Targeting the multidrug transporter Patched potentiates chemotherapy efficiency on adrenocortical carcinoma in vitro and in vivo
Anida Hasanovic1-4, Carmen Ruggiero1-4#, Sara Jung5”, Ida Rapa6, Laurie Signetti1-4, Monia Ben Hadj1-4, Massimo Terzolo’, Felix Beuschlein5, 7, Marco Volante’, Constanze Hantel5, 75, Enzo Lalli1-45 and Isabelle Mus-Veteau1-4*
1 Université Côte d’Azur, Sophia Antipolis, Valbonne, France
?CNRS UMR7275, Sophia Antipolis, Valbonne, France
3NEOGENEX CNRS International Associated Laboratory, Sophia Antipolis, Valbonne, France
4 Institut de Pharmacologie Moléculaire et Cellulaire, Sophia Antipolis, Valbonne, France 5 Endocrine Research Unit, Medizinische Klinik und Poliklinik IV, Ludwig-Maximilians- Universität, Munich, Germany
Department of Oncology, University of Turin at San Luigi Hospital, Orbassano, Turin, Italy 1Klinik für Endokrinologie, Diabetologie und Klinische Ernährung, Universitätsspital Zürich, Zurich, Switzerland
# These authors contributed equally
$ These authors contributed equally
* Corresponding author : Isabelle Mus-Veteau, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France
E-mail address: mus-veteau@ipmc.cnrs.fr
Running title: Inhibition of Patched drug efflux enhances chemotherapy
The authors declare no potential conflicts of interest
Novelty and Impact
The present study reports evidences that the drug efflux pump Patched is a major player of the doxorubicin resistance in adrenocortical carcinoma cells, and provides the first proof of concept demonstration that the combination of a Patched drug efflux inhibitor with a chemotherapeutic agent such as doxorubicin can significantly improve therapeutic treatment
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi:10.1002/ijc.31296
of adrenocortical carcinoma by increasing the content of chemotherapeutic agent specifically in tumors without obvious undesirable side effects.
Accepted Articles
Summary
One of the crucial challenges in the clinical management of cancer is the resistance to chemotherapeutics. We recently demonstrated that the Hedgehog receptor Patched, which is overexpressed in many recurrent and metastatic cancers, is a multidrug transporter for chemotherapeutic agents such as doxorubicin. The present work provides evidences that Patched is expressed in adrenocortical carcinoma (ACC) patients, and is a major player of the doxorubicin efflux and the doxorubicin resistance in the human ACC cell line H295R. We discovered that methiothepin inhibits the doxorubicin efflux activity of Patched. This drug- like molecule enhances the cytotoxic, pro-apoptotic, antiproliferative and anticlonogenic effects of doxorubicin on ACC cells which endogenously overexpress Patched, and thereby mitigates the resistance of these cancer cells to doxorubicin. Moreover, we report that in mice the combination of methiothepin with doxorubicin prevents the development of xenografted ACC tumors more efficiently than doxorubicin alone by enhancing the accumulation of doxorubicin specifically in tumors without obvious undesirable side effects. Our results suggest that the use of an inhibitor of Patched drug efflux such as methiothepin in combination with doxorubicin could be a promising therapeutic option for adrenocortical carcinoma, and most likely also for other Patched-expressing cancers. ☒
Keywords
Chemotherapy resistance; Patched; Hedgehog receptor; multidrug resistance; drug efflux pump; drug efflux inhibitor; cancers; adrenocortical carcinoma
Introduction
☒
Despite the major progresses in biomedical research and the development of novel therapeutic strategies, cancer is still among the dominant causes of death worldwide. One of the crucial challenges in the clinical management of cancer is primary (intrinsic) and secondary (acquired) resistance to both conventional and targeted chemotherapeutics. Multidrug resistance (MDR) has been intensively studied, and one of the most prominent mechanisms underlying MDR is overexpression of members of the family of ATP-binding cassette (ABC) transporters1,2. These transporters use energy derived from the hydrolysis of ATP to transport a wide range of substrates (endogenous toxicants and xenobiotics but also chemotherapeutic agents) across biological membranes against a concentration gradient. Since the discovery that the overexpression of ABC transporters in cancer cells can mediate resistance to anti- cancer drugs, research has been directed towards developing compounds that inhibit the efflux activity of ABC transporters and increase classical chemotherapy efficacy. However, to date, the Food and Drug Administration (FDA) has not approved the use of any ABC transporter