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The Triterpenoids of Hibiscus Syriacus Induce Apoptosis and Inhibit Cell Migration in Breast Cancer Cells

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The Triterpenoids of Hibiscus Syriacus Induce Apoptosis and Inhibit Cell Migration in Breast Cancer Cells

Ren-Jun Hsu et al. BMC Complement Altern Med.

Abstract

Background: Breast cancer-related mortality increases annually. The efficacy of current breast cancer treatments is limited, and they have numerous side effects and permit high recurrence. Patients with estrogen receptor (ER)-negative or triple-negative breast cancer are particularly difficult to treat. Treatment for this type of cancer is lacking, and its prognosis is poor, necessitating the search for alternative treatments.

Methods: This study screened Chinese herb Hibiscus syriacus extracts and identified a novel anti-cancer drug for patients with ER-negative breast cancer. The inhibitory effects on cell viability and migration were evaluated for each compound, and the molecular regulatory effects were evaluated on both mRNA and protein levels.

Result: We found several triterpenoids including betulin (K02) and its derivatives (K03, K04, and K06) from H. syriacus inhibited human triple-negative breast cancer cell viability and migration but revealed smaller cytotoxic effects on normal mammalian epithelial cells. Betulin and its derivatives induced apoptosis by activating apoptosis-related genes. In addition, they activated p21 expression, which induced cell cycle arrest in breast cancer cells. Betulin (K02) and betulinic acid (K06) had stronger inhibitory effects on cell viability and migration than K03 and K04.

Conclusions: H. syriacus extracts might inhibit breast cancer cell viability and induce apoptosis by activating p53 family regulated pathways and inhibiting AKT activation. H. syriacus extracts may provide important insight into the development of novel alternative therapies for breast cancer.

Figures

Figure 1
Figure 1
The effect of H. syriacus skin crude extracts and pure compounds on the cell viability of MDA-MB-231 and HBL100 cell lines. MDA-MB-231(A) and HBL100 (B) were treated with 0, 1, 10, 25, 50, or 100 μg/mL crude extracts for 48 h and subjected to MTT assay to analyze the cell viability of MDA-MB-231 and HBL100 cell lines. (A) MDA-MB-231 cells treated with 100 μg/ml HISY-F2 or HISY-F3 showed 20% cell viability compared with DMSO controls (0.1% DMSO). MDA-MB-231 cells treated with 50 μg/mL of HISY-F3 crude extract showed a cell viability of <50% compared with DMSO controls. (B) Cell viability of HBL-100 cells treated with 50 μg/mL of HISY-F2 or HISY-F3 was 20% of DMSO controls, and the cell viability of HBL-100 cells treated with 100 μg/mL of HISY-F2 or HISY-F3 was less than 20% of that of DMSO controls. (C) Cell viability decreased by 5 μg/mL treatment with K01, K02, K03, K04, or K06 compared with DMSO controls; 10 μg/mL of K02 or K06 reduced cell viability to <50% compared with DMSO controls. (D) Cell viability decreased by 1 μg/mL treatment with K01, K02, K03, K04, or K06 compared with DMSO controls; 10 μg/mL of K01, K02, K03, K04 or K06 reduced cell viability to <50% compared with DMSO controls.
Figure 2
Figure 2
Betulin and its derivatives cause morphological changes in HBL100 cells. After treating HBL-100 cells with 10 μg/mL of K02 (B), K03 (C), K04 (D) or K06 (E) for 48 h, cell morphology changed to star-shaped and linear-shaped with vacuolation, and cell density significantly decreased. In the DMSO control group (A, 0.1% DMSO), cell shapes were intact and flat with higher density.
Figure 3
Figure 3
Betulin and its derivatives inhibit cell migration in the MDA-MB-231 cell line. A wound healing assay was performed to determine the cell migration ability. MDA-MB-231 cells were treated with 0.1% DMSO (A, B, C) or 5 μg/mL K02 (D, E, F), K03 (G, H, I), K04 (J, K, L), or K06 (M, N, O) for 24 h. The cells were observed every 12 h under the same observation field for wound healing status. At 12 h, cell migratory abilities in K03- (H) and K04-treated (K) cells slightly decreased compared with DMSO controls (B). In K02- (F) and K06-treated (N) cells, cell migratory abilities more obviously decreased compared with DMSO controls (B). At 24 h, cell migratory abilities in the K02 (L), K03 (H), K04 (K), and K06 (O) groups significantly decreased compared with DMSO controls (B), and the wound area was larger than that of DMSO controls (C). The wound area was quantified by ImageJ software. Compared with 12 h of DMSO treatment, the wound area was slightly significantly larger (* p < 0.05) in the K03- and K04-treated groups and more significantly larger in the K02- and K06-treated groups (** p < 0.01). Compared with 24 h of DMSO treatment, the wound area was significantly larger (# p < 0.05) in the K03- and K04-treated groups and also significantly larger in the K02- and K06-treated groups (## p < 0.01).
Figure 4
Figure 4
K02, K03, K04, and K06 induce the expression of apoptosis-related genes in the HBL-100 cell line. (A) After treating HBL-100 cells with 10 μg/mL of K02, K03, K04, or K06 for 0, 12, 24, or 36 h, western blot was performed to analyze apoptosis-related proteins. β-Actin was used as the internal control for relative quantitation. Cleaved caspase-3 increased with treatment time to a peak at 24 h and decreased at 36 h under K02 and K06 treatment. Cleaved PARP also increased with treatment time to a peak at 24 h under K02 and K06 treatment. Bax expression was significantly increased at 36 h with all four compounds. Bcl-x expression decreased with treatment time, though without significance.(B) After treating HBL-100 cells with 10 μg/mL of K02, K03, K04, or K06 for 0, 12, 24, or 36 h, western blot was performed to analyze p53, p21, p-AKT and total AKT. p53 and p21 expression were induced after treatment with K02, K03 or K06 and increased with treatment time with all four compounds. p-AKT progressively decreased after treatment with K02 and K06, while total AKT showed no changes.

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