Akt promotes cisplatin resistance in human ovarian cancer cells through inhibition of p53 phosphorylation and nuclear function

Resistance to cisplatin-based chemotherapy is a major cause of treatment failure in human ovarian cancer. Wild-type TP53 status is often, but not always, associated with cisplatin sensitivity, suggesting that additional factors may be involved. Overexpression/activation of the phosphatidylinositol-3-kinase/Akt pathway is commonly observed in ovarian cancer, and Akt activation is a determinant of chemoresistance in ovarian cancer cells, an effect that may be due, in part, to its inhibitory actions on p53-dependent apoptosis. To that end, we examined the role and regulation of p53 in chemosensitive ovarian cancer cells, as well as in their chemoresistant counterparts, and investigated if and how Akt influences this pathway. Cisplatin induced apoptosis in chemosensitive, but not chemoresistant cells, and this was inhibited by downregulation of p53. Cisplatin upregulated PUMA in a p53-dependent manner, and the presence of PUMA was necessary, but not sufficient for cisplatin-induced apoptosis. p53 was phosphorylated on numerous N-terminal residues, including Ser15, Ser20, in response to cisplatin in chemosensitive, but not chemoresistant cells. Furthermore, activation of Akt inhibited the cisplatin-induced upregulation of PUMA, and suppressed cisplatin-induced p53 phosphorylation, while inhibition of Akt increased total and phospho-p53 contents and sensitized p53 wild-type, chemoresistant cells to cisplatin-induced apoptosis. Finally, mutation of Ser15 and/or Ser20, but not of Ser37, to alanine significantly attenuated the ability of p53 to facilitate CDDP-induced apoptosis, and this was independent of PUMA expression. These results support the hypothesis that p53 is a determinant of CDDP sensitivity, and suggest that Akt contributes to chemoresistance, in part, by attenuating p53-mediated PUMA upregulation and phosphorylation of p53, which are essential, but independent determinants of sensitivity to CDDP-induced apoptosis.     

曲古抑菌素A通过p53转录激活途径诱导尤文肉瘤细胞凋亡

目的：探讨组蛋白去乙酰化酶抑制剂曲古抑菌素A（trichostatin A,TSA）诱导尤文肉瘤细胞株WE-68和VH-64凋亡及作用机制.方法：四甲基偶氮唑蓝法（MTT法）测定细胞增殖抑制率.流式细胞计数法测量TSA给药后细胞周期中sub-G1含量的变化.免疫印迹法（Western-blot）检测细胞中活化型多聚ADP核糖多聚酶（cleaved-PARP）,p53-lys382残基乙酰化和p53蛋白总量的表达.实时定量PCR和siRNA转染技术测定p53多个下游基因的mRNA水平改变和p53表达下调对TSA诱导凋亡的影响.结果：TSA抑制了尤文肉瘤细胞的增殖,诱导细胞周期中sub-G1含量和凋亡终产物cleaved-PARP蛋白表达的增加.TSA给药后,p53-lys382残基乙酰化表达量呈浓度依存性增加,同时上调p53下游因子p21,mdm2,Bax和PUMA的mRNA水平.另一方面,p53蛋白表达的下调明显削弱了TSA介导的p21表达的上调和cleaved-PARP的产生.结论：组蛋白去乙酰化酶抑制剂TSA能够通过激活p53高乙酰化表达来恢复p53转录功能,从而诱导尤文肉瘤细胞株产生凋亡.     

p53 and redox state in etoposide-induced acute myeloblastic leukemia cell death

We investigated whether p53, being a redox-sensitive protein, has a role in the responsiveness of AML cells to etoposide. Two subclones of the OCI/AML-2 cell line, the etoposide-sensitive (ES) and the etoposide-resistant (ER), were used as models. Sensitivity to etoposide was measured by trypan blue and annexin V assays. Etoposide-induced peroxide formation was associated with the induction of cell death. Evident expression of mutated p53 was observed in both subclones in basal growth conditions as analysed by Western blotting and flow cytometry. After etoposide exposure for up to 24 hours, some nuclear accumulation of p53 was observed in the ER subclone, as analysed by Western blotting. The conformation of p53, however, was not changed from mutated toward wild-type during exposure in either of the subclones as analysed by flow cytometry. In conclusion, etoposide-induced change in cellular redox state was associated with apoptosis, but was not a sufficient stimulus for p53 to make its conformation active. Thus, mutated p53 seems to have no role in etoposide-induced apoptosis.     

