Anti-proliferative activity and possible mechanism of action of constituents identified in silkworm feces toward human lung carcinoma cell lines

Abstract

Lung cancer caused by diverse changes in cells resulted by exposure to carcinogens found in tobacco smoke, the environment, or sequential accumulation of genetic changes to the normal epithelial cells of the lung. Lung cancer is one of the most common malignancies. However, the fatality of lung cancer is highest than any other cancer in the whole world. Chemotherapy, radiotherapy, and targeted agents have been used for lung cancer treatment. Sometimes, there are serious side effects of these treatments occurred, such as bleeding, hair loss, vomiting, and difficulty getting and fatigue. Therefore, it needs to develop new impressive anticancer agents with effective target sites and low toxicity from natural products which have fewer side effects. In this study, an assessment is made of the anti-proliferative activity of constituents isolated from silkworm feces against 10 human cancer cell lines, including A549 and H727 non-small cell lung cancer cell(NSCLC) lines, using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results were compared with those of the commercially available anticancer agent with broad spectrum cisplatin. The air-dried silkworm feces (30.84 kg) yielded 660 g of a dark greenish ethanol extract. The ethanol extract of silkworm feces was proved to have anti-proliferative activity against all 10 species of human cancer cell lines including A549 and H727 lung, AGS stomach, PC-3 prostate, HeLa cervix, HT-29 colon, MCF-7 breast, SNU-213 pencreas, SK-Hep-1 liver, Hep-2 larynx and SK-OV-3 ovary cancer cell lines. The biologically active constituent was characterized as the Vomifoliol (Blumenol A) and Stigmasterol by spectroscopic analysis, including EI-MS and NMR. Vomifoliol (Blumenol A) and Stigmasterol were isolated from the silkworm feces as a new antiproliferative activity principle. Fifty percent inhibition concentration (IC50) values of the constituent against A549 and NCI-H727 were conducted. The IC50 of vomifoliol was 57.24 and 55.05 μM toward A549 and NCI-H727 cell lines, respectively. The IC50 of stigmasterol was 46.91 and 44.90 μM toward A549 and NCI-H727, respectively. These compounds were less toxic than cisplatin (IC50, 16.53 μM for A549

IC50, 13.50 μM for NCI-H727). The mRNA gene expression of Akt1 and Bcl-2 gene was investigated by real-time RT-PCR. At the presence of vomifoliol and stigmasterol, the mRNA gene expression level of Akt1 which is famous for anti-apoptosis gene was reduced. Also, the mRNA gene expression of Bcl-2 which is important factor in anti-apoptosis mechanism was inhibited by these two constituents, vomifoliol and stigmasterol identified in silkworm feces. In conclusion, global efforts to reduce the level of antitcancer agents justify further studies on the silkworm feces-derived materials containing Vomifoliol (Blumenol A), Stigmasterol as potential anticancer products or a lead molecule for the prevention or eradication from human lung cancer.