Involvement of sphingosine kinase 2 in p53-independent induction of p21 by the chemotherapeutic drug doxorubicin

Sphingosine-1-phosphate is a potent lipid mediator formed by phosphorylation of sphingosine, a metabolite of sphingolipids, catalyzed by two sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2. Expression of SphK2, which is enriched in the nucleus of MCF7 human breast cancer cells, increased expression of the cyclin-dependent kinase inhibitor p21 but had no effect on p53 or its phosphorylation. The anticancer drug doxorubicin is known to increase p21 via p53-dependent and p53-independent mechanisms. Down-regulation of endogenous SphK2 with small interfering RNA targeted to unique mRNA sequences decreased basal and doxorubicin-induced expression of p21 without affecting increased expression of p53. Down-regulation of SphK2 also decreased G(2)-M arrest and markedly enhanced apoptosis induced by doxorubicin. Moreover, siSphK2 reduced doxorubicin-induced p21 expression in p53-inactivated MCF7 cells. Likewise, in human wild-type p53- and p21-expressing HCT116 colon carcinoma cells, as well as in p53-null counterparts, down-regulation of SphK2 markedly reduced p21 induction by doxorubicin. Knockdown of SphK2 sensitized HCT116 cells to apoptosis induced by doxorubicin with concomitant cleavage of poly(ADP-ribose) polymerase. Collectively, our results show that endogenous SphK2 is important for p53-independent induction of p21 expression by doxorubicin and suggest that SphK2 may influence the balance between cytostasis and apoptosis of human cancer cells.     

Hsp27 modulates p53 signaling and suppresses cellular senescence

The small heat shock protein Hsp27 is expressed at high levels in many tumors and provides protection against anticancer drugs. Here, we show that expression of recombinant Hsp27 at elevated levels leads to protection of MCF10A human mammary epithelial cells from doxorubicin. The protection was associated with suppression of the doxorubicin-induced senescence, where Hsp27 inhibited p53-mediated induction of p21, the major regulator of the senescence program. Similarly, Hsp27 inhibited accumulation of p21 and suppressed senescence in response to the p53 activator nutlin-3, indicating that Hsp27 has a general effect on the p53 pathway. In line with these findings, down-regulation of Hsp27 in HCT116 human colon carcinoma cells that express this heat shock protein at high levels caused senescence in a population of cells and sensitized the rest of the cells to doxorubicin-induced senescence (at low doses) or apoptosis (at high doses of doxorubicin). Induction of senescence by Hsp27 down-regulation associated with activation of the p53 pathway and induction of p21. Interestingly, depletion of Hsp27 caused neither significant proteotoxic nor genotoxic stress, and therefore this heat shock protein seems to have a specific effect on the p53 signaling. Indeed, Hsp27 down-regulation was associated with destabilization of HDM2 and stabilization of p53. These data suggest that Hsp27 may play a general role in regulation of cellular senescence by modulating the p53 pathway.     

Identification of an ataxia telangiectasia-mutated protein mediated surveillance system to regulate Bcl-2 overexpression

Bcl-2 can both promote and attenuate tumorigenesis. Although the former function is relatively well characterized, the mechanism of the latter remains elusive. We report here that enforced Bcl-2 expression in MCF7 cells stabilizes p53, induces phosphorylation of p53 serine 15 (p53pSer15) and inhibits MCF7 cell growth. Consistent with p53 Ser15 being a target of ataxia telangiectasia mutated protein(ATM)/ATR (ATM- and rad3-related) in the DNA damage response, Bcl-2 activates ATM by inducing ATM Ser1981 phosphorylation, which is accompanied with the phosphorylaton of two additional ATM substrates, Chk2 Thr68 and H2AX Ser139. Downregulation of ATM using a specific small interference RNA fragment (ATMRNAi) abolished Bcl-2-induced p53pSer15 and Bcl-2-mediated growth inhibition of MCF7 cells. Ectopic expression of a dominant-negative p53 mutant, p53175H, partially rescued this growth inhibition. Taken together, these observations demonstrate the contribution of ATM-p53 function to Bcl-2-mediated inhibition of MCF7 cell growth, indicating an ATM-mediated surveillance system for regulating Bcl-2 overexpression. Consistent with this concept, we found that MCF7 cells express Bcl-2 heterogeneously with 34.5% of cells being Bcl-2 negative. In general, Bcl-2-positive MCF7 cells proliferate slower than those of Bcl-2 negative. Thus, we provide evidence suggesting that activation of ATM suppresses Bcl-2-induced tumorigenesis, and that attenuation of ATM function may be an important event in breast cancer progression.     

