Cisplatin Inhibits AhR Activation

The AhR binds to contain ligands, such as 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin, 3-methylcholantrene, or β-naphthoflavone. The activation mechanism of AhR is not yet fully understood, but it is known that AhR associates with the molecular chaperone HSP90 in the cytoplasm. There are a few reports about the association or dissociation of AhR and HSP90, and which domain of HSP90 binds to AhR. We reported the association and activation mechanisms between HSP90 and AhR-PAS or AhR-bHLH. In the current study, we found that cisplatin inhibits the AhR activation. Although ATP and 17-DMAG have no effect on the dissociation of HSP90 from AhR, some contents of HSP90 were dissociated from AhR in the presence of cisplatin. We could detect the increase of CYP1A in the presence of 3-MC. On the contrary, the induction of CYP1A1 was inhibited in the presence of cisplatin. We couldn’t detect AhR in the HeLa cell soluble fraction in the presence of 50 μM cisplatin. In the presence of MG-132, we could detect AhR. These results suggested that AhR was dissociated from the HSP90 chaperone complex and processed during the protein proteasome degradation system in the presence of cisplatin.


Introduction
The molecular chaperone HSP90 is an essential protein in eukaryotic cells.It interacts with more than 300 proteins, and regulates the physiological function in the cell [1] [2].HSP90 interacts with a cancer, and the inhibitor of HSP90 is ex- pected as an anticancer agent.17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) is one of the HSP90 inhibitors, and it inhibits the ATPase activity of HSP90.We reported that cisplatin, one of the anticancer agents, mainly binds to the C-terminal and also the N-terminal domain of HSP90 and inhibits the chaperon activity [3] [4].
After binding of a ligand, AhR is translocated into the nucleus and forms a complex with Arnt (AhR nuclear translocator).The AhR/Arnt heterodimer binds to the xenobiotic responsible element (XRE) and promotes the transcription of cytochrome P450 1A1 (CYP1A1), one of the xenobiotic metabolizing enzymes.Ligands of AhR cause toxicological effects, such as suppression of the immune response, impairment of reproduction, and promotion of carcinogenesis [11] [12].
AhR is composed of the basic helix-loop-helix (bHLH) domain and Per-Arnt-Sim homology (PAS) domain.The bHLH domain is a motif participating in DNA binding, HSP90 binding, and dimerization.The PAS domain is a ligand binding domain.It is made from PAS-A and PAS-B, and HSP90 binds to the PAS-B.The glutamine (Q)-rich region is near the carboxyl-terminal of AhR [10].Purification of the full-length AhR is difficult, and there are few studies that analyzed the direct interaction of HSP90 and AhR.Thus, we purified the AhR-PAS and AhR-bHLH domain, and analyzed the direct interaction of HSP90 and the AhR-PAS or AhR-bHLH domain in vitro.
Cisplatin (cis-Diamineplatium (II) dichloride) is a widely used antineoplastic drug for clinical cures.Cisplatin possesses the ability to bind DNA and inhibits the replication of DNA, resulted in the inhibition of tumor cell growth.Previously, we found that cisplatin directly binds HSP90 and inhibits its aggregation prevention activity in vitro [3].Cisplatin inhibits glucocorticoid receptor-dependent and androgen receptor-dependent transcriptional activities in a dose-dependent manner in human cultured cells [13].Inhibition of the HSP90 functions by cisplatin is considered to occur in vivo as well as in vitro.There is no report against the influence of cisplatin to AhR-HSP90 complex.We investigated the influence of cisplatin on the association or dissociation of AhR from HSP90-cochaperone complex.

Cell Culture
The human cervical cancer cell line, HeLa cells were cultured in Dullbecco's modified Eagle's medium (DMEM; SIGMA) with 10% fetal bovine serum (Equitech-Bio), 20 μg/ml streptomycin, and 20 unit/ml penicillin (GIBCO) at 37˚C and 5% CO 2 .For MG132 treatment, 20,000 cells were plated in 96 well E-plate and their growth was monitored for 24 h.Cells were treated with 50 μM cisplatin for 16 hr and 50 μM of MG132 was added for 1 h and fresh media was added following washing once with media.

Cell Viability Assay
Cells were cultured in 96-well plates at a density of 2500 per well in 100 μl of medium 24 hrs before addition of cisplatin.Cisplatin was added in concentrations of 0 -100 μM.To assess cell viability, 10 μl of MTT solution (Thiazolyl Blue Tetrazolium Bromide (SIGMA) 5 mg/ml in PBS (phosphate buffered saline (-)) was added after a 16 hrs exposure to cisplatin.After a 4 h incubation, medium was removed and precipitates were lysed with 0.04 N HCl in 2-propanol.Then, absorbance was measured using a plate reader (Infinite F200, Tecan) at 595 nm.

