MicroRNA-221/222对乳腺癌MDA-MB-231/DOX细胞阿霉素耐药性的影响

目的:探讨微小RNA-221/222(miR-221/222)对乳腺癌MDA-MB-231/阿霉素(DOX)细胞DOX耐药性的影响。方法:采用脂质体法转染miR-221/222抑制物(miR-221/222 inhibitor)至MDA-MB-231/DOX细胞内(Inhibitor组),同时设立空白对照组和转染无关序列的阴性对照组,采用实时荧光定量PCR (qRT-PCR)检测MDA-MB-231细胞株及MDA-MB-231/DOX细胞株的miR-221/222表达水平及转染效率;CCK-8法检测转染48 h后MDA-MB-231/DOX细胞对DOX药物敏感性的变化;流式细胞术(FCM)检测转染MDA-MB-231/DOX细胞的细胞凋亡率;蛋白免疫印迹实验(WB)检测转染后MDA-MB-231/DOX细胞内促凋亡蛋白p53上调凋亡调控因子(PUMA),Bcl2蛋白修饰因子(BMF)以及细胞周期蛋白激酶抑制因子p27(p27Kip1)的表达情况。结果:MDA-MB-231/DOX细胞中的miR-221/222表达水平高于亲本MDA-MB-231细胞(P0.05);MDA-MB-231/DOX细胞转染miR-221/222 inhibitor 96 h后,miR-221/222的表达水平低于空白对照组和阴性对照组(P0.05);与空白对照组相比,MDA-MB-231/DOX细胞转染miR-221/222 inhibitor 48h后,DOX继续处理48 h后,细胞的凋亡率明显升高,且细胞内的促凋亡蛋白PUMA,BMF以及p27Kip1的表达均增加(P0.05);DOX对inhibitor组耐药细胞的半数抑制浓度(IC50)显著低于空白对照组细胞及阴性对照组(P0.05)。结论:miR-221/222能够增加MDA-MB-231/DOX细胞对DOX的耐药性,这可能与下调促凋亡蛋白的表达有关;降低miR-221/222水平可诱导MDA-MB-231/DOX凋亡,并且上调促凋亡蛋白的表达,从而部分逆转MDA-MB-231/DOX对DOX的耐药性。     

The p53 pathway is synergized by p38 MAPK signaling to mediate 11,11'-dideoxyverticillin-induced G2/M arrest

The phytochemical 11,11'-dideoxyverticillin, derived from the fungus Shiraia bambusicola, has been shown to possess potent anticancer activity in vitro and in vivo. Here, we investigated the effect of 11,11'-dideoxyverticillin on cell cycle progression, and explored the potential mechanisms for this effect. A concentration- and time-dependent cell cycle blockade at G2/M phase was observed in human colon cancer cells (HCT-116) following 11,11'-dideoxyverticillin treatment and was associated with marked increases in levels of p53, phospho-p53(ser20) and phospho-Chk2(Thr 68). When wild type p53 expression was specifically inhibited by RNA interference, HCT-116 cells treated with 11,11'-dideoxyverticillin failed to arrest in G2/M and did not show increased phospho-Chk2(Thr 68). On the other hand, 11,11'-dideoxyverticillin treatment also elicited p38 MAP kinase activity and expression of phospho-p38 MAPK. Treatment with a specific p38 MAPK inhibitor (SB203580) successfully inhibited p38 MAPK and delayed the onset of G2/M arrest induced by 0.5 microM 11,11'-dideoxyverticillin after approximately 6 h, but did not abolish the induction of G2/M arrest. Additionally, SB203580 did not alter the levels of p53, phospho-p53 (ser20), or phospho-Chk2 (Thr68) proteins in 11,11'-dideoxyverticillin-treated cells. Together, these findings indicate that p53-mediated phosphorylation of Chk2 maybe plays a vital role in 11,11'-dideoxyverticillin-induced G2/M arrest, and that p38 MAPK might accelerate this progression. Our work suggests a new possibility of interactions among p53, Chk2 and p38 MAPK signaling in G2/M arrest.     

