TNFα induces Ca²⁺ influx to accelerate extrinsic apoptosis in hepatocellular carcinoma cells

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.

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.

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.

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 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.

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 was assayed using a fluorometric kit (Abcam, Cambridge, UK) according to the manufacturer’s protocol.

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.

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.

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).

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).

We firstly analyzed the level of cytosolic Ca²⁺ after TNFα treatment in SNU739 and HLF HCC cells, and found that fluorescence intensity of cytosolic Ca²⁺ indicator Fura-2 was obviously increased after TNFα treatment, which presented a dose-dependent manner (Fig. 1a-d). In contrast, Fura-2 fluorescence was not changed after TNFα treatment when HCC cells were cultured in calcium-free medium (Fig. 1e, f), which indicated that TNFα induced extracellular Ca²⁺ influx into HCC cells.

To explore whether tumor necrosis factor receptors (TNFRs) participated in TNFα-mediated Ca²⁺ influx in HCC cells, the expression of TNFR was measured at both the mRNA and protein level by real-time PCR and Western Blot, respectively. We found that TNFR1 but not TNFR2 was expressed in HCC cells (Additional file 2: Figure S1a, b). Moreover, we successfully silenced TNFR1 expression by siRNA in SNU739 and HLF cells, which was verified by real-time PCR and Western Blot. Our data showed that TNFR1 knockdown had no effect on the expression of TNFR2 at both the mRNA and protein levels in HCC cells, suggesting that a compensatory positive regulation of TNFR-2 expression may be excluded. We further confirmed that TRADD, which is a direct downstream molecular of TNFR1 and functions to transfer cell death signal after TNFα stimulation [14], was not able to effectively interact with TNFR1 after siTNFR1 treatment. These data further demonstrated that TNFR1 was successfully knocked down and TNFR1-induced classical extrinsic pathway was inactivated (Additional file 2: Figure S1c-e). Furthermore, our data indicated that the expression level of TNFR1 had no effect on the TNFα-mediated Ca²⁺ influx in HCC cells (Fig. 2a). These results indicate that TNFR pathway is not involved in the process of TNFα-mediated Ca²⁺ influx in HCC cells.

In recently years, 4 kinds of calcium channels in mammal cells have been identified, including voltage-gated calcium channels (VGCC), ligand-gated calcium channels (LGCC), transient receptor potential (TRP), and store-operated calcium channels (SOCE). As LGCCs are only expressed in excitable cells, and our data in Fig. 1e and f also showed that TNFα had no effect on the level of cytosolic Ca²⁺ in cells cultured in calcium-free medium, so the effects of LGCCs and SOCEs on TNFα-mediated Ca²⁺ influx in HCC cells were ruled out. We then explored whether VGCC and TRP channels participated in the process of TNFα-mediated Ca²⁺ influx in HCC cells. Our data showed that the extracellular Ca²⁺ influx in HCC cells was significantly inhibited by both two TRP channel blockers, CAI and SKF96365, whereas the VGCC channel blockers, verapamil and diltiazem had no effect on the TNFα-mediated Ca²⁺ influx in HCC cells (Fig. 2b, c), suggesting that TRP channels may participate in the process of TNFα-mediated Ca²⁺ influx in HCC cells.

It has been reported that a series of TRP channels, including TRPC1, TRPC6, TRPV5, TRPV6, TRPM3, TRPM7, and TRPP5, are implicated in cell apoptosis process [13‐15]. Our results demonstrated that TRPM7 was the main channel type expressed in HCC cells (Fig. 2d), which was also demonstrated by bioinformatic analysis based on public transcriptome sequencing data from The Cancer Genome Atlas (TCGA) database (Additional file 2: Figure S1 h). Moreover, we found that TRPM7 knockdown significantly inhibited TNFα-mediated Ca²⁺ influx in HCC cells (Fig. 2e, Additional file 2: Figure S1f, g). Furthermore, co-immunoprecipitation assay showed that TNFα did not directly interact with TRPM7 in SNU739 and HLF cells (Additional file 2: Figure S1i). These findings strongly suggest that TNFα-mediated Ca²⁺ influx may be mediated by TRPM7 and independent of TNFR.

