Abstract

A dramatic increase in the incidence of inflammatory bowel disease (IBD) has been observed in the past two decades, mainly in developed countries and also in developing regions. Necroptosis has been found to play an important role in the pathogenesis of IBD, suggesting its inhibitors are promising in clinic. However, clinical drugs targeting necroptosis are seriously lacking. Through screening a clinical compound library that contains 611 inhibitors, a pan-RAF inhibitor LY3009120 was found to be promising as a necroptosis inhibitor. LY3009120 inhibited necroptosis in vitro, and its inhibition against necroptosis was independent of its well-known activity to inhibit RAF. Surprisingly, LY3009120 prevented phosphorylation of receptor interacting serine/threonine kinase 1 (RIPK1) and subsequently phosphorylation of receptor interacting serine/threonine kinase 3 (RIPK3) and mixed lineage kinase domain like pseudokinase (MLKL) which happened during necroptosis. In vivo, LY3009120 significantly alleviated dextran sulfate sodium (DSS)-induced colitis as indicated by prevention of body weight loss, colon shortening, and decreased mortality. Furthermore, LY3009120 inhibited necroptosis of intestinal epithelial cells (IECs) and prevented intestinal barrier function loss. Consistently, LY3009120 decreased DSS-induced colonic inflammation, as indicated by decreased infiltration of macrophages and neutrophils, and decreased colonic TNF-α, IL-6, and IL-1β level in DSS treated mice. These results indicate that an anti-cancer pan-RAF inhibitor LY3009120 is a necroptosis inhibitor and may serve as a potential therapeutic drug for colitis.

Introduction

Inflammatory bowel diseases (IBD), namely Crohn’s disease (CD) and ulcerative colitis (UC), are chronic relapsing conditions that affect a growing number of children worldwide [1]. The pathogenesis is multifactorial, involving genetic predisposition, epithelial barrier defects, dysregulated immune responses, and environmental factors [2]. Necroptosis, a newly characterized programmed necrosis, has been found to play an important role in IBD [3]. However, clinical drugs that inhibit necroptosis are still lacking. Exploring clinical drugs that prevent necroptosis is promising for the treatment of IBD.

Necroptosis is a type of programmed cell death, which is independent of caspase but dependent on receptor interacting serine/threonine kinase 1 (RIPK1), receptor interacting serine/threonine kinase 3 (RIPK3), and the mixed lineage kinase domain like pseudokinase (MLKL) [4,5]. As one of the key molecules, RIPK1 plays important roles in regulating necroptosis. In TNF-induced necroptosis, together with TNF receptor 1, TNF receptor type 1-associated death domain (DD) protein (TRADD) and TNFR-associated factor 2 (TRAF-2), RIPK1 interacts with lots of proteins to form complex I by DD for mediating both apoptosis and necroptosis, and the switch mechanism depends on genetic deficiency or pharmacological inhibition of caspase 8, and the post-translational modification of RIPK1 [6–8]. Currently, the study about RIPK1 protein modification focused on ubiquitination, in which cellular inhibitors of apoptosis (c-IAPs) and Cylindromatosis (CYLD) play as important ubiquitination or de-ubiquitination regulators, and phosphorylation which is involved with kinases or auto-phosphorylation for following signaling regulation [9,10]. On the other hand, RIPK3 is also important to regulate necroptosis. RIPK3 is phosphorylated by RIPK1 at special Serine/Threonine sites, and associated with each other by the RIP homotypic interacting motif (RHIM) to form a special functional structure necrosome [11]. Necrosome is also an important checkpoint for necroptosis that MLKL binds to and be phosphorylated for trimerization and for following necroptotic performance [5,12,13]. Taken together, the regulation of necroptosis is such a complicated process that more in-depth investigations are required.

At present, more and more evidences have been discovered that necroptosis is involved in various diseases, including inflammation in respiratory, digestive, and nervous systems, and tumorigenesis [3,14]. Recent findings suggested a critical role of necroptosis in the pathogenesis of IBD. Caspase 8-deficiency in the intestinal epithelium led to TNF-α induced necroptosis of epithelial cells and induced terminal ileitis [15]. IEC-specific knockout of Fas-associated via DD (FADD), an adaptor protein required for death receptor-induced apoptosis, spontaneously developed epithelial cell necrosis, loss of Paneth cells, enteritis and severe erosive colitis, which was prevented by genetic deficiency of RIPK3 [16]. Consistently, Nec-1, an inhibitor of RIPK1, ameliorated dextran sulfate sodium (DSS)-induced colitis [17]. NSA, an MLKL inhibitor, has protective effect on intestinal epithelial cells (IECs) in a necroptosis model [18]. Increased necroptosis was found in colon tissues of CD and UC patients [19]. These findings suggest that pharmacologic inhibition of necroptosis may be a promising therapy for IBD.

