Qinghui Meng

BRCA1 and BRCA2 as molecular targets for phytochemicals indole-3-carbinol and genistein in breast and prostate cancer cells

Indole-3-carbinol (I3C) and genistein are naturally occurring chemicals derived from cruciferous vegetables and soy, respectively, with potential cancer prevention activity for hormone-responsive tumours (e.g., breast and prostate cancers). Previously, we showed that I3C induces BRCA1 expression and that both I3C and BRCA1 inhibit oestrogen (E2)-stimulated oestrogen receptor (ER-α) activity in human breast cancer cells. We now report that both I3C and genistein induce the expression of both breast cancer susceptibility genes (BRCA1 and BRCA2) in breast (MCF-7 and T47D) and prostate (DU-145 and LNCaP) cancer cell types, in a time- and dose-dependent fashion. Induction of the BRCA genes occurred at low doses of I3C (20u2009μM) and genistein (0.5–1.0u2009μM), suggesting potential relevance to cancer prevention. A combination of I3C and genistein gave greater than expected induction of BRCA expression. Studies using small interfering RNAs (siRNAs) and BRCA expression vectors suggest that the phytochemical induction of BRCA2 is due, in part, to BRCA1. Functional studies suggest that I3C-mediated cytoxicity is, in part, dependent upon BRCA1 and BRCA2. Inhibition of E2-stimulated ER-α activity by I3C and genistein was dependent upon BRCA1; and inhibition of ligand-inducible androgen receptor (AR) activity by I3C and genistein was partially reversed by BRCA1-siRNA. Finally, we provide evidence suggesting that the phytochemical induction of BRCA1 expression is due, in part, to endoplasmic reticulum stress response signalling. These findings suggest that the BRCA genes are molecular targets for some of the activities of I3C and genistein.

Role of NF-κB signaling in hepatocyte growth factor/scatter factor-mediated cell protection

The cytokine scatter factor/hepatocyte growth factor (HGF/SF) protects epithelial, carcinoma, and other cell types against cytotoxicity and apoptosis induced by DNA-damaging agents such as ionizing radiation and adriamycin (ADR, a topoisomerase IIα inhibitor). We investigated the role of nuclear factor kappa B (NF-κB) signaling in HGF/SF-mediated protection of human prostate cancer (DU-145) and Madin–Darby canine kidney (MDCK) epithelial cells against ADR. HGF/SF caused the rapid nuclear translocation of the p65 (RelA) subunit of NF-κB associated with the transient loss of the inhibitory subunit IκB-α. Exposure to HGF/SF caused the activation of an NF-κB luciferase reporter that was blocked or attenuated by the expression of a mutant ‘super-repressor’ IκB-α. Electrophoretic mobility shift assay supershift assays revealed that HGF/SF treatment induced the transient binding of various NF-κB family proteins (p65, p50, c-Rel, and RelB) with radiolabeled NF-κB-binding oligonucleotides. The HGF/SF-mediated protection of DU-145 and MDCK cells against ADR (demonstrated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays) was abrogated by the IκB-α super-repressor. The ability of HGF/SF to activate NF-κB signaling was dependent on c-Akt → Pak1 (p21-associated kinase-1) signaling (with Pak1 downstream of c-Akt) and was inhibited by the tumor suppressor PTEN (phosphatase and tensin homolog). Inhibitors of phosphatidylinositol-3′-kinase and Src family kinases significantly inhibited HGF/SF-mediated activation of NF-κB, while inhibitors of MEK, protein kinase C, and p70 S6 kinase had a modest effect or no effect on NF-κB activity. HGF/SF induced the expression of several known NF-κB target genes (cIAP-1 (cellular inhibitor of apoptosis-1), cIAP-2, and TRAF-2 (TNF receptor-associated factor-2)) in an NF-κB-dependent manner; HGF/SF blocked the inhibition of expression of these genes by ADR. Experimental manipulation of expression of these genes suggests that they (particularly TRAF-2 and cIAP-2) contribute to the protection against ADR by HGF/SF. These findings suggest that HGF/SF activates NF-κB through a c-Akt → Pak1 signaling pathway that is also dependent on Src, and that NF-κB contributes to HGF/SF-mediated protection against ADR.

