Ayami Sato

Yes is a central mediator of cell growth in malignant mesothelioma cells.

The constitutive activation of the Src family kinases (SFKs) has been established as a poor prognostic factor in malignant mesothelioma (MM), however, the family member(s) which contribute to the malignancy have not been defined. This study aimed to identify the SFK member(s) contributing to cell growth using RNA interference in various MM cell lines. Silencing of Yes but not of c-Src or Fyn in MM cells leads to cell growth suppression. This suppressive effect caused by Yes silencing mainly depends on G1 cell cycle arrest and partly the induction of apoptosis. Also, the knockout of Yes induces the inactivation of β-catenin signaling and subsequently decreases the levels of cyclin D necessary for G1-S transition in the cell cycle. In addition, Yes knockout has less effect on cell growth suppression in β-catenin-deficient H28 MM cells compared to other MM cells which express the catenin. Overall, we conclude that Yes is a central mediator for MM cell growth that is not shared with other SFKs such as c-Src.

Annatto Tocotrienol Induces a Cytotoxic Effect on Human Prostate Cancer PC3 Cells via the Simultaneous Inhibition of Src and Stat3

Prostate cancer is one of the most frequently occurring cancers and often acquires the potential of androgen-independent growth as a malignant phenotype. Androgen-independent prostate cancer has severe chemoresistance towards conventional chemotherapeutic agents, so a new treatment approach is required for curing such prostate cancer. In this context, the present study was undertaken to check if annatto tocotrienol (main component δ-tocotrienol) could suppress cell growth in human prostate cancer (PC3, androgen-independent type) cells via the inhibition of Src and Stat3. The tocotrienol showed cytotoxic effects on PC3 cells in a dose-dependent manner, and the effect depended on G1 arrest in the cell cycle and subsequent induction of apoptosis. In a cytotoxic dose, the tocotrienol suppressed cellular growth via the simultaneous inhibition of Src and Stat3. Similarly, the treatment combination of both Src and Stat3 inhibitors induced cytotoxic effects in PC3 cells in an additive manner compared to each by itself. With respect to cell cycle regulation and the induction of apoptosis, the combination treatment showed a similar effect to that of the tocotrienol treatment. These results suggest that annatto tocotrienol effectively induces cytotoxicity in androgen-independent prostate cancer cells via the suppression of Src and Stat3.

Redox-inactive Analogue of Tocotrienol as a Potential Anti-cancer Agent

Vitamins are prominent among natural or endogenous compounds that are considered to be beneficial for both prevention and therapy of various human ailments. The vitamin E group of compounds composed of tocopherol and tocotrienol isoforms, has been subsequently proven to have health benefits including antioxidant and related protective properties. However, individual isoforms exhibit a wide-range of antioxidant potencies. Tocotrienol (T3) displays powerful anticancer activity that is often not exhibited by tocopherols, by modulating multiple intracellular signaling pathways associated with tumor cell proliferation and survival. The anticancer effect of T3 remains not fully understood but generally is mediated independently of its antioxidant activity. Further we have synthesized a new redox-inactive analogue of T3, 6-O-carboxypropyl-α-tocotrienol (T3E) showing considerable promise for stronger anticancer potency than its mother compound. In this mini-review, we particularly focus upon the anticancer action of the above active components of vitamin E and describe current research on the anticancer effects of T3 irrespective of antioxidant activity.

Induction of the Connexin 32 Gene by Epigallocatechin-3-Gallate Potentiates Vinblastine-Induced Cytotoxicity in Human Renal Carcinoma Cells

