| Malignant tumor is a life threatening disease to human being. Current conventional treatment for cancer patients includes surgical operation, radiotherapy and chemotherapy. Most antineoplastic agents used in clinic belong to the category of cytotoxic drugs. These drugs often have poor selectivity, severe side effects and resistance is commonly observed after short term treatment. However, Chinese herb usually has little side effects and universal anti-tumor effects. Exploring the role of Chinese herb as second line anti-tumor drugs has become one of the hot spots in the field. Lung cancer is the most frequent malignant neoplasm in many countries. The incidence and mortality of lung cancer increased in the last decade worldwide. Efficacy of current therapeutic methods is usually not satisfactory and many of lung cancer patients died of early metastasis and rapid recurrence. Viscum, which blongs to Lorantbeceae, is consist of more than 30 species, 11 of them were identified in China. Only one of Viscum, named as Chinese mistletoe or North mistletoe, was included in Chinese Pharmacopoeia. It has been reported that mistletoe lectins and viscotoxins of Viscum have anti-tumor effect and one active form of mistletoe (Iscardo) is under clinical application as anti-tumor drug in Europe. In China, it also reported that mistletoe alkali from Chinese mistletoe has a strong anti-tumor effect. However, little is konwn about the mechanism of mistletoe alkali and its inhibitory effect on lung cancer. In this study, we investigated mechanisms of the in vitro and in vivo effects of mistletoe alkali on cell proliferation, apoptosis and invasion by using a lung adenocarcinoma cell line A549. Furthermore, acute toxicity of mistletoe alkali was also evaluated.Objective: To study in vitro and in vivo effects of mistletoe alkali on cell proliferation,invasion and apoptosis in A549 cells; explore mechanisms underlining above biological effects and evaluate toxicity of mistletoe alkali. Methods: The experiments were approached by pharmacodynamic and toxicologic methodes. Pharmacodynamic experiments were divided into two parts. In Vitro experiments include CCK-8 assay to evaluate the growth inhibition of A549 cells, flow Cytometry to detect cell cycle arrest and Immunocytochemical staining was used to evaluate PCNA, Caspase-3 and MMP-9 expression level. Meanwhile, apoptosis was detected by TUNEL, AO/EB staining, AnnexinⅤ-FITC&PI kit and cell invasion ability was observed in Boyden Chamber assay. In Vivo experiments includes establishment of human lung adenocarcinoma xenograft model by inoculating A549 cells subcutaneously in BALB/C nude mice at 6×106/mouse and drug treatment study which contains 8 mice in mistletoe alkali group, 6 mice in 5-fluorouracil group and 6 mice in vehicle (Sodium Chloride) control group. Treatment began at the second day after A549 inoculation with an everyday dosing schedule of mistletoe alkali 10mg·kg-1,5-fluorouracil 20mg·kg-1 and equal volume of Sodium Chloride through peritoneal injection (i.p.) for 21 days (mistletoe alkali and vehicle) or 10 days (5-fluorouracil), respectively. Due to the severe side effect of 5-fluorouracil, administration of 5-fluorouracil were stoped after 10 days of treatment. General body condition of all mice was observed every day. After formation of tumors, body weights and size of tumors were measured daily. Mice were then sacrificed after treatment and tumors were dissected for gross and macroscopic observation and immunohistochemical staining to examine PCNA, Caspase-3 and MMP-9 expression which reflect the proliferation, apoptosis and invasion of the tumors. Furthermore, TUNEL assay was also employed to detect apoptosis in tumors. LD50 was measured in acute toxicity test to evaluate toxicity of alkali group.Results:in vitro experiments: (1) Mistletoe alkali showed inhibitory effect on growth of A549 cells in dose dependent manner. IC50 of mistletoe alkali and 5-fluorouracil are 0.076mg·mL-1 and 0.01mg·mL-1, respectively. There is significant statistical difference in term of cell proliferation between mistletoe alkali and vehicle treatment group (P<0.001). (2) Flow