Acute ablation of survivin uncovers p53-dependent mitotic checkpoint functions and control of mitochondrial apoptosis

Survivin is a member of the Inhibitor of Apoptosis gene family that has been implicated in cell division and suppression of apoptosis. Here, we show that preferential ablation of the nuclear pool of survivin by RNA interference produces a mitotic arrest followed by re-entry into the cell cycle and polyploidy. Survivin ablation causes multiple centrosomal defects, aberrant multipolar spindle formation, and chromatin missegregation, and these phenotypes are exacerbated by loss of the cell cycle regulator, p21(Waf1/Cip1) in p21(-/-) cells. The mitotic checkpoint activated by loss of survivin is mediated by induction of p53 and associated with increased expression of its downstream target, p21(Waf1/Cip1). Accordingly, p53(-/-) cells exhibit reduced mitotic arrest and enhanced polyploidy upon survivin ablation as compared with their p53(+/+) counterparts. Partial reduction of the cytosolic pool of survivin by RNA interference sensitizes cells to ultraviolet B-mediated apoptosis and results in enhanced caspase-9 proteolytic cleavage, whereas complete ablation of cytosolic survivin causes loss of mitochondrial membrane potential and spontaneous apoptosis. These data demonstrate that survivin has separable checkpoint functions at multiple phases of mitosis and in the control of mitochondrial-dependent apoptosis.     

Gain-of-function mutant p53-R280K mediates survival of breast cancer cells

Missense mutations in TP53 resulting in the expression of p53-R175H, p53-R273H, or p53-R280K are frequently detected in human breast cancer. Currently, the role of mutant p53-R280K in breast cancer is relatively unknown, and therefore, the present study analyzed the function of mutant p53-R280K in breast cancer cell growth. To this end, we used small interfering RNA to study the role of mutant p53-R280K in MDA-MB-231 cells, which endogenously express the mutant protein. We found that curcumin induced apoptosis in MDA-MB-231 cells and downregulated mutant p53-R280K. We also observed that knockdown of mutant p53 by small interfering RNA induced apoptosis in MDA-MB-231 cells. Curcumin-induced apoptosis was further enhanced by the overexpression of wild-type p53, but was decreased by mutant p53-R280K overexpression. Our findings indicate that mutant p53-R280K has an important role in mediating the survival of triple-negative breast cancer MDA-MB-231 cells. Furthermore, this study suggests mutant p53-R280K could be used as a therapeutic target for breast cancer cells harboring this TP53 missense mutation.     

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.     

Developmental control of apoptosis by the immunophilin aryl hydrocarbon receptor-interacting protein (AIP) involves mitochondrial import of the survivin protein

Survivin is a multifunctional protein with essential roles in cell division and inhibition of apoptosis, but the molecular underpinnings of its cytoprotective properties are poorly understood. Here we show that homozygous deletion of the aryl hydrocarbon receptor-interacting protein (AIP), a survivin-associated immunophilin, causes embryonic lethality in mice by embryonic day 13.5-14, increased apoptosis of Ter119(-)/CD71(-) early erythropoietic progenitors, and loss of survivin expression in its cytosolic and mitochondrial compartments in vivo. In import assays using recombinant proteins, AIP directly mediated the import of survivin to mitochondria, thus enabling its anti-apoptotic function, whereas a survivin 1-141 mutant that does not bind AIP was not imported to mitochondria and failed to inhibit apoptosis. AIP-directed mitochondrial import of survivin did not affect cell division, was independent of the organelle transmembrane potential, did not require the chaperone Heat Shock Protein 90 (Hsp90), and was inhibited by cytosolic factor(s) present in normal cells. shRNA knockdown of the mitochondrial import receptor Tom20 abolished mitochondrial import of survivin and sensitized tumor cells to apoptosis, whereas silencing of Tom70 had no effect. Therefore, an AIP-Tom20 recognition contributes to cell survival in development and cancer by mediating the mitochondrial import of survivin.     

Silencing survivin gene expression promotes apoptosis of human breast cancer cells through a caspase-independent pathway

Survivin is recognized as an attractive target in cancer therapy because of its selective overexpression in the majority of tumors. Upregulated expression of this protein correlates with increased tumor grade, recurrence risk and decreased cancer patients survival. In this study, we assessed the efficacy of two survivin-specific small interfering RNA (siRNA) constructs to inhibit T47D human breast cancer cell growth. After siRNA transfection, T47D cells showed a significant reduction in proliferation and survival exhibiting clear signs of apoptosis. pSil_1 that targeted exon 1 exhibited a stronger inhibitory effect on cell growth, and increased cell apoptosis compared to pSil_30 that targeted exon 4. Cell apoptosis was found to be mediated by translocation of the mitochondrial apoptosis inducing factor (AIF), while no changes were observed in caspase-3 activation and Bid cleavage. Thus, silencing survivin expression using siRNA strategies represents a suitable therapeutic approach to selectively modulate the survival and growth of human breast cancer cells.     