Celecoxib induces p53-PUMA pathway for apoptosis in human colorectal cancer cells

Celecoxib, a clinical non-steroidal anti-inflammatory drug, displays anticarcinogenic and chemopreventive activities in human colorectal cancers, although the mechanisms of apoptosis by celecoxib are poorly understood. The existence of functional p53 but not securin in colorectal cancer cells was higher on the induction of cytotoxicity than the p53-mutational colorectal cancer cells following celecoxib treatment. The p53-wild type HCT116 cells were more susceptible to increase ∼25% cell death than the p53-null HCT116 cells after treatment with 100 μM celecoxib for 24 h. Transfection with a small interfering RNA of p53 reduced the celecoxib-induced cytotoxicity in the RKO (p53-wild type) colorectal cancer cells. Celecoxib (80-100 μM for 24 h) significantly increased total p53 proteins and the phosphorylated p53 proteins at serine-15, -20, -46, and -392 in RKO cells. However, the phospho-p53 (serine-15, -20, and -392) proteins were presented on the nuclei of cells but the phospho-p53 (serine-46) protein was located on the cytoplasma of apoptotic cells following treatment with celecoxib. Interestingly, the p53 up-regulated modulator of apoptosis (PUMA) protein, which located on the mitochondria, was induced by celecoxib in the p53-functional colorectal cancer cells but not in the p53-mutational cells. Together, this study provides the first time that celecoxib induces the various phosphorylated sites of p53 and activates p53-PUMA pathway, which potentiates the apoptosis induction in human colorectal cancer cells.     

Nek6 suppresses the premature senescence of human cancer cells induced by camptothecin and doxorubicin treatment

Cellular senescence plays an important role in tumor suppression. The mitotic kinase Nek6 has recently been shown to be overexpressed in various cancers and has been implicated in tumorigenesis. Previously, we reported that the down-regulation of Nek6 expression was required for p53-induced senescence. In this study, we examined the effect of Nek6 overexpression on the premature senescence of cancer cells induced by the anticancer drugs camptothecin (CPT) and doxorubicin (DOX). We found that CPT- and DOX-induced morphology changes and increases in senescence-associated β-galactosidase staining were significantly inhibited in EJ human bladder cancer cells and H1299 human lung cancer cells overexpressing HA-Nek6. DOX-induced G2/M cell cycle arrest and the reduction in cyclin B and cdc2 levels after DOX treatment were significantly reduced by Nek6 overexpression. In addition, an increase in the intracellular levels of ROS in response to DOX was also inhibited in cells overexpressing Nek6. These results suggest that the increased expression of Nek6 renders cancer cells resistant to premature senescence, and targeting Nek6 could be an efficient strategy for cancer treatment.     

Influence of p53 expression on sensitivity of cancer cells to bleomycin

In this study, we determined whether p53 expression affected the sensitivity of non–small cell lung cancer (NSCLC) and colon cancer cells to bleomycin (BLM). Two human NSCLC cell lines (A549 expressing wild‐type p53 and p53‐null H1299) and two colon cancer cell lines (HCT116 p53+/+ and its p53 deficient subline HCT116 p53?/?) were subjected to treatment with BLM. Cells were treated with various concentrations of BLM, and cellular viability was assessed by formazan assay. To investigate the role of p53 in BLM sensitivity, we transduced cells with adenovirus with wild‐type p53, dominant‐negative p53, and GFP control, and analyzed the effect on cellular viability. Cells expressing wild‐type p53 were more sensitive to BLM than p53‐null cells in both NSCLC and colon cancer cells. Sensitivity to BLM of the cells with wild‐type p53 was reduced by overexpression of dominant‐negative p53, while BLM sensitivity of p53‐null cells was increased by wild‐type p53 in both NSCLC cells and colon cancer cells. In conclusion, our data show that p53 sensitizes all four cancer cells lines tested to BLM toxicity and overexpression of p53 confers sensitivity to the cytotoxic activity of the anticancer agent in p53‐null cells. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:260–269, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20334     

TNF-alpha promotes Doxorubicin-induced cell apoptosis and anti-cancer effect through downregulation of p21 in p53-deficient tumor cells

p53 is a key regulator in cell apoptosis, and cancer cells deficient in p53 expression fail to respond to chemotherapy. Here we show that effective Doxorubicin (DOX)-induced apoptosis is p53-dependent. However, an alternative treatment of DOX/TNF-alpha/DOX restored sensitivity of p53-deficient cells to DOX-induced apoptosis. Treatment of cells with TNF-alpha resulted in a decrease of p21 (waf1/cip1/sdi1) expression following second dose of DOX. In previous work, we demonstrated that p21 suppressed DOX-induced apoptosis via its (cyclin-dependent kinase) CDK-binding and CDK-inhibitory activity. Thus, we propose that TNF-alpha enhances the anti-cancer effect of DOX through suppressing the anti-apoptotic activity of p21, and that a combined treatment TNF-alpha/Dox is an effective chemotherapeutic strategy for p53-deficient cancers.     

