| Paeonol(Pae,2-hydroxy-4-methoxyacetophenone),is isolated from the herb Pycnostelm paniculatum(Bunge)K.S.,and the root of the plant Paeonia Suffruticosa Andrew.It is a white needle crystal with a relatively low-melting point of 51℃-52℃.The molecular weight of Pae is 166.18 ku and the molecular formula is C9H10O3. In the past study indicated that Pae posseses extensive pharmacological activities such as sedation, hypnosis, antipyresis, analgesic, antioxidation, antiinflammation, inhibition of platelet aggregation and enhancement of cellular immune function. In our previous study,the antineoplastic activity of Pae has been demonstrated both in cell lines,such as human erythromyeloid cell line K562,breast cancer gene cell line T6-17, and human hepatoma cell line Bel-7404, and in animal models bearing HepA hepatocarcinoma. In the study of Pae on human esophageal cancer cell line Eca-109,we have not been seen.The inhibition of paeonol(Pae) on growth of human esophageal cancer cell line Eca-109 was studied by examining the influence of Pae in a different concentrations in vitro with MTT assay.The antitumor effect of Pae was investigated in nude mice implanted subcutaneous injection with human esophageal cancer cell line Eca-109.The nude mice were divided into 7 groups at random.Normal saline was given i.g. to the control group.Pae in different doses(25,50,100,200mg/kg) was given i.g. in the four groups.CDDP (5mg/kg) was given i.p. to treat the sixth group.The seventh group were treated with the combination of Pae(100mg/kg) and CDDP(5mg/kg).Pae was given i.g. 14 days,once a day. CDDP was injected i.p. twice a week, together 2 weeks. We observed the inhibition of tumor though weighing the tumor. The morphologic changes of tumor tissue were observed under light microscope and transmission electron microscope.The detecting of apoptosis index of caspase-3 and the expression of Bcl-2 and Bax in tissues of every group by TUNEL and immunochemistry techniques,we explored the antitumor activity of Pae and its chemanism.1. Effect of Pae on the proliferation of human esophageal cancer cell line Eca-109 The antiproliferative effect of Pae on Eca-109 cell line in vitro was measured by MTT assays.The results suggested that Pae at concentrations of 0.094～1.504mmol/L,had inhibitory effect on the proliferation of Eca-109 cells. It was observed that higher concentration of Pae,more inhibitory effect,suggesting obvious concentration-effect relationship of Pae.The r value of dose-effect reached curves for single-agent on Pae Eca-109 cell line was 0.964. The IC50 value of MTT was 0.342 mmol/L.2. Inhibition of tumor growth of Eca-109 by Pae in nude mice2.1 The influence of tumor growh of Eca-109 by Pae alone in nude mice In the model of human esophageal cancer xenografts in nude mice,Pae i.g suppressed the tumor growth significantly. The results suggested that the tumor-inhibitory rates were10.67%,23.54%,27.91% and 34.46% when Pae at doses of 25,50,100,200 mg/kg, respectively.Compared with the control group,Pae alone with 25 mg/kg had no significant effect on tumor growth in vivo. The results indicated that Pae at different doses of 50～200 mg/kg had an inhibitory effect.The higher the dose of Pae was,the stronger the antitumor effect reached,which suggested the dose-dependent manner of Pae. The r value of dose-effect reached curves for single-agent on Pae Eca-109 cell line was 0.974.2.2 The influence of tumor growth of Eca-109 by Pae and CDDP in nude mice To investigate the synergistically anti-tumor effect of Pae 100 mg/kg and CDDP 5mg/kg on the tumor growth of Eca-109 in nude mice.In vivo administration of Pae 100 mg/kg combined with cisplatin 5 mg/kg resulted in a significant inhibition of Eca-109 tumor growth with the inhibitory rate of 77.91%, compared with cisplatin used alone (58.71 % ). The coefficient of drug interaction(CDI)was used to analyse the synergistically anti-tumor effect of drug combination.The result indicated that the synergistic effect was significant when 100 mg/kg Pae combined with 5mg/kg CDDP.3. Apoptosis caused by human esophageal cancer Eca-109 cells in nude mice3.1 Morphological observation for Pae-induced apoptosis of Eca-109 cells in nude miceThe morphologic changes of tumor tissue were observed under light microscope and transmission electron microscope.The results suggested that the more apoptotic tumor cells can be seen under light microscope in theraperutic groups than that in control.The tumor cells of theraperutic groups showed the typically apoptotic changes,such as chromatin condensation, vacuolus of the nuclear, and the fragmentation of the nuclear,and so on. The apoptotic tumor cells are dispersed in tumor tissues. In tumor tissues,there are few necrotic cells. Changes of ultrastructure of tumor cells including concentration and side accumulation of the nuclear chromatin,and the fragmentation of the nuclear was observed under transmission electronic microscope. Apoptosis body was also found. 3.2 Apoptosis detected by TUNEL of human esophageal cancer Eca-109 xenografts in nude miceTerminal-deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique was performed to detect apoptosis.The apoptosis index(AI) was analyze the percent of apoptosic cells. It was found that the apoptotic cells were stained yellow by TUNEL techniques. The apoptosis index of every theraperutic groups were significantly different from the control (P<0.05 or P<0.01).4. Possible molecular mechanism of Pae-induced apoptosis of Eca-109 cellsImmuochemical S-P mothods were used to measure the expression of the protein including Bcl-2,Bax and caspase-3.Metamorph biological image software was performed to analyze the results.The results suggested that Pae could up-regulate the of Bax and caspase-3,and down-regulate the expression of Bcl-2.5.conclusionOur result indicated that Pae had anti-tumor effect in vivo and in vitro,and ally to the chemotherapeutic action of CDDP in vivo.These effects might be related to down-regulate the value of Bcl-2/Bax and active caspase-3.
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