Combining fisetin and ionizing radiation suppresses the growth of mammalian colorectal cancers in xenograft tumor models.

Fisetin (3,7,3',4'-tetrahydroxyflavone), which belongs to the flavonoid group of polyphenols and is found in a wide range of plants, has been reported to exhibit a number of biological activities in human cancer cells, including antioxidant, anti-inflammatory, antiangiogenic, anti-invasive and antiproliferative effects. Although previous in vitro studies have shown that fisetin treatment increases the apoptotic rate and enhances the radiosensitivity of human colorectal cancer cells, the in vivo effects of fisetin on tumor growth remain unclear. In the present study a murine xenograft tumor model was employed to investigate the therapeutic effects of fisetin in combination with radiation on CT-26 colon cancer cells and human HCT116 colorectal cancer cells. This revealed that intratumoral injection of fisetin significantly suppressed the growth of CT-26 tumors compared with the untreated control group, but had little effect on the growth of HCT116 tumors. However, fisetin in combination with 2-Gy radiation enhanced tumor suppressor activity in murine colon and human colorectal xenograft tumors, as compared with 2-Gy fractionated radiation administered alone for 5 days and fisetin alone. Interestingly, fisetin downregulated the expression of the oncoprotein securin in a p53-independent manner. However, securin-null HCT116 tumors showed only moderate sensitivity to fisetin treatment, and the combination of fisetin and radiation did not significantly suppress securin-null HCT116 tumor growth compared with normal HCT116 tumors. Therefore, the role of securin in mediating the effect of fisetin on colorectal cancer growth warrants further investigation. In conclusion, the results of the current study provide important preclinical data for evaluating the efficacy of fisetin and radiation combination treatment as an adjuvant chemoradiotherapy for human colorectal cancers.

miRNA-7/21/107 contribute to HBx-induced hepatocellular carcinoma progression through suppression of maspin.

Maspin suppresses tumor progression by promoting cell adhesion and apoptosis and by inhibiting cell motility. However, its role in tumorigenesis of hepatocellular carcinoma (HCC) remains unclear. The gene regulation of maspin and its relationship with HCC patient prognosis were investigated in this study. Maspin expression was specifically reduced in HBV-associated patients and correlated with their poor prognosis. Maspin downregulation in HCC cells was induced by HBx to promote their motility and resistance to anoikis and chemotherapy. HBx-dependent induction of microRNA-7, -107, and -21 was further demonstrated to directly target maspin mRNA, leading to its protein downregulation. Higher expressions of these microRNAs also correlated with maspin downregulation in HBV-associated patients, and were associated with their poor overall survival. These data not only provided new insights into the molecular mechanisms of maspin deficiency by HBx, but also indicated that downregulation of maspin by microRNAs confers HBx-mediated aggressiveness and chemoresistance in HCC.

Over-expression of cofilin-1 suppressed growth and invasion of cancer cells is associated with up-regulation of let-7 microRNA.

Cofilin-1, a non-muscle isoform of actin regulatory protein that belongs to the actin-depolymerizing factor (ADF)/cofilin family is known to affect cancer development. Previously, we found that over-expression of cofilin-1 suppressed the growth and invasion of human non-small cell lung cancer (NSCLC) cells in vitro. In this study, we further investigated whether over-expression of cofilin-1 can suppress tumor growth in vivo, and performed a microRNA array analysis to better understand whether specific microRNA would be involved in this event. The results showed that over-expression of cofilin-1 suppressed NSCLC tumor growth using the xenograft tumor model with the non-invasive reporter gene imaging modalities. Additionally, cell motility and invasion were significantly suppressed by over-expressed cofilin-1, and down-regulation of matrix metalloproteinase (MMPs) -1 and -3 was concomitantly detected. According to the microRNA array analysis, the let-7 family, particularly let-7b and let-7e, were apparently up-regulated among 248 microRNAs that were affected after over-expression of cofilin-1 up to 7 days. Knockdown of let-7b or let-7e using chemical locked nucleic acid (LNA) could recover the growth rate and the invasion of cofilin-1 over-expressing cells. Next, the expression of c-myc, LIN28 and Twist-1 proteins known to regulate let-7 were analyzed in cofilin-1 over-expressing cells, and Twist-1 was significantly suppressed under this condition. Up-regulation of let-7 microRNA by over-expressed cofilin-1 could be eliminated by co-transfected Twist-1 cDNA. Taken together, current data suggest that let-7 microRNA would be involved in over-expression of cofilin-1 mediated tumor suppression in vitro and in vivo.

