- Top of page
- Materials and Methods
This article has been retracted at the request of Editor-in-Chief & Author.
The following article from Cancer Science: Tetsuro Nakazato, Taeko Okudaira, Chie Ishikawa, Shinji Nakama, Shigeki Sawada, Mariko Tomita, Jun-nosuke Uchihara, Naoya Taira, Masato Masuda, Yuetsu Tanaka, Kazuiku Ohshiro, Nobuyuki Takasu, Naoki Mori. Anti-adult T-cell leukemia effects of a novel synthetic retinoid, Am80 (Tamibarotene), Cancer Science 2008; 99: 2286–2294, (doi: 10.1111/j.1349-7006.2008.00917.x), published online on 1 September 2008 on Wiley Interscience (http://www.interscience.wiley.com), now Wiley Online Library (http://onlinelibrary.wiley.com/), has been retracted by agreement between the authors, the journal Editor-in-Chief, Yusuke Nakamura, and Blackwell Publishing Asia Pty Ltd. All authors wish to retract this paper due to inappropriate image utilization in three of the figures within the article.
Yusuke Nakamura Editor-in-Chief Cancer Science
The Akt signaling pathway is important for survival and growth of cancer cells. In the present paper we show that the Akt signaling pathway is constitutively activated in human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines and in primary adult T-cell leukemia (ATL) cells. Curcumin, a natural compound present in turmeric, has been studied vigorously as a potent chemopreventive agent for cancer therapy because of its inhibitory effect on proliferation and induction of apoptosis in several tumor cell lines. We investigated the effect of curcumin on Akt activity in HTLV-I-infected T-cell lines and primary ATL cells. Phosphorylated PDK1 is an activator of Akt by phosphorylating Akt. Curcumin reduced phosphorylation of PDK1 and inhibited constitutive activation of Akt. Curcumin activated glycogen synthase kinase (GSK)-3β, a downstream target of Akt kinase, by inhibiting phosphorylation of this protein. Curcumin reduced the expression of cell cycle regulators, cyclin D1 and c-Myc proteins, which are both degraded by activated GSK-3β. Our results suggest that activation of the Akt signaling pathway plays an important role in ATL cell survival, and that curcumin may have anti-ATL properties mediated, at least in part, by inhibiting Akt activity. We propose that Akt-targeting agents could be useful for the treatment of ATL. In this regard, curcumin is a potentially promising compound for the treatment of ATL. (Cancer Sci 2006; 97: 322 – 327)
Human T-cell leukemia virus type I (HTLV-I) is the causative agent of adult T-cell leukemia (ATL), a malignant condition of mature CD4+ T-cells.(1–3) HTLV-I is also linked to the development of several chronic inflammatory diseases such as HTLV-I-associated myelopathy/tropical spastic paraparesis.(4,5) Presently, there is no accepted curative therapy for ATL and patients often progress to death, with a median survival time of 13 months for those with aggressive ATL.(6) Conventional therapies do not appear to prolong the life of patients with ATL and hence the establishment of new therapeutic strategies for ATL is necessary.
Akt is a serine/threonine protein kinase that functions as a critical regulator of cell survival and proliferation. A variety of growth factors and other extracellular stimuli can activate phosphatidylinositol 3-kinase (PI3K) through activation of their cognate receptors. The activated PI3K converts the plasma membrane lipid second messenger phosphatidylinositol-4,5-bisphosphate to phosphatidylinositlo-3,4,5-triphosphate (PIP3). Subsequently, PIP3 recruits downstream molecules, particularly Akt and phosphoinositide-dependent kinase (PDK)-1, via binding to their pleckstrinhomology domains.(7) At the membrane, Akt is activated through phosphorylation by PDK1.(8,9) Activated Akt in turn regulates a wide range of target proteins, such as the FOXO transcription factors, Bad, caspase-9, glycogen synthase kinase (GSK)-3β and the tuberin/hamartin complex, which regulate cell survival, proliferation and growth.