To find drugs that can inhibit necroptosis and have potential medical uses, we screened a clinical compound library that is used in clinical trials from MedChemExpress. The compound library contains 611 inhibitors that target several signal pathways, including RAF, PI3K, VEGFR etc. Through in vitro screening, LY3009120 was found to be the most potent inhibitor of necroptosis. LY3009120 inhibited necroptosis by preventing phosphorylation of RIPK1 and subsequently phosphorylation of RIPK3 and MLKL and this was independent of its ability to inhibit the kinase activity of RAF. In vivo, DSS-induced colitis was alleviated by LY3009120 treatment. Our study raises a possibility that LY3009120 may be used as a clinical drug to treat IBD.

Materials and methods

Cell lines

Human colorectal adenocarcinoma cell line HT-29 was maintained in McCoy’s 5a Medium Modified (Corning, NY, U.S.A.) supplemented with 10% fetal bovine serum (FBS, Gibco, NY, U.S.A.), penicillin (100 U/ml), and streptomycin (100 U/ml). Mouse fibroblast L929 was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Corning) supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 U/ml).

Reagents

The clinical compound library (HY-LD-000001189) was bought from MedChemExpress (Monmouth Junction, NJ, U.S.A.) in October 2017.

The antibodies used for immunoblotting included: mouse monoclonal antibody against GAPDH (RM2002, Beijing Ray, Beijing, China); rabbit monoclonal antibodies against p-RIPK1 (65746, Danvers, MA, CST, U.S.A.), RIPK1 (3493, CST), p-RIPK3 (93654, CST), human p-MLKL (91689, CST), human MLKL (ab184718, Abcam, Cambridge, MA, U.S.A.), and mouse p-MLKL (ab196436, Abcam); rabbit polyclonal antibodies against RIPK3 (ab56164, Abcam) and mouse MLKL (ab172868, Abcam); and goat anti-mouse (R3001, Beijing Ray) or goat anti-rabbit (R3002, Beijing Ray) HRP–conjugated secondary antibody.

The antibodies used for immunohistochemical staining included: MPO (ab9535, Abcam), F4/80 (ab111101, Abcam), and S100a9 (73425, CST).

Other reagents included: DSS (36000–50000 kDa, MP Biomedicals, Santa Ana, CA, U.S.A.), LY3009120, Ro 5126766, PLX8394, and RAF265 (MedChemExpress), Nec-1, BV-6 and Z-VAD (Selleck, Houston, TX, U.S.A.), mouse TNF-α (R&D, Minneapolis, MN, U.S.A.), Cell Counting Kit-8 (CCK8), FITC-dextran (4 kDa, Sigma, St. Louis, MO, U.S.A.).

Measurement of cell death

HT-29 or L929 cells were pretreated with dimethyl sulfoxide (DMSO) or inhibitors (MCE, U.S.A.) for 1 h, then stimulated with TNF-α (20 ng/ml) plus Smac mimetic (BV6, 2 μM) and Z-VAD (25 μM) for 8 h or TNF-α (1 ng/ml) plus Z-VAD (25 μM) for 3 h, respectively. For PI staining, cells were digested with trypsin containing 0.25 M EDTA, washed with cold 1× Assay buffer, stained with PI for 5 min and then analyzed by flow cytometry. For cell viability analysis, CCK8 was added to the well and incubated for 2 h and then OD450 was measured by Multi-Mode Microplate Reader (Varioskan Flash, Thermo, U.S.A.). Cell viability = (ODtarget − ODblank)/(ODcontrol − ODblank) × 100%. Target, cells treated with inhibitors plus T/S/Z or T/Z; control, cells with no treatment; blank, no cells.

SiRNA and gene knockdown

siRNAs were purchased from GenePharma (Shanghai, China). The siRNAs against A-Raf proto-oncogene, serine/threonine kinase (ARAF), B-Raf proto-oncogene, serine/threonine kinase (BRAF), Raf-1 proto-oncogene, serine/threonine kinase (CRAF), and RIPK1 targeted the mRNAs that coded for mouse Araf (NM_001159645.1), Braf (NM_139294.5), Craf (NM_029780.4), and human RIPK1 (NM_001354930.1), respectively. The negative control RNA duplex (NC) for siRNAs was non-homologous to any mouse or human genome sequences. Sequences of siRNAs are list in Supplementary Table S1. SiRAF-1 and siRAF-2 are two different sets of siRNAs which include three different siRNAs that target ARAF, BRAF, and CRAF, respectively. SiRNAs were transfected using Lipofectamine-RNAiMAX. Forty-eight hours after transfection, cells were harvested and total RNA was isolated by TRIzol. The levels of mRNA were determined by quantitative real-time PCR (qPCR).

Immunoblotting

Cell or tissue proteins were separated in a polyacrylamide gel and transferred to a methanol-activated PVDF membrane. The membrane was blocked for 1 h in Tris-buffered saline plus Tween-20 (TBST) containing 3% bovine serum albumin or 5% milk, and then immunoblotted sequentially with primary and secondary antibodies. The protein levels were detected using a Pierce ECL Western blotting Substrate.

