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Purpose: Previously, FTY720 was found to possess potent anticancer effects on various types of cancer. In the present study, we aimed to first verify the role of Runx2 in prostate cancer progression and metastasis, and, subsequently, assessed if FTY720 could modulate Runx2 expression, thus interfering downstream events regulated by this protein.Experimental Design: First, the association between Runx2 and prostate cancer progression was assessed using localized prostate cancer specimens and mechanistic investigation of Runx2-induced cancer aggressiveness was then carried out. Subsequently, the effect of FTY720 on Runx2 expression and transcriptional activity was investigated using PC-3 cells, which highly expressed Runx2 protein. Last, the involvement of Runx2 in FTY720-induced anticancer effects was evaluated by modulating Runx2 expression in various prostate cancer cell lines.Results: Runx2 nuclear expression was found to be up-regulated in prostate cancer and its expression could be used as a predictor of metastasis in prostate cancer. Further mechanistic studies indicated that Runx2 accelerated prostate cancer aggressiveness through promotion of cadherin switching, invasion toward collagen I, and Akt activation. Subsequently, we found that FTY720 treatment down-regulated Runx2 expression and its transcriptional activity, as well as inhibited its regulated downstream events. More importantly, silencing Runx2 in PC-3 enhanced FTY720-induced anticancer effects as well as cell viability inhibition, whereas overexpressing Runx2 in 22Rv1 that expressed very low endogenous Runx2 protein conferred resistance in the same events.Conclusion: This study provided a novel mechanism for the anticancer effect of FTY720 on advanced prostate cancer, thus highlighting the therapeutic potential of this drug in treating this disease.

Supplementary Data from Suppression of Androgen-Independent Prostate Cancer Cell Aggressiveness by FTY720: Validating Runx2 as a Potential Antimetastatic Drug Screening Platform

Supplementary Table 2 from MicroRNA-616 Induces Androgen-Independent Growth of Prostate Cancer Cells by Suppressing Expression of Tissue Factor Pathway Inhibitor TFPI-2

Rev7p has been suggested to play an important role in regulating DNA damage response in yeast, and recently, the human homologue (i.e., MAD2B) has been identified, which shares significant homology to the mitotic checkpoint protein MAD2. In this study, we investigated whether MAD2B played a key role in cellular sensitivity to DNA-damaging anticancer drugs by suppressing its expression using RNA interference in nasopharyngeal carcinoma cells. Using colony formation assay, we found that suppression of MAD2B conferred hypersensitivity to a range of DNA-damaging agents, especially DNA cross-linkers, such as cisplatin, and γ-irradiation. This effect was associated with reduced frequencies of spontaneous and drug-induced mutations, elevated phosphorylation of histone H2AX, and markedly increased chromosomal aberrations in response to DNA damage. In addition, there was also a significant decrease in cisplatin-induced sister chromatid exchange rate, a marker for homologous recombination-mediated post-replication repair in MAD2B-depleted cells. These results indicate that MAD2B may be a key factor in regulating cellular response to DNA damage in cancer cells. Our findings reveal a novel strategy for cancer therapy, in which cancer cells are sensitized to DNA-damaging anticancer drugs through inactivation of the MAD2B gene. (Cancer Res 2006; 66(8): 4357-67)

Supplementary Figure 3 from Sirtuin 1 Is Upregulated in a Subset of Hepatocellular Carcinomas where It Is Essential for Telomere Maintenance and Tumor Cell Growth

Supplementary Figure Legends 1-6, Table 1 from Sirtuin 1 Is Upregulated in a Subset of Hepatocellular Carcinomas where It Is Essential for Telomere Maintenance and Tumor Cell Growth

Supplementary Figure 5 from Sirtuin 1 Is Upregulated in a Subset of Hepatocellular Carcinomas where It Is Essential for Telomere Maintenance and Tumor Cell Growth

Supplementary Figure 4 from Sirtuin 1 Is Upregulated in a Subset of Hepatocellular Carcinomas where It Is Essential for Telomere Maintenance and Tumor Cell Growth

