目的 探究根皮素通過抑制丙酮酸脫氫酶激酶1（pyruvate dehydrogenase kinase 1，PDK1）-磷酸化丙酮酸脫氫酶E1亞基α 1（phosphorylation of pyruvate dehydrogenase E1 subunit alpha 1，p-PDHA1）進而影響谷氨酰胺（glutamine，Gln）代謝介導(dǎo)的前列腺癌的作用機制。方法 采用不同劑量根皮素（25、50、100 μmol/L）干預(yù)人前列腺癌PC-3、DU145、LNCaP細胞與人前列腺RWPE-1細胞，采用細胞計數(shù)試劑盒（cell counting kit-8，CCK-8）測定細胞活力。將人前列腺癌PC-3細胞分為對照（二甲基亞砜，dimethyl sulfoxide，DMSO）組、根皮素（100 μmol/L）組及順鉑（0.03 mmol/L）組，Transwell法檢測細胞侵襲能力、TUNEL法檢測細胞凋亡水平。根皮素及Gln單獨使用及聯(lián)合干預(yù)PC-3細胞，試劑盒測定Gln消耗水平、谷氨酸與腺嘌呤核苷三磷酸（adenosine triphosphate，ATP）產(chǎn)生水平，Western blotting法測定谷氨酰胺酶1（glutaminase 1，GLS1）蛋白表達水平，同時測定細胞增殖、侵襲、凋亡等細胞生物學(xué)行為變化。利用網(wǎng)絡(luò)藥理學(xué)及生物信息學(xué)分析根皮素、前列腺癌與Gln代謝相關(guān)基因的交集。Western blotting法測定各組細胞PDK1蛋白表達水平。將PC-3細胞分為空載體對照（pcDNA3.1）組、PDK1過表達載體（pcDNA3.1-PDK1）組、PDK1敲減載體（KD-PDK1）組及其對照（KD-Control）組、PDHA1過表達載體（pcDNA3.1-PDHA1）組、KD-PDK1＋pcDNA3.1-PDHA1組及其對照KD-PDK1＋pcDNA3.1組，以及根皮素（100 μmol/L）＋pcDNA3.1-PDK1組及其對照根皮素＋pcDNA3.1組，測定各組細胞增殖、侵襲、凋亡變化、Gln消耗水平、谷氨酸與ATP產(chǎn)生水平及GLS1蛋白表達水平。構(gòu)建前列腺癌移植瘤小鼠模型，通過根皮素干預(yù)治療，以順鉑作為陽性對照，探究根皮素對體內(nèi)腫瘤生長的影響。結(jié)果 根皮素對人前列腺RWPE-1細胞活力無顯著影響，但100 μmol/L根皮素可顯著抑制人前列腺癌PC-3、DU145、LNCaP細胞增殖（P＜0.05）。與對照組比較，根皮素組細胞增殖與侵襲能力顯著降低（P＜0.05）、凋亡水平顯著增加（P＜0.01），Gln消耗水平、谷氨酸和ATP產(chǎn)生水平顯著降低（P＜0.05）、GLS1蛋白表達水平顯著下降（P＜0.05）；Gln干預(yù)后可逆轉(zhuǎn)上述結(jié)果，且根皮素與Gln聯(lián)合干預(yù)PC-3細胞時，根皮素能夠抑制Gln的作用（P＜0.05）。網(wǎng)絡(luò)藥理學(xué)與生物信息分析表明，PDK1為根皮素通過Gln代謝途徑治療前列腺癌的關(guān)鍵靶點之一，且PDK1在PC-3細胞中高表達，根皮素可顯著抑制PDK1的表達（P＜0.05）。與pcDNA3.1組比較，進一步過表達PC-3細胞中的PDK1能夠促進細胞增殖與侵襲（P＜0.001）、抑制細胞凋亡（P＜0.001），增強細胞中的Gln代謝（P＜0.05）。與根皮素＋pcDNA3.1組比較，過表達PDK1能夠部分逆轉(zhuǎn)根皮素對PC-3細胞生物學(xué)行為及Gln代謝的影響（P＜0.05）。此外，與KD-Control組比較，敲減PC-3細胞中的PDK1有助于抑制細胞增殖與侵襲、促進細胞凋亡，減弱Gln代謝水平，然而與KD-PDK1＋pcDNA3.1組比較，聯(lián)合過表達PDHA1則能夠逆轉(zhuǎn)這一結(jié)果（P＜0.05）。體內(nèi)實驗表明，根皮素能夠顯著抑制腫瘤生長（P＜0.05）。結(jié)論 根皮素通過抑制PDK1-p-PDHA1軸進而影響Gln代謝介導(dǎo)的前列腺癌。

