P53-independent Pathway Research Articles

The MK5/PRAK Kinase and Myc Form a Negative Feedback Loop that Is Disrupted during Colorectal Tumorigenesis

Expression of the Myc oncoprotein is downregulated in response to stress signals to allow cells to cease proliferation and escape apoptosis, but the mechanisms involved in this process are poorly understood. Cell cycle arrest in response to DNA damage requires downregulation of Myc via a p53-independent signaling pathway. Here we have used siRNAscreening of the human kinome to identify MAPKAPK5 (MK5, PRAK) as a negative regulator of Myc expression. MK5 regulates translation of Myc, since it is required for expression of miR-34b and miR-34c that bind to the 3'UTR of MYC. MK5 activates miR-34b/c expression via phosphorylation of FoxO3a, thereby promoting nuclear localization of FoxO3a and enabling it to induce miR-34b/c expression and arrest proliferation. Expression of MK5 in turn is directly activated by Myc, forming a negative feedback loop. MK5 is downregulated in colon carcinomas, arguing that this feedback loop is disrupted during colorectal tumorigenesis.

Melatonin sensitizes Caki renal cancer cells to kahweol‐induced apoptosis through CHOP‐mediated up‐regulation of PUMA

Melatonin has recently gained attention as a regulator of biologic processes in addition to its effects on circadian rhythms. The mechanisms whereby melatonin regulates the apoptotic program remain poorly understood. In this study, we investigated the combined effect of melatonin and kahweol on apoptosis of cancer cells, but not in most normal human cell types, thus presenting an attractive novel strategy for cancer treatment. In our experiments, treatment with a combination of melatonin and kahweol induced apoptosis, stimulated DEVDase activity, and DNA fragmentation. Co-treatment with melatonin and kahweol induced up-regulation of p53-upregulated modulator of apoptosis (PUMA) while down-regulation of PUMA expression using small interfering RNAs attenuated melatonin plus kahweol-induced apoptosis. In addition, co-treatment with kahweol and melatonin induced PUMA up-regulation through endoplasmic reticulum stress-mediated C/EBP homologous protein induction and the p53-independent pathway. Our results collectively demonstrate that up-regulation of PUMA contributes to the sensitizing effect of melatonin plus kahweol on apoptosis in cancer cells.

Mechanisms of Apoptosis on Human Lens Epithelium after Ultraviolet Light Exposure

PurposeThe purpose of this study is to understand the mechanism of apoptosis occurring on a cultured human lens epithelial cell line after exposure to ultraviolet (UV) light. We intended to confirm the presence of cellular toxicity and apoptosis and to reveal the roles of p53, caspase 3 and NOXA in these processes.MethodsCells were irradiated with an ultraviolet lamp. Cellular toxicity was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hoechst staining and fluorescent anti-caspase 3 antibodies were used for apoptosis investigation. The quantities of p53, caspase 3, and NOXA were measured by Western blotting for to investigate the apoptosis pathway.ResultsCellular toxicity on the human lens epithelium markedly increased with time after UV exposure. On Hoechst staining, we found that apoptosis also remarkably increased after exposure to ultraviolet light, compared with a control group. In the immunochemical study using anti-caspase 3 antibodies, active caspase 3 significantly increased after exposure to ultraviolet light. On Western blotting, p53 decreased, while caspase 3 and NOXA increased.ConclusionsExposure of cultured human lens epithelial cell lines to ultraviolet light induces apoptosis, which promotes the expression of NOXA and caspase 3 increases without increasing p53. This may suggest that UV induced apoptosis is caused by a p53-independent pathway in human lens epithelial cells.

The RhoGAP domain-containing protein, Porf-2, inhibits proliferation and enhances apoptosis in neural stem cells

Neural stem cells (NSCs) are essential to developing and mature CNS. They shape the structural and functional layouts of the brain in developing CNS and continue to proliferate, generating new neurons in several adult brain regions. Preoptic regulatory factor-2 (Porf-2), a RhoGAP domain-containing protein expressed in CNS, has a role in gender-related brain development and function. Porf-2 expression was knocked down in C17.2, a mouse cerebellar multipotent cell line. This increased proliferation and decreased drug-induced apoptosis without affecting cell type distribution following differentiation induction. It lowered levels of cyclin kinase inhibitor p21, affected G1 to S phase cell cycle transition; partially blocked the elevation in p53 transcriptional activity, p21 and Bcl-2-associated X protein (Bax) levels caused by bleomycin, but had no influence on enhancement of Bax in response to staurosporine. Thus Porf-2 may inhibit NSC proliferation by enhancing p21 protein levels followed by G1 phase arrest; it plays pro-apoptotic roles in response to drug treatment through both p53 transcription-dependent and independent pathways. This is consistent with categorization of Porf-2 as a functional RhoGAP in CNS.

