Anti-inflammatory and anti-tumor activity of fucoxanthin from Ishige okamurae through MAPKs regulation

Abstract

Fucoxanthin has been reported to induce apoptosis in prostate cancer PC-3, DU145, LNCaP cells (Kotake-Nara et al., 2001), and leukemia (Kotake-Nara et al., 2005). In addition, fucoxanthin has been shown to cause cell cycle arrest in neuroblastoma cells (Okuzumi et al., 1990) and human hepatoma HepG2 cells (Das et al., 2008). However, there is not information available concerning the ability of fucoxanthin to inhibit B16F10 melanoma cells. Here, for the first time demonstrates that the mechanism of fucoxanthin-induced growth inhibition of B16F10 cells is due to a G0/G1 cell cycle arrest and apoptosis via downregulation of Akt and Bcl-xL. Futhermore, this author also fist showed that fucoxanthin suppressed in vivo growth of B16F10 melanoma in Balb/c mice. In the present study first examined the antiproliferative effect of fucoxanthin B16F10 melnoma cells at concentrations ranging from 6 to 100 μM for 72 h. Fucoxanthin significantly decreased the proliferation of B16F10 cells in a dose-dependent manner. More importantly, the fucoxanthin also exhibits effective anticancer properties in Balb/c mice. This was consistent with previous reports that fucoxanthin inhibits the growth of human prostate, leukemia, Neuroblastoma, and hepatoma cells (Das et al., 2008; Kotake-Nara et al., 2001; Kotake-Nara et al., 2005). Suppression of growth by fucoxanthin can be partially explained by the occurrence of arrest during the cell cycle. Cell cycle analysis using flow cytometry revealed that treatment with 50 μM fucoxanthin induced G0/G1 phase arrest of the cell cycle at 24 h. However, these concentrations did not induced similar level of cell death as evidenced by sub-G1 cells. These imply that the G0/G1 phase arrest of the cell cycle can be considered as one of the pathways by which the growth of B16F10 cells is inhibited. On the other hand, 100 and 200 μM of fucoxanthin caused the cell cycle arrest at 24 h and induced the apoptosis 48 h after treatment. Thus, this compound seems likely to cause cell cycle arrest at the low concentration (50 μM) and apoptosis at the high concentrations (100 and 200 μM) in B16F10 cells. pRb is the master switch regulating cell cycle progression, and its continual phosphorylation parallels cell transition through G1 and S (Weinberg, 1995). The overwhelming majority of invasive and metastatic melanoma specimens and cell lines express normal Rb protein (Albino et al., 2000). In the hypophosphorylated state, Rb family proteins associate with and inhibit the activity of E2F family transcription factors, which are involved in the transcription of key cell cycle regulators. Upon growth stimulation, the G1- specific CDKs/cyclins phosphorylate Rb proteins on multiple residues, resulting in the release of E2F family transcription factors (Roy et al., 2007). In the present study showed that hinokitiol treatment causes a dose-dependent decrease in the level of total Rb protein and inhibits Rb phosphorylation. Many studies have showed that the cyclins and CDKs control the G1/S transition in the cell cycle. In addition, the regulation for the cyclins and CDKs activity has turned out to be the most productive strategy for the discovery and design of novel anticancer agents targeting the cell cycle progression (Wu et al., 2006). Weinstein (2000) also reported that a family of CDK inhibitors plays a major role in the cell cycle regulation. In contrast, activation of CDKs at G1 phase are negatively regulated by two families of CDK inhibitors (CKIs): the kinase inhibitor protein (KIP) family including p21^(WAF1/Cip1), p27^(Kip1) and p57^(Kip2), and the inhibitors of CDK4 (INK4) family including p15^(INK4B), p16^(INK4A), p18^(INK4C), and p19^(INK4D) acting to inhibit CDK4 and CDK6 (Pei and Xiong, 2005). The cytostatic mechanism of fucoxanthin in B16F10 cells appeared to be related to the induction of cell cycle arrest at G1 phase. This arrest is associated with a negative control of the cell cycle machinery that inhibits G1-S transition. This results also indicate that fucoxanthin caused down-regulation of cyclin D1 and cyclin D2 expression, which was well correlated with decrease in expression levels of CDK 4. Concomitantly, the expression levels of p15^(INK4B) and p27^(Kip1) were upregulated in cells exposed to fucoxanthin. Elevation of these CDK inhibitors is suggested to act as negative regulators of G1 cell cycle progression by inhibiting the CDK activation. The Akt signal pathway plays critical roles in regulating cell survival and death in many physiological and pathological settings. Akt is involved in cell cycle regulation by preventing GSK-3β mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27^(Kip) (15) and p21^(WAF1/Cip1) (16). The present study sought to determine effects of Akt on fucoxanthin-induced cell arrest. This results demonstrated that fucoxanthin downregulates the Akt signaling pathway, and that the Akt pathway inhibition significantly increased fucoxanthin-induced cell arrest. These data strongly suggest that fucxoanthin-induced cell arrest is associated with the Akt pathway Apoptosis is an important way to maintain cellular homeostasis between cell division and cell death (Green and Reed, 1998; Hengartner, 2000; Kaufmann and Hengartner, 2001). Apoptosis is a cellular suicide or a programmed cell death that is mediated by the activation of an evolutionary conserved intracellular pathway (Bold, 1997). So, induction of apoptosis in cancer cells is one of the useful strategies for the development of anticancer drug (Hu and Kavanagh, 2003). Apoptosis is a tightly regulated process, which involves changes in the expression of distinct genes. Members of the Bcl-2 family (such as Bcl-2 and Bcl-xL) of proteins are critical regulators of the apoptotic pathway (Korsmeyer, 1999). Bcl-2 and BclxL is an upstream molecule in the apoptotic pathway and are identified as potent suppressors of apoptosis (Hockenbery, 1993). This data clearly demonstrated that fucoxanthin treatment to B16F10 cells resulted in a concentration dependent decrease in Bcl-xL levels. In addition, caspase activation is often regulated by various cellular proteins including members of the inhibitor of apoptosis (IAP; Deveraux and Reed 1999) or Bcl-2 families (Adams and Cory 1998; Antonsson and Martinou 2000). This data reveal that the protein expression of c-IAP-1, c-IAP-2, and XIAP proteins was decreased by fucoxanthin treatment. The cleavage of caspase-3 and -9 appears to be correlated with fucoxanthin-induced apoptosis in B16F10 cells. Caspase-3 and -9 are key components in the mitochondrial initiated pathway (Budihardjo et al., 1999). Once caspase is activated, a variety of cellular proteins are targeted, leading ultimately to apoptosis. PARP is the best known substrate of caspase and cleaved from 116 kDa intact form into 85 kDa fragment (Konopleva et al., 1999). This is important for cells to maintain their viaility; cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis (Oliver et al., 1998). The intact form of 116 kDa and cleaved form of 85 kDa of PARP were detected in fucoxanthine treated B16F10 cells. Hence, these data fist demonstrate that fucoxanthin induce apoptosis can be occurred through Bcl-xL and IAPs regulation. In conclusion, fucoxanthin had antiproliferative effects by inducing apoptosis and cell cycle arrest in melanoma B16F10 cells. Fucoxanthin increased the proportion of cells in the G1 phase of the cell cycle, which is associated with a decrease in Akt, cyclinD1, D2, and CDK4 expression and the induction of p15 and p27. Fucoxanthin-induced apoptosis might be related to caspase-3 and -9 activation and the down-regulation of Bcl-xL and IAPs expression (Fig. 4-11). Taken together, the data presented in this in vitro and in vivo study provide important insights into this cell-cycle-based therapeutic strategy and form a strong basis for the development of fucoxanthin as an anticancer agent.

