Background
TNFα is a 23KD type II transmembrane protein, which is arranged in stable homotrimers. It is primarily produced by macrophages and a variety of other cells, including NK cells, T lymphocytes, smooth muscle cells, fibroblasts and others [
1]. Many preclinical data suggest that TNFα may be used as a highly specific anti-cancer drug against many types of tumors [
1,
2]. Moreover, recombinant human TNFα (rhTNFα) has been tested as a systemic treatment of cancer patients in several phase I and phase II clinical trials [
3]. However, the initial enthusiasm for the development of TNFα as a systemic treatment has waned when facing significant toxicities and a lack of evidence for therapeutic benefit [
2]. How to increase the anti-cancer activity of TNFα at a low-dose condition is the key problem which needs to be solved urgently.
Ca
2+, as a common signal transduction factor, has played many important roles in the process of cell division, growth, and death [
4‐
6]. The cytosolic Ca
2+ level is always risen during the process of cell apoptosis [
7,
8]. Recent studies have revealed that TNFα is related with remodeling of cytosolic Ca
2+ homeostasis in a variety of human cells [
9,
10]. For example, a study with human pulmonary artery endothelial cells (HPAEC) has reported that TNFα exposure significantly increases TRPC1 expression level and thrombin-induced Ca
2+ influx in TNFα-stimulated HPAEC is two-fold greater than that in control cells [
11]. Moreover, Wang GJ et al. have reported that TNFα induces the increases of cytosolic Ca
2+ level, CaMKIIδB and CaN expression levels, and thus promotes cardiac hypertrophy [
12]. More recently, another study has showed that TNFα induces cell death by the activation of transient receptor potential melastatin (TRPM2) to increase cytosolic Ca
2+ level, followed by caspase activation and PARP cleavage [
13]. Although some studies have reported that the increase of cytosolic Ca
2+ level induced by TNFα plays important roles in varieties of physical activities, including cell death, it is still largely unknown about the functional roles and mechanisms underlying cytosolic Ca
2+ in TNFα-induced cell death and whether the remodeling of cytosolic Ca
2+ facilitates the pro-apoptotic effect of TNFα.
Here, we showed that TNFα induced extracellular Ca2+ influx in HCC cells. Importantly, the increased level of cytosolic Ca2+ mediated by TNFα was positively correlated with TNFα-induced cell apoptosis. The molecular mechanisms underlying the cytosolic Ca2+ in regulating TNFα-induced cell apoptosis were also deeply explored. Furthermore, combination with ionomycin was proven to be able to enhance the anti-cancer activity of TNFα in the mouse model.
Methods
Cell culture and public dataset collection
Human cell lines SNU739, SNU368, SNU354, SNU878, JHH-2, Huh-1, HLF, HLE, SMMC772, MHCC97H and QSG-7701 were routinely cultured. The authentication information of cell lines was provided in supplementary files. The detailed information about TCGA database was listed in Additional file
1: Table S3.
Knockdown, forced expression of target genes
Small interfering RNAs (siRNAs) were synthesized by GenePharma Company (Shanghai, China). The sequences of siRNA and primers were listed in Additional file
1: Table S2.
qRT-PCR and western blot
RNA extraction, cDNA synthesis and qPCR reactions were performed in Additional file
1: Supplementary Methods. Primers used in this study were list in Additional file
1: Table S2. The western blot assay was performed as described in Additional file
1: Supplementary Methods. The primary antibodies used in this study and the working concentration were listed in Additional file
1: Table S1.
Measurement of cytosolic Ca2+
Cells were loaded with Fura-2/AM (Invitrogen) for 30 min at 37 °C, and examined with a confocal laser scanning microscope FV1000 (Olympus, Tokyo, Japan). After 40 s of baseline recording, TNFα (100 ng/mL) was added where appropriate, and confocal images were recorded every 2 s.
Cell apoptosis assay
Cell apoptosis was measured by Annexin V-FITC detection Kit (BestBio, Shanghai, China) according to the manufacturer’s protocol. The percentages of total apoptotic cells (both early and late) defined as the AnnexinV-FITC-positive fraction were determined.
TUNEL assay
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (Roche Applied Science, Rotkreuz, Switzerland) was performed to analyze cell apoptosis in xenograft tissues according to the manufacturer’s protocol. Images of TUNEL/DAPI-stained sections were grabbed by a confocal laser scanning microscope FV1000 (Olympus). The apoptosis ratio was calculated as the percentage of both TUNEL and DAPI-positive nuclei after at least 500 cells were counted.
