KRAS mutation is a common driver of NSCLC, and there is a high proportion of lung cancer patients with KRAS G12C and G12D mutation. KRAS was previously considered an "undruggable" target, but the first KRAS G12C mutation-targeted drug AMG510, entered the market in 2021. However, treatments for G12D mutant tumors remain to be discovered. Salvianolic acid F (SalF), a monomer derived from the traditional Chinese medicine Salvia miltiorrhiza (SM), and KRAS had high binding affinity, especially for KRAS G12D. There is an urgent need to investigate effective and safe novel targeted therapies against KRAS G12D-driven NSCLC.To evaluate the anticancer effect of SalF, we used KRAS-overexpressing lung cancer cells in vitro, a subcutaneous transplant tumor model, and KRAS G12D mice model in vivo. Then, the binding effect of SalF and KRAS was investigated using molecular docking, proteolytic assays and protein thermal shift assays. More critically, the PI3K/AKT signaling pathway in the lung was investigated utilizing RT-qPCR and Western Blotting.This is the first study to evaluate the anticancer effect of SalF on KRAS-overexpressing lung cancer cells or KRAS G12D lung tumors in vivo. We demonstrated that SalF inhibits OE-KRAS A549 cell migration, proliferation and promotes apoptosis in vitro. In addition, we used a subcutaneous transplant tumor model to show that SalF suppresses the growth of lung cancer cells in vivo. Interestingly, our group found that SalF was strongly bound to G12D and could decrease the stability and promoted the degradation of the KRAS G12D mutant through molecular docking, proteolytic assays and protein thermal shift assays. Further research demonstrated that in the KrasG12D mice model, after SalF treatment, the number and size of mouse lung tumors were significantly reduced. More importantly, SalF can promote apoptosis by inhibiting downstream PI3K/AKT signaling pathway activation.SalF activated apoptosis signaling pathways, suppressed anti-apoptotic genes, and inhibited lung cancer cell growth. These datas suggested that SalF could effectively inhibit the growth of lung tumors with KRAS G12D mutation. SalF may be a novel inhibitor against KRAS G12D, providing a strong theoretical basis for the clinical treatment of lung cancer with KRAS mutations.
To elucidate whether microRNA-374b could participate in the development of lung cancer (LC) through downregulating PTEN (gene of phosphate and tensin homolog deleted on chromosome ten) expression via activating PI3K/Akt pathway.Expression levels of microRNA-374b and PTEN in LC tissues and adjacent normal tissues were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Moreover, the expression level of microRNA-374b in LC cell lines was detected as well. The microRNA-374b inhibitor was constructed and transfected to downregulate microRNA-374b expression in A549 and H358 cells. The regulatory effects of microRNA-374b on migratory and proliferative capacities of LC cells were explored by wound healing and cell counting kit-8 (CCK-8) assay, respectively. After co-transfection of microRNA-374b inhibitor and si-PTEN in LC cells, expression levels of PTEN/PI3K/Akt were determined by qRT-PCR and Western blot.QRT-PCR results showed that microRNA-374b expression was higher, while PTEN expression was lower in LC tissues than adjacent tissues. Identically, microRNA-374b was also highly expressed in LC cell lines. PTEN expression was negatively correlated with microRNA-374b expression in LC. The downregulation of microRNA-374b in A549 and H358 cells inhibited their migratory and proliferative potentials. Subsequently, we verified that microRNA-374b could bind to PTEN through dual-luciferase reporter gene assay. MicroRNA-374b could inhibit PTEN expression and activate the PI3K/Akt pathway. Furthermore, PTEN knockdown enhanced migratory and proliferative abilities of LC cells, which were attenuated by co-transfection of microRNA-374b inhibitor.MicroRNA-374b promotes the development of LC by downregulating PTEN expression through activating PI3K/Akt pathway.
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