Faculty of Pharmaceutical Sciences, Post Graduate Program in Biodiversity and Biotechnology of the Amazon (Bionorte), Federal University of Amazonas, Manaus, Amazonas, 69080-900, Brazil.
Drug Research and Development Center (NPDM), Federal University of Ceará, Fortaleza, Ceará, 60430-275, Brazil.
J Ethnopharmacol. 2021 Mar 1;267:113605. doi: 10.1016/j.jep.2020.113605. Epub 2020 Nov 21.
22β-hydroxytingenone (22-HTG) is a quinonemethide triterpene isolated from Salacia impressifolia (Miers) A. C. Smith (family Celastraceae), which has been used in traditional medicine to treat a variety of diseases, including dengue, renal infections, rheumatism and cancer. However, the anticancer effects of 22-HTG and the underlying molecular mechanisms in melanoma cells have not yet been elucidated.
The present study investigated apoptosis induction and antimetastatic potencial of 22-HTG in SK-MEL-28 human melanoma cells.
First, the in vitro cytotoxic activity of 22-HTG in cultured cancer cells was evaluated. Then, cell viability was determined using the trypan blue assay in melanoma cells (SK-MEL-28), which was followed by cell cycle, annexin V-FITC/propidium iodide assays (Annexin/PI), as well as assays to evaluate mitochondrial membrane potential, production of reactive oxygen species (ROS) using flow cytometry. Fluorescence microscopy using acridine orange/ethidium bromide (AO/BE) staining was also performed. RT-qPCR was carried out to evaluate the expression of BRAF, NRAS, and KRAS genes. The anti-invasiveness potential of 22-HTG was evaluated in a three-dimensional (3D) model of reconstructed human skin.
22-HTG reduced viability of SK-MEL-28 cells and caused morphological changes, as cell shrinkage, chromatin condensation, and nuclear fragmentation. Furthermore, 22-HTG caused apoptosis, which was demonstrated by increased staining with AO/BE and Annexin/PI. The apoptosis may have been caused by mitochondrial instability without the involvement of ROS production. The expression of BRAF, NRAS, and KRAS, which are important biomarkers in melanoma development, was reduced by the 22-HTG treatment. In the reconstructed skin model, 22-HTG was able to decrease the invasion capacity of melanoma cells in the dermis.
Our data indicate that 22-HTG has anti-tumorigenic properties against melanoma cells through the induction of cell cycle arrest, apoptosis and inhibition of invasiveness potential, as observed in the 3D model. As such, the results provide new insights for future work on the utilization of 22-HTG in malignant melanoma treatment.
22β-羟基金缕梅酮（22-HTG）是从 Salacia impressifolia（Miers）A.C.Smith（卫矛科）中分离出的一种醌甲醚三萜，传统医学中用于治疗多种疾病，包括登革热、肾感染、风湿和癌症。然而，22-HTG 的抗癌作用及其在黑色素瘤细胞中的潜在分子机制尚不清楚。
本研究旨在探讨 22-HTG 在 SK-MEL-28 人黑色素瘤细胞中的诱导凋亡和抗转移潜力。
首先，评估 22-HTG 在培养的癌细胞中的体外细胞毒性活性。然后，通过台盼蓝法测定黑色素瘤细胞（SK-MEL-28）的细胞活力，随后进行细胞周期、Annexin V-FITC/碘化丙啶（Annexin/PI）测定、线粒体膜电位以及使用流式细胞术评估活性氧（ROS）的产生。使用吖啶橙/溴化乙锭（AO/BE）染色进行荧光显微镜检查。进行 RT-qPCR 以评估 BRAF、NRAS 和 KRAS 基因的表达。通过重建人体皮肤的三维（3D）模型评估 22-HTG 的抗侵袭潜力。
22-HTG 降低了 SK-MEL-28 细胞的活力并引起形态变化，如细胞收缩、染色质浓缩和核碎裂。此外，22-HTG 引起细胞凋亡，这通过 AO/BE 和 Annexin/PI 染色增加来证明。这种凋亡可能是由于线粒体不稳定而没有 ROS 产生的参与。22-HTG 处理降低了黑色素瘤发生中重要的生物标志物 BRAF、NRAS 和 KRAS 的表达。在重建皮肤模型中，22-HTG 能够降低黑色素瘤细胞在真皮中的侵袭能力。
我们的数据表明，22-HTG 通过诱导细胞周期停滞、凋亡和抑制侵袭潜力对黑色素瘤细胞具有抗肿瘤特性，这在 3D 模型中得到了观察。因此，结果为未来利用 22-HTG 治疗恶性黑色素瘤提供了新的见解。