Supplementary Figure 6 from Sirtuin 1 Is Upregulated in a Subset of Hepatocellular Carcinomas where It Is Essential for Telomere Maintenance and Tumor Cell Growth

Expression of microRNA genes is profoundly altered in cancer but their role in the development of androgen-independent prostate cancer has received limited attention as yet. In this study, we report a functional impact in prostate cancer cells for overexpression of the microRNA miR-616, which occurred consistently in cells that were androgen-independent (AI) versus androgen-dependent (AD). miR-616 overexpression was confirmed in malignant prostate tissues as opposed to benign prostate specimens. Stable miR-616 overexpression in LNCaP cells by a lentiviral-based approach stimulated AI prostate cancer cell proliferation in vitro whereas concomitantly reducing androgen-induced cell growth. More importantly, miR-616 overexpressing LNCaP cells overcame castration resistance as shown by an enhanced ability to proliferate in vivo after bilateral orchiectomy. Conversely, antagonizing miR-616 in AI prostate cancer cells yielded opposite effects. Microarray profiling and bioinformatics analysis identified the tissue factor pathway inhibitor TFPI-2 mRNA as a candidate downstream target of miR-616. In support of this candidacy, we documented interactions between miR-616 and the 3′UTR of TFPI-2 and determined TFPI-2 expression to be inversely correlated to miR-616 in a series of prostate cell lines and clinical specimens. Notably, reexpression of TFPI-2 in LNCaP cells with stable miR-616 overexpression rescued the AD phenotype, as shown by a restoration of androgen dependence and cell growth inhibition. Taken together, our findings define a functional involvement for miR-616 and TFPI-2 in the development and maintenance of androgen-independent prostate cancer. Cancer Res; 71(2); 583–92. ©2011 AACR.

Supplementary Figure 1 from Inactivation of Human MAD2B in Nasopharyngeal Carcinoma Cells Leads to Chemosensitization to DNA-Damaging Agents

Supplementary Information from Sirtuin 1 Is Upregulated in a Subset of Hepatocellular Carcinomas where It Is Essential for Telomere Maintenance and Tumor Cell Growth

Supplementary Figure Legend from MicroRNA-616 Induces Androgen-Independent Growth of Prostate Cancer Cells by Suppressing Expression of Tissue Factor Pathway Inhibitor TFPI-2

Supplementary Figure 1 from Sirtuin 1 Is Upregulated in a Subset of Hepatocellular Carcinomas where It Is Essential for Telomere Maintenance and Tumor Cell Growth

Supplementary Figure Legend from Inactivation of Human MAD2B in Nasopharyngeal Carcinoma Cells Leads to Chemosensitization to DNA-Damaging Agents

Supplementary Figure S1 from Up-Regulation of TWIST in Prostate Cancer and Its Implication as a Therapeutic Target

Supplementary Figure 1 from MicroRNA-616 Induces Androgen-Independent Growth of Prostate Cancer Cells by Suppressing Expression of Tissue Factor Pathway Inhibitor TFPI-2

Supplementary Figure 2 from Sirtuin 1 Is Upregulated in a Subset of Hepatocellular Carcinomas where It Is Essential for Telomere Maintenance and Tumor Cell Growth

Androgen-independent metastatic prostate cancer is the main obstacle in the treatment of this cancer. Unlike a majority of solid cancers, prostate cancer usually shows poor response to chemotherapeutic drugs. In this study, we have shown a potential novel target, TWIST, a highly conserved bHLH transcription factor, in the treatment of prostate cancer. Using malignant and nonmalignant prostate tissues, we found that TWIST expression was highly expressed in the majority (90%) of prostate cancer tissues but only in a small percentage (6.7%) of benign prostate hyperplasia. In addition, the TWIST expression levels were positively correlated with Gleason grading and metastasis, indicating its role in the development and progression of prostate cancer. Furthermore, down-regulation of TWIST through small interfering RNA in androgen-independent prostate cancer cell lines, DU145 and PC3, resulted in increased sensitivity to the anticancer drug taxol-induced cell death which was associated with decreased Bcl/Bax ratio, leading to activation of the apoptosis pathway. More importantly, inactivation of TWIST suppressed migration and invasion abilities of androgen-independent prostate cancer cells, which was correlated with induction of E-cadherin expression as well as morphologic and molecular changes associated with mesenchymal to epithelial transition. These results were further confirmed on the androgen-dependent LNCaP cells ectopically expressing the TWIST protein. Our results have identified TWIST as a critical regulator of prostate cancer cell growth and suggest a potential therapeutic approach to inhibit the growth and metastasis of androgen-independent prostate cancer through inactivation of the TWIST gene.

