Tangeretin sensitises human lung cancer cells to TRAIL- induced apoptosis via ROS-JNK/ERK-CHOP pathway- mediated up-regulation of death receptor 5

Purpose: To investigate the efficacy of tangeretin, a bioactive flavonoid, as an enhancer of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human lung cancer cells (H1299 and H1975). Methods: H1299 and H1975 cells were exposed to tangeretin (25, 50, or 100 μM), and its effect on cell viability was assessed by MTT assay. The cytotoxic effects of tangeretin in combination with TRAIL (25 ng/mL) were determined by live/dead assay and annexin V staining. Intracellular levels of reactive oxygen species (ROS) were determined by DHFDA-flow cytometry analysis. Western blotting was performed to assess the expression of death receptors, apoptosis pathway proteins, JNK and ERK1/2. The cell surface expression of death receptors was determined. RT-PCR was performed to assess the expression of death receptor 5 and CEBP homologous protein (CHOP) mRNA. The cytotoxic effects of tangeretin in the presence of DR5, CHOP siRNA and TRAIL were also detected. Results: MTT assay revealed that tangeretin exerted potent cytotoxic effects and, in combination with TRAIL, exhibited striking apoptosis. Down-regulation of cell survival proteins (Bcl-2, Bcl-xL, survivin, xIAP, c-IAP-1, and c-IAP-2) and elevated expression of Bax and caspases 3, 8, and 9 were observed upon tangeretin exposure. The expression of death receptors DR4 and DR5 was highly up-regulated by tangeretin, while that of decoy receptors DcR1 and DcR2 was down-regulated. DR5 and CHOP siRNA transfection suggested the involvement of CHOP in tangeretin-mediated up-regulation of DR5, leading to more pronounced apoptotic effects. Tangeretin increased ROS levels and effectively caused activation of the mitogen-activated protein kinases JNK, ERK, and p38MAPKs. Expression analysis in the presence of specific MAPK inhibitors suggested the involvement of JNK and ERK in tangeretinmediated TRAIL sensitisation. Conclusions: Tangeretin induces death receptors and enhances TRAIL-induced apoptosis through upregulation of the ROS-JNK/ERK-CHOP pathways.


INTRODUCTION
Despite immense progression in the field of medicine, cancer remains a major threat to human health.Lung cancer is a prominent cause of cancer-related death worldwide [1].Non-small cell lung cancer (NSCLC), the primary type of lung cancer, accounts for approximately 85 % of all cases, and small cell lung cancer (SCLC) accounts for 15 % [2].Despite advancements in chemotherapy and radiation therapy, the long-term survival of patients with NSCLC remains low, with a 5-year survival rate of about 15 % [3].Induction of apoptosis is crucial in cancer therapy.Much research has recently focused on strategies that specifically target molecules directly involved in the apoptotic pathways, thereby enhancing cancer cell death.
The interaction of TRAIL with DR4 and DR5 leads to an association with Fas-associated death domain and caspase-8, forming a deathinducing signal complex that results in activation of an initiator caspase (caspase-8) and effector caspases (caspases-3, -6, -7, and -9), thereby inducing apoptosis [9,10].TRAIL-induced preferential apoptosis of cancer cells makes this agent crucial in cancer treatment.However, many cancer cells acquire resistance to TRAIL by multiple mechanisms [11].The mechanisms underlying TRAIL resistance include downregulation of DRs and up-regulation of decoy receptors [11,12] and various anti-apoptotic proteins, including cellular FLICE-like inhibitory protein (cFLIP(L)), which competes with caspase-8 [13] and inhibitor of apoptosis proteins (IAPs) [14].Thus, identification of TRAIL sensitizers that will overcome TRAIL resistance is a valuable approach in cancer therapy.

Cell lines
The human lung cancer cells H1299 and H1975 were obtained from ATCC and maintained according to the manufacturer's instructions.In brief, the cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum, 100 units/mL of penicillin, and 100 µg/mL of streptomycin.

