益瘿散结方含药血清对甲状腺乳头状癌TPC-1细胞增殖及有氧糖酵解的影响

The Effect of YiYingSanJie Formula Containing Serum on Proliferation and Aerobic Glycolysis of Papillary Thyroid Cancer TPC-1 Cells

关键词:  益瘿散结方  含药血清  甲状腺癌  有氧糖酵解

Key words:  YiYingSanJie Formula  drug-containing serum  thyroid cancer  aerobic glycolysis

摘要:

目的 观察益瘿散结方含药血清对人甲状腺乳头状癌细胞TPC-1增殖及有氧糖酵解的影响。方法 采用对雄性SD大鼠灌胃连续灌胃给药7d,制备益瘿散结方含药血清及空白血清。取对数期生长的TPC-1细胞,分为2.5%、5%、7.5%、10%含药血清组和空白血清组,每组3个复孔,分别用不同浓度含药血清和空白血清进行干预。采用CCK-8检测干预24h、48h、72h细胞增殖情况,并筛选益瘿散结方含药血清低、中、高剂量组同空白血清组进行后续实验；采用试剂盒检测干预48h后细胞乳酸外排量和三磷酸腺苷(Adenosine triphosphate,ATP)生成量情况；采用RT-PCR和Western Blot检测M2型丙酮酸激酶(Pyruvate kinase M2,PKM2)、已糖激酶2(Hexokinase 2,HK2)、乳酸脱氢酶A(Lactate Dehydrogenase A,LDHA)、c-Myc和缺氧诱导因子1α(Hypoxia-inducible factor 1-α,HIF-1α)mRNA和蛋白表达情况；采用可见分光光度法检测PK、HK和LDH酶活性表达情况。结果 与空白血清组比较,益瘿散结方含药血清可抑制TPC-1细胞增殖,以5%、7.5%和10%含药血清干预48h作为低、中、高剂量组进行后续实验。与空白血清组比较,各含药血清组细胞乳酸外排量和ATP生成量显著降低,且呈剂量依赖性(P<0.01或P<0.001)；高剂量组HK2、PKM2、LDHA、HIF-1α和c-Myc的mRNA和蛋白表达量降低(P<0.01或P<0.001)；中、高剂量组PK、HK和LDH酶活性降低(P<0.01或P<0.001)。结论 益瘿散结方含药血清能够抑制TPC-1细胞增殖、抑制细胞有氧糖酵解,其作用机制可能与其抑制c-Myc/ HIF-1α信号通路,下调PK、HK、LDH表达及酶活性,减少乳酸和ATP生成有关。

Abstract:

ABSTRACT: OBJECTIVE To observe the effects of YiYingSanJie Formula drug-containing serum on the proliferation and aerobic glycolysis of TPC-1 in human papillary thyroid cancer cells. METHODS Human thyroid cancer cell line TPC-1 was treated by gavage in male SD rats for 7 d. The TPC-1 cells were divided into 2.5%, 5%, 7.5%, 10% drug-containing serum and blank serum groups. Each group was treated with different concentrations of drug-containing serum and blank serum in 3 replicate wells. CCK-8 was used to detect the cell proliferation at 24h, 48h and 72h, and the low, medium and high dose groups of YI GAR Solute were screened for subsequent experiments; kits were used to detect lactate efflux and Adenosine triphosphate (ATP) production after 48h of intervention; RT-PCR and Western Blot were used to detect M2 acetone. Western Blot was used to detect M2 Pyruvate kinase M2 (PKM2), Hexokinase 2 (HK2), Lactate Dehydrogenase A (LDHA), c-Myc and Hypoxia-inducible factor 1α (HIF1). inducible factor 1-α (HIF-1α) mRNA and protein expression; PK, HK and LDH enzyme activity expression were detected by visible spectrophotometry. RESULTS Compared with the blank serum group, the drug-containing serum of YiYingSanJie Formula could inhibit the proliferation of TPC-1 cells. 5%, 7.5% and 10% drug-containing serum interventions for 48 h were used as the low, medium and high dose groups for the follow-up experiments. Compared with the blank serum group, cellular lactate efflux and ATP production were significantly reduced in each drug-containing serum group in a dose-dependent manner (P<0.01 or P<0.001); mRNA and protein expression of HK2, PKM2, LDHA, HIF-1α and c-Myc were reduced in the high dose group (P<0.01 or P<0.001); PK, HK and LDH enzyme activities were reduced in the medium and high dose groups (P<0.01 or P<0.001). CONCLUSION The drug-containing serum of YiYingSanJie Formula can inhibit the proliferation and aerobic glycolysis of TPC-1 cells, and its mechanism of action may be related to the inhibition of c-Myc/HIF-1α signaling pathway, down-regulation of PK, HK, LDH expression and enzyme activities, and reduction of lactate and ATP production.