Golden berry 4β-hydroxywithanolide E prevents tumor necrosis factor α-induced procoagulant activity with enhanced cytotoxicity against human lung cancer cells

Abstract

Inflammation in the tumor microenvironment is positively correlated with cancer progression and metastasis as well as the risk of thromboembolism in lung cancer patients. Here we show, in human non-small cell lung cancer (NSCLC) cell lines, the master inflammatory cytokine tumor necrosis factor (TNF-α) induced tissue factor expression and procoagulant activity, and these effects were potently inhibited by 4β-hydroxywithanolide E (4HW), a natural compound isolated from Physalis peruviana. Furthermore, combination of 4HW and TNF-α caused synergistic cytotoxicity against NSCLC cells by inducing caspase-dependent apoptosis. The underlying mechanism by which 4HW reverses the procoagulant effect of TNF-α but enhances its cytotoxic effect appears to be due to inhibition of NF-κB, which is a key switch for both inflammation-induced coagulation and cell survival. Our results suggest that 4HW may have a potential application for treating inflammation-derived cancer progression and cancer-associated hypercoagulable state.

Figure 1

4HW inhibits TNF-α-induced TF protein and mRNA expression in NSCLC cells. H1299 and A549 cancer cells were pretreated with DMSO (vehicle) or 4HW for 1 h, followed by incubation with or without TNF-α 20 ng/mL for 12 (A) or 2 h (B). The protein and mRNA levels of TF were evaluated by immunoblotting and real-time RT-PCR, respectively. Full-length blots are shown in the Supplementary information S1. All results are presented as mean ± SEM (n ≥ 3). *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the TNF-α control group. ^##P < 0.01, ^###P < 0.001 as compared with the untreated control group.

Figure 2

4HW inhibits TNF-α-induced procoagulant activity in NSCLC cells. H1299 and A549 cancer cells were pretreated with 4HW or DMSO (vehicle control) for 1 h, followed by incubation with or without TNF-α 20 ng/mL for 24 h. The treated cells were subjected to amidolytic assay (A), plasma clotting assay (B), and MTT assay (C) as described in “Methods”. The relative amidolytic activity is presented as a fold change relative to untreated control cells. Anti-TF antibody (20 μg/mL) was used as a positive control. Results are presented as mean ± SEM (n ≥ 3). **P < 0.01, ***P < 0.001 as compared with the TNF-α control group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 as compared with the untreated control group.

Figure 3

Combination of 4HW with TNF-α causes enhanced cytotoxicity against NSCLC cells. H1299 and A549 cancer cells were treated with 4HW alone or in combination of TNF-α for 48 h, and DMSO was used as a vehicle control. The cytotoxicity was evaluated by MTT assay (A), morphological changes (B), and annexin V/PI double staining assay (C), respectively. Apoptotic cells were indicated as annexin V-positive/PI-negative or -positive, while necrotic cells were indicated as annexin V-negative/PI-positive. Representative flow cytometry dot plots are shown in Supplementary Figure S1. Results are presented as mean ± SEM (n ≥ 3). *P < 0.05, ***P < 0.001 compared to the DMSO (vehicle) control group.

Figure 4

Cell death caused by 4-HW and TNF-α is mainly due to caspase-dependent apoptosis. (A) NSCLC cells were treated with 4HW or DMSO (vehicle control) in the presence or absence of TNF-α (20 ng/mL) for 24 h, the apoptosis-related biomarkers were then determined by immunoblotting. Full-length blots are presented in the Supplementary information S1. (B) NSCLC cells were pretreated with the pan-caspase inhibitor Z-VAD(OMe)-FMK (Z-VAD, 25 μM) or the necroptosis inhibitor necrostatin-1 (Nec, 20 μM), and treated with 4HW and/or TNF-α for 48 h. The apoptotic cells were evaluated by annexin V/PI double staining. Representative flow cytometry dot plots are shown in Supplementary Figure S2. Results are presented as mean ± SEM (n ≥ 3). *P < 0.05, ***P < 0.001 as compared with the indicated group. ^##P < 0.01, ^###P < 0.001 as compared with the untreated control group.

Figure 5

Inhibition of NF-κB reduces TNF-α-induced procoagulant activity and enhances cell death. NSCLC cells were pretreated Ro 106-9920 (10 μM), wortmannin (0.1 μM), or DMSO (vehicle control) for 1 h and then treated with TNF-α (20 ng/mL) for 12 (A), 24 (B), and 48 (C) hours, respectively. (A) The protein expression of TF was determined by immunoblotting. Full-length blots are presented in the Supplementary information S1. (B) Cancer cell-associated procoagulant activity was determined by plasma clotting assay. (C) Cell viability was assessed by MTT assay. Results are presented as mean ± SEM (n ≥ 3). *or ^#P < 0.05, **or ^##P < 0.01, ***P < 0.001.

Figure 6

The effect of 4HW on TNF-α-induced NF-κB signaling. (A) HEK 293T or H1299 cells transfected with a NF-κB luciferase reporter were pretreated with 4HW or DMSO (vehicle control) and then stimulated with TNF-α (20 ng/mL) for 6 h. NF-κB-dependent luciferase activity was determined as described in “Methods”. (B) NSCLC cells treated with 4HW (1 μM for H1299, 2 μM for A549) or DMSO (vehicle control) were stimulated with TNF-α for indicated periods, then protein expression of IκB was determined by immunoblot blotting. Full-length blots are shown in the Supplementary information S1. All results are presented as mean ± SEM (n ≥ 3). *or ^#P < 0.05, **P < 0.01, ***P < 0.001. (C,D) NSCLC cells were treated as (B), and nuclear translocation of NF-κB p65 (red) was determined by immunofluorescence microscopy. Nuclei were counterstained with DAPI (blue). The images were overlapped (merge) to determine translocation. LMB leptomycin B.