Chemicals and reagents
Tangeretin (Sigma-Aldrich, St. Louis, Mo, USA) and human trail (Peprotech, Rocky Hill, NJ, USA) were used.A cell apoptosis elisa detection kit was obtained from Roche.

Live/dead assay
A live/dead assay was performed to further assess apoptosis.The experiment was carried out using a Live/Dead assay kit (Invitrogen).The assay employs calcein-AM, a non-fluorescent polyanionic dye that determines the membrane integrity as a measure of cell viability.In brief, the cells with tangeretin (25, 50 or 100 µM) and/or TRAIL (25 ng/mL) were stained with Live/Dead reagent (5 μmol/L ethidiumhomodimer and 5 μmol/L calcein-AM).The cells were then incubated at 37 °C for 30 min.Apoptosis was assessed by measuring the fluorescence under a fluorescence microscope (Labophot-2; Nikon, Tokyo, Japan).Live cells retained the calcein-AM dye and produced intense green fluorescence, while the ethidiumhomodimerbecame bound to nucleic acids inside the dead cells and produced bright red fluorescence.

Analysis of apoptosis by Annexin V assay
Human lung carcinoma cells were incubated with tangeretin (50 or 100 µM) and/or TRAIL (25 ng/mL) for 24 h, and apoptosis was detected using an Annexin V-FITC detection kit II (BD Biosciences Pharmingen, San Diego, CA, USA).After incubation, the cells were treated with 5 μL annexin V-FITC reagent, incubated for 30 min at room temperature, and analysed using a flow cytometer (FACS Calibur; BD Biosciences).

Analysis of DR4 and DR5 expression
The influence of tangeretin (25,50, and 100 µM) on the cell-surface expression of death receptors (DR4 and DR5) in the lung cancer cells was assessed using mouse anti-human DR4 or DR5 monoclonal antibodies conjugated with phycoerythrin (R&D Systems,Minneapolis, MN, USA).Cells treated with tangeretin for 24 h were incubated with antibodies for 45 min at 4 °C.The expression was analysed by flow cytometry [20].

Transfection with siRNA
The influence of tangeretin on DR5 and CHOP expression following silencing with respective siRNA (Santa Cruz Biotechnology) was assessed.The H1299 and H1975 cells were transfected with siRNA oligonucleotides (30 nmol/L) using lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
Following transfection, the cells were treated with tangeretin (100 µM) for 12 h and incubated with TRAIL (25 ng/mL) for 24 h [22].After these treatments, the cells were collected and analysed for expression using western blotting, and apoptosis was assessed by the Live/Dead assay.

Measurement of reactive oxygen species
To detect intracellular reactive oxygen species (ROS) in the lung cancer cells, the cells were treated with 20 µM DCF-DA for 15 min at 37°C.Following DCF-DA treatment, the cells were incubated with various concentrations of tangeretin (25,50, or 100 µM) for 30 min.The increase in fluorescence resulting from the oxidation of DCF-DA to DCF was analysed by flow cytometry at 530 nm as previously described [23].Data were analysed for at least 10,000 cells at a flow rate of 250 to 300 cells/s.

Western blotting
The cells treated with tangeretin (25, 50, or 100 µM) and/or TRAIL (25 ng/mL) for 24 h were subjected to western blot analysis to assess the expression of proteins.Western blot analysis was carried out as previously described by Yang et al [24].Following treatment with tangeretin and TRAIL, the cells were incubated in 0.5 mL of icecold whole-cell lysate buffer (5 M NaCl, 10 % Nonidet P-40, 0.2 M sodium orthovanadate, 0.1 M EGTA, 0.5 M EDTA, 0.1 M phenylmethylsulfonyl fluoride, 1 M sodium fluoride, 1 M HEPES 2 µg/mL aprotinin, and 2 µg/mL leupeptin) on ice for 30 min.The isolated protein concentrations were determined using a Bio-Rad assay kit (Bio-Rad, Hercules, CA, USA).Equal amounts (60 µg) of isolated proteins were fractionated using SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), following incubation with respective antibodies.The immunoreactive bands were detected and analysed using an ECL Advance western blot analysis system (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA).The band densities were normalised to those of control β-actin using anti-β-actin antibody (Cell Signaling Technology).

