Inhibition of Lung Cancer by 2-Methoxy-6-Acetyl-7-Methyljuglone Through Induction of Necroptosis by Targeting Receptor-Interacting Protein 1

Abstract

Aims:

Most chemotherapeutic agents exploit apoptotic signaling to trigger cancer cell death, which frequently results in drug resistance. Necroptosis, a nonapoptotic form of regulated cell death, offers an alternative strategy to eradicate apoptosis-resistant cancer cells. We previously reported a natural necroptosis inducer 2-methoxy-6-acetyl-7-methyljuglone (MAM) in A549 lung cancer cells. The current study is designed to investigate the detailed necroptotic signaling and its cytotoxicity on drug-resistant cancer cells. Furthermore, in vivo anticancer effects were also evaluated in nude mice model.

Results:

MAM directly targets receptor-interacting protein 1 (RIP1) kinase in A549 and H1299 cells, which is responsible for reactive oxygen species (ROS, mainly hydrogen peroxide) generation. A positive feedback loop between calcium (Ca²⁺) and c-Jun N-terminal kinase (JNK) occurred following ROS generation, leading to lysosomal membrane permeabilization and mitochondrial dysfunction. MAM showed similar cytotoxic potency toward cisplatin-resistant A549 (A549/Cis) cells by inducing necroptosis as confirmed by the protective effect of 7-Cl-O-Nec-1 (Nec-1s) and by the morphological characteristics obtained via transmission electron microscopy. Interestingly, tumor necrosis factor alpha (TNFα) was not involved in this process. Intraperitoneal injection of MAM significantly suppressed tumor growth in A549 tumor xenograft without significant body weight loss and multiorgan toxicities.

Innovation and Conclusion:

Our findings demonstrate that MAM induces necroptosis in A549 and H1299 lung cancer cells by targeting RIP1 kinase and ROS in a TNFα-independent manner. MAM kills A549/Cis cells with similar potency through induction of necroptosis. MAM shows anticancer effect in animal model. The present study raises the therapeutic possibility and strategy to combat cancer by the induction of necroptosis.

Introduction

Lung cancer is by far the leading cause of cancer death among both men and women worldwide (30). Two main subtypes have been identified, namely non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). NSCLC, the most prevalent subtype, is less sensitive to chemotherapy and radiation compared with SCLC. Current chemotherapeutic agents for treatment of cancers, including lung cancer, mainly inhibit tumor growth by induction of apoptosis. However, defects in apoptotic signaling in cancer cells frequently lead to drug resistance, which has already been the main cause of chemotherapy failures (29). Several novel therapeutic strategies to overcome and/or avoid apoptotic resistance have been proposed (40). Among which, bypassing apoptosis by targeting nonapoptotic cell death might serve as an alternative approach to prevent drug resistance in cancer chemotherapy.

Innovation

Lung cancer is the leading cause of cancer death worldwide. Compared with small-cell lung cancer, non-small-cell lung cancer (NSCLC) is less sensitive to chemotherapy. In the current study, we revealed that 2-methoxy-6-acetyl-7-methyljuglone (MAM), a natural product, induced necroptosis by activating receptor-interacting protein 1 (RIP1) kinase in A549 and H1299 NSCLC cells. MAM also induced necroptosis in cisplatin-resistant A549 (A549/Cis) cells with similar potency to that of parental A549 cells. In addition, MAM inhibited tumor growth in mice possibly mediated by the induction of necroptosis. These findings provide experimental evidence for the application of necroptosis for anticancer drug research and development.

Necroptosis is a recently identified regulated form of necrosis with distinct molecular pathways from those of apoptosis. The necroptotic cell death signaling pathway is mostly well studied in tumor necrosis factor (TNF)-triggered model (Supplementary Fig. S1). The binding of TNF with its innate receptor TNFR1 forms a complex called complex I on the cell membrane, which includes TNFR-associated death domain protein (TRADD), receptor-interacting protein 1 (RIP1) kinase, TNF receptor associated factor 2 (TRAF2), and cellular inhibitor of apoptosis protein-1/-2 (cIAP1/2). This complex will be transformed to complex IIa, IIb, or IIc depending on different cellular contexts: In the presence of cycloheximide or absence of RIP1, complex IIa forms resulting in caspase-dependent apoptosis. If RIP1 is present, complex IIb forms causing RIP1-dependent apoptosis. However, when the activity of caspase-8 is blocked or absent, RIP1 will activate RIP3 and forms a complex with RIP3 termed necrosome (complex IIc). Necrosome further leads to the phosphorylation of mixed lineage kinase domain-like pseudokinase (MLKL). Then, MLKL translocates from the cytoplasm to the membrane, forms channels, and finally results in the rupture of plasma membrane and cell death (4, 45).

Accumulated studies showed that necroptosis offers potentials for the treatment of various diseases, such as ischemia and atherosclerosis. It is also considered as a novel strategy for cancer therapy by inducing direct cancer cell death and/or eradicating apoptosis-resistant cancer cells (8, 20, 49). However, compared with various necroptosis inhibitors (17), only few necroptosis inducers have been reported to engage necroptotic signaling to effectively kill malignant cells (35, 38). Especially, the feasibility of fighting cancer with necroptotic inducers remains largely unknown, and there are concerns and controversies on the proinflammatory effect of necroptosis in vivo (9, 14, 41).

Recently, we have shown that 2-methoxy-6-acetyl-7-methyljuglone (MAM), a naphthoquinone isolated from Polygonum cuspidatum (15), could induce nonapoptotic cell death in lung cancer A549 cells (32) and necroptosis in colon cancer HT-29 and HCT116 cells (33). MAM induced hydrogen peroxide (H₂O₂) generation, leading to c-Jun N-terminal kinase (JNK) activation. Then, inducible nitric oxide synthase expression increased resulting in release of nitric oxide (NO), which acts as an effector molecule. However, the detailed pronecroptotic signaling, the in vivo anticancer effect, and, especially, the in vivo pronecroptotic effect of MAM remain unclear. In the current study, we showed that MAM could directly target RIP1 for necroptosis induction. RIP1 is responsible for the downstream reactive oxygen species (ROS) generation, which leads to the occurrence of JNK/calcium (JNK/Ca²⁺) positive loop as intermediators, whereas the lysosomal membrane permeabilization (LMP) and subsequent mitochondrial dysfunction functioned as the executioners of necroptosis in response to MAM. Furthermore, MAM kills cisplatin-resistant A549 (A549/Cis) cells by the induction of necroptosis with similar potency to that of parental A549 cells. In addition, the in vivo anticancer evaluation shows no sign of apoptosis. Notably, our findings suggested the potential therapeutic application of necroptosis to combat cancer.