Background/Aim: Enforced expression of the connexin (Cx) 32 gene, a member of the gap junction gene family and a tumor suppressor gene in human renal cell carcinoma (RCC), enhanced vinblastine (VBL)-induced cytotoxicity in RCC cells due to suppression of multidrug resistance 1 (MDR1) expression. Furthermore, in RCC the Cx32 gene is silenced by hypermethylation of CpG islands in a promoter region of the Cx gene. In this study, we investigated if the green tea polyphenol epigallocatechin-3-gallate (EGCG) could enhance susceptibility of RCC cells (Caki-1, a human metastatic RCC cell) to VBL. Methods: The effects of EGCG on Caki-1 cells were estimated by WST-1 (cell viability), real-time RT-PCR (mRNA level) and immunoblotting (protein level). We estimated the methylation status in the promoter region of the Cx32 gene in RCC cells by methylation-specific PCR. Each protein function was inhibited by small interfering RNA (siRNA) and specific inhibitors. Results: The EGCG treatment elicited significant upregulation of Cx32 in Caki-1 cells, and the induction of the Cx led to the suppression of MDR1 mRNA expression through inactivation of Src and subsequent activation of c-Jun NH2-terminal kinase (JNK). Chemical sensitivity to VBL in Caki-1 cells was increased by EGCG pretreatment, and this effect was abrogated by siRNA-mediated knockdown of Cx32. Conclusion: This study suggests that the restoration of Cx32 by EGCG pretreatment improves chemical tolerance on VBL in Caki-1 cells via the inactivation of Src and the activation of JNK.

Inhibitory effect of a redox-silent analogue of tocotrienol on hypoxia adaptation in prostate cancer cells

Prostate cancer (PCa) is one of the most common cancers in Western countries and acquires a malignant phenotype, androgen-independent growth. PCa under hypoxia often has resistance to chemotherapy and radiotherapy. However, an effective therapy against PCa under hypoxia has not yet been established. In this report, we investigated the inhibitory effect of a redox-silent analogue of tocotrienol on the survival of a human androgen-independent PCa cell line (PC3) under hypoxia. We found that the redox-silent analogue exerted a cytotoxic effect on PC3 cells in a dose-dependent manner irrespective of either hypoxia or normoxia. Moreover, under hypoxia, the analogue dose dependently reduced the protein levels of hypoxia-inducible factor (HIF)-1&agr; and HIF-2&agr;. In addition, a specific inhibitor toward HIF-1&agr; induced cytotoxicity on PC3 cells, whereas selective inhibition of HIF-2&agr; exerted no effect. Furthermore, suppression of HIFs levels by the analogue in hypoxic PC3 cells was closely associated with the inactivation of Fyn, a member of the nonreceptor tyrosine kinase family, as confirmed by the action of a specific inhibitor toward the kinase (PP2). Taken together, these results suggest that the tocotrienol analogue could inhibit the survival of PC3 cells under hypoxia, mainly by the inhibition of Fyn/HIF-1&agr; signaling, and this may lead to the establishment of a new effective therapy for androgen-independent PCa.

A Succinate Ether Derivative of Tocotrienol Enhances Dickkopf-1 Gene Expression through Epigenetic Alterations in Malignant Mesothelioma Cells

Background: Wnt signaling plays an essential role in tumor cell growth, including the development of malignant mesothelioma (MM). Epigenetic silencing of negative Wnt regulators leading to constitutive Wnt signaling has been observed in various cancers and warrants further attention. We have reported that a succinate ether derivative of α-tocotrienol (T3E) has potent cytotoxic effects in MM cells. Thus, in this study, we investigated whether the anti-MM effect of T3E could be mediated via the epigenetic alteration of the Wnt antagonist gene, Dickkopf-1 (DKK1). Methods: WST-1 and cell analyzers were employed to analyze the effects of T3E on cell viability and apoptosis of human MM cell lines (H2452, H28). Real-time PCR and Western blot were performed to evaluate the expression at mRNA and protein levels. Methylation status and epigenetic modifications of DKK1’s promoter regions after T3E treatment in MM cells were studied using methylation-specific PCR and Chromatin immunoprecipitation. Small interfering RNA-mediated knockdown (siRNA), and specific inhibitors, were used to validate DKK1 as a target of T3E. Results: T3E markedly impaired MM cell viability, increased the expression of phosphorylated-JNK and DKK1 and suppressed cyclin D, a downstream target gene of Wnt signaling. Knockdown of DKK1 expression by siRNA or a specific JNK inhibitor confirmed the contribution of DKK1 and JNK to T3E-induced cytotoxicity in MM cells. On the other hand, cytoskeleton-associated protein 4 (CKAP4) expression, which promotes cell proliferation as a Wnt-independent DKK1 receptor was inhibited by T3E. Silencing CKAP4 by siRNA did not appear to directly affect MM cell viability, thereby indicating that expression of both DKK1 and CKAP4 is required. Furthermore, T3E-mediated inhibition of both DNA methyltransferases (DNMT1, 3A, and 3B) and histone deacetylases (HDAC1, 2, 3, and 8) in MM cells leads to increased DKK1 expression, thereby promoting tumor growth inhibition. MM cells treated with Zebularine (a DNMT inhibitor) and sodium butyrate (an HDAC inhibitor) exhibited cytotoxic effects, which may explain the inhibitory action of T3E on MM cells. In addition, an enhanced expression of DKK1 in MM cells following T3E treatment is positively correlated with the methylation status of its promoter; T3E decreased DNA methylation and increased histone acetylation. Moreover, T3E specifically increased histone H3 lysine 4 (H3K4) methylation activity, whereas no effects were observed on histone H3K9 and H3K27. Conclusions: Targeting the epigenetic induction of DKK1 may lead to effective treatment of MM, and T3E has great potential to induce anti-MM activity.

