Daxx regulates mitotic progression and prostate cancer predisposition.

Mitotic progression of mammalian cells is tightly regulated by the E3 ubiquitin ligase anaphase promoting complex (APC)/C. Deregulation of APC/C is frequently observed in cancer cells and is suggested to contribute to chromosome instability and cancer predisposition. In this study, we identified Daxx as a novel APC/C inhibitor frequently overexpressed in prostate cancer. Daxx interacts with the APC/C coactivators Cdc20 and Cdh1 in vivo, with the binding of Cdc20 dependent on the consensus destruction boxes near the N-terminal of the Daxx protein. Ectopic expression of Daxx, but not the D-box deleted mutant (DaxxΔD-box), inhibited the degradation of APC/Cdc20 and APC/Cdh1 substrates, leading to a transient delay in mitotic progression. Daxx is frequently upregulated in prostate cancer tissues; the expression level positively correlated with the Gleason score and disease metastasis (P = 0.027 and 0.032, respectively). Furthermore, ectopic expression of Daxx in a non-malignant prostate epithelial cell line induced polyploidy under mitotic stress. Our data suggest that Daxx may function as a novel APC/C inhibitor, which promotes chromosome instability during prostate cancer development.

Gamma-tocotrienol as an effective agent in targeting prostate cancer stem cell-like population.

Emerging evidence supports that prostate cancer originates from a rare subpopulation of cells, namely prostate cancer stem cells (CSCs). Conventional therapies for prostate cancer are believed to mainly target the majority of differentiated tumor cells but spare CSCs, which may account for the subsequent disease relapse after treatment. Therefore, successful elimination of CSCs may be an effective strategy to achieve complete remission from this disease. Gamma-tocotrienols (γ-T3) is one of the vitamin-E constituents, which have been shown to have anticancer effects against a wide range of human cancers. Recently, we have reported that γ-T3 treatment not only inhibits prostate cancer cell invasion but also sensitizes the cells to docetaxel-induced apoptosis, suggesting that γ-T3 may be an effective therapeutic agent against advanced stage prostate cancer. Here, we demonstrate for the first time that γ-T3 can downregulate the expression of prostate CSC markers (CD133/CD44) in androgen-independent prostate cancer cell lines (PC-3 and DU145), as evident from Western blotting analysis. Meanwhile, the spheroid formation ability of the prostate cancer cells was significantly hampered by γ-T3 treatment. In addition, pretreatment of PC-3 cells with γ-T3 was found to suppress tumor initiation ability of the cells. More importantly, although CD133-enriched PC-3 cells were highly resistant to docetaxel treatment, these cells were as sensitive to γ-T3 treatment as the CD133-depleted population. Our data suggest that γ-T3 may be an effective agent in targeting prostate CSCs, which may account for its anticancer and chemosensitizing effects reported in previous studies.

Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway.

Id-1 (inhibitor of differentiation or DNA binding) is a helix-loop-helix protein that is overexpressed in many types of cancer including esophageal squamous cell carcinoma (ESCC). We previously reported that ectopic Id-1 expression activates the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in human esophageal cancer cells. In this study, we confirmed a positive correlation between Id-1 and phospho-AKT (Ser473) expressions in ESCC cell lines, as well as in ESCC on a tissue microarray. To investigate the significance of Id-1 in esophageal cancer progression, ESCC cells with stable ectopic Id-1 expression were inoculated subcutaneously into the flank of nude mice and were found to form larger tumors that showed elevated Ki-67 proliferation index and increased angiogenesis, as well as reduced apoptosis, compared with control cells expressing the empty vector.The Id-1-overexpressing cells also exhibited enhanced metastatic potential in the experimental metastasis assay. Treatment with the PI3K inhibitor LY294002 attenuated the tumor promotion effects of Id-1, indicating that the effects were mediated by the PI3K/AKT signaling pathway. In addition, our in vitro experiments showed that ectopic Id-1 expression altered the expression levels of markers associated with epithelial-mesenchymal transition and enhanced the migration ability of esophageal cancer cells. The Id-1-overexpressing ESCC cells also exhibited increased invasive potential, which was in part due to PI3K/AKT-dependent modulation of matrix metalloproteinase-9 expression. In conclusion, our results provide the first evidence that Id-1 promotes tumorigenicity and metastasis of human esophageal cancer in vivo and that the PI3K inhibitor LY294002 can attenuate these effects.

