Yaxiong Tang

WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells

Epigenetic silencing of secreted wingless-type (Wnt) antagonists through hypermethylation is associated with tobacco smoking and with invasive bladder cancer. The secreted Wnt inhibitory factor-1 (WIF1) has shown consistent growth-inhibitory effect on various cancer cell lines. Therefore, we assessed the mechanisms of action of WIF1 by either restoring WIF1 expression in invasive bladder cancer cell lines (T24 and TSU-PR1) or using a recombinant protein containing functional WIF1 domain. Both ectopic expression of WIF1 and treatment with WIF1 domain protein resulted in cell growth inhibition via G1 arrest. The G1 arrest induced by WIF1 is associated with down-regulation of SKP2 and c-myc and up-regulation of p21/WAF1 and p27/Kip1. Conversely, reexpression of SKP2 in WIF1-overexpressing TSU-PR1 cells attenuated the WIF1-induced G1 arrest. Furthermore, inhibition of nuclear Wnt signaling by either dominant-negative LEF1 or short hairpin RNA of TCF4 also reduced SKP2 expression. The human SKP2 gene contains two TCF/LEF1 consensus binding sites within the promoter. Chromatin immunoprecipitation/real-time PCR analysis revealed that both WIF1 and dominant-negative LEF1 expression decreased the in vivo binding of TCF4 and β-catenin to the SKP2 promoter. Together, our results suggest that mechanisms of WIF1-induced G1 arrest include (a) SKP2 down-regulation leading to p27/Kip1 accumulation and (b) c-myc down-regulation releasing p21/WAF1 transcription. Additionally, we show that WIF1 inhibits in vivo bladder tumor growth in nude mice. These observations suggest a mechanism for transformation of bladder epithelium on loss of WIF1 function and provide new targets such as SKP2 for intervention in WIF1-deficient bladder cancer. [Mol Cancer Ther 2009;8(2):458–68]

The Wnt inhibitory factor 1 restoration in prostate cancer cells was associated with reduced tumor growth, decreased capacity of cell migration and invasion and a reversal of epithelial to mesenchymal transition

BackgroundAberrations in the Wnt pathway have been reported to be involved in the metastasis of prostate cancer (PCa) to bone. We investigated the effect and underlying mechanism of a naturally-occurring Wnt inhibitor, WIF1, on the growth and cellular invasiveness of a bone metastatic PCa cell line, PC3.ResultsThe WIF1 gene promoter was hypermethylated and its expression down-regulated in the majority (7 of 8) of PCa cell lines. Restoration of WIF1 expression in PC-3 cells resulted in a decreased cell motility and invasiveness via up-regulation of epithelial markers (E-cadherin, Keratin-8 and-18), down-regulation of mesenchymal markers (N-cadherin, Fibronectin and Vimentin) and decreased activity of MMP-2 and -9. PC3 cells transfected with WIF1 consistently demonstrated reduced expression of Epithelial-to-Mesenchymal Transition (EMT) transcription factors, Slug and Twist, and a change in morphology from mesenchymal to epithelial. Moreover, WIF1 expression significantly reduced tumor growth by approximately 63% in a xenograft mouse model. This was accompanied by an increased expression of E-cadherin and Keratin-18 and a decreased expression of vimentin in tumor tissues.ConclusionThese data suggest that WIF1 regulates tumor invasion through EMT process and thus, may play an important role in controlling metastatic disease in PCa patients. Blocking Wnt signaling in PCa by WIF1 may represent a novel strategy in the future to reduce metastatic disease burden in PCa patients.

Flavokawain B, a kava chalcone, induces apoptosis via up-regulation of death-receptor 5 and Bim expression in androgen receptor negative, hormonal refractory prostate cancer cell lines and reduces tumor growth.

