In Vitro and In Vivo Anti-Cancer Activity of Lasiokaurin in a Triple-Negative Breast Cancer Model

Abstract

Due to its intricate heterogeneity, high invasiveness, and poor prognosis, triple-negative breast cancer (TNBC) stands out as the most formidable subtype of breast cancer. At present, chemotherapy remains the prevailing treatment modality for TNBC, primarily due to its lack of estrogen receptors (ERs), progesterone receptors (PRs), and human epidermal growth receptor 2 (HER2). However, clinical chemotherapy for TNBC is marked by its limited efficacy and a pronounced incidence of adverse effects. Consequently, there is a pressing need for novel drugs to treat TNBC. Given the rich repository of diverse natural compounds in traditional Chinese medicine, identifying potential anti-TNBC agents is a viable strategy. This study investigated lasiokaurin (LAS), a natural diterpenoid abundantly present in Isodon plants, revealing its significant anti-TNBC activity both in vitro and in vivo. Notably, LAS treatment induced cell cycle arrest, apoptosis, and DNA damage in TNBC cells, while concurrently inhibiting cell metastasis. In addition, LAS effectively inhibited the activation of the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway and signal transducer and activator of transcription 3 (STAT3), thus establishing its potential for multitarget therapy against TNBC. Furthermore, LAS demonstrated its ability to reduce tumor growth in a xenograft mouse model without exerting detrimental effects on the body weight or vital organs, confirming its safe applicability for TNBC treatment. Overall, this study shows that LAS is a potent candidate for treating TNBC.

Keywords: PI3K/Akt/mTOR; STAT3; isodon; lasiokaurin; triple-negative breast cancer.

Figure 1

LAS inhibited breast cancer cell proliferation. (A) Chemical structure of LAS. (B–D) Cell viability of MDA-MB-231, MDA-MB-468, and MCF7 was separately measured by MTT assay after LAS treatment. (E) Cell viability of MDA-MB-231 was measured by MTT assay after oridonin treatment. (F) Colony formation ability of MDA-MB-231 cells treated with LAS for 13 days. Data are presented as means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to control.

Figure 2

LAS induced cell cycle arrest in MDA-MB-231 cells. MDA-MB-231 cells were stained with PI after LAS treatment and the cell cycle analyzed by flow cytometry. Representative DNA fluorescence histograms of cell cycle distribution after 24 h (A) and 48 h (C) treatment were presented. Bar charts showed the percentage of different phases after 24 h (B) and 48 h (D) treatment.

Figure 3

LAS induced apoptosis and DNA damage in MDA-MB-231 cells. MDA-MB-231 cells were treated with LAS for 24 h (A) and 48 h (B), stained with Annexin V-FITC/PI, and cell apoptosis was analyzed by flow cytometry. (C) Representative flow cytometry Annexin V/PI data. *** p < 0.001, compared to control. (D) Cell extracts were prepared from MDA-MB-231 cells and immunoblotted with the indicated antibodies. β-Actin was used as an internal control.

Figure 4

LAS inhibited the migration and invasion of MDA-MB-231 cells. (A) Cell migration was measured by wound-healing assay. (B) Cell invasion ability was assessed by transwell invasion assay.

Figure 5

LAS inhibited PI3K/Akt/mTOR pathway and STAT3 in MDA-MB-231 cells. MDA-MB-231 cells were treated with LAS at concentrations of 2.5, 5, 10 μM for 24 or 48 h. Cell pellets collected and immunoblotted with the indicated antibodies. β-Actin was used as an internal control. Quantitative analysis of protein expression was in the right panel. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to control.

Figure 6

LAS inhibited in vivo MDA-MB-231 xenograft tumor growth. A xenograft model was established by subcutaneous inoculation of MDA-MB-231 cells into BALB/c nude mice mammary fat pads. When the average tumor volumes reached 120 mm3, mice were randomly divided into four groups and administrated with vehicle (5% of Cremophor EL, 5% of ethanol in saline), LAS-LD (5 mg/kg), or LAS-HD (10 mg/kg) daily, or docetaxel (10 mg/kg) via i.p. injection. The treatment period lasted for 20 days and all mice were sacrificed. (A) Tumor volumes were measured throughout the experimental period. (B) Images of tumors at the end of experiment. (C) Mouse body weights throughout the experimental period. (D) Tumor weights at experimental endpoint. (E–J) Organ weights normalized to body weights and expressed at the percentage of body weight. Data are expressed as means ± SEM.* p < 0.05, ** p < 0.01, compared to the vehicle group.

Figure 7

Photomicrograph of H&E histology of tissues (tumor, liver, spleen, kidney, heart, lung) after LAS treatment. Representative images are shown (20×).