Rotenone inhibited osteosarcoma metastasis by modulating ZO-2 expression and location via the ROS/Ca2+/AMPK pathway

Abstract

Background: Pulmonary metastases in osteosarcoma (OS) are associated with a poor prognosis. Rotenone has shown anti-cancer activity. However, its effects on metastasis and the underlying mechanisms remain unknown. This study investigated the potential use of Rotenone for OS treatment.

Methods: The effect of Rotenone and ROS/Ca²⁺/AMPK/ZO-2 pathway on metastasis and EMT was evaluated by Western blot, Transwell and Wound healing. Flow cytometer was employed to measure the intracellular Ros and Ca²⁺ levels. The subcellular location of ZO-2 was detected by IF, interaction between AMPK and ZO-2 were examined by Co-IP. Then, subcutaneous tumor and metastasis models were used to evaluate the function of Rotenone in OS metastasis.

Results: Rotenone-induced ROS led to increased intracellular Ca²⁺, which promoted the EMT of OS cells through activation of AMPK and ZO-2 nuclear translocation. Inhibition of ROS production decreased intracellular Ca²⁺, restraining AMPK activity. Knock-down of ZO-2 significantly suppressed the anti-metastasis effects of Rotenone in OS cells. Moreover, Rotenone elevated p-AMPK and ZO-2 expression but inhibited EMT and lung metastasis in vivo.Conclusion These results provide evidence supporting an anti-metastatic effect of Rotenone. These findings support the use of Rotenone in the prevention of OS metastasis.

Keywords: AMPK; Ca2+‌; ROS; Rotenone; ZO-2; metastasis; osteosarcoma; translocation‌.

Figure 1.

Rotenone-induced ROS inhibited OS metastasis. (A) IC50 of Rotenone in MG-63, U2OS and 143B cells was measured by MTT assay (0.02, 1.09 and 1.35 μM, respectively). (B) Intracellular ROS levels after OS cells were treated with Rotenone (0.02 and 1.09 μM Rotenone for MG-63 and U2OS respectively for 48 h) or Rotenone + NAC (10 mM). (C–E) Metastasis inhibition effects of Rotenone were measured by wound-healing and transwell assays. (F) Rotenone modulated OS cell AMPK activity, ZO-2 expression, and EMT, as determined via Western blot analysis of EMT-related markers. (G) Bar graph depicting quantitative analysis of Western blots. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.

Figure 2.

Rotenone inhibited Ca²⁺/AMPK/ZO-2 signaling. (A) Intracellular Ca²⁺ levels after OS cells were treated with Rotenone (0.02 and 1.09 μM Rotenone for MG-63 and U2OS respectively for 48 h) or Rotenone + NAC (10 mM). (B–D) Metastasis capacity of different groups was measured by wound-healing and transwell assays. (E) AMPK activity, ZO-2 expression, and EMT-related markers were determined by Western blot analysis of OS cells treated with the indicated conditions. (F) Bar graph depicting quantitative analysis of Western blots. ***p < 0.001 vs. control.

Figure 3.

Rotenone and AMPK modulated ZO-2 subcellular location. (A, B) Immunoblotting and IF analysis of the subcellular distribution of ZO-2 after Rotenone treatment (0.02 and 1.09 μM Rotenone for MG-63 and U2OS respectively for 48 h). (C, D) AMPK inhibitor Compound C (10 μM for 48 h) modulated ZO-2 nuclear translocation, as measured by immunoblotting and IF. (E) Co-IP was employed to reveal the interaction between AMPK and ZO-2.

Figure 4.

Rotenone inhibited OS progression in vivo. (A, B) Rotenone suppressed tumor growth over time and showed a synergistic effect with Doxorubicin. Tumors were measured on the indicated days using Vernier calipers. Body weights of tumor-bearing mice were measured. (C, D) The expression of p-AMPK and ZO-2 in xenograft tumor tissues was determined by immunoblotting and IHC. (A, E) Rotenone suppressed lung metastasis and exhibited higher efficacy when combined with Doxorubicin. n = 5. The black dots on the surface of the lungs indicate metastasis foci. *p < 0.05, ***p < 0.001 vs. control. Rotenone and Doxorubicin administration was described in Materials and Methods.

Figure 5.

ZO-2 expression was correlated with p-AMPK and metastasis in OS patients. (A) Expression of p-AMPK and ZO-2 was measured by IHC in metastatic and non-metastatic OS specimens. (B) The GEPIA database showed a positive correlation between p-AMPK and ZO-2 in sarcoma samples. (C) Working model illustrating Rotenone modulation of ZO-2 expression and location via the ROS/Ca²⁺/AMPK pathway.