Background
Many experimental evidence indicates that TNF-α is associated with the survival of cancer cells [
1,
2]. TNF-α-mediated the killing of certain cancer cells has been demonstrated [
3,
4]. Although TNF-α itself was named for its ability to induce cell death, it has been known that TNF-α stimulation also can induce activation of the transcription factor NF-κB [
5‐
8]. Many normal cells are not killed by TNF-α and this may be related to NF-κB transactivation; blockade of NF-κB sensitizes cells to TNF-α and augments induced apoptotic cell death [
9]. TNF-α induced NF-κB transactivation through the pathway of IκB kinase complex phosphorylation, degradation of IκBα and release of cytoplasm-sequestered [
10]. TNF-α-induced NF-κB transactivation is mainly composed of a hetero-dimer of p65 (RelA) and a p50 subunits. NF-κB transactivation can activate expression of a wide variety of genes including the Ferritin heavy chain [
11,
12]. Recent studies have shown that NF-κB-regulated FHC can inhibit caspase activity and can prevent TNF-α-induced apoptosis [
13]. Additional studies have shown that suppression of IAP genes sensitized endothelial cells to TNF-α-induced apoptosis. We have previously shown that Hep3B and SMMC-7721 cells are resistant to serum starvation-induced cell death due to activation of NF-κB by TNF-α. In the present study, we show that serum starvation induced significant apoptosis in the Hep3B and SMMC-7721 cells, and this cell death was attenuated by pre-incubation of TNF-α via suppression of caspase activation and coincident with Ferritin heavy chain up-regulation. Inhibition of NF-κB transactivation using a pharmacological inhibitor of IKK abrogated the TNF-α-induced protection against serum starvation killing. We demonstrate that temporal TNF-α-mediated suppression of serum starvation-mediated apoptosis may be due to the transient up-regulation of FHC by TNF-α.
Methods
Cell culture and regent
Human hepatocellular carcinoma cell lines Hep3B and SMMC-7721 were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Human hepatocellular carcinoma cell lines Hep3B and SMMC-7721 were cultured at 37°C, with 5% CO2, in Dulbecco’s modified Eagle’s medium (high glucose) (GIBCO, Invitrogen) with 10% fetal bovine serum, supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100ug/ml streptomycin. Cells were subcultured every 3 days when they reached 70%-80% confluence. 3-Methyladenine (3-MA, Sigma-Aldrich) was dissolved in heated sterile double distilled water to make a 400 mM stock solution and then added to the medium after heating for a final concentration of 5 mM.
Western blot analysis
Total protein was extracted from cells using lysis buffer and the protein concentrations were measured by BCA protein assay. The cell lysates were loaded on SDS-PAGE, electrophoresed and transferred onto the PVDF membranes. The membranes were blocked in 5% non-fat dry milk in 0.01% Tween/PBS, incubated in primary antibody overnight at 4°C, then incubated in HRP-conjugated secondary antibodies and developed using ECL plus detection reagent. The primary antibodies used in this study are: P62 (#5114, Cell Signaling Technology), LC3 (#4108, Cell Signaling Technology), IκBα (#4812, Cell Signaling Technology), P65 (sc-8008, Santa Cruz Biotechnology, Inc), Cleaved caspase-3 (ab52293, Abcam), Caspase-8 (AP0358, Bioworld), FHC (ab65080, Abcam).
MTT assay
3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide assay (Sigma Aldrich) was used to determine cell survival. Cell count was adjusted to 1 × 104 cells/ml 100 μl of cells suspension was plated in each well of 96-well plate. At the end of the various treatment, the medium was removed and cells were immediately washed with PBS, then 150 μl/well of MTT solution was added. After 3 h, media containing MTT was removed and 100 μl of DMSO was added to each well to dissolve the formazan crystals. Absorbance was taken at 570 nm and 655 nm. Experiments were performed in triplicate and repeated three times.
