Dietary phytochemicals have been shown to be protective against various types of cancers. However, the precise underlying protective mechanisms are poorly understood. In the present study, we report that treatment of A549 cells with quercetin resulted in a dose-dependent reduction in cell viability and DNA synthesis with the rate of apoptosis equivalent to 1.2 +/- 0.8, 6.3 +/- 0.9, 16.5 +/- 1.5, 36.4 +/- 2.6 and 42.5 +/- 5.8% on treatment with 0.1% dimethylsulfoxide, 14.5, 29.0, 43.5 and 58.0 micro M quercetin, respectively. Concomitantly, quercetin treatments led to a 1.1-, 1.1-, 2.5- and 3.5-fold increase in Bax. Similar elevations were also observed in Bad, which increased 1.1-, 2.1-, 2.2- and 2.3-fold, respectively, as compared with control. While Bcl-2 was decreased by 30%, Bcl-x(L) was elevated in a dose-dependent fashion. Quercetin also induced the cleavage of caspase-3, caspase-7 and PARP (poly ADP-ribose polymerase). While Akt-1 and phosphorylated Akt-1 were inhibited, the extracellular signal-regulated kinase (ERK) was phosphorylated following quercetin treatment in a dose-dependent fashion. Phosphorylation of ERK and c-Jun occurred at 3 h and was sustained over 14 h. Phosphorylation of MEK1/2 was increased in concordance with ERK activation. Quercetin-induced phosphorylation of c-Jun N-terminal kinase (JNK) and cleavage of caspase-3 occurred 6 h after quercetin exposure and before cleavage of caspase-7 and PARP was detected. Inhibition of MEK1/2 but not PI-3 kinase, p38 kinase or JNK abolished quercetin-induced phosphorylation of c-Jun, cleavage of caspase-3 and -7, cleavage of PARP and apoptosis. Inhibition of caspase activation completely blocked quercetin-induced apoptosis. Expression of constitutively activated MEK1 in A549 cells led to activation of caspase-3 and apoptosis. The results suggest that in addition to inactivation of Akt-1 and alteration in the expression of the Bcl-2 family of proteins, activation of MEK-ERK is required for quercetin-induced apoptosis in A549 lung carcinoma cells.
Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies in South East Asia. Although activation of the MEK-MAPK is often associated with cellular growth, the role of MEK-MAPK in growth and survival of hepatocarcinoma cells has not been established.
Purpose: Our aims were to establish and characterize primary human hepatocellular carcinoma xenografts. They were used to screen new drugs and improve our current treatment regimens used in hepatocellular carcinoma. Experimental Design: Primary hepatocellular carcinomas were used to create the xenografts. Western blotting was used to determine the changes in proteins in these xenografts before and after therapies. Apoptotic and cell proliferation were analyzed by immunohistochemistry. Results: Seven lines of xenografts were established from primary human hepatocellular carcinomas. Lines 4-1318, 2-1318, 2006, and 26-1004 grew rapidly in severe combined immunodeficient (SCID) mice and doubled its volume every 48 to 72 hours. Series 5-1318 (5-1318, 30-1004, and 29-1104) grew relatively slowly in SCID mice and required ∼6 to 10 days to double its tumor volume. Western blot analysis revealed that the growth rate of these xenografts was associated with abnormal expression of proteins associated with the cell cycle, signaling pathways, and tumor suppressor genes. Although hepatocellular carcinoma xenografts expressed the receptors for androgens, estrogens, and progesterone, their growth rate was not affected by either castration or sex steroid hormone supplementation. Cisplatin, oxaliplatin, vitamin D analogue EB1089, and Iressa had no effects on the growth rate in SCID mice. Although 5-fluorouracil exerted mild growth inhibition of these xenografts, i.p. delivery of 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) or doxorubicin resulted in a significant growth inhibition. Doxorubicin-induced growth suppression was associated with elevation of p53 and p21Cip1/Waf1. In addition to up-regulation of p53 and p21Cip1/Waf1, SarCNU also increased the levels of phosphorylated cdc-2 at Tyr15. Conclusion: Hepatocellular carcinoma xenografts are powerful tools for screening drugs and SarCNU may be useful in the treatment of this fatal disease.
Purpose: Hepatocellular carcinoma (HCC) is the fifth most common primary neoplasm; surgery is the only curative option but 5-year survival rates are only 25% to 50%. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) are known to be involved in growth and neovascularization of HCC. Therefore, agents that target these pathways may be effective in the treatment of HCC. The aim of this study was to determine the antineoplastic activity of brivanib alaninate, a dual inhibitor of VEGF receptor (VEGFR) and FGF receptor (FGFR) signaling pathways. Experimental Design: Six different s.c. patient-derived HCC xenografts were implanted into mice. Tumor growth was evaluated in mice treated with brivanib compared with control. The effects of brivanib on apoptosis and cell proliferation were evaluated by immunohistochemistry. The SK-HEP1 and HepG2 cells were used to investigate the effects of brivanib on the VEGFR-2 and FGFR-1 signaling pathways in vitro. Western blotting was used to determine changes in proteins in these xenografts and cell lines. Results: Brivanib significantly suppressed tumor growth in five of six xenograft lines. Furthermore, brivanib–induced growth inhibition was associated with a decrease in phosphorylated VEGFR-2 at Tyr1054/1059, increased apoptosis, reduced microvessel density, inhibition of cell proliferation, and down-regulation of cell cycle regulators. The levels of FGFR-1 and FGFR-2 expression in these xenograft lines were positively correlated with its sensitivity to brivanib-induced growth inhibition. In VEGF-stimulated and basic FGF stimulated SK-HEP1 cells, brivanib significantly inhibited VEGFR-2, FGFR-1, extracellular signal-regulated kinase 1/2, and Akt phosphorylation. Conclusion: This study provides a strong rationale for clinical investigation of brivanib in patients with HCC.
Hepatocellular carcinoma (HCC) is a common malignancy in Asia and Africa. We previously reported that overexpression of extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2) and ERK1/2 was detected in HCC, and that their activation was required for liver cancer cell proliferation and survival. In the present study, we determined the efficacy of a specific MEK1/2 inhibitor AZD6244 (ARRAY-142886) in treatment of HCC. Treatment of primary HCC cells with AZD6244 led to growth inhibition, elevation of the cleavage of caspase-3 and caspase-7, and cleaved poly(ADP)ribose polymerase, but inhibition of ERK1/2 and p90RSK phosphorylation. Studying the protein expression profile of seven HCC xenografts revealed that their growth rate was positively correlated with the levels of phosphorylated MEK. AZD6244, when given p.o. to mice bearing these xenografts, resulted in a dose-dependent inhibition of tumor growth. AZD6244-induced growth suppression was associated with inactivation of ERK1/2 and p90RSK, and up-regulation of activated caspase-3 and caspase-7, and cleaved poly(ADP)ribose polymerase. Our data suggest that the MEK-ERK pathway plays an important role in the growth and survival of liver cancer cells and that the HCC xenograft models are excellent tools for screening preclinical drugs.
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