The Akt signaling pathway is not regulated in numerous tumors.(10) Manipulation of Akt activity resulted in altering the response of tumor cells to chemotherapy and irradiation.(11,12) Previous experiments showed that activation of PI3K-Akt signaling is involved in fibroblast Rat-1 transformation by the HTLV-I Tax protein.(13) These findings suggest that the Akt signaling pathway may be related to the cell transformation of HTLV-I-infected T-cells, and inhibitors of this pathway may be effective in the treatment of ATL.
Selective inhibitors of the Akt signaling pathway are not only potent tools for investigating the biological roles of this important signaling pathway,(14) but also potential therapeutic candidates for the treatment of Akt signaling-dependent tumors.(11,12) Although there is tremendous interest in the regulation of the Akt signaling pathway for the benefit of killing cancer cells,(15,16) not many inhibitors of this pathway have been identified since the first synthetic PI3K inhibitor LY294002 was reported.(14)
Curcumin (diferuloylmethane), the major yellow pigment in turmeric, which is commonly used as a flavoring and coloring agent in foods, drinks and cosmetics, is obtained from rhizomes of the plant Curcuma longa Linn. Several recent studies have shown that curcumin has anti-inflammatory, antioxidant and anticarcinogenic properties.(17,18) Curcumin suppresses tumor initiation and promotion in animal models and is a potent inhibitor of key molecules in oncogenic signaling pathways, including cycloxygenase-2,(19) lipoxygenase, ornithine decarboxylase,(20,21) c-Jun/AP-1,(22) NF-κB,(23) c-Jun N-terminal kinase(24) and protein kinase C.(25) Recently, we demonstrated that curcumin suppresses cell growth and survival of HTLV-I-infected T-cell lines and primary ATL cells by inhibiting the NF-κB and AP-1 signaling pathways.(26,27) However, little is known about the effect of curcumin on the Akt signaling pathway.
In the present study, we hypothesized that Akt signaling is necessary for malignant cell growth and survival of ATL cells, that curcumin could be effective against ATL, and that the anti-ATL effect of curcumin is mediated through the inhibition of Akt activity. To test our hypothesis, we evaluated Akt activity in HTLV-I-infected T-cell lines and primary ATL cells, and examined the effects of curcumin on Akt activity of these cells.
- Top of page
- Materials and Methods
The major findings of our study are: (i) the Akt signaling pathway is constitutively activated in HTLV-I-infected T-cell lines and primary ATL cells, but not in HTLV-I-negative PBMCs from healthy volunteers. (ii) Inhibition of the Akt signaling pathway by LY294002 leads to suppression of cell growth of HTLV-I-infected T-cell lines, but not that of HTLV-I-negative PBMCs from a healthy volunteer. (iii) Curcumin reduces phosphorylation of PDK1 and inhibits activation of the Akt signaling pathway. Because curcumin suppresses cell growth of HTLV-I-infected T-cell lines and primary ATL cells,(26,27) curcumin may suppress cell growth, at least in part, by inhibiting the Akt signaling pathway. Our results indicate that the constitutive active Akt signaling pathway may play a critical role in ATL cell survival.
We showed that Akt was constitutively activated in HTLV-I-infected T-cell lines and primary ATL cells. Several reports have suggested that Akt can be activated in a PI3K-independent manner. However, Akt activation is PI3K-dependent in HTLV-I-infected T-cell lines, because phosphorylation of Akt at Ser473 is sharply reduced in cells treated with the PI3K inhibitor LY294002 (Fig. 2a). These results suggest that HTLV-I-infection can induce constitutive Akt activation through the PI3K signaling pathway. PDK1 was also constitutively phosphorylated in HTLV-I-infected T-cell lines. Activation of PI3K leads to colocalization of Akt with PDK1 at the plasma membrane and PDK1 can phosphorylate Akt at Thr308. Although phosphorylation at Thr308 partially activates Akt,(36) full activation of Akt requires phosphorylation on Ser473. However, the mechanism mediating Ser473 phosphorylation remains controversial. Because Ser473 phosphorylation is dependent on PI3K, in addition to Thr308, PDK1 is assumed to be the kinase for Ser473 phosphorylation.(37) Therefore, constitutively phosphorylated PDK1 may enhance Akt kinase activity in HTLV-I-infected T-cell lines. Blocking Akt activity by LY294002 induced cell growth arrest in HTLV-I-infected T-cell lines (Fig. 2b), suggests that constitutive activated Akt plays a role in growth and survival signaling of HTLV-I-infected T-cells.