Berberine inhibits Rho GTPases and cell migration at low doses but induces G2 arrest and apoptosis at high doses in human cancer cells.

Berberine is an active ingredient extracted from Coptidis rhizoma which has been used for centuries as a traditional Chinese medicine for treatment of inflammatory diseases. Recent studies have indicated that berberine has anticancer properties. Berberine arrested cell growth and inhibited cell migration in various cancer cell lines. In this study, we examined the effects of berberine on HONE1 cells, which have been commonly used as a cell model for nasopharyngeal carcinoma. We observed the inhibitory effects of berberine on HONE1 cells at a high dosage (>150 microM). Berberine effectively induced the mitotic arrest of HONE1 cells at 300 microM which was associated with apoptosis. Berberine had differential intracellular localization at low and high doses. At a low dose (50 microM), berberine was localized in the mitochondria while at a high dose (300 microM), berberine was localized in the nucleus which may have induced mitotic arrest. Berberine effectively inhibited cell migration and invasion at low doses. Using a specific GST pull-down assay of activated Rho GTPases, we demonstrated that berberine suppressed the activation of Rho GTPases including RhoA, Cdc42 and Rac1. This indicates a novel function of berberine in the suppression of Rho GTPase signaling to mediate its inhibitory action on cell migration and motility. The potential of berberine to inhibit cancer metastasis in cancer warrants further investigation.

TWIST modulates prostate cancer cell-mediated bone cell activity and is upregulated by osteogenic induction.

TWIST, a helix-loop-helix transcription factor, is highly expressed in many types of human cancer. We have previously found that TWIST confers prostate cancer cells with an enhanced metastatic potential through promoting epithelial-mesenchymal transition (EMT) and a high TWIST expression in human prostate cancer is associated with an increased metastatic potential. The predilection of prostate cancer cells to metastasize to bone may be due to two interplaying mechanisms (i) by increasing the rate of bone remodeling and (ii) by undergoing osteomimicry. We further studied the role of TWIST in promoting prostate cancer to bone metastasis. TWIST expression in PC3, a metastatic prostate cancer cell line, was silenced by small interfering RNA and we found that conditioned medium from PC3 with lower TWIST expression had a lower activity on stimulating osteoclast differentiation and higher activity on stimulating osteoblast mineralization. In addition, we found that these effects were, at least partly, associated with TWIST-induced expression of dickkopf homolog 1 (DKK-1), a factor that promotes osteolytic metastasis. We also examined TWIST and RUNX2 expressions during osteogenic induction of an organ-confined prostate cancer cell, 22Rv1. We observed increased TWIST and RUNX2 expressions upon osteogenic induction and downregulation of TWIST through short hairpin RNA reduced the induction level of RUNX2. In summary, our results suggest that, in addition to EMT, TWIST may also promote prostate cancer to bone metastasis by modulating prostate cancer cell-mediated bone remodeling via regulating the expression of a secretory factor, DKK-1, and enhancing osteomimicry of prostate cancer cells, probably, via RUNX2.

MAD2 expression and its significance in mitotic checkpoint control in testicular germ cell tumour.