Induction of experimental DSS-induced colitis

Male C57BL/6 mice weighing 21–23 grams were purchased from Jinan Peng Yue Laboratory Animal Breeding Company Limited (China) and housed in specific-SPF facility with a 12:12-h light/dark cycle and ambient temperature of 22 ± 2°C at The Second Affiliated Hospital of Guangzhou Medical University (Guangzhou, China). DSS (3% wt/vol) was administered in drinking water ad libitum for 7 days (from days 0 to 7). DSS solution was replaced twice on days 2 and 4. For LY3009120 intervention experiments [20,21], mice were injected intraperitoneally with LY3009120 (20 mg/kg, dissolved in PBS containing 10% DMSO and 20% cyclodextrin) or vehicle (PBS containing 10% DMSO and 20% cyclodextrin), from days 0 to 9. For CEP-32496 and Ro 5126766 intervention experiments, CEP-32496 (30 mg/kg) [22], Ro 5126766 (1.5 mg/kg) [23] or vehicle (5% DMSO and 10% cyclodextrin solution in distilled water) was given orally everyday for 10 days from days 0 to 9. Mice weight and survival were recorded daily. Histologic scoring was conducted as previously described. For survival and body weight experiments, the experiment lasted 12 days, wherein inhibitors were injected i.p. ten-times from days 0 to 9. For colon length, HE, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) and immunoblotting, the experiments lasted for 7 days, wherein inhibitors were injected i.p. seven-times from days 0 to 6. All protocols involving animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publications Nos. 80–23, revised 1996) and under the approval of the Ethical Committee of Guangdong Provincial Animal Experiment Center.

TUNEL staining

Sections of formalin-fixed, paraffin-embedded tissues were deparaffinized with xylene, rehydrated through graded ethanol. Cell death was detected by TUNEL Apoptosis Detection Kit (FITC) (40306ES50, Yeasen, China) according to the manufacturer’s instructions. Five random fields (200×) were photographed and the average numbers of FITC positive cells per field were presented.

Measurement of intestinal permeability

The mice treated with DSS for 7 days were deprived of food for 4 h, given FITC-dextran (4 kDa, 0.6 mg/g body weight, dissolved in 0.1 ml PBS) intragastrically. Three hours later, hemolysis-free sera were collected and the fluorescence intensity of sera were detected by Multi-Mode Microplate Reader (excitation, 488 nm; emission, 520 nm).

ELISA

Three percent DSS was administered in drinking water to C57BL/6 mice for 7 days. LY3009120 (20 mg/kg mice) or vehicle was injected intraperitoneally everyday for 7 days. On day 7, colons were harvested, washed with PBS, sliced into small pieces approximately 1 mm3, and cultured with serum-free RPMI-1640 medium (1 ml/100 mg colon tissue) for 12 h. The supernatant was collected by sequential centrifugation at 500g for 10 min and 3000g for 10 min. The level of cytokine TNF-α, IL-6, and IL-1β were measured by BioLegend’s ELISA MAX™ Deluxe Sets according to manufacturer’s instructions.

Immunohistochemical staining

Sections of formalin-fixed, paraffin-embedded tissues were deparaffinized with xylene, rehydrated through graded ethanol, followed by quenching of endogenous peroxidase activity in 0.3% hydrogen peroxide, and antigen retrieval by microwave heating in 10 mM citrate buffer (pH 6.0) for S100a9 or in EDTA buffer (pH 9.0) for MPO and F4/80. Sections were incubated at 4°C overnight with rabbit polyclonal antibody against S100a9, MPO and F4/80, then immunostained by ChemMate DAKO EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse (DakoCytomation, Glostrup, Denmark). Subsequently, sections were counterstained with hematoxylin and mounted in non-aqueous mounting medium.

To detect the number of S100a9, MPO, or F4/80 positive cells, ten representative fields (200×) were photographed for each section and the average numbers of cells per field were presented.

Statistical analysis

Data from at least three independent experiments are shown as the mean ± standard error of the mean (S.E.M.). Unless otherwise noted, the differences between two groups were analyzed by unpaired Student’s t test. Analyses were performed with GraphPad Prism (Version 4.0, U.S.A.). All statistical tests were two-sided and P<0.05 was considered statistically significant.

Results

LY3009120 rescued cells from necroptosis independently of its RAF inhibition activity

To identify compounds that could prevent necroptosis, in vitro experiments were carried out to screen a clinical compound library that contained 611 compounds and was currently in clinical trials from MedChemExpress. Two cell lines, HT-29 and L929, which are commonly used for studying necroptosis were exploited for the screening. TNF-α plus Smac mimetic and Z-VAD (T/S/Z) was used to induce necroptosis in HT-29 cells while TNF-α plus Z-VAD (T/Z) was used to induce necroptosis in L929 cells [4,24]. As a positive control, Nec-1 decreased PI positive cells and prevented cell viability loss in T/Z treated L929 cells and T/S/Z treated HT-29 cells (Figure 1A–D). Among the library, some compounds were found to be able to inhibit necroptosis in both L929 and HT-29 cells (data not shown). LY3009120, a pan-RAF inhibitor, dramatically decreased necroptosis of L929 and HT-29 cells in a dose-dependent manner as shown by PI staining (Figure 1A,B) and CCK8 analysis (Figure 1C,D). However, three other RAF inhibitors CEP-32496, Ro 5126766, and PLX8394 did not inhibit necroptosis in L929 and HT-29 cells (Figure 2A,B), suggesting that LY3009120 prevented necroptosis independently of its ability to inhibit the kinase activity of RAF.