Supplementary References from MicroRNA-616 Induces Androgen-Independent Growth of Prostate Cancer Cells by Suppressing Expression of Tissue Factor Pathway Inhibitor TFPI-2

Supplementary Table 1 from Sirtuin 1 Is Upregulated in a Subset of Hepatocellular Carcinomas where It Is Essential for Telomere Maintenance and Tumor Cell Growth

Whether and how garlic-derived S-allylmercaptocysteine (SAMC) inhibits hepatocellular carcinoma (HCC) is largely unknown. In the current study, the role of low-density lipoprotein receptor (LDLR)-related protein 6 (LRP6) in HCC progression and the anti-HCC mechanism of SAMC was examined in clinical sample, cell model and xenograft/orthotopic mouse models. We demonstrated that SAMC inhibited cell proliferation and tumorigenesis, while induced apoptosis of human HCC cells without influencing normal hepatocytes. SAMC directly interacted with Wnt-pathway co-receptor LRP6 on the cell membrane. LRP6 was frequently over-expressed in the tumor tissue of human HCC patients (66.7% of 48 patients) and its over-expression only correlated with the over-expression of β-catenin, but not with age, gender, tumor size, stage and metastasis. Deficiency or over-expression of LRP6 in hepatoma cells could partly mimic or counteract the anti-tumor properties of SAMC, respectively. In vivo administration of SAMC significantly suppressed the growth of Huh-7 xenograft/orthotopic HCC tumor without causing undesirable side effects. In addition, stable down-regulation of LRP6 in Huh-7 facilitated the anti-HCC effects of SAMC. In conclusion, LRP6 can be a potential therapeutic target of HCC. SAMC is a promising specific anti-tumor agent for treating HCC subtypes with Wnt activation at the hepatoma cell surface., Graphical abstract Garlic-derived S-allylmercaptocysteine (SAMC) directly interacts with cancer cell membrane-bound receptor low-density lipoprotein receptor-related protein 6 (LRP6) to modulate the activation of the Wnt/β-catenin pathway. The apoptosis of cancer cell is promoted through the c-Myc- and Mcl-1-mediated pathways, while the proliferation of cancer cell is inhibited via the converged PCNA/p53/p21 regulations.fx1

Emerging evidence suggests that gamma-tocotrienol (&gamma, T3), a vitamin E isomer, has potent anti-cancer properties against a wide-range of cancers. &gamma, T3 not only inhibited the growth and survival of cancer cells in vitro, but also suppressed angiogenesis and tumour metastasis under in vivo conditions. Recently, &gamma, T3 was found to target cancer stem cells (CSCs), leading to suppression of tumour formation and chemosensitisation. Despite its promising anti-cancer potential, the exact mechanisms responsible for the effects of &gamma, T3 are still largely unknown. Here, we report the identification of Ang-1 (Angiopoietin-1)/Tie-2 as a novel &gamma, T3 downstream target. In prostate cancer cells, &gamma, T3 treatment leads to the suppression of Ang-1 at both the mRNA transcript and protein levels. Supplementing the cells with Ang-1 was found to protect them against the anti-CSC effect of &gamma, T3. Intriguingly, inactivation of Tie-2, a member receptor that mediates the effect of Ang-1, was found to significantly enhance the cytotoxic effect of &gamma, T3 through activation of AMP-activated protein kinase (AMPK) and subsequent interruption of autophagy. Our results highlighted the therapeutic potential of using &gamma, T3 in combination with a Tie-2 inhibitor to treat advanced prostate cancer.