inhibitor due to toxicity issues3,4. It has long been postulated that the multidrug efflux transporter P-glycoprotein (P-gp/ABCB1/MDR1) mediates the main mechanism of resistance within cancer cells5; however, we recently showed that the Hedgehog (Hh) receptor Patched, which is overexpressed in many recurrent and metastatic cancers, pumps chemotherapeutic agents such as doxorubicin (dxr) out of cancer cells thereby also contributing to chemotherapy resistance6. The Hh signaling pathway controls cell differentiation and proliferation. It plays a crucial role in embryonic development, in stem cell homeostasis and tissue regeneration in the adult7. However, Hh signaling is also involved in cancer development, progression, and metastasis. Indeed, aberrant activation of Hh signaling has been observed in many aggressive ☒ ☒ ☒ ☒ cancers®, in particular in cells exhibiting resistance to chemotherapy such as cancer stem cells ☒ ☒
or tumor-initiating cells’. The Hh receptor Patched, whose expression is induced upon activation of the Hh pathway, is overexpressed in many cancers such as lung, breast, prostate, ovary, colon, brain, melanoma8,10,11 and myeloid leukemia12,13 (see the Human Protein Atlas website http://www.proteinatlas.org/ENSG00000185920-PTCH1/cancer). Recent studies even suggested Patched as an early marker of gastric and thyroid cancers14,15. The discovery that Patched has a drug efflux activity led us to propose Patched as a new target to enhance the efficiency of classical chemotherapeutic treatments and to decrease the risk of recurrence and metastasis. We then developed screening tests to identify molecules able to inhibit the drug efflux activity of Patched16. Based on this screen, we have shown that a natural compound purified from a marine sponge increased the cytotoxicity of dxr in vitro on human melanoma cells which endogenously overexpress Patched. This compound was the first inhibitor of Patched dxr efflux activity to be described17.
☒
☒
Here, we report the discovery of a new inhibitor of Patched drug efflux activity identified by screening of a collection of 1200 small molecules. We show that methiothepin (also named P375 hereafter), a well known non-selective 5-HT transporter antagonist, enhances dxr efficiency both in vitro and in vivo against adrenocortical carcinoma (ACC) cells in which Patched is endogenously expressed and strongly contributes to the resistance to doxorubicin. These results provide a proof of concept demonstration that the combination of dxr with a Patched drug efflux inhibitor can significantly improve therapeutic treatment of adrenocortical carcinoma. ☒
☒
Materials and Methods
Chemical and biological Material
Methiothepin maleate (P375) was purchased from Santa Cruz: CAS number: 20229-30-5; MW: 472.62 ; molecular formula: C20H24N2S2.C4H4O4. Doxorubicin hydrochloride, Hoechst 33342, cisplatin and temozolomide were purchased from Sigma-Aldrich. PSC833 was
purchased from Tocris.
K699 Saccharomyces cerevisiae yeast strain (Mata, ura3 and leu 2-3, kindly donated by R. Arkowitz) was transformed with pYEP-hPtc-MAP (human Patched) or pYEP-mMyo-MAP (control) expression vector and grown at 18℃ until OD600 5-7 as described18.
☒
The human adrenocortical carcinoma cell line H295R was cultured in DMEM/F12 supplemented with 2% NuSerum (BD), 1% ITS+ (BD) and penicillin/streptomycin (Invitrogen) at 37°℃ in a 5% CO2/95% air water-saturated atmosphere. H295RdxrR were obtained by adding increasing concentrations of dxr until 0.2uM in the culture medium during 6 months.
MUC-1 cells were established and cultured as described in19.
The human colorectal carcinoma (HCT116), breast adenocarcinoma (MCF7) and melanoma (A375) cell lines were purchased from ATCC, and cultured in DMEM medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (Invitrogen) at 37°℃ in a 5% CO2/95% air water-saturated atmosphere.
Effect of molecules on the resistance to doxorubicin of yeast expressing Patched
☒
The yeast screening campaign was carried out as described in16. S. cerevisiae expressing human Patched were grown in 10 mL of minimal medium (supplemented with 2% of glucose and aminoacid cocktail without leucine) at 30°C. At OD600 between 5 and 7, yeast were diluted for a preculture in the same medium to OD600 = 1-2. Yeast were then diluted in rich medium containing 2% glucose in 96-well plates in triplicate. 10 uM of each molecule were added in all wells, and chemotherapeutic agent was added in half of the wells (dxr was added ☒
at 10 uM final concentration). Plates were incubated at 18℃ on a shaker at 1250 rpm (microtiter plate shaker SSL5 Stuart) and absorbance at 600 nm was recorded for about 72 hours.
