Induction of Apoptosis in TNF-Treated L929 Cells in the Presence of Necrostatin-1

It has been shown that necroptosis—caspase-independent programmed necrotic cell death—can be induced by treatment with tumor necrosis factor (TNF) in the L929 murine fibrosarcoma cell line, even in the absence of a caspase inhibitor. Although it was reported that necrostatin-1—a specific inhibitor of necroptosis—inhibited TNF-induced necroptosis in L929 cells, it has not been elucidated whether the cells eventually die by apoptosis in the presence of necrostatin-1. In this paper, induction of apoptosis was demonstrated in TNF-treated L929 cells in the presence of necrostatin-1. Co-treatment with cycloheximide expedited apoptosis induction in necrostatin-1/TNF-treated L929 cells: typical apoptotic morphological changes, including membrane blebbing and nuclear fragmentation, induction of caspase-3 activity, proteolytic activation of caspases-3, -8, and -9, and cleavage of poly(ADP-ribose) polymerase (PARP) (a well-known substrate of caspase-3) were observed. Moreover, co-treatment with Z-VAD-fmk (a pan-caspase inhibitor) inhibited apoptosis by completely inhibiting caspases, resulting in a shift from apoptosis to necroptosis. In contrast, co-treatment with Z-Asp-CH2-DCB (a caspase inhibitor preferential to caspase-3) inhibited apoptosis without expediting necroptosis. These results indicate that apoptosis can be induced in TNF-treated L929 cells when the cells are protected from necroptosis, and support the notion that partial activation of caspase-8 in the presence of a caspase inhibitor preferential to caspase-3 suppresses both apoptosis and necroptosis.


Introduction
Apoptosis and necrosis are two major forms of cell death with distinguishing morphological features: the former is characterized by cell shrinkage, fragmented nuclei with condensed chromatin, and the formation of apoptotic bodies, whereas the latter is characterized by swelling of the cells and organelles and loss of plasma membrane integrity [1][2][3]. The molecular mechanism of apoptosis was investigated in detail in the 1990s, and it has been established that caspases play crucial roles in the induction of apoptotic cell death [4]. In contrast to post-apoptotic secondary necrosis, caspase-independent programmed necrotic cell death-termed necroptosis-has been elucidated, owing to the identification of necrostatins-specific inhibitors of necroptosis-in the 2000s [5]. Thereafter, the molecular mechanism of necroptosis has been investigated in detail: receptor-interacting protein 1 (RIP1), whose kinase activity is inhibited by necrostatins [6], RIP3, and mixed lineage kinase domain-like protein (MLKL) are critically involved in necroptotic cell death [7][8][9]. Although apoptosis has been reported to be involved in various pathological conditions, recent studies have suggested that necroptosis plays a critical role in several disorders, including myocardial infarction, stroke, ischemia-reperfusion injury, and inflammatory bowel disease [10].
Whereas necroptosis has been induced in the presence of caspase inhibitors in many types of cells, it has been reported that necroptosis can be induced by the treatment with tumor necrosis factor (TNF) in the L929 murine fibrosarcoma cell line, even in the absence of caspase inhibitors [11,12]. Although it was reported that necrostatin-1 inhibited TNF-induced necroptosis in L929 cells [5,8], it has not been elucidated whether the cells treated with TNF in the presence of necrostatin-1 eventually die by apoptosis. In this paper, induction of apoptosis was demonstrated in TNF-treated L929 cells in the presence of necrostatin-1.

Induction of Necroptosis in TNF-Treated L929 Cells
As reported [11,12], necrotic cell death was induced in L929 cells by treatment with TNF. Morphologically, swelling of the cells without nuclear fragmentation was observed in TNF-treated L929 cells (Figure 1). TNF-induced necrotic cell death was expedited in the presence of Z-VAD-fmk (ZVAD), a pan-caspase inhibitor, and most of the cells became necrotic within 3 h of treatment with TNF/ZVAD. TNF-induced necrosis was also expedited in the presence of cycloheximide (CHX), a protein synthesis inhibitor. (TNF) in the L929 murine fibrosarcoma cell line, even in the absence of caspase inhibitors [11,12].
Although it was reported that necrostatin-1 inhibited TNF-induced necroptosis in L929 cells [5,8], it has not been elucidated whether the cells treated with TNF in the presence of necrostatin-1 eventually die by apoptosis. In this paper, induction of apoptosis was demonstrated in TNF-treated L929 cells in the presence of necrostatin-1.