Chloroquine-induced autophagic vacuole accumulation and cell death in glioma cells is p53 independent

Glioblastoma (GBM) is a high-grade central nervous system malignancy and despite aggressive treatment strategies, GBM patients have a median survival time of just 1 year. Chloroquine (CQ), an antimalarial lysosomotropic agent, has been identified as a potential adjuvant in the treatment regimen of GBMs. However, the mechanism of CQ-induced tumor cell death is poorly defined. We and others have shown that CQ-mediated cell death may be p53-dependent and at least in part due to the intrinsic apoptotic death pathway. Here, we investigated the effects of CQ on 5 established human GBM lines, differing in their p53 gene status. CQ was found to induce a concentration-dependent death in each of these cell lines. Although CQ treatment increased caspase-3–like enzymatic activity in all 5 cell lines, a broad-spectrum caspase inhibitor did not significantly attenuate death. Moreover, CQ caused an accumulation of autophagic vacuoles in all cell lines and was found to affect the levels and subcellular distribution of cathepsin D, suggesting that altered lysosomal function may also play a role in CQ-induced cell death. Thus, CQ can induce p53-independent death in gliomas that do not require caspase-mediated apoptosis. To potentially identify more potent chemotherapeutics, various CQ derivatives and lysosomotropic compounds were tested on the GBM cells. Quinacrine and mefloquine were found to be more potent than CQ in killing GBM cells in vitro and given their superior blood–brain barrier penetration compared with CQ may prove more efficacious as chemotherapeutic agents for GBM patients.     

Age dependent phosphorylation and deregulation of p53 in human vestibular schwannomas

Tumor-specific alterations at the p53 gene locus in 30 human vestibular schwannomas (VS) comprising 10 confirmed NF2 cases and 20 sporadic cases were analyzed. We found loss of heterozygosity (LOH) at the first intron of the p53 gene locus in 54% of the informative cases. This is the first report showing LOH at the p53 gene locus in a significant number of human VS and both sporadic and NF2 cases show the LOH event. Increased levels of normal size p53 mRNA and p53 protein were found in all the tumors analyzed. Thus p53 appears to be deregulated in all the tumors suggesting that p53 alterations may be associated with tumor progression in VS. There was a negative significant correlation of patients' age and percentage of Ser 392 phosphorylated p53 protein. The tumor samples obtained from younger patients of 35 yr and below showed higher percentage of Ser 392 phosphorylated p53 protein compared to the tumors of older patients. The increased percentage of Ser 392 phosphorylated p53 protein indicates that it could be involved in the acceleration of tumor growth in the younger patients. Our results suggest that age dependent phosphorylation of p53 protein and deregulation of p53 gene has a role in the development of human vestibular schwannomas.     

Anti-diol epoxide of benzo[a]pyrene induces transient Mdm2 and p53 Ser15 phosphorylation, while anti-diol epoxide of dibenzo[a,l]pyrene induces a nontransient p53 Ser15 phosphorylation

The polycyclic aromatic hydrocarbons (PAHs) dibenzo[a,l]pyrene (DBP) and benzo[a]pyrene (BP) are environmental contaminants and potent carcinogens. DBP is several orders of magnitude more mutagenic/carcinogenic than BP. This can be ascribed to differences in DNA binding efficiency of their ultimate carcinogenic bay- and fjord-region diol epoxide (DE) intermediates, differences in structural features of the DNA adducts and differences in DNA adduct recognition and the subsequent downstream signaling. In this study, we have characterized the effect of the ultimate carcinogenic DEs, (+)-anti-BPDE and (-)-anti-DBPDE following short exposure times, on Mdm2 and p53 pathway in A549 human lung epithelial carcinoma cells. In contrast to (-)-anti-DBPDE, (+)-anti-BPDE induces stabilization of phosphorylated Mdm2. (+)-anti-BPDE-induced effects on Mdm2 were transient and correlated with transient p53 Ser15 phosphorylation. DNA adducts of (-)-anti-DBPDE are more refractory to removal by nucleotide excision repair (NER) than adducts of (+)-anti-BPDE and do not induce Mdm2 phosphorylation. This suggests a role of phosphorylated Mdm2 in the repair process. In addition, (-)-anti-DBPDE, in contrast to (+)-anti-BPDE, induced prolonged p53 Ser15 phosphorylation as well as phosphorylation of p53 at Ser46, a phosphorylation site associated with apoptosis. It is also concluded that p53 Ser15 phosphorylation and antibody 2A10-site specific Mdm2 alterations are induced by nonidentical signaling pathways by the bay- and fjord-region DE. These differences may reflect the different carcinogenic potential of these compounds.     

