Differential activation of cell death pathways by novel tetrahydropyridinol derivatives and 5-fluorouracil in human hepatocellular carcinoma and breast carcinoma cells

Abstract

Among the different types of cancers, Hepatocellular carcinoma (HCC) is the most dangerous malignant disease and cause high rates of mortality. Factors that contribute increased risk of hepatocellular carcinoma are infection with hepatitis B and hepatitis C virus and interaction of aflatoxin B1 with chronic hepatitis B infection. The initial treatment is usually surgical excision, generally followed by chemotherapy when treating later stage. However, the prognosis for survival of the late stage of patient is poor. One of the major causes of high mortality associated with this disease is its poorly understood cellular and molecular basis. Analyses of different genetic alterations have lead to the identification of several major oncogenic pathways that are deregulated in HCC, including the p53, the Rb, the E-cadherin and the Wnt/β-catenin pathways. Wnt/ β-catenin signaling is frequently activated in many different types of cancer. Cytoplasmic β-catenin binds to the carboxyl terminus of E-cadherin at the plasma membrane, and this complex recruits α-catenin, and then further recruits other structural proteins to form the cell-cell junctions. In addition to its role as an adhesion protein, β-catenin can also be a transcription co-activator in the wnt signaling pathway. If Wnt/β-catenin pathway is activated inappropriately, then it might cause tumorigenesis. Apoptosis is a form of cell death that involves the consecutive action of a number of intracellular signaling pathways. Recent studies have concentrated on the endoplasmic reticulum (ER) as a third subcellular compartment implicated in apoptotic execution. Disruption of Ca2+ homeostasis and accumulation of misfolded proteins in the ER lumen cause ER stress and eventually leads to apoptotic cell death. Inducing apoptosis either by up-regulation of tumor suppressors or down-regulation of oncogenes might be good anticancer approaches to increase the efficacy of chemotherapeutic compounds. Therefore, in this study, the novel Tetrahydropyridinol derivatives (5a and 5b) and 5-Fluorouracil (known anti-cancer agent) was used to analyze the potential in the activation of various cell death pathways in human hepatocellular carcinoma cells. Compounds 5a and 5b were comparably and significantly cytotoxic to Sk-Hep1, Hep3B and MDA-MB-231 cells. Treatment of 5a and 5b at IC50 concentration to human cancer cells showed down-regulation of β-catenin only in Sk-Hep1 cells but not in Hep3B cells. Since β-catenin involved in Wnt/β-catenin signaling pathway, the impact of 5a and 5b was examined on β-catenin target proteins (c-Myc and Cyclin D1) and β-catenin signaling proteins (E-cadherin, Axin 1 and GSK-3β) in Sk-Hep1 and Hep3B cells. The expression of c-Myc and Cyclin D1 were down-regulated in Sk-Hep1 cells and the expression of E-cadherin, Axin1 were not altered by 5a and 5b compounds. But the GSK-3β expression was up-regulated only in Sk-Hep1 cells. This type of expression pattern was not observed in Hep3B cell line. In case of MDA-MB-231, the expression of β-catenin and GSK-3β was not altered. Instead, the expression of E-cadherin was up-regulated by 5a and 5b. In contrast, 5a and 5b induced G1 cell cycle arrest in Hep3B cells by modulating cell cycle regulatory molecule p21, p27 and cdk2 and which was confirmed by both western blot and flow cytometry. Moreover, these compounds significantly inhibited the invasion of Sk-Hep1 and MDA-MB-231 cells and which was confirmed by matrigel invasion assay. The alteration of MMP-2 expression was confirmed by western blot. Based on these results, compounds 5a and 5b suppress β-catenin signaling especially in highly invasive Sk-Hep1 and MDA-MB-231 cells and classical p53 independent cell cycle arrest in Hep3B cells (p53 null type). In another study, we have analyzed the molecular mechanism of 5-Fluorouracil (5-FU) in inducing cell death of human hepatocellular carcinoma Sk-Hep1 cells. In pursuit of novel effective strategy, we have evaluated the potential of 5-FU to promote the endoplasmic reticulum (ER) stress and autophagy in hepatocellular carcinoma Sk-Hep1 cells especially. We found that 5-FU profoundly induces ER stress in Sk-Hep1 cells and up-regulates p53 and activates CHOP/GADD153 and Caspase-12. Activation of CHOP/GADD153 and Caspase-12 promotes mitochondrial cell death in Sk-Hep1 cells followed by ER stress. Accumulation of misfolded proteins and changes in Ca2+ homeostasis in ER lumen results in ER stress and eventually leads to apoptotic cell death. In order to recover the ER from the accumulation of misfolded protein aggregates, the UPR may up-regulate the autophagy machinery. Our study demonstrates that 5-FU induced ER stress suppresses the autophagy and also down-regulates GRP78 expression. Activation of autophagy followed by ER stress could facilitate the cell survival response. Therefore, the inhibition of protective autophagy may provide a useful pharmacological target. These results propose that the 5-FU induced ER stress activates mitochondrial apoptotic cell death pathway by down-regulating GRP78 and protective autophagy proteins in human hepatocellular Sk-Hep1 cells.