Induction of Necroptosis in TNF-Treated L929 Cells
As reported [11,12], necrotic cell death was induced in L929 cells by treatment with TNF. Morphologically, swelling of the cells without nuclear fragmentation was observed in TNF-treated L929 cells (Figure 1). TNF-induced necrotic cell death was expedited in the presence of Z-VAD-fmk (ZVAD), a pan-caspase inhibitor, and most of the cells became necrotic within 3 h of treatment with TNF/ZVAD. TNF-induced necrosis was also expedited in the presence of cycloheximide (CHX), a protein synthesis inhibitor. Next, necrotic cell death of TNF-treated L929 cells was assessed by dual staining with Annexin V (AnV) and propidium iodide (PI) (Figure 2). An increase in AnV + /PI − cells and AnV + /PI + (necrotic) cells was observed in TNF-treated L929 cells within 3 h. Co-treatment with either ZVAD or CHX significantly expedited the appearance of AnV + /PI − , followed by AnV + /PI + cells. As previously reported [5,13,14], necrosis of TNF-treated L929 cells was completely inhibited in the presence of necrostatin-1, an inhibitor of RIP1 kinase activity (Figures 1 and 2). Furthermore, TNF/ZVAD-induced or TNF/CHX-induced L929 cell death was also almost completely inhibited by necrostatin-1. These results indicated that TNF-induced L929 cell death was necroptosis. Next, necrotic cell death of TNF-treated L929 cells was assessed by dual staining with Annexin V (AnV) and propidium iodide (PI) (Figure 2). An increase in AnV + /PI − cells and AnV + /PI + (necrotic) cells was observed in TNF-treated L929 cells within 3 h. Co-treatment with either ZVAD or CHX significantly expedited the appearance of AnV + /PI − , followed by AnV + /PI + cells. As previously reported [5,13,14], necrosis of TNF-treated L929 cells was completely inhibited in the presence of necrostatin-1, an inhibitor of RIP1 kinase activity (Figures 1 and 2). Furthermore, TNF/ZVAD-induced or TNF/CHX-induced L929 cell death was also almost completely inhibited by necrostatin-1. These results indicated that TNF-induced L929 cell death was necroptosis. Dual staining of L929 cells with Annexin V (AnV) and propidium iodide (PI). L929 cells were treated with 10 ng/mL TNF (T), 10 μM Z-VAD-fmk (V), 10 μg/mL cycloheximide (C), or 10 μM necrostatin-1 (N) for the indicated times. AnV + /PI − : early necroptosis; AnV + /PI + : necrosis (late necroptosis). The graph shows the average of three independent experiments for AnV + /PI + cells. * p < 0.05 compared to T2h; + p < 0.05 compared to T3h; # p < 0.05 compared to 3 h without necrostatin-1.

Induction of Apoptosis in TNF-Treated L929 Cells in the Presence of Necrostatin-1
Whether TNF-treated L929 cells eventually die by apoptosis in the presence of necrostatin-1 was investigated. To expedite cell death, L929 cells were treated with TNF and CHX in these experiments, since treatment of L929 cells with TNF in the presence of necrostatin-1 did not induce cell death, even after 24 h. Although TNF/CHX-induced necrotic cell death was almost completely inhibited in the presence of necrostatin-1 within 3 h (Figures 1 and 2), typical apoptotic changes such as cell shrinkage and membrane blebbing were observed at 4 h or later ( Figure 3). Dual staining of L929 cells with Annexin V (AnV) and propidium iodide (PI). L929 cells were treated with 10 ng/mL TNF (T), 10 µM Z-VAD-fmk (V), 10 µg/mL cycloheximide (C), or 10 µM necrostatin-1 (N) for the indicated times. AnV + /PI − : early necroptosis; AnV + /PI + : necrosis (late necroptosis). The graph shows the average of three independent experiments for AnV + /PI + cells. * p < 0.05 compared to T2h; + p < 0.05 compared to T3h; # p < 0.05 compared to 3 h without necrostatin-1.