Abstract B65: Cyclin-dependent kinase inhibitor p21 is a crucial molecular target for the potential antitumor efficacy of the dietary isothiocyanate erucin against prostate cancer

Abstract It is becoming increasingly clear that many dietary agents, such as isothiocyanates (ITCs) from cruciferous vegetables, can retard or prevent the process of prostate carcinogenesis by multiple mechanisms [1, 2]. Erucin (ER) is a dietary isothiocyanate present in cruciferous vegetables, which has been recently considered a new promising cancer chemopreventive phytochemical [2]. In this study, the potential protective activity of ER against prostate cancer was investigated using two different human prostatic cell lines, normal prostate epithelium (PNT1A) and prostate adenocarcinoma cells (PC3), in order to analyze its effects on molecular pathways involved in cell growth regulation, such as the cyclin-dependent kinase (CDKs) inhibitor p21WAF1/CIP1 (p21) pathway [3]. We have shown for the first time that the isothiocyanate ER may up-regulate significantly p21 protein expression in a dose-dependent manner to inhibit the proliferation of human adenocarcinoma prostate PC3 cells. Compared to the structurally related sulforaphane (SF), a well-studied broccoli-derived ITC, the isothiocyanate ER showed lower potency in inhibiting proliferation of PC3 cells, as well as in modulating p21 protein expression. Several studies have showed that the tumor suppressor protein p53 plays a key role in cellular response to DNA damage leading to apoptosis in case of unrepaired DNA damage, or indirectly to the block of cell cycle progression by transactivating p21 [4]. Because PC3 cells do not express functional p53, our data suggest that the up-regulation of p21 by ER occurs through a p53-independent pathway. Moreover, our results showed that ER and SF did not affect p21 protein levels in normal prostate epithelium PNT1A cells at all concentrations tested (0-25 µM). These preliminary data demonstrate that the above structurally related ITCs exert a specific action on oncogenic signaling pathways, such as p53/p21 pathways, that could explain the stronger cytotoxicity observed in cancer cells compared to non malignant cells. Although these dietary bioactive compounds have exhibited selectivity for oncogenically transformed cell lines, ITCs selectivity is an area of great interest that warrants further investigation.

Involvement of Lipogenic Pathway In 5-Azacytidine-Induced Cytotoxicity In Waldenstrom's Macroglobulinemia

AbstractAbstract 29915-azacitidine and 5-aza-2‘-deoxycytidine are demethylation agents used for the treatment of myelodysplastic syndrome (MDS). Despite a putative role for the reactivation of epigenetically silenced tumor-suppressor genes, the mechanism of action for these agents remains to be clarified. Recent studies suggest that 5-azacitidine may also act at the post-transcriptional level by insertion into RNA transcripts. As part of these efforts, we investigated the therapeutic potential of these agents in Waldenstrom's Macroglobulinemia (WM), and characterized molecular changes associated with treatment. We observed that 5-azacitidine was more potent than 5-aza-2‘-deoxycytidine in inducing dose-dependent cytotoxicity against BCWM.1 WM cells and primary WM lymphoplasmacytic cells. 5-azacitidine-induced cell cycle arrest at G1 was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p21 (Cip1/WAF1) through a p53-independent pathway. Activation of caspase-3, 7, 8, and 9, and PARP was also observed indicating involvement of both mitochondrial and death receptor pathways for 5-azacitidine-induced apoptosis. The BH3 domain proteins Bim and Puma were increased in WM cells treated with 5-azacitidine, and siRNA-mediated knockdown of Bim and Puma provided moderate protection from 5-azacitidine-induced apoptosis. In contrast, PI3K/AKT/mTOR and NF- B activities were not significantly altered by 5-azacitidine. To further characterize the molecular changes with 5-azacitidine, we profiled mRNA expression in the WM cells associated with 5-azacitidine treatment. The most strikingly down-regulated genes by 5-azacitidine include FASN and SCD1, which encode enzymes essential for lipogenesis. Importantly, by quantitative RT-PCR, FASN mRNA expression levels were significantly higher in WM patients versus healthy donors (p=0.001). WM patients who expressed higher levels of FASN mRNA showed greater bone marrow involvement (p=0.003). Importantly, siRNA-mediated knockdown of FASN or SCD1 induced significant growth arrest and apoptosis in WM cells. siRNA-mediated knockdown of the sterol regulatory element binding protein 1 (SREBP-1), a transcriptional activator for FASN and SCD1 was associated with reduced transcription of FASN and SCD1, along with increased cytotoxicity in WM cells. Inhibition of FASN activity by Cerulenin also induced significant apoptosis, and the combination of 5-azacitidine and Cerulenin resulted in a synergistic induction of apoptosis in WM cells. In conclusion, the results of this study support an important role for the lipogenic genes in the 5-azacitidine-induced cytotoxicity in WM cells, and highlight a novel mechanistic pathway for this agent, as well as novel targets for drug therapy in WM. Disclosures:Off Label Use: Role of 5-azacytidine in Waldenstrom's macroglobulinemia.

