Expression of Livin and the Inhibition of Tumor Progression by Livin Silencing in Laryngohypopharyngeal Cancer

Materials and Methods

Patients and tumor specimens. For evaluation of the protein expression of Livin, paraffin-embedded tissue sections were collected from 60 patients who underwent diagnostic biopsy for advanced LHSCC (stage III and IV) at the Chonnam National University Hwasun Hospital (Jeonnam, Korea) between July 2004 and February 2009. All 60 patients were treated with three cycles of cisplatin-, 5-fluorouracil-, and docetaxel (DCF)-based induction chemotherapy (IC), followed by cisplatin-based concurrent chemoradiation therapy (CCRT) in 56 patients with complete response (CR) or partial response (PR) after IC and two patients who refused operation despite of stable disease (SD) after IC. Another two patients with SD after IC underwent salvage total laryngectomy. CR was defined as no visible or palpable disease in the assessment by physical examination, laryngoscopy, and computed tomography. PR was defined as a >50% decrease in tumor size, compared with the initial measurement. SD was defined as stationary or progressive disease. The patients' clinicopathological characteristics were reviewed in hospital records. Tumors were staged according to the seventh edition of the American Joint Committee on Cancer staging system (11). Survival was measured from the start of chemotherapy to the date of death or date last seen. This study was approved by the Institutional Review Board of the Chonnam National University Hwasun Hospital.

Immunohistochemistry. Paraffin tissue sections were de-paraffinized, rehydrated, and treated with peroxidase-blocking solution (Dako, Carpinteria, CA, USA). The tissues were incubated with polyclonal rabbit anti-human Livin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing in Tris-buffered saline-Tween 20 buffer (TBST), tissues were stained using Dako RealTM Envision horseradish peroxidase (HRP)/3,3’-diaminobenzidine (DAB) detection system (Dako). Tissues were counter-stained with hematoxylin and mounted with coverslips. Two independent observers interpreted staining intensity of the specimens without any knowledge of the patients' clinical information. Assessment for staining intensity was performed as follows: 0, no staning of tumor cells; 1+, weak to comparable staining in cytoplasm and/or the nucleus compared to that of non-tumoral cells; 2+, readily appreciable or dark brown staining distinctly marking the tumor cell cytoplasm and/or nucleus. Specimens with 0 or 1+ staining were regarded as negative expression and those with 2+ staining were regarded as positive expression.

Cell culture and transfection. Cells of SNU 1041 and PCI 1 human LHSCC cell lines were provided from Dr. Sung MW (Seoul National University, Seoul, South Korea). Cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) in a humidified atmosphere of 5% CO₂ at 37°C. Small-interfering RNA (siRNA) was used to knockdown endogenous Livin gene expression in LHSCC cells. Cells were transfected with Livin-specific siRNA (Bioneer, Daejeon, Korea) and negative control siRNA (Qiagen, Valencia, CA, USA) using Lipofectamine™ 2000 (Invitrogen) for 48 h.

Cell invasion assay. Cell invasion ability was measured by the number of cells that invaded through a transwell invasion apparatus with 8.0 μm pore size (Costar, Cambridge, UK). Alive cells transfected with Livin siRNA or negative control siRNA were seeded at 3×10⁵ cells in 120 μl of a 0.2% bovine serum albumin (BSA) suspension in the upper chamber. Subsequently, 400 μl of 0.2% BSA containing 7 μg/ml fibronectin (Calbiochem, La Jolla, CA, USA) as the chemoattractant was loaded into the lower chamber. After incubation for 24 h, cells that had moved to the bottom surface of the Transwell were stained with Diff Quik solution (Sysmex, Kobe, Japan) and calculated in five random squares in the microscopic field of view. Results were expressed as the mean±standard error (SE) of the number of cells/field in three individual experiments.

Cell migration assay (wound healing assay). Cells transfected with Livin siRNA or negative control siRNA were seeded in each well of Culture-Inserts (Ibidi, Bonn, Germany) at 1.5×10⁵ cells/well. After incubation for 24 h, each insert was detached and the progression of cell migration was ascertained by photography at 0, 8, 12, and 24 h using an inverted microscope. The distance between gaps was normalized to 1 cm after capture of three random sites.

Cell apoptosis assay. Apoptosis was determined by an Annexin V-fluorescein isothiocyanate (FITC) assay. Forty eight hours after transfection, cells transfected with Livin siRNA or negative control siRNA were collected using trypsin, washed twice in phosphate buffered saline (PBS), and re-suspended in binding buffer (BD Biosciences, San Diego, CA, USA). Annexin V-FITC and 7-amino-actinomycin D (7-AAD; BD Biosciences) were added to the cells, which were then incubated in the dark for 15 min, then re-suspended in 400 ml of binding buffer. Cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Data analysis was performed using standard Cell Quest software (Becton Dickinson).

Protein isolation and western blot analysis. Cells were lysed in RIPA buffer (1 M Tris-HCl, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA) with 1 mM phenylmethanesulfonyl fluoride (PMSF), Halt™ phosphatase inhibitor and Halt™ protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). Resolved proteins (10-20 μg) were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Specific proteins were sequentially blotted with primary antibodies: Livin, cleaved caspase-3, cleaved caspase-7, cleaved poly ADP ribose polymerase (PARP), and β-actin (Cell Signaling Technology, Danvers, MA, USA) and polyclonal anti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology). Immunoreactive proteins were visualized on the enhanced chemiluminescence (ECL) detection system HRP substrate (Millipore) and the LAS-4000 luminescent image analyzer (Fujifilm, Tokyo, Japan).

RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR). Total RNA from cells was extracted using Trizol reagent (Invitrogen), reverse transcribed, and amplified using specific primers for LIVIN and GAPDH. The primer sequences were: Livin α and Livin β 5’-CAC ACA GGC CAT CAG GAC AAG-3’/ 5’-ACG GCA CAA AGA CGA TGG AC-3’ and GAPDH 5’-ACC ACA GTC CAT GCC ATC AC-3’/ 5’-TCC ACC ACC CTG TTG CTG TA-3’. The sizes of the amplified products were 456 bp for Livin α and 403 bp for Livin β. For cDNA synthesis, 1 μg mRNA was mixed with 50 ng/μl oligo-dT (Promega, Madison, WI, USA), Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Invitrogen), and RNAsin (Takara Bio, Shiga, Japan). PCR amplification of cDNA was performed using specific primers and GoTaq® DNA polymerase (Promega). PCR products were separated by electrophoresis on a 1% agarose gel containing ethidium bromide.

Statistical analyses. The relationship between Livin expression and various clinicopathologic parameters was compared using the χ² test and Fisher's exact test. Survival curves were calculated using the Kaplan-Meier method, and comparison of curves was performed using the log-rank test. Experimental differences between the Livin knockdown group and control group were tested with the Student's t-test. The Statistical Package for the Social Sciences (SPSS) version 21.0 (Microcal Software Inc, Chicago, IL, USA) was used for all analyses. A p-value <0.05 was considered statistically significant.

Ethical considerations. Local research ethics committee approval was obtained.