Skeletal metastases present a major clinical challenge for prostate cancer patient care, inflicting distinctive mixed osteoblastic and osteolytic lesions that cause morbidity and refractory skeletal complications. Macrophages are abundant in bone and bone marrow and can influence both osteoblast and osteoclast function in physiology and pathology. Herein, we examined the role of macrophages in prostate cancer bone lesions, particularly the osteoblastic response. First, macrophage and lymphocyte distributions were qualitatively assessed in patient's prostate cancer skeletal lesions by immunohistochemistry. Second, macrophage functional contributions to prostate tumour growth in bone were explored using an immune-competent mouse model combined with two independent approaches to achieve in vivo macrophage depletion: liposome encapsulated clodronate that depletes phagocytic cells (including macrophages and osteoclasts); and targeted depletion of CD169(+) macrophages using a suicide gene knock-in model. Immunohistochemistry and histomorphometric analysis were performed to quantitatively assess cancer-induced bone changes. In human bone metastasis specimens, CD68(+) macrophages were consistently located within the tumour mass. Osteal macrophages (osteomacs) were associated with pathological woven bone within the metastatic lesions. In contrast, lymphocytes were inconsistently present in prostate cancer skeletal lesions and when detected, had varied distributions. In the immune-competent mouse model, CD169(+) macrophage ablation significantly inhibited prostate cancer-induced woven bone formation, suggesting that CD169(+) macrophages within pathological woven bone are integral to tumour-induced bone formation. In contrast, pan-phagocytic cell, but not targeted CD169(+) macrophage depletion resulted in increased tumour mass, indicating that CD169(-) macrophage subset(s) and/or osteoclasts influenced tumour growth. In summary, these observations indicate a prominent role for macrophages in prostate cancer bone metastasis that may be therapeutically targetable to reduce the negative skeletal impacts of this malignancy, including tumour-induced bone modelling. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Ample evidence supports that prostate tumor metastasis originates from a rare population of cancer cells, known as cancer stem cells (CSCs). Unfortunately, little is known about the identity of these cells, making it difficult to target the metastatic prostate tumor. Here, for the first time, we report the identification of a rare population of prostate cancer cells that express the Tie-2 protein. We found that this Tie-2High population exists mainly in prostate cancer cell lines that are capable of metastasizing to the bone. These cells not only express a higher level of CSC markers but also demonstrate enhanced resistance to the chemotherapeutic drug Cabazitaxel. In addition, knockdown of the expression of the Tie-2 ligand angiopoietin (Ang-1) led to suppression of CSC markers, suggesting that the Ang-1/Tie-2 signaling pathway functions as an autocrine loop for the maintenance of prostate CSCs. More importantly, we found that Tie-2High prostate cancer cells are more adhesive than the Tie-2Low population to both osteoblasts and endothelial cells. Moreover, only the Tie-2High, but not the Tie-2Low cells developed tumor metastasis in vivo when injected at a low number. Taken together, our data suggest that Tie-2 may play an important role during the development of prostate tumor metastasis.

The phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR pathway is one of the most frequently activated signaling pathways in prostate cancer cells, and loss of the tumor suppressor PTEN and amplification of PIK3CA are the two most commonly detected mechanisms for the activation of these pathways. Aberrant activation of PI3K/Akt/mTOR has been implicated not only in the survival and metastasis of prostate cancer cells but also in the development of drug resistance. As such, selective inactivation of this pathway may provide opportunities to attack prostate cancer from all fronts. However, while preclinical studies examining specific inhibitors of PI3K or mTOR have yielded promising results, the evidence from clinical trials is less convincing. Emerging evidence from the analyses of some solid tumors suggests that a class of dual PI3K/mTOR inhibitors, which bind to and inactivate both PI3K and mTOR, may achieve better anti-cancer outcomes. In this review, we will summarize the mechanisms of action of these inhibitors, their effectiveness when used alone or in combination with other chemotherapeutic compounds, and their potential to serve as the next generation therapies for prostate cancer patients, particularly those who are resistant to the frontline chemotherapeutic drugs.