Figure 1

LY3009120 inhibited necroptosis in a dose-dependent manner

L929 or HT-29 cells were pretreated with DMSO or Nec-1 or LY3009120 with indicated concentrations for 1 h, then stimulated with TNF-α (1 ng/ml) plus Z-VAD (25 μM) for 3 h or TNF-α (20 ng/ml) plus Smac mimetic (2 μM) and Z-VAD (25 μM) for 8 h, respectively. PI positive cells were analyzed by flow cytometry (A,B), and cell viability was determined by CCK8 assay (C,D). Data from three independent experiments are shown. **P<0.01; ***P<0.001.

Figure 1

LY3009120 inhibited necroptosis in a dose-dependent manner

L929 or HT-29 cells were pretreated with DMSO or Nec-1 or LY3009120 with indicated concentrations for 1 h, then stimulated with TNF-α (1 ng/ml) plus Z-VAD (25 μM) for 3 h or TNF-α (20 ng/ml) plus Smac mimetic (2 μM) and Z-VAD (25 μM) for 8 h, respectively. PI positive cells were analyzed by flow cytometry (A,B), and cell viability was determined by CCK8 assay (C,D). Data from three independent experiments are shown. **P<0.01; ***P<0.001.

Figure 2

CEP-32496, Ro 5126766, and PLX8394 did not inhibit necroptosis in L929 and HT-29 cells

L929 or HT-29 cells were pretreated with DMSO or Nec-1 or indicated inhibitors for 1 h, then stimulated with T/Z for 3 h or T/S/Z for 8 h, respectively. Then cell viability was determined by CCK8 assay (A,B). Data from three independent experiments are shown.

Figure 2

CEP-32496, Ro 5126766, and PLX8394 did not inhibit necroptosis in L929 and HT-29 cells

L929 or HT-29 cells were pretreated with DMSO or Nec-1 or indicated inhibitors for 1 h, then stimulated with T/Z for 3 h or T/S/Z for 8 h, respectively. Then cell viability was determined by CCK8 assay (A,B). Data from three independent experiments are shown.

To confirm the role of RAF in necroptosis, we silenced ARAF, BRAF, and CRAF in L929 cells and stimulated L929 cells with T/Z. Two different sets of siRNAs significantly decreased mRNA levels of ARAF, BRAF, and CRAF in L929 cells (Figure 3A). However, silencing of RAF even increased necroptosis of L929 cells treated with T/Z (Figure 3B,C). Together, these results indicate that LY3009120 has the ability to prevent necroptosis, but this kind of inhibitory function is independent of RAF.

Figure 3

Silencing of RAF promoted necroptosis of L929 cells

(A) Silencing of ARAF, BRAF, and CRAF in L929 cells. L929 cells were transfected with negative control siRNA or siRNAs against ARAF, BRAF, and CRAF, respectively. Two different sets of siRNAs (siRAF-1 and siRAF-2) were used to knock down ARAF, BRAF, and CRAF. (B,C) Silencing of ARAF, BRAF, and CRAF promoted necroptosis of L929 cells. Forty-eight hours after transfection with indicated siRNAs, L929 cells were treated with T/Z for 3 h. PI positive cells were analyzed by flow cytometry (B), and cell viability was determined by CCK8 (C). Data from three independent experiments are shown. **P<0.01; ***P<0.001.

Figure 3

Silencing of RAF promoted necroptosis of L929 cells

(A) Silencing of ARAF, BRAF, and CRAF in L929 cells. L929 cells were transfected with negative control siRNA or siRNAs against ARAF, BRAF, and CRAF, respectively. Two different sets of siRNAs (siRAF-1 and siRAF-2) were used to knock down ARAF, BRAF, and CRAF. (B,C) Silencing of ARAF, BRAF, and CRAF promoted necroptosis of L929 cells. Forty-eight hours after transfection with indicated siRNAs, L929 cells were treated with T/Z for 3 h. PI positive cells were analyzed by flow cytometry (B), and cell viability was determined by CCK8 (C). Data from three independent experiments are shown. **P<0.01; ***P<0.001.