The Anti-Proliferative Effects of Tricyclic Coumarin GUT-70 as An Hsp90 Inhibitor In Mantle Cell Lymphoma

AbstractAbstract 3977Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor prognosis, requiring novel anticancer strategies. Since p53 inactivating mutations occur primarily in the aggressive and refractory MCL variants, targeting p53-independent signaling pathways is of considerable interest. We previously reported the cytotoxic efficacy of a newly discovered tricyclic coumarin GUT-70 (5-methoxy-2,2-dimethyl-6-(2-methyl-1-oxo-2-butenyl) -10-propyl-2H,8H-benzo[1,2-b;3,4-b]dipyran-8-one (C23H26O5) (synthesized at Nippon Shinyaku, Kyoto, Japan), originally derived from Calophyllum brasiliense, with more pronounced apoptotic effects in mutant-p53 MCL cells than in wild type-p53 cells (Jin et al., ASH abstract 2009). Some of the coumarin antibiotics are known to bind to chaperone molecule 90-kDa heat shock proteins (Hsp90) and induce degradation of Hsp90 client proteins including key components of multiple signaling pathways for cell proliferation and/or survival. In this study, the mechanisms of action of GUT-70 were investigated in MCL cell lines with known p53 mutation status (wt-p53: JVM-2, Granta-519, mt-p53: Jeko-1, MINO). GUT-70 demonstrated a dose-dependent binding affinity to Hsp90 (competitive binding assay using fluorescently labeled geldanamycin), increased ubiquitinated proteins accumulation, and further induced degradation of Hsp90 client proteins, including cyclin D1, Raf-1, Akt, and mt-p53, or increased Hsp70, a marker of Hsp90 inhibition. The downregulation of constitutively overexpressed cyclin D1 in MCL by GUT-70 was accompanied by p27 accumulation, decreased Rb phosphorylation, and impeded cell cycle progression in the wt-p53 JVM2 and Granta 519. However, GUT-70 induced apoptosis in mt-p53-bearing MINO and Jeko cells which was accompanied by only minimal cell cycle arrest. These findings suggest that apoptosis induction by GUT-70 in mt-p53 cells is in part independent from cell cycle arrest is known to protect cells from apoptosis. Moreover, the mt-p53 depletion by GUT-70 will further diminish its “gain of functions” for cell proliferation and anti-apoptosis.To determine if GUT-70 might potentiate the apoptotic effects of commonly used chemotherapeutic agents, we assessed the combination effects of GUT-70 with bortezomib (BTZ), a selective inhibitor of the 26S proteasome, and with doxoubicin (DOX), a conventional chemotherapeutic agent drug for MCL. Synergistic anti-proliferative effects of GUT-70 and BTZ or GUT-70 and DOX combinations were observed in both wt-p53 and mt-p53 MCL cells at 48 h post-exposure (combination index; GUT-70/Bortezomib; 0.59 for JVM2, 0.73 forMINO, GUT-70/Doxorubicin; 0.37 for JVM2, 0.35 for MINO). In conclusion, our results demonstrate that the novel anticancer agent tricyclic coumarin GUT-70, an Hsp90 inhibitor, has potential utility for mt-p53 bearing MCL cells, and in the combination therapy of MCL. Disclosures:No relevant conflicts of interest to declare.

