N6-Isopentenyladenosine promoted HeLa cell apoptosis through inhibitions of AKT and transforming growth factor β–activated kinase 1 activation

Abstract

N6-Isopentenyladenosine, a member of the family of plant hormones, possesses anti-cancer activities on a number of cancer cell lines. However, its mode of action in cervical cancer cell remains poorly understood. Our computational docking studies showed that N6-Isopentenyladenosine could bind with the really interesting new gene domain of tumor necrosis factor receptor–associated factor 6, which is an ubiquitination E3 ligase. Tumor necrosis factor receptor–associated factor 6–mediated ubiquitination is known to activate both protein kinase B (also known as AKT) and transforming growth factor β–activated kinase 1, and the really interesting new gene domain comprises the core of the ubiquitin ligase catalytic domain. First, we evaluated the effects of iPA on cervical cancer cell line HeLa using MTT and flow cytometry. Second, we examined the effects of iPA on activation of tumor necrosis factor receptor–associated factor 6–mediated downstream targets using western blot or immunoprecipitation. iPA could reduce HeLa cell proliferation through apoptosis, and such anti-cancer activity is associated with inhibitions of both AKT and transforming growth factor β–activated kinase 1 signaling pathways. In addition, suppression of the anti-apoptotic protein Bcl-2 and elevation of the pro-apoptotic protein Bax were also observed. Anti-proliferation properties of iPA are likely due to its binding at the really interesting new gene domain of tumor necrosis factor receptor–associated factor 6 and loss of AKT and transforming growth factor β–activated kinase 1 activities as a result of functional modulations of tumor necrosis factor receptor–associated factor 6. These results support the emerging notion that tumor necrosis factor receptor–associated factor 6 could serve as a viable target for developing new cancer therapeutics.

Introduction

Tumor necrosis factor receptor–associated factor 6 (TRAF6) is a unique member of the TRAF family of adaptor proteins and plays an important role in interleukin-1 receptor/Toll-like receptor (IL-1R/TLR)-mediated signaling pathways.¹ Recent reports have suggested that TRAF6 is an oncogene that not only regulates growth and survival of cancer cells but also overexpresses in many tumor tissues. Our previous study has shown that inhibition of AKT activation could be achieved through targeting TRAF6 using small molecules such as quinine.² Inhibition of AKT activation would result in timely apoptosis of several cancer cell lines such as HeLa and A549 in vitro and in vivo, thereby demonstrating that TRAF6 could serve as a viable target for developing new therapeutics for the treatment of cancer.³ TRAF6 consists of a C-terminus, which is known as the TRAF domain and is necessary for the association with upstream signaling proteins and other TRAFs.⁴ Its N-terminus contains a really interesting new gene (RING) domain, which is rich in cysteine and histidine residues, and this domain is followed by five zinc finger motifs.⁴ The RING domain of TRAF6 functions as an ubiquitin E3 ligase and is involved in a series of downstream signals.^5,6 Together with the dimeric E2 enzyme ubiquitin-conjugating enzyme-13 (Ubc13) and ubiquitin-conjugating E2 enzyme variant-1A (Uev1A), the RING domain of TRAF6 catalyzes the synthesis of Lys-63-linked polyubiquitin chains, leading to direct ubiquitination of protein kinase B (AKT).⁷ It is this ubiquitination that facilitates AKT membrane recruitment, induces AKT phosphorylations at Thr-308 and Ser-473, and renders its ultimate activation.⁷ AKT is essential to cell survival, metabolism, invasion, and metastasis because it mediates anti-apoptotic and proliferation signaling in response to essential cytokines. Its activation has also been implicated in the lipopolysaccharide (LPS)-induced activation of inhibitor of nuclear factor kappa-B kinase (IKK)/nuclear factor-κB (NF-κB) pathway⁸ and Bcl-2 family.⁹

