Arisaema heterophyllum Blume Monomer Stigmasterol Targets PPAR γ and Inhibits the Viability and Tumorigenicity of Lung Adenocarcinoma Cells NCI-H1975

Abstract

To clarify the regulatory effect and molecular mechanism of Arisaema heterophyllum Blume (AhBl) monomer stigmasterol on lung adenocarcinoma in human lung adenocarcinoma cells NCI-H1975 cultured in vitro and in nude mice. Oil red O staining, free fatty acid detection, adenosine triphosphate (ATP), and NADPH were applied to elucidate the regulatory effect of stigmasterol on the energy metabolism of NCI-H1975 cells. Simultaneously, colony formation assay and nude mouse tumorigenesis were performed to clarify the underlying mechanisms of stigmasterol on the proliferation and tumorigenesis of NCI-H1975 cells. Furthermore, peroxisome proliferator-activated receptor gamma (PPARγ) inhibitor GW9662 was supplemented to determine the expression changes of cyclins to clarify the regulation mechanism of stigmasterol. The results revealed that stigmasterol administration markedly inhibited the viability but promoted lipid deposition of NCI-H1975 cells. Meanwhile, the reduction of cell energy metabolism affected cell proliferation and colony formation. qPCR and western blot assays indicated that stigmasterol played a role in regulating the expression of cyclins and PPARγ signaling pathway proteins. Nude mouse tumorigenesis suggested that tumor size and weight in the stigmasterol-treated group were apparently lower as compared with the control group. Tumor tissue cells developed varying degrees of degeneration and large areas of ischemic necrosis presented in the central and peripheral cells. Immunohistochemistry results revealed that Ki67 expression in the stigmasterol group was substantially inhibited, while PPARγ expression was greatly elevated as compared with the control. GW9662 could mediate the inhibitory effect of stigmasterol on NCI-H1975 cells. The current study demonstrated that stigmasterol targeted PPARγ and inhibited the viability and tumorigenicity of lung adenocarcinoma cells NCI-H1975.

Figure 1

Network pharmacology and molecular docking analyzes the regulatory relationship between AhBl-stigmasterol-lung adenocarcinoma-PPARγ. (a) Venn diagram of AhBl drug targets and lung adenocarcinoma-related targets. (b) Network pharmacological interaction diagram. (c) Molecular docking results. Stigmasterol is the main active component of AhBl. TNX indicates AhBl; TNX1 is 8,11,14-docosatrienoic acid, methyl ester; TNX2 is [(2R)-2-[[[(2R)-2-(benzoylamino)-3-phenylpropanoyl]amino]methyl]-3-phenylpropyl] acetate; TNX4 is sitosterol.

Figure 2

Stigmasterol inhibits the viability and energy metabolism of NCI-H1975 cells. (a) MTT detection. (b) Oil red O staining, magnification of 100x. (c) Free fatty acid detection. (d) ATP detection. (e) NADPH detection. (f) Cell colony formation assay. Compared with the control group, ^#p < 0.05; ^##p < 0.01.

Figure 3

qPCR and western blot assays detects the expressions of cyclins and PPARγ. (a) qPCR detects the expressions of cyclinD1, CDK2, CDK4, CDK6, and p21 and the mRNA expressions of PPARγ-SIRT1 pathway proteins PPARγ and SIRT1. (b–c) Western blot detects the expressions of cyclinD1, CDK2, CDK4, CDK6, p21, and acetyl-p53 and the expressions of PPARγ-SIRT1 pathway proteins PPARγ and SIRT1. Compared with the control group, ^#p < 0.05; ^##p < 0.01.

Figure 4

Stigmasterol inhibits tumorigenesis of NCI-H1975 cells in nude mice. (a) Nude mouse tumorigenicity assay, vector, and tumors in vitro. (b) Tumor volume records and statistical results. (c) Tumor weight measurement results. (d) HE staining and immunohistochemical staining, magnification of 100x. Compared with the control group, ^#p < 0.05.

Figure 5

PPARγ inhibitor alleviates the anticancer effect induced by stigmasterol. The cells were co-treated with high concentrations of stigmasterol and GW9662 at 20 and 40 μmol/L, respectively, to detect the regulation of cell viability and metabolism. (a) Oil red staining, magnification of 100x. (b) Free fatty acid detection. (c) ATP detection. (d) NADPH detection. (e) Cell colony formation assay. (f) qPCR was applied to detect the expression of cyclinD, CDK2, CDK4, and CDK6. (g–h) Western blot was applied to detect the expression of cyclinD, CDK2. CDK4, and CDK6. Compared with the control group, ^#p < 0.05; ^##p < 0.01.