Folate Supplementation Induces Differential Dose-Dependent Modulation of Proliferative Phenotypes Among Cancerous and Noncancerous Oral Cell Lines In Vitro

ABSTRACTSufficient folate intake confers positive health benefits, while deficiency is linked with many health problems. Although the US policy of dietary folic acid fortification has reduced the incidence of these deficiency-related health problems, recent evidence has demonstrated an association between folic acid supplementation and increased colorectal cancer incidence. Few studies have explored the possibility that folate affects other slowly developing cancers. This study sought to determine whether folic acid supplementation is sufficient to alter the growth and development of existing oral cancers. A series of in vitro growth, viability, and adhesion assays were performed using the well-characterized human oral squamous cell carcinoma cell lines, CAL27 and SCC25, to determine the effects of folic acid supplementation. Folic acid administration significantly stimulated CAL27 and SCC25 proliferation in a dose-dependent manner, but it was not sufficient to increase proliferation at any concentration tested in the normal control cell line, HGF-1. Neither oral cancer cell line harbored the common C677T DNA polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene, which might reduce folate bioavailability. Overexpression of p53 mRNA was observed in both cancerous cell lines, but it was differentially altered by folic acid administration in only SCC25 cells. These findings suggest folic acid administration may significantly alter growth of oral cancers in vitro via p53-dependent and p53-independent pathways. As oral cancer rates continue to rise in specific geographic areas, and among specific subsets of the US population, understanding environmental mediators, such as folic acid supplementation, becomes increasingly important for nutrition and public health scientists.

Resveratrol-induced p53-independent apoptosis of human nasopharyngeal carcinoma cells is correlated with the downregulation of ΔNp63

ΔNp63, the N-terminal truncated isoform of p63, has been found to be overexpressed in several human epithelial cancers, including nasopharyngeal carcinomas (NPCs), suggesting a function in carcinogenesis. Trans-resveratrol (RSV) has been shown to exert proapoptotic activities through a p53-dependent or p53-independent pathway in various cancer cells. However, the effects of RSV on NPC are still unexplored. In this study, we investigated the apoptotic effects of RSV on ΔNp63-overexpressing NPC cell lines. We showed that RSV (12-100 μ) induced dose-dependent growth suppression, cell-cycle arrest in the S phase and caspase-dependent apoptosis in NPC-TW076 and NPC-TW039 cells. The RSV effect was accompanied by the downregulation of ΔNp63 and the upregulation of p53 protein in a dose-dependent manner. By using small-interfering RNA (siRNA) technology, we found that the targeted silencing of ΔNp63 induced apoptosis and sensitized the NPC cells to RSV-induced apoptosis through caspase-3 activation, whereas suppression of p53 by siRNA did not inhibit RSV-induced apoptosis. Furthermore, transfection with p53 siRNA or pretreatment with caspase inhibitors (Z-VAD-fmk or Z-DEVD-fmk) had no influence on the RSV downregulation of ΔNp63. Interestingly, ecoptic expression of ΔNp63 did not significantly block RSV-induced cell death and was also downregulated after RSV treatment. Downregulation of ΔNp63 by RSV was shown to occur at the mRNA transcript and post-translational levels. Importantly, RSV enhanced chemotheraptic drug-induced apoptosis in NPC and two human carcinoma cell lines, HT1376 and Hep3B cells. These results suggested that ΔNp63, but not p53, is a molecular target of RSV-induced apoptosis and the regulation of ΔNp63 expression by RSV may provide a therapeutic effect of RSV in human NPC.

Downregulation of Nucleostemin Causes G1 Cell Cycle Arrest via a p53-Independent Pathway in Prostate Cancer PC-3 Cells

Aims: To screen the genes and possible signal transduction pathways with which nucleostemin (NS) interacts and explore the mechanism of NS in prostate cancer. Methods: NS-specific short-hairpin RNA expression plasmid was used to downregulate the NS level in PC-3 cells and the changes of cell cycle were studied. After that, oligonucleotide DNA microarray was used to screen the genome changes in PC-3 cells and quantitative real-time PCR was used to further confirm the differentially expressed genes. Results: Detection of cell cycle showed a decrease of S stage and an increase of G1 stage after downregulation of NS. 219 differentially expressed genes were found and these genes were involved in cell cycle, cell proliferation, signal transduction, cell apoptosis and cell differentiation, and so on. Genes related to cell cycle were discussed emphatically. INK4 family genes (P15, P16, P18) were upregulated while cyclin D1 HDAC1 were downregulated. These genes were tightly related to CDK4/6-cyclin D and pRb-E2F1 complexes. Conclusion: NS is an important G1/S checkpoint regulator and it could regulate cell cycles via a p53-independent pathway in prostate cancer.