Chromosomal instability (CIN) is a common characteristic in testicular germ cell tumour (TGCT). A functional mitotic checkpoint control is important for accurate chromosome segregation during mitosis. Mitotic arrest deficient 2 (MAD2) is a key component of this checkpoint and inactivation of MAD2 is correlated with checkpoint impairment. The aim of this study was to investigate the function of mitotic checkpoint control in TGCT cells and to study its association with MAD2 expression using 8 TGCT cell lines as well as 23 TGCT tissue samples. We found that in response to microtubule disruption, 6 of 8 TGCT cell lines (75%) failed to arrest in mitosis demonstrated by the decreased mitotic index and aberrant expression of mitosis regulators, indicating that mitotic checkpoint defect is a common event in TGCT cells. This loss of mitotic checkpoint control was correlated with reduced MAD2 protein expression in TGCT cell lines implicating that downregulation of MAD2 may play a critical role in an impaired mitotic checkpoint control in these cells. In addition, immunohistochemistry studies on 23 seminomas and 12 normal testis tissues demonstrated that nuclear expression of MAD2 was much lower in seminomas (p<0.0001) but cytoplasmic MAD2 expression was higher in seminomas (p=0.06) than normal samples. Our results suggest that aberrant MAD2 expression may play an essential role in a defective mitotic checkpoint in TGCT cells, which may contribute to CIN commonly observed in TGCT tumours.

Evidence of a novel docetaxel sensitizer, garlic-derived S-allylmercaptocysteine, as a treatment option for hormone refractory prostate cancer.

The recent introduction of docetaxel in the treatment of hormone refractory prostate cancer (HRPC) has made a small but significant impact on patient survival. However, its effect is limited by intolerance and resistance. The aim of our study was to investigate if the garlic-derived compound, S-allylmercaptocysteine (SAMC), was able to act as a docetaxel sensitizing agent. First, the effect of SAMC on docetaxel sensitivity was examined on 3 HRPC cell lines by colony forming assay. We found that SAMC increased the efficacy of docetaxel on colony forming inhibition by 9-50% compared to single agent treatment. Second, using the HRPC CWR22R nude mice model, we found that the combination of SAMC and docetaxel was 53% more potent than docetaxel alone (p = 0.037). In addition, there was no additive toxicity in the mice treated with the combination therapy evidenced by histological and functional analysis of liver, kidney and bone marrow. These results suggest that SAMC is able to increase the anticancer effect of docetaxel without causing additional toxic effect in vivo. Third, flow cytometry and Western blotting analysis on HRPC cell lines demonstrated that SAMC promoted docetaxel-induced G2/M phase cell cycle arrest and apoptotic induction. In addition, immunohistochemistry on CWR22R xenograft revealed a suppression of Bcl-2 expression and upregulation of E-cadherin in the SAMC and docetaxel treated animals. These results suggest that SAMC may promote docetaxel-induced cell death through promoting G2/M cell cycle arrest and apoptosis. Our study implies a potential role for SAMC in improving docetaxel based chemotherapy for the treatment of HRPC.

Decreased adhesiveness, resistance to anoikis and suppression of GRP94 are integral to the survival of circulating tumor cells in prostate cancer.

The presence of circulating tumor cells (CTC) is common in prostate cancer patients, however until recently their clinical significance was unknown. The CTC stage is essential for the formation of distant metastases, and their continuing presence after radical prostatectomy has been shown to predict recurrent or latent disease. Despite their mechanistic and prognostic importance, due both to their scarcity and difficulties in their isolation, little is known about the characteristics that enable their production and survival. The aim of this study was to investigate the molecular mechanisms underlying the survival of CTC cells. A novel CTC cell line from the bloodstream of an orthotopic mouse model of castration-resistant prostate cancer was established and compared with the primary tumor using attachment assays, detachment culture, Western blot, flow cytometry and 2D gel electrophoresis. Decreased adhesiveness and expression of adhesion molecules E-cadherin, beta4-integrin and gamma-catenin, together with resistance to detachment and drug-induced apoptosis and upregulation of Bcl-2 were integral to the development of CTC and their survival. Using proteomic studies, we observed that the GRP94 glycoprotein was suppressed in CTC. GRP94 was also shown to be suppressed in a tissue microarray study of 79 prostate cancer patients, indicating its possible role in prostate cancer progression. Overall, this study suggests molecular alterations accounting for the release and survival of CTC, which may be used as drug targets for either anti-metastatic therapy or the suppression of latent disease. We also indicate the novel involvement of GRP94 suppression in prostate cancer metastasis.