RNA isolation and real-time PCR
Total cellular RNA was isolated from SMCs using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA was subjected to reverse transcription using Taqman reverse transcription kit (Applied Biosystems) following the manufacturer’s instructions. Real time PCR amplifications were performed using iQTM SYBR Green supermix (BIO-RAD). The relative quantities of mRNAs were obtained by using the comparative Ct method and were normalized with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Primers sequences for FHC were: forward 5′-ATTTCCCCATAGCCGTG -3′, reverse 5′-GCCTGGATAGATTTCTGATTC -3′.
The measurement of ROS accumulation
The intracellular ROS levels were detected by means of an oxidation-sensitive fluorescent probe (DCFH-DA). Briefly, the cells were cultured and treated with the indicated time intervals. Then, the cells were harvested, washed twice with PBS, incubated with DCFH-DA (1 μM) in serum-free DMEM at 37°C in a 5% CO2 incubator for 20 minutes, washed twice with PBS and analyzed by Immunofluorescence microscope.
Transient transfection and identification of autophagy
Hep3B and SMMC-7721 cells were seeded (5 × 104 cells/well) in 96-well plates for overnight, then GFP-LC3 expressing plasmids were transiently transfected into the cells using Fugene HD transfection reagent (Roche) according to the manu-facturer‘s instructions. After cultured for 24 h to ensure the expression of GFP-LC3, the cells were subjected to different treatment. At the end of the treatment, autophagy was detected by counting the percentage of cells with GFP-LC3-positive dots under fluorescence microscope. Aminimum of 200 cells per sample was counted in triplicate for each experiment.
Plasmid transfection
The site-specific, signal-induced degradation of IκBα depends on phosphorylation at Ser 32 and 36. Therefore, the pBαbe-SR-IκBα plasmid that consisted of a double point mutation (Ser to Lactamine) was thus resistant to phosphorylation. The mutant and control plasmids were transiently transfected into Hep3B and SMMC-7721 cells by Lipofectamine. Hep3B and SMMC-7721 cells were removed by trypsin/EDTA treatment and seeded at a density of 2x105 cells/ml in 6-cm culture dishes. Cells were grown to 90% confluence and subjected to 24-h synchronization in serum-free medium. Hep3B and SMMC-7721 cells were transfected with 4 μg of the pBαbe-SR-IκBα or control pBαbe plasmid per dish with the use of Lipofectamine. After incubation for 6 h, the transfection medium was replaced by fresh medium for an additional 48-h incubation to allow for gene expression to occur.
Short hairpin RNA
shRNA candidate target sequences to Beclin1 is 5′-GCAGATGAGGAAGATCGCCTT-3′. The oligonucleotides encoding the shRNA sequence were inserted into the GFP express vector pGCL-GFP (Shanghai GeneChem, shanghai). SCR-shRNA was used as a negative RNAi control. The recombinant virus was packaged using Lentivector Expression Systems (Shanghai GeneChem).
Apoptosis assays
Hep3B and SMMC-7721 cells were plated (1 × 106 cells/well) onto 6-well plates for overnight in incubator to resume exponential growth. At the end of the various treatment, every sample were removed from the medium and washed with PBS twice. Then the cells were stained with FITC-conjugated Annexin V and propidium iodide (PI), using Annexin V-FITC Apoptosis Detection kit and according to manufacturer‘s recommendation (Calbiochem). Flow cytometry (BD Biosciences, USA) was used to determine the percentage of apoptotic cells.
NF-κB-dependent reporter gene assay
NF-κB luciferase reporter assays were performed as described previously [
14]. Briefly, cells were co-transfected with a pNF-κB-luc reporter construct and a renilla luciferase-expressing plasmid (internal control to normalize for transfection efficiency) using Lipofectamine 2000 according to the manufacturer’s instructions. At the end of the various treatment, firefly and renilla luciferase activities were assessed using a dual luciferase reporter gene assay kit. NF-κB transcriptional activity = relative light units of firefly luciferase/relative light units of renilla luciferase.