Effect of P375 on doxorubicin cytotoxicity
Cells were seeded in 96-well plates in triplicate and grown in medium to achieve 70% to 80% confluence. Medium was then removed and replaced with 100 uL/well of complete medium containing P375 or DMSO as control. After 2 hours, 100 uL of complete medium containing serial dilutions of dxr was added. For P375 IC50 calculation, cells were incubated with serial dilutions of P375 2 hours before addition of medium containing 2uM of dxr or PBS. Plates were incubated at 37℃ and 5% CO2. After 24 or 48 hours, cells were incubated 3 hours at 37°℃ with 100 uL/well neutral red (NR) solution (50ug/mL in medium) or MTT (Sigma- Aldrich) following the manufacturer’s protocols. Measurements were made in microplate readers (Multiskan Go Microplate Spectrophotometer from Thermo Scientific and SPECTRA from Tecan). IC50 was defined as the concentration that resulted in a 50% decrease in the number of live cells, and IC50 values were calculated using GraphPad Prism 6 software.
Isobologram analysis for determination of synergy
Isobologram analysis was performed as described in20,21. To establish the mode of interaction between dxr and P375, the cells were treated with different concentrations of dxr and P375. Analysis was done on the basis of dose-response curves of cell viability treated with dxr or P375 alone or their combination for 48 h, and isobolograms were created using Compusyn Software (Version 1.0 downloaded from www.combosyn.com,22). These equations provide the theoretical basis for the combination index (CI)-isobologram equation that allows quantitative determination of drug interactions, where CI < 1 (below the diagonal), = 1
(diagonal), and > 1 (above the diagonal) indicate synergism, additive effect, and antagonism, respectively. Dose-reduction index (DRI) is reported. ☒
Apoptosis measurements
☒
Cells were seeded at a density of 7000 cells per well in a 96-well white polystyrene plate (Falcon Corning 96 Well Plate) in triplicate and cultured overnight at 37℃ and 5% CO2. After removal of the medium, cells were treated 48 hours with medium alone or with medium containing dxr alone, P375 alone, or dxr and P375 together. Quantification of caspase 3/7 activity was performed using the luminescent assay Caspase-Glo 3/7 (Promega) and a luminometer (Glomax 96 Microplate Luminometer Promega) following the manufacturer’s protocol.
Proliferation
Cells were seeded at a density of 5000 cells per well in 96-well plates (Falcon 96 Well Clear Microplate, Corning Inc) in triplicate and grown for 24 hours at 37°℃ and 5% CO2. After removal of the medium, cells were treated with medium containing serial dilutions of dxr in the presence or the absence of P375. DMSO was added in control wells. After 7 days at 37℃ and 5% CO2, NR test was used for quantification of living cells. IC50 was defined as the concentration that resulted in a 50% decrease in the number of live cells, and IC50 values were calculated using GraphPad Prism 6 software.
Clone formation
Cells were seeded in 24 well plates (Falcon from Corning Inc) at a density of 5000 cells per well in triplicate, treated with DMSO as control, P375 alone, dxr alone or a combination of P375 and dxr, and incubated at 37℃ and 5% CO2. After 7 days, 500 uL of medium containing the respective molecules was added to the wells. After 14 days cells were fixed ☒
with 4% PFA and incubated with 0.4% crystal violet. After 1 hour, cells were washed quickly with PBS and pictures of the wells were taken. Cells were solubilized with 1% SDS and absorbance was read in a microplate reader at 550 nm (Multiskan Go Microplate Spectrophotometer, Thermo Scientific).
Patched and P-gp knock-down
H295R cells were transfected with Patched Silencer® Select Pre-designed siRNA (Ambion,
#4392420, s11441 (sense: 5’GCACUUACUUUACGACCUAtt3’, as: 5’UAGGUCGUAAAGUAAGUGCtg3’) or P-gp (MDR1) siRNA (sense: 5’GUAUUGACAGCUAUUCGAAdTdT3’, as: 5’UUCGAAUAGCUGUCAAUACdTdT3’23) or control (medium GC) siRNA oligos (Invitrogen) using the Amaxa nucleofection technique (Lonza), as described24. 5x106 cells per condition were electroporated with 400 pmol siRNA following the manufacturer’s protocol, then seeded in 24-well plates and incubated at 37℃ and 5% CO2 during 16 hours before western blotting and dxr efflux measurements.