The medicinal mushroom Trametes versicolor has been well recognized for its activity in maintaining the general health of the population and in managing and treating human diseases in various cultures. Its use has been recently gaining acceptance and popularity in Western countries. The reported health benefits of T. versicolor led to a search for the identity of its bioactive ingredients. These efforts have resulted in the isolation of the polysaccharopeptide PSP from cultured mycelia of strain Cov-1, which expresses large amounts of PSP. The availability of highly purified PSP was followed by studies of its biological activities using tissue culture models and limited human clinical trials. In this review we summarize recent advances in the antitumorigenic and immunomodulatory effects of PSP, elimination of prostate cancer stem cells and control of the intestinal microbiome, and its interplay with host cells as a prebiotic. These findings may have implications for widening and repurposing the use of PSP.

Caveolin-1 has a complex role in prostate cancer and has been suggested to be a potential biomarker and therapeutic target. As mature caveolin-1 resides in caveolae, invaginated lipid raft domains at the plasma membrane, caveolae have been suggested as a tumor-promoting signaling platform in prostate cancer. However, caveola formation requires both caveolin-1 and cavin-1 (also known as PTRF; polymerase I and transcript release factor). Here, we examined the expression of cavin-1 in prostate epithelia and stroma using tissue microarray including normal, non-malignant and malignant prostate tissues. We found that caveolin-1 was induced without the presence of cavin-1 in advanced prostate carcinoma, an expression pattern mirrored in the PC-3 cell line. In contrast, normal prostate epithelia expressed neither caveolin-1 nor cavin-1, while prostate stroma highly expressed both caveolin-1 and cavin-1. Utilizing PC-3 cells as a suitable model for caveolin-1-positive advanced prostate cancer, we found that cavin-1 expression in PC-3 cells inhibits anchorage-independent growth, and reduces in vivo tumor growth and metastasis in an orthotopic prostate cancer xenograft mouse model. The expression of α-smooth muscle actin in stroma along with interleukin-6 (IL-6) in cancer cells was also decreased in tumors of mice bearing PC-3-cavin-1 tumor cells. To determine whether cavin-1 acts by neutralizing caveolin-1, we expressed cavin-1 in caveolin-1-negative prostate cancer LNCaP and 22Rv1 cells. Caveolin-1 but not cavin-1 expression increased anchorage-independent growth in LNCaP and 22Rv1 cells. Cavin-1 co-expression reversed caveolin-1 effects in caveolin-1-positive LNCaP cells. Taken together, these results suggest that caveolin-1 in advanced prostate cancer is present outside of caveolae, because of the lack of cavin-1 expression. Cavin-1 expression attenuates the effects of non-caveolar caveolin-1 microdomains partly via reduced IL-6 microenvironmental function. With circulating caveolin-1 as a potential biomarker for advanced prostate cancer, identification of the molecular pathways affected by cavin-1 could provide novel therapeutic targets.

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.

Obesity has long been linked with prostate cancer progression, although the underlying mechanism is still largely unknown. Here, we report that adipocytes promote the enrichment of prostate cancer stem cells (CSCs) through a vicious cycle of autocrine amplification. In the presence of adipocytes, prostate cancer cells actively secrete the peptide hormone cholecystokinin (CCK), which not only stimulates prostate CSC self-renewal, but also induces cathepsin B (CTSB) production of the adipocytes. In return, CTSB facilitates further CCK secretion by the cancer cells. More importantly, inactivation of CCK receptor not only suppresses CTSB secretion by the adipocytes, but also synergizes the inhibitory effect of CTSB inhibitor on adipocyte-promoted prostate CSC self-renewal. In summary, we have uncovered a novel mechanism underlying the mutual interplay between adipocytes and prostate CSCs, which may help explaining the role of adipocytes in prostate cancer progression and provide opportunities for effective intervention.