Cell Treatment
HeLa cells were trypsinized and harvested in 35-mm dishes at a density of 1.5 × 10 5 cells, allowed to adhere for 24 hrs.Then, cells were treated by addition of 2 ml fresh DMEM containing samples at desired concentrations in DMSO.The cells were incubated in a certain time at 37˚C and 5% CO 2 .

Immunoblotting
Cells were rinsed three times with cold PBS, and collected using cell scraper (iwaki).The cell lysates were centrifuged at 2000 rpm at 4˚C for 5 min.The supernatants were removed and then lysed in lysis buffer (50 mM HEPES-NaOH, 5% Glycerol, 1% NP-40, 5 mM EDTA, 100 mM NaCl, 1 mM PMSF).After 10min incubation on ice, the lysates were centrifuged at 15,000 rpm at 4˚C for 15 min and then the supernatants were used.Total protein concentrations of supernatants were determined by using BCA Protein Assay Kit (Thermo Fisher Scientific).Cellular proteins were separated by 7% (for AhR and HSP90) and 13% (for XAP2 and p23) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDE-PAGE), transferred onto PVDF membranes (BIO-RAD).Mem-American Journal of Molecular Biology branes were blocked by 3% skim milk in TBS for 1 h at room temperature and proved with specific primary antibodies overnight at room temperature.AhR diluted 1:1000, β-actin diluted 1:3000, HSP90 diluted 1:1000, XAP2 diluted 1:500, p23 diluted 1:3000.Washing was twice for 5 min and membranes were proved with the secondary antibody for 1 h at room temperature.Anti-Rabbit IgG (whole molecule)-Alkaline Phosphatase against AhR, HSP90, XAP2, and p23 was diluted 1:2000.Anti-Mouse IgG (whole molecule)-Alkaline Phosphatase against β-actin was diluted 1:3000.Washing was twice for 5 min and membranes were detected by labeling with alkaline phosphatase.The protein contents were quantified using Image J software (Drop of Wisdom).

Recombinant Protein Contract, Expression and Purification
The total RNA was isolated from the HeLa cells using the RNeasy Mini Kit A full length cDNA of HSP90α was kindly provided by Dr. Yokoyama (RIKEN, Japan).To generate the HSP90 recombinant protein expression, the HSP90 cDNA was amplified by PCR (iCycler, BioRad) using the forward primers 5'-GGATCCATGCCTGAGGAAACCCAGACC-3' and reverse primer

GST Pull-Down Assay
For the GST pull-down assay, the concentration of ATP, 17-DMAG, and cisplatin were according to previous reports All the GST pull-down samples were separated by SDS-PAGE (9% or 11% gel) and detected by Coomassie Brilliant Blue R-250 staining.

Influence of Cisplatin on HeLa Cell Viability
We investigated the effect of cisplatin on the HeLa cell viability.In the present study, we did the evaluation using human epitheloid cervix carcinoma HeLa cells.The cell viability was reduced to about 70% in a cisplatin dependent manner (0 -250 μM) (Figure 1).We focused on 50 μM cisplatin in which the cell viability was about 80%.

Effects of Cisplatin on AhR and Chaperone Complex
We analyzed the effects of cisplatin (0 -50 μM) on the contents of AhR, HSP90, XAP2, and p23 in the cells.No changes were observed for the proteins contents of HSP90, XAP2, and p23 on immunoblotting (Figure 2

Effects of Cisplatin on AhR Activation
We next investigated CYP1A1 expression level.Cyp1A1 mRNA was analyzed by RT-PCR in the absence (Figure 3

Influence of ATP, Cisplatin, and 17-DMAG on AhR-HSP90 Complex
We investigated the GST pull-down assay using GST-bHLH and HSP90.GST pull-down assay was performed in the presence or absence of 50 μM cisplatin, 50

