| Objection:To study the antitumor effects and side effects of erlotinib with different dosing time in Lewis tumor-bearing mice, and investigate its underlying mechanism via EGFR-AKT-CyclinD-CDK-4pathway.Methods:1. The mouse model of Lewis lung cancer xenografts was established. The animals were randomly divided into three dose groups (15,30,60mg·kg-1) and one model group, and each group was divided into6time groups including8:00,12:00,16:00,20:00,24:00, and04:00.2. The mice in the6time groups were administered by gavage with15mg·kg-1,30mg·kg-1,60mg·kg-1of erlotinib suspension at different circadian times:8:00,12:00,16:00,20:00,24:00and04:00. The mice in the model group and the blank control group received the same volume of sodium carboxymethyl cellulose and distilled water.3. The mice were observed for diet, exercise and mental status during the experiment. Tumor volume was measured with calipers every four days and the tumor growth rate was expressed as the tumor volume change. The tumor growth curves were drawn. Blood samples were taken from the retroorbital sinus vein after21days of administration. Leukocytes, neutrophils, lymphocytes and hemoglobin were analyzed with automatic blood analyzer.4. The expression of EGFR, AKT1, CDK-4and CyclinD1in tumor tissue were detected by qRT-PCR.5. The expression of protein AKT, P-AKT and CyclinD1in tumor tissue were detected by Western-blot.Results:1. Tumor growth after initiation of erlotinib treatment (15,30,60mg·kg-1) was significantly suppressed compared with that in the model group given sodium carboxymethyl cellulose (P<0.05). The tumor growth of the30mg·kg-1and60 2. mg·kg-1groups was significantly different from that of the15mg·kg-1group. However, no significant difference of tumor growth was found between30mg·kg-1and60mg·kg-1groups.3. Tumor growth in groups8:00,12:00,16:00in the light phase was significantly suppressed when compared with that in the dark phase (groups20:00,24:00,04:00) with the effect in group16:00being the most effective (P<0.05).3. The effect of erlotinib on mean leukocytes, neutrophils, lymphocytes and hemoglobin count depended on dosing time of the drug (P<0.05). Leukocytes, neutrophils and lymphocytes of groups24:00and04:00were significantly elevated when compared with the model group (P<0.05). Hemoglobin of groups16:00and20:00was significantly reduced when compared with the model group (P<0.05).4. The tumor weights of group8:00,12:00,16:00,20:00,04:00was significantly suppressed when compared with the model (P<0.05), and the group16:00was the best.5. In the model group, the tumor cells arranged normally and almost no necrosis was observed. Large areas of necrosis, and inflammatory cell infiltration and bleeding were observed in groups8:00,12:00,16:00,20:00and small focal necrosis and inflammatory cell infiltration were observed in groups24:00and04:00. The tumor cell apoptosis of the erlotinib groups was significantly obvious than that of the model group, and that of the light phase changed significantly when compared with the dark phase.6. The expression of EGFR in groups8:00,12:00,16:00was significantly lower when compared with that of the model group (P<0.05), and that of group20:00,24:00and04:00changed no significantly (P>0.05) when compared with the model group. The expression of AKT1in groups8:00,12:00,16:00and20:00was significantly lower when compared with the model group (P<0.05), the group16:00was the best (P<0.05), and the group24:00and04:00changed no significantly (P>0.05) when compared with the model group. The expression of CDK-4in all groups was no significantly lower when compared with that of the model group (P>0.05). The expression of CyclinDl in groups8:00,12:00,16:00and20:00was significantly 7. lower when compared that of the model group (P<0.05), and that of group24:00and04:00changed no significantly (P>0.05) when compared with the model group.8. The P-AKT protein level in groups12:00and16:00was significantly lower when compared with the model group (P<0.05), and it was significantly different when compared between groups12:00and16:00, while the level of AKT remained unchanged (P>0.05). The Cyclin D1protein level in groups8:00,12:00,16:00and04:00was significantly lower when compared with the model group (P<0.05).Conclusion:Erlotinib has significant antitumor effect on xenografts of non-small cell lung cancer in mice, and its efficacy and toxicity shows circadian rhythm. Its mechanism of action might be related to the EGFR-AKT-CyclinD-CDK-4pathway. |
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