Hepatocellular carcinoma (HCC) is a highly malignant tumor with a poor prognosis. Treatment of HCC is complicated by the fact that the disease is often diagnosed at an advanced stage when it is no longer amenable to curative surgery, and current systemic chemotherapeutics are mostly inefficacious. Sirtuin 1 (SIRT1) is a class III histone deacetylase that is implicated in gene regulations and stress resistance. In this study, we found that SIRT1 is essential for the tumorigenesis of HCC. We showed that although SIRT1 was expressed at very low levels in normal livers, it was overexpressed in HCC cell lines and in a subset of HCC. Tissue microarray analysis of HCC and adjacent nontumoral liver tissues revealed a positive correlation between the expression levels of SIRT1 and advancement in tumor grades. Downregulation of SIRT1 consistently suppressed the proliferation of HCC cells via the induction of cellular senescence or apoptosis. SIRT1 silencing also caused telomere dysfunction–induced foci and nuclear abnormality that were clearly associated with reduced expressions of telomerase reverse transcriptase (TERT), and PTOP, which is a member of the shelterin complex. Ectopic expression of either TERT or PTOP in SIRT1-depleted cells significantly restored cell proliferation. There was also a positive correlation between the level of induction of SIRT1 and PTOP in human HCC. Finally, SIRT1-silencing sensitized HCC cells to doxorubicin treatment. Together, our findings reveal a novel function for SIRT1 in telomere maintenance of HCC, and they rationalize the clinical exploration of SIRT1 inhibitors for HCC therapy. Cancer Res; 71(12); 4138–49. ©2011 AACR.

One of the common features in advanced prostate cancer is bone metastasis. In this study, we investigated the clinical relevance of a bone factor, MSX2, in predicting the metastatic ability of prostate adenocarcinoma. Evaluation of MSX2 expression was performed using prostate cell lines as well as patient specimens. A sharp decrease in MSX2 was found in primary prostate cancer cells, 22Rv1, when compared with the non-malignant counterparts, followed by a gradual increase in more aggressive prostate cancer cell lines. Interestingly, the MSX2 protein was upregulated and predominantly expressed in the nucleus in aggressive prostate cancer cell line, C4-2b, compared with the less aggressive 22Rv1. Consistent with the in vitro results, MSX2 nuclear expression was significantly higher in nodular hyperplasia when compared with high-grade prostatic intraepithelial neoplasia (PIN), while MSX2 nuclear expression in prostate adenocarcinoma was higher than that in high-grade PIN. Importantly, MSX2 expression was increased significantly in tumors with metastasis compared with those without metastasis. Finally, MSX2 nuclear scores were significantly increased in patients with preoperative serum PSA >20 ng/mL. No correlation between MSX2 nuclear score and Gleason score was found. Taken together, MSX2 may serve as a potential biomarker in predicting primary prostate tumors with higher metastatic capability.

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 (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.

To date, the most effective cure for metastatic melanoma remains the surgical resection of the primary tumor. Recently, tocotrienol-rich-fraction has shown antiproliferative effect on cancer cells. To elucidate this anticancer property in malignant melanoma, this study aimed, first, to identify the most potent isomer for eliminating melanoma cells and second to decipher the molecular pathway responsible for its activity. Results showed that the inhibitory effect of gamma-tocotrienol was most potent, which resulted in induction of apoptosis as evidenced by activation of procaspases and the accumulation of sub-G1 cell population. Examination of the prosurvival genes revealed that the gamma-tocotrienol-induced cell death was associated with suppression of NF-kappaB, EGF-R, and Id family proteins. Meanwhile, gamma-tocotrienol treatment also resulted in induction of JNK signaling pathway, and inhibition of JNK activity by selective inhibitor was able to partially block the effect of gamma-tocotrienol. Interestingly, gamma-tocotrienol treatment led to suppression of mesenchymal markers and the restoration of E-cadherin and gamma-catenin expression, which was associated with suppression of cell invasion capability. Furthermore, synergistic effect was observed when cells were cotreated with gamma-tocotrienol and chemotherapy drugs. Together, our results demonstrated for the first time the anti-invasion and chemonsensitization effect of gamma-tocotrienol against human malignant melanoma cells.