Secretory expression of p53(N15)-Ant following lentivirus-mediated gene transfer induces cell death in human cancer cells

p53 (N15)-Ant 32-peptide has been considered a novel and effective peptide for cancer therapy. To further enhance its anticancer effect and overcome the limitation of peptide therapy, a recombinant lentivirus was constructed in this study with the following strategies: the secretory expression of therapeutic peptide and lentivirus gene transfer system. The results demonstrated that LV-NT4(Si)-p53 (N15)-Ant could significantly suppress cell growth and induce rapid cell death in MCF-7 (overexpressed wild-type p53), HepG2(wide-type p53), OVCAR-3 (mutant type p53) and H1299 (null p53)cells in time-dependent manners through successful gene transfer and secretory expression of therapeutic peptide at 48 h post-infection. Transmission electron microscopy and flow cytometric analysis revealed that LV-NT4(Si)-p53 (N15)-Ant could induce two different kinds of cell death (necrosis and apoptosis) by two different mechanisms, since p53 (N15)-Ant peptide has the potential of blocking interaction of mdm-2 with other protein target, and on the other hand, it could form S-shape helix-loop-helix structures, which is able to rapidly disrupt cancer cell membranes. Based on these finding, LV-NT4 (Si)-p53 (N15)-Ant may be a novel recombinant virus because it induces cell death by two different pathways.     

p53在调控DNA损伤所致MDA-MB-231细胞死亡中的作用及其机制

目的：研究p53在调控DNA损伤所致乳腺癌MDA-MB-231细胞死亡中发挥的作用及其相关机制。方法：采用5 J/m²短波紫外线UVC体外照射MDA-MB-231细胞建立DNA损伤模型,通过Western blot检测磷酸化H2AX以鉴定DNA损伤程度,并采用Westernblot检测细胞死亡相关蛋白p21、PARP、磷酸化p53和p53,以及核因子NF-90表达的变化。结果：与对照组比较,5 J/m² UVC处理细胞0.5 h后即检测到明显的H2AX磷酸化（P < 0.05）,表明成功建立了DNA损伤模型;同时,p21发生降解并持续保持低表达状态,p53开始发生磷酸化（p-p53增加,P < 0.05）,处理8 h后观察到PARP的剪切增加（P < 0.05）,而p53和NF-90蛋白表达未发现明显改变。结论：MDA-MB-231细胞通过p21-PARP途径发生死亡,而磷酸化p53的增加则可以促进细胞存活,从而抑制DNA损伤引起的细胞死亡。     

p53与化疗敏感性

本文归纳了p53涉及化疗敏感性的功能,汇总了近年来p53与化疗敏感性有关的基础和临床研究资料,阐述了p53调节化疗敏感性的可能机制,展望了对基因预测化疗敏感性的研究和应用前景.     

The predictive value of p53, p53R2, and p21 for the effect of chemoradiation therapy on oesophageal squamous cell carcinoma