Immunofluorescence
Immunofluorescence staining was performed according to standard protocol (Santa Cruz Biotechnology). Hep3B and SMMC7721 cells were seeded (1 × 105 cells/well) on a 48-well plate, cultured in DMEM without FBS and Antibiotic for 6 h, and 3-MA was added to the cell culture at the same time. Then cells were treated with or without TNF-α (10 ng/ml) for 24 h, then the cells were washed twice with PBS, and fixed in 4% paraformaldehyde and 0.1% Triton X 100 in PBS buffer at 4°C for 30 minutes. After being washed with PBS, the cells were incubated with the blocking solution (10% goat serum in PBS), and then incubated overnight with the primary antibodies, washed with PBS, and finally incubated with secondary antibodies at 37°C for 2 hours. After being stained with DAPI, all matched samples were photographed using an immunofluorescence microscope and identical exposure times.
Statistical analysis
All the experiments were performed at least three times. Student’s t-test was used for all the statistical analyses, and the differences were considered significant if the p value was less than 0.05.
Discussion
TNF-α is an extraordinarily pleiotropic cytokine produced mainly by activated macrophages and a few by several other types of cell [
27,
28]
. Under normal physiological conditions TNF-α plays a part in maintenance and homeostasis of host defence and the immune system; on the other side, its “inappropriate” overexpression is implicated in malignant disease and pathological injury, such as insulin resistance, autoimmunity, septic shock, allergy and allograft rejection [
28,
29]. TNF-α is well known for the anticancer properties and is being an effective anticancer agent for the treatment of patients with locally advanced solid tumors [
29]. However, TNF-α is a double-edged sword for cancer. TNF-α could be an endogenous tumor promoter in tumor microenvironment, because TNF-α stimulates cancer cells’ growth, proliferation, invasion and metastasis, and tumor angiogenesis [
30‐
32]. It is of significance to research the relationship between TNF-α and HCC, which is one of the 10 most common human carcinomas in the world. Past studies have shown that TNF-α contributes to liver carcinogenesis early in the preneoplastic phase through driving oval cell proliferation [
33]. In the model of p-glyco-protein 2 (Mdr2)-knockout mice, TNF-α and the activation of NF-кB accelerated the process of tumor in the occurrence of HCC [
34]. In our study, it is consistent with previous studies [
4,
29], high doses of TNF-α (100 or 1000 ng/ml) enhanced serum starvation-induced apoptosis, which maybe due to the direct killing effect of TNF-α on tumor cells. Interestingly, low doses of TNF-α (0.1, 1 and 10 ng/ml) attenuated serum starvation-induced apoptosis, especially in the 10 ng/ml TNF-α group (Figure
1). The results suggested that TNF-a may play an important role in HCC survival.
In the present study, we examine the TNF-α effect on serum starvation-mediated apoptosis of HCC cells in light of the potential importance of TNF-α in HCC survival, we examine whether the TNF-α effect on serum starvation-induced apoptosis of HCC cells via autophagy. Autophagy can also be considered a temporary survival mechanism during periods of serum starvation where self-digestion provides an alternative energy source and also may facilitate the disposal of unfolded proteins under stress conditions [
35]. Autophagy has a dynamic role in cancer—both as a tumor suppressor early in progression and later as a protumorigenic process, critical for tumor maintenance and therapeutic resistance [
36‐
38]. Evolving tumors develop regions of hypoxia and nutrient limitation, where elevated autophagy activation has been found previously to promote tumor survival. Moreover, the role for the TNF-α-induced NF-κB pathway in autophagy was supported by the evidence that the IKK complex was necessary for the stimulation of autophagy by several factors [
18,
39,
40]. Therefore, it is necessary to examine whether the TNF-α effect on serum starvation-induced apoptosis of HCC cells via autophagy. In the present study, we showed that 3-Methyladenine (3-MA) attenuated TNF-α protection against serum starvation-mediated apoptosis (Figure
2). This suggested that autophagy conferred the TNF-α protection against serum starvation-mediated apoptosis.
