis in HepG2 cells through both extrinsic and intrinsic apoptotic pathways in a p53-dependent manner. Mechanistically, DMFC activated caspases 3, 8 and 9, depolarized mitochondrial membrane potential and induced cytochrome c and apoptosis-inducing factor release. DMFC-induced apoptosis was also characterized by DNA fragmentation, phosphatidylserine externalization and sub-G1 peak in DNA histograms. Moreover, both caspase 8 and 9 inhibitors suppressed the apoptosis induced by DMFC. Western blot analyses revealed that DMFC also significantly increased the expression levels of p53, Fas death receptor, Fas-associated death domain protein and proapoptotic Bcl-2 family members such as Bax, Bad and tBid, as well as decreased the levels of pro-survival members such as Bcl-2 and Bcl-xl., Conclusion: DMFC is potentially an effective therapeutic agent in liver cancer therapy., (Copyright © 2011 S. Karger AG, Basel.)

Aim: We investigated the angiogenesis and density of newly formed blood vessels in embryonal tumors in relation to Ki-67, bcl-2, p-53 and p-27 expression., Methods: Forty-five children with embryonal tumors were enrolled in the study. Forty patients had a medulloblastoma (MB) and 5 patients had atypical teratoid/rhabdoid tumor (AT/RT)., Results: In MB, the 5-year PFS and OS was 62.5 and 70%, respectively. Patients with Ki-67 index >50%, bcl-2 index >30% and higher density of new vessels were associated with worse survival. In the multivariate analysis, Ki-67 index was identified as a factor with independent prognostic power. In AT/RTs, high density of new vessels (>25 HRF) was observed in 3 patients and Ki-67 index over 25% was found in 4 patients., Conclusion: Increased Ki-67, bcl-2 and density of new vessels are of prognostic value for the disease outcome in MB., (Copyright © 2012 S. Karger AG, Basel.)

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumors including malignant glioblastoma. The mechanism of TMZ-induced glioblastoma cell death and apoptosis, however, is not fully understood. Here, we tested the potential involvement of AMP-activated protein kinase (AMPK) in this process. We found that methylating agents TMZ and N-methyl-N'-nitro-N-nitrosoguanidine induce AMPK activation in primary cultured human glioblastoma and glioblastoma cell lines. TMZ-induced O(6)-methylguanine production is involved in AMPK activation. O(6)-benzylguanine, an O(6)-methylguanine-DNA methyltransferase inhibitor, enhances TMZ-induced O(6)-methylguanine production, leading to enhanced reactive oxygen species production, which serves as an upstream signal for AMPK activation. Activation of AMPK is involved in TMZ-induced glioblastoma cell death and apoptosis. AMPK inhibitor (Compound C) or AMPKα siRNA knockdown inhibits TMZ-induced glioblastoma cell death and apoptosis, whereas AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside enhances it. In further studies, we found that activation of AMPK is involved in TMZ-induced p53 activation and subsequent p21, Noxa, and Bax up-regulation. Activation of AMPK by TMZ also inhibits mTOR complex 1 (mTORC1) signaling and promotes anti-apoptosis protein Bcl-2 down-regulation, which together mediate TMZ-induced pro-cell apoptosis effects. Our study suggests that activation of AMPK by TMZ contributes to glioblastoma cell apoptosis, probably by promoting p53 activation and inhibiting mTORC1 signaling.

The purpose of the study was to determine the frequency of expression p53 and p16INK4a proteins and bcl-2 oncoprotein in malignant skin melanoma and to determine their correlation with the proliferative index and tumor thickness. The study involved 53 patients: 27 (51%) male and 26 (49%) female. Mitotic index showed a correlation with p53 protein expression, a negative correlation with p16INK4a protein expression. Statistically significant correlations were determined between the Breslow tumor thickness, Clark invasion level and p53 protein expression, as well as Breslow tumor thickness and bcl-2 oncoprotein expression (p<0.05), whereas there was no correlation between the p16INK4a protein expression and melanoma thicknes and Clark invasion level. Overexpression p53 protein and bcl-2 oncoprotein, with the loss p16INK4a protein of expression in the nodular melanoma, confirms a frequent loss of function of these tumor suppressor gene and oncogene, and indicates a vertical tumor growth phase. The loss of tumor suppression function the p53 protein and bcl-2 oncoprotein overexpression in cutaneous melanoma correlates with larger tumor thickness, whereas the overexpression of mutated p53 protein and loss p16INK4a protein of expression indicate a higher proliferative tumour potential. Therefore, these evaluated proteins may be the aggressive biological tumour activity markers.