Evaluation of the therapeutic and diagnostic effects of PEGylated liposome-embedded 188Re on human non-small cell lung cancer using an orthotopic small-animal model.

UNLABELLED: Non-small cell lung cancer (NSCLC) is a highly morbid and mortal cancer type that is difficult to eradicate using conventional chemotherapy and radiotherapy. Little is known about whether radionuclide-based pharmaceuticals can be used for treating NSCLC. Here we embedded the therapeutic radionuclide (188)Re in PEGylated (PEG is polyethylene glycol) liposomes and investigated the biodistribution, pharmacokinetics, and therapeutic efficacy of this nanoradiopharmaceutical on NSCLC using a xenograft lung tumor model and the reporter gene imaging techniques. METHODS: Human NSCLC NCI-H292 cells expressing multiple reporter genes were used in this study. (188)Re was conjugated to N,N-bis(2-mercaptoethyl)-N',N'-diethylethylenediamine (BMEDA) and loaded into the PEGylated liposome to form a (188)Re-liposome. The tumor growth rates and localizations were confirmed using bioluminescent imaging and SPECT/CT after the (188)Re-BMEDA or (188)Re-liposome was intravenously injected. The accumulation of the nanodrug in various organs was determined by the biodistribution analysis and the nano-SPECT/CT system. The pharmacokinetic and dosimetric analyses were further determined using WinNonlin and OLINDA/EXM, respectively. RESULTS: The biodistribution and nano-SPECT/CT imaging showed that PEGylated (188)Re-liposome could efficiently accumulate in xenograft tumors formed by NCI-H292 cells that were subcutaneously implanted in nude mice. Pharmacokinetic analysis also showed that the retention of (188)Re-liposome was longer than that of (188)Re-BMEDA. In an orthotopic tumor model, ex vivo γ counting revealed that the uptake of (188)Re-liposome was detected in tumor lesions but not in surrounding normal lung tissues. Moreover, we evaluated the therapeutic efficacy using bioluminescent imaging and showed that the lung tumor growth was suppressed but not eradicated by (188)Re-liposome. The life span of (188)Re-liposome-treated mice was 2-fold longer than that of untreated control mice. CONCLUSION: The results of biodistribution, pharmacokinetics, estimated dosimetry, nano-SPECT/CT, and bioluminescent imaging suggest that the PEGylated liposome-embedded (188)Re could be used for the treatment of human lung cancers.

Autophagy promotes radiation-induced senescence but inhibits bystander effects in human breast cancer cells.

Ionizing radiation induces cellular senescence to suppress cancer cell proliferation. However, it also induces deleterious bystander effects in the unirradiated neighboring cells through the release of senescence-associated secretory phenotypes (SASPs) that promote tumor progression. Although autophagy has been reported to promote senescence, its role is still unclear. We previously showed that radiation induces senescence in PTTG1-depleted cancer cells. In this study, we found that autophagy was required for the radiation-induced senescence in PTTG1-depleted breast cancer cells. Inhibition of autophagy caused the cells to switch from radiation-induced senescence to apoptosis. Senescent cancer cells exerted bystander effects by promoting the invasion and migration of unirradiated cells through the release of CSF2 and the subsequently activation of the JAK2-STAT3 and AKT pathways. However, the radiation-induced bystander effects were correlated with the inhibition of endogenous autophagy in bystander cells, which also resulted from the activation of the CSF2-JAK2 pathway. The induction of autophagy by rapamycin reduced the radiation-induced bystander effects. This study reveals, for the first time, the dual role of autophagy in radiation-induced senescence and bystander effects.

