Laboratory of Cell and Molecular Tumor Biology and Bioactive Compounds, Post Graduate Program in Health Science, São Francisco University, Bragança Paulista 12916-900, São Paulo, Brazil.
Laboratory of Multidisciplinary Research, Post Graduate Program in Health Science, São Francisco University, Bragança Paulista 12916-900, São Paulo, Brazil.
Int J Mol Sci. 2023 Jun 27;24(13):10716. doi: 10.3390/ijms241310716.
The enzymatic hydrolysis of the extract of by two glycosyl hydrolases (hesperidinase and galactosidase) was performed in order to obtain kaempferol (KPF)-enriched extract with an enhanced anticancer activity. The current study examined the effectiveness of both extracts (before (KPF-BBR) and after (KPF-ABR) bioconversion reactions) in reducing cell viability and inducing apoptosis in human high-degree gliomas in vitro. Cytotoxicity was determined using an MTT assay. The effects of both compounds on the proliferation of glioma cell lines were measured using trypan blue exclusion, flow cytometry for cell cycle, wound healing (WH), and neurosphere formation assays. Cellular apoptosis was detected by DNA fragmentation and phosphatidylserine exposure. qPCR and luciferase assays evaluated NF-kB pathway inhibition. The survival rate of NG-97 and U-251 cells significantly decreased in a time- and dose-dependent manner after the addition of KPF-BBR or KPF-ABR. Thus, a 50% reduction was observed in NG-97 cells at 800 µM (KPF-BBR) and 600 µM (KPF-ABR) after 72 h. Both compounds presented an IC50 of 1800 µM for U251 after 72 h. The above IC50 values were used in all of the following analyses. Neither of the KPF presented significant inhibitory effects on the non-tumoral cells (HDFa). However, after 24 h, both extracts (KPF-BBR and KPF-ABR) significantly inhibited the migration and proliferation of NG-97 and U-251 cells. In addition, was downregulated in glioma cells stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) plus KPF-BBR and TPA+KPF-ABR compared with the TPA-treated cells. Both KPF-BBR and KPF-ABR significantly inhibited the proliferation of glioma stem cells (neurospheres) after 24 h. DNA fragmentation assays demonstrated that the apoptotic ratio of KPF-ABR-treated cell lines was significantly higher than in the control groups, especially NG-97, which is not TMZ resistant. In fact, the flow cytometric analysis indicated that KPF-BBR and KPF-ABR induced significant apoptosis in both glioma cells. In addition, both KPF induced S and G2/M cell cycle arrest in the U251 cells. The qPCR and luciferase assays showed that both KPFs downregulated , and , indicating an inhibitory effect on the NF-kB pathway. Our findings suggest that both KPF-BBR and KPF-ABR can confer anti-tumoral effects on human cell glioma cells by inhibiting proliferation and inducing apoptosis, which is related to the NF-κB-mediated pathway. The KPF-enriched extract (KPF-ABR) showed an increased inhibitory effect on the cell migration and invasion, characterizing it as the best antitumor candidate.
为了获得具有增强抗癌活性的富含山柰酚（KPF）的提取物，使用两种糖苷水解酶（橙皮苷酶和半乳糖苷酶）对 的提取物进行酶解。本研究检测了两种提取物（生物转化反应前后（KPF-BBR 和 KPF-ABR））在体外降低人高级别神经胶质瘤细胞活力和诱导细胞凋亡的有效性。使用 MTT 测定法测定细胞毒性。使用台盼蓝排除法、细胞周期流式细胞术、划痕愈合（WH）和神经球形成测定法测量两种化合物对神经胶质瘤细胞系增殖的影响。通过 DNA 片段化和磷脂酰丝氨酸暴露检测细胞凋亡。qPCR 和荧光素酶测定法评估 NF-kB 途径抑制作用。NG-97 和 U-251 细胞的存活率在添加 KPF-BBR 或 KPF-ABR 后呈时间和剂量依赖性显著降低。在 72 小时后，NG-97 细胞中观察到 50%的减少，在 800 µM（KPF-BBR）和 600 µM（KPF-ABR）。两种化合物在 72 小时后对 U251 的 IC50 均为 1800 µM。在以下所有分析中均使用上述 IC50 值。两种 KPF 对非肿瘤细胞（HDFa）均无明显抑制作用。然而，在 24 小时后，两种提取物（KPF-BBR 和 KPF-ABR）均显著抑制 NG-97 和 U-251 细胞的迁移和增殖。此外，与 TPA 处理的细胞相比，用 12-O-十四烷酰佛波醇-13-乙酸酯（TPA）加 KPF-BBR 和 TPA+KPF-ABR 刺激的神经胶质瘤细胞中下调了 。在 24 小时后，KPF-BBR 和 KPF-ABR 均显著抑制神经球（神经胶质瘤干细胞）的增殖。DNA 片段化分析表明，KPF-ABR 处理的细胞系的细胞凋亡率明显高于对照组，特别是对 TMZ 耐药的 NG-97。事实上，流式细胞术分析表明，KPF-BBR 和 KPF-ABR 诱导两种神经胶质瘤细胞发生明显的细胞凋亡。此外，两种 KPF 均诱导 U251 细胞中的 S 和 G2/M 细胞周期停滞。qPCR 和荧光素酶测定表明，两种 KPF 均下调 、 和 ，表明对 NF-kB 途径有抑制作用。我们的研究结果表明，KPF-BBR 和 KPF-ABR 均可通过抑制增殖和诱导凋亡对人神经胶质瘤细胞发挥抗肿瘤作用，这与 NF-κB 介导的途径有关。富含 KPF 的提取物（KPF-ABR）对细胞迁移和侵袭的抑制作用增强，表明其是最佳的抗肿瘤候选物。