Comparative study of p53 expression in human carcinoma cell lines A549 and MCF7 under anticancer drug treatment

The effect of anticancer drugs on the expression of p53 protein in tumor cells was studied using the Western Blot analysis. Human lung carcinoma cell line A549 and human breast carcinoma cell line MCF7 sensitive (WT) and resistant (DOX/R) to doxorubicin were used. An increase in p53 protein expression was found in A549 and MCF7 (WT) cells treated with cisplatin, methotrexate, and doxorubicin, whereas the level of p53 was not statistically significantly changed in the MCF7 DOX/R cells. In the untreated MCF7 DOX/R cells the level of p53 protein was markedly higher than in the untreated WT MCF7 cells. A potential role of p53 protein in the development of doxorubicin-resistance in carcinoma cells is discussed.     

SN2 DNA-alkylating agent-induced phosphorylation of p53 and activation of p21 gene expression

p53 is an important player in the cellular response to genotoxic stress whose functions are regulated by phosphorylation of a number of serine and threonine residues. Phosphorylation of p53 influences its DNA-binding and gene regulation activities. This study examines p53 phosphorylation in HCT-116 (MMR-deficient) and HCT-116+ch3 (MMR-proficient) human colon cancer cells treated with a S(N)2 DNA-alkylating agent, methylmethane sulfonate (MMS). MMS induces phosphorylation of p53 on Ser15 and Ser392 in a dose- and time-dependent manner. MMS-induced p53 phosphorylation is independent of DNA mismatch repair (MMR) activity. Nuclear extracts from MMS-treated HCT-116 cells had higher p21WAF1/Cip1 (p21) promoter DNA-binding activity in vitro opposed to untreated cells. After MMS treatment, the activation of the cloned p21 promoter in a transient transfection assay and endogenous p21 mRNA levels in HCT-116(p53+/+) versus HCT-116(p53-/-) cells increased, which correlates with an increased levels of phospho-p53(Ser15) and phospho-p53(Ser392). These results suggest that SN2 DNA-alkylating agent-induced phosphorylation of p53 on Ser15 and Ser392 increases its DNA-binding properties to cause an increased expression of p21 that may play a role in cell cycle arrest and/or apoptosis of HCT-116 cells.     

肺癌组织中PCNA、p63和p53蛋白的表达及临床意义

目的:检测蛋白增殖细胞核抗原(PCNA)、p63和p53在肺癌组织中的表达情况,以探讨三者在肺癌的发生、发展中的生物学作用和临床意义。方法:选取195例肺癌组织(其中57例有癌旁组织),应用组织芯片技术和免疫组织化学方法观察三种蛋白的表达情况,并研究三者之间及其与临床病理参数的关系。结果:PCNA、p63和p53蛋白在肺癌组织中的阳性表达率分别为96.41%、38.46%及58.46%,但三者在癌旁组织中均无表达,差异有统计学意义(均P0.05);在肺癌组织中,PCNA、p63和p53蛋白的表达情况均与组织分型有关(P0.05),且PCNA、p53蛋白表达与分化程度有关(P0.05),分化越差,表达越高;p53表达与PCNA表达呈正相关(r=0.352,P=0.043),p63与p53、PCNA的表达不相关(P0.05)。结论:肺癌组织中PCNA、p63和p53蛋白的表达升高,三者均在肺癌的发生、发展中发挥着重要作用,并且临床可通过检测三者的蛋白水平,作为鉴别肺鳞状细胞癌与其他类型癌的重要参考指标,为病理诊断提供依据。     

The effect of doxorubicin and retinoids on proliferation, necrosis and apoptosis in MCF-7 breast cancer cells