Results

MAM induces nonapoptotic cell death

We previously reported that MAM could induce nonapoptotic cell death in lung cancer A549 cells (32). In the current study, we further determined the cytotoxic effect of MAM on another lung cancer cell line, H1299 cells. The cell viability of H1299 decreased dramatically in a concentration-dependent manner in response to MAM (Supplementary Fig. S2). After treatment with MAM (7.5 μM) for 8 h, necrotic but not apoptotic cell death characteristics, such as swollen mitochondria and damaged cell membrane, were observed in A549 cells (Supplementary Fig. S3). A time-lapse confocal imaging in live cells showed less affected nuclei stained by Hoechst 33258 and increased cell membrane permeability as indicated by enhanced SYTOX Green fluorescence intensity (Supplementary Video S1). Furthermore, no cleavage of caspase 3, 8, 9, and poly(ADP-ribose) polymerase (PARP), the general signs of apoptosis, was observed in both cell lines (Fig. 1A). Especially, downregulated activities of caspase 3/7, 8, and 9 were observed after MAM exposure (Fig. 1B). Neither the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (z-VAD-fmk), nor the H₂O₂-induced necrosis inhibitor, 2-(1H-indol-3-yl)-3-pentylamino-maleimide (IM54), could reverse MAM-induced cytotoxicity (Fig. 1C, D). Collectively, these data support that MAM induced nonapoptotic cell death in lung cancer cell lines.

FIG. 1.

MAM induced nonapoptotic cell death in A549 and H1299 cells. (A) Expression levels of caspase 3, 8, 9, and PARP in MAM-treated A549 and H1299 cells (7.5 and 2.5 μM, respectively) were determined by Western blotting. *Nonspecific bands. (B) Caspase activities were determined with Caspase-Glo Assay Kits after MAM treatment for 8 h (A549 cells) or 6 h (H1299 cells). ***p < 0.001, analyzed by Student's t-test. A549 cells were pretreated with z-VAD-fmk (C) or IM54 (D) for 1 h, followed by MAM treatment for another 8 h (A549 cells) or 6 h (H1299 cells), and cell viability was measured by the MTS assay (A549 cells) and ATP assay (H1299 cells). Data are presented as means ± SD of triplicate experiments. n.s., no significance, analyzed by two-way ANOVA with Bonferroni post hoc test. ANOVA, analysis of variance; IM54, 2-(1H-indol-3-yl)-3-pentylamino-maleimide; MAM, 2-methoxy-6-acetyl-7-methyljuglone; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PARP, poly(ADP-ribose) polymerase; SD, standard deviation; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone.

MAM induces TNFα- and MLKL-independent necroptosis

Phosphorylation of RIP1, RIP3, and MLKL represents the hallmarks of necroptosis induction (11). In the current study, time-dependent phosphorylation of RIP1, RIP3, and MLKL at Ser166, Ser227, and T357/S358, respectively, were observed after MAM treatment in both cell lines (Fig. 2A). The formation of RIP1/RIP3 complex was also detected, which was dramatically inhibited by 7-Cl-O-Nec-1 (Nec-1s), a highly selective RIP1 inhibitor (Fig. 2B). Furthermore, blocking the RIP1 activity by Nec-1s and silence of RIP1 and RIP3 partially reversed the cell viability (Fig. 2C and Supplementary Fig. S4), indicating the essential roles of RIPs in mediating MAM-induced necroptosis. By contrast, the MLKL inhibitor, necrosulfonamide (NSA), which targets the N-terminal coiled-coil domain of MLKL without interfering with phosphorylation by RIP3 (31), showed no effect (Fig. 2D). Mutations targeting the kinase activities of RIP1 and RIP3 could also significantly rescue cell death induced by MAM (Fig. 2E, F), suggesting that MAM-elicited necroptosis was RIP1/RIP3 dependent. As TNFα was a common necroptosis trigger and its autocrine was involved in necroptosis (44), the TNFα levels after MAM treatment was detected. No TNFα accumulation was determined after 8 h treatment (below the minimum detection limit, data not shown). Overall, these data support that MAM induces RIP1/RIP3-dependent necroptosis without the involvement of TNFα and MLKL in lung cancer cells.

FIG. 2.

MAM induced necroptosis in A549 and H1299 cells. (A) A549 and H1299 cells were treated with MAM (7.5 and 2.5 μM, respectively), and the protein expression was determined by Western blotting analysis. (B) RIP1/RIP3 complex formation by immunoprecipitation using antibody against RIP1 in MAM-treated cells for 8 h (A549 cells) and 4 h (H1299 cells). Immunoblots determining the RIP1 and RIP3 expression in whole-cell lysates and immunoprecipitates were included. (C, D) Cells were pretreated with RIP1 inhibitor, Nec-1s (20 μM), or MLKL inhibitor, NSA (5 μM), for 1 h, followed by MAM challenge for another 8 h (A549 cells) or 6 h (H1299 cells). Cell viability was evaluated. Kinase dead RIP1 (K45A) (E) and RIP3 (D160N) (F) mutants protect cells from necroptosis. Cells were transiently transfected with kinase dead or wild-type plasmid constructs, followed by MAM treatment for 8 h (A549 cells) or 6 h (H1299 cells). Cell viability was determined. Data are presented as means ± SD of triplicate experiments. n.s., no significance, *p < 0.05, **p < 0.01, ***p < 0.001, analyzed by two-way ANOVA with Bonferroni post hoc test. MLKL, mixed lineage kinase domain-like pseudokinase; Nec-1s, 7-Cl-O-Nec-1; NSA, necrosulfonamide; RIP1, receptor-interacting protein 1.

Positive JNK/Ca²⁺ feedback loop functions downstream of RIP1/ROS signaling

Previously, we have found that glutathione (GSH) depletion causing oxidative stress is an early upstream event of MAM-induced necroptosis in A549 cells (32). The relationship between RIP1 and ROS generation was further explored in the current study. Pretreatment with Nec-1s could significantly reverse MAM-induced ROS generation in both cell lines (Fig. 3A), indicating that RIP1 functions at the upstream of ROS.

