Zhiwei He

Direct Inhibition of Insulin-Like Growth Factor-I Receptor Kinase Activity by (−)−Epigallocatechin-3-Gallate Regulates Cell Transformation

Insulin-like growth factor-I receptor (IGF-IR) has been implicated in cancer pathophysiology. Furthermore, impairment of IGF-IR signaling in various cancer cell lines caused inhibition of the transformed phenotype as determined by the inhibition of colony formation in soft agar and the inhibition of tumor formation in athymic nude mice. Thus, the IGF-IR might be an attractive target for cancer prevention. We showed that the tea polyphenol, (−)−epigallocatechin-3-gallate (EGCG), is a small-molecule inhibitor of IGF-IR activity (IC50 of 14 μmol/L). EGCG abrogated anchorage-independent growth induced by IGF-IR overexpression and also prevented human breast and cervical cancer cell phenotype expression through inhibition of IGF-IR downstream signaling. Our findings are the first to show that the IGF-IR is a novel binding protein of EGCG and thus may help explain the chemopreventive effect of EGCG on cancer development. (Cancer Epidemiol Biomarkers Prev 2007;16(3):598–605)

Caffeine Inhibits Cell Proliferation by G0/G1 Phase Arrest in JB6 Cells

Caffeine is a major biologically active constituent in coffee and tea. Because caffeine has been reported to inhibit carcinogenesis in UVB-exposed mice, the cancer-preventing effect of caffeine has attracted considerable attention. In the present study, the effect of caffeine in quiescent (G0 phase) cells was investigated. Pretreatment with caffeine suppressed cell proliferation in a dose-dependent manner 36 h after addition of fetal bovine serum as a cell growth stimulator. Analysis by flow cytometry showed that caffeine suppressed cell cycle progression at the G0/G1 phase, i.e., 18 h after addition of fetal bovine serum, the percentages of cells in G0/G1 phase in 1 mM caffeine-treated cells and in caffeine-untreated cells were 61.7 and 29.0, respectively. The percentage of cells in G0/G1 phase at 0 h was 75.5. Caffeine inhibited phosphorylation of retinoblastoma protein at Ser780 and Ser807/Ser811, the sites where retinoblastoma protein has been reported to be phosphorylated by cyclin-dependent kinase 4 (cdk4). Furthermore, caffeine inhibited the activation of the cyclin D1-cdk4 complex in a dose-dependent manner. However this compound did not directly inhibit the activity of this complex. In addition, caffeine did not affect p16INK4 or p27Kip1 protein levels, but inhibited the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3beta. Our results showed that caffeine suppressed the progression of quiescent cells into the cell cycle. The inhibitory mechanism may be due to the inhibition of cell growth signal-induced activation of cdk4, which may be involved in the inhibition of carcinogenesis in vivo.

Fyn is a novel target of (−)‐epigallocatechin gallate in the inhibition of JB6 Cl41 cell transformation

The cancer preventive action of (−)‐epigallocatechin gallate (EGCG), found in green tea, is strongly supported by epidemiology and laboratory research data. However, the mechanism by which EGCG inhibits carcinogenesis and cell transformation is not clear. In this study, we report that EGCG suppressed epidermal growth factor (EGF)‐induced cell transformation in JB6 cells. We also found that EGCG inhibited EGF‐induced Fyn kinase activity and phosphorylation in vitro and in vivo. Fyn was implicated in the process because EGF‐induced JB6 cell transformation was inhibited by small interfering RNA (siRNA)‐Fyn‐JB6 cells. With an in vitro protein‐binding assay, we found that EGCG directly bound with the GST‐Fyn‐SH2 domain but not the GST‐Fyn‐SH3 domain. The Kd value for EGCG binding to the Fyn SH2 domain was 0.367 ± 0.122 µM and Bmax was 1.35 ± 0.128 nmol/mg. Compared with control JB6 Cl41 cells, EGF‐induced phosphorylation of p38 MAP kinase (p38 MAPK) (Thr180/Tyr182), ATF‐2 (Thr71) and signal transducer and activator of transcription 1 (STAT1) (Thr727) was decreased in siRNA‐Fyn‐JB6 cells. EGCG could inhibit the phosphorylation of p38 MAPK, ATF‐2, and STAT1. The DNA binding ability of AP‐1, STAT1, and ATF‐2 was also decreased in siRNA‐Fyn‐JB6 cells. Overall, these results demonstrated that EGCG interacted with Fyn and inhibited Fyn kinase activity and thereby regulated EGF‐induced cell transformation. Inhibition of Fyn kinase activity is a novel and important mechanism that may be involved in EGCG‐induced inhibition of cell transformation.

