ABSTRACT
Hepatocellular carcinoma (HCC) is a leading cancer worldwide. Advanced HCCs are usually resistant to anticancer drugs, causing unsatisfactory chemotherapy outcomes. In this study, we showed that a 4-phenoxyphenol derivative, 4-[4-(4-hydroxyphenoxy)phenoxy]phenol (4-HPPP), exerts an inhibitory activity against two HCC cell lines, Huh7 and Ha22T. We further investigated the anti-HCC activities of 4-HPPP, including anti-proliferation and induction of apoptosis. Our results showed that higher dosage of 4-HPPP downregulates the expression of α-tubulin and causes nuclear enlargement in both the Huh-7 and Ha22T cell lines. Interestingly, the colony formation results showed a discrepancy in the inhibitory effect of 4-HPPP on HCC and rat liver epithelial Clone 9 cells, suggesting the selective cytotoxicity of 4-HPPP toward HCC cells. Furthermore, the cell proliferation and apoptosis assay results illustrated the differences between the two HCC cell lines. The results of cellular proliferation assays, including trypan blue exclusion and colony formation, revealed that 4-HPPP inhibits the growth of Huh7 cells, but exerts less cytotoxicity in Ha22T cells. Furthermore, the annexin V assay performed for detecting the apoptosis showed similar results. Western blotting results showed 4-HPPP caused the increase of pro-apoptotic factors including cleaved caspase-3, Bid and Bax in HCC cells, especially in Huh-7. Furthermore, an increase of autophagy-associated protein microtubule-associated protein-1 light chain-3B (LC3B)-II and the decrease of Beclin-1 and p62/SQSTM1 were observed following 4-HPPP treatment. Additionally, the level of γH2A histone family, member X (γH2AX), an endogenous DNA damage biomarker, was dramatically increased in Huh7 cells after 4-HPPP treatment, suggesting the involvement of DNA damage pathway in 4-HPPP-induced apoptosis. On the contrary, the western blotting results showed that treatment up-regulates pro-survival proteins, including the phosphorylation of protein kinase B (Akt) and the level of survivin on Ha22T cells, which may confer a resistance toward 4-HPPP. Notably, the blockade of extracellular signal-regulated kinases (ERK), but not Akt, enhanced the cytotoxicity of 4-HPPP against Ha22T cells, indicating the pro-survival role of ERK in 4-HPPP-induced anti-HCC effect. Our present work suggests that selective anti-HCC activity of 4-HPPP acts through induction of DNA damage. Accordingly, the combination of ERK inhibitor may significantly enhance the anti-cancer effect of 4-HPPP for those HCC cells which overexpress ERK in the future.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , Hepatocytes/drug effects , Phenyl Ethers/pharmacology , Tubulin/genetics , Animals , Antineoplastic Agents/chemical synthesis , BH3 Interacting Domain Death Agonist Protein/genetics , BH3 Interacting Domain Death Agonist Protein/metabolism , Beclin-1/genetics , Beclin-1/metabolism , Caspase 3/genetics , Caspase 3/metabolism , Cell Line , Cell Line, Tumor , DNA Damage , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation , Hepatocytes/metabolism , Hepatocytes/pathology , Histones/genetics , Histones/metabolism , Humans , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Organ Specificity , Phenyl Ethers/chemical synthesis , Rats , Sequestosome-1 Protein/genetics , Sequestosome-1 Protein/metabolism , Signal Transduction , Tubulin/metabolism , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Lung cancer is a leading cause of cancer death worldwide, and non-small-cell lung cancer (NSCLC) accounts for 85% of lung cancer, which is highly metastatic, leading to the poor survival rate of patients. We recently reported that 4-[4-(4-hydroxyphenoxy)phenoxy]phenol (4-HPPP), a phenoxyphenol, exerts antihepatoma effects by inducing apoptosis and autophagy. In this study, we further examined the effect of 4-HPPP and its analogs on NSCLC cells. Colony formation assays showed that 4-HPPP exerts selective cytotoxicity against NSCLC H1299 cells; furthermore, the inhibitory effect of 4-HPPP on the proliferation and migration of NSCLC cells was validated using an in vivo zebrafish-based tumor xenograft assay. The flow cytometry-based dichlorofluorescein diacetate (DCF-DA) assays indicated that 4-HPPP caused an increase in reactive oxygen species (ROS) in NSCLC cells, and Western blot assays showed that the major ROS scavenging enzymes superoxide dismutases- (SODs-) 1/2 were upregulated, whereas peroxidase (PRX) was downregulated. Furthermore, 4-HPPP caused both aneuploidization and the accumulation of γH2AX, a sensor of DNA damage, as well as the activation of double-strand break (DSB) markers, especially Ataxia-telangiectasia-mutated and Rad3-related (ATR) in NSCLC cells. Our present work suggests that the antiproliferative effects of 4-HPPP on lung cancer cells could be due to its phenoxyphenol structure, and 4-HPPP could be a candidate molecule for treating NSCLC by modulating ROS levels and lowering the threshold of polyploidy-specific cell death in the future.