Ginsenoside compound K exerts antitumour effects in renal cell carcinoma via regulation of ROS and lncRNA THOR

Figures

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  Figure 1.. CK inhibits the proliferation of RCC cells. (A) The viability of Caki-1 and 786-O cells was measured via an MTT assay after exposure to various doses of CK for different times. (B) A colony formation assay was performed to measure the proliferative activity of RCC cells after treatment with various doses of CK. The data are presented as the mean ± SD of at least three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control group. CK, ginsenoside compound K; RCC, renal cell carcinoma.

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  Figure 2.. CK inhibits the migration and invasion of RCC cells and induces cell cycle arrest. (A) Wound healing assays were performed to measure the effects of CK on the migration of RCC cells. (B) Transwell invasion assays were performed to measure the effects of CK on the invasion of RCC cells. (C) Cells were treated with different doses of CK for 24 h, and the total cellular lysates were subjected to western blot analysis with the indicated antibodies. (D) Cells were treated with different doses of CK for 24 h, and cell cycle distribution was assayed by PI staining and flow cytometry analysis. The data are presented as the mean ± SD of at least three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control group. CK, ginsenoside compound K; RCC, renal cell carcinoma.

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  Figure 3.. CK induces caspase-dependent apoptosis of renal cell carcinoma cells. (A) Cells were treated with different doses of CK for 24 h, and cellular apoptosis was measured. (B) After incubation with different doses of CK, the expression levels of apoptosis-related proteins were measured via western blotting. (C) Cells were treated with different doses of CK for 12 h, and caspase-3 activity was measured. (D) Cells were treated with CK (40 µM) with or without z-VAD (10 µM) for 24 h, and cellular apoptosis was measured. The data are presented as the mean ± SD of at least three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control group. CK, ginsenoside compound K.

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  Figure 4.. CK treatment leads to the generation of ROS in renal cell carcinoma cells. (A) Intracellular ROS levels were measured by flow cytometry. (B) Cell viability was measured via an MTT assay. (C) Cell migration was measured by a wound healing assay. (D) Cell invasion was measured via a Transwell invasion assay. (E) Cellular apoptosis was measured. (F) The indicated proteins were measured via western blotting. (G) Caspase-3 activity was analysed. The data are presented as the mean ± SD of at least three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control group or as indicated. CK, ginsenoside compound K; ROS, reactive oxygen species.

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  Figure 5.. lncRNA THOR is downregulated in RCC cells after exposure to CK. (A) Expression of 15 lncRNAs in HK-2, Caki-1 and 786-O cells. (B) Expression levels of 15 lncRNAs in RCC cells before or after treatment with CK (40 µM). The data are presented as the mean ± SD of at least three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control group. NS, not significant; lncRNA, long non-coding RNA; CK, ginsenoside compound K; RCC, renal cell carcinoma; THOR, testis associated oncogenic.

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  Figure 6.. Silencing of THOR inhibits the tumorigenesis of renal cell carcinoma cells. (A) Cells were transfected as indicated for 24 h, and the expression of THOR was measured. (B) Cell viability was measured via an MTT assay after transfection. (C) Migration and invasion were measured after transfection. (D) Cellular apoptosis was measured after transfection. (E) Cellular lysates were subjected to western blotting analysis with the indicated antibodies after transfection. (F) Cell cycle distribution was analysed after transfection. The data are presented as the mean ± SD of at least three independent experiments. **P<0.01 and ***P<0.001 vs. control group. si-, small interfering RNA; NC, negative control; THOR, testis associated oncogenic.

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  Figure 7.. Upregulation of THOR inhibits the effects of CK on renal cell carcinoma cells. (A) Cells were transfected as indicated for 24 h, and the expression of THOR was measured. (B) Cell viability was measured via an MTT assay after transfection. (C) Migration and invasion were measured after transfection. (D) Cellular apoptosis was measured after transfection. (E) Cellular lysates were subjected to western blotting analysis with the indicated antibodies after transfection. (F) Cell cycle distribution was analysed after transfection. The data are presented as the mean ± SD of at least three independent experiments. *P<0.05, **P<0.01 and ***P<0.001. CK, ginsenoside compound K; THOR, testis associated oncogenic.

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  Figure 8.. CK inhibits the growth of RCC cells in tumour xenografts. BALB/c mice were ectopically implanted with RCC cells, and when the tumour size reached ~100 mm3, the mice were injected with CK (intraperitoneal) every 3 days for 30 days. (A) Effects of CK on tumour volume. (B) Effects of CK on mouse body weight. (C) H&E staining and immunohistochemical analysis of changes in Ki-67 expression in the harvested tumours after different treatments as indicated. Representative images are shown (magnification, ×40). (D) Western blotting analysis of cleaved caspase-3 expression in tumours collected from different groups. *P<0.05 and **P<0.01 vs. control group. CK, ginsenoside compound K; RCC, renal cell carcinoma; H&E, haematoxylin and eosin.