LY3009120 decreased p-RIPK1, p-RIPK3, and p-MLKL level in T/S/Z treated HT-29 cells

T/S/Z induced necroptosis in HT-29 cells is RIPK1 dependent. Three different siRNAs against RIPK1 significantly decreased RIPK1 protein level in HT-29 cells (Figure 4A). Silencing of RIPK1 prevented HT-29 cells from T/S/Z induced necroptosis (Figure 4B). To find the target of LY3009120, the levels of total and phosphorylated RIPK1, RIPK3, and MLKL were detected by immunoblotting. Phosphorylated RIPK1 increased 2 h after T/S/Z stimulation and reached a peak 4 h post stimulation (Figure 4C). Interestingly, total RIPK1 decreased gradually upon T/S/Z stimulation. Subsequently, RIPK3 and MLKL became phosphorylated 4 h post T/S/Z stimulation. Pretreated with LY3009120 blocked phosphorylation of RIPK1, RIPK3, and MLKL induced by T/S/Z, but did not affect RIPK1 degradation (Figure 4C). These results indicate that LY3009120 inhibits necroptosis by preventing phosphorylation of RIPK1 and subsequently phosphorylation of RIPK3 and MLKL.

Figure 4

LY3009120 inhibited necroptosis by preventing phosphorylation of RIPK1, RIPK3, and MLKL

(A) Silencing effect of siRNAs against RIPK1 in HT-29 cells. Forty-eight hours after transfection with indicated siRNAs, proteins were detected by immunoblotting. (B) Silencing of RIPK1 prevented necroptosis in T/S/Z treated HT-29 cells. Forty-eight hours after transfection with indicated siRNAs, HT-29 cells were treated with T/S/Z for 8 h. Cell viability was determined by CCK8. (C) LY3009120 decreased phosphorylation of RIPK1, RIPK3, and MLKL in T/S/Z treated HT-29 cells. HT-29 cells were pretreated with DMSO or LY3009120 for 1 h, then treated with T/S/Z for 0, 2, 4, and 6 h, respectively. Indicated proteins were detected by immunoblotting. For (A,C), representative images from three independent experiments are shown. For (B), data from three independent experiments are shown. *, P<0.05; **, P<0.01; ***P<0.001.

Figure 4

LY3009120 inhibited necroptosis by preventing phosphorylation of RIPK1, RIPK3, and MLKL

(A) Silencing effect of siRNAs against RIPK1 in HT-29 cells. Forty-eight hours after transfection with indicated siRNAs, proteins were detected by immunoblotting. (B) Silencing of RIPK1 prevented necroptosis in T/S/Z treated HT-29 cells. Forty-eight hours after transfection with indicated siRNAs, HT-29 cells were treated with T/S/Z for 8 h. Cell viability was determined by CCK8. (C) LY3009120 decreased phosphorylation of RIPK1, RIPK3, and MLKL in T/S/Z treated HT-29 cells. HT-29 cells were pretreated with DMSO or LY3009120 for 1 h, then treated with T/S/Z for 0, 2, 4, and 6 h, respectively. Indicated proteins were detected by immunoblotting. For (A,C), representative images from three independent experiments are shown. For (B), data from three independent experiments are shown. *, P<0.05; **, P<0.01; ***P<0.001.

LY3009120 ameliorates DSS-induced colitis in vivo

Previous studies suggest that necroptosis of IECs is an important process that leads to disruption of the intestinal barrier and contributes to the development of IBD. To verify the anti-necroptosis and anti-colitis activity of LY3009120 in vivo, DSS-induced colitis was established. Compared with the control treatment (DMSO) in which DSS treatment led to a rapid body weight loss from days 4 to 12, administration of LY3009120 intraperitoneally significantly decreased body weight loss (Figure 5A). Furthermore, LY3009120 dramatically reduced DSS-induced mortality and shortening of colon length (Figure 5B,C). Moreover, H&E staining showed that UCF-101 significantly decreased tissue damage and infiltration of inflammatory cells in colons of DSS-treated mice (Figure 5D,E). Similarly, LY3009120 decreased body weight loss of TNBS treated mice (Supplementary Figure S1). Taken together, these results imply that LY3009120 ameliorates DSS- or TNBS-induced colitis in mice.

Figure 5

LY3009120 ameliorated DSS-induced colitis in vivo

Three percent DSS was administered in drinking water to C57BL/6 mice for 7 days and replaced with fresh water thereafter. LY3009120 (20 mg/kg) or vehicle was injected intraperitoneally everyday for 10 days. Body weight (A) and survival rate (B) were determined, n=8 mice/group. Mice were killed on day 7 to measure the colon length (C); colon tissues from mice on day 7 were evaluated by haematoxylin and eosin (H&E) staining (E) and histologic scoring analysis (D), n=4 mice/group. Scale bar, 100 μm. In (A,C,D), data are presented as mean ± S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001 (two-tailed unpaired Student’s ttest).

Figure 5

LY3009120 ameliorated DSS-induced colitis in vivo

Three percent DSS was administered in drinking water to C57BL/6 mice for 7 days and replaced with fresh water thereafter. LY3009120 (20 mg/kg) or vehicle was injected intraperitoneally everyday for 10 days. Body weight (A) and survival rate (B) were determined, n=8 mice/group. Mice were killed on day 7 to measure the colon length (C); colon tissues from mice on day 7 were evaluated by haematoxylin and eosin (H&E) staining (E) and histologic scoring analysis (D), n=4 mice/group. Scale bar, 100 μm. In (A,C,D), data are presented as mean ± S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001 (two-tailed unpaired Student’s ttest).