Objective To explore whether phloretin can affect the biological behavior of prostate cancer cells mediated by glutaminolysis by inhibiting pyruvate dehydrogenase kinase 1 (PDK1)-phosphorylation of pyruvate dehydrogenase E1 subunit alpha 1 (p-PDHA1). Methods Human prostate cancer cell lines PC-3, DU145, LNCaP, and human prostate epithelial cell line RWPE-1 were treated with different concentrations of phloretin (25, 50, 100 μmol/L), and cell proliferation was assessed using the cell counting kit-8 (CCK-8). PC-3 cells were divided into control (0.1% DMSO), phloretin (100 μmol/L), and cisplatin (DDP, 0.03 mmol/L) groups, and cell invasion was detected using the Transwell method while cell apoptosis was detected using the TUNEL method. PC-3 cells were treated with phloretin and/or glutamine (Gln) separately or in combination, and Gln levels, glutamate and adenosine triphosphate (ATP) production levels were measured using kits, while glutaminase 1 (GLS1) protein expression was determined by Western blotting in cell proliferation, invasion, and apoptosis were also assessed. Network pharmacology and bioinformatics were used to analyze the intersection of phloretin, prostate cancer, and Gln-related genes. Western blotting was used to measure PDK1 protein expression levels in RWPE-1, PC-3, and PC-3 + phloretin groups. Furthermore, The PC-3 cells were divided into control pcDNA3.1 group, pcDNA3.1-PDK1 group, the KD-PDK1 group and its control KD-Control group, pcDNA3.1-PDHA1 group, KD-PDK1 + pcDNA3.1-PDHA1 group and its control KD-PDK1 + pcDNA3.1 group, and phloretin + pcDNA3.1-PDK1 group and its control phloretin + pcDNA3.1 group, Changes in cell proliferation, invasion, apoptosis, Gln levels, glutamate and ATP production levels, and GLS1 protein expression in each group were measured. A prostate cancer xenograft mouse model was established, and phloretin intervention therapy was administered, with DDP treatment serving as a positive control, to investigate the effect of phloretin on tumor growth in vivo.Results Phloretin had no significant effect on the proliferation of RWPE-1 cells but significantly inhibited the proliferation of PC-3, DU145, and LNCaP cells, with the most significant effect on PC-3 cells at a concentration of 100 μmol/L (P < 0.05). Compared with control group, the phloretin group showed significantly reduced cell proliferation and invasion ability (P < 0.05), increased apoptosis levels (P < 0.05), decreased Gln levels, reduced glutamate and ATP production levels (P < 0.05), and weakened GLS1 protein expression (P < 0.05). Gln intervention yielded opposite results, and when phloretin and Gln were co-administered to PC-3 cells, phloretin inhibited the Gln-induced effects (P < 0.05). Network pharmacology and bioinformatics analysis indicated that PDK1 is one of the key targets for phloretin in the treatment of prostate cancer through the Gln metabolism pathway. Moreover, PDK1 was highly expressed in PC-3 cells and was inhibited by phloretin (P < 0.05). Compared with pcDNA3.1 group, overexpression of PDK1 in PC-3 cells promoted cell proliferation and invasion (P < 0.05), inhibited cell apoptosis (P < 0.05), but enhanced Gln metabolism in cells (P < 0.05). Compared with the phloretin + pcDNA3.1 group, overexpression of PDK1 partially reversed the effects of phlorizin on the biological behavior and Gln metabolism of PC-3 cells (P < 0.05). Additionally, compared with the KD-Control group, knockdown of PDK1 in PC-3 cells contributed to the inhibition of cell proliferation and invasion, promotion of cell apoptosis, and reduction of Gln metabolism levels. However, compared with the KD-PDK1 + pcDNA3.1 group, co-overexpression of PDHA1 reversed these effects (P < 0.05). In vivo experiments showed that phloretin significantly inhibited tumor growth in mice (P < 0.05). Conclusion Phloretin affects Gln metabolism-mediated prostate cancer progression by inhibiting the PDK1-PDHA1 axis.

R285.5

天津市科技計劃項目（20JCQNJC00550）