The etiology of heart failure in dilated cardiomyopathy involves multiple agents. The purpose of this study was to investigate the presence of apoptosis-related proteins p53, bcl-2, and the defects of force transmission in end-stage dilated cardiomyopathy. We studied myocardial samples from 20 hearts with histologic findings of dilated cardiomyopathy. Myocardial samples obtained from 10 normal hearts were used as controls. An immunohistochemical method was performed with the use of desmin, N-cadherin, p53, and bcl-2 antibodies. The expression of desmin and N-cadherin was much more pronounced in dilated cardiomyopathy, and both of them were arranged disorderly. On the other hand, increased expression of p53 is associated with progressive loss of myocytes by apoptosis in heart failure, and increased expression of bcl-2 represents a possible compensatory antiapoptotic mechanism. The increased amount and the irregular distribution of desmin and N-cadherin in dilated cardiomyopathy may compensate for the loss of cellular stability due to the loss of contractile material. These alterations contribute to the deterioration of contractile function in heart failure. Furthermore, the prevalence of an apoptotic or compensatory antiapoptotic mechanism may influence the evolution of heart failure in dilated cardiomyopathy., (Copyright 2010 Elsevier GmbH. All rights reserved.)

Background/aims: The endoscopic and histologic findings of gastroesophageal reflux disease are usually indistinct. The current study was designed to define accurately the histology in gastroesophageal reflux disease and to develop a hypothesis that reflux produces immunohistochemical changes., Methods: The study was based on the examination of endoscopic esophageal biopsy specimens obtained from 20 patients with evidence of reflux with 24-hour pH-meter monitoring and from 20 control subjects without clinical or endoscopic reflux. The pathogenesis of reflux esophagitis was discussed by comparing the histopathologic changes with determined Ki-67, p53 and Bcl-2 immunoreactivity., Results: In this study, the presence of esophagitis was determined endoscopically in only 55% of the patients with gastroesophageal reflux disease, while microscopic esophagitis was detected in 60% of them. No correlation was found between presence of endoscopic esophagitis and microscopic esophagitis in the patients with gastroesophageal reflux disease. There was a significant difference between control cases and the patients according to histological parameters, which included basal activity (p=0.006), height of papillae (p=0.006), intraepithelial neutrophils (p=0.000), intraepithelial eosinophils (p=0.006), congestion (p=0.001), and dilated intercellular spaces (p=0.006). Immunohistochemically, there was a significant difference in the expression of p53 and Ki-67 between the three study groups (patients with/without microscopic esophagitis, controls) (p<0.05). However, there was no difference in Bcl-2 between the patients with reflux and control cases., Conclusions: In this study, we considered that microscopic esophagitis does not always accompany reflux, and the lack of reliable diagnostic histologic criteria is still a serious problem for pathologists. Immunohistochemically, an increase in cell proliferative activity and p53 protein accumulation to repair oxidative DNA damage related to reflux were observed. However, the close Bcl-2 immunoreactivity in all groups that was indicated by a weak positivity suggests that the inhibition of apoptosis may not be involved in reflux esophagitis.