Mitotic checkpoint defects in human cancers and their implications to chemotherapy.

The mitotic checkpoint, also known as spindle assembly checkpoint, is to ensure accurate chromosome segregation by inducing mitotic arrest when errors occur in the spindle structure or in the alignment of the chromosomes on the spindle. Loss of mitotic checkpoint control is a common event in human cancer cells, which is thought to be responsible for chromosome instability frequently observed in cancer cells. Several reports have shown that cells with a defective mitotic checkpoint are more resistant to several types of anticancer drugs from microtubule disruptors to DNA damaging agents. In addition, inactivation of key mitotic checkpoint proteins such as BUB (budding uninhibited by benzimidazole) and MAD (mitotic arrest deficient ) is influential in drug resistance in mitotic checkpoint defective cancer cells. The mitotic checkpoint has also been linked to DNA damage response and a defective mitotic checkpoint confers cancer cells resistance to certain DNA damaging anticancer drugs. This review presents recent evidence on mitotic checkpoint defects in human cancers and their association with resistance to anticancer drugs. In addition, the clinical importance and potential therapeutic implications of targeting the mitotic checkpoint to reverse drug resistance in cancer cells are also discussed.

The prognostic significance of BMP-6 signaling in prostate cancer.

The importance of bone-morphogenetic proteins in prostate cancer is well recognized. Bone-morphogenetic protein-6 overexpression has been shown to increase the aggressiveness and invasiveness of prostate cancer cells. Recent studies on noggin and sclerostin, potent inhibitors of bone-morphogenetic protein signaling, have found that noggin also modifies the ability of prostate cancer cells to metastasize to bone. Taken together, these results suggest that bone-morphogenetic protein-6 signaling is important in prostate cancer progression. Our study investigated the expression of bone-morphogenetic protein-6, noggin and sclerostin in human prostate specimens (n=136) by immunohistochemical staining. We found that bone-morphogenetic protein-6 was increased (P<0.001), whereas sclerostin was decreased (P=0.004) in prostate cancer compared with nodular hyperplasia. In addition, significantly higher level of bone-morphogenetic protein-6 expression was observed in high-grade prostate cancer with Gleason score >or=7 (P=0.027). Bone-morphogenetic protein-6, noggin and sclerostin alone could not predict the development of distant metastasis in our patient cohort. However, high level of bone-morphogenetic protein-6 and low level of noggin, or high level of bone-morphogenetic protein-6 and low level of both noggin and sclerostin expression in primary prostate cancer significantly predicted development of distant metastasis. The predictive value was still valid when only high-grade prostate cancers were included or when patients with secondary lesion other than bone were excluded. Taken together, these results suggest that a high level of bone-morphogenetic protein-6 signaling, resulting from increased expression of bone-morphogenetic protein-6 and decreased expression of its inhibitors, might promote the development of prostate cancer metastases. Our results also imply the potential use of bone-morphogenetic protein-6, noggin and sclerostin expression together as a prognostic predictor for metastatic progression of prostate cancer.

Effect of a prodrug of the green tea polyphenol (-)-epigallocatechin-3-gallate on the growth of androgen-independent prostate cancer in vivo.