We next investigated the roles of TNFα-mediated Ca²⁺ influx in the apoptosis of HCC cells. As shown in Additional file 3: Figure S2a and c, both cytosolic Ca²⁺ scavenger (BAPTA-AM) and Ca²⁺-binding protein parvalbumin (PV) efficiently reduced the level of [Ca²⁺]_c after TNFα treatment in HCC cells. We further found that either Ca²⁺ scavengers or PV reduced the percentage of TNFα-induced apoptotic HCC cells (Fig. 3a, b, Additional file 3: Figure S2b, d). We then investigated whether TRP channels mediated the effects of TNFα-mediated Ca²⁺ influx on cell apoptosis. Our data showed that the apoptotic rates of SNU739 and HLF cells were significantly decreased after treatment of TRP channel blocker, CAI or SKF96365 (Fig. 3c, Additional file 3: Figure S2f), and the same effect was also observed after knockdown of TRPM7 in SNU739 and HLF cells (Fig. 3d, Additional file 3: Figure S2 g). Taken together, our results demonstrate that inhibiting the elevation of TNFα-mediated cytosolic Ca²⁺ attenuates TNFα-induced apoptosis in vitro.

To evaluate the role of TNFα-mediated Ca²⁺ influx in tumor growth in vivo, we established the subcutaneous nude mice model. The mice treated with TNFα exhibited significantly decreased growth capacity than those in the control group (Fig. 3e). Moreover, our results also indicated that the growth capacity of xenograft tumors developed from SNU739-PV cells were significantly enhanced than xenograft tumors developed from SNU739-EV cells with TNFα treatment. These results strongly suggest that PV protein may attenuate TNFα-induced cell apoptosis via buffering the TNFα-mediated Ca²⁺ influx in HCC cells. We further confirmed the effect of PV on TNFα-mediated cell apoptosis in xenograft tumor tissues using TUNEL staining (Fig. 3f). These in vivo data support the evidence that extracellular Ca²⁺ influx contributes to the TNFα-induced HCC cell apoptosis.

Furthermore, we investigated whether increased extracellular Ca²⁺ influx enhanced TNFα-induced apoptosis in HCC cells. Ionomycin (an ionophore with high affinity to calcium) and IP₃ (an agonist to promote calcium releasing from ER) were used to rapidly elevate [Ca²⁺]_c as described previously [16, 17]. As shown in Additional file 4: Figure S3a and d, the optimal concentrations of ionomycin and TNFα were arbitrarily determined as 1 μM and 100 ng/mL, respectively, based on apoptosis rate of less than 10% in normal hepatic cell QSG-7701. Our results showed that ionomycin (1 μM) treatment remarkably increased the level of [Ca²⁺]_c in HLF and QSG-7701 cells after TNFα treatment (Additional file 4: Figure S3b). The similar effects were observed in HLF and QSG-7701 cells after IP₃ (10 μM) treatment (Additional file 4: Figure S3c). Our data further demonstrated that TNFα-induced cell apoptosis was significantly increased at a dose-response manner in HLF and QSG-7701 cells with treatment of either 1 μM ionomycin or 10 μM IP₃ (Fig. 4a, b, Additional file 4: Figure S3e, f). Taken together, these results indicate that the increased cytosolic Ca²⁺ sensitizes HCC cells and normal hepatocytes to TNFα-induced apoptosis. Moreover, our data indicated that the treatment with a combination of 100 ng/mL TNFα and 1 μM ionomycin exhibited an apoptosis percentage of about 37% in HCC cell HLF, but only about 14% in normal hepatocyte QSG-7701, which is two times as treatment with TNFα (100 ng/mL) only (Additional file 4: Figure S3d, e). We have evaluated the toxicity of TNFα only and the combination of TNFα with ionomycin in normal hepatic cell QSG-7701. Our data showed that treatment with 100 ng/mL TNFα and a combination of 100 ng/mL TNFα and 1 μM ionomycin exhibited an apoptosis percentage of 6.5% and 14.6%, respectively, in normal hepatic cell QSG-7701 (Additional file 4: Figure S3d, e), suggesting that the possible toxicity of TNFα may be increased by a combined use of ionomycin, although the pro-apoptotic effect can be improved.

To evaluate whether ionomycin could sensitize HCC cells to TNFα-induced apoptosis in vivo, we treated xenograft tumors developed from SNU739 cells with ionomycin and TNFα. As shown in Fig. 4c, a significant decrease of growth capacity was observed in xenograft tumors treated with the combination of TNFα (40 μg/kg) and ionomyin (3 mg/kg) for one month when compared with xenograft tumors with treatment of TNFα (40 μg/kg) alone. We further confirmed the effect of combined TNFα and ionomyin treatment on cell apoptosis in xenograft tumor tissues using TUNEL staining (Fig. 4d). These in vivo data further indicated that the increased Ca²⁺ influx sensitizes HCC cells to TNFα-mediated cell apoptosis and the induction of Ca²⁺ influx by ionomycin may synergize the apoptotic effect of TNFα.