FIG. 3.

Positive JNK/Ca²⁺ feedback loop functions downstream of RIP1/ROS signaling. (A) Cells were pretreated with or without Nec-1s (20 μM) for 1 h, followed by MAM challenge for another 1 h. ROS levels were determined using DCFH₂-DA (A549 cells) and DHE (H1299 cells) staining by flow cytometry. (B) Cells were treated with MAM (7.5 and 2.5 μM, respectively) followed by Fluo-3/AM staining and flow cytometry analysis. (C) Cells were pretreated with or without various inhibitors (Nec-1s, 20 μM; catalase, 2000 U/mL; SP600125, 20 μM; BAPTA-AM, 1 μM) for 1 h, followed by MAM insult for another 4 h. Intracellular Ca²⁺ levels were determined. (D) Cells were pretreated with BAPTA-AM (1 μM) for 1 h, followed by MAM treatment (7.5 and 2.5 μM, respectively) for another 8 h (A549 cells) or 6 h (H1299 cells). Cell viability was evaluated. (E) Cells were treated as described in (C), followed by MAM insult for another 1 h. Expression levels of JNK1/2 and phosphorylated JNK1/2 were determined by Western blotting. Data are presented as means ± SD of triplicate experiments. **p < 0.01, analyzed by two-way ANOVA with Bonferroni post hoc test. Ca²⁺, calcium; DCFH₂-DA, 2′,7′-dichlorofluorescin-diacetate; DHE, dihydroethidium; JNK, c-Jun N-terminal kinase; ROS, reactive oxygen species. Color images are available online.

We also reported the critical role of Ca²⁺ in MAM-induced colon cancer cell death (33). Similarly, the cytosolic Ca²⁺ concentration increased after MAM treatment for 4 and 2 h in A549 and H1299 cells, respectively (Fig. 3B). The elevation of cytosolic Ca²⁺ in A549 cells was further confirmed by GCaMP3 (Supplementary Fig. S5), a genetically encoded calcium indicator (34). The GCaMP3 fluorescent intensity was remarkably increased at 4 h after MAM treatment. We also demonstrated that a high Ca²⁺ concentration resulted in JNK activation in colon cancer cells (33). Pretreatment with SP600125, a JNK inhibitor, could completely reverse MAM-induced Ca²⁺ elevation, which is comparable with the effect of cell-permeant Ca²⁺ chelator, BAPTA-AM. Both Nec-1s and catalase could dramatically decrease the Ca²⁺ concentrations, indicating that RIP1/ROS function at the upstream of Ca²⁺ elevation (Fig. 3C). Furthermore, MAM-induced JNK activation was significantly reversed by Nec-1s, BAPTA-AM, SP600125, and catalase (Fig. 3E). In addition, BAPTA-AM could confer protection against MAM-induced cytotoxicity (Fig. 3D). Collectively, these results suggested the existence of a positive JNK/Ca²⁺ feedback loop downstream of RIP1/ROS.

Involvement of LMP and mitochondrial dysfunction in the execution of necroptosis

Considering that Ca²⁺-induced LMP through calpain activation critically participate in the execution of programmed necrosis to impair cell integrity (37), we examined whether LMP was involved in MAM-induced necroptosis. LysoTracker Red, which accumulates inside acidic organelles in normal cells and diffuses after LMP, was used to assess LMP. A significant decrease of fluorescence intensity was observed after MAM treatment for 2 h (Fig. 4A). Since decreased LysoTracker Red fluorescence intensity may also be attributed to mere increase in lysosomal pH, acridine orange (AO), which would shift from red to green when LMP occurs, was applied (1). MAM-treated cells displayed increased green fluorescence and reduced red fluorescence (Supplementary Fig. S6). To further confirm the involvement of LMP, fluorescent dextran was loaded into lysosomes, which exhibits punctate structures representing intact lysosomes that diffuse throughout the cytoplasm after LMP-inducing insults (6). As expected, punctate staining was observed in the control group, whereas a diffused pattern was observed throughout the cells upon MAM treatment for 2 h (Fig. 4B). LMP would result in translocation of soluble lysosomal components (such as cathepsins) from the lysosomal lumen to the cytosol. Cathepsin B-specific immunostaining was confined in cytoplasmic punctate structures in control cells, which diffused throughout the entire cells after MAM treatment (Fig. 4C). Thus, these results indicated the involvement of LMP in MAM-induced necroptosis.

FIG. 4.

Involvement of LMP downstream of RIP1/ROS in MAM-induced necroptosis. (A) A549 and H1299 cells were treated with MAM (7.5 and 2.5 μM, respectively) followed by LysoTracker Red staining and flow cytometry analysis. (B) A549 and H1299 cells were incubated with dextran for 24 h, followed by MAM (7.5 and 2.5 μM, respectively) insult for 2 h. Images were captured with a confocal microscope. (C) A549 and H1299 cells were treated with or without MAM treatment for 2 h. Cathepsin B was visualized using immunofluorescence staining. Scale bar: 10 μm. (D) A549 cells were pretreated with various inhibitors (SP600125, 20 μM; BAPTA-AM, 1 μM; DFO, 100 μM; Nec-1s, 20 μM; catalase, 2000 U/mL; Hegb, 10 μM) for 1 h, followed by MAM insult for another 2 h. Cells were stained with AO, and images were captured with an IN Cell Analyzer 2000.(E) A549 and H1299 cells were treated with MAM (7.5 and 2.5 μM, respectively) for 2 h with or without pretreatment with various inhibitors for 1 h, followed by LysoTracker Red staining and flow cytometry analysis. (F) A549 and H1299 cells were pretreated with DFO (100 μM) for 1 h, followed by MAM (7.5 and 2.5 μM, respectively) insult for another 8 and 6 h. Cell viability was measured. Data are presented as means ± SD of triplicate experiments. ***p < 0.001, analyzed by two-way ANOVA with Bonferroni post hoc test. AO, acridine orange; DFO, deferoxamine mesylate salt; Hegb, hemoglobin; LMP, lysosomal membrane permeabilization. Color images are available online.