Calpain activity assay
Calpain activity was assayed using a fluorometric kit (Abcam, Cambridge, UK) according to the manufacturer’s protocol.
Nude mice xenograft model
Nude mice xenograft model was used to assess in vivo tumor growth as described in Additional file
1: Supplementary Methods. TNFα (40 μg/Kg) only, or ionomycin (3 mg/Kg) only, or TNFα (40 μg/Kg) combined with ionomycin (3 mg/Kg) were administered by tail vein injection every three days with vehicle (40% (wt/vol.) of 2-hydroxyproplyl-β-cyclodextrin for one month.
Immunoprecipitation
For immunoprecipitation experiments, total cell protein or synthesized TNFα were incubated with 200 μL protein A beads (BEAVER, Suzhou, China) supplemented with an antibody overnight at 4 °C. After washing the protein A beads, normalized amounts of total lysates or immunoprecipitated samples were analyzed by SDS-PAGE and western blot.
Detection of mitochondrial membrane potential
Mitochondrial membrane potential was measured using the fluorescence probe TMRM (Invitrogen) according to the manufacturer’s protocol. Cells were incubated with 10 nM TMRM for 10 min at 37 °C in the dark and images were captured by laser confocal microscope (FV1000, Olympus) and analyzed by ImagePro image analysis software (Media Cybernetics, Silver Spring, MD, USA).
Detection of cytochrome c release
Cytochrome c release was measured by immunofluorescence staining assay. Briefly, cells were incubated with Mito-Tracker Red (1 μM) for 45 min at 37 °C in the dark. After washing the dyes, cell samples were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.2% Triton X-100 for 10 min, blocked by 2% BSA for 30 min and incubated with Cytochrome c antibody (1:100) overnight at 4 °C. Cell samples were then incubated with fluorophore-conjugated secondary antibody (1:200) and visualized by a confocal laser scanning microscope FV1000 (Olympus).
Statistical analysis
SPSS 17.0 software (SPSS, Chicago, IL) was used for all statistical analyses and P values less than 0.05 was considered to be statistically significant. Unpaired t-tests were used for comparisons between two groups where appropriate after checking for normal distribution and equal variance of the data. One-way ANOVA were used for comparisons among three or more groups. Correlations between measured variables were tested by Spearman’s rank correlation analyses.
Discussion
TNFα has been proven to be an effective anticancer agent in a series of preclinical studies [
2]. However, the promise of systemic TNFα has not translated to patient therapy and the enthusiasm had been cubed due to the toxicity profile and lack of efficacy at maximum tolerated dose (MTD) of the patients [
2]. In the past decades, lots of researchers have focused on the modifications of TNFα structure so as to increase its anti-tumor activity, although these modifications are not remarkably effective [
1]. Recently, TNFα has been described to be related with levels of cytosolic Ca
2+ in various cell types [
7,
19], although its effect remains largely unclear. In this study, we for the first time demonstrated that TNFα induced extracellular Ca
2+ influx in HCC cells. Bellomo et al. have reported that TNFα induces a sustained increase in intracellular free Ca
2+ concentration in mammary adenocarcinoma [
19]. Chang et al. have reported that the combined treatment of TNFα and IFN-γ significantly increases the cytosolic Ca
2+ concentration in pancreatic β cell [
7]. However, Carrasquel et al. have reported that TNFα increases the basal level of [Ca
2+]
c after a Ca
2+ pulse in human sperm [
20]. Motagally et al. have reported that the incubation with TNFα decreases depolarization-induced Ca
2+ influx in postganglionic sympathetic neurons [
10]. Moreover, a recent report has provided further supporting evidence, showing that the level of cytosolic Ca
2+ is decreased in cardiocytes after TNFα treatment [
12]. These findings suggest that the level of cytosolic Ca
2+ is regulated by TNFα, but the dual roles of TNFα in regulating the level of cytosolic Ca
2+ may be context dependent or cell type specific, which needs more comprehensive investigation.