Hypoxia-induced nucleophosmin protects cell death through inhibition of p53

Nucleophosmin (NPM) is a multifunctional protein that is overexpressed in actively proliferating cells and cancer cells. Here we report that this proliferation-promoting protein is strongly induced in response to hypoxia in human normal and cancer cells. Up-regulation of NPM is hypoxia-inducible factor-1 (HIF-1)-dependent. The NPM promoter encodes a functional HIF-1-responsive element that can be activated by hypoxia or forced expression of HIF-1alpha. Suppression of NPM expression by small interfering RNA targeting NPM increases hypoxia-induced apoptosis, whereas overexpression of NPM protects against hypoxic cell death of wild-type but not p53-null cells. Moreover, NPM inhibits hypoxia-induced p53 phosphorylation at Ser-15 and interacts with p53 in hypoxic cells. Thus, this study not only demonstrates hypoxia regulation of a proliferation-promoting protein but also suggests that hypoxia-driven cancer progression may require increased expression of NPM to suppress p53 activation and maintain cell survival.     

Radicicol, an inhibitor of Hsp90, enhances TRAIL-induced apoptosis in human epithelial ovarian carcinoma cells by promoting activation of apoptosis-related proteins

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various cancer cells. Hsp90 is known to be involved in cell survival and growth in tumor cells. Nevertheless, Hsp90 inhibitors exhibit a variable effect on the cytotoxicity of anticancer drugs. Furthermore, the combined effect of Hsp90 inhibitors on TRAIL-induced apoptosis in epithelial ovarian cancer cells has not been determined. To assess the ability of an inhibitor of Hsp90 inhibitor radicicol to promote apoptosis, we investigated the effect of radicicol on TRAIL-induced apoptosis in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. TRAIL induced a decrease in Bid, Bcl-2, Bcl-xL, and survivin protein levels, increase in Bax levels, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases (-8, -9, and -3), cleavage of PARP-1 and an increase in the tumor suppressor p53 levels. Radicicol enhanced TRAIL-induced apoptosis-related protein activation, nuclear damage and cell death. These results suggest that radicicol may potentiate the apoptotic effect of TRAIL on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-dependent pathway and the mitochondria-mediated apoptotic pathway, leading to caspase activation. Radicicol may confer a benefit in the TRAIL treatment of epithelial ovarian adenocarcinoma.     

Blockage of NF-κB Induces Serine 15 Phosphorylation of Mutant p53 by JNK Kinase in Prostate Cancer Cells

The p53 tumor suppressor gene plays an important role during induction of apoptosis in cancer. In contrast, NF-κB prevents apoptosis in response to chemotherapeutic agents and is a critical regulator of cell survival. Despite the riches of information on the regulation of wild-type p53 function by phosphorylation, nothing is known about the modulation of mutant p53 activity by phosphorylation. Here we report that inhibition of NF-κB in DU145 prostate cancer cells results in p53 mutant phosphorylation at serine 15 (Ser15), leading to an increase of p53 stability, DNA binding and gain of function. Serine 15-phosphorylation is due to GADD45a-dependent induction of JNK kinase, which can be blocked by SP600125, a JNK kinase inhibitor. Furthermore, inhibition of GADD45a by small interfering RNA blocks JNK activation and abrogates Ser15 phosphorylation. Together, these results highlight the importance of Ser15 phosphorylation in regulating the oncogenic function of mutant p53 and apoptosis induction in the context of the NF-κB/IκB signaling pathway.     

18β-Glycyrrhetinic acid potentiates Hsp90 inhibition-induced apoptosis in human epithelial ovarian carcinoma cells via activation of death receptor and mitochondrial pathway