Whether JNK-Ca²⁺ loop is responsible for LMP induction was explored. AO and LysoTracker Red staining revealed that inhibition of JNK activation by SP600125 and calcium elevation by chelator BAPTA-AM could reverse MAM-induced LMP (Fig. 4D, E). These findings suggested the contribution of JNK/Ca²⁺ loop to LMP. Interestingly, Nec-1s, catalase, and hemoglobin (Hegb, a NO scavenger) could also reverse LMP (Fig. 4D, E), indicating that RIP1/ROS function upstream of LMP, and NO might also be an initiator of LMP. Lysosomes were the intracellular compartment for the Fenton reaction because of the enriched reduced iron and permeability to H₂O₂. Oxidative stress-induced LMP generally could be suppressed by iron [mainly Fe(III)] chelator deferoxamine mesylate salt (DFO) (19, 36). As expected, DFO pretreatment substantially protected cells against MAM-induced LMP in both assays (Fig. 4D, E). Furthermore, DFO protected cells from MAM-induced cell death (Fig. 4F).

We previously reported the mitochondrial ROS generation and ultrastructure alterations induced by MAM in A549 cells (32). Here, we found that overexpression of mitochondrial antioxidant protein manganese superoxide dismutase (MnSOD), which could modulate the cellular redox status by converting superoxide to H₂O₂ and dioxygen, significantly prevented A549 cells from MAM-induced mitochondrial ROS generation and cell death (Fig. 5A, B). In addition to mitochondrial ROS generation, MAM induced severe mitochondrial membrane potential (ΔΨm) collapse as determined by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and tetramethylrhodamine methyl ester (TMRM) staining (Fig. 5D and Supplementary Video S1). Interestingly, Nec-1s, catalase, SP600125, BAPTA-AM, and DFO could substantially prevent MAM-induced mitochondrial ROS generation and ΔΨm collapse (Fig. 5C, D).

FIG. 5.

Involvement of mitochondrial dysfunction as an executioner of necroptosis. Effects of overexpression of MnSOD or empty vector on MAM-induced cytotoxicity by the MTS assay (A) and mitochondrial ROS generation by flow cytometry analysis (B). (C) A549 and H1299 cells were pretreated with various inhibitors (Nec-1s, 20 μM; catalase, 2000 U/mL; SP600125, 20 μM; BAPTA-AM, 1 μM; DFO, 100 μM) for 1 h, followed by MAM (7.5 and 2.5 μM, respectively) for another 4 h. Cells were stained with MitoSox Red before flow cytometry analysis. (D) A549 cells were treated with MAM for 4 h with or without pretreatment of various inhibitors for 1 h, followed by JC-1 staining. Images were captured with an IN Cell Analyzer 2000. *p < 0.05, analyzed by two-way ANOVA with Bonferroni post hoc test. JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MnSOD, manganese superoxide dismutase. Color images are available online.

Mitochondrial permeability transition pore (MPTP) opening elicited by Ca²⁺ overload has been reported to result in ΔΨm collapse (18). We then examined whether MPTP is responsible for ΔΨm loss in our setting. MPTP inhibitor, cyclosporin A (CSA), pretreatment showed no effect on either ΔΨm collapse or cytotoxicity induced by MAM staining (Fig. 5D and Supplementary Fig. S7). Thus, these data suggested that MPTP-independent mitochondrial dysfunction is a downstream executioner of necroptosis.

MAM induced necroptosis in A549/Cis cells

Shikonin, a natural naphthoquinone, induces necroptosis in several types of cancer cell lines (7, 28, 39). Moreover, shikonin and its analogs were reported to circumvent cancer drug resistance via the induction of necroptosis (10, 13, 43). Considering the structural similarity between MAM and shikonin, we assume that MAM possesses similar properties. As shown in Figure 6A, although A549/Cis cells were more resistant to MAM than the parental A549 cells at early time points, this difference nearly disappeared after 48 h or beyond. Specifically, the half maximal inhibitory concentration (IC₅₀) values at 72 h for A549 and A549/Cis cells were 4.8 and 5.8 μM, respectively (Fig. 6B). Nec-1s pretreatment also significantly prevent A549/Cis cells from MAM-induced cytotoxicity (Fig. 6C). Furthermore, typical necrotic features, such as extensive vesiculation of cytoplasmic organelles and damaged mitochondria showing swelling of cristae, were observed while the cell nuclei remained intact (Fig. 6D). Overall, these data suggested the induction of necroptosis in A549/Cis cells by MAM.

FIG. 6.

MAM induced necroptosis in A549/Cis cells. (A) A549 and A549/Cis cells were treated with MAM (10 μM) for the indicated time points followed by the MTS assay. (B) A549 and A549/Cis cells were treated with MAM for 72 h, followed by the MTS assay. (C) A549/Cis cells were pretreated with Nec-1s (20 μM) for 1 h, followed by MAM insult for another 72 h. Cell viability was measured by the MTS assay. (D) Morphological analysis of A549/Cis cells with or without MAM (10 μM) treatment by TEM, showing intact nuclei and swollen mitochondria with disrupted cristae. Enlarged images of the boxed areas shown in (b) and (c). Scale bar: 5 μm (a, b); 1 μm (c). Data are presented as means ± SD of triplicate experiments. **p < 0.01, analyzed by two-way ANOVA with Bonferroni post hoc test. A549/Cis, cisplatin-resistant A549; TEM, transmission electron microscopy.

Activation of RIP1 by MAM

To explore whether MAM can directly activate RIP1, we performed a Kinase-Glo Luminescent Kinase Assay based on the amount of ATP consumed during kinase reaction (5). As shown in Figure 7A, autophosphorylation and intrinsic ATPase activities of recombinant RIP1 were measureable because the luminescent signal was reduced to ∼74% of the ATP remaining in the control group. The addition of Nec-1s substantially inhibited the autophosphorylation reaction. Nec-1s at the concentration of 5 μM nearly abolished the reaction. By contrast, MAM at the concentration of 0.1 μM enhanced the autophosphorylation level of RIP1 as indicated by the response rate of 50%. To further explore the interaction between MAM and RIP1, a molecular docking assay was performed. MAM is buried in a relatively hydrophobic pocket between the N-lobe and the C-lobe (Fig. 7B). The activation pocket includes the following amino acids: Met67, Leu70, Val75, Val76, Leu78, Met92, Leu129, Val134, His136, Asp156, Ser161, and Phe162, which are quite similar to those of Nec-1s binding with RIP1 (42). In this activation pocket, MAM can interact with Met67, Leu70, Val75, Leu129, Val134, and His136 through van der Waals contacts, and MAM could also form H-bonds with Ser161 and Ile154 on the activation loop (Fig. 7C). Overall, these results suggested that MAM could directly interact with RIP1.