Triptolide induces Bcl-2 cleavage and mitochondria dependent apoptosis in p53-deficient HL-60 cells

Triptolide, a bioactive component of the Chinese medicinal herb Tripterygium wilfordii Hook F., induces p53-mediated apoptosis in cancer cells. This study demonstrated that triptolide activated an alternative p53-independent apoptotic pathway in HL-60 cells. In the absence of an intact p53 and without changing Bax level, at nM range triptolide induced apoptosis with concomitant DNA fragmentation, S phase cell cycle arrest, mitochondrial cytochrome c release and the activation of caspases. Besides, both caspases 8 and 9 were activated and the simultaneous inhibition of both was required to completely block triptolide's apoptotic effect. Importantly, triptolide induced the appearance of a truncated 23kD Bcl-2 which was inhibited by the general caspase inhibitor Z-VAD-FMK. In the MCF-7 cells that possessed the wild type p53 but lacked caspases 3, triptolide induced cell death with an increase in p53 but Bcl-2 remained unaltered. On the other hand, transfected cells overexpressing the 28kD Bcl-2 became more resistant to triptolide and upon triptolide treatment accumulated in the G(1) instead of S phase. After 36h treatment, triptolide activated JNK pathways, at the same time inactivated the ERK and p38 pathways. However, SP600125, a specific JNK inhibitor, could not inhibit the triptolide-mediated cleavage of caspase 3, indicated that activation of JNK might not be related to the apoptotic effects of triptolide. Our data suggest that in the absence of an intact p53 and without altering Bax level triptolide induces apoptosis activates a positive amplification loop involving caspase-mediated Bcl-2 cleavage/activation, mitochondrial cytochrome c release and further activation of caspases.     

Regulation of p53 target gene expression by cisplatin-induced extracellular signal-regulated kinase

The extracellular signal-regulated kinase (ERK) pathway is among several signal transduction pathways that are activated in response to exposure to the DNA damage-inducing chemotherapeutic agent cisplatin. We have previously reported that inhibition of cisplatin-induced ERK activity enhances sensitivity to cisplatin. Furthermore, we have demonstrated that cisplatin-induced ERK activation is required for optimal p53 protein accumulation following cisplatin-induced DNA damage. In the present study, we expanded our investigations to examine the effect of cisplatin-induced ERK activation on the expression of p53-targeted genes that have been shown to be important in the cellular response to DNA damage including Bax, Bcl-2, Bcl-x1, Cyclin G, Gadd45, p21WAF1, and Mdm2. In the ovarian carcinoma cell line A2780, cisplatin was shown to induce expression of p21WAF1, Gadd45 and Mdm2, but cisplatin had no effect on expression of Bax, Bcl-2, Bcl-x1, or Cyclin G. Inhibition of cisplatin-induced ERK activity by PD98059 resulted in decreased levels of p21WAF1, Gadd45 and Mdm2. These results provide evidence that ERK activity during the cisplatin DNA damage response, regulates in part, these cell cycle control (p21WAF1, Gadd45), DNA repair (Gadd45) and p53-regulatory (Mdm2) proteins.     

Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53

The anti-cancer agent paclitaxel (Taxol) stabilizes microtubules leading to G2/M cell cycle arrest and apoptotic cell death. In order to analyse the molecular mechanisms of Taxol-induced cytotoxicity, we studied the involvement of mitogen-activated protein kinases (MAPK) ERK and p38 as well as the p53 pathways in Taxol-induced apoptosis. The human breast carcinoma cell line MCF7 and its derivatives, MCF7/HER-2 and MDD2, were used in the study. We found that Taxol treatment strongly activated ERK, p38 MAP kinase and p53 in MAP kinase MCF7 cells prior to apoptosis. PD98059 or SB203580, specific inhibitors of ERK and p38 kinase activities, significantly decreased apoptosis, leaving the surviving cells arrested in G2/M. These inhibitors did not significantly affect Taxol-induced alterations in the cell cycle regulatory proteins Rb, p53, p21/Waf1 and Cdk-2. In addition, inactivation of p53 did not affect cellular sensitivity to Taxol killing. However, cells with inactivated p53, unlike cells harboring wild type p53, failed to arrest in G2/M after treatment with Taxol and continued to divide or go into apoptosis. Our data show that both ERK and p38 MAP kinase cascades are essential for apoptotic response to Taxol-induced cellular killing and are independent of p53 activity. However, p53 may serve as a survival factor in breast carcinoma cells treated with Taxol by blocking cells in G2/M phase of the cell cycle.     

Raf-1 and Bcl-2 induce distinct and common pathways that contribute to breast cancer drug resistance.

Overexpression of Bcl-2 plays a role in the development of drug resistance in leukemia and other apoptosis-prone tumors. Raf isoforms areserine/threonine kinases that act as signal transducers in cascades initiated by many growth factors and mitogens. Raf isoform activation has been linked to drug resistance in leukemia. In this study we investigated effects of Bcl-2 and Raf-1 on doxorubicin-induced growth inhibition of MCF-7 breast cancer cells. In the absence of doxorubicin, overexpression of Bcl-2 or a constitutively active form of Raf-1 in MCF-7 cells did not affect proliferation rate. Overexpression of Bcl-2 increased resistance of MCF-7 cells to doxorubicin in 2-day, 5-day, and 8-week assays. Analysis of doxorubicin sensitivity of individual MCF/Bcl-2 clones showed that doxorubicin resistance was positively correlated with level of Bcl-2 overexpression. Overexpression of constitutively active Raf-1 also increased resistance to doxorubicin. Induction of Raf-1 activity in MCF-7 cells overexpressing Bcl-2 resulted in greater doxorubicin resistance than induction of Raf-1 activity in MCF-7 cells lacking Bcl-2 overexpression. Furthermore, levels of P-glycoprotein mRNA were increased in MCF-7 cells overexpressing a constitutively active Raf-1. MCF-7 cells overexpressing constitutively active Raf-1 were also more resistant to paclitaxel, which, like doxorubicin, is a substrate of P-glycoprotein. These observations suggest both independent and overlapping roles for Raf-1 and Bcl-2 oncogenes in the resistance to growth inhibition by doxorubicin.     

Hsp90-inhibitor geldanamycin abrogates G2 arrest in p53-negative leukemia cell lines through the depletion of Chk1

Checkpoint protein Chk1 has been identified as an Hsp90 client. Treatment with 100 nM geldanamycin (GM) for 24 h markedly reduced the Chk1 amount in Jurkat and ML-1 leukemia cell lines. Because Chk1 plays a central role in G2 checkpoint, we added GM to G2-arrested Jurkat and HL-60 cells pretreated with 50 nM doxorubicin for 24 h. GM slowly released both cell lines from doxorubicin-induced G2 arrest into G1 phase. GM also abrogated ICRF-193-induced decatenation G2 checkpoint in Jurkat and HL-60 cells. Western blot analysis showed that addition of GM attenuates doxorubicin- and ICRF-193-induced Chk1 phosphorylation at Ser345. GM, however, failed to abrogate G2 arrest in p53-positive ML-1 cells maybe due to the p21 induction. GM released HeLa cells from doxorubicin-induced G2 arrest but trapped them at M phase. Flow cytometric analysis showed that addition of GM converted doxorubicin-induced necrosis into apoptosis in Jurkat cells. Colony assay indicated that although GM has a weak cytotoxic effect as a single agent, it dramatically intensifies the cytotoxicity of doxorubicin and ICRF-193 in Jurkat and HL-60 cells. These results suggest that abrogation of G2 checkpoint by GM may play a central role in sensitizing p53-negative tumor cells to DNA-damaging and decatenation-inhibiting agents.     