The Hsp90 inhibition has been shown to induce apoptosis in various cancer cells. The licorice compounds may enhance the anti-cancer drug effect. However, effect of the licorice compounds on the Hsp90 inhibition-induced apoptosis in ovarian cancer cells has not been studied. To assess the ability of 18β-glycyrrhetinic acid to promote apoptosis, we examined whether 18β-glycyrrhetinic acid potentiated the Hsp90 inhibitor-induced apoptosis in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. Radicicol and geldanamycin induced a decrease in Bid, Bcl-2, Bcl-xL and survivin protein levels, an increase in Bax levels, the mitochondrial transmembrane potential loss, cytochrome c release, activation of caspases (-8, -9, and -3), cleavage of PARP-1, and an increase in the tumor suppressor p53 levels. 18β-Glycyrrhetinic acid enhanced Hsp90 inhibitor-induced apoptosis-related protein activation, nuclear damage, and cell death. The results suggest that 18β-glycyrrhetinic acid may potentiate the Hsp90 inhibition-induced apoptosis in ovarian carcinoma cell lines via the activation of the caspase-8- and Bid-dependent pathways and the mitochondria-mediated cell death pathway, leading to activation of caspases. Combination of Hsp90 inhibitors and 18β-glycyrrhetinic acid may confer a benefit in the treatment of epithelial ovarian adenocarcinoma.     

Geldanamycin analog 17-DMAG limits apoptosis in human peripheral blood cells by inhibition of p53 activation and its interaction with heat-shock protein 90 kDa after exposure to ionizing radiation

Exposure to ionizing radiation induces p53, and its inhibition improves mouse survival. We tested the effect of 17-dimethylamino-ethylamino-17-demethoxygeldanamycin (17-DMAG) on p53 expression and function after radiation exposure. 17-DMAG, a heat-shock protein 90 (Hsp90) inhibitor, protects human T cells from ionizing radiation-induced apoptosis by inhibiting inducible nitric oxide synthase (iNOS) and subsequent caspase-3 activation. Using ex vivo human peripheral blood mononuclear cells, we found that ionizing radiation increased p53 accumulation, acute p53 phosphorylation, Bax expression and caspase-3/7 activation in a radiation dose- and time postirradiation-dependent manner. 17-DMAG inhibited these increases in a concentration-dependent manner (IC(50) = 0.93 ± 0.01?μM). Using in vitro models, we determined that inhibition of p53 by genetic knockout resulted in lower levels of caspase-3/7 activity 1?day after irradiation and enhanced survival at 10?days. Analysis of p53-Hsp90 interaction in ex vivo cell lysates indicated that the binding between the two molecules occurred after irradiation but 17-DMAG prevented the binding. Taken together, these results suggest the presence of p53 phosphorylation and Hsp90-dependent p53 stabilization after acute irradiation. Hsp90 inhibitors such as 17-DMAG may prove useful with radiation-based cancer therapy as well as for general radioprotection.     

Buxus alkaloid compound destabilizes mutant p53 through inhibition of the HSF1 chaperone axis

BackgroundP53 is the most frequently mutated gene in most tumour types, and the mutant p53 protein accumulates at high levels in tumours to promote tumour development and progression. Thus, targeting mutant p53 for degradation is one of the therapeutic strategies used to manage tumours that depend on mutant p53 for survival. Buxus alkaloids are traditionally used in the treatment of cardiovascular diseases. We found that triterpenoid alkaloids extracted from Buxus sinica found in the Yunnan Province exhibit anticancer activity by depleting mutant p53 levels in colon cancer cells.PurposeTo explore the anticancer mechanism of action of the triterpenoid alkaloid KBA01 compound by targeting mutant p53 degradation.Study design and methodsDifferent mutant p53 cell lines were used to evaluate the anticancer activity of KBA01. MTT assay, colony formation assay and cell cycle analysis were performed to examine the effect of KBA01 on cancer cell proliferation. Western blotting and qPCR were used to investigate effects of depleting mutant p53, and a ubiquitination assay was used to determine mutant p53 ubiquitin levels after cells were treated with the compound. Co-IP and small interfering RNA assays were used to explore the effects of KBA01 on the interaction of Hsp90 with mutant p53.ResultsThe triterpenoid alkaloid KBA01 can induce G2/M cell cycle arrest and the apoptosis of HT29 colon cancer cells. KBA01 decreases the stability of DNA contact mutant p53 proteins through the proteasomal pathway with minimal effects on p53 mutant protein conformation. Moreover, KBA01 enhances the interaction of mutant p53 with Hsp70, CHIP and MDM2, and knocking down CHIP and MDM2 stabilizes mutant p53 levels in KBA01-treated cells. In addition, KBA01 disrupts the HSF1-mutant p53-Hsp90 complex and releases mutant p53 to enable its MDM2- and CHIP-mediated degradation.ConclusionOur study reveals that KBA01 depletes mutant p53 protein in a chaperone-assisted ubiquitin/proteasome degradation pathway in cancer cells, providing insights into potential strategies to target mutant p53 tumours.     