Berberine Inhibits Metastasis of Nasopharyngeal Carcinoma 5-8F Cells by Targeting Rho Kinase-mediated Ezrin Phosphorylation at Threonine 567

Ezrin is highly expressed in metastatic tumors and is involved in filopodia formation as well as promotion of tumor metastasis. Thus, Ezrin may serve as a potential target for anti-metastatic therapy. This study demonstrates that berberine reduces filopodia formation of a nasopharyngeal carcinoma (NPC) cell line, 5-8F, at non-cytotoxic concentrations. Furthermore, invasion and motility of 5-8F cells are decreased in a dose- and time-dependent manner, resulting in 73.0% invasion and 67.0% motility inhibition at 20 μm. The inhibitory effects of berberine on 5-8F cell metastasis were further confirmed in a mouse model of metastasis. Berberine treatment in vivo resulted in a 51.1% inhibition of tumor metastasis to the lymph nodes and decreased Ezrin phosphorylation at threonine 567 in metastatic samples. Berberine suppressed the presence of phosphorylated Ezrin (phospho-Ezrin) in a dose- and time-dependent manner but had no effect on total Ezrin protein expression at non-cytotoxic concentrations. Furthermore, the inhibitory effects of berberine on phospho-Ezrin were dependent on the suppression of Rho kinase activity. Reduction of Ezrin phosphorylation at Thr567 by berberine was associated with its inhibitory effect on filopodia formation in 5-8F cells. However, berberine did not effectively inhibit the motility and invasion of NPC cells containing Ezrin Thr567 mutants. These results confirm that berberine inhibits Ezrin phosphorylation at Thr567. Nonetheless, berberine reduces motility and invasion of cells and inhibits tumor metastasis. The reduction of Rho kinase-mediated Ezrin phosphorylation mediated by berberine may be a novel anti-metastatic pathway in NPC 5-8F cells.

The p53 protein is a novel substrate of ribosomal S6 kinase 2 and a critical intermediary for ribosomal S6 kinase 2 and histone H3 interaction.

The tumor suppressor p53 protein is one of the most highly connected nodes in cellular signal transduction pathways and acts as a central regulatory switch in networks controlling cell proliferation and apoptosis. It is involved in the activation of genes that maintain control over cellular responses to DNA errors such as DNA repair, chromosomal recombination, and chromosome segregation. Here we show that ribosomal S6 kinase 2 (RSK2) activates and phosphorylates p53 (Ser15) in vitro and in vivo and colocalizes with p53 in the nucleus. Deficiency of p53 diminishes RSK2-mediated phosphorylation of histone H3 (Ser10) and adding back p53 to p53-/- embryonic fibroblasts restored phosphorylation of histone H3 at Ser10. These results show that the p53 protein is an important substrate of RSK2 and a critical intermediary in the RSK2 and histone H3 interaction. The RSK2-p53-histone H3 complex may likely contribute to chromatin remodeling and cell cycle regulation.