To confirm that LY3009120 inhibited colitis independently of RAF, we detected the effect of other two RAF inhibitors, CEP-32496 and Ro 5126766, on DSS-induced colitis. As shown in Supplementary Figure S2, CEP-32496 and Ro 5126766 did not have protective effect on DSS-induced colitis, suggesting a RAF-independent regulation of necroptosis by LY3009120 in DSS-induced colitis.

LY3009120 decreases necroptosis and intestinal barrier disruption in colons of DSS-treated mice

Next, we examined whether LY3009120 decreased necroptosis of colonic epithelial cells in DSS-induced colitis. Massive death of intestinal cells was found in DSS-treated mice as shown by TUNEL staining, but treatment with LY3009120 significantly decreased TUNEL positive cells in the colon (Figure 6A,B). Since TUNEL staining is not able to distinguish necroptosis from apoptosis, we further detected protein level of p-MLKL (indicator of necroptosis) and cleaved caspase-3 (indicator of apoptosis). In isolated colonic epithelial cells of DSS-treated mice, p-MLKL was increased, but little cleaved caspase-3 was detected (Figure 6C), suggesting that necroptosis, but not apoptosis, contributed to DSS-induced colitis. In colon of LY3009120-treated mice, much less p-MLKL was detected (Figure 6C), demonstrating a suppression of necroptosis by LY3009120 in DSS-induced colitis.

Figure 6

LY3009120 decreased necroptosis and intestinal barrier function loss in colons of DSS-treated mice

Three percent DSS was administered in drinking water to C57BL/6 mice for 7 days. LY3009120 (20 mg/kg) or vehicle was injected intraperitoneally everyday for 7 days, n=4 mice/group. (A,B) Sections of colon on day 7 were subjected to TUNEL staining. Scale bar, 50 μm. Representative images (A) and numbers of TUNEL positive cells per field (B) are presented. (C) Colonic proteins on day 7 were tested by immunoblotting to detect p-MLKL, MLKL, and cleaved caspase-3 with corresponding antibodies. GAPDH was used as an internal control. Three individual mice from each group are shown. (D,E) Intestinal barrier permeability was detected by intragastrical injection of FITC-Dextran. Colon tissues were sliced and representative images of colons from indicated groups were detected by fluorescent microscope (D), and FITC-Dextran levels in hemolysis-free sera from indicated groups were detected with spectrophotometer (E). Scale bar, 50 μm. In (B,E), data are presented as mean ± S.E.M. **, P<0.01; ***, P<0.001 (two-tailed unpaired Student’s t test).

Figure 6

LY3009120 decreased necroptosis and intestinal barrier function loss in colons of DSS-treated mice

Three percent DSS was administered in drinking water to C57BL/6 mice for 7 days. LY3009120 (20 mg/kg) or vehicle was injected intraperitoneally everyday for 7 days, n=4 mice/group. (A,B) Sections of colon on day 7 were subjected to TUNEL staining. Scale bar, 50 μm. Representative images (A) and numbers of TUNEL positive cells per field (B) are presented. (C) Colonic proteins on day 7 were tested by immunoblotting to detect p-MLKL, MLKL, and cleaved caspase-3 with corresponding antibodies. GAPDH was used as an internal control. Three individual mice from each group are shown. (D,E) Intestinal barrier permeability was detected by intragastrical injection of FITC-Dextran. Colon tissues were sliced and representative images of colons from indicated groups were detected by fluorescent microscope (D), and FITC-Dextran levels in hemolysis-free sera from indicated groups were detected with spectrophotometer (E). Scale bar, 50 μm. In (B,E), data are presented as mean ± S.E.M. **, P<0.01; ***, P<0.001 (two-tailed unpaired Student’s t test).

Massive death of epithelial cells leads to the disruption of intestinal barrier function. We further explored the protective effect of LY3009120 on intestinal barrier function in colitis. On day 7 of DSS induction, mice were given FITC-dextran tracer intragastrically. Increased FITC-Dextran was found in the colon and serum of Vehicle-treated mice, but it was significantly decreased in LY3009120-treated mice (Figure 6D,E), suggesting that the increased intestinal permeability seen after DSS induction could be diminished by LY3009120. These results suggest that LY3009120 decreases necroptosis and intestinal barrier disruption in colons of DSS-treated mice.

LY3009120 decreased colonic inflammation in DSS-treated mice

Inflammation is a major hallmark of IBD and can be induced by necroptosis. Next, we examined whether LY3009120 decreased inflammation in DSS-induced colitis. As expected, LY3009120 significantly decreased tissue damage and infiltration of inflammatory cells in colons of DSS-treated mice as shown by H&E staining (Figure 5E). Consistently, pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, were significantly restrained in LY3009120-treated mice (Figure 7A–C). In DSS-induced colitis, increased numbers of macrophages (F4/80 positive) and neutrophils (MPO positive) infiltrated into the mucosa and epithelial layer of the damaged colon (Figure 7D,E). The infiltration of macrophages and neutrophils in the colon was dramatically decreased in LY3009120-treated mice (Figure 7D,E). The same phenomenon was observed with the infiltration of S100a9 positive cells, a marker of inflammation (Figure 7F). Taken together, these results suggest that LY3009120 ameliorated DSS-induced colonic inflammation in vivo.