FIG. 7.

Effects of Nec-1s and MAM on recombinant GST-RIP1 kinase activity. (A) The experiment was performed as described in the Materials and Methods section using Kinase-Glo assay. Data are presented as means ± SD of triplicate experiments. **p < 0.01, analyzed by one-way ANOVA followed by Dunnett's post hoc test. (B) Binding mode of MAM in RIP1 crystalline structures obtained with a molecular docking calculation. (C) Close-up view of the interactions between MAM and surrounding residues in RIP1. The proteins are represented by a green cartoon. MAM and the residues in the active site are shown in sticks. Hydrogen bonds are represented by red dashed lines. Color images available online.

MAM inhibited lung cancer in xenograft model

The in vivo effect of MAM against lung cancer was further evaluated in A549-derived xenograft nude mice model. MAM treatment (1 and 2 mg/kg) dramatically inhibited tumor growth (Fig. 8A). The tumor weights decreased by ∼80%, which was comparable with that of cisplatin (5 mg/kg) (Fig. 8B). Furthermore, no sign of body weight loss was observed (Fig. 8C). The average tumor sizes also decreased significantly after MAM treatment, and high dosage of MAM obtained an inhibitory effect comparable with that of cisplatin (Fig. 8D). Histopathological examination of the main organs (lung, liver, spleen, kidney, and heart) showed that MAM has no significant toxicity at both dosages (Fig. 8E). These data indicated that MAM is efficacious in inhibiting lung cancer growth without obvious toxicity.

FIG. 8.

Anticancer effect of MAM on A549-derived xenograft mice model. A549 cells were transplanted to BALB/c nude mice for 11 days. Mice were intraperitoneally injected with DMSO in saline (vehicle), MAM (1 and 2 mg/kg), or cisplatin (5 mg/kg) for 18 consecutive days. The tumor masses (A) were photographed at 18 days postadministration. (B) Final average tumor weight in each group (n = 8). The effects of MAM on the body weights and tumor volumes of mice during the 18-day treatment are shown in (C and D), respectively. *p < 0.05, **p < 0.01, n.s., no significance, analyzed by one-way ANOVA with Dunnett's post hoc test. (E) H&E staining for the major organs in MAM-treated mice. Scale bar: 100 μm. DMSO, dimethyl sulfoxide; H&E, hematoxylin and eosin. Color images available online.

To explore MAM-induced cell death type in vivo, tumor tissues were further examined. Strong transferase dUTP nick end labeling (TUNEL) signals were observed in cisplatin-treated tumors (Fig. 9A and Supplementary Fig. S8), indicating the presence of apoptosis. By contrast, MAM-treated tumors showed very weak TUNEL signals, suggesting the minor contribution of apoptosis. Transmission electron microscopy (TEM) analysis revealed that MAM-treated tumors exhibit intact nuclei and damaged mitochondria. Classical apoptotic features, such as nuclear chromatin condensation and apoptotic body formations, were only found in cisplatin-treated group (Fig. 9C). Furthermore, increased expression of RIP1 and phosphorylated RIP3 were detected in MAM-treated tumors (Fig. 9B). Thus, these data revealed that MAM might exhibit in vivo anticancer activity via necroptosis.

FIG. 9.

MAM induced nonapoptotic cell death in tumor tissues. (A) Tumor tissue sections were analyzed by TUNEL staining. Scale bar: 100 μm. (B) Western blotting assay of RIPs expression in tumor tissues. Tumor tissues from two mice in each group were chosen for the experiment. (C) Morphological observations of tumor tissues by TEM analysis. Note the normal nucleus (b) and damaged mitochondria ( red arrows in c) in MAM-treated group. For the cisplatin-treated group, early apoptotic features of nuclear chromatin condensation and margination ( white arrows in d) and late apoptotic features of apoptotic bodies ( yellow arrows in d) were observed. Scale bar: 2 μm (a, b, d); 0.5 μm (c). TUNEL, transferase dUTP nick end labeling. Color images available online.

Discussion

RIP1 kinase, which contains an N-terminal Ser/Thr kinase and a C-terminal death domain, plays important roles in both cell survival and death (3). Its kinase domain is responsible for necroptosis induction, and the death domain can engage it to the intracellular domain of TNFR1. An intermediate domain of RIP1, which regulates NF-κB activation and promote cell survival, also exists.

Because RIP1 kinase is a key upstream regulator that controls the activation of multiple cell death pathways, it is considered a potential target for the treatment of both acute and chronic diseases (24). Several small molecular RIP1 inhibitors, such as Nec-1, Nec-1s, PN10, and Cpd27, have been identified and have demonstrated protective effect against TNF-induced systemic inflammatory response syndrome in vivo (4). However, as far as we know, no direct RIP1 activator was reported. Here, we found that MAM could directly enhance the autophosphorylation level of RIP1 in vitro. Furthermore, blocking the RIP1 kinase activity using either the small-molecule inhibitor Nec-1s or genetic approach of kinase inactivation could significantly prevent MAM-induced necroptosis. The molecular docking assay also showed that MAM could directly bind to RIP1. Thus, these data suggested that MAM might target RIP1 kinase for necroptosis induction. The in vitro kinase assay results support our previous finding of MAM-induced necroptosis in human colon cancer cells (33) but are inconsistent with MAM-induced apoptosis in MCF7 and B16F10 cells (32). This inconsistency might be attributed to the genotypic differences among these cells. For example, the expression of RIP3 mRNA and protein in MCF7 cells was not detected (22), which impaired the formation of RIP1/RIP3 complex, the core structure for necroptosis.