DNA Adducts of Decarbamoyl Mitomycin C Efficiently Kill Cells without Wild-Type p53 Resulting from Proteasome-Mediated Degradation of Checkpoint Protein 1

The mitomycin derivative 10-decarbamoyl mitomycin C (DMC) more rapidly activates a p53-independent cell death pathway than mitomycin C (MC). We recently documented that an increased proportion of mitosene1-β-adduct formation occurs in human cells treated with DMC in comparison to those treated with MC. Here, we compare the cellular and molecular response of human cancer cells treated with MC and DMC. We find the increase in mitosene 1-β-adduct formation correlates with a condensed nuclear morphology and increased cytotoxicity in human cancer cells with or without p53. DMC caused more DNA damage than MC in the nuclear and mitochondrial genomes. Checkpoint 1 protein (Chk1) was depleted following DMC, and the depletion of Chk1 by DMC was achieved through the ubiquitin proteasome pathway since chemical inhibition of the proteasome protected against Chk1 depletion. Gene silencing of Chk1 by siRNA increased the cytotoxicity of MC. DMC treatment caused a decrease in the level of total ubiquitinated proteins without increasing proteasome activity, suggesting that DMC mediated DNA adducts facilitate signal transduction to a pathway targeting cellular proteins for proteolysis. Thus, the mitosene-1-β stereoisomeric DNA adducts produced by the DMC signal for a p53-independent mode of cell death correlated with reduced nuclear size, persistent DNA damage, increased ubiquitin proteolysis and reduced Chk1 protein.

Major heat shock protein Hsp72 controls oncogene‐induced senescence

Various heat shock proteins, including Hsp72, are strongly upregulated in cancers, but their significance for tumor emergence and growth is poorly understood. Here we review recent data from several labs to indicate that Hsps, including Hsp72, are critical for growth of transformed but not normal cells. By manipulating expression and activity of Hsp72 and several oncogenes, it was shown that Hsp72 suppresses oncogene-induced senescence, thus allowing proliferation of cancer cells. Importantly, Hsp72 is able to suppress both p53-dependent and p53-independent senescence pathways. We propose that targeting Hsp72 may be a promising approach toward development of novel cancer therapies.

Abstract 838: Mdm2 and oncogenic mutant p53 as biomarkers for potential drug targets in estrogen receptor positive and triple negative breast cancer

Abstract The study of Mdm2 and oncogenic p53 as biomarkers for potential drug targets in breast cancers has not been developed. Triple negative breast cancers often have gain-of-function oncogenic mutant p53 while estrogen receptor positive breast cancers retain wild-type p53 and over-express Mdm2. We are investigating how to inhibit the growth of various types of breast cancer cell lines when p53 function is compromised. Here we report two approaches. The first approach utilizes the nucleoside analogue 8-aminoadenosine (8-amino-Ado) and the second approach utilizes inducible RNA interference for oncogenic mdm2 or p53. 8-amino-Ado is under pre-clinical investigation for the treatment of hematological malignancies. We investigated if 8-amino-Ado could inhibit breast cancer cell growth and promote cell death in a manner that did not require functional p53 protein and compared this to gemcitabine (a nucleoside analogue used to treat breast cancer). We used the triple negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 as well as the estrogen receptor positive breast cancer cell line MCF-7. 8-amino-Ado promoted death of the triple negative mutant p53 breast cancers as well, or better, than it did for the estrogen receptor positive wild-type p53 breast cancer and in all cases was superior to gemcitabine. For the second approach we constructed cell lines using an inducible shRNA vector that contained a naturally occurring miR sequence. We previously showed that mutant p53 is required for the survival of MDA-MB-231 cells in the absence of serum. Using an inducible p53 shRNA we see that the mutant p53 found in MDA-MB-231 and MDA-MB-468 gives these cells a survival advantage in the absence of serum and when p53 is knocked down the cells die without PARP cleavage, suggesting non-apoptotic cell death. Estrogen increases the expression of Mdm2 in MCF-7 cells and therefore we investigated if the growth activation feature of estrogen in MCF-7 cells required Mdm2 protein. Induction of mdm2 shRNA reduced the Mdm2 protein level and increased the p21 protein level without a detectable change in p53. Induction of mdm2 shRNA caused a corresponding decrease in estrogen-activated MCF-7 cell growth. Inducible expression of shRNA to p53 or the control construct had no effect on the growth of MCF-7 cells. Our data suggest that 8-amino-Ado induced death occurs via a p53-independent pathway and thus might be beneficial for the treatment of all breast cancers. Additionally Mdm2 and oncogenic mutant p53 are biomarkers that can be used to screen for interfering RNA based and 8-amino-Ado therapeutic approaches for estrogen receptor positive and triple negative breast cancers. This work was supported by The Breast Cancer Research Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 838.