Involvement of the Akt/mTOR pathway on EGF-induced cell transformation

Our previous study demonstrated that phosphatidylinositol 3‐kinase (PI3K) is necessary for epidermal growth factor (EGF)–induced cell transformation in mouse epidermal JB6 cells. Akt and the mammalian target of rapamycin (mTOR) are regarded as PI3K downstream effectors. Therefore, in this study, we investigated the role of Akt and mTOR on EGF‐induced cell transformation in JB6 cells using rapamycin, a specific mTOR inhibitor, and cells expressing dominant negative mutants of Akt1 (DNM‐Akt1). We found that the treatment of cells with rapamycin inhibited EGF‐induced cell transformation but only slightly inhibited JB6 cell proliferation at 72 h. Although LY294002, a PI3K inhibitor, attenuated EGF‐induced activator protein 1 (AP‐1) activation, treatment with rapamycin did not affect AP‐1 activity. Treatment with rapamycin inhibited EGF‐induced phosphorylation and activation of ribosomal p70 S6 protein kinase (p70 S6K), an mTOR downstream target, but had no effect on phosphorylation and activation of Akt. Rapamycin also had no effect on EGF‐induced phosphorylation of extracellular signal–regulated protein kinases (ERKs). We showed that introduction of DNM‐Akt1 into JB6 mouse epidermal Cl 41 (JB6 Cl 41) cells inhibits EGF‐induced cell transformation without blocking cell proliferation. The expression of DNM‐Akt1 also suppressed EGF‐induced p70 S6K activation as well as Akt activation. These results indicated an involvement of the Akt/mTOR pathway in EGF‐induced cell transformation in JB6 cells.

Correlation analysis of JAK-STAT pathway components on prognosis of patients with prostate cancer.

Janus kinases (JAK)/signal transducers and activator of transcription (STAT) pathway is activated constitutively in prostate cancer (PCa). Despite previous reports implying a role of this pathway in the development of clinical hormone-refractory PCa, the correlation of pathway members with the clinicopathologic features and prognosis of patients with PCa has not been elucidated. To address this problem, pJAK-1Tyr1022/1023 and pSTAT-3Tyr705 were evaluated by immunostaining in needle biopsies of the prostate from 202 PCa patients treated by definitive therapy (105 cases) or hormonal therapy (97 cases). The correlation of two protein expression with the clinicopathologic features and the prognosis of PCa were subsequently assessed. The expression levels of pJAK-1Tyr1022/1023 and pSTAT-3Tyr705 were both positively correlated with Gleason score and clinical stage of patients with PCa. Their expression was also significantly higher in patients with biochemical (prostate-specific antigen, PSA) failure than that in those with no PSA failure (both P < 0.001). In all patients, the recurrence-free survival (RFS) rates were significantly higher in those with low pJAK-1Tyr1022/1023 and pSTAT-3Tyr705 expression than that in those with high expression (both P < 0.001). Moreover, for patients treated by definitive or hormonal therapy, the RFS rates in those with lower pJAK-1Tyr1022/1023 (P < 0.001 and 0.012, respectively) and pSTAT-3Tyr705 expression (P < 0.001 and 0.015, respectively) were significantly higher than in those with higher expression. Cox multivariate analysis showed that the expression levels of pJAK-1Tyr1022/1023 (P = 0.002) and pSTAT-3Tyr705 (P = 0.005) were prognostic factors for PCa in addition to extraprostatic extension (P = 0.026) and Gleason score (P = 0.018). The results of pJAK-1Tyr1022/1023 and pSTAT-3Tyr705 immunostainings in needle-biopsy specimens are prognostic factors for PCa.

Preparation of a ligustrazine ethosome patch and its evaluation in vitro and in vivo

Background The purpose of this study was to develop a transdermal ligustrazine patch containing a stable formulation and with good entrapment efficiency, release rate, and transdermal absorption. Methods Ligustrazine ethosomes were prepared by ethanol injection-sonication, with entrapment efficiency as an indicator. Using acrylic resin as the primary constituent, the ligustrazine ethosome patch was prepared by adding succinic acid as a crosslinking agent and triethyl citrate as a plasticizer. In vitro release and transdermal permeation studies were carried out. Finally, a pharmacokinetic study was carried out in rats to explore relative bioavailability. The formulations of ligustrazine ethosome were 1% (w/v) phospholipid, 0.4% (w/v) cholesterol, and 45% (v/v) ethanol. Results Ligustrazine ethosomes were obtained with an average particle size of 78.71 ± 1.23 nm and an average entrapment efficiency of 86.42% ± 1.50%. In vitro transdermal testing of the ligustrazine ethosome patches showed that the cumulative 24-hour amount of ligustrazine was up to 183 ± 18 μg/cm2. The pharmacokinetic results revealed that the relative bioavailability was 209.45%. Conclusion Compared with conventional ligustrazine administration, ligustrazine ethosome patches could promote better drug absorption and increase bioavailability. This study demonstrates that the transdermal action of the ligustrazine ethosome patch was comparatively good.

