Fucoidan에 의한 HT-29 인간 대장암 세포 사멸 유도 기전

Abstract

Fudoidan is the collective name for algal sulfated polysaccharides extracted from the edible brown seaweeds. Fucoidan has been extensively studied because of its many biological properties including anti-coagulant, anti-viral, anti-inflammatory and anti-tumor activities. The present study examined whether fucoidan inhibits HT-29 human colon cancer cell growth and the mechanisms underlying its effects. HT-29 cells were incubated in serum-free medium with various concentrations of fucoidan (0~1,000 μg/mL). Fucoidan decreased cell growth in a concentration- and time-dependent manner. Fucoidan markedly inhibited [3H]thymidine incorporation into DNA in HT-29 cells. Following fucoidan treatment, the cells showed apoptotic bodies, typical of cells undergoing apoptosis, morphological changes, and H33342 staining. Fluorescence-activate cell sorting analysis results revealed that the apoptotic cell percentage was increased after treatment with fucoidan. Western blot analysis revealed that fucoidan decreased levels of pro-caspase proteins, such as caspase-9, -8, -7 and -3. Furthermore, bcl-2 and bcl-xL protein levels were decreased, but bax, t-bid, and Fas protein levels were increased by fucoidan treatment. These results indicate that fucoidan induced apoptosis through both the extrinsic and intrinsic pathways. Fucoidan also induced sub-Gl phase arrest in the cell cycle, and Gl cell cycle protein expression levels were decreased by down-regulating retinoblastoma protein (pRB) and E2F. Nuclear factor kappa B (NF-κB) regulates cell growth, differentiation, immune responses, and inflammatory responses. The present study examined the effects of fucoidan on the NF-κB signaling pathway. Western blot analysis of cytosolic fractions revealed that fucoidan decreased IKK, IκB and phosphorylated IκB protein levels. NF-κB protein in the cytosol and the nuclear fraction was also decreased by fucoidan treatment. These results indicate that fucoidan induced apoptosis involved decreased NF-κB expression levels in the cytosolic and nuclear fractions. NF-κB protein levels were decreased as a result of down-regulating the phosphorylation of p38, c-jun N-terminal kinases (JNK), i nitric oxide synthesis (iNOS) and cyclooxygenase-2 (COX-2). Insulin-like growth factor (IGF-I) regulates the growth of colon cancer cells by an autocrine mechanism. The present study examined the effects of fucoidan on the IGF-I receptor (IGF-IR) signaling pathway. Western blot analysis of total cell lysates revealed that fucoidan decreased IGF-IR, phospho-tyrosine, insulin receptor substrate (IRS)-1, AKT and extracellular-signal regulated kinase (ERK) protein levels in a dose-dependent manner. Immunoprecipitation/ Western blot studies showed that fucoidan decreaed IGF-I-induced phosphorylation of IGF-IR. However, the expression of IGF-I-induced phosphorylation of AKT and ERK were inceresed by fucoidan treatment. These results suggest that fucoidan inhibited cell proliferation and stimulated apoptosis of HT-29 cells via inhibition of IGF-IR signaling pathway, partly. Over-expression of ErbB2 and ErbB3 genes is a frequent event in several human cancers. The present study also determined whether the growth inhibitory effect of fucoidan was related to changes in ErbB protein levels and the ErbB receptor signaling pathway in HT-29 cells. Fucoidan decreased ErbB2 and ErbB3 and phospho-tyrosine protein levels. Immunoprecipitation/Western blot studies revealed that fucoidan were decreased heregulin (HRG)-induced phosphorylation of ErbB3 but, increased phosphorylation of AKT and ERK. These results indicate that down-regulation of ErbB2/ErbB3 signaling may be a mechanism by which fucoidan inhibits HT-29 cell growth. Thus, fucoidan induced apoptosis, markedly decreasing DNA synthesis and cell growth through sub-G1 cell cycle arrest, the extrinsic and intrinsic pathways, and the NF-κB signaling pathway, involves down-regulation of IGF-IR signaling and the ErbB2/ErbB3 signaling pathway, partly.