Abstract 3162: MDM2 regulates MYCN mRNA stabilization and translation in human neuroblastoma cells

Abstract The MYCN gene plays a critical role in determining the clinical behavior of neuroblastoma. Genomic amplification occurs in high-risk subsets, but it remains unclear how MYCN expression is regulated in human neuroblastomas. Here, we report that the expression of MYCN is regulated by the oncoprotein MDM2 at the post-transcriptional level. Ectopic overexpression of MDM2 or an increase in the cytoplasmic levels of endogenous MDM2 by inhibition of PI3K/Akt activity enhanced both MYCN mRNA and protein expression. UV cross-linking and RNA-protein binding assays demonstrated that the C-terminal RING domain of MDM2 bound to AU-rich elements within the 3’-untranslated region (3’-UTR) of MYCN mRNA. Furthermore, gene transfection and reporter assay confirmed that MDM2 increased MYCN 3’-UTR-mediated mRNA stability and induced translation of reporter luciferase. Conversely, MDM2 silencing by specific siRNA rendered the MYCN mRNA unstable, reducing the abundant MYCN protein found in MYCN-overexpressing neuroblastoma cell lines, which are a consequence of gene amplification. Importantly, silencing of MDM2 in MYCN-amplified neuroblastoma cell lines resulted in a remarkable inhibition of cell growth and induction of cell death, through a p53-independent pathway. These results indicate that MDM2 plays a p53-independent role in regulating MYCN mRNA stabilization and protein translation, and suggest that MDM2-mediated MYCN expression is an important mechanism for tumor growth and for disease progression, specifically in MYCN-associated neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3162.

Abstract 5048: TAp73a enhances the cellular chemosensitivity to cisplatin in ovarian cancer cells

Abstract Ovarian cancer is the most lethal gynecological malignancy. Most ovarian cancer patients relapse and subsequently die due to the development of resistance to chemotherapy. Strategies for overcoming resistance to chemotherapy and improving prognosis of ovarian cancer patients are very critical. P73, one member of the tumor suppressor p53 family, shares highly structural and functional similarity to p53. Established evidence showed that p73 mediated cellular sensitivity to chemotherapeutic agents in some human tumor cells. However, the role of p73 in the regulation of cellular response to DNA damaging agents in ovarian cancer is still poorly understood, and the function of the transcriptionally active isoform TAp73 as a tumor suppressor is still controversial. The aim of the present study was to examine the effect of TAp73a on cellular response to commonly used chemotherapeutic agent cisplatin in ovarian cancer cells. We established TAp73a overexpressed stable clones in two ovarian cancer cell lines: OVCA433 (wild-type p53) and SKOV3 (null p53). We found that TAp73a enhanced chemosensitivity of ovarian cancer cells to cisplatin by inhibiting cell growth and inducing cell cycle arrest and apoptosis in wild-type p53 OVCA433 cells. Additionally, TAp73a up-regulated the p53 expression and its downstream genes: PUMA, NOXA, p53AIP1, and down-regulated the phosphorylation of Akt as assessed by western blotting. The accumulation of p53 protein induced by TAp73a was found at the post-transcriptional level. Co-immunoprecipitation assay demonstrated that TAp73a directly bound to p53 protein, suggesting that such interaction might block the degradation of p53 protein in OVCA433 cells. In p53-null SKOV3 cells, the increase of chemosensitivity induced by TAp73a was also observed, indicating that TAp73a also functioned independent of p53 by up-regulating the cell cycle inhibitor p21 expression. In conclusion, these results suggested that TAp73a might play an important role in the enhancement of cellular sensitivity to chemotherapeutic drug cisplatin in ovarian cancer cells through p53 dependent and independent pathways. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5048.