Androgen receptor (AR) is a ligand-dependent transcription factor and its activity is regulated by numerous AR coregulators. Aberrant expression of AR coregulators in prostate cancer cells has an important role in the development and progression of prostate cancer. We report here that CDC25A, a cell cycle-promoting phosphatase over-expressed in a number of cancers, functions as an AR coregulator suppressing the AR transcriptional activity. In this study, we found that CDC25A is upregulated in human prostate cancer and its expression level is positively associated with the Gleason score and disease metastasis. More importantly, we showed that CDC25A can physically interact with AR through its putative catalytic domain. In addition, ectopic expression of CDC25A in prostate cancer cell lines suppresses PSA and Probasin promoter activities significantly, indicating that CDC25A may function as an AR corepressor. This was further confirmed by knockdown of endogenous CDC25A expression using small interfering RNA (siRNA), which resulted in upregulation of PSA promoter activity. Moreover, a truncated mutant that does not interact with AR fails to suppress the PSA promoter activity, indicating that CDC25A downregulates androgen-responsive promoter by physically interacting with AR. Taken together, our results demonstrated a novel function of CDC25A in the regulation of androgen signaling in human prostate cancer cells.

Tocotrienol-rich fraction (TRF) has demonstrated antiproliferative effect on prostate cancer (PCa) cells. To elucidate this anticancer property in PCa cells, this study aimed, first, to identify the most potent isomer for eliminating PCa cells; and second, to decipher the molecular pathway responsible for its activity. Results showed that the inhibitory effect of gamma-tocotrienol was most potent, which resulted in induction of apoptosis as evidenced by activation of pro-caspases and the presence of sub-G(1) cell population. Examination of the pro-survival genes revealed that the gamma-tocotrienol-induced cell death was associated with suppression of NF-kappaB, EGF-R and Id family proteins (Id1 and Id3). Meanwhile, gamma-tocotrienol treatment also resulted in the induction of JNK-signalling pathway and inhibition of JNK activity by a specific inhibitor (SP600125) was able to partially block the effect of gamma-tocotrienol. Interestingly, gamma-tocotrienol treatment led to suppression of mesenchymal markers and the restoration of E-cadherin and gamma-catenin expression, which was associated with suppression of cell invasion capability. Furthermore, a synergistic effect was observed when cells were co-treated with gamma-tocotrienol and Docetaxel. Our results suggested that the antiproliferative effect of gamma-tocotrienol act through multiple-signalling pathways, and demonstrated for the first time the anti-invasion and chemosensitisation effect of gamma-tocotrienol against PCa cells.

Accumulating epidemiological data suggest that Asian men have lower incidences of prostate cancer and benign prostate hyperplasia (BPH) compared with American and European populations and may have benefited from their higher intake of phytoestrogens in their diet. However, how these phytochemicals affect prostatic diseases is still unclear. In this study, we isolated six lignans from a plant, Campylotropis hirtella (Franch.) Schindl., which has been used as a folk medicine for treatment of BPH in China, through bioassay guided fractionation. They were dehydrodiconiferyl alcohol (C1), 4-[(-6-hydroxy-2,3-dihydro-1-benzofuran-3-yl)methyl]-5-methoxybenzene-1,3-diol (C2), erythro-guaiacylglycerol-beta-O-4'-coniferyl ether (C3), threo-guaiacylglycerol-beta-O-4'-coniferyl ether (C4), secoisolariciresinol (C5), and prupaside (C6), where C2 was identified as a new lignan analog. Their IC50 values for inhibition of prostate specific antigen (PSA) secretion were 19, 45, 110, 128, 137, and 186 microM, respectively, from C1 to C6 in LNCaP cells. Further study showed that C1-5 down-regulated cellular PSA expression and C1-4 also decreased androgen receptor (AR) expression in LNCaP cells. Furthermore, we investigated the proapoptotic effect of C1 on LNCaP cells. The active forms of caspase 3 associated with the specific proteolysis of poly (ADP-ribose) polymerase (PARP) were detected, and the antiapoptotic protein Bcl-2 was down-regulated after the treatment with C1. These results collectively indicated that these lignans may have chemopreventive or therapeutic actions for prostate cancer through suppressing AR signaling pathway and inducing apoptosis.