Arsenite inhibits p53 phosphorylation, DNA binding activity, and p53 target gene p21 expression in mouse epidermal JB6 cells.

Epidemiologic investigations demonstrated that arsenite exposure increases the risk of various human cancers, including skin, lung, bladder, and kidney cancers. However, oral administration of arsenite alone has failed to induce tumors in animal models, suggesting that arsenic may act to enhance mutagenicity induced by other carcinogens. Arsenite may function as a co‐carcinogen, acting by inhibiting repair of carcinogen‐induced DNA damage mediated by p53 and p21, a p53 target gene. To elucidate the interaction between arsenite and p53 tumor suppressor protein, we studied the effect of arsenite on ultraviolet B (UVB)‐induced p53 phosphorylation, p53 DNA binding activity, and p53‐induced target gene transactivation in the JB6 Cl41 mouse epidermal skin cell model. Our results indicated that arsenite suppressed UVB‐induced p53 phosphorylation and p53 DNA binding activity. Arsenite also inhibited casein kinase 2 (CK2) activity and decreased p53‐regulated p21 protein expression. These data suggest that the direct inhibition of p53 functional activation is one of the mechanisms through which arsenite interferes with p53 function, and thus may be a significant mechanism for the co‐carcinogenic effects of arsenite.

Increased phosphorylation of histone H3 at serine 10 is involved in Epstein-Barr virus latent membrane protein-1-induced carcinogenesis of nasopharyngeal carcinoma

BackgroundIncreased histone H3 phosphorylation is an essential regulatory mechanism for neoplastic cell transformation. We aimed to explore the role of histone H3 phosphorylation at serine10 (p-H3Ser10) in Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1)-induced carcinogenesis of nasopharyngeal carcinoma (NPC).MethodsThe expression of p-H3Ser10 was detected by the immunohistochemical analysis in NPC, chronic nasopharyngitis and normal nasopharynx tissues, and its correlation with LMP1 was analyzed in NPC tissues and cell lines. Using the small interfering RNA (siRNA)-H3 and histone H3 mutant (S10A), the effect of histone H3 Ser10 motif on LMP1-induced CNE1 cell proliferation, transformation and activator protein-1 (AP-1) activation were evaluated by CCK-8, focus-forming and reporter gene assay respectively. Mitogen- and stress-activated kinase 1 (MSK1) kinase activity and phosphorylation were detected by in vitro kinase assay and western blot. Using MSK1 inhibitor H89 or siRNA-MSK1, the regulatory role of MSK1 on histone H3 phosphorylation and AP-1 activation were analyzed.ResultsImmunohistochemical analysis revealed that the expression of p-H3Ser10 was significantly higher in the poorly differentiated NPC tissues than that in chronic nasopharyngitis (p <0.05) and normal nasopharynx tissues (p <0.001). Moreover, high level of p-H3Ser10 was positively correlated with the expression of LMP1 in NPC tissues (χ2=6.700, p =0.01; C=0.350) and cell lines. The knockdown and mutant (S10A) of histone H3 suppressed LMP1-induced CNE1 cell proliferation, foci formation and AP-1 activation. In addition, LMP1 could increase MSK1 kinase activity and phosphorylation. MSK1 inhibitor H89 or knockdown of MSK1 by siRNA blocked LMP1-induced phosphorylation of histone H3 at Ser10 and AP-1 activation.ConclusionEBV-LMP1 can induce phosphorylation of histone H3 at Ser10 via MSK1. Increased phosphorylation of histone H3 at Ser10 is likely a crucial regulatory mechanism involved in LMP1-induced carcinogenesis of NPC.