Discussion
It has been reported that AhR is composed of three domains (b-HLH, PAS, and TAD; transactivation domains) [14] [15] [16].AhR forms a complex with the molecular chaperone HSP90, co-chaperone p23, and the hepatitis B virus X-associated protein XAP2 in the cytoplasm [14] [15] [16].The molecular chaperone HSP90 regulates the physiological functions of more than 300 proteins including the steroid hormone receptors in the cells [1] [2].We reported the activation mechanisms of AHR by HSP90 [9].We also reported that AhR binds to the HSP90 N-domain [10].We speculated that the bHLH binding site of the HSP90 N-domain may be the neighbor of the M-domain.On the contrary, the PAS binding site of the HSP90 N-domain may be the end of the N-domain.
In the present study, we investigated the influence of cisplatin on the AhR activation.We reported that cisplatin binds to HSP90 and inhibits HSP90 chaperone activity [3] [4].Cisplatin binds to the HSP90 C-domain following the N-domain.The AhR contents in the HeLa cells were strongly reduced in the presence of 50 μM cisplatin at 16 hrs after addition of the drug.However, we could detect AhR in the presence of MG132 during immunoblotting.AhR plays the role as a transcription factor in the nuclei and induces CYP1A1 [17].In the presence of 50 μM cisplatin, the CYP1A1 mRNA level was strongly reduced.
These results suggested that AhR was dissociated from the HSP90 chaperone complex when in the presence of cisplatin.AhR is a very unstable protein, thus the released unstable AhR will be degraded through the 26S proteasome.
It is difficult to purify the full length AhR because of its unstability.The Q-rich transactivation domain of AhR is toxic to E. coli.We investigated the influence of cisplatin and 17-DMAG (both compounds are HSP90 inhibitors) on the bHLH-HSP90 complex using a pull-down assay.No changes were shown in 17-DMAG in the presence or absence of ATP.On the other hand, the bHLH bound HSP90 was reduced by cisplatin.We reported that AhR-bHLH is also the HSP90 binding domain, the same as the AhR-Pas domain [10].We investigated the influence of cisplatin on the ligand-bound AhR-HSP90 using AhR-ΔAD.
In our present study, 17-DMAG didn't affect the AhR-HSP90 complex in the presence or absence of ATP.However, cisplatin affects the AhR-HSP90 complex.
Based on these results, AhR may bind to the HSP90 N-domain, the cisplatin binding site, but not the ATP-binding site.Although 17-DMAG has no effect on the AhR activation, cisplatin inhibits the AhR activation.This is a first report of

(
Qiagen, Valencia, CA).The amount and purity of the total RNA was estimated by spectrophotometric analysis at A260 and A280.The RNA quality was determined by agarose gel electrophoresis following ethidium bromide staining.Aliquots of the total RNA were diluted in diethylpyrocarbonated (DEPC)-treated water and stored at −80˚C.RNA (4 μg) was used to synthesize the first strand complementary DNA (cDNA) with Super Script III First-Strand (Invitrogen) under the following general conditions: denaturation at 94˚C for 15 seconds, annealing at 55˚C for 30 seconds, and extension at 68˚C for 30 seconds for up to 40 cycles using an iCycler (BioRad).The cDNAs were PCR-amplified by iCycler (BioRad) with the primers of AhR[9] [10].The AhR-bHLH domain was amplified by PCR (iCycler, BioRad) using the forward primer 5'-GTCGACATGGCT GAAGGAATCAAGTCAA-3' and reverse primer 5'-GCGGCCGCTCAATCA AAGAAGCTCTTGGCTCT-3'.The resulting PCR products were inserted into the SalI/NotI sites of the pGEX-5X-3 vector (Takara Bio, Japan).The constructs were confirmed by DNA sequencing (PRISM 3100, ABI).The AhR-ΔAD domain was amplified by PCR (iCycler, BioRad) using the forward primer 5'-GTC GACATGAACAGCAGCAGCCGCCAAC-3' and reverse primer 5'-CTCGAG CTATTTTCGTAAATGCTCTGTTCC-3'.The resulting PCR products were in-American Journal of Molecular Biology serted into the SalI/XhoI sites of the pGEX-5X-3 vector (Takara Bio, Japan).The constructs were confirmed by DNA sequencing (PRISM 3100, ABI).