In a related manner, TRAF6-catalyzed ubiquitination plays a critical role in activating transforming growth factor β–activated kinase 1 (TAK1) signaling pathway.^10–12 TAK1 is a member of the MAP kinase kinase kinase (MAPKKK) family and is closely associated with cell survival as well as differentiation and inflammation via a number of specific transcription factors, including activator protein-1 (AP-1) and NF-κB.¹³ In particular, TRAF6 could interact with TAK1 and three proteins that are complexed to it: TAK1-associated binding protein 1, 2, 3 (TAB1, 2, 3). The TRAF6/TAK1/TAB1/TAB2 complex could subsequently bind to the ubiquitin ligases Ubc13 and Uev1A. This process leads to TAK1 ubiquitination and triggers TAK1 autophosphorylations at Thr-184 and Thr-187 and its activation.^13–15 More importantly, like AKT, activated TAK1 can also mediate the activation of IKK/NF-κB, which could suppress cell apoptosis. Consequently, in TRAF-mediated signaling pathways, ubiquitination plays a vital role in the progression of several tumor types.¹⁴

N6-Isopentenyladenosine (iPA) is a plant hormone that regulates plant cell growth and differentiation.¹⁶ Its anti-cancer properties have also been documented. The effect of iPA on human colon cancer cell line DLD1 has been studied extensively because c-Jun N-terminal kinase (JNK) activation is closely associated with cell apoptosis.¹⁷ It is also known to suppress cell proliferation and inhibits DNA synthesis in lung cancer cells,¹⁸ and that it reduces cell growth of human breast cancer through inhibition of AKT and prevention of translocation of NF-κB into the nucleus.¹⁹ Reports have implicated that iPA could influence cytotoxic activity and cytokines production in natural killer (NK) cells and exert topical anti-inflammatory activity in mice.²⁰ Intriguingly, TRAF6 is upstream of JNK as well as AKT and NF-κB in IL-1R/TLR-mediated signaling pathways, its potential involvement in iPA-induced anti-cancer activities is not clear. Through virtual screening and computation docking, we found that iPA could be a high-affinity ligand for the RING domain of TRAF6 (Figure 1(b)). Thus, we investigated anti-proliferation activity of iPA using HeLa cells and its relations with TRAF6-mediated activation and associated downstream targets. We wish to communicate our findings herein.

Figure 1.

(a) The chemical structure of N6-Isopentenyladenosine (iPA). (b) The predicated binding model of iPA to TRAF6.

[Figure omitted. See PDF]

Materials and methods

Materials

The cervical cancer cell line HeLa was kindly provided from Tianjin International Joint Academy of Biomedicine (Tianjin, China). RPMI-1640 and fetal calf serum were purchased from Tianjin Hope Biotechnology Co., Ltd (Corning, Tianjin, Australia). Rabbit polyclonal antibodies against B cell lymphoma 2 (Bcl-2; Cat. No. 2876), Bcl-2-associated X protein (Bax; Cat. No. 2772), total TAK1 (cat. no. 4505), phospho-TAK1 (Thr184/187; Cat. No. 4531s), rabbit polyclonal antibodies against total AKT (Cat. No. 4691), phospho-AKT (Ser-473; Cat. No. 4051s), phospho-AKT (Thr-308; Cat. No. 4056s), and mouse polyclonal antibodies against total AKT (Cat. No. 2920) were purchased from Cell Signaling Technology (CST; Danvers, Massachusetts). Anti-ubiquitin antibody was purchased from Santa Cruz Biotechnology (Cat. No. sc-8409; Santa Cruz, CA, USA). Rabbit polyclonal antibody against β-actin was purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd (Cat. No. PR0255; Beijing, China). Horseradish peroxidase (HRP)-conjugated goat polyclonal antibody against rabbit or mouse was received from Shanghai Abmart Biotechnology Co., Ltd (Cat. No. M21003; Shanghai, China). Radioimmunoprecipitation assay (RIPA) buffer and protease inhibitors were purchased from Beijing Kangwei Biotechnology Co., Ltd (Beijing, China). Phosphatase inhibitors were purchased from Sigma Co. (St. Louis, Missouri). iPA of analytical grade was purchased from Shanghai Jianglai Biochemical Technology Co., Ltd (Shanghai, China). Protein A Sepharose beads were purchased from Beijing Kangwei Biotechnology Co., Ltd (Pierce, Beijing). All the chemicals and solvents were of analytical grade.