Doxorubicin (Adriamycin) is the most active drug in the treatment of breast cancer. The aim of this study was to investigate the interaction of doxorubicin and retinoids in the inhibition of proliferation of hormone sensitive (ER+) human breast cancer cell line MCF-7 and to find out whether this combination can result in the enhancement of its therapeutic effect. As a comparison we also used estradiol and tamoxifen. We also made an attempt to elucidate the effect of these compounds on the stimulation of the apoptotic pathway in breast cancer cells. Cell proliferation in a 24-hour culture was assessed by [3H] thymidine incorporation into cancer cells and by immunocytochemical analysis of cellular cycle-related PCNA and Ki-67 antigens expression, after the incubation of the cell culture with 10, 20 and 50 nM doxorubicin (DOX), 2 nM estradiol (E2), 10 microM tamoxifen (TAM) and 1 nM, 0.01, 0.1, 1 and 10 microM of all-trans retinoid acid (ATRA). The assessment of cell viability and analysis of apoptotic and necrotic cells were performed after the 72-hour incubation of the culture with the examined substances and following apoptosis induction using acridine orange and ethidine bromide. Of the doxorubicin concentrations used in the study, 20 nM inhibited thymidine incorporation to 84.83 +/- 10.00% (control=100%). In the same culture conditions, 2 nM E2 stimulated cancer cells to 157.09 +/- 8.84%. Concentrations of 10 microM TAM and 10 microM ATRA inhibited the proliferation to 63.16 +/- 7.85% and 52.19 +/- 3.21%, respectively. A statistically significant reduction of these values was observed when 20 nM DOX was added to medium with E2 - 39.24 +/- 7.6%, TAM - 48.34 +/- 2.05% and ATRA - 21.98 +/- 1.69%, respectively; the percentage of PCNA- and Ki-67-positive cells was also reduced. Despite high antiproliferative efficacy of 20 nM DOX and 10 microM ATRA combination, the percentage of apoptotic cells was only 25 +/- 0.81%, being similar to that obtained in the culture with 20 nM DOX. The concentrations of 10, 20 and 50 nM DOX that were used to inhibit the proliferation of MCF-7 cell line were not particulary effective. The inhibitory effect was obtained when 20 nM of DOX and E2, TAM or ATRA were used simultaneously. The use of E2 caused a two-fold decrease in the percentage of proliferating cells. It was also shown that the effectiveness of DOX in combination with ATRA is significantly higher than that of DOX combined with TAM, which might suggest a valuable approach to the treatment of breast cancer.     

Survivin and p53 modulate quercetin-induced cell growth inhibition and apoptosis in human lung carcinoma cells

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells. However, the regulation of survivin and p53 on the quercetin-induced cell growth inhibition and apoptosis in cancer cells remains unclear. In this study, we investigated the roles of survivin and p53 in the quercetin-treated human lung carcinoma cells. Quercetin (20-80 mum for 24 h) induced the cytotoxicity and apoptosis in both A549 and H1299 lung carcinoma cells in a concentration-dependent manner. Additionally, quercetin inhibited the cell growth, increased the fractions of G(2)/M phase, and raised the levels of cyclin B1 and phospho-cdc2 (threonine 161) proteins. Moreover, quercetin induced abnormal chromosome segregation in H1299 cells. The survivin proteins were highly expressed in mitotic phase and were located on the midbody of cytokinesis; however, the survivin proteins were increased and concentrated on the nuclei following quercetin treatment in the lung carcinoma cells. Transfection of a survivin antisense oligodeoxynucleotide enhanced the quercetin-induced cell growth inhibition and cytotoxicity. Subsequently, quercetin increased the levels of total p53 (DO-1), phospho-p53 (serine 15), and p21 proteins, which were translocated to the nuclei in A549 cells. Treatment with a specific p53 inhibitor, pifithrin-alpha, or transfection of a p53 antisense oligodeoxynucleotide enhanced the cytotoxicity of the quercetin-treated cells. Furthermore, transfection of a small interfering RNA of p21 enhanced the quercetin-induced cell death in A549 cells. Together, our results suggest that survivin can reduce the cell growth inhibition and apoptosis, and p53 elevates the p21 level, which may attenuate the cell death in the quercetin-treated human lung carcinoma cells.     

Quercetin Potentiates Doxorubicin Mediated Antitumor Effects against Liver Cancer through p53/Bcl-xl

Background

The dose-dependent toxicities of doxorubicin (DOX) limit its clinical applications, particularly in drug-resistant cancers, such as liver cancer. In this study, we investigated the role of quercetin on the antitumor effects of DOX on liver cancer cells and its ability to provide protection against DOX-mediated liver damage in mice.