Chemosensitivity of human hepatocellular carcinoma cell line QGY-7703 is related to bcl-2 protein levels.

Bcl-2 protein is one of the major apoptosis regulators. The study examines the effect of Bcl-2 protein on the chemosensitivity of a human hepatocellular carcinoma cell line, QGY-7703. Western blot analysis showed that Bcl-2 and Bax proteins were expressed in QGY-7703 cells. Characteristic features of Taxol- and doxorubicin-induced apoptosis were evidenced by the Annexin-V binding assay, TUNEL and DAPI staining. At constant Bax protein levels, stable sense and antisense gene-transfected QGY-7703 cells showed that constitutive expression of Bcl-2 could render the cells more resistant to Taxol and doxorubicin. Contrarily, decreased Bcl-2 levels caused the cells to be more sensitive to the drugs. As Bcl-2 levels are directly proportional to the resistance of QGY-7703 cells to Taxol and doxorubicin, manipulation of Bcl-2 could be performed to enhance the sensitivity of liver cancer to chemotherapeutic agents. Copyright Copyright 1999 S. Karger AG, Basel     

The acetyltransferase p300/CBP-associated factor is a p53 target gene in breast tumor cells

p300/CBP-associated factor (PCAF) is a coactivator of the tumor suppressor, p53. PCAF participates in p53's transactivation of target genes through acetylation of both bound p53 and histones within p53 target promoters. Using microarrays, we discovered that PCAF itself is induced by p53 in a panel of breast tumor cell lines. Two p53 mutant breast tumor cell lines, BT-549 and UACC-1179, were chosen for further study of PCAF induction by wild-type p53. PCAF induction following adenoviral transduction of p53 expression was confirmed with real-time polymerase chain reaction in a time course experiment. Chromatin immunoprecipitation experiments then showed that PCAF induction was associated with increased p53 binding to the PCAF promoter, which contains p53 consensus-binding sites. PCAF induction by p53 activity was further demonstrated in wild-type p53 MCF10A cells when PCAF expression was induced following activation of endogenous wild-type p53 with doxorubicin in a dose- and time-dependent manner. Furthermore, the doxorubicin-induced increase in PCAF expression was blocked by pretreatment of the MCF10A cells with siRNA (small interfering RNA) targeted against p53 mRNA. Taken together, the results show that PCAF expression can be induced by wild-type p53.     

Involvement of ERK1/2 and p38 MAP kinase in doxorubicin-induced uPA expression in human RC-K8 lymphoma and NCI-H69 small cell lung carcinoma cells

We previously demonstrated the doxorubicin-induced urokinase-type plasminogen activator (uPA) expression in human RC-K8 lymphoma cells and NCI-H69 small cell lung carcinoma cells in which reactive oxygen species might be involved. Western blotting analysis revealed phosphorylation/activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) 1/2, p38 MAP kinase and stress-activated protein kinase/c-jun N-terminal protein kinase (SAPK/JNK) in doxorubicin-treated RC-K8 and H69 cells, and, therefore, we attempted to identify the MAP kinases implicated in doxorubicin-induced uPA expression by the use of their specific inhibitors. U0126, SB202190 and JNKI-1, inhibitors for MAPK kinase, (MEK) 1/2, p38 MAP kinase and SAPK/JNK, respectively, specifically and clearly inhibited their corresponding kinases. U0126 and SB202190, but not JNKI-1, almost completely inhibited the doxorubicin-induced uPA expression in both RC-K8 and H69 cells. However, U0126 rather enhanced the doxorubicin-induced activation of caspase-3 and poly ADP-ribose polymerase (PARP), and U0126 itself activated caspase-3 and PARP. Interestingly, JNKI-1 inhibited the doxorubicin-induced activation of caspase-3 and PARP. Therefore, doxorubicin treatment activates the above three kinases, but different MAP kinase signaling is responsible in the doxorubicin-induced caspase activation and expression of uPA. Thus, we could possibly manipulate the direction of doxorubicin-induced MAP kinase activation and the effects of doxorubicin on the tumor cell biology by the use of MAP kinase inhibitors.     