Nucleolin is important for Epstein-Barr virus nuclear antigen 1-mediated episome binding, maintenance, and transcription.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for EBV episome maintenance, replication, and transcription. These effects are mediated by EBNA1 binding to cognate oriP DNA, which comprise 20 imperfect copies of a 30-bp dyad symmetry enhancer and an origin for DNA replication. To identify cell proteins essential for these EBNA1 functions, EBNA1 associated cell proteins were immune precipitated and analyzed by liquid chromatography-tandem mass spectrometry. Nucleolin (NCL) was identified to be EBNA1 associated. EBNA1's N-terminal 100 aa and NCL's RNA-binding domains were critical for EBNA1/NCL interaction. Lentivirus shRNA-mediated NCL depletion substantially reduced EBNA1 recruitment to oriP DNA, EBNA1-dependent transcription of an EBV oriP luciferase reporter, and EBV genome maintenance in lymphoblastoid cell lines. NCL RNA-binding domain K429 was critical for ATP and EBNA1 binding. NCL overexpression increased EBNA1 binding to oriP and transcription, whereas NCL K429A was deficient. Moreover, NCL silencing impaired lymphoblastoid cell line growth. These experiments reveal a surprisingly critical role for NCL K429 in EBNA1 episome maintenance and transcription, which may be a target for therapeutic intervention.

mPlum-IFP 1.4 fluorescent fusion protein may display Förster resonance energy transfer associated properties that can be used for near-infrared based reporter gene imaging.

Bacteriophytochrome infrared fluorescent protein (IFP) has a long emission wavelength that is appropriate for detecting pathophysiological effects via near-infrared (NIR) based imaging. However, the brightness and photostability of IFP are suboptimal, although an exogenous supply of biliverdin (BV) IXα is able to enhance these properties. In this study, we fused a far red mPlum fluorescent protein to IFP 1.4 via a linker deoxyribonucleic acid (DNA) sequence encoding eight amino acids. The brightness of mPlum-IFP 1.4 fusion protein at the IFP emission channel was comparable to that of native IFP 1.4 protein when fusion protein and IFP 1.4 were excited by 543 and 633 nm using confocal microscopy, respectively. Visualization of IFP 1.4 fluorescence by excitation of mPlum in mPlum-IFP 1.4 fusion protein is likely to be associated with Förster resonance energy transfer (FRET). The FRET phenomenon was also predicted by acceptor photobleaching using confocal microscopy. Furthermore, the expression of mPlum-IFP 1.4 fusion protein could be detected in cell culture and in xenograft tumors in the absence of BV using in vivo imaging system, although the BV was still essential for detecting native IFP 1.4. Therefore, this innovative-fluorescent fusion protein would be useful for NIR-based imaging in vitro and in vivo.

Radiation-induced senescence in securin-deficient cancer cells promotes cell invasion involving the IL-6/STAT3 and PDGF-BB/PDGFR pathways.

Securin overexpression correlates with poor prognosis in various tumours. We have previously shown that securin depletion promotes radiation-induced senescence and enhances radiosensitivity in human cancer cells. However, the underlying molecular mechanisms and the paracrine effects remain unknown. In this study, we showed that radiation induced senescence in securin-deficient human breast cancer cells involving the ATM/Chk2 and p38 pathways. Conditioned medium (CM) from senescent cells promoted the invasion and migration of non-irradiated cancer and endothelial cells. Cytokine assay analysis showed the up-regulation of various senescence-associated secretory phenotypes (SASPs). The IL-6/STAT3 signalling loop and platelet-derived growth factor-BB (PDGF-BB)/PDGF receptor (PDGFR) pathway were important for CM-induced cell migration and invasion. Furthermore, CM promoted angiogenesis in the chicken chorioallantoic membrane though the induction of IL-6/STAT3- and PDGF-BB/PDGFR-dependent endothelial cell invasion. Taken together, our results provide the molecular mechanisms for radiation-induced senescence in securin-deficient human breast cancer cells and for the SASP responses.

Enhanced cellular radiosensitivity induced by cofilin-1 over-expression is associated with reduced DNA repair capacity.

PURPOSE: A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity. MATERIALS AND METHODS: Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by Comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632. RESULTS: When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation-induced double-strand break (DSB) repair systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1. CONCLUSION: CFL-1 over-expression enhances radiosensitivity and this is associated with reduced DNA repair capacity. Although phosphorylated CFL-1 seems to be involved in radiosensitivity, further studies are required to address the importance of CFL-1 activity to the regulation of radiosensitivity.