Induction of Apoptosis in TNF-Treated L929 Cells in the Presence of Necrostatin-1
Whether TNF-treated L929 cells eventually die by apoptosis in the presence of necrostatin-1 was investigated. To expedite cell death, L929 cells were treated with TNF and CHX in these experiments, since treatment of L929 cells with TNF in the presence of necrostatin-1 did not induce cell death, even after 24 h. Although TNF/CHX-induced necrotic cell death was almost completely inhibited in the presence of necrostatin-1 within 3 h (Figures 1 and 2), typical apoptotic changes such as cell shrinkage and membrane blebbing were observed at 4 h or later ( Figure 3).     Furthermore, changes in caspase-3 activity were examined. As shown in Figure 5, a slight increase in caspase-3 activity was detected at 4 h in the cells treated with necrostatin-1/TNF/CHX, and thereafter caspase-3 activity was increased in a time-dependent manner. In contrast, caspase-3 activity was not apparently increased in TNF/CHX-treated L929 cells in the absence of necrostatin-1.
Furthermore, changes in caspase-3 activity were examined. As shown in Figure 5, a slight increase in caspase-3 activity was detected at 4 h in the cells treated with necrostatin-1/TNF/CHX, and thereafter caspase-3 activity was increased in a time-dependent manner. In contrast, caspase-3 activity was not apparently increased in TNF/CHX-treated L929 cells in the absence of necrostatin-1. Moreover, activation of caspases was assessed by Western blot analysis ( Figure 6). When the cells were treated with necrostatin-1/TNF/CHX for 5 h, reduction of procaspases-3, -8, and -9 with concomitant appearance of cleaved caspases-3 and -9 was observed. The level of PARP-a well-known substrate of caspase-3-was also reduced, with the concomitant appearance of cleaved PARP in necrostatin-1/TNF/CHX-treated cells, indicating the activation of caspase-3.

Differential Effects of Caspase Inhibitors on Necrostatin-1/TNF-Treated L929 Cells
Dual staining with AnV and PI demonstrated the appearance of AnV + /PI − cells followed by AnV + /PI + cells in necrostatin-1/TNF/CHX-treated cells at 4 h or later (Figure 7), suggesting that these AnV + /PI − and AnV + /PI + cells were early and late apoptotic, respectively. It was previously reported that ZVAD inhibited apoptosis but induced necroptosis, whereas Z-Asp-CH2-DCB (ZAsp)-a caspase Moreover, activation of caspases was assessed by Western blot analysis ( Figure 6). When the cells were treated with necrostatin-1/TNF/CHX for 5 h, reduction of procaspases-3, -8, and -9 with concomitant appearance of cleaved caspases-3 and -9 was observed. The level of PARP-a well-known substrate of caspase-3-was also reduced, with the concomitant appearance of cleaved PARP in necrostatin-1/TNF/CHX-treated cells, indicating the activation of caspase-3. Furthermore, changes in caspase-3 activity were examined. As shown in Figure 5, a slight increase in caspase-3 activity was detected at 4 h in the cells treated with necrostatin-1/TNF/CHX, and thereafter caspase-3 activity was increased in a time-dependent manner. In contrast, caspase-3 activity was not apparently increased in TNF/CHX-treated L929 cells in the absence of necrostatin-1. Moreover, activation of caspases was assessed by Western blot analysis ( Figure 6). When the cells were treated with necrostatin-1/TNF/CHX for 5 h, reduction of procaspases-3, -8, and -9 with concomitant appearance of cleaved caspases-3 and -9 was observed. The level of PARP-a well-known substrate of caspase-3-was also reduced, with the concomitant appearance of cleaved PARP in necrostatin-1/TNF/CHX-treated cells, indicating the activation of caspase-3.

Differential Effects of Caspase Inhibitors on Necrostatin-1/TNF-Treated L929 Cells
Dual staining with AnV and PI demonstrated the appearance of AnV + /PI − cells followed by AnV + /PI + cells in necrostatin-1/TNF/CHX-treated cells at 4 h or later (Figure 7), suggesting that these AnV + /PI − and AnV + /PI + cells were early and late apoptotic, respectively. It was previously reported that ZVAD inhibited apoptosis but induced necroptosis, whereas Z-Asp-CH2-DCB (ZAsp)-a caspase