Statistical analysis
SPSS software (ver.22.0; IBM Corp., Armonk, NY, USA) was used for analysis.The experimental data are given as means ± SD (n = 3 or 6).The means of the various groups were compared by one-way ANOVA followed by Duncan's multiple range test (DMRT) as a posthoc analysis.Differences with a p value of < 0.05 were considered statistically significant.

Tangeretin enhances TRAIL-induced apoptosis of lung cancer cells
We also examined whether tangeretin was able to enhance and sensitise lung cancer cells to TRAIL-induced apoptosis by the Live/Dead assay and Annexin V staining.The Live/Dead assay was performed after exposure to 25, 50 or 100 µM tangeretin.Exposure to the 100-µM concentration resulted in a drastic increase in apoptosis (69.09 % of H1299 cells and 58.93 % of H1975 cells) (Fig. 2).Tangeretin at the 100-µM concentration induced higher apoptotic cell counts than did lower concentrations.Interestingly, however, combined exposure to TRAIL and tangeretin at 100 µM resulted in a multi-fold increase in cytotoxicity.The apoptosis percentage increased to 81.19 % in H1299 cells and 84.90 % in H1975 cells (Figure 2), while TRAIL alone induced apoptosis of about 37.18 and 40.24 % in H1299 and H1975 cells, respectively.
We further analysed the efficacy of tangeretin in enhancing TRAIL-induced apoptosis by Annexin V staining assay.The assay examines the integrity of the cell membrane as a measure of cell viability.The translocation of phosphatidylserine from the cytoplasmic interface to the extracellular surface of the membrane is a preliminary indicator of apoptosis.The results presented in Figure 3 show that tangeretin was able to markedly (p < 0.05) increase the apoptosis percentage with combined exposure to TRAIL and tangeretin at 50 and 100µM compared with exposure to either TRAIL or tangeretin.These observations suggest that tangeretin at the tested doses significantly enhanced sensitivity to TRAIL and improved TRAIL-induced apoptosis.

Tangeretin potentially up-regulates the expression of DR4 and DR5
Studies have shown that down-regulation or loss of DR4 and DR5 expression is a major mechanism involved in TRAIL resistance [10,11].We observed significantly (p < 0.05) enhanced cell surface expression of DR4 and DR5 upon treatment with tangeretin in a dose-dependent manner (Fig. 4a).Furthermore, expression at the protein level was detected by western blot analysis.The results revealed similarly increased expression of DR4 and DR5 upon exposure to tangeretin (Fig. 4b), suggesting the efficacy of tangeretin in up-regulating the expression of DR4 and DR5, thereby increasing apoptosis.Additionally, 100 µM of tangeretin increased the expression when compared with lower doses.

Influence of tangeretin on the expression of decoy receptors
The enhanced expression of antagonistic decoy receptors of TRAIL (DcR1 and DcR2) is reportedly involved in TRAIL resistance [12].We observed that tangeretin, at all tested doses, was able to potentially supress the expression of DcR1 and DcR2.
However, the effects were more pronounced on DcR1 than DcR2, and the 100-µM dose exhibited maximal effects (Fig. 5).Thus, by downregulating DcR1 and DcR2 and inducing DR4/5 expression, tangeretin effectively aids in activating apoptosis of lung cancer cells.