Docking study

Structure of the compound was generated using a two- or three-dimensional (2D/3D) editor-sketcher and was minimized to a local energy minimum using the Chemistry at Harvard Macromolecular Mechanics (CHARMM) like force field implemented within the Catalyst 4.11 software. The 3D structure of TRAF6 (residues 50–159, RING and zinc finger) was retrieved from the Protein Data Bank with an access code of 3HCT, as the docking receptor. The docking area was defined as a dimension of 60 × 60 × 60 points with grid spacing of 0.375 Å. The grid box was centered on the binding site of the Ubc13. Both the TRAF6 crystal structure and candidate ligands were prepared using AutoDock Tools v.1.5.2 software. The docking results were ranked according to the binding free energy. The conformations from the docking experiments were analyzed using Chimera, which also identifies the hydrophobic interactions between the receptor and the ligands.

Cell culture

The cervical cancer cell line HeLa was maintained in RPMI-1640 that is supplemented with 10% fetal bovine serum. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

Cell proliferation assay

According to our previous study, the cells were seeded into 96-well plates at densities of 4 × 10³ per well and allowed to adhere overnight.² The cells were then treated with different concentrations of iPA (1–500 µM) for 48, 72, or 96 h. Cis-platinum (16.7 µM) was used as a positive control. After treatment, 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma) assay is used for detection of cellular viability. The MTT assay result was measured at 490 nm using ST-360 Microplate Reader (Shanghai Kehua Bio-Engineering Co., Ltd, Shanghai, China). The ‘cell inhibition ratio’ was calculated using the following formula

$$\mathrm{Cell\; inhibition\; ratio}\left(\mathrm{\%}\right)=\frac{1-\mathrm{OD\; treated}}{\mathrm{OD\; control}}\times 100\%$$

Half-maximal inhibitory concentration (IC₅₀) was calculated by regression curve of ultraviolet at different concentrations.

Detection of apoptosis by flow cytometric analysis

Cells (6 × 10⁵) were seeded into a 60-mm plate and allowed to attach overnight. After treatment with different concentrations of iPA (5, 10, and 50 µM) for 48 h, cells were then harvested and labeled with Annexin V–fluorescein isothiocyanate (FITC) and propidium iodide (PI), and the resulting mixture was incubated for 15 min at room temperature (RT) in the dark. The apoptotic index was measured using Becton-Dickinson FACS Calibur Flow Cytometer.

Western blot analysis

Cells were incubated with 10 µM iPA in 60 mm dishes. Then cells were lysed with RIPA buffer supplemented with 1% Phenylmethanesulfonyl fluoride (PMSF) and 0.5% phosphatase inhibitors for 30 min on ice. After centrifugation at 12,000 r/min for 10 min at 4°C, protein concentrations were determined using a BCA Protein Assay Kit (Cwbio, Beijing, China) and equal amounts of protein from the cell lysates were electrophoresed on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then, proteins were transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). Subsequently, the membranes were blocked with 5% nonfat dry milk or 5% bovine serum albumin (BSA) which supplemented in phosphate-buffered saline with Tween 20 (TBS-T) for 2 h at RT. The membranes were then washed with TBS-T and incubated with appropriate primary antibodies for β-actin (1:1500), AKT (1:1000), p-AKT (1:1000), TAK1 (1:1000), p-TAK1 (1:1000), Bax (1:1000), or Bcl-2 (1:1000) overnight at 4°C. The membrane was incubated with a secondary antibody (1:10,000 dilution of goat anti-rabbit or mouse immunoglobulin G (IgG)-HRP, Abmart, Shanghai China) for 1 h after washing. Antibody binding was detected using the electrochemiluminescence (ECL) as per the manufacturer’s protocol (CWBIO, China).