SDS-PAGE and Western Blotting
Total RIPA (Radioimmunoprecipitation assay buffer) extracts from cells were prepared. Protein concentrations were determined by the DC Protein Assay (Biorad). Samples (50 to 80 ug) were separated on 7.5% SDS-PAGE and transferred to nitrocellulose membranes (Amersham) using standard techniques. After 1 hour at room temperature in blocking buffer (20 mmol/L Tris-HCI pH 7.5, 45 mmol/L NaCl, 0.1%Tween-20, and 5% non-fat milk), nitrocellulose membranes were incubated overnight at 4℃ with rabbit anti-Patched antibody (Abcam ab53715; 1/1000), rabbit anti-Smo antibody (gently provided by M. Ruat; 1/1000), rabbit anti-Gli2 antibody (Abcam ab26056; 1/1000), rabbit anti-Shh antibody (gently provided by M. Ruat; 1/1000), mouse anti-P-gp antibody (Abcam ab3366; 1/2000) or mouse anti-ß-tubulin antibody (Sigma; 1/1000). After 3 washes, membranes were incubated 45 min. ☒ ☒
☒
with anti-rabbit (1:2000) or anti-mouse (1:5000) immunoglobulin coupled to horseradish peroxidase (Dako). Detection was carried out with an ECL Prime Western Blotting detection reagent (Amersham) on a Fusion FX imager (Vilber Lourmat).
Immunohistochemistry on tumors
Immunohistochemistry for Patched and Smoothened was performed using a monoclonal mouse antibody for Patched (PTCH Antibody, clone 5c7, NBP1-47945, Novus Biologicals, Littleton, CO, USA) and a rabbit polyclonal antibody for Smoothened (ab72130, Abcam, Cambridge. UK) in an automated system (BenchMark XT platform, Ventana Medical Systems, Inc. Tucson, AZ, USA) on a series of 70 ACC tissue samples collected from the archives of the Pathology Unit at San Luigi Hospital, Orbassano (Italy), having detailed clinical and pathological information available. The H-score represents the product of the IHC intensity score (0, 1+, 2+, and 3+) and the percentage of positive cells, ranging from 0 to 300.
Doxorubicin efflux
Dxr efflux measurements were carried out as described in17.
On yeast: yeast expressing Patched (hPtc) or control yeast were grown to an OD600 of 5, centrifuged, washed with cold water, resuspended at an OD600 of 10 in HEPES-NaOH buffer (pH 7.0) supplemented with 5 mM 2-deoxy-D-glucose, and incubated with 10 uM dxr for 2 hours at 4℃ in the cold room on a rotating wheel protected from light. Yeast were centrifuged and the supernatant was removed. One sample was immediately fixed with 4% PFA for dxr loading control. The other samples were resuspended in HEPES-NaOH buffer (pH 7.0) containing 5 mM 2-deoxy-D-glucose supplemented with DMSO, 10 uM P375 or 2.5 uM PSC833, and incubated 10 min. at 20℃ with gentle shaking in a Benchmark Multi-therm shaker (Innova 2000, New Brunswick Scientific) protected from light. Samples were centrifuged for 1 min. at 18,000g, supernatants were removed and yeast were fixed with 4% ☒ ☒ ☒
PFA. 10 µL of each sample were deposited on a coverslip and mounted in SlowFade Gold antifade reagent with DAPI (Invitrogen). ☒
☒
On cells: cells were seeded on coverslips in 24-well plates and allowed to grow to 80% confluence. Coverslips were incubated at 37℃ and 5% CO2 with 10 uM dxr in physiological buffer (140 mM NaCl, 5 mM KCI, 1 mM CaCl2, 1 mM MgSO4, 5 mM glucose, 20 mM HEPES, pH 7.4). After 2 hours, three coverslips were immediately fixed 10 min. with 4% PFA for dxr loading control, rapidly washed with PBS and mounted in SlowFade Gold antifade reagent with DAPI (Invitrogen). The other coverslips (a triplicate per condition) were incubated with physiological buffer supplemented with DMSO, 10 uM P375 or PSC833 for 30 min. under gentle shaking at room temperature and protected from light. They were then immediately fixed with 4% PFA, washed and mounted as described above.