Epigallocatechin-3-gallate (EGCG) is the major and most potent polyphenol compound of green tea that has been shown to have anticancer effects against various types of cancers. In this study, in addition to the EGCG compound, a synthetic derivative, the peracetate of EGCG (EGCG-P), was used to investigate the inhibitory effects on growth of androgen-independent prostate cancer in vivo. The advantage of EGCG-P is that it may act as a prodrug, leading to higher bioavailability than EGCG itself. The aim of our study was to compare the differences between EGCG and EGCG-P on their inhibitory effect on androgen-independent prostate cancer, CWR22R, xenograft model in nude mice. The mice were administrated daily with solvent dimethyl sulfoxide, EGCG, and EGCG-P separately through intraperitoneal injection for 20 days. Tumor volume and body weight of nude mice were recorded daily. Serum prostate-specific antigen (PSA) levels were also measured before and after the treatment. The effects of both EGCG and EGCG-P on tumor cell proliferation were assessed by immunohistochemical (IHC) method using antibodies against Ki-67 and proliferating cell nuclear antigen. The apoptotic effect was evaluated by IHC against B-cell non-Hodgkin lymphoma-2 and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay by in situ apoptosis detection kit. Moreover, the potential suppression of angiogenesis by EGCG and EGCG-P on prostate cancer was examined by IHC against CD31. Our results revealed that treatment of EGCG and EGCG-P compounds suppressed the growth of CWR22R xenografts without causing any detectable side effects in nude mice. The suppression of growth of the tumor was correlated with the decrease of serum PSA level together with the reduction in tumor angiogenesis and an increase in apoptosis on prostate cancer cells. The results showed that treatment of EGCG and EGCG-P inhibited tumor growth and angiogenesis while promoting apoptosis of the prostate cancer cells in vivo. Our results suggest that EGCG-P may be a more stable and useful compound for increasing the therapeutic anticancer effects in androgen-independent prostate cancer.

Garlic-derived S-allylmercaptocysteine is a novel in vivo antimetastatic agent for androgen-independent prostate cancer.

PURPOSE: There is epidemiologic evidence that high garlic consumption decreases the incidence of prostate cancer, and compounds isolated from garlic have been shown to have cancer-preventive and tumor-suppressive effects. Recent in vitro studies in our laboratory have shown that garlic-derived organosulfur compound S-allylmercaptocysteine suppresses invasion and cell motility of androgen-independent prostate cancer cells via the up-regulation of cell-adhesion molecule E-cadherin. S-allylmercaptocysteine is therefore a potential antimetastatic drug with broad clinical applications that we tested in vivo for the first time in this study. EXPERIMENTAL DESIGN: We used a newly established fluorescent orthotopic androgen-independent prostate cancer mouse model to assess the ability of S-allylmercaptocysteine to inhibit tumor growth and dissemination. RESULTS: We showed that oral S-allylmercaptocysteine not only inhibited the growth of primary tumors by up to 71% (P < 0.001) but also reduced the number of lung and adrenal metastases by as much as 85.5% (P = 0.001) without causing notable toxicity. This metastatic suppression was accompanied by a 91% reduction of viable circulating tumor cells (P = 0.041), suggesting that S-allylmercaptocysteine prevents dissemination by decreasing tumor cell intravasation. CONCLUSIONS: Our results provide in vivo evidence supporting the potential use of S-allylmercaptocysteine as an E-cadherin up-regulating antimetastatic agent for the treatment of androgen-independent prostate cancer. This is the first report of the in vivo antimetastatic properties of garlic, which may also apply to other cancer types.

Inactivation of ID-1 gene induces sensitivity of prostate cancer cells to chemotherapeutic drugs.

Resistance to anticancer drugs is one of the major reasons of treatment failure for androgen-independent prostate cancer (PC). Increase in expression of Id-1 has been reported in several types of advanced cancer including PC. It has been suggested that overexpression of Id-1 may provide an advantage for cancer cell survival and thus inactivation of Id-1 may be able to increase the susceptibility of cancer cells to apoptosis. In this study, using small RNA interfering (siRNA) technology, we inactivated the Id-1 gene in two androgen-independent PC cell lines, DU145 and PC3, and investigated whether down-regulation of Id-1 could lead to increased sensitivity of these PC cells to a commonly used anticancer drug, taxol (Tx). Our results showed that inactivation of Id-1 by siId-1 resulted in decrease in both colony forming ability and cell viability in prostate cancer cells after Tx treatment. Furthermore, the siId-1 induced sensitization to Tx was associated with activation of apoptotic pathway. In addition, c-Jun N-terminal kinase (JNK), one of the common pathways responsible for Tx-induced apoptosis, was also activated in the si-Id-1 transfected cells. Inhibition of JNK activity by a specific inhibitor, SP600125, blocked the siId-1-induced sensitivity to Tx. These results indicate that increased Id-1 expression in PC cells may play a protective role against apoptosis, and down-regulation of Id-1 may be a potential target to increase sensitivity of Tx-induced apoptosis in PC cells.