The nuclear chaperone nucleophosmin escorts an Epstein-Barr Virus nuclear antigen to establish transcriptional cascades for latent infection in human B cells.

Epstein-Barr Virus (EBV) is an oncogenic γ-herpesvirus that capably establishes both latent and lytic modes of infection in host cells and causes malignant diseases in humans. Nuclear antigen 2 (EBNA2)-mediated transcription of both cellular and viral genes is essential for the establishment and maintenance of the EBV latency program in B lymphocytes. Here, we employed a protein affinity pull-down and LC-MS/MS analysis to identify nucleophosmin (NPM1) as one of the cellular proteins bound to EBNA2. Additionally, the specific domains that are responsible for protein-protein interactions were characterized as EBNA2 residues 300 to 360 and the oligomerization domain (OD) of NPM1. As in c-MYC, dramatic NPM1 expression was induced in EBV positively infected B cells after three days of viral infection, and both EBNA2 and EBNALP were implicated in the transactivation of the NPM1 promoter. Depletion of NPM1 with the lentivirus-expressed short-hairpin RNAs (shRNAs) effectively abrogated EBNA2-dependent transcription and transformation outgrowth of lymphoblastoid cells. Notably, the ATP-bound state of NPM1 was required to induce assembly of a protein complex containing EBNA2, RBP-Jκ, and NPM1 by stabilizing the interaction of EBNA2 with RBP-Jκ. In a NPM1-knockdown cell line, we demonstrated that an EBNA2-mediated transcription defect was fully restored by the ectopic expression of NPM1. Our findings highlight the essential role of NPM1 in chaperoning EBNA2 onto the latency-associated membrane protein 1 (LMP1) promoters, which is coordinated with the subsequent activation of transcriptional cascades through RBP-Jκ during EBV infection. These data advance our understanding of EBV pathology and further imply that NPM1 can be exploited as a therapeutic target for EBV-associated diseases.

Securin enhances the anti-cancer effects of 6-methoxy-3-(3',4',5'-trimethoxy-benzoyl)-1H-indole (BPR0L075) in human colorectal cancer cells.

BPR0L075 [6-methoxy-3-(3',4',5'-trimethoxy-benzoyl)-1H-indole] is a novel anti-microtubule drug with anti-tumor and anti-angiogenic activities in vitro and in vivo. Securin is required for genome stability, and is expressed abundantly in most cancer cells, promoting cell proliferation and tumorigenesis. In this study, we found that BPR0L075 efficiently induced cell death of HCT116 human colorectal cancer cells that have higher expression levels of securin. The cytotoxicity of BPR0L075 was attenuated in isogenic securin-null HCT116 cells. BPR0L075 induced DNA damage response, G(2)/M arrest, and activation of the spindle assembly checkpoint in HCT116 cells. Interestingly, BPR0L075 induced phosphorylation of securin. BPR0L075 withdrawal resulted in degradation of securin, mitotic exit, and mitotic catastrophe, which were attenuated in securin-null cells. Inhibition of cdc2 decreased securin phosphorylation, G(2)/M arrest and cell death induced by BPR0L075. Moreover, BPR0L075 caused cell death through a caspase-independent mechanism and activation of JNK and p38 MAPK pathways. These findings provided evidence for the first time that BPR0L075 treatment is beneficial for the treatment of human colorectal tumors with higher levels of securin. Thus, we suggest that the expression levels of securin may be a predictive factor for application in anti-cancer therapy with BPR0L075 in human cancer cells.

Dietary flavonoid fisetin targets caspase-3-deficient human breast cancer MCF-7 cells by induction of caspase-7-associated apoptosis and inhibition of autophagy.

