Pharmacological anti-tumor effects of natural Chamaecyparis obtusa (siebold & zucc.) endl. Leaf extracts on breast cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Chamaecyparis obtusa (C. obtusa, cypress species) is a plant that grows mainly in the temperate Northern Hemisphere and has long been used as a traditional anti-inflammatory treatment in East Asia. C. obtusa contains phytoncides, flavonoids, and terpenes, which have excellent anti-cancer effects and have been reported to prevent the progression of various cancers. However, the detailed mechanisms underlying the anti-cancer effects of C. obtusa extracts are unknown. AIM OF THE STUDY: We sought to confirm the anti-cancer effects of C. obtusa leaf extracts and to reveal the mechanism of action, with the possibility of its application in the treatment or prevention of cancer. MATERIAL &METHODS: The cytotoxicity of C. obtusa leaf extracts was confirmed using an MTT assay. Intracellular changes in protein levels were measured by immunoblotting, and mRNA levels were measured with qRT-PCR. Wound healing assay and transwell migration assay were used to evaluate the metastatic potential of breast cancer cells. The extract-induced apoptosis was observed using IncuCyte Annexin V Red staining analysis. A syngeneic breast cancer mouse model was established by injecting 4T1-Luc mouse breast cancer cells into the fat pad of female BALB/c mice, and the extract was administered orally. Luciferin solution was injected intraperitoneally to assess primary tumor development and metastasis by bioluminescence. RESULTS: C. obtusa leaf extracts were extracted with boiling water, 70% EtOH, and 99% EtOH. Among the extracts, the 99% EtOH extract of C. obtusa leaf (CO99EL) most clearly inhibited the tyrosine phosphorylation of Signal Transducer and Activator of Transcription 3 (pY-STAT3) in MDA-MB-231 breast cancer cells at a concentration of 25 and 50 µg/mL. In addition, CO99EL strongly inhibited not only endogenous pY-STAT3 levels but also IL-6-induced STAT3 activation in various types of cancer cells, including breast cancer. CO99EL inhibited metastatic potential by downregulating the expression of N-cadherin, fibronectin, TWIST, MMP2, and MMP9 in MDA-MB-231 breast cancer cells. CO99EL also induced apoptotic cell death by increasing cleaved caspase-3 and decreasing anti-apoptotic proteins Bcl-2 and Bcl-xL. In an in vivo syngeneic breast cancer mouse model, 100 mg/kg CO99EL suppressed tumor growth and induced apoptosis of cancer cells. Moreover, CO99EL significantly inhibited lung metastasis from primary breast cancer. CONCLUSIONS: Our study demonstrated that 100 mg/kg CO99EL has potent anti-tumor effects against breast cancer, thus suggesting that 100 mg/kg CO99EL has potential applications in the treatment and prevention of breast cancer.

Pygenic Acid A (PA) Sensitizes Metastatic Breast Cancer Cells to Anoikis and Inhibits Metastasis In Vivo.

Metastasis is the main cause of cancer-related deaths. Anoikis is a type of apoptosis caused by cell detachment, and cancer cells become anoikis resistant such that they survive during circulation and can successfully metastasize. Therefore, sensitization of cancer cells to anoikis could prevent metastasis. Here, by screening for anoikis sensitizer using natural compounds, we found that pygenic acid A (PA), a natural compound from Prunella vulgaris, not only induced apoptosis but also sensitized the metastatic triple-negative breast cancer cell lines, MDA-MB-231 cells (human) and 4T1 cells (mouse), to anoikis. Apoptosis protein array and immunoblotting analysis revealed that PA downregulated the pro-survival proteins, including cIAP1, cIAP2, and survivin, leading to cell death of both attached and suspended cells. Interestingly, PA decreased the levels of proteins associated with anoikis resistance, including p21, cyclin D1, p-STAT3, and HO-1. Ectopic expression of active STAT3 attenuated PA-induced anoikis sensitivity. Although PA activated ER stress and autophagy, as determined by increases in the levels of characteristic markers, such as IRE1α, p-elF2α, LC3B I, and LC3B II, PA treatment resulted in p62 accumulation, which could be due to PA-induced defects in autophagy flux. PA also decreased metastatic characteristics, such as cell invasion, migration, wound closure, and 3D growth. Finally, lung metastasis of luciferase-labeled 4T1 cells decreased following PA treatment in a syngeneic mouse model when compared with the control. These data suggest that PA sensitizes metastatic breast cancer cells to anoikis via multiple pathways, such as inhibition of pro-survival pathways and activation of ER stress and autophagy, leading to the inhibition of metastasis. These findings suggest that sensitization to anoikis by PA could be used as a new therapeutic strategy to control the metastasis of breast cancer.