Helichrysetin was supplied by Professor Jingshan Shen (Shanghai Institute of Material Medica, Chinese Academy of Sciences, Shanghai, China), and high-performance liquid chromatography analysis was performed as previously described (16) to confirm that it had a purity of >95%. Recombinant human TNF-α (cat. no. 210-TA) was purchased from R&D Systems, Inc. Primary antibodies against caspase-3 (cat. no. 9665s), PARP (cat. no. 9532s), TAK1 (cat. no. 5206s), pTAK1 (cat. no. 4536s), TAK1 binding protein 1 (TAB1) (cat. no. 3226s), TAB2 (cat. no. 3745s), IKKβ (cat. no. 8943s), phosphorylated (p)IKKα/β (cat. no. 2697s), EGFR (cat. no. 4267s), pEGFR-S1046/7 (cat. no. 2238s), pNF-κB p65-S536 (cat. no. 3033s) and β-actin (cat. no. 4970s) were purchased from Cell Signaling Technology, Inc. Primary antibodies against IKKα (cat. no. c0514) and NF-κB p65 (cat. no. k0515) were obtained from Santa Cruz Biotechnology, Inc. Cell Counting Kit-8 (CCK-8) and Annexin V-FITC Apoptosis Detection kit (cat. no. AD10) were purchased from Dojindo Molecular Technologies, Inc. The Hoechst 33258 staining kit (cat. no. MA0160) was purchased from Beyotime Institute of Biotechnology.

Human cervical cancer (HeLa) and human glioma (T98G) cells were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and cultured in DMEM (high glucose) containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin (all Gibco; Thermo Fisher Scientific, Inc.), in a humidified 5% CO₂ atmosphere at 37°C.

HeLa cells were transfected with a luciferase reporter plasmid p65 NF-κB under the control of 4× κB sites; the luciferase reporter plasmid was provided by Professor Hiroaki Sakurai (University of Toyama, Toyama, Japan) and also contained a neo resistance gene. A stable clone (HeLa-κB) was isolated in medium containing 500 μg/ml G418. Cells (1.6×10⁴) were seeded in a 96-well plate at 37°C for 48 h. After pretreatment with 50 μM helichrysetin at 37°C for 30 min, cells were stimulated with TNF-α (20 ng/ml) at 37°C for another 6 h. Luciferase activity was detected using the ONE-Glo™ Luciferase Assay System (Promega Corporation) and measured using a microplate reader.

HeLa cells were seeded at 1.6×10⁴ cells/well, and T98G cells were seeded at 2×10⁴ cells/well in 96-well plates and cultured overnight to adhere at 37°C. After pretreatment with helichrysetin at 37°C for 30 min, cells were stimulated with TNF-α (20 ng/ml) at 37°C for another 24 h. Subsequently, cells were incubated with CCK-8 solution (10 μl CCK-8 added to 90 μl medium per well) at 37°C for 1 h. Absorbance was detected at 450 nm on a Varioskan Flash microplate reader (Thermo Fisher Scientific, Inc.). The cell viability rate (%) was calculated as follows: (absorbance of drug-treated sample-blank)/(absorbance of control sample-blank) ×100.

HeLa or T98G cells were seeded in 96-well culture plates and cultured overnight to adhere at 37°C. After pretreatment with helichrysetin at 37°C for 30 min, cells were stimulated with TNF-α (20 ng/ml) for 24 h at 37°C. Subsequently, cells were fixed with 4% paraformaldehyde at room temperature for 10 min and washed with PBS. Cells were incubated with 50 μl Hoechst 33258 staining solution at room temperature for 5 min and then washed twice with PBS. Cell morphology was observed and captured under a fluorescence microscope (magnification, ×200).

An Annexin V-FITC Apoptosis Detection kit was used for apoptosis assays. HeLa cells were plated at 3.2×10⁵ cells/well and T98G cells were plated at 4×10⁵ cells/well in 35-mm cell culture dishes. Cells were pretreated with helichrysetin at 37°C for 30 min, followed by stimulation with TNF-α at 37°C for 24 h. Subsequently, the cells were harvested and washed with PBS, and the cell number was adjusted to 1×10⁶ cells/well. After collection by centrifugation at 300 × g at 4°C for 5 min, the cells were resuspended in 1× Binding Buffer, and stained with Annexin V for 15 min and PI for 5 min at room temperature in the dark. Apoptosis was analyzed on a CytoFlex S flow cytometer (Beckman Coulter, Inc.) using the CytExpert v2.3 software (Beckman Coulter, Inc.).