The outcome of producing apoptotic defects in cancer cells is the primary obstacle that limits the therapeutic efficacy of anticancer agents, and hence the development of novel agents targeting novel non-canonical cell death pathways has become an imperative mission for clinical research. Fisetin (3,3',4',7-tetrahydroxyflavone) is a naturally occurring flavonoid commonly found in fruits and vegetables. In this study, we investigated the potential anticancer effects of fisetin on breast cancer cells. The result showed fisetin induced higher cytotoxicity in human breast cancer MCF-7 than in MDA-MB-231 cells otherwise it did not exert any detectable cytotoxicity in non-tumorigenic MCF-10A cells. We found fisetin can trigger a novel form of atypical apoptosis in caspase-3-deficient MCF-7 cells, which was characterized by several apoptotic features, including plasma membrane rupture, mitochondrial depolarization, activation of caspase-7, -8 and -9, and PARP cleavage; however, neither DNA fragmentation and phosphotidylserine (PS) externalization was observed. Although p53 was also activated by fisetin, the fisetin-induced apoptosis was not rescued by the p53 inhibitor pifithrin-α. In contrast, the fisetin-induced apoptosis was abrogated by pan-caspase inhibitor z-VAD-fmk. Furthermore, inhibition of autophagy by fisetin was shown as additional route to prompt anticancer activity in MCF-7 cells. These data allow us to propose that fisetin appears as a new potential anticancer agent which can be applied to develop a clinical protocol of human breast cancers.

Curcuminoids promote neurite outgrowth in PC12 cells through MAPK/ERK- and PKC-dependent pathways.

Curcuminoids, the predominant polyphenolic compounds in the rhizome of Curcuma longa Linn., consist of curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). They exhibit multiple desirable characteristics for a neuroprotective agent including antioxidant, anti-inflammatory, and antiamyloid activities. In this work, we report the first investigation of the neurotrophic action and mechanism of curcuminoids in PC12 cells, which respond to nerve growth factor (NGF) and therefore serve as a model system for primary neuronal cells. The percentages of neurite-bearing cells for those treated with 20 µM curcumin, DMC, and BDMC for 72 h reached 21.6 ± 2.0%, 16.3 ± 2.4%, and 19.9 ± 2.5%, respectively, and were significantly higher than that of the negative control (2.0 ± 0.3%, p < 0.05). In parallel, increased expression of the neuronal differentiation markers, growth-associated protein-43 (GAP-43), and neurofilament-L (NF-L) was found in curcuminoid-treated cells. All three curcuminoids (20 µM) activated extracellular signal-regulated protein kinase 1/2 (ERK1/2) and protein kinase C (PKC) signalings, and inhibition of these kinases with the respective pharmacological inhibitors effectively attenuated curcuminoid-induced neurite outgrowth. Furthermore, our results show that both curcumin and DMC, but not BDMC, induced phosphorylation of cAMP response element-binding protein (CREB) and CRE-reporter gene activity significantly (p < 0.05). These inductions were markedly attenuated by the addition of MEK/ERK or PKC inhibitor; as a consequence, ERK- and PKC-dependent pathways may be involved in curcuminoid-mediated neuritogenesis in PC12 cells. Moreover, activation of CREB coupling with CRE-dependent gene transcription may play a vital role for curcumin- or DMC-induced PC12 differentiation.

Antimigratory Effects of the Methanol Extract from Momordica charantia on Human Lung Adenocarcinoma CL1 Cells.

Momordica charantia has been found to exhibit anticancer activity, in addition to its well-known therapeutic functions. We have demonstrated that the leaf extract of Momordica charantia (MCME) induces apoptosis in several human cancer cells through caspase- and mitochondria-dependent pathways. In this study, a different susceptibility to MCME was found in human lung adenocarcinoma CL1 cells with different metastatic ability, leading to the significant difference of cell viability and invasiveness between MCME-treated CL1-0 and CL1-5 cells. MCME was found to upregulate the expression of Wnt-2 and affect the migratory and invasive ability of CL1 cells through suppressed MMP-2 and MMP-9 enzymatic activities. We proposed that MCME mediates inhibition against migration of CL1 cells by reducing the expression and activation of Src and FAK to decrease the expression of downstream Akt, ß-catenin, and MMPs.

Antiproliferative effects of N-heterocyclic indolyl glyoxylamide derivatives on human lung cancer cells.