ROS and reactive nitrogen species, two classes of active small molecules, actively participate in the execution of necroptosis and apoptosis (37). For example, TNF increases mitochondrial ROS generation, which facilitates necrosome formation (27). Here, we found that RIP1 kinase is responsible for ROS (mainly H₂O₂) induction, which is consistent with TNF-induced necrotic cell death (12). The impaired caspase 8 activity would prevent RIP1 and RIP3 from cleavage and facilitate downstream ROS induction. Excessive ROS can not only impair the lysosomal and mitochondrial membrane but also function as a second messenger for downstream signal transduction. The mitochondrial ROS were elevated and could be inhibited by Nec-1s. A recent study showed that mitochondrial ROS could promote RIP1 autophosphorylation and facilitate RIP3 recruitment into necrosome (46). Thus, the detailed link of mitochondrial ROS and necroptosis in our setting needs further study.

Sustained JNK activation was a key event downstream of RIP1 in TNFα-induced necroptosis (47). Accumulation of cytosolic Ca²⁺ was reported to either induce RIP1 kinase activation or function downstream of membrane localization of MLKL (2, 23). Here, a JNK/Ca²⁺ feedback loop was detected downstream of ROS production. However, robust JNK phosphorylation was observed 1 h after MAM treatment, which occurred much earlier than Ca²⁺ elevation. Thus, accumulated Ca²⁺ might be responsible for the prolonged JNK activation, thereby generating a positive feedback loop. However, our previous study (33) revealed a different pattern in which Ca²⁺ precedes JNK phosphorylation in colon cancer cells. This may due to the varied signaling threshold of different cell lines toward MAM.

LMP results in the release of enzymes, mainly cathepsins, from the lysosomal lumen to the cytosol (25). LMP may be induced by various stimuli, including oxidative stress. Depending on the different levels of permeabilization, distinct cell death types may occur. Moderate release of lysosomal enzymes leads to apoptosis, whereas vast release of enzymes can result in necrosis (16). In this study, ROS promoted LMP directly or through the downstream JNK/Ca²⁺ loop indirectly. Furthermore, NO downstream of JNK might also partially stimulate LMP. Thus, the severe LMP and extensive lysosomal cathepsin B release might contribute to the final necroptosis. Apart from LMP, mitochondrial dysfunction is another executioner of necroptosis (21). MAM-induced mitochondrial dysfunction was demonstrated by TEM analysis, redox imbalance, and ΔΨm loss. The inhibitory effect of various upstream inhibitors indicates that mitochondrial dysfunction occurs at the late stage of necroptosis.

The antitumor effect of MAM was further confirmed in vivo. We found for the first time that MAM could significantly inhibit tumor growth, which showed similar efficacy to that of cisplatin. Analyses of body weights and histopathological assay of main organs suggested that MAM showed no significant toxicity. Furthermore, weak TUNEL staining and TEM analysis indicated that the in vivo anticancer effect of MAM was apoptosis-independent. The increased expression of RIPs in tumor tissues supports the involvement of necroptosis. Thus, MAM could inhibit tumor growth mediated not only by apoptosis but also possibly by necroptosis.

In summary, as depicted in Figure 10, MAM induced necroptosis in lung cancer cells by targeting the RIP1 kinase. The following ROS generation initiated a JNK/Ca²⁺-positive feedback loop and led to LMP and mitochondrial dysfunction, which finally resulted in necroptosis. Furthermore, MAM induced A549/Cis cell death with similar potency to that of parental A549 cells. Especially, MAM inhibited tumor growth in mice possibly through necroptosis induction. Our findings highlight the potential application of small-molecule necroptosis inducers in anticancer drug research and development.

FIG. 10.

Schematic representation of the pronecroptotic effect of MAM in vitro and in vivo. MAM triggered RIP1/RIP3 complex formation by directly targeting RIP1 and impairing Casp 8 activity in the initiation phase. ROS production and JNK/Ca²⁺ positive loop functioned as the downstream effector of RIP1/RIP3 complex. LMP and subsequent mitochondrial dysfunction (ΔΨm collapse and ROS elevation) were the final executioners of necroptosis. For the in vivo effect, MAM decreased tumor weight and tumor volume possibly through the induction of necroptosis. ΔΨm, mitochondrial membrane potential; Casp, caspase; CAT, catalase; CTSB, cathepsin B. Color images available online.

Materials and Methods

Materials

NSA was obtained from EMD Millipore Corporation (Darmstadt, Germany). IM54 was obtained from Abcam (Cambridge, MA). Nec-1s was obtained from BioVision (Milpitas, CA). SP600125, DFO, Hegb, and AO were purchased from Sigma–Aldrich (St Louis, MO). z-VAD-fmk was obtained from Selleckchem (Houston, TX). CSA, JC-1, catalase, and LysoTracker Red were obtained from Beyotime (Shanghai, China). MitoSox Red was purchased from Invitrogen (Carlsbad, CA). BAPTA-AM, 2′,7′-dichlorofluorescin-diacetate (DCFH₂-DA), dihydroethidium (DHE), Fluo-3/AM ester, TMRM, and Hoechst 33258 and Hoechst 33342 were obtained from Molecular Probes (Eugene, OR).

Cell lines and cell culture

A549 and H1299 cells were purchased from ATCC and cultivated with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. A549/Cis cells were obtained from the Cell Resource Center, IBMS, CAMS/PUMC (Beijing, China) and cultivated with McCoy's 5A medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. All the cells were maintained in humidified air with 5% CO₂ at 37°C.

Cell viability assay

Cell viability was measured with CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) Kit, which contains the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS], according to the manufacturer's protocols. Absorbance at 490 nm measured using a microplate reader (FlexStation 3 microplate reader; Molecular Devices, Sunnyvale, CA) was recorded for the calculation of cell viability. Cell viability was also evaluated using the CellTiter-Glo Luminescent Assay (Promega) according to the manufacturer's instructions. To confirm the cytotoxicity, the ATP assay (Promega) was also used according to the manufacturer's instructions in H1299 cells.

Western blot analysis

Cell lysate and xenograft tumor tissue lysate preparation, protein concentration determination, electrophoresis, and immunoblotting were performed as described previously (32, 48). The following antibodies were purchased from Cell Signaling Technology (Beverly, MA): caspase 3 (9665S), caspase 8 (9746s), caspase 9 (9502s), GAPDH (5174S), JNK (9252S), MLKL (14993S), p-JNK1/2 (4668S), p-RIP1 (65746S), RIP1 (3493S, 3494T), RIP3 (13526S, 95702S), RARP (9542S), and horseradish peroxidase-conjugated secondary antibodies (7074V). pMLKL (ab187091) and pRIP3 (ab209384, 57220S) were obtained from Abcam (Cambridge, United Kingdom). Signals of chemiluminescence intensity were acquired using a ChemiDoc^™ MP Imaging System and analyzed with Image Lab 5.1 software (Bio-Rad, Hercules, CA).