Combination Effect of δ-Tocotrienol and γ-Tocopherol on Prostate Cancer Cell Growth

Tocotrienols (T3s) and tocopherols (Tocs) are both members of the vitamin E family. It is known that δ-tocotrienol (δ-T3) has displayed the most potent anti-cancer activity amongst the tocotrienols. On the other hand, γ-tocopherol (γ-Toc) is reported to have a protective effect against prostate cancer. Therefore, we investigated whether the combination of γ-Toc and δ-T3 could strengthen the inhibitory effect of δ-T3 on prostate cancer cell growth. In this study the effect of combined δ-T3 (annatto T3 oil) and γ-Toc (Tmix, γ-Toc-rich oil) therapy was assessed against human androgen-dependent prostate cancer cells (LNCaP). We found that combined treatment of δ-T3 (10 μM) and γ-Toc (5 μM) resulted in reinforced anti-prostate cancer activity. Specifically, cell cycle phase distribution analysis revealed that in addition to G1 arrest caused by the treatment with δ-T3, the combination of δ-T3 with γ-Toc induced G2/M arrest. Enhanced induction of apoptosis by the combined treatment was also observed. These findings indicate that combination of δ-T3 and γ-Toc significantly inhibits prostate cancer cell growth due to the simultaneous cell cycle arrest in the G1 phase and G2/M phase.

Abstract 2624: Possibility of prostate cancer prevention based on cancer stem cell theory

Cancer stem cells are considered to be one of the causes of cancer. In recent years, stemness (stem like) of gene expression patterns in the development process of cancer has been observed. Stemness and pathology of cancer are closely related. Elucidation of the stemness regulation of cancer is an important key to the development of new cancer prevention methods. However, as far as we know, there exist no cancer prevention studies focusing on cancer stem cells. In this study, we have investigated the possibility of prostate cancer prevention with a focus on cancer stem cells. We utilized prostate cancer cell lines PC3, DU145 (androgen-independent) and LNCaP (androgen-dependent) cells. Then, using spheroid formed cells, mRNA expression levels of cancer stem cells markers were estimated by RT-real time-PCR. Cancer cells have several malignant phenotypes under hypoxia, and we investigated for the cancer stem cells induced malignant alteration under hypoxia. In addition, under hypoxia, we investigated cell viability effect under tocotrienol-rich fraction (TRF) from annatto. TRF almost consists of delta- tocotrienol (δ-T3) that has the strongest anti-cancer activity. We observed spheroid formation capacity and compared the spheroid sizes and mRNA levels of DU145, PC3 and LNCaP cells. Size of DU145 spheroid is smaller in comparison to spheroids of PC3 and LNCaP. mRNA expression levels of cancer stem cells marker were elevated in PC3 and LNCaP cells. This means that PC3 and LNCaP cells have higher concentration of cancer stem cells. Furthermore, stemness decreases with increase of passage number, the number of cultures. We confirmed the presence of stemness in the formation capacity of spheroid until passage 4. Under hypoxia, cell viability of cancer stem cells was elevated compared to normal cultured prostate cancer cells. In addition, under hypoxia and TRF treatment, cell viability of cancer stem cells decreased with increase in TRF concentration compared to control (0 μg/ml). Our results suggest that TRF suppress cell viability of prostate cancer stem cells under hypoxic states and is effective in prostate cancer prevention. Citation Format: Nobuya Shiozawa, Ryosuke Sugahara, Ayami Sato, Masako Ota, Tomohiro Yano. Possibility of prostate cancer prevention based on cancer stem cell theory. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2624.