Methodology and Results

The MTT and Annexin V/PI staining assay demonstrated that quercetin selectively sensitized DOX-induced cytotoxicity against liver cancer cells while protecting normal liver cells. The increase in DOX-mediated apoptosis in hepatoma cells by quercetin was p53-dependent and occurred by downregulating Bcl-xl expression. Z-VAD-fmk (caspase inhibitor), pifithrin-α (p53 inhibitor), or overexpressed Bcl-xl decreased the effects of quercetin on DOX-mediated apoptosis. The combined treatment of quercetin and DOX significantly reduced the growth of liver cancer xenografts in mice. Moreover, quercetin decreased the serum levels of alanine aminotransferase and aspartate aminotransferase that were increased in DOX-treated mice. Quercetin also reversed the DOX-induced pathological changes in mice livers.

Conclusion and Significance

These results indicate that quercetin potentiated the antitumor effects of DOX on liver cancer cells while protecting normal liver cells. Therefore, the development of quercetin may be beneficial in a combined treatment with DOX for increased therapeutic efficacy against liver cancer.     

Altered Expression of Proliferation-Inducing and Proliferation-Inhibiting Genes Might Contribute to Acquired Doxorubicin Resistance in Breast Cancer Cells

This study was designed to investigate the molecular changes that may develop during exposure of breast cancer cells to anticancer agents and that may lead to acquired resistance. We used two breast cancer cell lines, a parental (MCF7/WT) and a doxorubicin-resistant (MCF7/DOX) one. Cell survival, cell cycle distribution and RT-PCR expression level of genes involved in DNA damage response, MDR1, GST and TOPOIIα were measured. MCF7/DOX cells were five-fold more resistant to doxorubicin (DOX) than the MCF7/WT cells. DOX treatment causes arrest of MCF7/DOX cells in G1 and G2 phases of cell cycle whereas MCF7/WT cells were arrested in S-phase. The molecular changes in both cell lines due to DOX treatment could be classified into: (1) the basal level of p53, p21, BRCA1, GST and TOPOIIα mRNA was higher in MCF7/DOX than MCF7/WT. During DOX treatment, the expression level of these genes decreased in both cell lines but the rate of down-regulation was faster in MCF7/WT than MCF7/DOX cells. (2) The expression level of MDR1 was the same in both cell lines but 48 and 72 h of drug treatment, MDR1 disappeared in MCF7/WT but still expressed in MCF7/DOX. (3) There was no change in the expression level of BAX, FAS and BRCA2 in both cell lines. Conclusively, after validation in clinical samples, overexpression of genes like BRCA1, p53, p21, GST, MDR1 and TOPOIIα could be used as a prognostic biomarker for detection of acquired resistance in breast cancer and as therapeutic targets for the improvement of breast cancer treatment strategies.     

The Effect of TIGAR Knockdown on Apoptotic and Epithelial‐Mesenchymal Markers Expression in Doxorubicin‐Resistant Non‐Small Cell Lung Cancer A549 Cell Lines

Resistance to chemotherapeutic drugs is a critical problem in cancer therapy, but the underlying mechanism has not been fully elucidated. TP53‐induced glycolysis regulatory phosphatase (TIGAR), an important glycolysis and apoptosis regulator, plays a crucial role in cancer cell survival by protecting cells against oxidative stress‐induced apoptosis. In the present study, we investigated whether TIGAR is involved in epithelial‐mesenchymal transition (EMT) in doxorubicin (DOX)‐resistant human non‐small cell lung cancer (NSCLC), A549/DOX cells. We found that the expression of TIGAR was significantly higher in A549/DOX cells than in the parent A549 cell lines. siRNA‐mediated TIGAR knockdown reduced migration, viability and colony survival of doxorubicin‐resistant lung cancer cells. Also, TIGAR knockdown decreased pro‐survival protein Bcl‐2 and increased pro‐apoptotic Bax and cleaved poly (ADP‐ribose) polymerase (PARP). Moreover, TIGAR depletion significantly up‐regulated both caspase‐3 and caspase‐9 expression. Furthermore, TIGAR depletion up‐regulated the expression of E‐cadherin and down‐regulated the expression of vimentin. These results indicate that TIGAR knockdown may inhibit EMT in doxorubicin (DOX)‐resistant human NSCLC and may represent a therapeutic target for a non‐small lung cancer cells chemoresistance.     