Molecular downstream events and induction of thymidylate synthase in mutant and wild-type p53 colon cancer cell lines after treatment with 5-fluorouracil and the thymidylate synthase inhibitor raltitrexed

Inhibition of the key enzyme in DNA synthesis, thymidylate synthase (TS), by 5-fluorouracil (5-FU) and the novel antifolate raltitrexed (Tomudex; ZD1694), induces dTTP depletion, resulting in DNA strand breaks, which can initiate pathways leading to an apoptotic mode of cell death. We studied 5-FU- and ZD1694-induced TS inhibition in relation to the expression of p53, p21, Bcl-2 and Bax in six colon carcinoma cell lines, two with a wild-type (wt) p53 (Lovo, LS174T) and four with a mutant (mt) p53 (WiDr, WiDr/F, HT29 and SW948) phenotype. In untreated cells, a reciprocal correlation between p53 and Bcl-2 was found: in cells with a low wt p53, Bcl-2 expression was present; whilst in cells with mt p53, Bcl-2 expression was not detectable. Exposure to 5-FU (50 and 100 microM) and ZD1694 (50 and 100 nM) for 24 and 48 h induced p53 and p21 expression in wt p53 cells, but not in mt p53 cells. TS was induced approximately 2-10-fold in all cell lines. TS induction was highest after ZD1694 exposure in the mt p53 cells HT29 and WiDr/F (6-10-fold). After 5-FU treatment, TS was present both as the free enzyme and in the ternary complex; however, predominantly as the ternary complex between TS, FdUMP and 5,10-methylenetetrahydrofolate. In wt p53 cells, both drugs increased Bax expression up to 5-fold, whereas in mt p53 cells, only a very slight induction was found. In wt p53 cells, Bcl-2 expression hardly changed after drug treatment. These results indicate a p53-independent induction of TS but a regulatory role of wt p53 in the synthesis of Bax in the colon carcinoma cell lines after TS inhibition.     

EBV-miR-BART5-3p靶向p53对鼻咽癌细胞放射敏感性的影响

目的:研究爱泼斯坦-巴尔病毒（EBV）-miR-BART5-3p对鼻咽癌细胞放射敏感性的影响,并探讨其作用机制。方法:体外培养人EBV阳性鼻咽癌细胞（C666-1）和人鼻咽癌细胞（CNE-2Z）,将CNE-2Z组细胞设置为正常组,C666-1细胞随机分为对照组、EBV-miR-BART5-3p NC组、EBV-miR-BART5-3p mimics组和EBV-miR-BART5-3p inhibitor组。用实时荧光定量PCR（RT-qPCR）法检测各组细胞及EBV阴性鼻咽癌患者和EBV阳性鼻咽癌患者癌组织中EBV-miR-BART5-3p表达情况,噻唑蓝（MTT）法检测各组细胞存活率,平板克隆实验评估各组放疗敏感性变化情况,膜联蛋白V-异硫氰酸荧光素/碘化丙啶（Annexin V-FITC/PI）法检测各组细胞凋亡敏感性变化,蛋白免疫印迹分析法检测转染后各组细胞p53、Bcl-2相关X蛋白（Bax）、半胱天冬氨酸酶-3（caspase-3）和Bcl-2蛋白表达情况,双荧光素酶报告实验验证EBV-miR-BART5-3p与p53的靶向关系。结果:与EBV阴性组相比,EBV阳性组鼻咽癌组织中EBV-miR-BART5-3p表达水平显著升高（P＜0.05）。与正常组相比,对照组EBV-miR-BART5-3p表达、存活率、克隆数量和Bcl-2蛋白表达水平显著升高（P＜0.05）,凋亡率、p53、Bax和caspase-3蛋白表达水平显著降低（P＜0.05）。与对照组和EBV-miR-BART5-3p NC组相比,EBV-miR-BART5-3p mimics组EBV-miR-BART5-3p表达水平、存活率、克隆数量和Bcl-2蛋白表达水平显著升高（P＜0.05）,凋亡率、p53、Bax和caspase-3蛋白表达水平显著降低（P＜0.05）;与对照组和EBV-miR-BART5-3p NC组相比,EBV-miR-BART5-3p inhibitor组EBV-miR-BART5-3p表达水平、存活率、克隆数量和Bcl-2蛋白表达水平显著降低（P＜0.05）,凋亡率、p53、Bax和caspase-3蛋白表达水平显著升高（P＜0.05）。双荧光素酶报告实验结果显示,与TP53-3' UTR-WT+EBV-miR-BART5-3p NC组比较,TP53-3' UTR-WT+EBV-miR-BART5-3p inhibitor组荧光素酶活性降低（P＜0.05）。结论:下调EBV-miR-BART5-3p可能通过靶向促进p53蛋白表达,提高人EBV阳性鼻咽癌细胞的放射敏感性。     