HeLa and T98G cells were cultured in 35-mm dishes at 37°C overnight, then incubated with fresh culture medium containing 0.5% FBS at 37°C for another 24 h. Cells were then pretreated with helichrysetin at 37°C for 30 min, followed by stimulation with TNF-α at 37°C for 5 min, 10 min, 6 h or 12 h. Cells were harvested and lysed in CelLytic™ MT Cell Lysis Reagent (cat. no. C3228; Sigma-Aldrich; Merck KGaA) containing protease and phosphatase inhibitors (cat. nos. 04693116001 and 04906837001; Roche Diagnostics). The protein concentration was determined by BCA assay. A total of 20 μg protein from each sample was separated by standard SDS-PAGE (7.5, 10 or 12.5%) and transferred to Immobilon-P membranes (EMD Millipore) by semi-dry transfer. The membranes were incubated with SuperBlock™ (PBS) Blocking Buffer (Thermo Fisher Scientific, Inc.) at room temperature for 2 h, and then rinsed twice with 1× PBS-Tween (PBST) containing 1% Tween-20. Membranes were incubated with primary antibodies (1:1,000) against caspase-3, PARP, TAK1, pTAK1, TAB1, TAB2, pIKKα/β, EGFR, pEGFR-s1046/7, pNF-κB p65-s536, β-actin, IKKα, IKKβ and NF-κB p65 overnight at 4°C. After washing twice with PBST, membranes were incubated with HRP-conjugated anti-rabbit secondary antibody (1:5,000; cat. no. 122107; Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. The target protein bands were visualized with Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore) using a Tanon-5200 chemiluminescent imaging system (cat. no. 20182351; Tanon Science and Technology Co., Ltd.). For quantification, target proteins were normalized to β-actin within the same sample using ImageJ v1.52a (National Institutes of Health).

HeLa and T98G cells cultured in 24-well plates were pretreated with helichrysetin at 37°C for 30 min and then stimulated with TNF-α at 37°C for 4 h. Total RNA was isolated from the harvested cells using an RNA Faster 2000 kit (cat. no. 220011; Fastagen) according to the manufacturer's protocol. cDNA was reverse transcribed from RNA (1 μg) using PrimeScript™ RT Master Mix (Perfect Real Time) (cat. no. RRD36A; Takara Bio, Inc.) according to the manufacturer's protocol. Quantitative PCR was performed using TB Green^® Premix Ex Taq™ II (Tli RNaseH Plus), ROX plus (Takara Bio, Inc.) on a Quant Studio 6 Flex System (Thermo Fisher Scientific, Inc.) under the following conditions: 95°C for 30 sec; 40 cycles at 95°C for 5 sec and 60°C for 30 sec; 95°C for 15 sec; 60°C for 1 min; and 95°C for 15 sec. Quantification of target genes was determined using the 2^−ΔΔCq method (24). The relative expression of individual target genes was normalized to that of GAPDH in the same sample. The sequences of the primers (Generay Biotech Co., Ltd.) used are listed in Table I.

HeLa and T98G cells were cultured in 100-mm dishes at 37°C overnight and then incubated with fresh culture medium containing 0.5% FBS at 37°C for another 24 h. After treatment with helichrysetin at 37°C for 30 min, cells were collected and washed with PBS, then resuspended in PBS with protease inhibitors. The cell suspension was evenly distributed into PCR tubes and heated at 4, 40, 43, 46, 49 and 52°C for 3 min, and then cooled for another 3 min at room temperature. Subsequently, cells were lysed by rapid freeze-thawing. The lysates were centrifuged at 20,000 × g for 20 min at 4°C. Soluble fractions were transferred to new tubes and samples were prepared for western blot analysis as aforementioned.

All data are presented as the mean ± SD. Differences between 2 groups were analyzed via Student's unpaired t-test, and differences among ≥3 groups were analyzed via one-way ANOVA with Tukey's post-hoc test using GraphPad 7.0 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Helichrysetin inhibits NF-κB nuclear translocation in mouse pancreatic β cells (17). However, the effect of helichrysetin on NF-κB activation in cancer cells has not been clarified. Therefore, the present study measured the effect of helichrysetin on the transcriptional activity of NF-κB and the phosphorylation of NF-κB in human cancer cells. HeLa-κB cells were firstly used to measure the inhibitory effect of helichrysetin on the transcriptional activity of NF-κB induced by TNF-α. HeLa-κB cells were pretreated with 50 μM helichrysetin for 30 min and then stimulated with TNF-α for 6 h. Helichrysetin significantly inhibited the transcriptional activity of NF-κB induced by TNF-α (Fig. 1B). Phosphorylation of NF-κB at Ser-536 is crucial for its transcriptional activity. Thus, the phosphorylation of NF-κB p65 at Ser-536 was analyzed. In order to detect the effect of helichrysetin on the NF-κB and EGFR signaling pathways, T98G and HeLa cells were used for further experiments, since in the present study, both HeLa and T98G cells had a good response upon TNF-α stimulation, which strongly induces NF-κB activation, and both of them express wild-type EGFR. As expected, although helichrysetin exhibited no effect on the protein levels of p65 NF-κB, it inhibited the phosphorylation of p65 NF-κB induced by TNF-α stimulation in both HeLa and T98G cells (Fig. 1C and D). Overall, helichrysetin inhibited NF-κB activation in HeLa and T98G cells.

