Study On The Apoptosis Induced In Human Colon Cancer Cells By SFPS

Background Sargassum fusiforme (Harv.) Setchess are algae of temperate zone, which are mainly rich in the eastern part of north pacific ocean. The algae have been known since ancient times and played a pivotal role in health improvement and pharmaceutic application. Sargassum fusiforme (Harv.) Setchess and the extracts have been confirmed to possess anti-tumor, immunity enhancing, anti-lipidperoxidation effects. Recently, with the increased occurrence of colon cancer and the decreased curability in our country, the prevention and treatment has played an all-important role in public health. And in order to explore the therapeutic agents and provide a great opportunity for high curability, the low toxicity and anti-tumor effects of Chinese medicine have been a hotspot in research of tumor therapy.Recent reports have demonstrated the extracts from Sargassum fusiforme (Harv.) Setchess have been suggested to have cytotoxic effect or apoptosis-inducing activity in tumor cell lines.SFPS, which contain alginate, fucoidan (FCD) and laminaran, are the polysaccharides extracted from Sargassum fusiforme. As main component of water extraction from Sargassum fusiforme range from 3500 to 10000, which can pass the cell membrane freely. As polysaccharides from marine brown algae, SFPS have a wide spectrum of activity in biological systems. Besides their well-attested anticoagulant and antithrombotic activity, they act on the inflammation and immune systems, have antitumor and anti-lipid-peroxidation effect, protect cells from viral infection, and so on.But these studies were limited on the animals to explore the effects to immunesystems, the free radicals removed and anti-lipid-peroxidation effects. And the reseach related to the apoptosis induced by SFPS and mechanism in human colon cancer cells (RKO and lovo cells ) in vitro has not been reported yet. The study may be helpful to search for natural potential antitumor drugs and provide a great opportunity to facilitate the development of Chinese medicine and health.Objective In this study, we chose human colorectal cancer cells, lovo and RKO, as objects to explore the antitumor effects and mechanism of SFPS and investigate the apoptosis of lovo cells and RKO cells induced by SFPS.Methods Inhibition of proliferation was measured by MTT assay. DNA fragmentation of colon cancer cells treated with SFPS was examined by DNA electrophoresis. Morphological alterations of colon cancer cells treated with SFPS were observed by electron microscopy. Analysis of apoptotic cells and cell cycle distribution by flow cytometry (Annexin V/PI or PI). Caspase-3 mRNA measurement by RT-PCR. Caspase-3, 8, 9 were examined by western blot and caspase activity kit.Results The potential of SFPS in inhibiting lovo and RKO cells viability was assessed by MTT assay. SFPS exhibited significant antiproliferative activity which depended on dosage. After incubation with 300 mg/L SFPS for 24 hours, both of the two cells showed a ladder-like pattern of DNA fragmentation on agarose gel electrophoresis, and the fragments were integral of 180-200 bp. When the concentration was increased to 1000 mg/1, DNA ladder disappeared entirely and both of the two cells lines showed smear. Morphological examination by electron microscopy showed lovo and RKO cells with chromatin condensation and margination, cell shrinkage and swollen mitochondria. And then the nucleus curled up, the chromatin fragmented and accumulated into denser blocks to form crescent-shaped chromatin associated with the nuclear membrane, though the plasma membrane remained well defined. The apoptotic ratio of colon cancer cells treated with SFPS and the overall effect of SFPS on the cell cycle distribution was examined by using flow cytometry. It suggests that the apoptotic ratio was concentration-and time- dependent, which is to a certain extent consistent with the inhibition of colon cancer cells. However, we also found that SFPS could arrest the human colon cancer cell line RKO at Go/Gi phase, and the RKO cells at S phase decreased significantly, whilethere shows no observed changes of cell cycle distribution in SFPS treated lovo cells. It was demonstrated by western blot analysis and caspase activity assay that SFPS induced differential activation of caspases in lovo and RKO cells, of which caspase-3 and -9 were mainly activated in lovo cells, while caspase-3, -8 and -9 were activated significantly in RKO cells.Conclusions (1) SFPS is an effective anti-tumor medicine and the antitumor effect of SFPS is relative to cell apoptosis. The antitumor effect of SFPS on RKO cells is relative to the blocking of Go/Gi period cells, while it is not relative to cell cycle when inducing lovo cells. (2) The antitumour effect of SFPS is dosage- and time-dependent. (3) The suppressive effects of SFPS on lovo cells in vitro may be through the caspase-3-dependent apoptotic cell death, which involve the initiator caspases (caspase-9) activation. (4) SFPS induces caspase-3-dependent apoptotic cell death, which involve the caspase-8, -9 activation in RKO cells. And the suppression of antiproliferation on RKO cells by SFPS appears to be caspase cascade activation supported by Go/Gi arrest.