Network pharmacology study to explore the multiple molecular mechanism of SH003 in the treatment of non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the leading causes of human death worldwide. Herbal prescription SH003 has been developed to treat several cancers including NSCLC. Due to the multi-component nature of SH003 with multiple targets and pathways, a network pharmacology study was conducted to analyze its active compounds, potential targets, and pathways for the treatment of NSCLC. METHODS: We systematically identified oral active compounds within SH003, employing ADME criteria-based screening from TM-MC, OASIS, and TCMSP databases. Concurrently, SH003-related and NSCLC-associated targets were amalgamated from various databases. Overlapping targets were deemed anti-NSCLC entities of SH003. Protein-protein interaction networks were constructed using the STRING database, allowing the identification of pivotal proteins through node centrality measures. Empirical validation was pursued through LC-MS analysis of active compounds. Additionally, in vitro experiments, such as MTT cell viability assays and western blot analyses, were conducted to corroborate network pharmacology findings. RESULTS: We discerned 20 oral active compounds within SH003 and identified 239 core targets shared between SH003 and NSCLC-related genes. Network analyses spotlighted 79 hub genes, including TP53, JUN, AKT1, STAT3, and MAPK3, crucial in NSCLC treatment. GO and KEGG analyses underscored SH003's multifaceted anti-NSCLC effects from a genetic perspective. Experimental validations verified SH003's impact on NSCLC cell viability and the downregulation of hub genes. LC-MS analysis confirmed the presence of four active compounds, namely hispidulin, luteolin, baicalein, and chrysoeriol, among the eight compounds with a median of > 10 degrees in the herb-compounds-targets network in SH003. Previously unidentified targets like CASP9, MAPK9, and MCL1 were unveiled, supported by existing NSCLC literature, enhancing the pivotal role of empirical validation in network pharmacology. CONCLUSION: Our study pioneers the harmonization of theoretical predictions with practical validations. Empirical validation illuminates specific SH003 compounds within NSCLC, simultaneously uncovering novel targets for NSCLC treatment. This integrated strategy, accentuating empirical validation, establishes a paradigm for in-depth herbal medicine exploration. Furthermore, our network pharmacology study unveils fresh insights into SH003's multifaceted molecular mechanisms combating NSCLC. Through this approach, we delineate active compounds of SH003 and target pathways, reshaping our understanding of its therapeutic mechanisms in NSCLC treatment.

Depletion of phosphatidylinositol 4-phosphate at the Golgi translocates K-Ras to mitochondria.

Ras proteins are small GTPases localized to the plasma membrane (PM), which regulate cellular proliferation, apoptosis and differentiation. After a series of post-translational modifications, H-Ras and N-Ras traffic to the PM from the Golgi via the classical exocytic pathway, but the exact mechanism of K-Ras trafficking to the PM from the ER is not fully characterized. ATP5G1 (also known as ATP5MC1) is one of the three proteins that comprise subunit c of the F0 complex of the mitochondrial ATP synthase. In this study, we show that overexpression of the mitochondrial targeting sequence of ATP5G1 perturbs glucose metabolism, inhibits oncogenic K-Ras signaling, and redistributes phosphatidylserine (PtdSer) to mitochondria and other endomembranes, resulting in K-Ras translocation to mitochondria. Also, it depletes phosphatidylinositol 4-phosphate (PI4P) at the Golgi. Glucose supplementation restores PtdSer and K-Ras PM localization and PI4P at the Golgi. We further show that inhibition of the Golgi-localized PI4-kinases (PI4Ks) translocates K-Ras, and PtdSer to mitochondria and endomembranes, respectively. We conclude that PI4P at the Golgi regulates the PM localization of PtdSer and K-Ras.This article has an associated First Person interview with the first author of the paper.

Inhibition of Acid Sphingomyelinase Depletes Cellular Phosphatidylserine and Mislocalizes K-Ras from the Plasma Membrane.