Previous studies showed that HTLV-I transforming protein Tax induces PI3K signaling pathway activation and is associated with cell transformation of fibroblast cell line stably expressing the Tax protein.(13) During preparation of this article, Jeong et al. showed that the Akt signaling pathway is activated in HTLV-I-transformed cells, and Tax can activate this signaling pathway by inducing Akt phosphorylation.(38) These observations indicate that the Tax protein plays a critical role in constitutive activation of Akt in HTLV-I-infected T-cell lines. Although we confirmed the activation of Akt in HTLV-I-infected T-cell lines, phosphorylation of Akt was also observed in some primary ATL cells (Fig. 3c), in which expression of the Tax protein was not detected (data not shown). These results suggest that Tax-independent mechanisms that can activate the Akt signaling pathway may exist. We are currently investigating the mechanisms of Tax-independent Akt activation in ATL cells.
It has been shown that curcumin and its derivatives inhibit Akt in human renal and prostate cancer cell lines.(39–41) Consistent with these observations, curcumin suppressed Akt activity of HTLV-I-infected T-cell lines and primary ATL cells (Fig. 3a,c). Curcumin also reduced constitutive phosphorylation of PDK1, suggesting that curcumin inhibits Akt activity by reducing phosphorylation of PDK1.
Although components of the PI3K-Akt signaling pathway present promising targets for therapeutic intervention, non-specific drug toxicity is one of the major problems in anticancer drug development. Because the PI3K-Akt pathway is involved in the survival, growth and proliferation of normal cells, a reasonable therapeutic index would depend on tumors being more sensitive to inhibitors of this pathway than normal tissues. Curcumin hardly inhibited the survival of normal PBMCs and systemic treatment of curcumin caused no obvious toxicity in mice.(26) Moreover, phase I clinical trials indicate that curcumin is tolerated in extremely large oral doses without apparent toxicity in humans.(42) These results indicate that curcumin is a pharmacologically safe agent. Therefore, curcumin is a potentially useful anti-ATL drug.
Multiple oncogenic signals such as NF-κB,(43) AP-1,(44) Jak-Stat(45) and Akt pathways may be involved in leukemogenesis of ATL. Curcumin has been reported to regulate these signaling pathways.(22,23,39–41,46,47) Recently, we demonstrated that curcumin suppresses cell growth and survival of HTLV-I-infected T-cell lines and primary ATL cells by inhibiting the NF-κB and AP-1 signaling pathways.(26,27) Because curcumin inhibits multiple signaling pathways which are associated with leukemogenesis of ATL, we propose that curcumin is a potentially promising compound for the treatment of ATL.
In summary, we demonstrated that Akt signaling is constitutively activated in HTLV-I-infected T-cell lines and primary ATL cells, and activation of Akt is linked to cell growth of HTLV-I-infected T-cell lines. Curcumin, which is known to suppress cell growth and survival of HTLV-I-infected T-cell lines and primary ATL cells, inhibited activation of the Akt pathway and reduced the expression of cyclin D1 and c-Myc proteins, downstream targets of the Akt signaling pathway. Our results highlight the importance of the PI3K-Akt signaling pathway as a new target for the development of therapeutic strategies against ATL. We propose that curcumin is a potentially promising compound for the treatment of ATL.