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. 2025 May 27;16(8):2567-2577.
doi: 10.7150/jca.111718. eCollection 2025.

Theaflavin-3,3'-digallate triggers apoptosis in osteosarcoma cells via the caspase pathway

Yat-Yin Law  1   2   3 , Yi-Hsien Hsieh  1   4 , Yih-Shou Hsieh  2 , Yi-Hsun Lee  1 , Chia-Ling Tang  5 , Chia-Yi Lee  1 , Shun-Fa Yang  1   4 , Shu-Chen Chu  5 , Pei-Ni Chen  1   4
Affiliations

Theaflavin-3,3'-digallate triggers apoptosis in osteosarcoma cells via the caspase pathway

Yat-Yin Law et al. J Cancer. .

Abstract

Osteosarcoma is a cancer associated with a guarded prognosis. Various compounds can induce apoptosis in osteosarcoma cells. Theaflavin-3,3'-digallate (TF3) has been demonstrated to alter cell growth and induce apoptosis in various cancer cells. The present study investigated the apoptotic effect of TF3 on osteosarcoma cells. It further explored key apoptotic pathways activated by TF3. Viability of 143B and U2OS osteosarcoma cells after TF3 treatment was assessed. The effects of TF3 on key apoptotic pathways were analyzed. Furthermore, a xenograft mouse model of osteosarcoma was established for in vivo experiments. The results indicated that TF3 significantly reduced the viability of 143B and U2OS cells. Western blotting revealed that TF3 upregulated the expression of cleaved caspase-3 and cleaved caspase-9 in osteosarcoma cells. In addition, TF3 increased the levels of phosphorylated histone H2Ax, Bax, Bak1, and cytochrome c, while reducing the levels of Mcl-1 and survivin in osteosarcoma cells. Furthermore, TF3 significantly reduced the average tumor volume in the xenograft model. Overall, this study suggests that TF3 induces apoptosis in osteosarcoma cells, primarily by regulating the caspase pathway.

Keywords: apoptosis; caspase; osteosarcoma; reactive oxygen species; theaflavin-3,3'-digallate.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
The cell viability of osteosarcoma cells after TF3 treatment with different analysis. (A) The cell viability of 143B osteosarcoma cells after TF3 treatment according to MTT assay. (B) The cell viability of U2OS osteosarcoma cells after TF3 treatment according to MTT assay. (C) The apoptotoic effect of 143B osteosarcoma cells after TF3 treatment according to PI/AnnexinV flow cytometry. (D) The apoptotoic effect of U2OS osteosarcoma cells after TF3 treatment according to PI/AnnexinV flow cytometry. **P < 0.01 as compared with DMSO treatment (the 0 μM). ***P < 0.001 as compared with DMSO treatment (the 0 μM). ###P < 0.001 as compared with DMSO treatment (the 0 μM).
Figure 2
Figure 2
The mitochondria membrane potential changes of osteosarcoma cells after TF3 treatment. (A) The mitochondria membrane potential change of 143B osteosarcoma cells after TF3 treatment according to JC-1 green fluorescence stain. (B) The mitochondria membrane potential change of U2OS osteosarcoma cells after TF3 treatment according to JC-1 green fluorescence stain. **P < 0.01 as compared with DMSO treatment (the 0 μM). ***P < 0.001 as compared with DMSO treatment (the 0 μM).
Figure 3
Figure 3
The level of reactive oxygen species in osteosarcoma cells after TF3 treatment. (A) The reactive oxygen species amount of 143B osteosarcoma cells after TF3 treatment according to DHE assay. (B) The reactive oxygen species amount of U2OS osteosarcoma cells after TF3 treatment according to DHE assay. **P < 0.01 as compared with DMSO treatment (the 0 μM). ***P < 0.001 as compared with DMSO treatment (the 0 μM).
Figure 4
Figure 4
The cell viability with N-acetyl-L-cysteine treatment of osteosarcoma cells after TF3 treatment. (A) The cell viability with or without NAC treatment of 143B osteosarcoma cells after TF3 treatment according to MTT assay. (B) The cell viability with or without NAC treatment of U2OS osteosarcoma cells after TF3 treatment according to MTT assay.
Figure 5
Figure 5
The apoptotic pathway of osteosarcoma cells after TF3 treatment according to Western blotting analysis. (A) The expression of cleaved-caspase-3 and -9 of 143B osteosarcoma cells after TF3 treatment. (B) The expression of cleaved-caspase-3 and 9 of U2OS osteosarcoma cells after TF3 treatment. (C) The expression of phosphorylated p53 and phosphorylated H2Ax of 143B osteosarcoma cells after TF3 treatment. (D) The expression of phosphorylated p53 and phosphorylated H2Ax of U2OS osteosarcoma cells after TF3 treatment. (E) The expression of Bax, Bak1 and cytochrome C of 143B osteosarcoma cells after TF3 treatment. (F) The expression of Bax, Bak1 and cytochrome C of U2OS osteosarcoma cells after TF3 treatment. (G) The expression of Mcl-1 and survinin of 143B osteosarcoma cells after TF3 treatment. (H) The expression of Mcl-1 and survinin of U2OS osteosarcoma cells after TF3 treatment. *P < 0.05 as compared with DMSO treatment (the 0 μM). **P < 0.01 as compared with DMSO treatment (the 0 μM). ***P < 0.001 as compared with DMSO treatment (the 0 μM).
Figure 6
Figure 6
The cell viability and caspase-3 activity with caspase inhibitor treatment of osteosarcoma cells after TF3 treatment. (A) The cell viability with or without caspase inhibitor Z-VAD-FMK treatment of 143B and U2OS osteosarcoma cells after TF3 treatment according to MTT assay. (B) The caspase-3 activity with or without caspase inhibitor Z-VAD-FMK treatment of 143B and U2OS osteosarcoma cells after TF3 treatment according to MTT assay.
Figure 7
Figure 7
The xenograft model for the osteosarcoma proliferation in mice after TF3 treatment. (A) The average body weight change of mice after the xenograft of 143B osteosarcoma cells and TF3 treatment. (B) The average tumor volume change of mice after the xenograft of 143B osteosarcoma cells and TF3 treatment. (C) The gross appearance of 143B osteosarcoma tumor after TF3 treatment. (D) The appearance of 143B osteosarcoma tumor in mice after TF3 treatment. (E) The average tumor weight of mice after the xenograft of 143B osteosarcoma cells and TF3 treatment. (F) The Ki-67 expression of mice after the xenograft of 143B osteosarcoma cells and TF3 treatment. **P < 0.01 as compared with placebo group. ***P < 0.001 as compared with placebo group (the 0 μM).
Figure 8
Figure 8
The schematic graph of TF3-related pathway for apoptosis in osteosarcoma.
See this image and copyright information in PMC

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