K-Ras must localize to the plasma membrane for biological activity; thus, preventing plasma membrane interaction blocks K-Ras signal output. Here we show that inhibition of acid sphingomyelinase (ASM) mislocalizes both the K-Ras isoforms K-Ras4A and K-Ras4B from the plasma membrane to the endomembrane and inhibits their nanoclustering. We found that fendiline, a potent ASM inhibitor, reduces the phosphatidylserine (PtdSer) and cholesterol content of the inner plasma membrane. These lipid changes are causative because supplementation of fendiline-treated cells with exogenous PtdSer rapidly restores K-Ras4A and K-Ras4B plasma membrane binding, nanoclustering, and signal output. Conversely, supplementation with exogenous cholesterol restores K-Ras4A but not K-Ras4B nanoclustering. These experiments reveal different operational pools of PtdSer on the plasma membrane. Inhibition of ASM elevates cellular sphingomyelin and reduces cellular ceramide levels. Concordantly, delivery of recombinant ASM or exogenous ceramide to fendiline-treated cells rapidly relocalizes K-Ras4B and PtdSer to the plasma membrane. K-Ras4B mislocalization is also recapitulated in ASM-deficient Neimann-Pick type A and B fibroblasts. This study identifies sphingomyelin metabolism as an indirect regulator of K-Ras4A and K-Ras4B signaling through the control of PtdSer plasma membrane content. It also demonstrates the critical and selective importance of PtdSer to K-Ras4A and K-Ras4B plasma membrane binding and nanoscale spatial organization.

Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function.

Aberrant signaling by oncogenic mutant rat sarcoma (Ras) proteins occurs in ∼15% of all human tumors, yet direct inhibition of Ras by small molecules has remained elusive. Recently, several small-molecule ligands have been discovered that directly bind Ras and inhibit its function by interfering with exchange factor binding. However, it is unclear whether, or how, these ligands could lead to drugs that act against constitutively active oncogenic mutant Ras. Using a dynamics-based pocket identification scheme, ensemble docking, and innovative cell-based assays, here we show that andrographolide (AGP)--a bicyclic diterpenoid lactone isolated from Andrographis paniculata--and its benzylidene derivatives bind to transient pockets on Kirsten-Ras (K-Ras) and inhibit GDP-GTP exchange. As expected for inhibitors of exchange factor binding, AGP derivatives reduced GTP loading of wild-type K-Ras in response to acute EGF stimulation with a concomitant reduction in MAPK activation. Remarkably, however, prolonged treatment with AGP derivatives also reduced GTP loading of, and signal transmission by, oncogenic mutant K-RasG12V. In sum, the combined analysis of our computational and cell biology results show that AGP derivatives directly bind Ras, block GDP-GTP exchange, and inhibit both wild-type and oncogenic K-Ras signaling. Importantly, our findings not only show that nucleotide exchange factors are required for oncogenic Ras signaling but also demonstrate that inhibiting nucleotide exchange is a valid approach to abrogating the function of oncogenic mutant Ras.

Therapeutic levels of the hydroxmethylglutaryl-coenzyme A reductase inhibitor lovastatin activate ras signaling via phospholipase D2.

Hydroxmethylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) lower serum cholesterol but exhibit pleiotropic biological effects that are difficult to ascribe solely to cholesterol depletion. Here, we investigated the effect of lovastatin on protein prenylation and cell signaling. We show that high concentrations (50 µM) of lovastatin inhibit Ras, Rho, and Rap prenylation but that therapeutic levels of lovastatin (50 nM to 500 nM) do not. In contrast, depletion of cellular cholesterol by therapeutic levels of lovastatin increased Ras GTP loading and mitogen-activated protein kinase (MAPK) activation in human umbilical vein endothelial cells and rodent fibroblasts. Elevated Ras signaling was not seen in statin-treated cells if cholesterol levels were maintained by supplementation. Activation of Ras-MAPK signaling was a consequence of, and dependent on, activation of phospholipase D2 (PLD2). Expression of dominant interfering PLD2 or biochemical inhibition of PLD2 abrogated Ras and MAPK activation induced by lovastatin. In contrast, ectopic expression of wild-type PLD2 enhanced Ras and MAPK activation in response to therapeutic levels of lovastatin. Statin-induced cholesterol depletion also modestly activated the epidermal growth factor receptor (EGFR), resulting in downregulation of EGFR expression. These results suggest that statins modulate key cell signaling pathways as a direct consequence of cholesterol depletion and identify the EGFR-PLD2-Ras-MAPK axis as an important statin target.