Limited success has been achieved in extending the survival of patients with metastatic and hormone‐refractory prostate cancer (HRPC). There is a strong need for novel agents in the treatment and prevention of HRPC. We have shown that flavokawain B (FKB), a kava chalcone, is about 4‐ to 12‐fold more effective in reducing the cell viabilities of androgen receptor (AR)‐negative, HRPC cell lines DU145 and PC‐3 than AR‐positive, hormone‐sensitive prostate cancer cell lines LAPC4 and LNCaP, with minimal effect on normal prostatic epithelial and stromal cells. FKB induces apoptosis with an associated increased expression of proapoptotic proteins: death receptor‐5, Bim and Puma and a decreased expression of inhibitors of apoptosis protein: XIAP and survivin. Among them, Bim expression was significantly induced by FKB as early as 4 hr of the treatment. Knockdown of Bim expression by short‐hairpin RNAs attenuates the inhibitory effect on anchorage‐dependent and ‐independent growth and caspase cleavages induced by FKB. These findings suggest that the effect of FKB, at least in part, requires Bim expression. In addition, FKB synergizes with TRAIL for markedly enhanced induction of apoptosis. Furthermore, FKB treatment of mice bearing DU145 xenograft tumors results in tumor growth inhibition and increases Bim expression in tumor tissues. Together, these results suggest robust mechanisms for FKB induction of apoptosis preferentially for HRPC and the potential usefulness of FKB for prevention and treatment of HRPC in an adjuvant setting.

Frzb, a secreted Wnt antagonist, decreases growth and invasiveness of fibrosarcoma cells associated with inhibition of Met signaling.

Soft tissue sarcomas (STS) have a strong propensity for aggressive growth and metastasis. We showed that the secreted Wnt antagonist Frzb exhibited potent antitumor activity against prostate cancer, an epithelial type of malignancy. In this study, we further showed the antitumor efficacy of Frzb in STS, a mesenchymal group of cancer. Frzb transfection of HT1080 (fibrosarcoma) and SW872 (liposarcoma) cell lines and their conditioned media resulted in a significant reduction in cellular invasion, motility, and colony formation in soft agar compared with vector control-transfected cells. In a xenograft mouse model, Frzb dramatically suppressed tumor growth of HT1080 cells in nude mice. In a tail-vein injection metastatic model, Frzb-transfected HT1080 cells formed fewer and smaller lung nodules than vector control cells. In addition, we identified new mechanisms for Frzb antitumor activities. Frzb reduced c-Met expression and inhibited Met-mediated signaling, associated with up-regulation of epithelial markers (i.e., keratins 8 and 18) and down-regulation of mesenchymal markers (i.e., vimentin, N-cadherin, fibronectin, Slug, and Twist). Similar to Frzb, silencing of c-Met by short hairpin RNA or using a dominant-negative LRP5 receptor also suppressed Met signaling, leading to reduced cellular motility, invasion, and in vivo tumor growth. Given recent studies indicating an important role of c-Met in sarcoma development and progression, our data showed that Frzb expression was significantly inversely correlated with Met expression in both STS cell lines and tissues. These results suggested the usefulness of Frzb in modulating Met signaling as a new treatment strategy for STS.

Effects of the Kava Chalcone Flavokawain A Differ in Bladder Cancer Cells with Wild-type versus Mutant p53

Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain As action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G1 arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation of SKP2, which then increased the stability of the p27/KIP1 protein. The accumulation of p21/WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G2-M arrest in six p53 mutant-type, high-grade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation, which then led to a G2-M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G2-M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G2-M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer.

Flavokawain B induces apoptosis of non-small cell lung cancer H460 cells via Bax-initiated mitochondrial and JNK pathway

Flavokawain B (FKB) possesses strong anti-neoplastic activity against many cancer cells. Here we assessed its antitumor activity and molecular mechanisms in lung cancer H460 cells in vitro. FKB significantly inhibited cell proliferation and caused arrest of the cell cycle G2-M of H460 cells in a dose-dependent manner. FKB also inducted apoptosis, which was associated with cytochrome c release, caspase-7 and caspase-9 activation and Bcl-xL/Bax dys-regulation. FKB significantly down-regulated survivin and XIAP, and the inhibitory effect induced by FKB was greatly attenuated by through over-expression of survivin or Bax−/− MEFs. Furthermore, FKB activated the mitogen-activated protein kinases and the JNK inhibitor SP600125 significantly decreased the growth-inhibitory and apoptotic effects of FKB. Together, these results suggest the anti-lung cancer potential of flavokawain B for the prevention and treatment of lung cancer.