Abstract 729: Mechanism of action of a p53-derived peptide that targets a novel death pathway inducing selective necrosis in cancer cells

Abstract In a series of preliminary in vitro and in vivo studies we have shown that p53-derived peptides consisting of residues 12-26 and 17-26 from the murine double minute binding domain (MDM-2) possess anti-cancer activity. These peptides are linked to a trans-membrane penetrating sequence (penetratin) derived from the antennapedia homeodomain of Drosophila and are designated PNC-27 and PNC-28, respectively. We also found that the previously determined 3D structure of these PNC residues are directly superimposeable on the same residues bound to MDM-2, suggesting that these peptide may target MDM-2 in cancer cells. These peptide constructs appear to promote cancer cell death through a novel pathway that leads to necrosis rather than apoptosis while sparing their untransformed counterparts. In our initial experiments, the critical role of the MDM-2 oncoprotein as a potential target molecule for these PNC peptides was elucidated when a sequence specific MDM-2 antibody blocked PNC-27 activity on cancer cells. To further elucidate this anti-cancer mechanism we then tested parent molecule PNC-27 against selected cancer cell lines (Panc-02, pancreatic; HT1080, fibrosarcoma; and MCF-7, breast) plus respective untransformed counterparts (BMRPA1, BJ, and MCF-10) which were treated with PNC-27 and control peptide. Subsequently, treatment with fluorescent labeled peptide, immunoprecipitation (IP), confocal, and time-lapse electron microscopy (EM) was employed as well as transfection of untransformed cells with the MDM-2 gene to study the anti-cancer mechanism. PNC-27 did not cause elevation of pro-apoptotic proteins but induced rapid dose-dependent tumor cell death with release of lactate dehydrogenase (LDH). This specific anti-cancer mechanism, in part, may be due to observed increased expression of MDM-2 found in cancer cells compared to their normal cellular counterparts which is consistent with abrogation of MDM-2 and stabilization of p53 protein levels after PNC-27 treatment. Moreover, following IP of fluorescent labeled PNC-27 from treated cancer cells; labeled MDM-2 was detected. Furthermore, confocal microscopy and EM revealed co-localization of PNC-27 and MDM-2 along with cancer cell membrane pore formation, respectively. Remarkably, transfection of the MDM-2 gene resulted with increased expression of MDM-2 and now untransformed cell susceptibility to PNC-27. These results suggest a mechanism of PNC-27 binding to MDM-2 in the cancer cell membrane, leading to pore formation and membrane disruption; a p53-independent pathway consistent with rapid selective cancer cell necrosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 729.

Inhibitory effects of fucoidan on ICAM‐1 expression induced by inflammatory cytokine p53‐independent pathway in SH‐SY‐5Y

Fucoidans that are high‐molecular‐weight sulfated polysaccharides extracted from brown seaweeds have been known to have various biological activities. It is well known that the upregulation of adhesion molecules on endothelial cell surface is involved in inflammation responses, so we examined the effect of fucoidan on ICAM‐1 expression induced by inflammatory cytokines, fnTNF‐α combined with IFN‐γ in SH‐SY‐5Y cells. Both NO production and ICAM‐1 expression which were induced by inflammatory cytokines were decreased by treatment of fucoidan in SH‐SY‐5Y. We detected ICAM‐1 expression in same condition which was using p53 mutant SH‐SY‐5Y cells to investigate mechanisms involved in down regulation of ICAM‐1 expression. Because ICAM‐1 expression induced by inflammatory cytokines was not detected in p53 mutant SH‐SY‐5Y, we postulated p53 gene is necessary for induction of ICAM‐1 expression. But, decreasing effects of fucoidan on ICAM‐1 expression induced by inflammatory cytokinesfnin SH‐SY‐5Y did not compare with decrease of p53 expression in dose‐dependent manners. From these results, we suggested that ICAM‐1 expression induced by inflammatory cytokines is decreased through p53 independent pathway by treatment of fucoidan in SH‐SY‐5Y. (This work was supported by grant No. RTI05‐01‐02 from the Regional Technology Innovation Program of the Ministry of Knowledge Economy(MKE))