Involvement of p53 in gemcitabine mediated cytotoxicity and radiosensitivity in breast cancer cell lines

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdCyd) is one of the anti-metabolites drugs that target DNA replication. We evaluated dFdCyd cytotoxicity and its radiosensitizing ability in human breast cancer cell lines, MCF-7 (wild-type p53) and MDA-MB-231 (mutant-type p53) along with normal mammary epithelial cell line (MCF-12) for comparison. Radiosensitivity and cytotoxicity were measured by the clonogenic survival assays. DNA DSBs was studied by Pulse Field Gel Electrophoresis (PFGE) and cell cycle distribution was analyzed by flow cytometry. MDA-MB-231 cells were the most sensitive to the cytotoxicity of dFdCyd (IC(50) 5 nM) then MCF-7 (IC(50) 10nM), whereas MCF-12 cells were the most resistant to the cytotoxicity of dFdCyd (IC(50) 70 nM). MCF-12 and MCF-7 cell lines did not show any radiosensitization to dFdCyd, whereas the MDA-MB-231 cells showed significantly increased radioresistant to dFdCyd at equimolar concentration (p=0.002) and at IC(50) concentration (p<0.001). The DNA double strand breaks (DSBs) repair showed that dFdCyd neither increases DNA DSBs nor decreases the rate of their repair in MCF-12 and MCF-7 cell lines, while the same treatment in MDA-MB-231 cell line led to decrease the rate of DSBs or increase the rate of DNA repair (p=0.034). Therefore, dFdCyd is a cytotoxic agent, especially in the cancer cells irrespective of having wild-type or mutated p53 protein, but it is not effective as radiosensitizer in the cell lines used in this study. dFdCyd combined with radiation reduces the efficacy of chemo-radiotherapy in p53 mutated cells. Therefore, p53-mutated cancer could be a counter-indication for radiation-gemcitabine combined treatment.     

The Role of Kif4A in Doxorubicin-Induced Apoptosis in Breast Cancer Cells

This study was to investigate the mechanism and role of Kif4A in doxorubicin-induced apoptosis in breast cancer. Using two human breast cancer cell lines MCF-7 (with wild-type p53) and MDA-MB-231 (with mutant p53), we quantitated the expression levels of kinesin super-family protein 4A (Kif4A) and poly (ADP-ribose) Polymerase-1 (PARP-1) by Western blot after doxorubicin treatment and examined the apoptosis by flow cytometry after treatment with doxorubicin and PARP-1 inhibitor, 3-Aminobenzamide (3-ABA). Our results showed that doxorubicin treatment could induce the apoptosis of MCF-7 and MDA-MB-231 cells, the down-regulation of Kif4A and upregulation of poly(ADP-ribose) (PAR). The activity of PARP-1 or PARP-1 activation was significantly elevated by doxorubicin treatment in dose- and time-dependent manners (P < 0.05), while doxorubicin treatment only slightly elevated the level of cleaved fragments of PARP-1 (P > 0.05). We further demonstrated that overexpression of Kif4A could reduce the level of PAR and significantly increase apoptosis. The effect of doxorubicin on apoptosis was more profound in MCF-7 cells compared with MDA-MB-231 cells (P < 0.05). Taken together, our results suggest that the novel role of Kif4A in doxorubicin-induced apoptosis in breast cancer cells is achieved by inhibiting the activity of PARP-1.     

Pifithrin-alpha protects against doxorubicin-induced apoptosis and acute cardiotoxicity in mice

The present experiments were designed to evaluate the effects of pifithrin-alpha (PFT-alpha), which is a p53 inhibitor, on doxorubicin (DOX)-induced apoptosis and cardiac injury. Administration of DOX (22.5 mg/kg ip) in mice upregulated the mRNA levels of Bax and MDM2, whereas PFT-alpha attenuated those levels when administered at a total dose of 4.4 mg/kg at 30 min before and 3 h after DOX challenge. DOX treatment led to an upregulation of p53 protein levels, which was preceded by elevated levels of phosphorylated p53 at Ser15. PFT-alpha had no effect on the level of p53 or its phosphorylated form. The protein levels of Bax and MDM2 were elevated by DOX and attenuated by PFT-alpha. DOX gave rise to increased apoptosis-positive nuclei in cardiac cells, elevated serum creatine phosphokinase, ultrastructural alterations, and cardiac dysfunction. PFT-alpha offered protection against all of the aforementioned changes. Finally, PFT-alpha did not interfere with the antitumor potency of DOX. This study demonstrates that PFT-alpha effectively inhibits DOX-induced cardiomyocyte apoptosis, which suggests that PFT-alpha has the potential to protect cancer patients against DOX-induced cardiac injury.     

