KHF16 is a Leading Structure from Cimicifuga foetida that Suppresses Breast Cancer Partially by Inhibiting the NF-κB Signaling Pathway

Abstract

Triterpenoids extracted from Cimicifuga foetida have been reported to inhibit cancer by inducing cell cycle arrest and apoptosis. In this study, KHF16 (24-acetylisodahurinol-3-O-β-D-xylopyranoside), a cycloartane triterpenoid isolated from the rhizomes of C. foetida, showed potent anti-cancer activity in multiple ERα/PR/HER2 triple-negative breast cancer (TNBC) cell lines. KHF16 significantly induces cell cycle G2/M phase arrest and apoptosis in both MDA-MB-468 and SW527 TNBC cell lines. KHF16 reduces the expression levels of XIAP, Mcl-1, Survivin and Cyclin B1/D1 proteins. Importantly, KHF16 inhibits TNFα-induced IKKα/β phosphorylation, IKBα phosphorylation, p65 nuclear translocation and NF-κB downstream target gene expression, including XIAP, Mcl-1 and Survivin, in TNBC cells. These results suggest that KHF16 may inhibit TNBC by blocking the NF-κB signaling pathway in part.

Keywords: Apoptosis; Cell cycle; Cycloartane triterpenoid; KHF16; NF-κB.; Triple negative breast cancer.

Figure 1

The identification of KHF16 as a potent anti-cancer compound in TNBC cell lines. A. The MCF7 and MDA-MB-231 breast cancer cell lines were treated with 42 different compounds (10 μM) extracted from Cimicifuga for two days. Cell viability was measured with the SRB assay. DMSO was used as the negative control. Doxorubicin was used as the positive control. The six compounds labeled with an asterisk were selected for further study. B. The chemical structure of KHF16. C. Five different TNBC breast cancer cell lines and MCF7 were treated with different concentrations (0-10 μM) of KHF16 for 48 h. Cell viability was measured with the SRB assay.

Figure 2

KHF16 suppresses DNA synthesis in the MDA-MB-468 and SW527 cell lines. A. KHF16 (5/10/20 μM) was used to treat the MDA-MB-468 and SW527 cells for 24 h. DNA synthesis was measured by the EdU assay. B. The quantitative data of panel A (MDA-MB-468 cells). Percentages of EdU-positive proliferating cells vs. total cells are shown. * p < 0.05, ** p < 0.01, t-test. C. Quantitative data of panel A (SW527 cells).

Figure 3

KHF16 induces cell cycle G2/M arrest in MDA-MB-468 and SW527. A. MDA-MB-468 and SW527 cells were treated with KHF16 (5/10/20 μM) for 24 h. The cell cycle distribution was analyzed by FlowJo software (version 7.6). B. KHF16 (10/20 μM) significantly increased the percentage of G2/M phase MDA-MB-468 cells compared to DMSO. ** p < 0.01. C. KHF16 (10/20 μM) significantly increased the percentage of G2/M phase SW527 cells compared to DMSO. ** p < 0.01.

Figure 4

KHF16 induces apoptosis in MDA-MB-468 and SW527. A. MDA-MB-468 and SW527 cell morphology changed dramatically after the cells were treated with KHF16 (20 μM) for 24 h. B. MDA-MB-468 and SW527 cells were treated with KHF16 (5/10/20 μM) for 24 h, stained with Annexin V/7AAD and analyzed by flow cytometry. Doxorubicin was used as the positive control. C. The quantitative data of panel B (MDA-MB-468 cells). The percentages of Annexin V-positive cells are shown. ** p < 0.01. D. The quantitative data of panel B (SW527 cells).

Figure 5

KHF16 decreases the protein expression levels of multiple cell cycle and apoptosis regulators in MDA-MB-468 and SW527. A. MDA-MB-468 and SW527 cells were treated with KHF16 (5/10/20 μM) for 24 h. Cell lysates were collected for WB to detect cleaved Caspase-3, -7, -8 and PARP, Survivin, XIAP, Mcl-1, Bcl-X_L, Cyclin B1, D1 and E1, p21, p27, p15 and p53. β-actin was used as the loading control. The quantification data is listed in Supplementary Figure 1. B. MDA-MB-468 and SW527 cells were treated with KHF16 (10 μM) for 0, 4, 8, 12 and 24 h. The cell lysates were collected for WB to detect cleaved Caspase-3 and PARP, XIAP, Mcl-1, Survivin, Bcl-X_L, Cyclin B1, D1, and E1, p21, p27, p15 and p53. β-actin was used as the loading control. The quantification data is listed in Supplementary Figure 1.

Figure 6

KHF16 blocks the TNFα-induced NF-κB signaling pathway and anti-apoptosis protein expression in MDA-MB-468 and SW527. A. MDA-MB-468 and SW527 cells were first treated with or without KHF16 (10 μM) for 4 h. Following that, TNFα (10 ng/ml) was added for 5 to 120 min. The cell lysates were collected for WB to detect the protein levels of p-IKKα/β, IKKα, IKKβ, p-IκBα(Ser32), IκBα, XIAP, Mcl-1, Survivin, Bcl-2, RelB, p65 and A20. GAPDH was used as the loading control. The quantification data is listed in Supplementary Figure 2. B. MDA-MB-468 and SW527 cells were treated with KHF16 and TNFα as described above, except that the TNFα treatment time points were different. p-JNK(Thr183/Tyr185), JNK, p-STAT3 (Tyr705), STAT3, p-AKT(Ser473), AKT, p-ERK(Thr202/Tyr204), ERK, p-p38(Thr180/Tyr182) and p38 protein levels were detected. The pAKT protein level was undetectable in SW527. The quantification data is listed in Supplementary Figure 2.

Figure 7

KHF16 decreases p-65 (RelA) nuclear translocation and blocks NF-κB activation. A. MDA-MB-468 and SW527 cells were first treated with or without KHF16 (10 μM) for 4 h. Then, TNFα (10 ng/ml) was added for 60 and 120 min. The nuclear and cytoplasmic fractions were collected for p-65 detection. Tubulin and Histone H3 were used as cytoplasmic and nuclear protein controls. B. Immunofluorescence staining of p-65 (RelA) in SW527 cells. SW527 cells were stimulated with TNFα (10 ng/ml) for 6 h. KHF16 (10 μM) blocked p-65 (red) nuclear translocation. Nucleic acid was stained with DAPI (blue). C. KHF16 (10 μM) blocked TNFα (10 ng/ml)-induced NF-κB activation. Luciferase reporter assays were used to detect the NF-κB activity in MDA-MB-468 and SW527 cells. ** p < 0.01.