Caspase activity

Caspase 3/7, 8, and 9 activities were determined with the Caspase-Glo Assay Kit (Promega) according to the manufacturer's instructions.

ROS determination

Cells were pretreated with or without Nec-1s for 1 h and followed by MAM insult for 1 h. Then, the cells were loaded with DCFH₂-DA (10 μM) or DHE (10 μM) for 30 min at 37°C. After washing twice with phosphate-buffered saline (PBS), fluorescence signal was collected with the FACScan^™ flow cytometer (BD Biosciences) using the fluorescein isothiocyanate (FITC) channel. At least 1 × 10⁴ cells were collected in each sample.

TEM imaging

After MAM treatment for 8 h, samples used for TEM analysis were prepared as described previously (32) and examined under TEM (H-7650; Hitachi) at 80 kV.

Monitoring necroptosis using high-resolution time-lapse imaging

A549 cells were incubated with TMRM (100 nM) for 30 min followed by MAM treatment (10 μM) to monitor ΔΨm. To monitor cell membrane permeability, SYTOX Green (1 μM) was also preloaded. Hoechst 33258 at 5 μg/mL was used for nuclei counterstaining. Image acquisition and data analysis were performed as described previously (33).

Measurement of TNFα

Quantitative determination of TNFα after MAM treatment for 8 h was performed using a commercial enzyme-linked immunosorbent assay (ELISA) Kit (EHC103a; eBioscience, Shenzhen, China) according to the manufacturer's instructions.

Measurement of LMP

After MAM treatment for the indicated time points, LysoTracker Red (50 nM) was incubated for another 1 h at 37°C before analyzing by flow cytometry. AO was another probe applied to monitor LMP. AO (10 μg/mL) was loaded and incubated for 15 min before obtaining images with an IN Cell Analyzer 2000 (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). LMP was also evaluated by loading lysosomes with fluorescent dextran (Lucifer Yellow, 10,000 MW, anionic, Lysine Fixable, D-1825; Molecular Probes). Briefly, 0.1 mg/mL dextran was incubated with cells for 24 h to allow sufficient accumulation in the lysosomal compartment. After extensive washing, cells were cultivated in standard medium to equilibrate for 2 h. Then, cells were treated with or without MAM for another 2 h before image acquisition on the Leica TCS SP8.

Immunofluorescence using antibodies against cathepsin B was also performed, which could reveal lysosomal proteases redistribution after LMP. Briefly, cells were cultured in cover glass-bottom dish (SPL, Pocheon, Korea) overnight and treated with or without MAM for another 2 h. Then, cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 min at 4°C, followed by permeabilization with 0.1% Triton X-100 in PBS for 30 min and blocking with PBS containing 5% nonfat milk for 1 h at room temperature. After blocking, cells were stained with cathepsin B dilution buffer for 2 h and fluorochrome-conjugated secondary antibody for 1 h before analyzing by confocal microscopy.

Overexpression of MnSOD

A549 cells were transiently transfected with pMnSOD plasmid (OriGene) tagged with Myc-DDK at the C-terminal or an empty vector using Lipofectamine 3000 (Life Technologies) according to the manufacturer's protocol.

Calcium measurement by Fluo-3/AM ester and GCaMP3

Cells were loaded with Fluo-3/AM ester (5 μM, 30 min, 37°C) after MAM insults in the presence or absence of various inhibitors, followed by flow cytometry analysis.

To further validate the involvement of Ca²⁺, transfection of GCaMP3 plasmid was performed as described previously (33). After MAM treatment, image acquisition was performed by confocal laser scanning microscopy (Leica, SP8).

Measurement of ΔΨm

For the measurement of ΔΨm, JC-1 (2 μg/mL, 30 min, 37°C) was loaded and images were acquired with an IN Cell Analyzer 2000. In addition, TMRM (100 nM, 15 min, 37°C) was another fluorescent probe used for ΔΨm measurement, followed by flow cytometry analysis.

MitoSox Red staining

To determine the mitochondrial superoxide levels, MitoSox Red (5 μM) was incubated with cells after MAM treatment in the presence or absence of various inhibitors for 1 h at 37°C. Fluorescent signals were determined by a flow cytometry (Becton-Dickinson, Oxford, United Kingdom).

Measurement of RIP1/RIP3 complex by co-immunoprecipitation

RIP1/RIP3 complex formation was validated by co-immunoprecipitation as described previously (33).

Transfections

Cells were transiently transfected with plasmid constructs using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. The following plasmids (Addgene, Cambridge, MA) were used: hRIP1 HA GFP wt (No. 41396; Addgene), hRIP1 HA GFP K45A (kinase dead, No. 41389; Addgene), hRIP3 GFP wt (No. 41387; Addgene), and hRIP3 GFP D160N (kinase dead, No. 41386; Addgene).

For small interfering RNA (siRNA) knockdown, cells were transfected with siRNA targeting RIP1 or RIP3, or negative control siRNA using Lipofectamine 3000. All siRNAs were purchased from GenePharma Company (Shanghai, China), and the specific sequences used were as follows (5′-3′): RIP1 target sequence, AUCAAUCUGAGACUGUGUGAAGCCCdTdT; RIP3 target sequence, GCAGUUGUAUAUGUUAACGAGCGGUCGdTdT.

Animal xenograft model

Six-week-old male BALB/c nude mice, weighing 18–21 g, were used in this study. Mice were housed in a laminar flow under sterilized condition. A549 cells were trypsinized and washed twice with PBS. Then, 0.2 mL of cells at 5 × 10⁷ cells/mL suspended in RPMI 1640 were injected into the right sides of mice. Eleven days later, the average tumor volume reached ∼100 mm³. The mice were randomly divided into four groups as follows: vehicle group, low-dose MAM (1 mg/kg) group, high-dose MAM (2 mg/kg) group, and cisplatin group (5 mg/kg) (n = 8). Drug treatment was performed intraperitoneally daily for 18 consecutive days. Tumor volumes and body weights were measured every 3 days. Tumor volume was measured with a Vernier caliper and calculated using the following equation: volume = (width² × length)/2. At the last day, mice were euthanized and tumors were isolated and weighed. Tumor tissues and visceral of the mice were excised for terminal deoxynucleotidyl TUNEL, hematoxylin and eosin (H&E) staining, and Western blotting assay.