Figure 7

LY3009120 decreased inflammation in colon of DSS-induced colitis

Three percent DSS was administered in drinking water to C57BL/6 mice for 7 days. LY3009120 (20 mg/kg) or vehicle was injected intraperitoneally everyday for 7 days, n=4 mice/group. (AC) On day 7, mice were killed and TNF-α, IL-6, and IL-1β levels in supernatant of cultured colon tissues were measured by ELISA. (DF) Colons were harvested and sections of colon tissues were immunohistochemically stained for F4/80 (D), MPO (E), and S100a9 (F) with corresponding antibodies. Scale bar, 50 μm. Ten random fields (200×) were photographed for each section. The average numbers of positive cells per field are presented. Data are presented as mean ± S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001 (two-tailed unpaired Student’s t test).

Figure 7

LY3009120 decreased inflammation in colon of DSS-induced colitis

Three percent DSS was administered in drinking water to C57BL/6 mice for 7 days. LY3009120 (20 mg/kg) or vehicle was injected intraperitoneally everyday for 7 days, n=4 mice/group. (AC) On day 7, mice were killed and TNF-α, IL-6, and IL-1β levels in supernatant of cultured colon tissues were measured by ELISA. (DF) Colons were harvested and sections of colon tissues were immunohistochemically stained for F4/80 (D), MPO (E), and S100a9 (F) with corresponding antibodies. Scale bar, 50 μm. Ten random fields (200×) were photographed for each section. The average numbers of positive cells per field are presented. Data are presented as mean ± S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001 (two-tailed unpaired Student’s t test).

Discussion

In this article, we reveal that an anti-cancer drug LY3009120 inhibits necroptosis independently of its RAF inhibition activity and ameliorates DSS-induced colitis. Our data raise a possibility that LY3009120 may be used for anti-colitis treatment.

Necroptosis has been implicated in several pathologies based on disease models, which include IBD, retinal degeneration, brain impact trauma, cerulein-induced pancreatitis, ethanol-induced liver injury etc [25,26]. However, clinical drugs targeting necroptosis are rare. Nec-1, GSK′842, and Necrosulfonamide are well-known inhibitors for RIPK1, RIPK3, and human MLKL, respectively [3,13]. But, none of these inhibitors have been used in clinical trials. Dabrafenib is a B-Raf inhibitor which has been used as an anti-cancer drug in Phase IV clinical trials. It was recently found to be able to inhibit RIPK3 independently of its B-Raf inhibition activity and alleviate acetaminophen-induced liver injury in mouse model [27]. LY3009120 is a pan-RAF inhibitor and has been used for anti-cancer treatment in Phase I clinical trials. Herein, we found that LY3009120 inhibited necroptosis and alleviated DSS-induced colitis for the first time.

LY3009120 inhibited necroptosis independently of RAF. LY3009120 is a pan-RAF inhibitor and inhibits necroptosis of HT-29 and L929 cells in vitro. However, other RAF inhibitors did not inhibit necroptosis in vitro, which is consistent with one previous study [27]. These results suggest that the kinase activity of RAF may not be involved in the regulation of TNF-α induced necroptosis of HT-29 and L929 cells. Furthermore, we found that silencing of RAF even promoted T/Z induced necroptosis in L929 cells. Since RAF is involved in the regulation of cell survival and mice deficient in RAF showed a lethal phenotype at embryonic period [28], silencing of RAF may affect the survival of L929 cells. However, the exact role of RAF in necroptosis needs further exploration.

RIPK1/RIPK3/MLKL signal transduction is necessary for TNF-α induced necroptosis [29,30]. Herein, we found phosphorylation of RIPK1, RIPK3, and MLKL was completely blocked by LY3009120, suggesting that the target of LY3009120 is upstream of RIPK1. Interestingly, we found that RIPK1 protein level decreased upon T/S/Z treatment and inhibition of RIPK1 phosphorylation by LY3009120 did not prevent degradation of RIPK1, implying that RIPK1 phosphorylation is not required for its degradation. We also observed that LY3009120 decreased phosphorylation and degradation of MLKL, suggesting that MLKL phosphorylation may be required for its degradation.

Excessive cell death has been found in IBD [31,32]. Interestingly, increased p-MLKL but not cleaved caspase-3 was found in colons of DSS-treated mice, suggesting that necroptosis but not apoptosis is the major form of cell death in DSS-induced colitis. Interestingly, the level of p-MLKL and cleaved caspase-3 was inversely correlated, suggesting there is reciprocal inhibition between these two types of cell death. Impaired intestinal permeability has been found in IBD patients [33,34]. Our data showed that intestinal permeability positively correlated with the level of p-MLKL, indicating that necroptosis contributes to the increased intestinal permeability in DSS-induced colitis.