Apoptosis-related proteins play an important role in lymphoma cell death during chemotherapy. In our study, we investigated the prognostic significance of CD95, BCL2, and P53 expression in extranodal non-Hodgkin's lymphoma (NHL). We examined 71 patients with extranodal NHL [45 diffuse large B-cell lymphomas (DLBCLs) and 26 mucosa-associated lymphoid tissue lymphomas (MALTLs)], 35 male and 36 female, with a median age of 65.8 years. The most common site of origin was the stomach (N = 31). Paraffin-embedded specimens were analyzed immunohistochemically for CD95, BCL2, and P53 expression. Multivariate analysis revealed that in DLBCLs, positive CD95 and negative BCL2 expression were independent prognostic factors for overall survival. We reached the same conclusion for MALTLs, with positive CD95 and negative P53 expression. In DLBCLs, the 5-year overall survival rate was 71.5% for the CD95-positive cases and 35% for the CD95-negative cases (p = 0.004) and the 5-year overall survival was significantly better in BCL2-negative cases (70.8%) when compared to BCL2-positive cases (37%; p = 0.009). In MALTLs, the 5-year overall survival rate for the CD95-positive and CD95-negative groups was 89.5% and 42.9%, respectively (p = 0.004) and the 5-year overall survival rate was 50% for the P53-positive cases and 88.9% for the P53-negative cases (p = 0.016). In conclusion, positive CD95 expression proved to be a good prognostic factor of overall survival in both extranodal DLBCLs and MALTLs. In contrast, positive expression of BCL2 and P53 was found to be unfavorably associated with survival in extranodal DLBCLs and MALTLs, respectively.

We have previously described a mechanism through which the high-mobility group A1 (HMGA1) proteins inhibit p53-mediated apoptosis by delocalizing the p53 proapoptotic activator homeodomain-interacting protein kinase 2 from the nucleus to the cytoplasm. By this mechanism, HMGA1 modulates the transcription of p53 target genes such as Mdm2, p21(waf1), and Bax, inhibiting apoptosis. Here, we report that HMGA1 antagonizes the p53-mediated transcriptional repression of another apoptosis-related gene, Bcl-2, suggesting a novel mechanism by which HMGA1 counteracts apoptosis. Moreover, HMGA1 overexpression promotes the reduction of Brn-3a binding to the Bcl-2 promoter, thereby blocking the Brn-3a corepressor function on Bcl-2 expression following p53 activation. Consistently, a significant direct correlation between HMGA1 and Bcl-2 overexpression has been observed in human breast carcinomas harboring wild-type p53. Therefore, this study suggests a novel mechanism, based on Bcl-2 induction, by which HMGA1 overexpression contributes to the escape from apoptosis leading to neoplastic transformation., (Copyright 2010 AACR.)

Objective: Studies exploring roles of p53 and bcl-2 in neuroprotection by hypothermia in focal cerebral ischemia have not provided consistent results. In the present study, we determined whether p53 and bcl-2 are involved in the hypothermia-induced neuroprotection., Methods: Male Sprague-Dawley rats were divided into four groups: normothermic (37-38 degrees C) ischemia, hypothermic (31-32 degrees C) ischemia, hyperthermic (41-42 degrees C) ischemia and sham-operated group. Global cerebral ischemia was established for 20 minutes using the Pulsinelli four-vessel occlusion model and the brain temperature was maintained at defined levels for 60 minutes following the 20 min ischemia. The mortality in rats was evaluated at 72 hour and 168 hour reperfusion. The expression of p53 and bcl-2 proteins was detected at 24, 48 and 72 hours after reperfusion. At the same intervals, neuron necrosis and apoptosis in brain regions was also detected using hematoxylin and eosin (HE) staining and terminal deoxynucleotldyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL)., Results: The mortalities of rats in normothemia, hypothermia and hyperthermia groups was 33.3, 16.7 and 50% at 72 hour reperfusion. At 168 hours of reperfusion, the mortality in the three groups was 58.3, 25 and 100%, respectively. In light microscopy studies, necrotic neurons and apoptotic neurons were found in the hippocampus after global cerebral ischemia. Surviving neurons in hippocampus was increased in mild hypothermic ischemia group (p<0.05) and decreased in hyperthermia ischemia group (p<0.01) at 24, 48 and 72 hour reperfusion. TUNEL-positive neurons in hippocampus decreased in hypothermic ischemia group (p<0.05 or p<0.01) and increased in hyperthermic ischemia group (p<0.01) at 24, 48 and 72 hour reperfusion. The expression of p53 and bcl-2 proteins was found in the neurons of cerebral cortex after global cerebral ischemia. P53 decreased and bcl-2 increased in hypothermia group., Conclusion: Hypothermia reduces ischemic neuronal necrosis and apoptosis by reducing p53 and increasing bcl-2 expression. Hyperthermia accelerated ischemic neuronal injury by increasing p53 and reducing bcl-2 expression.