Plasmid construction of 10His-GST-RIP1 kinase domain

The complementary DNA (cDNA) of human RIP1 kinase domain (amino acids 1–375) was synthesized by Genewiz, Inc. (Suzhou, China), which was digested with NcoI and StuI restriction enzymes and ligated into the same sites in the plasmid pFastbacT1-N-10His-GST.

Protein expression and purification on 10His-GST-RIP1 kinase domain fusion

Spodoptera frugiperda (Sf9) insect cells were cultivated in Sf-900 II SFM medium (Gibco/Invitrogen) at 27°C. Recombinant baculovirus was generated using the Bac-to-Bac System (Invitrogen) according to the manufacturer's specifications. Briefly, recombinant 10His-GST-RIP1 bacmid was transformed into DH10Bac competent cells. Positive (white) colonies were selected and further confirmed by polymerase chain reaction. The recombinant bacmids DNA were extracted and transfected into the Sf9 insect cells seeded in six-well plates at 0.5 × 10⁶ cells/mL using Cellfectin reagent. After 72 h of transfection, recombinant baculovirus was collected.

For protein expression, Sf9 cells were grown in ESF921 Protein Free medium (Expression Systems) by shaking (150 rpm) at 27°C to a density of 2 × 10⁶ cells/mL and infected with passage two baculovirus to express 10His-GST-RIP1 recombinant protein. When the viability of the cell reached 80%, the infected cells were harvested and resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, Cocktail protease inhibitors). Cells were disrupted by sonication and centrifugation. Protein was purified using 5 mL of Ni-chelating beads (GE Healthcare). 10His-GST-RIP1 fractions were combined, and the buffer was changed to the final buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.25 mM DTT, 25% glycerol) by dialysis. After dialysis, the protein was concentrated, and the concentration was determined by the Bradford method.

In vitro kinase assay

This assay was performed using Kinase-Glo Luminescent Kinase Assay Kit (Cat. No. V6711; Promega) as described previously (5) with minor modifications. Briefly, 2 μL of 5 × kinase buffer (100 mM HEPES, pH 7.3, 5 mM MgCl₂, 5 mM MnCl₂, 750 mM NaCl, 0.5% BAS) was added to a 384-well white plate (PerkinElmer, Waltham, MA). Then, 4 μL of 5.9 μM GST-RIP1 1-375 and 2 μL of Nec-1s or MAM (diluted in 1 × kinase buffer) were added. After incubation for 10 min at room temperature, 2 μL of 100 μM ATP (Cat. No. A7699; Sigma), diluted in 1 × kinase buffer, was added into the reaction system and incubated for another 90 min. Then, 10 μL of Kinase-Glo reagent was added to terminate the reaction. The luminescence signal was collected with a microplate reader (FlexStation 3 microplate reader; Molecular Devices) with integration time of 1 s. The degree of response (inhibition or activation) was calculated by subtracting the value of the control protein sample from the values in all other samples except for the ATP alone sample. The background signal from the corresponding control compound sample was also subtracted from the values in groups including compound addition. The response rate was calculated using the formula: Response rate (%) = (luminescent signal [Nec-1s or MAM]/luminescent signal [ATP alone]) × 100%.

Molecular docking assay

The crystal structures of RIP1 were retrieved from the Protein Data Bank (PDB ID: 4ITH). The structure of MAM was downloaded from PubChem Database (PubChem CID: 158739). RIP1 and MAM were docked with AutoDock 4.2 (26). AutoDockTools were used to prepare the protein and ligand. Grid box settings were centered at −32.174, −0.391, and 5.552; grid spacing was 0.375 Å. The results were analyzed using Chimera and PyMOL visualization software.

Statistical analysis

Two-sided Student's t-test was used to compare differences between two groups. One-way analysis of variance (ANOVA) followed by Dunnett's post hoc test or two-way ANOVA followed by Bonferroni post hoc test was used to compare differences in more than two groups. A p-value of <0.05 was considered statistically significant.

Footnotes

Acknowledgments

This study was supported by the Science and Technology Development Fund, Macao S.A.R. (078/2016/A2), and the Research Fund of University of Macau [MYRG118(Y3-L4)-ICMS13-CXP, MYRG2016-00043-ICMS-QRCM].

Author Disclosure Statement

No competing financial interests exist.

Supplementary Material

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Figure S5

Supplementary Figure S6

Supplementary Figure S7

Supplementary Figure S8

Supplementary Video S1

Abbreviations Used

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Supplementary Material

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Inhibition of Lung Cancer by 2-Methoxy-6-Acetyl-7-Methyljuglone Through Induction of Necroptosis by Targeting Receptor-Interacting Protein 1

Abstract

Aims:

Results:

Innovation and Conclusion:

Introduction

Results

MAM induces nonapoptotic cell death

MAM induces TNFα- and MLKL-independent necroptosis

Positive JNK/Ca2+ feedback loop functions downstream of RIP1/ROS signaling

Involvement of LMP and mitochondrial dysfunction in the execution of necroptosis

MAM induced necroptosis in A549/Cis cells

Activation of RIP1 by MAM

MAM inhibited lung cancer in xenograft model

Discussion

Materials and Methods

Materials

Cell lines and cell culture

Cell viability assay

Western blot analysis

Caspase activity

ROS determination

TEM imaging

Monitoring necroptosis using high-resolution time-lapse imaging

Measurement of TNFα

Measurement of LMP

Overexpression of MnSOD

Calcium measurement by Fluo-3/AM ester and GCaMP3

Measurement of ΔΨm

MitoSox Red staining

Measurement of RIP1/RIP3 complex by co-immunoprecipitation

Transfections

Animal xenograft model

Plasmid construction of 10His-GST-RIP1 kinase domain

Protein expression and purification on 10His-GST-RIP1 kinase domain fusion

In vitro kinase assay

Molecular docking assay

Statistical analysis

Footnotes

Acknowledgments

Author Disclosure Statement

Supplementary Material

Abbreviations Used

References

Supplementary Material

Positive JNK/Ca²⁺ feedback loop functions downstream of RIP1/ROS signaling