The p53 family regulates cell-cycle arrest, triggers apoptosis or is involved in repair of DNA damage. In the present study, we analysed the expression of some p53 family proteins and their responses to chemoradiation therapy (CRT) in cases of oesophageal squamous cell carcinoma (ESCC). We immunohistochemically investigated the relationship between p53, p53R2, and p21 expression in biopsy specimens of untreated primary tumours and their clinical and histological responses to CRT in 62 patients with ESCC. Chemoradiation therapy consisted of 5-fluorouracil plus cisplatin and 40 Gy of radiation. The rates of clinical and histological responses (complete or partial) to CRT were 71.0% (clinical) and 52.8% (histological). The rate of positive expression was 43.5% for p53, 37.1% for p53R2, and 54.8% for p21 expression. Statistically significant correlations were found between p53 or p53R2 expression and favourable response to CRT (P=0.0001 or 0.041 clinical, P=0.016 or 0.0018 histological, respectively). Furthermore, in p53-negative tumours, CRT was more effective in tumours with p53R2 negative expression than those with p53R2 positive expression (P=0.0014). We demonstrated that the negative expression of p53 and p53R2 expression was closely related to the effect of CRT and should predict the CRT outcome in patients with ESCC.     

p53蛋白表达和基因突变在所谓肺硬化性血管瘤组织中的意义

背景与目的所谓肺硬化性血管瘤（so-called pulmonary sclerosing hemangioma，PSH）是一种至今不能肯定其来源及性质的少见肺肿瘤。虽然目前普遍认为PSH是一种良性肿瘤，但却发现有少数PSH可发生浸润和转移。p53蛋白表达和基因突变是反映肿瘤生物学行为的有用指标。本研究的目的是检测PSH组织中p53基因突变及p53蛋白的表达情况及其意义。方法应用免疫组化S-P法、激光捕获显微切割（laser capture microdissection，LCM）技术、单链构象多态性（single-stranded conformation polymorphism，SSCP）及DNA测序分析方法（5～8外显子）检测19例PSH中多角形细胞和表面立方细胞的p53蛋白表达和基因突变情况。结果19例PSH组织中p53蛋白阳性表达率为21．1％（4／19），SSCP及测序分析检测p53基因突变率分别为26．3％（5／19）和42．1％（8／19）。p53蛋白阳性表达的4例PSH组织，测序分析显示3例标本有突变发生，2例为错义突变，1例同时发生错义突变和移码突变；而p53蛋白阴性表达的15例PSH组织中，5例标本经测序证实亦发生了突变，4例为移码突变，1例为错义突变。在有突变的8例PSH组织中，单一多角形细胞突变5例，单一立方细胞突变2例，多角形细胞和立方细胞同时突变1例。结论PSH组织p53蛋白阳性表达并不能完全反映p53基因突变的真实情况；p53基因突变或蛋白表达异常均可发生在PSH组织中的两种不同的细胞内；p53基因的高突变率提示PSH可能具有潜在的恶性生物学行为。     

ＰＴＴＧ和ｐ５３在非小细胞肺癌中的表达及其意义

目的：探讨非小细胞肺癌（ＮＳＣＬＣ）组织中垂体肿瘤转化基因（ＰＴＴＧ）和ｐ５３的表达及其与ＮＳＣＬＣ发生、发展之间的关系。方法：用免疫组化法测定６１例ＮＳＣＬＣ手术切除的癌组织标本及２０例正常肺组织中ＰＴＴＧ、ｐ５３的表达。结果：ＮＳＣＬＣ组织中ＰＴＴＧ、ｐ５３的表达率分别为６８.８５％和７３.７７％,明显高于正常肺组织的１５.００％和１０.００％（Ｐ均＜０.０１）;在ＮＳＣＬＣ不同组织学类型间,其表达率差异无统计学意义（Ｐ＞０.０５）;在ＮＳＣＬＣ不同分化程度之间,其表达率差异有统计学意义（Ｐ＜０.０５）;在ＮＳＣＬＣ是否伴淋巴结转移之间,其表达率差异亦有统计学意义（Ｐ＜０.０５）。ＰＴＴＧ与ｐ５３的表达率呈显著正相关（Ｐ＜０.０５）。结论：ＰＴＴＧ、ｐ５３在ＮＳＣＬＣ组织中有较高的表达,有可能作为评估预后、指导治疗的参考指标。     

Hibiscus polyphenol-rich extract induces apoptosis in human gastric carcinoma cells via p53 phosphorylation and p38 MAPK/FasL cascade pathway