We then investigated the relationship between TNFα-induced cell apoptosis and the level of extracellular Ca²⁺ influx in 10 HCC cell lines with TNFα treatment (Additional file 5: Figure S4a, b). Our results showed a different degree of TNFα-mediated extracellular Ca²⁺ influx in 10 HCC cells, suggesting that TNFα-mediated extracellular Ca²⁺ influx was a common phenomenon in HCC cells (Fig. 5a). Moreover, we found that TNFα-induced cell apoptosis was obviously positively correlated with the level of extracellular Ca²⁺ influx (Fig. 5b, c).

Previous studies have demonstrated that calpain, a class of Ca²⁺-dependent cysteine proteases, and one of its main substrates the inhibitor apoptosis protein (IAP) plays critical roles in TNFα-induced extrinsic apoptotic pathway [18]. Therefore, we investigated the effect of TNFα-mediated extracellular Ca²⁺ influx on calpain/cIAP/caspase3 pathway. Western blot analysis showed that TNFα had no effect on the expression of calpain. However, the enzymatic activity of calpain was significantly increased in HCC cells with TNFα treatment when compared with control group (Fig. 6a). Our data also showed that the expression of cIAP2 and XIAP was significantly decreased by TNFα treatment, whereas the expression of cIAP1 was not affected. In addition, the expression of the cleaved caspase3 was significantly increased by TNFα treatment (Fig. 6b).

To further investigated the TNFR1-independent mechanism, we re-evaluated the effect of TNFα-meditated Ca²⁺ influx on TNFα-mediated cell apoptosis in HCC cells with TNFR1 knockdown. Moreover, the calpain activity was significantly inhibited by calpeptin (40 μM) treatment, whereas TNFR1 knockdown had no effect on the calpain activity, suggesting that the activation of calpain was independent of TNFR1 (Fig. 6c). In contrast, the expression levels of cIAP2 and XIAP were remarkably increased and the cleaved caspase 3 was decreased by TNFR1 knockdown in HCC cells (Fig. 6d). In addition, the suppression of calpain activity by calpeptin significantly increased the expression of cIAP2 and XIAP and decreased the expression of cleaved caspase 3 in HCC cells with TNFR1 knockdown (Fig. 6d). Besides, similar results were found after the decrease of cytosolic [Ca²⁺] through the forced expression of PV (Fig. 6e, f). In contrast, ionomycin treatment, which can cause the increase of cytosolic [Ca²⁺], significantly inhibited the expression of cIAP2 and XIAP and increased the calpain activity and expression of cleaved caspase 3 in HCC cells with TNFR1 knockdown (Fig. 6g, h). Overall, these results clearly show that cytosolic Ca²⁺ participates in the process of TNFα-induced cell apoptosis through calpain/IAP/caspase3 pathway in HCC cells and demonstrate that TNFα-induced cell apoptosis is affected by both the expressions of TNFR1 and the levels of cytosolic Ca²⁺ in HCC cells. Furthermore, we have also investigated the effect of TNFα on the extracellular Ca²⁺ influx, TRPM7 activity, and activation of calpain signaling in normal hepatic cell QSG-7701. As shown in Additional file 6: Figure S5a, the cytosolic Ca²⁺ level was significantly increased after TNFα treatment in QSG-7701 cells. The extracellular Ca²⁺ influx was significantly inhibited after TRPM7 knockdown, indicating that TNFα-mediated Ca²⁺ influx is also dependent on TRPM7 channel in QSG-7701 cells (Additional file 6: Figure S5b, c). In addition, our results also showed that the activity of calpain was significantly increased after TNFα treatment in normal hepatic cells (Additional file 6: Figure S5d).

To investigate whether TNFα-mediated Ca²⁺influx was associated with mitochondrial-dependent intrinsic apoptosis, we evaluated the mitochondrial membrane potential (MMP), the subcellular location of cytochrome c (Cyto c), and the translocation of Bcl-2 family members in HCC cells after TNFα treatment. As shown in Fig. 7a, TNFα-mediated Ca²⁺ influx had no effect on the MMP of HCC cells. There was also no obvious release of Cyto c from mitochondria to cytoplasm (Fig. 7b), and no obvious translocation of Bax and Bak from the cytoplasm to mitochondria after TNFα treatment (Fig. 7c). Overall, these data indicate that cell apoptosis induced by TNFα-mediated Ca²⁺ influx is independent of intrinsic apoptosis pathway.