Studies on the Effects of Bufalin on Apoptosis and Metastasis of NCI-H460 cells

• Main Authors: Shin-Hwar Wu, 吳莘華
• Other Authors: Wu-Huei Hsu
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/6u6fm3

博士 === 中國醫藥大學 === 臨床醫學研究所博士班 === 102 === We evaluated bufalin’s effects on human lung cancer cell NCI-H460 both in vitro and in vivo. Bufalin exerted a significant cytotoxicity on NCI-H460 cell in concentration as low as 1 μM. DNA condensation in a dose-dependent manner was observed in bufalin-treated cells stained by 4,6-diamidino-2-phenylindole (DAPI). A decrease of mitochondrial membrane potential and release of reactive oxygen species in bufalin-treated NCI-H460 cells were detected by flow cytometry. Western blotting showed several pro-apoptotic proteins-- Fas, Fas-ligand, Bax, cytochrome c, apoptosis protease activating factor-1, endonuclease G, caspase 3 and 9 were up-regulated after bufalin treatment. However, anti-apoptotic B-cell lymphoma 2 protein was down-regulated. RT-PCR studies demonstrated decreased glucose regulated protein-78 gene and increased growth arrest- and DNA damage-inducible gene 153 after bufalin treatment. Intraperitoneal injection of bufalin reduced the tumor sizes of BALB/C nu/nu mice implanted with NCI-H460 cells in a dose dependent way. Except for high dose of 0.4 mg/Kg, bufalin injection did not produce significant drug-related toxicity in experimental animals. Following apoptosis, DNA damage response (DDR) usually will be elicited to counteract the damage. We demonstrated bufalin not only induce apoptosis and DNA damage, but also disrupt NCI-H460 cell’s DDR. DNA damage and condensation in NCI-H460 cell treated with bufalin were displayed by comet assay and DAPI staining. Western blotting indicated that bufalin suppressed the proteins levels associated with DNA damage and repair such as DNA dependent serine/threonine protein kinase (DNA-PK), DNA repair proteins breast cancer 1, early onset (BRCA1), 14-3-3 sigma, mediator of DNA damage checkpoint 1 (MDC1), O6-methylguanine-DNA methyltransferase (MGMT) and p53 (tumor suppressor protein). DNA damage in NCI-H460 cells after treated with bufalin caused up-regulates its ATM and ATR genes, which encode proteins functioning as sensors in DDR and also up-regulated gene expression (mRNA) of BRCA1, DNA-PK and but bufalin suppressed the gene expression (mRNA) of p53 and 14-3-3 sigma, an important checkpoint keeper of DDR, and led to potential mitotic catastrophe. By cDNA microarray hybridization, we found 165 apoptosis-related genes were affected. CASP9 was up-regulated by 5.51 fold and THAP1 by 2.75-fold while CCAR1 was down-regulated by 2.24 fold. 107 genes related to DNA damage/repair were affected. MDC1 was down-regulated by 2.22-fold, DDIT4 by 2.52 fold while GADD45B up-regulated by 3.72 fold. 201 genes related to cell cycles were affected. CCPG1 was down-regulated by 2.11 fold and CDCA7L by 2.71 fold. Under sub-lethal concentrations (from 25 up to 100 nM), bufalin significantly inhibit the invasion and migration nature of H460 by Matrigel Cell Migration Assay and Invasion System. Bufalin also suppressed the enzymatic activity of matrix metallo-proteinase (MMP) -9 on gelatin zymography. Western blotting revealed depressed several key metastasis-related proteins, such as NF-κB, MMP-2, MMP-9, protein kinase C, phosphatidylinositol 3-kinase (PI3-K), phosphorylated Akt, growth factor receptor-bound protein 2 (GRB2), phosphorylated extracellular signal-regulated kinase (ERK), phosphorylated p38 and phosphorylated c-Jun NH2-terminal kinase (JNK) following bufalin treatment. As evidenced by immunostaining and electrophoretic mobility shift assay, bufalin induced not only a decreased cytoplasmic NF-κB production, but also a decreased its nuclear translocation. Several metastasis-related genes, including Rho-associated, coiled-coil-containing protein kinase 1 and focal adhesion kinase, were down-regulated after bufalin treatment. In conclusion, very low concentration of bufalin can induce apoptosis of human lung cancer cell NCI-H460 in vitro. Bufalin also reduce tumor sizes of mice with NCI-H460 cell xerografts without significant drug-related toxicity. Bufalin also disrupt DDR following DNA damage and suppress the metastatic behavior of NCI-H460 cell. These results suggest bufalin has a potential to develop into a clinically useful therapy for human non-small cell lung cancer.