푸코잔틴은 카로티노이드 색소류 잔토필의 일종으로 갈조식물과 황색식물에만 함유되어 있는 물질이다. 푸코잔틴은 항암과 항산화 및 항비만 등 다양한 생리활성이 있다고 알려져 있으나, 항염증과 항암활성의 메커니즘에 대한 연구는 미흡한 실정이다. 이 연구에서는 패(갈조류)로부터 푸코잔틴을 분리하고, 분리된 푸코잔틴이 항염증활성과 HL-60(백혈병 세포)과 B16F10(피부암 세포) 세포에 대한 항암활성을 측정하였고, 그 작용 메커니즘을 조사하였다. 1. 푸코잔틴은 LPS로 자극된 RAW 264.7 세포에서 염증성 매개 인자의 생성에 미치는 영향을 조사 한 결과, 푸코잔틴은 염증성 매개인자인 TNF-α, IL-1β, IL-6와 NO의 생성을 농도의존적으로 억제함을 알 수 있었다. 항염증 활성의 작용기전 규명을 위해, LPS로 자극된 NF-κB와 MAPK 활성화에 미치는 영향을 조사한 결과는 NF-κB의 전사활성 억제와 MAPK 인산화를 억제함을 확인 되었다. 이러한 결과는 푸코잔틴이 NF-κB와 MAPK 조절함으로써 항염증활성을 나타내는 것으로 생각된다. 2. 푸코잔틴의 HL-60 세포에 대한 항암활성은 세포성장을 억제시키고 apoptosis로 세포사멸을 일으켰다. 세포사멸의 작용기전은 푸코잔틴을 처리 하였을 때 MAPK 활성화를 조사한 결과, p38과 JNK가 활성화를 보였다. 그리고, 암세포 사멸에 영향을 미치는 ROS의 발생은 푸코잔틴을 처리하였을 때 ROS가 발생하는 것을 확인하였다. 이 ROS 발생이 apoptosis와 MAPK에 미치는 영향은 항산화제인 NAC을 푸코잔티과 같이 처리하였을 때 apoptosis 유도가 억제되고 MAPK이 활성이 억제되는 것을 확인하였다. 이러한 결과는 ROS발생에 의해 MAPK가 조절되면서 HL-60 세포를 apoptosis로 유도하여 항암활성이 나타나는 것으로 생각된다. 3. 푸코잔틴의 B16F10 세포에 대한 항암활성은 푸코잔틴을 처리하였을 때 B16F10 세포의 증식이 유의적으로 감소하였다. 세포 증식은 cell arrest 또는 apoptosis에 의해 억제되는데, 푸코잔틴을 처리하였을 때 G0/G1기 arrest와 apoptosis가 유도 되는 것이 cell cycle과 형태적 관찰을 통해 확인되었다. G0/G1기 arrest 관련 단백질 측정으로 확인된 arrest 작용 기전은 푸코잔틴을 처리 하였을 때 Akt 활성화가 억제되고 p15와 p27 단백질이 증가 되었다. 또한 apoptosis유도 기전으로 Bcl-xL 단백질이 감소 함으로써 apoptosis가 유도 되는 것을 확인 할 수 있었다. 이와 같은 결과로 볼 때 Akt와 Bcl-xL 조절에 의해 B16F10 세포의 cell arrest와 apoptosis가 유도 됨으로써 항암활성을 나타내는 것으로 생각된다. 이 모든 결과를 종합해 볼 때, 푸코잔틴이 항염과 항암활성에 의해 산업적 용도가 매우 다양할 것으로 생각되며 특히 식품산업으로서의 이용 가능성을 높일 수 있을 것이라 판단된다.