Noscapine Induced Apoptosis via Downregulation of Survivin in Human Neuroblastoma Cells Having Wild Type or Null p53

Neuroblastoma is the most common extracranial solid tumor of childhood. It accounts for 15% of pediatric cancer deaths. Chemotherapy is the mainstay of treatment in children with advanced neuroblastoma. Noscapine, a nontoxic natural compound, can trigger apoptosis in many cancer types. We now show that p53 is dispensable for Noscapine-induced cell death in neuroblastoma cell lines, proapoptotic response to this promising chemopreventive agent is mediated by suppression of survivin protein expression. The Noscapine treatment increased levels of total and Ser(15)-phosphorylated p53 protein in SK-SY5Y cells, but the proapoptotic response to this agent was maintained even after knockdown of the p53 protein level. Exposure of SK-SY5Y and LA1-5S cells to Noscapine resulted in a marked decrease in protein and mRNA level of survivin as early as 12 hours after treatment. Ectopic expression of survivin conferred statistically significant protection against Noscapine-mediated cytoplasmic histone-associated apoptotic DNA fragmentation. Also, the Noscapine-induced apoptosis was modestly but statistically significantly augmented by RNA interference of survivin in both cell lines. Furthermore, Noscapine-induced apoptotic cell death was associated with activation of caspase-3 and cleavage of PARP. In conclusion, the present study provides novel insight into the molecular circuitry of Noscapine-induced apoptosis to indicate suppression of survivin expression as a critical mediator of this process.     

The protective role of a small GTPase RhoE against UVB-induced DNA damage in keratinocytes

RhoE, a p53 target gene, was identified as a critical factor for the survival of human keratinocytes in response to UVB. The Rho family of GTPases regulates many aspects of cellular behavior through alterations to the actin cytoskeleton, acting as molecular switches cycling between the active, GTP-bound and the inactive, GDP-bound conformations. Unlike typical Rho family proteins, RhoE (also known as Rnd3) is GTPase-deficient and thus expected to be constitutively active. In this study, we investigated the response of cultured human keratinocyte cells to UVB irradiation. RhoE protein levels increase upon exposure to UVB, and ablation of RhoE induction through small interfering RNA resulted in a significant increase in apoptosis and a reduction in the levels of the pro-survival targets p21, Cox-2, and cyclin D1, as well as an increase of reactive oxygen species levels when compared with control cells. These data indicate that RhoE is a pro-survival factor acting upstream of p38, JNK, p21, and cyclin D1. HaCat cells expressing small interfering RNA to p53 indicate that RhoE functions independently of its known associates, p53 and Rho-associated kinase I (ROCK I). Targeted expression of RhoE in epidermis using skin-specific transgenic mouse model resulted in a significant reduction in the number of apoptotic cells following UVB irradiation. Thus, RhoE induction counteracts UVB-induced apoptosis and may serve as a novel target for the prevention of UVB-induced photodamage regardless of p53 status.     

Oxaliplatin down-regulates survivin by p38 MAP kinase and proteasome in human colon cancer cells

Oxaliplatin, a platinum derivative cancer drug, has been used for treating human colorectal cancers. Survivin has been proposed as a cancer target, which highly expressed in most cancer cells but not normal adult cells. In this study, we investigated the regulation of survivin expression by exposure to oxaliplatin in human colon cancer cells. Oxaliplatin (3–9 μM for 24 h) markedly induced cytotoxicity, proliferation inhibition and apoptosis in the human RKO colon cancer cells. The survivin protein expression of RKO cells is dramatically reduced by oxaliplatin; however, the survivin gene expression is slightly altered. The survivin blockage of oxaliplatin elevated caspase-3 activation and apoptosis in RKO cells. Over-expression of survivin proteins by transfection with a survivin-expressed vector resisted the oxaliplatin-induced cancer cell death. Meantime, oxaliplatin elicited the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB202190, a specific p38 MAP kinase inhibitor, restored the survivin protein level and attenuated oxaliplatin-induced cancer cell death. In addition, oxaliplatin increased the levels of phospho-p53 (Ser-15) and total p53 proteins. Inhibition of p53 expression by a specific p53 inhibitor pifithrin-α reduced the phosphorylated p38 MAP kinase and active caspase-3 proteins in the oxaliplatin-exposed RKO cells. In contrast, SB202190 did not alter the oxaliplatin-induced p53 protein level. Furthermore, treatment with a specific proteasome inhibitor MG132 restored survivin protein level in the oxaliplatin-treated colon cancer cells. Taken together, our results demonstrate for the first time that survivin is down-regulated by p38 MAP kinase and proteasome degradation pathway after treatment with oxaliplatin in the human colon cancer cells.