Ultraviolet radiation down-regulates expression of the cell-cycle inhibitor p21WAF1/CIP1 in human cancer cells independently of p53

PURPOSEnTo investigate the regulation of G1 cyclin-dependent kinase inhibitor p21WAF1/CIP1 by ultraviolet (UV) radiation in human carcinoma cells.nnnMATERIALS AND METHODSnHuman cancer cell lines were irradiated with UV-C (254 nm) radiation, and their responses were characterized by Western blotting, Northern blotting, semi-quantitative RT-PCR analysis, trypan blue staining and flow cytometric cell cycle analysis.nnnRESULTSnAt 24 h after UV irradiation, p21 expression was down-regulated in various cancer cell types (breast, prostrate, cervix, colon, glioma, squamous cancers), independently of their p53 genetic and functional status. UV-mediated down-regulation of p21 was dose- and time-dependent, was observed at the protein and mRNA levels, and did not correlate with cytotoxicity. Reduction of p21 protein levels required about 4 and 1 h, respectively, in MCF-7 and MDA-MB-231 breast cancer cells; some of the UV-induced decreases in p21 levels in these cell lines was due to enhanced proteasomal degradation. Despite decreased p21 levels, UV-irradiated breast cancer cells with wild-type p53 (MCF-7) retained the capacity for G1 cell-cycle arrest, whereas UV-treated cells with mutant p53 (MDA-MB-231) accumulated in S phase, suggesting a p53-dependent G1 checkpoint in MCF-7. UV treatment caused other alterations in cell-cycle regulatory, DNA repair and tumour suppressor genes, as described in this report.nnnCONCLUSIONSnIn contrast to X-rays, UV causes down-regulation of the cell-cycle inhibitor p21 in tumour cells. It is postulated that this may be an adaptation to promote the growth and survival of transformed cells.

Sanguinarine inhibits growth of human cervical cancer cells through the induction of apoptosis

Sanguinarine, a natural benzophenanthridine alkaloid, has been shown to possess anticancer activity inxa0vitro and inxa0vivo. In the present study, we demonstrated that sanguinarine caused a dose-dependent inhibition of growth in HeLa and SiHa human cervical cancer cells, i.e., 2.43xa0µmol/l (IC50) in HeLa cells and 3.07xa0µmol/l in SiHa cells. Cell cycle analysis revealed that sanguinarine significantly increased the sub-G1 population, from 1.7 to 59.7% in HeLa cells and from 1.7 to 41.7% in SiHa cells. Sanguinarine caused a dose-dependent decrease in Bcl-2 and NF-κB protein expression and a significant increase in Bax protein expression. Our findings indicate that sanguinarine as an effective anticancer drug candidate inhibits the growth of cervical cancer cells through the induction of apoptosis.

Polydatin inhibits growth of lung cancer cells by inducing apoptosis and causing cell cycle arrest

Polydatin (PD), a small natural compound from Polygonum cuspidatum, has a number of biological functions. However, the anticancer activity of PD has been poorly investigated. In the present study, thiazolyl blue tetrazolium bromide assay was used to evaluate the inhibitory effect of PD on cell growth. Cell cycle distribution and apoptosis were investigated by flow cytometry. In addition, the expression of several proteins associated with apoptosis and cell cycle were analyzed by western blot analysis. The results demonstrated that PD significantly inhibits the proliferation of A549 and NCI-H1975 lung cancer cell lines and causes dose-dependent apoptosis. Cell cycle analysis revealed that PD induces S phase cell cycle arrest. Western blot analysis showed that the expression of Bcl-2 decreased as that of Bax increased, and the expression of cyclin D1 was also suppressed. The results suggest that PD has potential therapeutic applications in the treatment of lung cancer.

Ras effector pathways modulate scatter factor-stimulated NF-κB signaling and protection against DNA damage

Scatter factor (SF) (hepatocyte growth factor) is a pleiotrophic cytokine that accumulates within tumors in vivo and protects tumor cells against cytotoxicity and apoptosis due to DNA damaging agents in vitro. Previous studies have established that SF-mediated cell protection involves antiapoptotic signaling from its receptor (c-Met) to PI3 kinase → c-Akt → Pak1 (p21-activated kinase -1) → NF-κB (nuclear factor-kappa B). Here, we found that Ras proteins (H-Ras and R-Ras) enhance SF-mediated activation of NF-κB and protection of DU-145 and MDCK (Madin–Darby canine kidney) cells against the topoisomerase IIα inhibitor adriamycin. Studies of Ras effector loop mutants and their downstream effectors suggest that Ras/PI3 kinase and Ras/Raf1 pathways contribute to SF stimulation of NF-κB signaling and cell protection. Further studies revealed that Raf1 positively regulates the ability of SF to stimulate NF-κB activity and cell protection. The ability of Raf1 to stimulate NF-κB activity was not due to the classical Raf1 → MEK1/2 → ERK1/2 pathway. However, we found that a MEK3/6 → p38 pathway contributes to SF-mediated activation of NF-κB. In contrast, RalA, a target of the Ras/RalGDS pathway negatively regulated the ability of SF to stimulate NF-κB activity and cell protection. Ras, Raf1 and RalA modulate SF stimulation of NF-κB activity, in part, by regulating IκB kinase (IKK)-β kinase activity. These findings suggest that Ras/Raf1/RalA pathways may converge to modulate NF-κB activation and SF-mediated survival signaling at the IKK complex and/or a kinase upstream of this complex.