Differential Effects of Caspase Inhibitors on Necrostatin-1/TNF-Treated L929 Cells
Dual staining with AnV and PI demonstrated the appearance of AnV + /PI − cells followed by AnV + /PI + cells in necrostatin-1/TNF/CHX-treated cells at 4 h or later (Figure 7), suggesting that these AnV + /PI − and AnV + /PI + cells were early and late apoptotic, respectively. It was previously reported that ZVAD inhibited apoptosis but induced necroptosis, whereas Z-Asp-CH 2 -DCB (ZAsp)-a caspase inhibitor preferential to caspase-3-suppressed apoptosis without inducing necroptosis in TNF-treated U937 human monocytic leukemia cells [15]. As shown in Figure 3, necrotic cells showing cell swelling without apoptotic morphology were observed in L929 cells treated with necrostatin-1/TNF/CHX in the presence of ZVAD. Moreover, co-treatment with ZVAD expedited the appearance of necrotic AnV + /PI + cells in necrostatin-1/TNF/CHX-treated cells (Figure 7), suggesting the induction of necroptosis due to the inhibition of apoptosis by ZVAD. In contrast, co-treatment with ZAsp at least partially inhibited apoptosis without expediting necrotic cell death (Figure 3), and inhibited the appearance of both AnV + /PI − and AnV + /PI + cells in necrostatin-1/TNF/CHX-treated cells (Figure 7). These results indicated that ZAsp inhibited apoptosis without expediting necroptosis, which is consistent with the previous report [15]. As shown in Figure 6, proteolytic activation of caspases-9 and -3 (which depends on activated caspase-8 in the death-receptor pathway [4]) was completely inhibited by ZVAD, whereas cleaved caspases-9 and -3 were slightly detected in the presence of ZAsp. These results are also consistent with the previous report [15], suggesting that partial activation of caspase-8 in the presence of ZAsp may suppress the induction of necroptosis. inhibitor preferential to caspase-3-suppressed apoptosis without inducing necroptosis in TNF-treated U937 human monocytic leukemia cells [15]. As shown in Figure 3, necrotic cells showing cell swelling without apoptotic morphology were observed in L929 cells treated with necrostatin-1/TNF/CHX in the presence of ZVAD. Moreover, co-treatment with ZVAD expedited the appearance of necrotic AnV + /PI + cells in necrostatin-1/TNF/CHX-treated cells (Figure 7), suggesting the induction of necroptosis due to the inhibition of apoptosis by ZVAD. In contrast, co-treatment with ZAsp at least partially inhibited apoptosis without expediting necrotic cell death (Figure 3), and inhibited the appearance of both AnV + /PI − and AnV + /PI + cells in necrostatin-1/TNF/CHX-treated cells (Figure 7). These results indicated that ZAsp inhibited apoptosis without expediting necroptosis, which is consistent with the previous report [15]. As shown in Figure 6, proteolytic activation of caspases-9 and -3 (which depends on activated caspase-8 in the death-receptor pathway [4]) was completely inhibited by ZVAD, whereas cleaved caspases-9 and -3 were slightly detected in the presence of ZAsp. These results are also consistent with the previous report [15], suggesting that partial activation of caspase-8 in the presence of ZAsp may suppress the induction of necroptosis.

