Metochalcone induces senescence-associated secretory phenotype via JAK2/STAT3 pathway in breast cancer

Abstract

Breast and lung cancers are the leading causes of mortality and most frequently diagnosed cancers in women and men, respectively, worldwide. Although the antitumor activity of chalcones has been extensively studied, the molecular mechanisms of isoliquiritigenin analog 2', 4', 4-trihydroxychalcone (metochalcone; TEC) against carcinomas remain less well understood. In this study, we found that TEC inhibited cell proliferation of breast cancer BT549 cells and lung cancer A549 cells in a concentration-dependent manner. TEC induced cell cycle arrest in the S-phase, cell migration inhibition in vitro, and reduced tumor growth in vivo. Moreover, transcriptomic analysis revealed that TEC modulated the activity of the JAK2/STAT3 and P53 pathways. TEC triggered the senescence-associated secretory phenotype (SASP) by repressing the JAK2/STAT3 axis. The mechanism of metochalcone against breast cancer depended on the induction of SASP via deactivation of the JAK2/STAT3 pathway, highlighting the potential of chalcone in senescence-inducing therapy against carcinomas.

Keywords: Breast cancer; JAK2/STAT3; Lung cancer; Metochalcone; SASP.

Figure 1. TEC inhibited cell proliferation and migration, and reduced cell cycle arrest in BT549 and A549 cells. (A) The molecular structure of TEC (PubChem CID: 6063342). (B and C) The CCK-8 method showed TEC inhibited proliferation at 24 h and 48 h. (D and E) TEC induced cell cycle arrest in BT549 and A549 cells at 24 h. (F and G) TEC suppressed cell migration in wound healing assays at low concentrations (200×). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2. TEC inhibited migration (A), invasion (B) in BT549 and A549 cells. The red scale bar represents 10 μM. ***p < 0.001.

Figure 3. The results of RNA-sequencing in BT549 and A549 cells at TEC (20 μM) for 24 h treatment. (A and B) Volcano map of different expression genes (DEGs) in BT549 and A549 cells, with the threshold | log₂(fold change) | > 2 and adjusted p value < 0.05. (C) Veen diagram showed the common DGEs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis via R package clusterProlifer was implemented to the DGEs in BT549 (D) and A549 cell (E), respectively. Veen diagram implied common shared KEGG pathways between BT549 and A549 after TEC treatment. (F) Veen diagram implied common shared KEGG pathways. The pathway activity analysis with two-tail t-test of expression matrix in BT549 (G) and A549 cells (H), respectively. Color gradient in the heatmap represents the pathway activity score.

Figure 4. TEC inhibited JAK2/STAT3 signaling cascade. (A and B) Western blotting showed the protein expression after TEC treatment for 24 h in BT-549 and A549 cells. (C) The affinity heatmap between STAT3 proteins and STAT3 inhibitors were revealed by Autodock Vina. (D and E) The docking pattern between TEC (pink) and STAT3 proteins (cyan) was visualized by Pymol. (F) MST analysis revealed the affinity of TEC towards STAT3 protein (Kd = 9.85 ± 4.38 μM). (G) RT-PCR analysis of STAT3 mRNA expression with or without TEC treatment. ***p < 0.001.

Figure 5. TEC induced senescence-associated secretory phenotype via JAK2/STAT3 pathway. (A) β-galactosidase staining (200×). (B) ELISA detected senescence associated secretions in cell supernatant after TEC treatment for 24 h. (C) β-galactosidase staining after TEC treatment (20 μM) with or without IL-6 (50 ug/mL) or STATTIC (STA, 2 μM), 200×. (D) Pharmacological intervention of the JAK2/STAT3 pathway altered the TEC-mediated proliferating-inhibition effect with or without IL-6 (50 ug/mL) or STATTIC (STA, 2 μM). (E–G) ELISA detected senescence associated secretions in cell supernatant after TEC treatment and/or pharmacological treatment. (H and I) TEC enhanced expression of SASP molecular markers: P16, P21, P53, and JAK2/STAT3 signaling axis. **p < 0.01; ^#p < 0.0001.

Figure 6. TEC reduced tumor growth in the BT549 cells xenograft mice model (A–E). (A) The tumor photo of the TEC (40 mg/kg) and control groups (8 mice per group). (B–D) The tumor weights, tumor volumes and body weight of mice in two groups. (E) HE and IHC staining of tumor tissue sections (Scale bar = 100 μm). TEC shrinks tumor growth in A549 cells-derived xenograft mice model (F–J). (F) The tumor photo of the TEC and control groups (6 mice per group). (G–I) The tumor weights, tumor volumes and body weight of mice in two groups. (J) HE and IHC staining of tumor tissue sections (Scale bar = 100 μm). ns, p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.