CHOP mediates tangeretin-induced upregulation of death receptors
It has been demonstrated that the induction of DRs by various stimulants is mediated through the activation of CHOP [25,26].We investigated whether tangeretin influenced CHOP expression and whether CHOP is also involved in DR5 expression and tangeretin-induced apoptosis.RT-PCR analysis revealed enhanced DR5 and CHOP mRNA levels (Fig. 6a and b).In line with the CHOP mRNA levels, western blot analysis also revealed significantly (p < 0.05) enhanced CHOP protein expression.These observations indicate a positive influence of tangeretin on CHOP expression.siRNA was used to further confirm whether tangeretin was able to mediate DR5 expression via CHOP and to determine whether CHOP-DR5 is involved in tangeretin-mediated apoptosis.Interestingly, DR5 expression decreased upon transfection with CHOP siRNA (Fig. 7), and this decreased expression markedly affected the apoptosis percentage (Figs.8 and 9).Significantly reduced apoptosis of lung cancer cells was observed on transfection with both CHOP siRNA and DR5 siRNA.However, tangeretin treatment considerably enhanced DR5 expression even in the presence of CHOP siRNA and enhanced the apoptosis percentage to some extent, while the percentage was lower than that in the absence of siRNA.These observations indicate that tangeretin-induced apoptosis occurs in part through direct stimulation of apoptosis and is in part mediated by CHOP.Additionally, CHOP is critical in DR5-mediated TRAIL-induced apoptosis.

Tangeretin modulates expression of various cell survival proteins
Numerous studies have demonstrated that overexpression of cell survival proteins, such as IAPs, survivin, cFLIP, Bcl-2, and Bcl-xL, is involved in the development of TRAIL resistance [27,28].We observed significantly (p < 0.05) increased expression of cell survival proteins in H1299 and H1975 cells (Figure 10).However, tangeretin treatment at 25, 50, or 100 µM resulted in a decline in the expression levels in a dose-dependent manner.

Tangeretin up-regulates expression of proapoptotic proteins
We observed a remarkable increase (p < 0.05) in the expression of caspase-3, -8 and -9 in H1299 and H1975 cells on exposure to tangeretin (Fig. 11); pro-apoptotic protein Bax was also upregulated.The expression was dose-dependently regulated by tangeretin.

Tangeretin-induced up-regulation of TRAIL receptors is mediated by MAPKs
We investigated whether activation of MAPKs (JNK, ERK, and p38MAPK) is involved in tangeretin-induced DR5 induction.We observed a significant (p < 0.05) increase in the activation of ERK and JNK, as evidenced by raised phosphorylation levels.The 50-and 100-µM doses were more effective in activating JNK and ERK than was the 25-µM dose.Further, ERK levels were higher as against JNK.While the levels of p38 were noticeably increased by tangeretin, there was no significant increase, suggesting that the activation of JNK and ERK is involved more critically in tangeretin-mediated apoptosis than p38.
We also observed a drastic reduction in the expression of DR5 and DR4 in the presence of inhibitors of JNK (SP600125) and ERK1/2 (PD98059).However, the expression was not significantly affected by the presence of an inhibitor of p38 MAPK (SB202190).Tangeretin induced the expression of TRAIL receptors, even in the presence of inhibitors, in a dosedependent manner.These observations illustrate that the activation of JNK and ERK is more closely involved in the up-regulated expression of DR4 and DR5.

Involvement of ROS in tangeretin-mediated TRAIL-induced apoptosis
ROS are reportedly involved in the induction of DRs [20].We observed enhanced ROS levels upon exposure to tangeretin (Fig. 13).The increase, however, was dose-dependent.These observations suggest the possible involvement of ROS in up-regulation of DRs and ERK/JNK MAPKs.

