Study on the regulatory mechanism of luteolin inhibiting WDR72 on the proliferation and metastasis of non small cell lung cancer

Abstract

Non-small cell lung cancer (NSCLC) is a major cause of cancer-related mortality worldwide. Understanding molecular mechanisms and identifying potential therapeutic targets are crucial for improving treatment outcomes. This study aims to explore the effect of luteolin on NSCLC progression by regulating WDR72 and to investigate the related molecular mechanisms using cellular and animal models. The study employed a comprehensive set of experiments to evaluate the impact of luteolin and WDR72 on NSCLC cell proliferation and metastasis. Techniques included the CCK- 8 assay, colony formation assay, scratch test, and Transwell assay. Molecular docking experiments were performed to validate the binding interaction between luteolin and WDR72. Experimental groups included OE-WDR72, OE-WDR72 + Luteolin, Control, Control + Luteolin, and sh-WDR72. Western blot analysis was used to examine protein expression related to apoptosis, epithelial-mesenchymal transition (EMT), AKT signaling, and other markers. Additionally, a nude mouse subcutaneous tumor model was established to assess the in vivo tumor-forming ability of NSCLC cells under different treatments. Luteolin significantly inhibited the proliferation, invasion, and migration of NSCLC cell lines (H1299 and A549) and reduced tumor formation in nude mice. Molecular docking demonstrated strong binding affinity between luteolin and WDR72. Overexpression of WDR72 promoted NSCLC cell proliferation and migration, while WDR72 silencing showed the opposite effects. Western blot analysis revealed that WDR72 overexpression increased phosphorylated AKT and Bcl- 2 levels while decreasing caspase- 3. In contrast, silencing WDR72 reduced these protein levels. Luteolin treatment in WDR72-overexpressing cells resulted in decreased phosphorylated AKT, increased apoptosis, and suppressed EMT. Tumor transplantation experiments indicated that tumors in the OE-WDR72 group exhibited the fastest growth, while the sh-WDR72 group showed the slowest growth. Luteolin treatment significantly reduced WDR72 expression, suggesting a regulatory role in NSCLC progression. Luteolin effectively inhibits EMT, invasion, and migration of NSCLC cells by modulating WDR72. WDR72 plays a pivotal role in stimulating the proliferation and metastasis of NSCLC cells. By downregulating WDR72, luteolin suppresses NSCLC progression, potentially through modulation of the PI3 K/AKT/EMT signaling pathway. These findings highlight luteolin as a promising therapeutic agent for NSCLC treatment.

Keywords: Epithelial-mesenchymal transition; Luteolin; Metastasis; Non-small cell lung cancer; Proliferation; Signaling pathway; WDR72.

Fig. 1

Molecular structural formula of luteolin and its inhibitory effect on non-small cell lung cancer cells. (A) Molecular structural formula and three-dimensional structure of luteolin. (B) IC50 of luteolin at different time points and cell proliferation at different concentrations for 24 h. (C) Morphological changes of non-small cell lung cancer cells induced by luteolin.

Fig. 2

Luteolin demonstrates the ability to inhibit the invasion and migration of non-small cell lung cancer cells. A) The study shows alterations in invasion and migration capability following luteolin treatment. B) The scratch test reveals changes in migration ability before and after luteolin administration.

Fig. 3

In vivo experiments verify the inhibitory effect of luteolin on non-small cell lung cancer. A)nude mouse tumor model was used to demonstrate the inhibitory effect of luteolin on non-small cell lung cancer. B) In vivo verification showed that luteolin does not cause damage to major organs after its action.

Fig. 4

Luteolin directly targets the WDR72 protein and can influence changes in its expression. (A) Molecular docking of WDR72 and luteolin, (B) Expression of WDR72 in cellular proteins following the addition of varying concentrations of luteolin, (C) Subcellular localization and expression of fluorescently labeled WDR72 in cells, (D) Western blot and RT-PCR experiments used to identify the shRNA sequence with the highest silencing efficiency, (E) Western blot and RT-PCR experiments confirming a significant increase in WDR72 expression after overexpression.

Fig. 5

Silencing or overexpressing WDR72 can impact the proliferation and invasion of non-small cell lung cancer cells. (A) CCK8 assay was used to assess changes in the proliferation capacity of these cells following WDR72 silencing and overexpression. (B) Cell clone assay was employed to evaluate the clone-forming ability of lung cancer cells after WDR72 silencing and overexpression. (C) Transwell chamber assay was conducted to measure alterations in the invasion and migration abilities of non-small cell lung cancer cells post WDR72 silencing and overexpression. (D) Scratch assay was utilized to observe changes in the migration ability of these cells after WDR72 silencing and overexpression.

Fig. 6

Luteolin inhibits EMT, AKT and promotes apoptosis in non-small cell lung cancer cells by inhibiting the expression of WDR72.

Fig. 7

Effect of luteolin on H1299 xenograft model, (A) Gross appearance and tumor volume curve of nude mice, (B) Tumor appearance and weight histogram, (C) Relative expression level of WDR72 mRNA in transplanted tumors of different groups, (D) Immunohistochemical detection of WDR72 expression in Control + Luteolin group and Control group.

Fig. 8

Model diagram of luteolin regulating WDR72 to affect the proliferation and metastasis of non-small cell lung cancer through the PI3 K/AKT/EMT axis.