Anti-tumor Activity Of Fucoxanthin, An Active Ingredient Of Kelp Extract, Against Human NSCLC And Its Mechanism

BackgroundLung cancer is the most common cause of cancer deaths around the world. The incidence continues to rise at a rate of approximately 0.5% per year. Despite recent progress in the diagnosis and the multimodality treatments, prognosis for lung cancer, still remains unsatisfactory with the overall five-year survival rate only at 8 to 15 percent. Non-small cell lung cancer (NSCLC) accounts for 75 to 80 percent of all lung cancers. At present, the remission rate of the most effective chemotherapy regimen is only about 30% in advanced NSCLC. It is therefore, urgent to improve the therapeutic effect, to research and to discover a new effective drug in lung cancer therapy.Kelp has been a common component of Chinese traditional medicine in anti-cancer prescription for more than two thousand years. Fucoxanthin, a major carotenoid in Kelp, has been reported to demonstrate anti-carcinogenesis effect in various cancer cells, but the anti-cancer mode of action on NSCLC is still unknown. There have recently been several reports that fucoxanthin induced cell cycle arrest and apoptosis in several cancer cell lines, but the mechanism is still unclear.In this research, I have investigated the mechanism by which fucoxanthin elicits the in vitro and in vivo anti-cancer effects in NSCLC in culture system and human A549 lung cancer cell transplanted model, by means of various techniques, including trypan bule exclusion, cell counting kit-8, flow cytometry, Real time quantitative PCR, western blot, colorimetric method, HE staining, immunohistochemistry and TUNEL staining.Methods1. Cancer cells and normal cell:chronic myelogenous leukemia cells (K562), human acute promyelocytic leukemia cells (HL-60), human breast adenocarcinoma cells (MCF-7), human hepatoma cells (HepG2), human cervix tumor cells (Hela), human T cell leukemia cancer line (Jurkat), human histiocytic lymphoma cells (U937) and normal hepatic cell (L-02) were cultured. Cell viability was determined by a dye exclusion test using Trypan blue after tumor cells were treated with media containing different concentrations of fucoxanthin or vehicle following 24 h. In K562, U937 and Jurkat, cells were treated for 24, 48 and 72 h.2. The effect of fucoxanthin on the cell cycle in K562, U937 and Jurkat cells was investigated by flow cytometry in the PI-stained cells at 48 h after different concentrations of fucoxanthin treated for 48 h.3. The apoptosis rate of Jurkat, U937 and K562 cells were determined by Annexin V-FITC and PI staining flow cytometry after these cells were treated with different concentrations of fucoxanthin for 48 h.4. Four NSCLC cell lines:human lung adenocarcinoma A549 cells, human lung adenocarcinoma SPC-A1 cells, human lung large cells carcinoma NCI-H460 cells, p53-null lung adenocarcinoma H1299 cells, were cultured. Cell viability was determined by cell counting kit-8 after tumor cells were treated with media containing different concentrations of fucoxanthin for 24,48 and 72 h.5. The effect of fucoxanthin on the cell cycle distribution of A549 and H1299 cells was investigated by flow cytometry in the PI-stained cells at 48 h after different concentrations of fucoxanthin treated for 48 h.6. The apoptosis rate of A549 and H1299 cells were detected by Annexin V-FITC and PI staining flow cytometry after the cells were treated with different concentrations of fucoxanthin for 48 h.7. The mRNA expressions of p53, p21WAF1/CIP1, PUMA, Bcl-2 and Fas in A549 and H1299 cells were detected by real-time quantitative PCR after the cells were treated with different concentrations of fucoxanthin for 48 h.8. The expressions of p53, p-p53, p21 WAF1/CIP1, PUMA, Bcl-2 and Fas proteins in A549 and H1299 cells were detected by western blotting after the cells were treated with different concentrations of fucoxanthin for 48 h.9. The activity of Caspase-3 and Caspas-8 were detected by colorimetric method after A549 and H1299 cells were treated with different concentrations of fucoxanthin for 48 h.10. Different dosages (5 mg/kg,15 mg/kg and 50 mg/kg) of fucoxanthin were