CHX and Lexa Combination-Induced Apoptosis in HCC fragmentation (nuclear morphology changes). (Apoptotic cells were marked with arrows). C, Huh7 cells were treated with DMSO (Con), CHX (10 mg/ ml), Lexa (1 mg/ml), or combination treatment of Lexa (1 mg/ml) and CHX (10 mg/ml) and apoptosis was evaluated through Annexin V and PI double staining based FACS analysis using the Annexin-V assay kit (see ’materials and methods’). D, The percentage of apoptotic cells were characterized as those that stained with Annexin-V. Data represent the mean values of three independent experiments (*p,0.05). E, LH86 cells were treated with DMSO (Con), CHX (10 mg/ml), Lexa (1 mg/ml), or a combination of Lexa (1 mg/ml) and CHX (10 mg/ml). Apoptosis was measured as in Fig. 1B (Apoptotic cells were marked with arrows). F, LH86 cells were treated with various stimuli as in Fig. 1E. Apoptosis was measured through FACS analysis as in Fig. 1C and 1D and statistical analysis was performed to show percentage of Annexin-V positive cells for apoptosis ratio. Data represent the mean values of three independent experiments (**p,0.05). G, Huh7 cells were treated with Lexa (1 mg/ml) or combination of Lexa (1 mg/ml) and CHX (10 mg/ml) for up to 6 h and harvested. Cell lysates were prepared for Western blotting to detect the cleavages of caspase 8, caspase 10, caspase 3, and Bid. b-actin protein levels were set up as loading control for equal total protein amounts. H, Huh7 cells treated with indicated conditions for 6 h were harvested and cell lysates were prepared and subjected to Western blotting analysis. Caspase 3 activation was evaluated through detecting cleaved bands with specific antibody. b-actin protein levels were set up as loading control for equal total protein amounts. Each experiment was conducted in triplicate or duplicate and repeated twice independently. doi:10.1371/journal.pone.0016966.g001 HSP90 mRNA were from Cell Signaling Technology (Beverly, MA). HCC cells were plated at a density of 16106 cells/well in 6- well plates (BD Bioscience, San Diego, CA). Next day, cells were transfected with 100 nM siRNA duplex mixtures for 24 h in the presence of lipo-fectamine RNAiMax (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. A non-specific random siRNA (Cell Signaling Biotechnology, Beverly, MA) was also transfected at the same concentration as control. Subcellular fractionation This assay was performed as described previously [22]. Cells were harvested and resuspended in three volumes of hypotonic buffer (210 mM sucrose, 70 mM mannitol, 10 mM HEPES (pH 7.4), 1 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 50 mg/ml trypsin inhibitor, 10 mg/ml leupeptin, 5 mg/ml aprotinin, and 10 mg/ml pepstatin. After gentle homog- enization with a Dounce homogenizer, cell lysates were centri- fuged at 10006g for 5 min to remove unbroken cells and nuclei. The postnuclear supernatant was centrifuged at 10,0006g to pellet the mitochondria-enriched heavy membrane fraction. The supernatant was further centrifuged at 100,0006g to obtain the cytosolic fraction. A 30-ml aliquot from the 500 ml cytosolic fraction was analyzed by Western blotting with anti-cytochrome c monoclonal antibody. Detection of Bax conformation change This assays was performed as described previously [23]. Cells were lysed with Chaps lysis buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, and 1% Chaps) containing 1 mM phenylmethyl- sulfonyl fluoride (PMSF), 50 mg/ml trypsin inhibitor, 10 mg/ml leupeptin, 1 mg/ml aprotinin, and 5 mg/ml pepstatin. The cell lysates were normalized for protein content and 500 mg of total protein was incubated with 2 mg of anti-Bax 6A7 monoclonal antibody in 500 ml of Chaps lysis buffer at 4uC for 4 h. Then, 40 ml of protein G plus-agarose were added into the reactions and incubated at 4uC for an additional 2 h. Following three washings in Chaps lysis buffer, beads were boiled in loading buffer, and the conformationally changed Bax protein in the immunoprecipitates was subjected to SDS–PAGE (15% gel) and immunoblot analysis with anti-Bax polyclonal antibody as described above. Results Lexa induces apoptosis in HCC cells in the presence of CHX Increasing evidence indicated that many cancer types including breast cancer cells, lung cancer cells, and hepatoma cells, could develop resistance to TRAIL-induced apoptosis through DR5- mediated activation of NF-kB signal pathway [24–26]. Here, to investigate whether Lexa triggers NF-kB activation in HCC cell lines, Huh7 cells were treated with TNF-a or Lexa for the indicated times. Western blotting results indicated that TNF-a induced Ik-B-a decrease after treatment for 15 min and Lexa did that with treatment for 60 min. However, compared with TNF-a, Lexa could not promote Ik-B-a phosphorylation at 60 min (Fig. 1A); thus Lexa, a DR5 specific monoclonal antibody unlike multifunctional TNF-a, did not activate the anti-apoptotic NF-kB signal pathway through binding to DR5. Then, we tested whether HCC cells are sensitive to Lexa-induced cell death. Huh7 and LH86 cells were treated with Lexa (1 mg/ml) for up to 6 h. Apoptosis assays indicated that both HCC cell lines were resistant to Lexa (see Lexa-treated groups in Fig. 1B, 1C, and 1D for Huh7 cells, and Fig. 1E and 1F for LH86 cells). It has been shown that chemotherapeutic reagent CHX, a protein synthesis inhibitor, can enhance TRAIL-induced apoptosis in tumor cells such as renal cancer [27], colon cancer [28], and lung carcinoma [29]. As cells did not undergo apoptosis with Lexa single treatment, we examined whether CHX can alter the insensitivity of HCC cells to Lexa. Apoptosis assays indicated that pre-treatment with CHX (10 mg/ml for 30 min) followed by Lexa (1 mg/ml for 6 h) could significantly enhance apoptosis induction in both Huh7 and LH86 cells (Lexa and CHX combination treated groups in Fig. 1B, 1C, and 1D for Huh7 cells, and Fig. 1E and 1F for LH86 cells). To further confirm Lexa-induced apoptosis could be enhanced by CHX pre-treatment, we evaluated caspase activation and the cleavage of the pro-apoptotic protein Bid through Western blotting analysis. Our data indicated that co- treatment with Lexa and CHX for 6 h significantly induced proteolytic processing of caspase 8, 10, and 3, as well as Bid (Fig. 1G, right panel), but single treatment with either Lexa or CHX could not result in cleaved bands or active subunits from procaspase 8, procaspase 10, or procaspase 3. Additionally, Bid cleavage was not progressively processed after 3 h (Fig. 1G, left panel), supporting the assumption that these liver cancer cells are resistant to Lexa-induced apoptosis. To investigate the possible signaling pathways underlying the resistance of HCC cells to Lexa, we used the specific inhibitors NBD, PD98059, SB203580, and SP600125 to respectively inhibit the IKK, MAPK, P38, and JNK signaling pathways. As shown in Fig. 1H, in comparison with positive control that CHX and Lexa combination treatment- induced caspase 3 cleavages, the other four inhibitors did not reverse the insensitivity of HCC cells to Lexa-induced apoptosis. These results suggest that Lexa and CHX combination treatment synergistically induces apoptosis in HCC cells. Lexa and CHX combination treatment induces caspase dependent apoptosis Death receptor-mediated apoptosis can be classified as either caspase dependent or caspase independent [30,31]. To examine whether Lexa and CHX combination-induced apoptosis is PLoS ONE | www.plosone.org 4 February 2011 | Volume 6 | Issue 2 | e16966