Discussion
TNF induces caspase-dependent apoptosis in many types of cells, whereas it induces caspase-independent necroptosis in the presence of caspase inhibitors such as Z-VAD-fmk. In contrast, TNF induces necroptosis even in the absence of caspase inhibitors in L929 cells. Although it was reported that necrostatin-1 (an inhibitor of necroptosis) inhibited TNF-induced necroptosis in L929 cells, it remained to be determined whether L929 cells treated with necrostain-1/TNF eventually die by apoptosis. In this paper, it is demonstrated that apoptosis can be induced in TNF-treated L929 cells in the presence of necrostatin-1. Necrostatin-1 has been identified as a specific inhibitor of RIP1 kinase activity [6]. It has been demonstrated that RIP1 is a pleiotropic adaptor protein mediating the activation of NF-κB and mitogen-activated protein kinase (MAPK), apoptosis, and necroptosis [16]. It was reported that RIP1 depletion blocked TNF-induced necroptosis in the presence of ZVAD in L929 cells [8,14], although it did not protect L929 cells from TNF-induced cell death in the absence of ZVAD due to a switch from necroptosis to apoptosis [14]. Necrostatin-1 induced a shift from necroptosis to apoptosis in TNF-treated L929 cells in this study, suggesting that the inhibition of RIP1 kinase activity, but not the depletion of RIP1, is sufficient to induce a switch from necroptosis to apoptosis. A recent report showed that depletion of RIP3 or MLKL induced a switch from necroptosis to apoptosis in TNF-treated L929 cells [17], consistent with the notion that RIP1 depletion is not necessary for the shift from necroptosis to apoptosis.
It was previously reported that co-treatment with Z-Asp-CH 2 -DCB (ZAsp, a caspase inhibitor preferential to caspase-3) inhibited TNF-induced apoptosis without expediting necroptosis in U937 cells, while co-treatment with ZVAD (a pan-caspase inhibitor) induced necroptosis by inhibiting apoptosis [15]. In this paper, ZAsp inhibited necrostatin-1/TNF/CHX-induced apoptosis without expediting necroptosis in 929 cells, while ZVAD induced a switch from apoptosis to necrosis in necrostatin-1/TNF/CHX-treated cells, which is consistent with the previous report [15]. These results support the notion that partial activation of caspase-8 in the presence of ZAsp may suppress necroptosis, possibly due to the cleavage of proteins involved in necroptosis, such as RIP1 [18], RIP3 [19], and CYLD [20], by activated caspase-8.
It is generally thought that apoptosis is tolerogenic (non-immunogenic), whereas necroptosis is immunogenic, although apoptosis can be immunogenic depending on the stimuli [21,22]. Therefore, a shift from apoptosis to necroptosis or vice versa may greatly affect immunological responses to dead cells. For example, a switch from apoptosis to necroptosis by caspase inhibitors such as ZVAD might augment immunological responses against malignant tumor cells treated with anticancer drugs, and a shift from necroptosis to apoptosis by necrostatins might reduce detrimental immunological responses against myocardial infarction and stroke. Further studies are required to elucidate the immunological effect of pharmacological manipulation of apoptosis and necroptosis on various pathological conditions.
In conclusion, apoptosis can be induced in TNF-treated L929 cells when the cells are protected from necroptosis by necrostatin-1. It is recognized that TNF induces both apoptotic and necroptotic signaling. In many cells treated with TNF, apoptotic signaling occurs more rapidly than necroptotic signaling. Moreover, activated caspase-8 in apoptotic signaling cleaves proteins involved in necroptotic signaling, such as RIP1, RIP3, and CYLD, resulting in the inhibition of necroptosis. In contrast, necroptotic signaling occurs more rapidly than apoptotic signaling in TNF-treated L929 cells. Therefore, the inhibition of necroptotic signaling by necrostatin-1 induces apoptosis at a later time point in L929 cells. The mechanism by which necroptotic signaling occurs more rapidly than apoptotic signaling in TNF-treated L929 cells remains to be determined.

Dual Staining with Annexin V and Propidium Iodide
The cells were trypsinized and inoculated in non-treated 24 well plates (0.5 mL in each well). After 1 h, the cells were treated with 10 ng/mL TNF in the presence or absence of 10 µM necrostatin-1, 10 ng/mL cycloheximide, 10 µM Z-VAD-fmk, or 100 µM Z-Asp-CH 2 -DCB for the indicated times. Then, the cells were collected by pipetting only (without trypsinization). After centrifugation, the cells were incubated in 100 µL binding buffer (10 mM Hepes/KOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl 2 ) containing 5 µL FITC-conjugated Annexin V and 2.5 µg/mL PI for 30 min at 4 • C. The cells were analyzed using a FACS Calibur flow cytometer and Cell Quest software (BD Biosciences, San Jose, CA, USA). As was previously reported [23,24], not only early apoptotic cells but also early necroptotic cells are Annexin V + /PI − before becoming PI + , which can be discriminated by co-treatment with necrostatin-1: the former are only slightly affected, whereas the latter are almost completely inhibited by necrostatin-1. Apoptosis and necroptosis are primarily judged from morphology. Annexin V/PI double staining is used to detect early apoptotic or early necroptotic cells, both of which are Annexin V + /PI − .

Hoechst 33342 Staining
The cells were fixed with 4% paraformaldehyde in PBS at room temperature for 5 min. Then the cells were stained with 10 µM Hoechst 33342 for 5 min, and nuclear morphology was observed under fluorescent microscope, BZ-9000 (Keyence, Osaka, Japan).

Analysis of Cellular DNA Content
The cells were trypsinized and inoculated in non-treated 24 well plates (0.5 mL in each well). After 1 h, the cells were treated with 10 µM necrostatin-1, 10 ng/mL TNF, and 10 ng/mL cycloheximide overnight. The cells were collected by pipetting only, and fixed with 1% paraformaldehyde in phosphate-buffered saline (PBS) containing 0.5% saponin for 5 min at 4 • C. After centrifugation, the cells were incubated in buffer containing 5 µg/mL propidium iodide and 1 mg/mL ribonuclease A (Nacalai Tesque, Kyoto, Japan) for 10 min at 4 • C. Then, the cells were analyzed using FACS Calibur flow cytometer and Cell Quest software.