Lunasin結合短期飢餓加強對人類乳癌細胞之細胞毒性及氧化壓力

Abstract

乳癌是全球女性中最常見的癌症，也是女性癌症相關死亡的主要原因之一。研究證據顯示，短期飢餓 (short-term starvation, STS) 可以透過誘導代謝壓力選擇性抑制癌細胞的存活與增殖，進而延緩腫瘤進展。Lunasin是一種子胜肽，由43 個胺基酸所組成，已證實其具有抗發炎、抗氧化以及抗腫瘤等生理功能。然而，lunasin結合STS的合併處理是否能增強對乳癌細胞的作用，目前尚未明確。本研究旨在探討lunasin結合STS是否會影響對TNBC (Triple negative breast cancer) 型乳癌細胞MDA-MB-231和Luminal A型乳癌細胞MCF-7的生長，進而增強抑制效果，並分析細胞生長、存活與相關訊號路徑。實驗中的STS模式採用十六小時調降培養基中營養成分 (血清1% 及葡萄糖1 g/L)，另八小時則維持正常培養基 (血清10% 及葡萄糖4.5 g/L) 。結果顯示，單獨lunasin及STS相較於正常組，均可降低乳癌細胞MDA-MB-231和MCF-7的存活率，而合併處理相較於單獨處理組則增強抑制效果。在細胞移行及聚落形成實驗中，合併處理具有抑制癌細胞遷移與聚落形成的趨勢。而在正常乳腺上皮細胞MCF-10A中，lunasin處理可以減緩因STS而造成的細胞損傷。在氧化壓力方面，合併處理顯著增加癌細胞內活性氧 (reactive oxygen species, ROS) 的生成，並顯著降低細胞內GSH/GSSG比值；而在MCF-10A細胞中，合併處理則可降低STS所誘導的ROS上升，並且降低GSSG，表現出對正常細胞的保護效應。細胞凋亡分析顯示，lunasin、STS及合併處理均可顯著增加癌細胞內的總凋亡比例。在細胞凋亡週期的部分，三種處理皆促使MDA-MB-231和MCF-7停滯於G2/M期；而STS及合併處理則會使MCF-10A細胞週期停滯在G0_G1期，伴隨S期比例顯著下降。此外，也觀察到STS會誘導癌細胞Nrf2上調並抑制GPX4，尤以MDA-MB-231最為明顯。相較之下，STS並未誘導MCF-10A的Nrf2上調，顯示正常細胞的氧化還原調控較為穩定。綜合上述，lunasin合併STS可能藉由選擇性調控細胞內氧化壓力與凋亡路徑，誘導乳癌細胞的細胞毒性，同時減輕對正常細胞的傷害，突顯了lunasin與STS聯合應用作為乳癌的潛在化學預防策略。

Breast cancer is the most common cancer among women worldwide and is the leading cause of cancer-related deaths in females. Short-term starvation (STS) can selectively retard or inhibit cancer progression by inducing metabolic stress. Lunasin, a peptide derived from seeds, exhibits multiple bioactivities including anti-carcinogenesis, anti-inflammation, and anti-oxidation. However, the combined effect of lunasin and STS in breast cancer cells remains unclear. The study aims to investigate the impact of lunasin combined with STS on human breast cancer cell lines MDA-MB-231 and MCF-7, and to examine the related signaling pathways. In this study, the program of STS model was used, as nutrient-reduced medium (1% serum and 1g/L glucose) for 16 hours, followed by complete medium (10% serum and 4.5g/L glucose) for 8 hours. The results showed that both lunasin and STS alone significantly reduced cancer cell viability compared to the control group, and their combination exhibited a stronger inhibitory effect. Similarly, the combination treatment effectively suppressed cell migration and colony-forming ability compared to single treatments. Interestingly, in normal mammary epithelial cells MCF-10A, lunasin treatment was able to retard the cellular damage caused by STS. Regarding oxidative stress, the combination treatment significantly increased intracellular reactive oxygen species (ROS) levels and reduced the GSH/GSSG ratio in cancer cells. Conversely, in MCF-10A cells, the combination treatment reduced STS-induced ROS accumulation and lowered GSSG levels, supporting its protective effect on normal cells. Apoptosis assays revealed that lunasin, STS, and their combination significantly increased apoptosis in cancer cells, whereas lunasin reduced STS-induced apoptosis in MCF-10A. Cell cycle analysis further showed that G2/M phase arrest in cancer cells after all treatments, whereas in MCF-10A, STS, and the combination treatment induced G0/G1 arrest and decreased the S phase. In addition, we observed that STS upregulated Nrf2 expression and suppressed GPX4 in MDA-MB-231 cells. In contrast, STS did not increase Nrf2 in MCF-10A cells, indicating that normal cells may have a stable redox regulatory capacity. These findings suggest that lunasin combined with STS selectively enhances oxidative stress and apoptotic signaling in breast cancer cells while protecting normal cells, highlighting that lunasin combined with STS is a potential chemopreventive strategy against breast cancer.