In our study, Pre-induction of cells with the pharmacological inhibitor-Bay11-7082, inhibited NF-κB transactivation, attenuated TNF-α protection against serum starvation-mediated apoptosis in Hep3B and SMMC-7721 cells. Our results suggested that TNF-α induced transactivation of NF-κB in favor of survival or anti-apoptotic signaling in Hep3B and SMMC-7721 cells (Figure
3 and Figure
4). Ferritin heavy chain (FHC) is one of the NF-κB-regulated genes that counteract apoptotic signaling by TNF-α in a number of cells [
26]. It is identified that FHC as a pivotal effector of the antioxidant and protective actions of NF-κB downstream of TNF-Rs [
41]. FHC is upregulated by TNF-α through a mechanism controlled by NF-κB, which is required for inhibition of TNF-α-induced killing and blocking PCD in NF-κB-deficient cells [
26]. FHC is one of several acute-phase proteins, induced in the liver during the organismal response to stress, injury and infection. FHC might also play a prominent role in NF-κB-dependent oncogenesis, tumor progression and cancer chemo- and radio-resistance. High levels of FHC have in fact been found in several tumors and have been associated with resistance to anti-cancer treatment and an aggressive malignant phenotype [
42]. In our Hep3B and SMMC-7721 cells, FHC was present at very low detectable level endogenously, but increased significantly 24 h after TNF-α treatment. The kinetics of TNF-α-induced FHC expression was directly correlated with kinetics of TNF-α protection against serum starvation-induced apoptosis in Hep3B and SMMC-7721 cells. In order to confirm that FHC contributes to apoptosis resistance in Hep3B and SMMC-7721 cells, we suppressed FHC expression in the cells by small interfering RNA (siRNA) and assayed their apoptosis sensitivity. Apoptotic cell population in Hep3B and SMMC-7721 cells transfected with FHC siRNA was larger than that transfected with control siRNA at concentration of 10 ng/ml TNF-α exposure (Figure
5). These results suggested that FHC prevents apoptosis induced by serum starvation.
The observation that FHC protected HCC cells from serum starvation-induced apoptosis prompted us to investigate whether FHC mediated the inhibition of ROS by serum starvation. Human mesothelial cells stably over-expressing FHC generated less H
2O
2 when challenged by asbestos and were resistant to apoptosis induced by oxidant stimuli compared with control cells [
43]. It suggested FHC reduced intracellular oxidative stress triggered by asbestos exposure in mesothelial cells and contributes to apoptosis resistance by diminishing ROS generation. Our results are consistent with the results obtained by others, using Hep3B and SMMC-7721 cells. Recently, it is reported the key role of FHC in regulating apoptosis during inflammation. They showed that FHC was required to prevent sustained c-Jun N-terminal kinase cascade activation, thus inhibiting apoptosis induced by TNF-α. FHC-driven inhibition of c-Jun N-terminal kinase signaling depends on suppressing ROS generation and is achieved through kllits ability to sequester iron. Our results showed HCC cells over-expressing FHC generated less ROS when challenged by TNF-α and were resistant to apoptosis induced by serum starvation. These results suggested that autophagy conferred the TNF-α protection against starvation-mediated apoptosis of hepatocellular carcinoma cells, the process involved with transactivation of NF-κB, up-regulation of anti-apoptotic FHC, reactive oxygen species and caspase suppression. Understanding the contribution of TNF-α-mediated cell survival may be relevant to the therapy of hepatocellular carcinoma.
Acknowledgements
This project was supported by a grant from Key Basic Research Project of China (2011CB966200, 2010CB945600, 2012CBA01303, 2011CB965100); Key project of National Natural Science Foundation of China ( 81030041); National Natural Science Foundation of China (31171321, 81101622, 81201584); Special Funds for National key Sci-Tech Special Project of China (2012ZX10002-016, 2012ZX10002011-011); Shanghai Science and Technology Committee (10ZR1439600, 11ZR1449500, 12431900802, 12ZR1454200, 11 nm0504700, 09QA1407200); Shanghai Municipal Health Bureau (XYQ2011044, 20114004); Science Fund for Creative Research Groups, NSFC, China (81221061).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
XRK, YYJ, WJD and LXW participated in the design and performance of the study. KS, ZPH and FY performed statistical analysis. GFY, QMF, LG, XZ, QDZ and RL carried out cell culture and molecular studies. MCW and LXW conceived of the study and participated in its design and coordination. The manuscript was drafted by XRK, YYJ and WJD, and reviewed by all authors. All authors approved the final version of the manuscript to be published. All authors read and approved the final manuscript.