Control of p53 nuclear accumulation in stressed cells

Wild-type p53 accumulates in the nucleus following stress. Current models suggest this nuclear accumulation involves phosphorylation at p53 N-terminal sites, and inhibition of murine double minute (MDM)2-dependent nuclear export. We monitored the effects of stress on MDM2-dependent nuclear export of wild-type p53 and a mutant lacking N-terminal phosphorylation sites. Etoposide and ionizing radiation inhibited nuclear export of wild-type p53 and the phosphor-mutant to comparable extents, indicating nuclear export inhibition does not require N-terminal phosphorylation. Cytoplasmic p53 accumulated in the nucleus of transfected cells treated with the nuclear export-inhibitor leptomycin B (LMB). Interestingly, LMB caused less p53 nuclear accumulation than stress treatment, suggesting stress-induced nuclear accumulation of p53 does not result solely from inhibited nuclear export.     

p53 acetylation enhances Taxol-induced apoptosis in human cancer cells

Microtubule inhibitors (MTIs) such as Taxol have been used for treating various malignant tumors. Although MTIs have been known to induce cell death through mitotic arrest, other mechanisms can operate in MTI-induced cell death. Especially, the role of p53 in this process has been controversial for a long time. Here we investigated the function of p53 in Taxol-induced apoptosis using p53 wild type and p53 null cancer cell lines. p53 was upregulated upon Taxol treatment in p53 wild type cells and deletion of p53 diminished Taxol-induced apoptosis. p53 target proteins including MDM2, p21, BAX, and β-isoform of PUMA were also upregulated by Taxol in p53 wild type cells. Conversely, when the wild type p53 was re-introduced into two different p53 null cancer cell lines, Taxol-induced apoptosis was enhanced. Among post-translational modifications that affect p53 stability and function, p53 acetylation, rather than phosphorylation, increased significantly in Taxol-treated cells. When acetylation was enhanced by anti-Sirt1 siRNA or an HDAC inhibitor, Taxol-induced apoptosis was enhanced, which was not observed in p53 null cells. When an acetylation-defective mutant of p53 was re-introduced to p53 null cells, apoptosis was partially reduced compared to the re-introduction of the wild type p53. Thus, p53 plays a pro-apoptotic role in Taxol-induced apoptosis and acetylation of p53 contributes to this pro-apoptotic function in response to Taxol in several human cancer cell lines, suggesting that enhancing acetylation of p53 could have potential implication for increasing the sensitivity of cancer cells to Taxol.     

Δ133p53 decreases the chemosensitivity of carcinoma cell line H1299

The research evaluated the effect of Δ133p53 on the chemosensitivity of lung adenocarcinoma cell line H1299. By this study, the drug‐resistant molecular marker and a new target for cancer therapy could be provided. Δ133p53 or negative control plasmid were transferred into H1299 cells by lentivirus vector. The expression of Δ133p53 in transfected cells was examined using immunofluorescence. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) method and colony formation test were applied to detect drug sensitivity after cisplatin or 5‐fluorouracil (5‐FU) treatment. After cisplatin (CDDP)/FU treatment, MTT assay demonstrated that the inhibition rate of H1299/Δ133p53 cell was reduced compared with that of the H1299 and H1299/NEG cells at the same concentration of drug. The 50% inhibitory concentrations (IC ₅₀) of CDDP and 5‐FU rose by 36.1 and 30.2%, respectively (P < 0.05). The colony formation assay suggested that the cell proliferation ability of H1299/Δ133p53 cell was prominently increased when compared with that of control group H1299 and H1299 /NEG cells (P < 0.05). The present study demonstrated that the transfection of the Δ133p53 gene in H1299 cells led to the reduction of chemosensitivity.