An Anti-Cancer Peptide LVTX-8 Inhibits the Proliferation and Migration of Lung Tumor Cells by Regulating Causal Genes' Expression in p53-Related Pathways

Abstract

Spider venom has been found to show its anticancer activity in a variety of human malignancies, including lung cancer. In this study, we investigated the anti-cancer peptide toxin LVTX-8, with linear amphipathic alpha-helical conformation, designed and synthesized from the cDNA library of spider Lycosa vittata. Multiple cellular methods, such as CCK-8 assay, flow cytometry, colony formation assay, Transwell invasion and migration assay, were performed to detect peptide-induced cell growth inhibition and anti-metastasis in lung cancer cells. Our results demonstrated that LVTX-8 displayed strong cytotoxicity and anti-metastasis towards lung cancer in vitro. Furthermore, LVTX-8 could suppress the growth and metastasis of lung cancer cells (A549 and H460) in nude mouse models. Transcriptomics, integrated with multiple bioinformatics analysis, suggested that the molecular basis of the LVTX-8-mediated inhibition of cancer cell growth and metastasis manifested in two aspects: Firstly, it could restrain the activity of cancer cell division and migration through the functional pathways, including "p53 hypoxia pathway" and "integrin signaling". Secondly, it could regulate the expression level of apoptotic-related proteins, which may account for programmed apoptosis of cancer cells. Taken together, as an anticancer peptide with high efficiency and acceptable specificity, LVTX-8 may become a potential precursor of a therapeutic agent for lung cancer in the future.

Keywords: LVTX-8; cytotoxicity; migration; nude mouse model; peptide; transcriptomics.

Figure 1

Purification and molecular mass determination of LVTX-8. (A) The purification of LVTX-8 using RP-HPLC. (B) The molecular weight of LVTX-8 was determined by MALDI-TOF MS.

Figure 2

The effect of LVTX-8 on cell cytotoxicity and growth. Effect of LVTX-8 on A549, H460 (A) and HEK293T cells (B). The cytotoxic activities were detected by CCK-8 assay after treatment with LVTX-8 for 24 h (C). A549 and H460 cells apoptosis treatment with LVTX-8 was achieved by flow cytometry. Colony formation of A549 and H460 cells treated with LVTX-8 was evaluated (D). The situation of A549 and H460 colony formation with 2.5 μM or 5 μM LVTX-8 treatment. Quantitative results of A549 (E) and H460 (F) colony are illustrated (n = 3).

Figure 3

LVTX-8 inhibited A549 and H460 cells’ migration and invasion, detected by Transwell chamber assay. Representative images and statistical analysis of migrated A549 cells (A) and H460 cells (C) in the Transwell migration assay (n = 3). Representative images and statistical analysis of invaded A549 cells (B) and H460 cells (D) in the Transwell invasion assay (n = 3).

Figure 4

The influence of LVTX-8 on the growth of A549 and H460 in xenograft tumors. (A) Images of the nude mice and their xenograft tumors at 32 d after injection (n = 5). (B) In situ labeling (TUNEL) examination of nude mice tumor tissues. Dynamic volume of xenograft tumors at different times after injection, for A549 xenograft model (C) and H460 xenograft model (D). Weight of xenograft tumors at the 32nd day after injection, A549 (E) and H460 (F).

Figure 5

LVTX-8 inhibited metastatic lesion formation. (A) Representative lung imaging of nude mice injected with H460 (upper) and A549 (lower) cells. (B) H&E examination of nude mice lung tissues that received different treatments. Phosphate buffer saline (PBS) (left), LVTX-8 (right). (C,D) Quantification of H460 (left grouping) and A549 (right grouping) lung metastatic colony formation of the lung metastasis in the nude mice model (n = 5). (E,F) The Kaplan–Meier method is used to assess the survival time of animals. The survival times of H460 (left grouping) and A549 (right grouping) mice in the LVTX-8-treated group were significantly longer than those in control group (n = 6).

Figure 6

Enrichment analysis based on differentially expressed genes (DEGs) identified in 2 µM and 5 µM LVTX-8-treated samples. (A,C) show the enrichment analysis results based on DEGs between 2 µM LVTX-8-treated and control samples, while (B,D) show the enrichment analysis results based on DEGs between 5 µM LVTX-8-treated and control sample.

Figure 7

Result of Reactome pathway analysis. (A,B) showed the main biological processes and hierarchical structure of Reactome pathways (p < 0.05) after 2 µM and 5 µM LVTX-8 treatment, respectively.

Figure 8

Result of gene set enrichment analysis (GSEA). (A) Enrichment plot of P53 hypoxia between 2 µM LVTX-8-treated and control samples. (B) Heatmap of get sets in P53 hypoxia pathway based on transcripts expression. (C) Enrichment plot of apoptotic cleavage of cellular proteins between 5 µM LVTX-8-treated and control samples. (D) Heatmap of get sets in apoptotic cleavage of cellular proteins based on transcript expression. (E) Enrichment plot of integrin signaling between 5 µM LVTX-8-treated and control samples. (F) Heatmap of get sets in integrin signaling based on transcript expression.