Role of Src Signal Transduction Pathways in Scatter Factor-mediated Cellular Protection

Scatter factor (SF) (hepatocyte growth factor) is a pleiotrophic cytokine that accumulates in tumors, where it may induce invasion, angiogenesis, and chemoresistance. We have studied the mechanisms by which SF and its receptor (c-Met) protect cells against the DNA-damaging agent adriamycin (ADR) as a model for chemoresistance of SF/c-Met-overexpressing tumors. Previous studies identified a phosphatidylinositol 3-kinase/c-Akt/Pak1/NF-κB cell survival pathway in DU-145 prostate cancer and Madin-Darby canine kidney epithelial cells. Here we studied Src signaling pathways involved in SF cell protection. Src enhanced basal and SF stimulated NF-κB activity and SF protection against ADR, in a manner dependent upon its kinase and Src homology 3 domains; and endogenous Src was required for SF stimulation of NF-κB activity and cell protection. The ability of Src to enhance SF stimulation of NF-κB activity was due, in part, to its ability to stimulate Akt and IκB kinase activity; and Src-mediated stimulation of NF-κB was due, in part, to a Rac1/MKK3/6/p38 pathway and was Akt-dependent. SF caused the activation of Src and the Rac1 effector Pak1. Furthermore, SF induced activating phosphorylations of MKK3, MKK6, and p38 within the c-Met signalsome in an Src-dependent manner. The NF-κB-inducing kinase was found to act downstream of TAK1 (transforming growth factor-β-activated kinase 1) as a mediator of SF- and Src-stimulated NF-κB activity. Finally, the Src/Rac1/MKK3/6/p38 and Src/TAK1/NF-κB-inducing kinase pathways exhibited cross-talk at the level of MKK3. These findings delineate some novel signaling pathways for SF-mediated resistance to ADR.

Adenovirus-mediated expression of UHRF1 reduces the radiosensitivity of cervical cancer HeLa cells to γ-irradiation

AbstractAim:An in vitro study was carried out to determine the effect of UHRF1 overexpression on radiosensitivity in human cervical cancer HeLa cells using adenovirus-mediated UHRF1 gene transfer (Ad5-UHRF1).Methods:Cell survival was evaluated using the clonogenic survival assay and the MTT assay; apoptosis and cell cycle distribution were monitored by flow cytometry. Protein levels were measured by Western blotting. Silencing XRCC4 expression was performed by transfection of small interfering RNA (siRNA).Results:Increased expression of UHRF1 by Ad5-UHRF1 significantly reduced the radiosensitivity of HeLa cells. The UHRF1-mediated radioresistance was correlated with increased DNA repair capability and increased expression of the DNA damage repair protein, XRCC4. Knocking down XRCC4 expression in the cells using XRCC4 siRNA markedly reduced the UHRF1-mediated radioresistance.Conclusion:These results provide the first evidence for revealing a functional role of UHRF1 in human cervical cancer cells as a negative regulator of radiosensitivity.

BTG2 is an LXXLL-dependent co-repressor for androgen receptor transcriptional activity

The tumor suppressor gene, BTG2 has been down-regulated in prostate cancer and the ectopic expression of this gene has been shown to inhibit prostate cancer cell growth. Sequence analysis revealed that the BTG2 protein contains two leucine-rich motifs ((20)LxxLL(24) and (92)LxxLL(96)), which are usually found in nuclear receptor co-factors. Based on this, we postulated that there will be an association between BTG2 and AR. In this study, we discovered that BTG2 directly bound to the androgen receptor (AR) in the absence of 5α-dihydrotestosterone (DHT), and in the presence of the androgen, this interaction was increased. BTG2 bearing the mutant (20)LxxLL(24) motif bound to AR equally efficient as the wild-type BTG2, while BTG2 bearing the mutant (92)LxxLL(96) motif failed to interact with AR. Functional studies indicated that ectopic expression of BTG2 caused a significant inhibition of AR-mediated transcriptional activity and a decreased growth of prostate cancer cells. Androgen-induced promoter activation and expression of prostate-specific antigen (PSA) are significantly attenuated by BTG2. The intact (92)LxxLL(96) motif is required for these activities. These findings, for the first time, demonstrate that BTG2 complexes with AR via an LxxLL-dependent mechanism and may play a role in prostate cancer via modulating the AR signaling pathway.

Effect of Akt inhibition on scatter factor-regulated gene expression in DU-145 human prostate cancer cells.

The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected±a dominant-negative mutant Akt, treated±SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription–polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIα inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.