Lupulone triggers p38 MAPK-controlled activation of p53 and of the TRAIL receptor apoptotic pathway in human colon cancer-derived metastatic cells

We previously reported that the chemopreventive agent lupulone induces apoptosis through activation of the extrinsic pathway via TRAIL DR4/DR5 death receptors overcoming SW620 cell resistance to TRAIL. However, the underlying molecular mechanisms remain unknown. Since the mitogen-activated protein kinases (MAPKs), Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 control fundamental cellular processes such as apoptosis, we determined the role of these MAPKs in lupulone-triggered apoptosis. We analyzed the effects of JNK, ERK and p38 MAPK inhibitors on lupulone-induced apoptosis by flow cytometry using specific antibodies and real-time RT-PCR. Our data showed that among the MAPKs, only p38 played a major role in lupulone-triggered apoptosis. In contrast to JNK and ERK inhibition, the specific inactivation of p38 inhibited the lupulone-triggered up-regulation of p53 and TRAIL-death receptor DR4/DR5 expression, and prevented DNA fragmentation. Lupulone treatment enhanced the expression of the anti-apoptotic Mcl-1 protein by 60% favoring the preservation of mitochondrial integrity. The inactivation of p38 initiated a 50% reduction in Mcl-1, Bcl-2 and Bax expression without changing the Mcl-1/Bax ratio suggesting that p38 was not involved in the protective effect of lupulone on mitochondria. Our data support the view that the lupulone-triggered enhanced expression of p38 plays a major role in the activation of p53 and of the TRAIL-death receptor apoptotic pathway in SW620 human colon cancer-derived metastatic cells.     

Mitochondrial p53 phosphorylation induces Bak-mediated and caspase-independent cell death

Chemoresistance in cancer has previously been attributed to gene mutations or deficiency. Caspase mutations or Bax deficiency can lead to resistance to cancer drugs. We recently demonstrated that Bak initiates a caspase/Bax-independent cell death pathway. We show that Plumbagin (PL) (5-hydroxy-2-methyl-1,4-napthoquinone), a medicinal plant-derived naphthoquinone that is known to have anti-tumor activity in a variety of models, induces caspase-independent cell death in HCT116 Bax knockout (KO) or MCF-7 Bax knockdown (KD) cells that express wild-type (WT) Bak. The re-expression of Bax in HCT116 Bax KO cells fails to enhance the PL-induced cell death. Additionally, Bak knockdown by shRNA efficiently attenuates PL-induced cell death. These results suggest that PL-induced cell death depends primarily on Bak, not Bax, in these cells. Further experimentation demonstrated that p53 Ser15 phosphorylation and mitochondrial translocation mediated Bak activation and subsequent cell death. Knockdown of p53 or a p53 Ser15 mutant significantly inhibited p53 mitochondrial translocation and cell death. Furthermore, we found that Akt mediated p53 phosphorylation and the subsequent mitochondrial accumulation. Taken together, our data elaborate the role of Bak in caspase/Bax-independent cell death and suggest that PL may be an effective agent for overcoming chemoresistance in cancer cells with dysfunctional caspases.