Cyproheptadine inhibits in vitro and in vivo lung metastasis and drives metabolic rewiring

Abstract

Background: Non-small cell lung cancer (NSCLC) accounts for 81% of lung cancer cases, among which over 47% presented with distant metastasis at the time of diagnosis. Despite the introduction of targeted therapy and immunotherapy, enhancing the survival rate and overcoming the development of resistance remain a big challenge. Thus, it is crucial to find potential new therapeutics and targets that can mitigate lung metastasis and investigate its effects on biomarkers, such as cellular metabolomics. In the current study, we investigated the role of cyproheptadine (CPH), an FDA-approved anti-histamine drug in lung metastasis in vitro and in vivo.

Methods and results: CPH showed potent cytotoxicity on different lung cancer cell lines in vitro. Moreover, CPH decreased invasion and migration of LLC1 and A549 cells in Matrigel invasion transwell and plate scratch assays. The in vivo LLC1 syngeneic lung cancer model found decreased number of metastatic nodules on the surface of lungs of Setd7 KO mice compared to SETD7 WT. CPH treatment resulted in decreased growth of LLC1 subcutaneous tumors compared to untreated SETD7 WT. Finally, metabolomic study of tumor tissues showed rewiring of metabolomic pathways and downregulation of amino acids, such as arginine, serine, and glycine) in Setd7 KO and WT treated with CPH compared to untreated Setd7 WT mice.

Conclusion: These findings identify CPH as a potential therapeutic agent to block metastasis in advanced NSCLC and suggest SETD7 as a potential target for the prevention of lung metastasis.

Keywords: Lung cancer; Metabolomics; Metastasis; Syngeneic mouse model.

Fig. 1

CPH-mediated cytotoxicity and regulation of Setd7-target genes in different lung cancer cell lines. a Cell viability of LLC1 cells after various concentrations of CPH treatment for 24-, 48- and 72-h; b Cell viability of the A549 cells after various concentrations of CPH treatment for 24-, 48- and 72-h. Cytotoxicity was determined by the MTS assay; c SETD7 protein expression in different lung cell lines quantified by Western Blot. The protein expressions were shown as fold expression compared to the normal lung cell lines Beas-2b. GAPDH was used as the housekeeping protein for normalization. All data are presented as the means ± SD of three independent experiments. *P < 0.05 indicates significant differences between the treatment groups and the control group. The student’s t-test was used to calculate the significance of the differences compared with the control

Fig. 2

CPH inhibits cell migration and invasion and down-regulates MMPs gene expression in LLC1 and A549 cell lines. a Cell migration of LLC1 and A549 cells treated with different concentrations of CPH for 24 h using plate scratch assay (n = 3); b a representative image of plate scratch assay for LLC1 cells treated with different concentrations of CPH for 24 h. The average width of the scratch line was calculated with Image-J; c Transwell Matrigel cell invasion assay of serum-starved LLC1 and A549 cells after treatment of different concentrations of CPH for 24 h. Cell invasion was calculated by fluorescence plate reader of the eluted cells from the membrane and normalized against the control (0.1% DMSO). The assay was repeated 3 times (n = 3) and the mean ± SD; d MMPs mRNA expression in LLC1 cells treated with different concentrations of CPH for 24 h measured by RT-PCR. mRNA expressions were shown as fold change compared to the control group (0.1% DMSO). All data are presented as the means ± SD of three independent experiments. *P < 0.05 indicates significant differences between the treatment groups and the control group. The student’s t-test was used to calculate the significance of the differences compared with the control

Fig. 3

Setd7 KO and CPH regulate tumor growth and metastasis in the lungs in vivo. a Exponential tumor growth developed by LLC1 cells s.c. injection in Setd7 KO and Setd7 WT ± CPH presented as mm³ volume measured by caliper. Tumor volume was calculated following the formula: ½ × length × (width)² (n = 8); b Tumor weight at the end of the study (n = 8); c Representative images for tumors of Setd7 WT mice treated with CPH vs vehicle; d Number of surface metastatic nodules on left lung counted under microscope (n = 8); e Representative images of H&E stained lung tissues of Setd7 WT and KO mice under electron microscope with 2× and 10× magnification power. Setd7 WT lung tissues show upregulated cell growth and large tumor/metastatic nodules (red arrows) compared to Setd7 KO lung tissues; f mRNA expression of metastasis-related genes for tumor tissues of Setd7 WT (control group), Setd7 KO, and Setd7 WT treated with CPH. mRNA expression was measured by RT-PCR and shown as fold change compared to Setd7 WT (control group). mRNA expression was measured for three samples per group (n = 3), and the mean ± SD were plotted. (Color figure online)

Fig. 4

Setd7 KO drives metabolomic rewiring in tumor tissues. a Heatmap showing the top regulated metabolites in tumor tissues of Setd7 KO vs WT; b Metabolism pathway analysis; c Metabolites of glycerophospholipid metabolism pathway; d Quantified amino acids level in tumor tissues. All metabolites were assayed using LC/MS/MS of three replicates (n = 3). The metabolomic study was analyzed using Metaboanalyst 5 software

Fig. 5

Setd7 inhibitor CPH drives metabolomic rewiring in tumor tissues. a Heatmap showing the top regulated metabolites in tumor tissues of Setd7 WT treated with vehicle and CPH; b Metabolism pathway analysis; c Quantified amino acids level in tumor tissues. All metabolites were assayed using LC/MS/MS of three replicates (n = 3). The metabolomic study was analyzed using Metaboanalyst 5 software