Introduction Hepatocellular carcinoma HCC is

Introduction Hepatocellular carcinoma (HCC) is one of the most difficult cancers to treat, and it threatens human health severely [1]. Although various interventions, including surgical operation, radiofrequency ablation, and chemotherapy, have been endorsed, the prognosis of HCC patients remains poor due to metastasis and a high recurrence rate [2]. Gene therapy is a potentially new treatment modality for cancer patients [3]. Recently, p53, a well-known tumor suppressor, was designed to be expressed by an engineered recombinant replication-defective adenovirus (rAd-p53), encouraging clinical responses [4]. rAd-p53 has been used as a gene therapy to cure many different tumors, including HCC [5]; however, rAd-p53 did not achieve the desired effect to cure HCC [5]. Thus, new gene therapies are required to be explored for HCC treatment. As an ancient phylogenetically conserved flavoprotein, apoptosis-inducing factor mitochondrion-associated 1 (AIFM1) possesses both NADH oxidizing and apoptosis-inducing activities [[6], [7], [8]]. AIFM1 is first expressed as a precursor of 67 kDa that contains three domains, including an N-terminal mitochondrial localization sequence (MLS) of 100 amino acids, a spacer of 27 nkcc inhibitor and a C-terminal oxidoreductase domain of 485 amino acids (including a nuclear localization sequence, NLS) [9]. Once the precursor is translocated to the mitochondrial intermembrane space by its N-terminal MLS, it is processed to a mature form of 62 kDa by proteolytic cleavage [6]. Under physiological conditions, the 62-kDa AIFM1 is tethered to the inner mitochondrial matrix, where it plays an important role in regulating electron transport, ferredoxin metabolism, reactive oxygen species generation and ATP production [10]. However, upon induction of apoptosis, the 62-kDa AIFM1 form will be cleaved by activated calpains and/or cathepsins to produce truncated AIFM1 with a molecular weight of 57 kDa [6,7]. Truncated AIFM1 is released from the mitochondria to the cytosol and nucleus to induce apoptosis in a caspase-independent manner [11]. Additionally, truncated AIFM1-induced apoptosis is characterized by chromatin condensation and large-scale 50-kb DNA fragmentation [6,7].
Materials and methods Cell culture and treatment. Two HCC cell lines, HepG2 and Hep3B, were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. HepG2 and Hep3B cells were grown in Dulbecco's modified Eagle's medium (DMEM). DMEM was supplemented with 10% fetal bovine serum (FBS). shRNA-mediated interference was used to knock down caspase3, DRAM and AIFM1 in HepG2 and Hep3B cells. Briefly, cells were transfected with lentiviral constructs expressing shRNAs for caspase3, DRAM and AIFM1 (OriGene, MD, USA) or sh Ctrl for 24 h. Positive cells were selected with puromycin for 14 days. Recombinant adenovirus AIFM1 (rAd-AIFM1) encoding full-length AIFM1 was used to infect HepG2 and Hep3B cells. Briefly, HepG2 and Hep3B cells were washed in 1 × PBS three times and were then cultured in serum-free DMEM medium; rAd-AIFM1 was added into the serum-free DMEM for 24–48 h. A recombinant adenovirus vector was used as a control for rAd-AIFM1. Real-time PCR assay. The RNeasy Mini Kit (Qiagen, Hilden, Germany) was used to isolate total RNA from the cultured cells. Reverse transcription was used to synthesize first-strand cDNA using the Superscript II First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA). SYBR Green was used to detect the dsDNA products during the real-time PCR. The mRNA content was normalized to the expression of the housekeeping gene β-actin. The following specific primer sequences were used for real-time PCR: β-actin, 5′-GCCCTGAGGCACTCTTCCA-3’ (forward) and 5′-CGGATGTCCACGTCACACTT-3’ (reverse). The caspase3 and DRAM primers were obtained from the PrimePCR TM SYBR Green® Assay kit (Bio-RAD). Immunoblot assay. Cell lysates were subjected to immunoblot analysis, as previously described [12]. Briefly, total cellular lysates were separated on 10% SDS-PAGE gels, and the separated proteins were then transferred to PVDF membranes. The protein blots were blocked with 5% non-fat milk and sequentially probed with specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies. The detection of specific proteins on the blots was achieved using an enhanced chemiluminescence system (Pierce SuperSignal, Thermo Fisher Scientific Inc. Rockford, IL), and the results were captured on X-ray film.