Role of p14ARF in TWIST-mediated senescence in prostate epithelial cells.

Recently, TWIST, a basic helix-loop-helix transcription factor, is suggested to be an oncogene because of its over-expression in many types of human cancer and its positive role in promoting cell survival. The aim of this study was to investigate the role of TWIST on the growth of human epithelial cells. Using two immortalized human prostate epithelial cell lines, we demonstrated that inactivation of TWIST by small RNA technology led to the promotion of cellular senescence and growth arrest, suggesting that TWIST plays a key role in the continuous proliferation of immortalized cells. Over-expression of TWIST, in contrast, resulted in suppression of cellular senescence in response to genotoxic damage and promotion of cell proliferation with DNA damage accumulation, indicating that TWIST promotes genomic instability. In addition, we also found that the TWIST-mediated cellular senescence was regulated through its negative effect on p14(ARF) and subsequent suppression of MDM2/p53 and Chk1/2 DNA damage response pathways. Our results suggest that over-expression of TWIST results in down-regulation of p14(ARF), which leads to the impairment of DNA damage checkpoint in response to genotoxic stress. This negative effect of TWIST on DNA damage response facilitates uncontrolled cell proliferation with genomic instability and tumorigenesis in non-malignant cells.

Id-1 activation of PI3K/Akt/NFkappaB signaling pathway and its significance in promoting survival of esophageal cancer cells.

Inhibitor of differentiation or DNA binding (Id-1) is a helix-loop-helix protein that is over-expressed in many types of cancer including esophageal cancer. This study aims to investigate its effects on the phosphatidylinositol-3-kinase (PI3K)/Akt/ nuclear factor kappa B (NFkappaB) signaling pathway and the significance in protecting esophageal cancer cells against apoptosis. We found elevated expression of phosphorylated forms of Akt, glycogen synthase kinase 3beta and inhibitor of kappa B, as well as increased nuclear translocation of NFkappaB subunit p65 and NFkappaB DNA-binding activity, in esophageal cancer cells with stable ectopic Id-1 expression. Transient transfection of Id-1 into HEK293 cells confirmed activation of PI3K/Akt/NFkappaB signaling and the effects were counteracted by the PI3K inhibitor LY294002. Treatment with tumor necrosis factor-alpha (TNF-alpha) elicited a significantly weaker apoptotic response, following a marked and sustained activation of Akt and NFkappaB in the Id-1-over-expressing cells, compared with the vector control. The effects of Id-1 on the PI3K/Akt/NFkappaB signaling pathway and apoptosis were reversed in esophageal cancer cells transfected with siRNA against Id-1. In addition, inhibition of PI3K or NFkappaB signaling using the PI3K inhibitor LY294002 or the NFkappaB inhibitor Bay11-7082 increased the sensitivity of Id-1-over-expressing esophageal cancer cells to TNF-alpha-induced apoptosis. Our results provide the first evidence that Id-1 induces the activation of PI3K/Akt/NFkappaB signaling pathway, and protects esophageal cancer cells from TNF-alpha-induced apoptosis in vitro. Inactivation of Id-1 may provide us with a novel strategy to improve the treatment and survival of patients with esophageal cancer.

Increased Id-1 expression is significantly associated with poor survival of patients with prostate cancer.