Furthermore, both TRP channel inhibitor and TRPM7 knockdown significantly inhibited TNFα-mediated Ca
2+ influx in HCC cells, indicating that the activity of TRPM7 is regulated by TNFα. Our results further showed that TNFα did not directly interact with TRPM7, indicating that TNFα was not a direct activator of TRPM7 channel. Numata et al. have reported that TRPM7 channel is activated by membrane stretch which is produced by negative pressure (3 cm H
2O) or hypotonic solution [
21]. Several organic molecules have also been identified as the activators of TRPM7, including naltriben, mibefradil and bradykinin [
22,
23]. Furthermore, Desai et al. have found that TRPM7 channel is activated by caspase-dependent cleavage [
24]. However, our results showed that TRPM7 channel was immediately activated upon TNFα stimulation and no direct interaction was observed between TNFα and TRPM7. Therefore, the exact mechanism underlying the activation of TRPM7 channel by TNFα needs to be explored in future study.
Changes in the levels of intracellular Ca
2+ provide dynamic and highly versatile signals that control kinds of cellular processes, although their importance is perhaps most strikingly exemplified by their functional role in life-and-death decisions [
5]. Accumulating evidence have demonstrated the increased levels of cytosolic Ca
2+ plays a critical role in cell death [
8,
19]. Bellomo et al. have reported that the increased intra-nuclear free Ca
2+ induced by TNFα enhances the activity of Ca
2+-dependent endonuclease, resulting in DNA fragmentation and cell apoptosis [
19]. In consistence with above-mentioned study, we found that the decreased cytosolic Ca
2+ level attenuates TNFα-induced cell apoptosis, whereas the increased cytosolic Ca
2+ sensitizes HCC cells to TNFα-induced apoptosis, strongly suggesting that TNFα-induced apoptosis may be positively correlated with the level of extracellular Ca
2+ influx in HCC cells.
Lots of proteases have been identified as the downstream molecules activated by cytosolic Ca
2+ to trigger cell death, such as calpain, calcineurin, and DAP kinase [
8,
25]. Calpain is a Ca
2+-activated cysteine protease localized to the cytosol and mitochondria, which has been shown to regulate apoptosis and necrosis [
25]. It has been demonstrated that calpain mediates the cisplatin-induced apoptosis in human lung adenocarcinoma cells through truncating Bid to tBid and then inducing the mitochondrial apoptotic pathway [
26]. Recently, more and more substrates have been identified to be hydrolyzed specifically by calpain during apoptosis, such as human DNA polymerase epsilon, cain/cabin1, fodrin, p53, caspase 7 and caspase 3 [
26‐
30]. Moreover, inhibitor apoptosis proteins (IAPs) has been found to be hydrolyzed by calpain to promote TNFα-induced classical extrinsic apoptosis [
3,
18]. In the present study, we first reported that cytosolic Ca
2+/calpain/IAPs pathway plays a critical role in synergizing the pro-apoptotic effect of TNFα.
It is generally accepted that mitochondrial Ca
2+ uptake functions in cell proliferation [
5]. However, excessive Ca
2+ load to the mitochondria may induce apoptosis [
8]. Accumulated Ca
2+ within mitochondria regulates production of ATP, and activates of metabolism-related enzymes involved in cell proliferation [
5]. In contrast, mitochondrial Ca
2+ loading also causes PTP opening to irreversibly commit cells to death by causing IMM depolarization, matrix swelling, release of stored Ca
2+ and apoptogenic proteins [
8]. These findings highlight a dual role of mitochondrial Ca
2+ in energy provision and induction of cell death, which depend on the amount of mitochondrial Ca
2+ uptake [
31]. Actually, our data showed that MMP, subcellular location of cytochrome c, and the translocation of Bcl-2 family proteins were not obviously changed by TNFα treatment, strongly suggesting that TNFα-induced extracellular Ca
2+ influx may not trigger mitochondria-dependent cell death in HCC.
Ionomycin is antibiotic produced by
Streptomyces conglobatus, which is characterized as a calcium ionophore used to increase the cytosolic Ca
2+ concentration in numerous studies, but the usage of ionomycin in vivo is rare [
32]. Recently, Zheng et al. has reported that ionomycin treatment effectively improves the hyperglycaemia and insulin resistance in the mouse model of diabetes [
33]. In the present study, our data showed that the combination of TNFα with ionomycin significantly improves the pro-apoptotic effect in HCC cells, although the possible toxicity of TNFα may be also increased. In future study, suitable chemical or biological reagents that specifically increase cytosolic Ca
2+ need to be further screened and tested for better combination effect with TNFα in HCC treatments.