DISCUSSION
Despite the promising effects of TRAIL in cancer cell therapy, many reports have described human tumours developing resistance to TRAIL [10,11] through various mechanisms.Altered expression of DRs and the Fas-associated death domain,as well as overexpression of antiapoptotic proteins, such as Bcl-2, survivin, cFLIP [11,13,27,28], and xIAP, have been observed in many tumour cell lines exhibiting TRAIL resistance [28].
Thus, strategies to overcome this dysregulation and sensitise cells to TRAIL and possibly enhance its apoptotic effects are crucial and of tremendous value.Compounds of plant origin are known to effectively sensitise cells to TRAILinduced cancer cell cytotoxicity [15,20].Here, we investigated whether tangeretin was able to sensitise and enhance TRAIL-induced apoptosis in the human lung cancer cells H1299 and H1975.
Treatment with tangeretin at 25 to 100 µM reduced the viability of H1299 and H1975 cells, indicating its potent anti-proliferative efficacy.
The combined exposure to TRAIL and tangeretin dramatically increased the apoptotic cell counts compared with treatment with tangeretin or TRAIL separately.This suggests that tangeretin was able to effectively enhance the effects of TRAIL; in addition, tangeretin down-regulated the cell survival proteins Bcl-2, Bcl-xL, xIAP, cFLIP, cIAP-1, cIAP-2, and survivin.These proteins have been reported to be associated with TRAIL resistance [27,28].Thus, decreased expression of anti-apoptotic proteins aids in TRAIL-induced apoptosis.Previous studies have demonstrated that down-regulation of survivin, Bcl-xL, and Bcl-2 promotes sensitivity to TRAIL [27,28].
xIAP contributes to TRAILresistance by inhibiting caspase-3, -7, and -9 [28], and cFLIP inhibits caspase activation by competing for the Fasassociated death domain [13].Thus, effective suppression of cFLIP and IAPs by tangeretin could potentially contribute to sensitising cancer cells to TRAIL.In the present study, elevated levels of caspase-3, -8 and -9 were observed upon tangeretin exposure.This up-regulated expression could be due to the inhibition of xIAP and cFLIP.Increased expression of the proapoptotic protein Bax also aids in enhancing TRAIL-mediated apoptosis.The modulations observed in the expression patterns were dosedependent, with 100-µM of tangeretin exhibiting maximal effects; further combination with TRAIL showed an additional influence.Phytochemicals, such asguggulsterone and nimbolide [15,25], enhanced TRAIL-mediated cell death via upregulation of caspases and suppression of cell survival proteins.
Binding of TRAIL with the death receptors DR4 and DR5 triggers TRAIL-induced apoptotic signalling, leading to the activation of executor caspase.Dysregulated expression levels have been reported in several TRAIL-resistant cancers [29], and subsequent up-regulation of the receptors promotes apoptosis [11,25].Significantly up-regulated cell surface expression of DR4 and DR5 was observed upon tangeretin treatment.While tangeretin exposure caused elevated expression of DR4 and DR5, the expression of TRAIL decoy receptors DcR1 and DcR2 was dose-dependently reduced at the protein level.Mellier et al [30] showed that sensitivity to TRAIL-mediated apoptosis is influenced by the over-expression of TRAILdecoy receptors.The down-regulated expression of DcR1 and DcR2, along with elevated DR4 and DR5 expression upon exposure to tangeretin, effectively contributes to enhance TRAILmediated apoptosis.Furthermore, DR5 expression was up-regulated by tangeretin even at the gene level, as evidenced by increased DR5mRNA levels.The mRNA levels of CHOP were also enhanced upon tangeretin exposure.CHOP is a key transcriptional factor that regulates DR5.CHOP binds to the DR5 promoter region and upregulates DR5 expression [17].The enhanced CHOP levels on tangeretin exposure suggest that tangeretin modulates DR5 expression via CHOP.This was further confirmed when CHOP expression was silenced using CHOP siRNA, which resulted in significant inhibition of DR5 expression; however, tangeretin was able to considerably enhance DR5 expression even with CHOP siRNA.Silencing of DR5 and CHOP genes resulted in a sharp decline in apoptosis levels, reflecting the vital roles of DR5 and CHOP in TRAIL-mediated cell death.To an extent, tangeretin exposure was able to increase the apoptosis levels even in the presence of DR5 siRNA and CHOP siRNA.These observations suggest that tangeretin was able to potentiate the apoptosis-mediating effects of TRAIL by modulating DR5 and CHOP.
We also observed that tangeretin was able to induce ROS production in H1299 and H1975 cells.The observed ROS levels could have also induced CHOP expression, because CHOP is a major stress-regulated protein [25].Several studies have reported that chemotherapeutic agents enhance DR5 expression and TRAILmediated apoptosis via ROS-dependent pathways [15,25,26].