Immunoprecipitation

The cells were collected and lysed in buffer A (50 mM hydroxyethyl piperazine ethanesulfonic acid (HEPES), pH 7.4, 150 mM NaCl, and 1% Nonidet P-40 (NP-40)) supplemented with 1% protease inhibitor cocktail (Roche, Shanghai, Germany). The supernatants were pre-cleared using Protein A Sepharose beads (Pierce, USA) at 4°C for 1 h followed by centrifugation at 12,000 r/min for 20 min. Next, 2 µL of the primary antibody was added to the pre-cleared lysate and then incubated under constant rotation at 4°C overnight. Next day, 20 µL of Protein A Sepharose beads was added to the immunecomplexes at 4°C for 5 h. Subsequently, the beads were washed for three times with buffer A and the sample was then detected by western blot assay. The following antibodies were used for immunoprecipitation (IP) and immunoblotting (IB): anti-ubiquitin antibody (IB: 1:1000), anti-AKT antibody (IP: 1:200; IB: 1:1000), and anti-β-actin antibody (IB: 1:1500).

Statistical analysis

Statistical data were shown as means ± standard deviation (SD) from three separate experiments. The statistical significance of differences between the control and treated groups was determined by Dunnett’s t-test (*p < 0.05 and **p < 0.01 vs control).

Results

Docking study and identification of iPA

In this mode, iPA was surrounded by the residues Tyr57–Glu69. iPA made three hydrogen bonds with the residues Phe60 and Leu64 in TRAF6 (shown in Figure 1(b)). The alkyl chain at the terminal of the compound prevented the formation of salt-bridge bonds between Asp57 of TRAF6 and Lys10 of Ubc13, which are the key interactions between TRAF6 and Ubc13 reported by Yin’s group.¹²

iPA suppresses proliferation of HeLa cells

HeLa cells expressing high levels of endogenous TRAF6 were treated with iPA (0–500 µM) at different time intervals (48, 72, and 96 h). As shown in Figure 2, analysis of dose-dependent effects of iPA on HeLa cells proliferation by MTT assay revealed almost no significant effects, when treating with 1 µM of iPA up to 96 h. Whereas at 5 µM or higher doses, a significant dose-dependent inhibition of cell proliferation was revealed relative to the control (p < 0.01). In addition, prolonged incubation with iPA also increased the inhibitory rate. Based on these results, the half maximal effective concentration was found to be 10 µM and all following assays were performed with 10 µM iPA. MTT assay revealed that iPA treatment inhibits HeLa cell proliferation in a concentration-dependent and time-dependent manner.

Figure 2.

The inhibitory effects of iPA on HeLa cell proliferation. HeLa cells were incubated with different doses of iPA for different time intervals (48, 72, and 96 h). Cell proliferation was assessed with MTT. Results are shown as the mean ± SD from six separate experiments.

*p < 0.05 and **p < 0.01 versus control.

[Figure omitted. See PDF]

iPA induces apoptosis in HeLa cells

To determine whether reduced proliferation is associated with actual apoptosis, HeLa cells were treated with various concentrations of iPA for 48 h before flow cytometry assays. As illustrated in Figure 3, the apoptosis rate of untreated HeLa cells was less than 5% (Annexin V⁻/PI⁻, 94.46 ± 1.16%; Annexin V⁺/PI⁻, 2.57 ± 0.92%; Annexin V⁺/PI⁺, 1.23 ± 0.54%; Annexin V⁻/PI⁺, 1.55 ± 0.05%). Cis-platinum, the positive control, reduced the number of viable cells (Annexin V⁻/PI⁻, 42.74 ± 1.16%) and gave a concomitant increase in the fraction of early apoptotic cells (Annexin V⁺/PI⁻, 51.12 ± 2.98%; p < 0.01). After iPA treatment, the fraction of early apoptosis cells (Annexin V⁺/PI⁻) was significantly higher than that in control groups (iPA: 5 µM, 17.46 ± 0.04%; 10 µM, 31.66 ± 1.15%; 50 µM, 37.34 ± 0.41%; p < 0.01; Figure 3). However, it was found that iPA had no significant effects on late apoptosis or necrosis (Annexin V⁺/PI⁺; Annexin V⁻/PI⁺; p > 0.05). These results revealed that iPA-induced early apoptosis improved gradually with an increase in the iPA concentration, thereby suggesting that iPA had triggered an apoptotic response in HeLa cells.

Figure 3.

