Abstract
BACKGROUND: In our previous study, we provided evidence that iRGD-modified co-loaded curcumin piperine liposomes (iRGD-LP-CUR-PIP) have in vitro and in vivo anti-non-small cell lung cancer (NSCLC) activity. However, the mechanism of action of CUR-PIP on NSCLC is unclear; therefore, this study aimed to investigate the mechanism of CUR-PIP combination anti-tumor therapy by inhibiting angiogenesis. METHODS: The target binding effect of iRGD on the integrin avβ3 receptor was observed by cellular uptake assay. Western blot (WB) and immunofluorescence analyses were performed to investigate the effect of iRGD-LP-CUR-PIP on the VEGFR2/P38/MK2 pathway in vivo. In addition, a new A549 + HUVEC co cultured cell model was established and characterized for migration analysis.WB and immunofluorescence were used to detect the effect of iRGD-LP-CUR-PIP on the VEGFR2/P38/MK2 pathway in vitro, and the effect of iRGD-LP-CUR-PIP on the VEGFR2/P38/MK2 pathway was verified by constructing plasmids transfected to knock down VEGFR2 expression. RESULTS: Our data showed that iRGD-LP could bind to the αvβ3 receptor on the cell membrane surface; with increasing uptake time, A549 cells could easily enter. The results of in vitro and in vivo mechanism experiments showed that iRGD-LP-CUR-PIP and iRGD-LP-CUR both reduced the levels of VEGF and VEGFR2 proteins. However, for downstream proteins (p-P38MAPK and p-MK2), iRGD-LP-CUR-PIP was more effective in reducing protein levels than iRGD-LP-CUR. In addition, VEGFR2 silencing almost completely inhibited the phosphorylation of P38 and MK2, while iRGD-LP-CUR-PIP did not enhance the inhibition of P38 and MK2 phosphorylation after VEGFR2 silencing. CONCLUSION: iRGD-LP-CUR-PIP reduces tumor angiogenesis and affects tumor growth by inhibiting the VEGFR2/P38/MK2 pathway. Among them, CUR affects VEGF and PIP may affect CTR1, which in turn affects CTR1-VEGFR2 co-internalization and downstream signaling pathways. These changes result in enhanced anti-tumor activity by CUR-PIP binding, and the best anti-tumor activity was observed in the iRGD-LP-CUR-PIP group compared with the other groups.