The levels of Id-1 (inhibitor of DNA binding or inhibitor of cell differentiation) expression in a series of prostate cell lines and in an archival set of prostate tissues were examined. Western blot analysis showed that the level of Id-1 expressed in the androgen sensitive cell line LNCaP was 1.2 +/- 0.2 times that detected in the benign cell line PNT-2. The level of Id-1 increased further to 1.8 +/- 0.2 and 2.9 +/- 0.3 in the androgen-insensitive cell lines Du-145 and PC-3, respectively. Immunohistochemical staining with Id-1 antibody performed on 113 cases of prostate tissues showed that among the 7 normal cases, 6 (86%) stained either negative or weakly positive whereas only 1 (14%) stained moderately positive. Among the 36 benign prostatic hyperplasia (BPH) samples, 34 (94%) stained either negative or weakly positive; only 1 (3%) stained moderately and 1 (3%) stained strongly. Of the 70 carcinomas, 8 (11.5%) stained weakly, 34 (48.5%) stained moderately, and 28 (40%) stained strongly positive. The intensity of Id-1 staining in carcinomas was significantly stronger than that detected in the normal prostate and BPH (chi(2) test, P < .001) and it was significantly increased as the increasing malignancy of carcinomas measured by Gleason score (chi(2) test, P < .001). The intensity of Id-1 staining was partially associated with the levels of prostate-specific antigen, but not related to the level of androgen receptor. Kaplan-Meier survival curve analysis showed that, similar to Gleason scores, overexpression of Id-1 was significantly associated with the reduced length of patient survival (log-rank test, P = .01). These results suggest that Id-1 is a useful prognostic marker to predict the outcomes of patients with prostate cancer.

Upregulation of Twist in oesophageal squamous cell carcinoma is associated with neoplastic transformation and distant metastasis.

BACKGROUND: The antiapoptotic and epithelial-mesenchymal transition activities of Twist have been implicated in the neoplastic transformation and the development of metastasis, respectively. Upregulation of Twist, described in several types of human cancer, also acts as a prognostic marker of poor outcome. AIM: To investigate Twist expression in oesophageal squamous cell carcinoma (SCC) and its prognostic value in a Chinese cohort of patients with oesophageal SCC. METHODS: Twist expression in primary oesophageal SCC of 87 Chinese patients was investigated by immunohistochemical staining. Twist protein level in one immortalised normal oesophageal epithelial cell line and six oesophageal SCC cell lines was measured by western blot analysis. Twist mRNA level in 30 pairs of frozen specimens of primary oesophageal SCC and non-neoplastic oesophageal epithelium from the upper resection margin of corresponding oesophagectomy specimen was also determined by semiquantitative reverse transcription-PCR. RESULTS: It was found that Twist was upregulated in oesophageal SCC cell lines, and its mRNA and protein levels were both increased in oesophageal SCC and the non-neoplastic oesophageal epithelium (p<0.001). In addition, a high level of Twist expression in oesophageal SCC was significantly associated with a greater risk for the patient of developing distant metastasis within 1 year of oesophagectomy (OR 3.462, 95% CI 1.201 to 9.978; p=0.022). CONCLUSIONS: Our results suggest that upregulation of Twist plays a role in the neoplastic transformation to oesophageal SCC and subsequent development of distant metastasis. Twist may serve as a useful prognostic marker for predicting the development of distant metastasis in oesophageal SCC.

Regulation of angiogenesis by Id-1 through hypoxia-inducible factor-1alpha-mediated vascular endothelial growth factor up-regulation in hepatocellular carcinoma.

PURPOSE: Metastasis is commonly associated with poor prognosis of hepatocellular carcinoma (HCC). Being an important angiogenic factor, vascular endothelial growth factor (VEGF) plays a central role in HCC growth and metastasis. Recently, Id-1 (inhibitor of differentiation/DNA synthesis) has been suggested to be a key factor in cancer progression but the molecular mechanism remains unknown. EXPERIMENTAL DESIGN: We first showed that overexpression of Id-1 was correlated with HCC metastasis (P < 0.001) and its expression was significantly correlated with VEGF expression by tissue microarray. By ectopic transfection of Id-1 into HCC cells, Id-1 was able to induce VEGF secretion through activation of VEGF transcription. RESULTS: Increased VEGF secretion in Id-1 transfectants induced morphologic change and proliferation of human umbilical vascular endothelial cell resulting in promotion of angiogenesis. Id-1 induced transcriptional activation of VEGF by stabilizing hypoxia-inducible factor-1alpha protein. Down-regulation of Id-1 by antisense approach led to suppression of hypoxia-inducible factor-1alpha-mediated VEGF production. In addition, Id-1 suppression resulted in retardation of cell invasion through down-regulation of VEGF. CONCLUSIONS: Id-1 is a novel angiogenic factor for HCC metastasis and down-regulation of Id-1 may be a novel target to inhibit HCC metastasis through suppression of angiogenesis.