given to mice and they were investigated for 14 days to study the toxicity effects.11. Human A549 tumor models were established in 4-6 weeks old athymic BABL/c nude mice (SPF grade). Athymic nude mice were inoculated with A549 cells/0.2ml (1.0×107) s.c. After treatment for 5 days, the mice were randomly assigned into each group (n= 8 mice/group). Treatments were given i.g. with blank soybean oil, fucoxanthin at different doses of 5 to 50 mg/kg for 35 days, or i.p. with cisplatin at 1 mg/kg for 10 days. Tumor size was determined by caliper measurement of the largest and perpendicular diameters 3 times a week. Tumor volume was calculated according to the formula V=π×ab2/6. After 40 days, the nude mice were put to death and the tumor weight was determined to calculate the rate of inhibitory Effects of fucoxanthin.12. The tumor tissues were stained with HE to study the pathological changes after treatment of different doses of fucoxanthin.13. The protein expressions of P53, Bcl-2 and PUMA in tumor tissues were determined by immunohistochemistry after treatment of different doses of fucoxanthin.14. TUNEL method was used to investigate the degree of apoptosis tumor tissue after fucoxanthin treatment.Results1. In HL-60, MCF-7, HepG2 and Hela cells treated with fucoxanthin for 24 h, viable cell numbers were decreased in a concentration-dependent manner. The sensitivity to fucoxanthin was tumor cell specific, hematological system tumor cells were more sensitive to fucoxanthin than solid tumor cells. The anti-proliferative activity of fucoxanthin against tumor cell lines was much higher than that of normal cells.The inhibitory efect of fucoxanthin in K562, Jurkat and U937 cells was concentration-and time-dependent. 2. Fucoxanthin caused a significant accumulation of cells in G0/G1 phase compared with vehicle-treated cells. In K562 cells, the percentage of G0/G1 phase increased from 34.4±4.34% (control) to 38.0±1.41% (Fuco-6.25),39.4±1.79%(Fuco-12.5),41.4±3.79% (Fuco-25) and 50.5±7.86% (Fuco-50). In U937cells, the percentage of G0/G1 phase increased from 30.2±7.39% (control) to 39.6±4.14% (Fuco-6.25),42.4±0.76% (Fuco-12.5),47.2±3.00% (Fuco-25) and 49.8±6.14% (Fuco-50). In Jurkat cells, the percentage of G0/G1 phase increased from 43.4±5.34% (control) to 51.3±4.53% (Fuco-6.25),52.3±5.1% (Fuco-12.5),56.1±3.5% (Fuco-25) and 58.4±4.49%(Fuco-50).3. Fucoxanthin caused a significant induction of apoptosis in K562, U937 and Jurkat cells. In K562 cells, apoptosis rate increased from 4.73±2.60% (control) to 18.6±10.3% (Fuco-6.25),24.0±12.3% (Fuco-12.5) and 30.8±9.59%(Fuco-25, P<0.05 vs control). In U937 cells, apoptosis rate increased from 4.13±1.36% (control) to 14.3±0.60% (Fuco-6.25, P<0.01 vs control),25.0±13.6%(Fuco-12.5,P<0.01 vs control) and 65.3±12.3% (Fuco-25, P<0.01 vs control). In Jurkat cells, apoptosis rate increased from 2.43±0.21% (control) to 13.1±4.35% (Fuco-6.25, P<0.05 vs control),28.7±15.2%(Fuco-12.5μM, P<0.01 vs control) and 88.4±0.69% (Fuco-25, P<0.01 vs control).4. The significant inhibitory effect was observed in the four NSCLC cell lines (A549, H460, SPC-A1 and H1299 cells), and exited a concentration-and time-dependent manner. A549, H460 and SPC-A1 cells (wild-type p53) were more sensitive to fucoxanthin than H1299 (p53-null).5. After treated with fucoxanthin of 12.5,25 and 50μm for 48 h, the number of A549 and H1299 increased gradually in the stage of G0/G1, presenting an arrest in the stage of G0/G1. In A549 cells, the percentage of G0/G1 phase increased from 45.7±5.33% (control) to 61.0±4.46% (Fuco-12.5, P＜0.05 vs control),61.3±3.95%(Fuco-25, P＜0.01 vs control) and 66.3±4.55% (Fuco-50, P＜0.01 vs control). While in H1299 cells, the percentage of G0/G1 phase increased from 39.0±6.51% to 51.3±4.85% (Fuco-12.5),56.2±4.62% (Fuco-25, P＜0.05 vs control) and 57.3±6.42% (Fuco-50, P＜0.05 vs control) 6. Fucoxanthin caused a significant induction of apoptosis in A549 and H1299 cells. In A549 cells, apoptosis rate increased from 1.70±1.13% (control) to 26.4±2.96% (Fuco-12.5, P<0.01 vs control),38.6±7.13% (Fuco-25, P<0.01 vs control) and 40.6±9.72% (Fuco-50, P<0.01 vs control). In H1299 cells, apoptosis rate increased from 