5 '
-TCTAGATTAGTCTAGTTCTTCCATGCG-3'.The resulting PCR products were inserted into the BamHI/XbaI sites of the pColdI vector (Takara Bio, Japan).The generated pColdI-HSP90 constructs were confirmed by DNA sequencing (PRISM 3100, ABI).HSP90 and the GST-tagged AhR bHLH domain was expressed in previously reported[9] [10].The GST-tagged AhR-ΔAD domain was expressed in an Escherichia coli BL21 (DE3) Arctic Competent Cell (Stratagene).The cells were grown at 30˚C in LB BROTH medium (Invitrogen) supplemented with 100 μg/ml ampicillin for 3 hr, then cultured at 10˚C for 30 min.The cells were next induced by the addition of 0.5 mM IPTG and the cultures were incubated at 10˚C for an additional 24 hr.The cells were harvested by centrifugation at 20,000 xg for 15 min at 4˚C, and the cell pellets were suspended in 10 mM Tris-HCl, pH 7.4.The cells were sonicated, centrifuged at 20,000 xg for 10 min at 4˚C and the formed pellets collected.The collected pellets were suspended in buffer (1 M Arginine, 10 mM Tris-HCl, pH 7.4), then dialyzed with 10 mM Tris-HCl, pH7.4,overnight to remove the Arginine.After dialysis, the lysates were cleared by centrifugation at 20,000 xg for 10 min at 4˚C.The supernatant was applied to glutathione columns (Glutathione Sepharose 4B, GE Healthcare Life Science), washed with 10 mM Tris-HCl, pH7.4,and then eluted with elution buffer (20 mM Glutathione, 10 mM Tris-HCl, pH7.4).The eluted proteins were concentrated by ultrafiltration.
[9] [10].2.5 μM GST-bHLH or GST-AhR-ΔAD or GST protein was added to a solution of 2.5 μM HSP90 in the presence or absence of 3 μM 3MC, 50 μM cisplatin, 50 μM 17-DMAG, 1 mM ATP and 150 μl buffer A (0.1 M KCl, 10 mM MgCl 2 , 20 mM Na 2 MoO 4 , 0.6 M NaCl, 5% Glycerol, 0.1% NP-40 in 25 mM HEPES-KOH pH 7.4).The total volumes of the sample was 300 μl by adding buffer B (5% Glycerol, 0.1% NP-40 in 25 mM HEPES-KOH pH 7.4) and incubated using a rotator with gentle rotation for 15 min at 37˚C.The samples were loaded onto a GST resin equilibrated with buffer C (50 mM KCl, 5 mM MgCl 2 , 10 mM Na 2 MoO 4 , 0.3 M NaCl, 5% Glycerol, 0.1% NP-40 in 25 mM HEPES-KOH pH 7.4) and incubated for 15 min at 4˚C with gentle rotation followed by spinning at 5000 rpm for 10 sec at 4˚C to remove the supernatant.The beads were washed three times with buffer C and American Journal of Molecular Biology bound proteins were eluted by boiling at 100˚C for 5 min in SDS sample buffer.
(a)).On the contrary, the contents of AhR were reduced at 50 μM cisplatin.At 50 μM cisplatin, about 80% of the AhR contents was reduced (Figure 2(b)).We investigated the effects of MG132 (proteasome inhibitor) on the AhR contents in the presence of cisplatin.In the presence of 50 μM cisplatin, we could detect faintly AhR protein band in the absence of MG132.On the contrary, the AhR protein bands were clearly shown in the presence of 50 μM cisplatin and MG132 (Figure 2(c)).

Figure 1 .
Figure 1.Effects of cisplatin on HeLa cell viability.HeLa cells were cultured in the presence or absence of cisplatin (5 -250 μM) for 16 hrs.The cell viability was measured using the MTT assay.The cell viability was about 80% at 50 μM cisplatin.In the present study, we used 50 μM of cisplatin.

Figure 2 .
Figure 2. Effects of cisplatin on AhR.(a) HeLa cells were cultured in the presence or absence of cisplatin (10, 25, and 50 μM) for 16 hrs.The HeLa cell lysates were analyzed by SDS-PAGE following immunoblotting using antibodies against AhR, HSP90, XAP2, p23, and β-actin.The AhR protein level based on (a) was quantified using Image J software (b).(c) HeLa cells were cultured in the presence or absence of 50 mM cisplatin for 16 hrs and 50 μM MG132 added for more than 1 hr.The HeLa cells lysate were analyzed by SDS-PAGE following immunoblotting using antibodies against AhR and β-actin.
(a)) or presence of 3MC (Figure 3(b)) from 0 to 8 hrs.CYP1AI was increased from 2 hrs and the maximum occurred at 4 hr (Figure 3(b)).We next analyzed the influence of cisplatin on the CYP1A1 expression.In the presence of 3MC, the indicated cisplatin was added to the HeLa cells and the CYP1A1 mRNA was analyzed by RT-PCR and agarose gel electrophoresis (Figure 3(c)).The CYP1A1/β-actin ratio based on Figure 3(c) was quantified.At 50 μM cisplatin, the CYP1A mRNA level was low until about 20% (Figure 3(d)).

Figure 4 .
Figure 4. GST pull-down assay.(a) GST pull-down assays were investigated in the presence of 50 μM cisplatin or 50 μM 17-DMAG and 1 mM ATP using GST-bHLH and HSP90 on SDS-PAGE (11% gel).(b) GST pull-down assays were analyzed using GST or GST-AhR-ΔAD and HSP90 in the presence or absence of 3 μM 3MC on SDS-PAGE (9% gel).(c) GST pull-down assays were analyzed using GST or GST-AhR-ΔAD and HSP90 in the presence or absence of 3 μM 3MC, 50 μM cisplatin and 1 mM ATP on SDS-PAGE (9% gel).