BACKGROUND: N-Heterocyclic indolyl glyoxylamide compounds are derived from the antimicrotubule agent D-24851, which exhibits anticancer activity after oral administration. The actions of these compounds on lung cancer cells are still unknown. Here, we investigated the effects of two N-heterocyclic indolyl glyoxylamides, BPR0C259 and BPR0C123, on non-small human lung cancer cells. MATERIALS AND METHODS: 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the half maximal inhibitory concentration (IC(50)), cell viability and radiation response of A549 cells and H1299 cells. Apoptosis was determined by sub-G(1) ratio, colony formation assay and caspase-3 activation. Cell cycle distribution was detected using flow cytometry. RESULTS: Both compounds were able to inhibit the viability of human lung cancer cells, although the IC(50) of BPR0C123 was lower than that of BPR0C259. Both compounds induced significant sub-G1 and caspase-3 activation as low as 0.1 µM in both cell lines. These effects were independent of p53 activation because the level of serine-15 phosphorylated p53 was not affected after drug treatment. Furthermore, both compounds induced similar levels of G(2)/M phase arrest and radiosensitivity in these lung cancer cells. CONCLUSION: Current data suggest that N-heterocyclic indolyl glyoxylamides can suppress the proliferation of and potentially increase radiosensitivity of human lung cancer cells.

Securin depletion sensitizes human colon cancer cells to fisetin-induced apoptosis.

Securin is highly-expressed in various tumors including those of the colon. In this study, the role of securin in the anticancer effects of fisetin on human colon cancer cells was investigated. Fisetin-induced apoptosis in HCT116 cells as indicated by TUNEL assay, Annexin V-FITC/PI double staining, Ser15-phosphorylation of p53, and cleavages of procaspase-3 and PARP. These effects were enhanced in HCT116 securin-null cells or in wild-type cells in which securin was knockdown by siRNA, but attenuated when wild-type or non-degradable securin was reconstituted. Moreover, fisetin did not induce apoptosis in HCT116 p53-null and HT-29 p53-mutant cells. Knockdown of securin in HCT116 p53-null cells potentiated fisetin-induced cytotoxicity by induction of apoptosis. Our results provide the first evidence to support that securin depletion sensitizes human colon cancer cells to fisetin-induced apoptosis.

Enhancement of p53-mutant human colorectal cancer cells radiosensitivity by flavonoid fisetin.

PURPOSE: The aim of this study was to investigate whether fisetin is a potential radiosensitizer for human colorectal cancer cells, which are relatively resistant to radiotherapy. METHODS AND MATERIALS: Cell survival was examined by clonogenic survival assay, and DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The effects of treatments on cell cycle distribution and apoptosis were examined by flow cytometry. Western blot analysis was performed to ascertain the protein levels of gamma-H2AX, phospho-Chk2, active caspase-3, PARP cleavage, phospho-p38, phospho-AKT, and phospho-ERK1/2. RESULTS: Fisetin pretreatment enhanced the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells but not human keratocyte HaCaT cells; it also prolonged radiation-induced G(2)/M arrest, enhanced radiation-induced cell growth arrest in HT-29 cells, and suppressed radiation-induced phospho-H2AX (Ser-139) and phospho-Chk2 (Thr-68) in p53-mutant HT-29 cells. Pretreatment with fisetin enhanced radiation-induced caspase-dependent apoptosis in HT-29 cells. Fisetin pretreatment augmented radiation-induced phosphorylation of p38 mitogen-activated protein kinase, which is involved in caspase-mediated apoptosis, and SB202190 significantly reduced apoptosis and radiosensitivity in fisetin-pretreated HT-29 cells. By contrast, both phospho-AKT and phospho-ERK1/2, which are involved in cell proliferation and antiapoptotic pathways, were suppressed after irradiation combined with fisetin pretreatment. CONCLUSIONS: To our knowledge, this study is the first to provide evidence that fisetin exerts a radiosensitizing effect in p53-mutant HT-29 cells. Fisetin could potentially be developed as a novel radiosensitizer against radioresistant human cancer cells.

Depletion of securin induces senescence after irradiation and enhances radiosensitivity in human cancer cells regardless of functional p53 expression.