0.93±0.60% (control) to 18.1±4.38%(Fuco-12.5, P<0.01 vs control),23.8±6.51% (Fuco-25,P<0.01 vs control) and 27.9±2.07%(Fuco-50, P<0.01 vs control).7. The real time quantitative PCR results showed fucoxanthin could increase the mRNA expression of genes which could promote apoptosis, like p53, PUMA and Fas, p21 WAF1/CIP1 which can arrest tumor cells at the G0/G1 phase. Only in A549 cells, p53 mRNA expression was increased after fucoxanthin treatment. The mRNA expression of Bcl-2, an apoptosis inhibitory gene was down-regulated by fucoxanthin.8. The western blot results showed fucoxanthin could increase the protein expression of PUMA, p21 WAF1/CIP1 and Fas in A549 and in H1299 cell. Only in A549 cells, the p53 and p-p53 protein expression was increased after fucoxanthin treatment.9. After treatment of fucoxanthin at the concentration of 12.5μM,25μM and 50μM, the activity of Caspase-3 and Caspase-8 increased significantly in a dose-dependent manner.10. In the dosage range between 538 to 3200 mg/kg, fucoxanthin is safe when intragastric administrated to mice for one time.11. A549 mice transplanted tumor models were successfully set up. Tumor volume assay showed that both fucoxanthin and DDP resulted in the antitumor activity (P<0.05 or P<0.01 vs. vehicle control). But the tumor growth rates in the fucoxanthin group were higher than the DDP group (P<0.05 or P<0.01). Tumor weight assay resulted that the inhibitiory rate of fucoxanthin 50 mg/kg group was 78.1%, fucoxanthin 15 mg/kg group is 69.9%, fucoxanthin 5 mg/kg group is 62.0% and DDP group was 84.6%. At the end of the experiment, the body weight was significantly increased in the fucoxanthin and vehicle control group, but in the DDP group, the body weight of mice was not increased. And the major organ/tissue like liver, spleen and kidney had no change in size and morphology.12. The HE staining results showed that in the fucoxanthin and DDP groups, cancer cell population decreased and necrosis cells increased. The apoptosis tumor cells showed contraction, karyopyknosis and blue-dyeing morphology changes.13. The immunohistochemistry assay showed that fucoxanthin and DDP could decrease expression of P53 and Bcl-2 proteins in tumor tissues and increased PUMA protein expression.14. TUNEL assay results showed that fucoxanthin and DDP could significantly increase the apoptotic ratio of tumor tissue in A549 transplanted nude mice. The apoptosis rate was 5.40±2.90% (vehicle control),50.2±14.4% (Fuco-50),35.5±10.7% (Fuco-15), 22.9±3.97% (Fuco-5) and 58.6±11.9% (DDP). Conclusion:1. Fucoxanthin can inhibit the growth of human leukemia, hepatoma, breast adenocarcinoma and cervix tumor cells in vitro.2. Fucoxanthin can induced G0/G1 phase arrest and apoptosis in K562, U937 and Jurkat cells.3. Fucoxanthin can inhibit growth of four human NSCLC cells in vitro and exhibit a concentration-and time-dependent manner.4. Fucoxanthin can induce G0/G1 phase arrest and apoptosis in A549 and H1299 cells in a concentration-dependent manner.5. Fucoxanthin can increase the expression of phosphorylated p53 and p53 in A549 cells. The p53 signal pathway may involved in the cell cycle arrest and apoptosis induction effect of fucoxanthin since the p53 target genes like p21WIP1/CIF1, PUMA and Fas were increased and the Bcl-2 expression was decreased. The activity of Caspase-3 and Caspase-8 in A549 cells were also elevated.6. Although p53 is null in H1299 cells, fucoxanthin still could induce G0/G1 arrest and apoptosis, this may be mediated though p53-independent pathway to increase the expression of p21WIP1/CIF1, PUMA and Fas and down-regulation the expression of Bcl-2.7. In the dose range between 538 to 3200 mg/kg, fucoxanthin is safe when intragastric administrated to mice.8. After intragastric administration of fucoxanthin at the dosage of 5 to 50 mg/kg for 5 weeks, the growth of A549 lung cancer xenograft tumor was significant inhibited and it was well tolerated.9. Fucoxanthin can induce apoptosis in tumor tissue and decrease expression of P53 and Bcl-2 protein; and can increase the expression of Caspase-3. These proteins may involve in the mechanism of fucoxanthin for apoptosis induction in tumor tissues.