Dirisina et al. [35] reported an increased cleaved caspase-3 on day 4 in 1.5% DSS-treated mice. Tambuwala et al. [36] and Kolachala et al. [37] reported an increased cleaved caspase-3 in 3 and 5% DSS-treated mice, respectively, but the exact time points for detecting cleaved caspase-3 were lacking. However, we observed a slightly decreased cleaved caspase-3 in 3% DSS-treated mice on day 7. Therefore, we propose that cleaved caspase-3 is increased on early phase of DSS-treated mice, and on later phase apoptosis decreases while necroptosis arises.

We observed decreased p-MLKL and increased cleaved caspase-3 in colon of LY3009120-treated mice, while TUNEL staining was decreased. As shown in Figure 5A,C, cleaved caspase-3 caused little apoptosis (few TUNEL-positive cells) in H2O-treated mice, while p-MLKL caused massive necroptosis (many TUNEL-positive cells) in Vehicle+DSS treated mice. Even though increased cleaved caspase-3 was found in colon of LY3009120+DSS-treated mice, the dramatic decrease in p-MLKL resulted in a decrease in TUNEL positive cells in colon of LY3009120-treated mice.

Inflammation is a hallmark of IBD, while necroptosis is thought to be highly pro-inflammatory [38,39]. In DSS-induced colitis, increased TNF-α, IL-6, and IL-1β was found in the supernatant of inflamed colon. When necroptosis was blocked by LY3009120, the level of these inflammatory cytokines were dramatically decreased. These data strongly suggest that necroptosis is a highly pro-inflammatory process in vivo.

IBD is an important risk factor in the pathogenesis of colon cancer, namely, colitis-associated cancer (CAC) [40–42]. Necroptosis is a highly pro-inflammatory type of programmed cell death and plays important roles in the development of IBD. Apart from its anti-cancer activity by inhibiting RAF, LY3009120 may also decease the incidence of CAC by inhibiting necroptosis of IECs and the downstream inflammation.

Collectively, we found that an anti-cancer pan-RAF inhibitor LY3009120 could inhibit necroptosis in vitro by targeting RIPK1 phosphorylation and ameliorated DSS-induced colitis in vivo. Our study raises a possibility that LY3009120 may be used for anti-colitis treatment.

Clinical perspectives

  • Necroptosis has been found to play an important role in the pathogenesis of numerous diseases, including IBD. However, clinical drugs targeting necroptosis are seriously lacking.

  • We found that an anti-cancer drug LY3009120 inhibited necroptosis independently of its RAF inhibition activity and ameliorated DSS-induced colitis.

  • Our data raise a possibility that LY3009120 may serve as a potential therapeutic drug for colitis.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the National Science and Technology Major Project of China [grant number 2016ZX08011-005]; and the Scientific and Technological Project of Guangzhou [grant numbers 201604020008, 201804020042].

Author contribution

J.Y. is the principal investigator responsible and takes full responsibility for the paper. C.Z. contributed to generation of hypothesis and experimental design. C.Z., Y.L., Q.H., S.L., and A.H. contributed to the execution of experiments. C.Z., Y.L., Q.H., and S.L. contributed to data analyses and literature review. C.Z. and J.Y. were responsible for manuscript writing and final manuscript approval.

Abbreviations

     
  • ARAF

    A-Raf proto-oncogene, serine/threonine kinase

  •  
  • BRAF

    B-Raf proto-oncogene, serine/threonine kinase

  •  
  • CAC

    colitis-associated cancer

  •  
  • CCK8

    cell counting kit-8

  •  
  • CD

    Crohn’s disease

  •  
  • CRAF

    Raf-1 proto-oncogene, serine/threonine kinase

  •  
  • DD

    death domain

  •  
  • DMSO

    dimethyl sulfoxide

  •  
  • DSS

    dextran sulfate sodium

  •  
  • FBS

    fetal bovine serum

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • HRP

    horseradish peroxidase

  •  
  • H&E

    haematoxylin and eosin

  •  
  • IBD

    inflammatory bowel disease

  •  
  • IEC

    intestinal epithelial cell

  •  
  • IL

    interleukin

  •  
  • i.p.

    intraperitoneally

  •  
  • MLKL

    mixed lineage kinase domain like pseudokinase

  •  
  • OD

    optical density

  •  
  • PI

    propidium iodide

  •  
  • PI3K

    phosphatidylinositol-4,5-bisphosphate 3-kinase

  •  
  • RIPK1

    receptor interacting serine/threonine kinase 1

  •  
  • RIPK3

    receptor interacting serine/threonine kinase 3

  •  
  • SPF

    specific-pathogen free

  •  
  • TNBS

    trinitro-benzene-sulfonic acid

  •  
  • TNF

    tumor necrosis factor

  •  
  • TUNEL

    terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling

  •  
  • UC

    ulcerative colitis

  •  
  • VEGFR

    kinase insert domain receptor

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