Twist overexpression correlates with hepatocellular carcinoma metastasis through induction of epithelial-mesenchymal transition.

PURPOSE: Hepatocellular carcinoma (HCC) is a rapidly growing tumor associated with a high propensity for vascular invasion and metastasis. Epithelial-mesenchymal transition (EMT) is a key event in the tumor invasion process. Recently, Twist has been identified to play an important role in EMT-mediated metastasis through the regulation of E-cadherin expression. However, the actual role of Twist in tumor invasiveness remains unclear. The purpose of this study is to investigate the expression and possible role of Twist in HCC. EXPERIMENTAL DESIGN: We evaluated Twist and E-cadherin expression in HCC tissue microarray of paired primary and metastatic HCC by immunohistochemical staining. The role of Twist in EMT-mediated invasiveness was also evaluated in vitro in HCC cell lines. RESULTS: We first showed that overexpression of Twist was correlated with HCC metastasis (P=0.001) and its expression was negatively correlated with E-cadherin expression (P=0.001, r=-0.443) by tissue microarray. A significant increase of Twist at the mRNA level was also found in metastatic HCC cell lines MHCC-97H, MHCC-97L, and H2M when compared with nonmetastatic Huh-7, H2P, and PLC cell lines. The MHCC-97H cell line, which has a higher metastatic ability, was found to have a higher level of Twist than MHCC-97L. Accompanied with increased Twist expression in the metastatic HCC cell lines when compared with the nonmetastatic primary ones, we found decreased E-cadherin mRNA expression in the metastatic HCC cell lines. By ectopic transfection of Twist into PLC cells, Twist was able to suppress E-cadherin expression and induce EMT changes, which was correlated with increased HCC cell invasiveness. CONCLUSION: This study shows that Twist overexpression was correlated with HCC metastasis through induction of EMT changes and HCC cell invasiveness.

Mitotic arrest deficient 2 expression induces chemosensitization to a DNA-damaging agent, cisplatin, in nasopharyngeal carcinoma cells.

Recently, mitotic arrest deficient 2 (MAD2)-mediated spindle checkpoint is shown to induce mitotic arrest in response to DNA damage, indicating overlapping roles of the spindle checkpoint and DNA damage checkpoint. In this study, we investigated if MAD2 played a part in cellular sensitivity to DNA-damaging agents, especially cisplatin, and whether it was regulated through mitotic checkpoint. Using nine nasopharyngeal carcinoma (NPC) cell lines, we found that decreased MAD2 expression was correlated with cellular resistance to cisplatin compared with the cell lines with high levels of MAD2. Exogenous MAD2 expression in NPC cells also conferred sensitivity to DNA-damaging agents especially cisplatin but not other anticancer drugs with different mechanisms of action. The increased cisplatin sensitivity in MAD2 transfectants was associated with mitotic arrest and activation of apoptosis pathway evidenced by the increased mitotic index and apoptosis rate as well as decreased Bcl-2 and Bax ratio and expression of cleaved poly(ADP-ribose) polymerase and caspase 3. Our results indicate that the MAD2-induced chemosensitization to cisplatin in NPC cells is mediated through the induction of mitotic arrest, which in turn activates the apoptosis pathway. Our evidence further confirms the previous hypothesis that spindle checkpoint plays an important part in DNA damage-induced cell cycle arrest and suggests a novel role of MAD2 in cellular sensitivity to cisplatin.