Apoptotic effects of iPA on HeLa cells. HeLa cells were pretreated with iPA (concentration: 5, 10, and 50 µM). Negative control cells received no treatment of iPA, and positive control cells were treated with cis-platinum (concentration: 16.7 µM). After 48 h incubation, rate of apoptosis was detected by flow cytometry with Annexin-V/PI double staining. Results are shown as the mean ± SD from three separate experiments.

*p < 0.05 and **p < 0.01 versus control.

[Figure omitted. See PDF]

iPA treatment attenuates ubiquitination of AKT and reduces the phosphorylation level of AKT and TAK1

To identify downstream mediators of TRAF6 in HeLa cell apoptosis, we turned to AKT, which is associated with many proteins that contribute to cell survival.²¹ We assessed AKT ubiquitination and AKT phosphorylation in the HeLa cell line following LPS treatment. LPS is a strong inducer of AKT activity that can increase levels of AKT ubiquitination and AKT phosphorylation. Using HeLa cells, we performed IP assays to measure AKT ubiquitination following iPA treatment. As shown in Figure 4(a), we found that iPA could diminish the effect of LPS on AKT ubiquitination in a time-dependent manner. Furthermore, the levels of AKT phosphorylation at Thr-308 and Ser-473 were significantly attenuated by iPA at all time points comparing to untreated controls (Figure 4(b)). Moreover, given that TAK1 was identified as another direct target of TRAF6, we also determined the effect of iPA on the phosphorylation level of TAK1 after being stimulated with LPS. We found that TAK1 phosphorylation was significantly lower in the iPA treatment group than the control group (Figure 4(c)).

Figure 4.

Effects of iPA on AKT and TAK1 activation in HeLa cells. (a) HeLa cells were pretreated with 10 µM of iPA for the indicated times and then treated with LPS (20 µg/mL) for 1 h prior. The level of AKT ubiquitination was studied by immunofluorescence assay and western blot assay. Total cellular protein lysates were subjected to western blot. (b and c) HeLa cells were pretreated with 10 µM of iPA for the indicated times and then treated with LPS (20 µg/mL) for 3 h prior to western blot analysis for p-AKT (Thr-308 and Ser-473) and p-TAK1. A representative plot of three independent experiments is presented here. Results are shown as the mean ± SD from three separate experiments.

*p < 0.05 and **p < 0.01 versus control.

[Figure omitted. See PDF]

iPA changes the expression of apoptotic and anti-apoptotic proteins

Although our data above showed that iPA treatment could inhibit activation of AKT and TAK1, the underlying mechanism for the iPA-induced apoptosis remained unclear. To preliminarily explore possible mechanisms, we assessed the expression levels of apoptotic and anti-apoptotic proteins. As seen in Figure 5, treatment of HeLa cells with iPA caused a significant increase in the expression of apoptotic protein Bax. While the level of anti-apoptotic protein Bcl-2 was remarkably reduced in 10 µM iPA-treated HeLa cells in a time-dependent manner. These results indicated that iPA exposure could induce HeLa apoptosis through modulations of these key apoptosis–regulating proteins.

Figure 5.

iPA induces the regulation of Bcl-2 family protein expression in HeLa cells. HeLa cells were incubated with 10 µM of iPA within different culture durations. Expression levels of Bax and Bcl-2 were detected using western blot analysis. A representative plot of three independent experiments is presented here. Results are shown as the mean ± SD from three separate experiments.

*p < 0.05 and **p < 0.01 versus control.

[Figure omitted. See PDF]

Discussion

Cervical cancer is the second most common cause of death among women. Studies have shown that HeLa cell lines express high levels of endogenous TRAF6,²² which plays a critical role in regulating many genes that are involved in cellular proliferation, apoptosis, and immune responses to invasions.^23–25 Recently, TRAF6 has emerged as a potential therapeutic target for treatment of human cancers²⁶ because its inhibition has led to reduced cancer cell proliferation and tumor formation.²⁷ Binding of Ubc13 at the RING domain of TRAF6 has been implicated to accelerate Lys-63-dependent ubiquitination and activate AKT and TAK1 pathways.^12,28 It is noteworthy that both AKT and TAK1 pathways are important in regulating cellular processes, and that TRAF6, acting as an ubiquitin E3 ligase, is considered to be at the branching point or linchpin of the two pathways. Based on these understandings, we believe that targeting the RING domain of TRAF6 could cumulate a new therapeutic approach for the treatment of cancer.