To mimic the tumor microenvironment, HeLa and T98G cells were treated with TNF-α and the synergistic effect of helichrysetin and TNF-α on the apoptosis of cancer cells was measured. Cells were pretreated with 50 μM helichrysetin for 30 min and then stimulated with TNF-α for 24 h. The results revealed that helichrysetin, but not TNF-α, had an inhibitory effect on cell viability in both cell lines; additionally, the combination of helichrysetin and TNF-α synergistically decreased cell viability (Fig. 2A and B).

Consistent with the CCK-8 assay results, Hoechst 33258 staining demonstrated that the combination of helichrysetin and TNF-α synergistically increased the number of T98G and HeLa cells with dense stained nuclei, compared with cells treated with helichrysetin alone (Fig. 2C and D).

For apoptosis analysis, cells were pretreated with 50 μM helichrysetin for 30 min and then stimulated with TNF-α for 8 (for HeLa cells) or 24 h (for T98G cells). Annexin V/PI staining detected by flow cytometry demonstrated that helichrysetin, but not TNF-α, significantly enhanced apoptosis, and that the combination of helichrysetin and TNF-α synergistically increased the ratio of apoptotic cells in both cell lines (Fig. 2E and F).

Next, the activity of apoptosis-associated proteins was determined. As demonstrated in Fig. 3, HeLa and T98G cells were pretreated with 50 μM helichrysetin for 30 min and then stimulated with TNF-α for 6 or 12 h. After stimulation with TNF-α for 6 h, compared with the control group, helichrysetin, but not TNF-α, significantly increased the protein expression levels of cleaved PARP in both cell lines. The combination of helichrysetin and TNF-α synergistically enhanced this increase. Additionally, helichrysetin significantly increased the protein expression of cleaved caspase-3 in HeLa but not T98G cells. After stimulation with TNF-α for 12 h, helichrysetin, but not TNF-α, increased the protein expression levels of cleaved PARP, and the combination of helichrysetin and TNF-α synergistically enhanced the protein levels of cleaved PARP and cleaved caspase-3 in both cell lines (Fig. 3). Overall, these results demonstrated that the combination of helichrysetin and TNF-α had a synergistic promoting effect on apoptosis.

To elucidate the detailed molecular mechanisms for the observed effects, the phosphorylation of TAK1 and IKKs was analyzed. As shown in Fig. 4, TNF-α stimulation significantly upregulated the phosphorylation of TAK1 and IKKα/β in both cell lines. Although helichrysetin alone had no effect on the phosphorylation of these molecules, it significantly counteracted the phosphorylation induced by TNF-α in both cell lines (Fig. 4).

Activation of NF-κB promotes the expression levels of many proinflammatory factors, such as TNF-α, IL1β, CCL2, CCL5 and CXCL10 (25-27). To further confirm the inhibitory effect of helichrysetin on NF-κB activation induced by TNF-α, HeLa and T98G cells were pretreated with 50 μM helichrysetin for 30 min and then stimulated with TNF-α for 4 h. After TNF-α stimulation, the mRNA expression levels of TNF-α, IL1β, CCL2, CCL5 and CXCL10 were significantly increased in both cell lines; this was completely reversed by helichrysetin treatment (Fig. 5). Overall, these results indicated that helichrysetin blocked TAK1/IKK/NF-κB signaling pathway.

Finally, whether helichrysetin affected EGFR phosphorylation at Ser-1046/7 was analyzed. As shown in Fig. 6, helichrysetin alone had no effect on the phosphorylation of EGFR Ser-1046/7, but it significantly inhibited TNF-α-induced EGFR phosphorylation.

Whether helichrysetin could directly bind to the TAK1/TAB1/TAB2 complex to inhibit TAK1 activity was further analyzed using a thermal shift assay. When compound-protein interactions exist, the stability of the complex will be increased compared with that of a single protein at certain temperatures. In other words, the expression of the complex will be higher than that of a single protein in a thermal shift assay. As shown in Fig. 7, TAK1, TAB1 and TAB2 expression was not increased in helichrysetin-treated cells compared with that in helichrysetin-untreated cells. Therefore, it was demonstrated that helichrysetin did not directly bind to the complex.