In view of the continuing need for effective anticancer agents, and the association of diet with reduced cancer risk, edible plants are increasingly being considered as sources of anticancer drugs. Hibiscus sabdariffa Linne (Malvaceae), an attractive plant believed to be native to Africa, is cultivated in the Sudan and Eastern Taiwan. Polyphenols had been demonstrated previously to possess antioxidative and antitumor promoting effects. In this study, investigations were conducted to examine the mechanism of the anticancer activity of H. sabdariffa L., Hibiscus polyphenol-rich extracts (HPE). Using HPLC assay, HPE was demonstrated to contain various polyphenols. HPE induced cell death of eight kinds of cell lines in a concentration-dependent manner. Among them human gastric carcinoma (AGS) cells were the most susceptible to HPE (0.95 mg/mL HPE inhibited its growth by 50%). Our results revealed that AGS cells underwent DNA fragmentation, and had an increase in the distribution of hypodiploid phase (apoptotic peak, 52.36%) after a 24-h treatment with HPE (2.0 mg/mL). This effect of HPE in AGS cells might be mediated via p53 signaling and p38 MAPK/FasL cascade pathway, as demonstrated by an increase in the phosphorylation of p53 and the usage of a specific p38 inhibitor, SB203580. Thus, our data present the first evidence of HPE as an apoptosis inducer in AGS cells and these findings may open interesting perspectives to the strategy in human gastric cancer treatment.     

Aurora-A induces cell survival and chemoresistance by activation of Akt through a p53-dependent manner in ovarian cancer cells

Aurora-A is frequently altered in epithelial malignancies. Overexpressing Aurora-A induces centrosome amplification and G2/M cell cycle progression. We have previously shown elevated level of Aurora-A in ovarian cancer and activation of telomerase by Aurora-A in human mammary and ovarian epithelia. Here we report that Aurora-A protects ovarian cancer cells from apoptosis induced by chemotherapeutic agent and activates Akt pathway in a p53-dependent manner. Ectopic expression of Aurora-A renders cells resistant to cisplatin (CDDP), etoposide and paclitaxel-induced apoptosis and stimulates Akt1 and Akt2 activity in wild-type p53 but not p53-null ovarian cancer cells. Aurora-A inhibits cytochrome C release and Bax conformational change induced by CDDP. Knockdown of Aurora-A by RNAi sensitizes cells to CDDP-induced apoptosis and decreases phospho-Akt level in wild-type p53 cells. Reintroduction of p53 decreases Akt1 and Akt2 activation and restores CDDP sensitivity in p53-null but not p53-null-Aurora-A cells. Inhibition of Akt by small molecule inhibitor, API-2, overcomes the effects of Aurora-A-on cell survival and Bax mitochondrial translocation. Taken collectively, these data indicate that Aurora-A activates Akt and induces chemoresistance in a p53-dependent manner and that inhibition of Akt may be an effective means of overcoming Aurora-A-associated chemoresistance in ovarian cancer cells expressing wild-type p53.     

p53 Expression in Non-Hodgkin's Lymphomas: A Marker of p53 Inactivation?

The p53 gene located in the short arm of chromosome 17 at position 17p13, is involved in the negative regulation of cellular growth. p53 mutation seems to be the most frequent genetic alteration found in human cancer. Mutant conformation of the p53 gene is associated with cell proliferation and tumour progression, and in most cases implies p53 stabilization, which renders the p53 protein detectable through the use of immunohistochemical techniques. p53 expression is a frequent finding in high grade lymphomas of either B or T cell lineage, having been detected in 30% of cases in our series. The focal presence of p53+ cells was seen in a wide range of low and high grade lymphomas, including lymphadenitis and reactive tonsils.

In 37.5% of cases this increased expression of p53 was secondary to mutation in highly conserved regions (exons 5-8). Unlike findings reported in other tumours, in lymphomas, p53 expression seems to be secondary to genetic alterations other than p53 mutation. Initial data suggest that the MDM2 protein could be involved in inactivating p53 protein in most of these cases.