While anti-proliferative activity of iPA has been documented,¹⁸ very little is known about its exact role in cervical cancer cell. We pursued a computational docking study and found that iPA could complex with the RING domain of TRAF6 and disrupt the natural TRAF6–Ubc13 interaction (Figure 1(b)). Thus, iPA represents a potential candidate for modulating TRAF6-mediated activation of downstream target proteins, and to examine this connection, we elected to evaluate specifically anti-proliferation effects of iPA on HeLa cells. MTT assay revealed that iPA could indeed inhibit the proliferation of HeLa cells in a concentration-dependent as well as time-dependent manner (Figure 2). Subsequently, the effect of iPA on apoptosis of HeLa cells was determined using a flow cytometry. The fraction of early apoptosis cells was distinctly higher than untreated controls (Figure 3), thereby suggesting that the iPA-promoted reduction of cell proliferation was due to apoptosis.

AKT, a serine–threonine kinase, is associated with a variety of cellular processes required for cell growth and plays an important role in the regulation of cellular apoptosis and proliferation. Phosphorylations at Ser-473 of the C-terminal hydrophobic region and at Thr-308 of the catalytic domain are key modifications required for the AKT activation. These phosphorylations are accomplished by phosphoinositide-dependent kinase-2 (PDK-2) for Ser-473 and by PDK-1 for Thr-308.²⁹ Western blot analysis indicated that iPA significantly inhibited phosphorylations of AKT at Thr-308 and Ser-473 (Figure 4(b)). Although phosphorylations normally regulate the activation of protein kinases, Yang et al.⁷ demonstrated that TRAF6 promotes the Lys-63-dependent ubiquitination of oncogenic AKT, which apparently contributes to increased AKT membrane localization and is responsible for the enhanced AKT phosphorylation. Therefore, we performed IP assays and found that iPA actually suppressed the level of AKT ubiquitination with a time-dependent manner (Figure 4(a)), which is consistent with Yang et al.’s⁷ work. Given that downregulation of AKT activity generally results in cell death,⁸ and that a significant decrease in AKT activity is observed in our studies, iPA-induced HeLa cell apoptosis likely occurred through suppressing the AKT signaling pathway.

To further strengthen our model for the role of TRAF6 in iPA-induced apoptosis, we examined TAK1, another well-established downstream target of TRAF6. We found that the phosphorylation pattern of TAK1 was similar to AKT and it was significantly inhibited by iPA at all time points (Figure 4(c)). Studies have found that downregulation of TAK1 activity has a greater anti-tumorigenic response,^30,31 and that inhibition of transforming growth factor β (TGF-β) signaling leads to suppression of TAK1 and its downstream survival pathway. In addition, Ying et al.³² confirmed that inhibition of the TAK1 pathway through a novel TAK1 inhibitor LYTAK1 reduced the growth of ovarian cancer cells. Our studies demonstrated that downregulation of TAK1 activity by iPA produced an apoptotic effect on cervical cancer HeLa cells. Collectively, it could be suggested that anti-proliferation activities of iPA are due to inhibitions of both AKT and TAK1 signaling pathways. Based on our molecular docking study, iPA could bind to the RING domain of TRAF6 and competitively interfere with the natural interaction of Ubc13 with TRAF6 required for ubiquinating and activating AKT and TAK1 signaling pathways. Thus, anti-proliferation activities of iPA are likely associated with its ability to modulate TRAF6-mediated ubiquitination.

Moreover, having confirmed that iPA was able to induce apoptosis of HeLa cells by flow cytometry (Figure 3), we further explored expressions of proteins relevant to tumor apoptosis. Bax and Bcl-2, members of the Bcl-2 family, regulate programmed cell death required for maintaining an appropriate number of cells in the body. Furthermore, reports have indicated that AKT could directly or indirectly affect mitochondrion-dependent apoptosis through modulations of the Bcl-2 family proteins such as Bax and Bcl-2. Recent evidence also links the change in the Bcl-2 expression directly to phosphorylation of TAK1.³³ Accordingly, western blot was utilized to analyze the levels of Bcl-2 and Bax in HeLa cells. As expected, iPA treatment led to a marked increase in the protein expression of pro-apoptotic Bax and a clear reduction in the expression of anti-apoptotic Bcl-2 in a time-dependent manner (Figure 5). Therefore, these results further suggest that anti-cancer activities of iPA are through apoptosis and associated with AKT and TAK1 activities.