Id-1 (Inhibitor of DNA binding/differential-1) plays a positive role in tumorigenesis through regulation of multiple signaling pathways. Recently, it is suggested that upregulation of Id-1 in cancer cells promotes chromosomal instability. However, the underlying molecular mechanism is not known. In this study, we report a novel function of Id-1 in regulation of mitosis through physical interaction with Cdc20 (cell division cycle protein 20) and Cdh1 (Cdc20 homolog 1). During early mitosis, Id-1 interacts with Cdc20 and RASSF1A (Ras association domain family 1A), leading to enhanced APC(Cdc20) activity, which in turn promotes cyclin B1/securin degradation and premature mitosis. During late mitosis, Id-1 binds to Cdh1 and disrupts the interaction between Cdh1 and APC, resulting in suppression of APC(Cdh1) activity. On the other hand, overexpression of Cdh1 leads to Id-1 protein degradation, suggesting that Id-1 may also act as a substrate of APC(Cdh1). The negative effect of Id-1 on APC(Cdh1) results in suppression of APC(Cdh1)-induced Aurora A and Cdc20 degradation, leading to failure in cytokinesis. As a result, overexpression of Id-1 in human prostate epithelial cells leads to polyploidy in response to microtubule disruption, and this effect is abolished when Id-1 expression is suppressed using antisense technology. These results demonstrate a novel function of Id-1 in promoting chromosomal instability through modification of APC/C activity during mitosis and provide a novel molecular mechanism accounted for the function of Id-1 as an oncogene.

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.

The mitotic arrest deficient 2 (MAD2) is suggested to play a key role in a functional mitotic checkpoint because of its inhibitory effect on anaphase-promoting complex/cyclosome (APC/C) during mitosis. The binding of MAD2 to mitotic checkpoint regulators MAD1 and Cdc20 is thought to be crucial for its function and loss of which leads to functional inactivation of the MAD2 protein. However, little is known about the biological significance of this MAD2 mutant in human cells. In this study, we stably transfected a C-terminal-deleted MAD2 gene (MAD2DeltaC) into a human prostate epithelial cell line, Hpr-1 and studied its effect on chromosomal instability, cell proliferation, mitotic checkpoint control and soft agar colony-forming ability. We found that MAD2DeltaC was able to induce aneuploidy through promoting chromosomal duplication, which was a result of an impaired mitotic checkpoint and cytokinesis, suggesting a crucial role of MAD2-mediated mitotic checkpoint in chromosome stability in human cells. In addition, the MAD2DeltaC-transfected cells displayed anchorage-independent growth in soft agar after challenged by 7,12-dimethylbenz[A]anthracene (DMBA), demonstrating a cancer-promoting effect of a defective mitotic checkpoint in human cells. Furthermore, the DMBA-induced transformation was accompanied by a complete loss of DNA damage-induced p53 response and activation of the MAPK pathway in MAD2DeltaC cells. These results indicate that a defective mitotic checkpoint alone is not a direct cause of tumorigenesis, but it may predispose human cells to carcinogen-induced malignant transformation. The evidence presented here provides a link between MAD2 inactivation and malignant transformation of epithelial cells.

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

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.

Mushroom extracts have been extensively studied for their medicinal effects. They can stimulate immune responses and thus have been explored in cancer treatment. Recently, it has also been shown that some mushroom extracts can produce direct cytotoxic effect on cancer cells. In this review, we summarize the cytotoxic effect of mushroom extracts in cancer treatment revealed by both in vitro and in vivo studies. We also summarize the current understanding of the mechanisms associated with such an effect with an emphasis on the mitochondrial apoptotic pathway. The recent finding that mushroom extracts have direct cytotoxic effects supplements their known immune stimulating effects. Thus, novel anticancer agents based on new findings from mushroom extracts may soon be added to the present pool of anticancer drugs. Specifically, we propose that nanodelivery of the bioactive compounds of mushroom extracts to mitochondria will further increase their potential treatment efficacy.

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-1α protein. Down-regulation of Id-1 by antisense approach led to suppression of hypoxia-inducible factor-1α–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.