ROS
reportedly trigger various signal transduction pathways that are involved in cell growth, differentiation, or death.MAPKs are important downstream mediators of ROSinduced signalling [26].ERK, JNK, and p38 MAPK have been found to be activated in TRAILmediated apoptosis [15,30] and are involved in TRAIL receptor induction [15,26].In our study, the significant increases in the activation of JNK and ERK, along with a considerable increase in p38 MAPK activation on exposure to tangeretin, could have been either due to direct stimulation of tangeretin and/or in part indirectly due to ROS.We also observed suppression of DR4 and DR5 in the presence MAPK inhibitors, which was noticeably enhanced by tangeretin.

CONCLUSION
Tangeretin up-regulates death receptors and enhances TRAIL-mediated apoptosis via ROS-CHOP signalling and MAPKs.Thus, tangeretin should be further explored as a potent candidate for combined therapy with TRAIL for induction of apoptosis of cancer cells.

Figure 1 :
Figure 1: Cytotoxicity of various concentrations of tangeretin.Results are expressed as means ± SD with n = 6.p < 0.05 versus control, denoted by a ; b-e denotes mean values within the same group that differ from each other at p < 0.05 on one-way ANOVA and Duncan's multiple range test (DMRT) analysis

Figure 2 :
Figure 2: Tangeretin enhances apoptosis of H1299 and H1975 cells.Tangeretin in combination with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) enhanced apoptosis in a dose-dependent manner.Results are expressed as means ± SD with n = 6.p < 0.05 versus control, denoted by a ;b-e denotes mean values within the same group that differ from each other at p < 0.05 on one-way ANOVA and DMRT analysis

Figure 3 :Figure 4 :
Figure 3: Tangeretin enhanced TRAIL-induced apoptosis.Results are expressed as means ± SD with n = 6.p< 0.05 versus control, denoted by a ;b-f denotes mean values within the same group that differ from each other at p < 0.05 on ANOVA and DMRT analysis

Figure 5 :
Figure 5: Tangeretin modulated decoy receptor expressions.Tangeretin effectively down-regulated the expressions of DcR1 and DcR2 dose-dependently.Results are expressed as means ± SD with n = 3.p < 0.05 versus control, denoted by a ; b-e denotes mean values within the same group that differ from each other at p < 0.05 on one-way ANOVA and DMRT analysis

Figure 6 :
Figure 6: Influence of tangeretin on DR5 and CHOP expression.Tangeretin significantly up-regulated the mRNA levels of both CHOP and DR5 (a and b).Results are expressed as means ± SD with n = 6.p < 0.05 versus control, denoted by a ; b-e denotes mean values within the same group that differ from each other at p < 0.05 on one-way ANOVA and DMRT analysis (L1, Control; L2, 25 µM tangeretin; L3, 50 µM tangeretin; L4, 100 µM tangeretin)

Figure 7 :
Figure 7: Tangeretin enhanced DR5 expression after transfection with CHOP siRNA.Results are expressed as means ± SD with n = 6.p < 0.05 versuscontrol, denoted by a ;b-g denotes mean values within the same group that differ from each other at p < 0.05 on one-way ANOVA and DMRT analysis

Figure 8 :
Figure 8: Tangeretin exposure increased the apoptosis percentage of human lung cancer cells following transfection with CHOP siRNA.Results are expressed as means ± SD with n = 6.p < 0.05 versus control, denoted by a ; b-g denotes mean values within the same group that differ from each other at p < 0.05 on one-way ANOVA and DMRT analysis

Figure 9 :
Figure 9: Tangeretin exposure significantly increased the apoptosis percentage following transfection with DR5 siRNA.Results are expressed as means ± SD with n = 6.p < 0.05 versus control, denoted by a ; b-g denotes mean values within the same group that differ from each other at p < 0.05 on one-way ANOVA and DMRT analysis