Suppression of B-cell lymphomagenesis by the BH3-only proteins Bmf and Bad

AbstractOncogenic c-Myc is known to balance excessive proliferation by apoptosis that can be triggered by p53-dependent and p53-independent signaling networks. Here, we provide evidence that the BH3-only proapoptotic Bcl-2 family members Bcl-2 modifying factor (Bmf) and Bcl-2 antagonist of cell death (Bad) are potent antagonists of c-Myc–driven B-cell lymphomagenesis. Tumor formation was preceded by the accumulation of preneoplastic pre-B and immature immunoglobulin M–positive (IgM+) B cells in hematopoietic organs of Eμ-myc/bmf−/− mice, whereas Eμ-myc/bad−/− mice showed an increase of pre-B cells limited to the spleen. Although the loss of Bad had no impact on the tumor immunophenotype, Bmf deficiency favored the development of IgM+ B cell over pre-B cell tumors. This phenomenon was caused by a strong protection of immature IgM+ B cells from oncogene-driven apoptosis caused by loss of bmf and c-Myc–induced repression of Bmf expression in premalignant pre-B cells. Steady-state levels of B-cell apoptosis also were reduced in the absence of Bad, in support of its role as a sentinel for trophic factor-deprivation. Loss of Bmf reduced the pressure to inactivate p53, whereas Bad deficiency did not, identifying Bmf as a novel component of the p53-independent tumor suppressor pathway triggered by c-Myc.

Boswellic Acid Fractions Induces Apoptosis And Cell Cycle Arrest In Hepatocellular Carcinoma Cell Line (HEP-G2) Through P53 Accumulation

There has been growing interest in naturally occurring compounds with anti-cancer potential. Boswellic acid fractions (BA) are the bioactive constituent of the oleogum resin of Boswellia carterii Birdwood (Bursearceae). It has been shown to exert anti-neoplastic and anti-inflammatory effects. The antitumor activity molecular pathways of BA action are not clear. Nevertheless, BA is known to induce apoptosis by p53-dependent and p53-independent pathways in cancer cell lines. Growth inhibition is associated with induction of cellcycle arrest. In this study, the anticancer effect of BA in human HepG-2 cancercells line was investigated. BA has exhibited effective cell growth inhibition by inducing cancer cells to undergo G2/M phase arrest and apoptosis. Blockade of cell cycle was associated with increased levels of p21. BA treatment triggered the apoptotic pathway indicated by a change in caspase-8 and caspase-3 activation. We also found that, BAinduced cell growth inhibition as a result of increase in the expression level of p53. These results confirm a critical role of p53 in BAinduced G2/M arrest and apoptosis of human hepatoma cancer cells.

Bortezomib suppresses focal adhesion kinase expression via interrupting nuclear factor-kappa B

Aims Bortezomib is a potent proteasome inhibitor currently used to treat various malignancies with promising results. To explore the role of bortezomib in reducing cancer cell migration and inducing apoptosis, we evaluated the effects of bortezomib on the expression of focal adhesion kinase (FAK). Main methods Various types of cancer cells including lung cancer A549, H1299; a breast cancer MCF7; a hepatocellular carcinoma Huh7, and a tongue squamous cell carcinoma SCC-25 were treated with different concentrations of bortezomib or MG-132 as indicated for 24 h. Protein and mRNA levels were determined by Western blotting and real-time PCR. Apoptosis was analyzed by caspase 3 cleavage and activity. FAK promoter and NFκB binding activities were measured by luciferase-reporter method. NFκB subunit p65 binding capacity was determined by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis. Key findings Both bortezomib and another proteasome inhibitor, MG-132, significantly reduced FAK expression, suppressed cancer cell migration and increased cell apoptosis. Results of real-time PCR and promoter activity assay revealed that bortezomib decreased FAK expression through transcriptional inactivation. Results of FAK promoter activity and ChIP assays in A549 and H1299 cells indicated that bortezomib suppressed FAK activity through a p53-independent pathway. Furthermore, reduction of NFκB binding capacity demonstrated by EMSA and ChIP assay suggested that NFκB plays an important role in bortezomib suppressing FAK expression. Significance These results suggested that FAK is downregulated by bortezomib through a proteasome-dependent NFκB inhibitory mechanism. Thus, FAK could be a potential molecular target of bortezomib for therapeutic strategy.