PURPOSE: Radiotherapy is one of the best choices for cancer treatment. However, various tumor cells exhibit resistance to irradiation-induced apoptosis. The development of new strategies to trigger cancer cell death besides apoptosis is necessary. This study investigated the role of securin in radiation-induced apoptosis and senescence in human cancer cells. METHODS AND MATERIALS: Cell survival was determined using clonogenic assays. Western blot analysis was used to analyze levels of securin, caspase-3, PARP, p53, p21, Rb, gamma-H2AX, and phospho-Chk2. Senescent cells were analyzed using a beta-galactosidase staining assay. A securin-expressed vector (pcDNA-securin) was stably transfected into securin-null HCT116 cells. Securin gene knockdown was performed by small interfering RNA and small hairpin RNA in HCT116 and MDA-MB-231 cells, respectively. RESULTS: Radiation was found to induce apoptosis in securin wild type HCT116 cells but induced senescence in securin-null cells. Restoration of securin reduced senescence and increased cell survival in securin-null HCT116 cells after irradiation. Radiation-induced gamma-H2AX and Chk2 phosphorylation were induced transiently in securin-wild-type cells but exhibited sustained activation in securin-null cells. Securin gene knockdown switches irradiation-induced apoptosis to senescence in both HCT116 p53-null and MDA-MB-231 cells. CONCLUSIONS: Our results demonstrated that the level of securin expression plays a determining role in the radiosensitivity and fate of cells. Depletion of securin impairs DNA repair after irradiation, increasing DNA damage and promoting senescence in the residual surviving cells regardless of functional p53 expression. The knockdown of securin may contribute to a novel radiotherapy protocol for the treatment of human cancer cells that are resistant to irradiation.

Rosiglitazone enhances the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells.

Combined-modality treatment has improved the outcome in cases of various solid tumors, and radiosensitizers are used to enhance the radiotherapeutic efficiency. Rosiglitazone, a synthetic ligand of peroxisome proliferator-activated receptors gamma used in the treatment of type-2 diabetes, has been shown to reduce tumor growth and metastasis in human cancer cells, and may have the potential to be used as a radiosensitizer in radiotherapy for human colorectal cancer cells. In this study, rosiglitazone treatment significantly reduced the cell viability of p53-wild type HCT116 cells but not p53-mutant HT-29 cells. Interestingly, rosiglitazone pretreatment enhanced radiosensitivity in p53-mutant HT-29 cells but not HCT116 cells, and prolonged radiation-induced G(2)/M arrest and enhanced radiation-induced cell growth inhibition in HT-29 cells. Pretreatment with rosiglitazone also suppressed radiation-induced H2AX phosphorylation in response to DNA damage and AKT activation for cell survival; on the contrary, rosiglitazone pretreatment enhanced radiation-induced caspase-8, -9, and -3 activation and PARP cleavage in HT-29 cells. In addition, pretreatment with a pan-caspase inhibitor, zVAD-fmk, attenuated the levels of caspase-3 activation and PARP cleavage in radiation-exposed cancer cells in combination with rosiglitazone pretreatment. Our results provide proof for the first time that rosiglitazone suppresses radiation-induced survival signals and DNA damage response, and enhances the radiation-induced apoptosis signaling cascade. These findings can assist in the development of rosiglitazone as a novel radiosensitizer.

Upregulated claudin-1 expression confers resistance to cell death of nasopharyngeal carcinoma cells.

Accumulating evidence reveals that aberrant expression of claudins manifests in various tumors; however, their biological functions are poorly understood. Here, we report on the elevated expression of claudin-1 in nasopharyngeal carcinoma (NPC) cell lines under serum deprivation or fluorouracil (5-FU) treatment. Interestingly, an increase in expression of claudin-1 considerably reduced apoptosis rather than enhancing cell proliferation. However, claudin-1 expression and activity were unaffected by external stimuli or Akt and NF-kappaB activation. Notably, predominant cytoplasmic and nuclear localization of claudin-1 in NPC cells reflected the aforementioned feature. On the other hand, loss of epithelial morphology and E-cadherin expression was associated with serum withdrawal in NPC cells. Interestingly, restoration of E-cadherin inhibited the protein elevation and antiapoptotic activity of claudin-1. In conclusion, our data demonstrate the regulation and novel biological function of claudin-1 and indicate the important role of claudin-1 in NPC tumorigenesis.