Like AKT, activated TAK1 also mediates activation of IKK/NF-κB, which may be due to upregulation of inflammatory cytokine expression and inhibition of apoptosis. TRAF6 can also ubiquitinate STAT3, which is also associated with apoptosis. Other reports indicate that TRAF6 inhibits cell proliferation by preventing cell cycle arrest. The scope of this study is to demonstrate that (a) whether iPA could inhibit the binding of TRAF6 to Ubc13; (b) whether such impediment would lead to loss of AKT and TAK1 activities, and ultimate inhibition of cancer cell growth; and (c) whether TRAF6 could serve to be a potential anti-tumor target. In the near future, we intend to use an iPA substrate that is substituted with a fluorescent probe to observe whether it could directly bind to TRAF6 in cells and enter the nucleus, and we also intend to investigate its anti-cancer effect in vivo.

Conclusion

We have demonstrated here that iPA possesses anti-cancer effect on cervical cancer cell line HeLa. We found that iPA-induced apoptosis is associated with loss of both AKT and TAK1 activities, and that the level of anti-apoptotic protein Bcl-2 was reduced, while that of the pro-apoptotic protein Bax was elevated. Given the computational evidence that iPA could bind at the RING domain of TRAF6 and interrupt the natural complexation of Ubc13 with TRAF6, and given the role of TRAF6 in ubiquitination and activation of AKT and TAK1 signaling pathways, the observed anti-proliferation effect of iPA on HeLa cells is most likely a result of TRAF6 modulation. These results further support the emerging notion that TRAF6 could serve as a viable target for developing new cancer therapeutics.

Future perspective

However, we suggest further investigation of this compound, as the detailed mechanism remains largely unknown. Our docking studies suggested that the compound would bind to the RING domain of TRAF6, thus blocking the interaction between TRAF6 and Ubc13. Our lab has confirmed that the anti-apoptotic effects of iPA on HeLa cells are mediated via the AKT and TAK1 pathways regulated by TRAF6. The TRAF6/TAK1/TAB1/TAB2 complex binds to the ubiquitin ligases Ubc13 and Uev1A. This leads to the ubiquitination of TRAF6, triggering TAK1 autophosphorylation at Thr-184 and Thr-187 as well as its subsequent activation. Further studies are needed to determine whether iPA-reduced activation of TAK1 was due to reduction of ubiquitination of TRAF6. Moreover, inflammatory conditions represent a well-known risk factor for cancer development. Numerous studies have shown that the activation of both TAK1 and AKT attribute to the upregulation of inflammatory cytokine expression such as IL-6 and IL-1.

Summary points

• iPA possesses an anti-cancer effect on cervical cancer cell line HeLa.

• iPA has anti-proliferative activity on cervical cancer cell line HeLa.

• iPA-reduced cell proliferation was due to the occurrence of apoptosis.

• iPA could target to the RING finger domain of TRAF6.

• iPA could disrupt the TRAF6–Ubc13 interaction.

• iPA could significantly suppress AKT ubiquitination and its phosphorylation at Thr-308 and Ser-473.

• iPA could also decrease the level of TAK1 phosphorylation, another well-established downstream target of TRAF6.

• iPA treatment led to a marked increase in the protein expression of pro-apoptotic Bax and a clear reduction in the expression of anti-apoptotic Bcl-2 in a time-dependent manner.

The authors are grateful to Professor Richard P Hsung of University of Wisconsin–Madison for invaluable discussions and preparation of this manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by the National Natural Science Foundation of China (81641132).

Informed consent

Informed consent was obtained from all individual participants included in the study.

Research involving human participants and/or animals

This article does not contain any studies with human participants or animals performed by any of the authors.