Shikonin and 4-hydroxytamoxifen synergistically inhibit the proliferation of breast cancer cells through activating apoptosis signaling pathway in vitro and in vivo

Abstract

Background: Tamoxifen (TAM) is a cell type-specific anti-estrogen and is applied to improve the survival of patients with estrogen receptor positive (ER +) breast cancer. However, long-term TAM use can induce serious drug resistance, leading to breast cancer recurrence and death in patients. Further, it is almost useless among patients with estrogen receptor negative (ER −) breast cancer. Shikonin (SK) is a natural product broadly explored in cancer therapy. Some studies have demonstrated the combined treatment of SK and clinical anticancer drugs including TAM on various tumors. However, the combined effect of SK and 4-hydroxytamoxifen (4-OHT) on ER-breast cancer is not known. The current study aimed to assess the combination effects of SK and 4-OHT on human breast cancer cells, MCF-7 (ER +) and MDA-MB-435S (ER −), in vitro and in vivo and to investigate the underlying mechanisms. Methods: CCK-8 assays and flow cytometry were conducted to determine the cell viability and apoptotic profiles of human breast cancer cell lines (MCF-7 and MDA-MB-435S) treated with SK, 4-OHT, and the combination. ROS and JC-1 assays were used to determine ROS level and mitochondrial membrane potential. Western blot analysis was performed to investigate proteins that are associated with apoptosis. Haematoxylin & Eosin (HE) staining was used to detect the tumor and kidney morphology of mice. TUNEL and immunohistochemical staining were performed to detect Ki67 expression level and cell apoptotic profile in tumor tissues. Results: SK and 4-OHT synergistically inhibited MCF-7 and MDA-MB-435S cell proliferation and promoted apoptosis by reducing mitochondrial membrane potential and increasing the intracellular ROS level. The combination of SK and 4-OHT activated the mitochondrial-dependent apoptosis and the death receptor pathways, significantly regulating the PI3K/AKT/Caspase 9 signaling pathway. Compared with SK and 4-OHT alone, the combination of SK and 4-OHT could better inhibit tumor growth in mice. Conclusion: The combination of SK and 4-OHT shows highly efficient anticancer effects on breast cancer therapy. SK may be a promising candidate as an adjuvant to 4-OHT for breast cancer treatments, especially for ER-breast cancer.

Combinatorial Cytotoxic Effects of 2,3-Dichloro-5,8-dimethoxy-1,4-naphthoquinone and 4-hydroxytamoxifen in Triple-negative Breast Cancer Cell Lines

“…MDA-MB-231 demonstrated an antagonistic response with the compound combination. Lin et al, also observed some antagonistic responses when treating this cell line with shikonin and 4-OHT, shikonin being a naphthoquinone (55). This response may indicate a functional independence of the two compounds on cell proliferation.…”

Shikonin Inhibits Cell Growth of Sunitinib-Resistant Renal Cell Carcinoma by Activating the Necrosome Complex and Inhibiting the AKT/mTOR Signaling Pathway

“…Several reports described that SHI induced apoptosis [ 15 ] in colon cancer cell lines [ 63 ], pancreatic cancer [ 64 ], and non-small cell lung bronchial carcinoma tumor cells [ 65 ]. Inhibition of proliferation in breast cancer cell lines has also been attributed to apoptosis induction [ 66 ], and in colorectal cancer cells, administration of SHI increased ROS levels, which in turn induced intrinsic apoptosis [ 46 ].…”

Long Noncoding RNA OIP5‐AS1 Promotes the Disease Progression in Nasopharyngeal Carcinoma by Targeting miR‐203

“…It has been well established that cancer cells are characterized by uncontrolled cell proliferation and decreased apoptosis [ 30 , 31 ]. In this research, we found that OIP5-AS1 knockdown significantly suppressed the colony formation, proliferation, and cell cycle progression in vitro .…”