Finally, p53 expression has been found to be a poor prognostic marker in high grade B-cell lymphomas in a large series of cases. High p53 expression was associated with a short survival, this relation being stronger in cases with simultaneous bcl2 expression.     

p53与血管生长抑素基因共转染对SG7901细胞生长的抑制作用

背景与目的：p53基因为抑癌基因，血管生长抑素（angiostatin，Angio，AS）基因能抑制血管内皮细胞生长和血管新生，间接抑制肿瘤的生长。本研究探讨以pV1TRO2质粒为载体共转染野生型人p53和人Angio基因（pV1TRO2-hp53-hAngio）对人胃癌细胞生长的影响。方法：用脂质体转染法将pV1TRO2-hp53-h-Angio转染人胃癌细胞系SG7901。以RT—PCR法检测转染后细胞目的基因的表达，通过细胞集落形成试验、MTT生长曲线、流式细胞仪调亡检测观察其生物学特性变化。结果：野生型p53基因或AS基因转染后均能抑制转染细胞的生长（细胞集落形成试验及MMT，P〈0．05），而共转染两种基因的细胞生长抑制诱导凋亡效果优于单个甚因转染（细胞集落形成试验及MMT，P〈0．05）。结论：p53和AS基因具有协同抑制肿瘤细胞生长作用，提示联合基因可能有利于肿瘤的基因治疗。     

非小细胞肺癌的CT增强值与p53表达的关系研究

目的:探索非小细胞肺癌中CT增强值与微血管密度及突变型p53表达的关系.方法:对54例非小细胞肺癌进行了CT扫描和因子、p53表达的免疫组化染色(S-P法),因子用来标记微血管内皮细胞.结果:54例非小细胞肺癌(NSCLC)中,CT增强值与微血管密度(MVD)呈正相关,γ=0.631 3;p53阳性组的平均MVD、平均CT增强值显著高于p53阴性组,P=0.007、P=0.000.结论:非小细胞肺癌的CT增强值与微血管密度有关,也与p53表达有关.     

Expression of p53R2, newly p53 target in oral normal epithelium,epithelial dysplasia and squamous cell carcinoma

Recently, the p53R2 gene has been isolated and shown to play a crucial role in DNA repair after DNA damage. The p53R2 gene encodes the p53 inducible ribonucleotide reductase small subunit 2 homologue, which is part of the p53 pathway. However, the function of p53R2 in human cancer is still unclear. We investigated p53R2 mRNA expression in human oral normal epithelium, epithelial dysplasias and squamous cell carcinomas (SCCs). Surgical or biopsy-proven specimens of 10 normal epithelium, 48 epithelial dysplasias and 63 SCCs were collected in our department. Then, p53R2 was identified by in situ hybridization to visualize and localize the expression of specific mRNAs. The authors examined the p53 gene mutation by polymerase chain reaction-single strand conformation polymorphism analysis. p53, mdm2, p21^WAF1/CIP1 and Ki-67 expression was detected by immunohistochemistry. p53R2 expression was detected in none of ten normal epithelium (0%), ten of 48 dysplasias (20.8%) and 33 of 63 SCCs (52.4%). In oral SCC, the expression of p53R2 was significantly associated with tumor size, lymph node metastasis and histological differentiation (P=0.014, 0.046 and 0.022, respectively). p53R2 expression was significantly associated with p53 abnormality in epithelial dysplasia and SCC (P=0.034 and 0.009, respectively). Of 63 patients, 37 received preoperative radiochemotherapy. p53R2 mRNA expression was significantly associated with the pathologic response to radiochemotherapy (P=0.031). This study suggested that p53R2 expression could be associated with oral carcinogenesis. The presence of p53R2 mRNA expression would be a predictive factor for tumor development, tumor cell differentiation and the sensitivity to radiochemotherapy in oral SCC.     

p53与卵巢癌的基因治疗

卵巢癌是妇科恶性肿瘤的首要死亡原因,为了提高5年生存率,卵巢癌的生物治疗已经成为学者们关注的治疗方法.卵巢癌中p53的突变率最高,p53与卵巢癌密切相关.卵巢癌的p53基因治疗已兴起十余年,本文着重从体外到临床试验阐述了p53基因与卵巢癌发病相关性,以及p53基因治疗在卵巢癌的应用和仍然存在的问题.目前重组人p53腺病毒注射液作为首个基因治疗产品已经问世,在美国、欧洲等国家仍有50多个临床试验正在实施,基因治疗有望成为继手术治疗、化学治疗、放射治疗之后的又一种治疗卵巢癌的有效方法.