| Cancer, a fatal disease to human life and health, is the top one factor for mortality in many countries. Oral cancer accounts for 3%, 5%, 40% in all sorts of cancers in Sweden, USA, Indian and Southeast-Asia, respectively. And that the 80% of the oral cancer is oral squamous cell carcinoma. Proanthocyanidins play chemopreventive or chemotherapeutic role to oral cancer, skin tumor, breast cancer, pancreatic tumor, prostatic cancer, liver cancer, lung carcinoma, gastric adenocarcinoma and colon tumor, thanks to their antioxidant and free radical scavenging abilities, as well as remarkable biological, pharmacological and therapeutic activities. Developed on our Lab., LSPC (Procyanidin of Lotus Seedpod) has been verified for a variety of pharmacological properties. This study is aimed at investigating the pharmacological effects of LSPC on oral cancer at the level of sub-cell, cell and animals, at widing the application of LSPC and providing the scientific basis for chemoprevention and chemotherapeuty to oral cancer.The results are as follows: 1. Effects of LSPC on KB cell growthIn order to find out the optimal working concentration of LSPC's effects on KB (human nasopharyngeal epidermoid carcinoma) cell, MTT assay was applied to testthe inhibitory effects of LSPC on KB cell proliferation and its cytotoxity against L929 cell. When LSPC's concentration was 100-1000 ug/ml, it remarkably inhibited KB cell growth in relation of dosage effect, while it obviously promoted KB cell growth if its concentration was 50 (ag/ml. Coincidently, LSPC inhibited L929 cell growth at the concentration of 400-1 OOOug/ml, but failed to affect L929 cell growth when its concentration was 100~300u.g/ml, while it could inhibit L929 cell growth if the concentration was 50 ug/ml. It was safe to say that there was an optimal dosage range, which was 200~400ug/ml for LSPC's cytotoxity against cancer cell lines and protection on normal cells.2. Effects of LSPC on KB cell morphologyKB cell's morphological alteration caused by LSPC with different concentration was observed under upside-down microscopy, HE staining and transmission electron microscopy (TEM). For inverted microscopy, it was apparent that KB cell was getting round and small, its membrance was transparent and the cell was floating, all of which indicated that the KB cell fission phase decreased and its growth was inhibited. For HE staining, KB cell nuclear was getting round and small with wrinkle, as well as broken, which indicated that LSPC was of cytotoxity and inducing apoptosis on KB cell. For TEM, there were classical chatacteristics of cell apoptosis, which was bridge structure disappeared, cell withered , chromosome aggregated and broken and there was bubbles in cell. All of these showed that LSPC was able to inhibit KB cell growth and change its morphology, as well as induce apoptosis.3. LSPC's inducing KB cell apoptosisBased on Flow Cytometry (FCM), including PI staining and FITC-annexin V/PI, LSPC's inducing KB cell apoptosis at different concentration was investigated, and the apoptotic pathway was studied by inspecting the expression amount of surface protein CD95 on KB cell. PI staining indicated that 7.5%, 8.3% and 17.5% of KB cell was under apoptosis or necrosis or the mixtrue when LSPC's concentration was 200u.g/ml, 300ug/ml and 400ug/ml. FITC-annexin V/PI assay showed that a part of KB cell was under the early phase of apoptosis when LSPC's concentration was 200ug/ml, while a part of KB cell was under the late stage of apoptosis or necrosis, orthe blending when LSPC's concentration was 300ug/ml , 400ug/ml. It followed that LSPC could induce KB cell apoptosis and necrosis with dosage effect. However, CD95 expression amount decreased with the increment of LSPC's concentration, indicating that apoptotic KB cell declined through Fas pathway, which was in controversy to the result of LSPC's inducing KB cell apoptosis. One possoble answer to this problem was the linkage between LSPC and CD95 which limited the linkage between LSPC and CD95-PE antibody, a... |
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