Cytotoxic Sesquiterpene Lactones from Aerial Parts of Xanthium sibiricum

Chemical investigation of the aerial parts of Xanthium sibiricum led to the isolation of four new xanthanolide-type sesquiterpene lactones, including two xanthanolide dimers, pungiolide D (1) and pungiolide E (2), and two xanthanolide monomers, 8-epi-xanthatin-1α,5α-epoxide (3) and 1β-hydroxyl-5α-chloro-8-epi-xanthatin (4), together with four known compounds, pungiolide A (5), 8-epi-xanthatin-1β,5β-epoxide (6), xanthatin (7), and 11α,13-dihydro-8-epi-xanthatin (8). The structures of these compounds were elucidated on the basis of spectroscopic data analysis. Pungiolide D (1) displayed an unusual structure featuring a 5/5/6-fused tricyclic system in the unit B. Compound 4 was shown to be a rare sesquiterpene lactone containing halogen, and its absolute configuration was determined by X-ray crystallographic analysis. The evaluation of the cytotoxic activities of the isolated new compounds against the SNU387 liver and A-549 lung human cancer cell lines showed that compound 4 possessed significant in vitro cytotoxicity with an IC50 value of 5.1 µM against SNU387 liver cells.

HBx affects CUL4–DDB1 function in both positive and negative manners

Hepatitis B virus (HBV) infection is a major public health problem by affecting 350 million people worldwide. The mechanisms that regulate HBV gene expression and viral replication remain poorly understood. HBx is known as the central regulator for HBV replication and is associated with the CUL4-DDB1 ubiquitin ligase through H-box motif. Here, we show that blocking the activity of DDB1 by RNA interfering inhibited viral production and gene expression of HBV, and direct association of HBx with DDB1 promoted viral activities, indicating that DDB1 function is required for viral production. On the other hand, HBx interfered with DDB1-dependent polyubiquitination of PRMT1, arginine methyltransferase 1, suggesting that HBx can also block the function of a subset of CUL4-DDB1 E3 ligases. Thus, we conclude that HBx regulates the function of DDB1 in both positive and negative manners in the context of distinct CUL4-DDB1 complexes and plays different roles in HBV replication cycle.

Loss of Smu1 function de-represses DNA replication and over-activates ATR-dependent replication checkpoint.

Smu1 is an evolutionarily conserved gene that encodes a member of the WD40-repeat protein family. Disruption of Smu1 function leads to multiple cellular defects including chromosomal instability, aberrant DNA replication and alternative RNA splicing events. In this paper, we show that Smu1 is a chromatin-bound protein that functions as a negative regulator of DNA replication. Knockdown of Smu1 gene expression promotes excessive incorporation of dNTP analogue, implicating the acceleration of DNA synthesis. Smu1-silenced cells show an excessive activation of replication checkpoint in response to ultraviolate (UV) or hydroxyurea treatment, indicating that abnormal stimulation of DNA replication leads to instability of genomic structure. Hence, we propose that Smu1 participates in the protection of genomic integrity by negatively regulating the process of DNA synthesis.

(−)-Gochnatiolide B, synthesized from dehydrocostuslactone, exhibits potent anti-bladder cancer activity in vitro and in vivo

With limited success achieved in bladder cancer patient management, novel agents are in urgent need for the purpose of therapy and prevention. As a sesquiterpenoid dimmer isolated from Gochnatia pomculat, (−)-gochnatiolide B has been bio-mimetically synthesized in multiple steps with a poor yield, which heavily limited the further research and clinical application. Herein, (−)-gochnatiolide B was synthesized beginning with dehydrocostuslactone in four steps with a total yield of 26%. MTT assays showed that (−)-gochnatiolide B inhibited the growth of vast majority of human cancer cells especially bladder cancer cells. Mechanistically, (−)-gochnatiolide B induced the increased expression of pro-apoptotic proteins and the decreased expression of anti-apoptosis proteins and further resulted in apoptosis of T24 cells. (−)-Gochnatiolide B induced G1 arrest which associated with SKP2 downregulation, leading to p27/Kip1 accumulation and downregulation of cyclin D1 in T24 cells. Furthermore, in vivo studies showed that (−)-gochnatiolide B remarkably inhibited tumor growth by 81% compared with vehicle control. Taken together, (−)-gochnatiolide B exhibits inhibitory activity against bladder cancer cells both in vitro and in vivo by inducing apoptosis, which suggests that (−)-gochnatiolide B could be an important candidate compound for prevention and treatment of bladder cancer.