[Effect of Fei-Liu-Ping ointment combined with cyclophosphamide on lung cancer cell proliferation and acidic microenvironment].

Objective: To observe the effects of Fei-Liu-Ping ointment and chemotherapy on mice with lung cancer, and to explore the inherent mechanism of action from the point of acidic microenvironment and apoptosis.

Methods: First of all, the Lewis lung cancer transplanted mouse model was established. Therefore, they were treated by Fei-Liu-Ping ointment, cyclophosphamide, Fei-Liu-Ping ointment + cyclophosphamide and the saline as control. All the groups' tumor size, tumor growth rate and food consumption were recorded. The mice were sacrificed and the tumors were took out after 15 days' interventions. Then lactate relative concentrations were detected with lactate kits and the protein expressions of glucose transporter 4 (GLUT4), hexokinase 1 (HK1), glucose-regulated protein 78 (GRP78), carbonic anhydrase-IX (CA-IX) were detected through immunohistochemical staining. Flow cytometry was adopted to detect the percentage of apoptotic tumor cells and regulatory T cells (Treg), and the expression of hypoxia-inducible factor-1α (HIF-1α), Bcl-2, Bax, Caspase-3, interleukin-2 (IL-2) were tested through western blot.

Results: The strongest inhibition effect and the lowest tumor growth rate was found in Fei-Liu-Ping ointment + cyclophosphamide group. There were significant differences between Fei-Liu-Ping ointment + cyclophosphamide group and saline group(P<0.05). And the highest food consumption was found in Fei-Liu-Ping ointment + cyclophosphamide group while there were no significant differences between Fei-Liu-Ping ointment + cyclophosphamide group and saline group (P>0.05). Further molecular biological detections found that the lowest lactate level and regulatory T cells ratio were found in Fei-Liu-Ping ointment + cyclophosphamide group and these expressions of GLUT4, HK1, GRP78, CA-IX were suppressed. There were significant differences between Fei-Liu-Ping ointment+cyclophosphamide group and saline group (P<0.05). In addition, the Fei-Liu-Ping ointment + cyclophosphamide group's cell apoptosis increased significantly compared with saline group and there were significant differences on expressions of HIF-1α, Bcl-2, Bax, Caspase-3, IL-2 for this group compared with saline group.

Conclusion: Chemotherapy and Fei-Liu-Ping ointment had the synergistic effect on inhibiting tumor growth and improving the general conditions of tumor-bearing mice. The effect was partly owed to the improvement on tissue hypoxia, the inhibition of HIF-1α expression and the regulations on its downstream proteins, such as GLUT4, HK1, GRP78, and CA-IX. And then all these alterations led to the modulation tumor acidic microenvironment, the induced tumor cells apoptosis and suppression of T cells to regulatory T cells differentiation.