Cytometry revealed cell cycle arrest in G0/G1 Phase by Medium dose of mistletoe alkali, S phase fraction (SPF) and Proliferous index (PI) of mistletoe alkali treated cells are also lower than that of 5-fluorouracil and vehicle treated cells. (3) Immunocytochemical staining indicated dose dependent inhibition of PCNA expression by mistletoe alkali. Comparing with vehicle control, high dose of mistletoe alkali and Medium dose of mistletoe alkali were efficient to induce statistically significant inhibition on PCNA expression (P<0.001). Moreover, inhibition of high dose of mistletoe alkali on PCNA expression is also statistically superior to that of 5-fluorouracil (P<0.05). All three dosage of mistletoe alkali induced statistically significant elevation of Caspase-3 expression when compared with vehicle control group (P<0.001), while high,medium dose of mistletoe alkali also resulted in statistically significant higher Caspase-3 expression than that of 5-fluorouracil (P<0.01). All 3 doses of mistletoe alkali inhibited MMP-9 expression when compared that of vehicle control (P<0.05). No statistical significance could be drawn in comparison with 5-fluorouracil group (P>0.05). (4) Results of TUNEL assay only showed weak fluorescence in control group, while mistletoe alkali induced dramatically increased fluorescence in dose dependent manner (P<0.01). High dose of mistletoe alkali also induced higher fluorescence intensity than 5-fluorouracil (P<0.01). (5) AO/EB staining indicated that the apoptosis rate of three mistletoe alkali groups were higher than that of control (P<0.001), while apoptosis rate of high dose of mistletoe alkali was higher than that of 5-fluorouracil group (P<0.01). (6) Apoptosis rate determined by AnnexinⅤ-FITC&PI kit via Flow Cytometry showed that high,medium dose of mistletoe alkali led to an increase in total apoptosis rate when compared with that of vehicle control (P<0.01), and an increase in early apoptosis rate when high dose of mistletoe alkali compared with that of 5-fluorouracil (P<0.05). (7) In Boyden Chamber assay, medium dose of mistletoe alkali resulted in much fewer A549 cells that could pass through the artificial basement membrane when compared with that of Control group (P<0.01) and 5-fluorouracil group (P<0.05). The invasion distance of medium dose of mistletoe alkali treated cells was also dramatically shorter than that of Control (P<0.001) and 5-fluorouracil (P<0.001) treated cells.in vivo Experiments: (1) General body condition of mice in mistletoe alkali and NS group were good, but mice in 5-FU group lost more than 10% of body weight after treatment for one week, possibly due to side effect of 5-FU. (2) Weight of tumors in mistletoe alkali group was less than that of NS and 5-FU group. The inhibitory rates of mistletoe alkali and 5-FU group were 75.85% and 47.86%, respectively. Average tumor size of mistletoe alkali group was smaller that that of other 2 groups (P<0.01). (3) HE staining of tumors under mistletoe alkali administration was characterized with obvious necrosis. While necrosis in 5-fluorouracil treated tumors was occasionally observed, little necrosis could be found in NS group. (4) Immunohistochemistry staining analysis revealed inhibition of PCNA, MMP-9 and elevation of Caspase-3 expression level in mistletoe alkali group, similar to the results of in vitro experiments. (5) TUNEL assay showed higher apoptosis rate in mistletoe alkali group than that in NS group (P<0.001) and 5-fluorouracil group (P<0.05).LD50 of mistletoe alkali to ICR mouse was 490.09mg·kg-1, as measured in acute toxicity test.Conclusions: (1) IC50 of mistletoe alkali to A549 cells were 0.076mg·mL-1. (2) Mistletoe alkali inhibits growth and proliferation of A549 cells in vitro and in vivo via down regulation of PCNA expression and subsequent reduction of DNA synthesis. (3) Mistletoe alkali induces apoptosis of A549 cells in vitro and in vivo by activating Caspase-3 cascade. (4) Mistletoe alkali inhibits invasion of A549 cells, possibly through down regulation of MMP-9 expression. (5) LD50 of mistletoe alkali to ICR mouse was 490.09mg·kg-1.
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