☒
☒
Images for dxr quantification were acquired with a Zeiss Axioplan 2 fluorescence microscope coupled to a digital charge-coupled device camera using a 63X objective for yeast or a 40X /1.3 Plan NeoFluar objective for cells and filters for Alexa 594. Dxr fluorescence was quantified using ImageJ software. Sampling of yeast/cells was performed randomly. About 100 yeast/cells (from three wells) were scored per condition per experiment.
On tumors and heart slides: images for dxr quantification were acquired on slides with a Zeiss Axioplan 2 fluorescence microscope coupled to a digital charge-coupled device camera using a 10X/1.5 Plan NeoFluar objective and a filter for Alexa 594. Dxr fluorescence was quantified using ImageJ software.
☒
Pharmacokinetic study
☒
P375 was formulated at 0.4 mg/mL (for IV) and 1 mg/mL (for PO) in DMSO/Solutol® HS15/ phosphate buffered saline (PBS) (5/5/90, v/v/v, and administered to non-fasted male ICR
mice (weighing 20 - 30 g) intravenously (IV) at the dosing volume of 5 ml/kg and orally (PO) at 10 ml/kg. Serial plasma samples were harvested via the facial vein at 3, 10, 30, 60, 120, 240, 360 and 1440 min. post dosing for IV group, and at 10, 30, 60, 120, 240, 360, 480 and 1440 min. for PO group (3 mice per time point). Plasma was prepared from blood aliquots (50 µL) collected via facial vein sampling from individual mice, in tubes coated with K2EDTA, mixed gently, then kept on ice and centrifuged at 2500g for 15 min. at 4℃, within 1 hour of collection. For control animals, blood was collected by cardiac puncture. The plasma samples were processed using acetonitrile precipitation and analyzed by LC-MS/MS. A plasma calibration curve was generated. Aliquots of drug-free plasma were spiked with the test compound at the specified concentration levels. The spiked plasma samples were processed together with the unknown plasma samples using the same procedure. The processed plasma samples were stored at -70℃ until LC-MS/MS analysis, at which time peak areas were recorded, and the concentrations of the test compound in the unknown plasma samples were determined using the respective calibration curve. Plots of plasma concentration versus time were constructed and the pharmacokinetic parameters of P375 after IV and PO dosing were obtained from the analysis of the plasma data. ☒
Animal experiments
For therapeutic experiments female athymic NMRI nu/nu mice (6 - 8 weeks) were purchased from Charles River (Sulzfeld, Germany) and housed under pathogen-free conditions. All experiments were carried out following protocols approved by the Regierung von Oberbayern and in accordance with the German guidelines for animal studies. H295R xenografts were induced as described before25,26. For short-term experiments, tumor-bearing mice (n= 4-5) were treated with one therapeutic cycle which consisted of either liposomal doxorubicin (lipdxr) (6 mg/kg body weight, intravenously on days 4 and 10), P375 (2 mg/kg body weight,
intraperitoneally on days 1 to 11) or a combination of lipdxr and P375 (P375: 2 mg/kg body weight intraperitoneally on days 1 to 11 plus lipdxr 6 mg/kg body weight intravenously on days 4 and 10). 48 hours after the last administration, the short-term study was terminated and animals sacrificed. Afterwards, tumors were excised and subsequently paraffin embedded. The slide preparation of these paraffin embedded tissues as well as the quantification of the number of apoptotic tumor cells, using the colorimetric DeadEND TUNEL System (Promega, Mannheim, Germany), were performed as described elsewhere25. For long-term therapeutic protocols, mice (n= 7-8) received two treatment cycles with a therapy-free interval of six days in between. Animals were monitored daily and tumor sizes measured every second day (as tumor length x width [cm2]). At day 40 after tumor induction, when first tumors reached a longest tumor diameter of 1.5 cm, the long term therapeutic study was terminated, tumors and hearts were excised and subsequently paraffin embedded. ☒ ☒
☒
Statistical analysis
All results represent at least three independent replications. Data are shown as mean value ± SEM. Statistical analyses were performed with Prism 6 software (GraphPad) using one-way analysis of variance (ANOVA) followed by Bonferroni’s or Dunnett’s (TUNEL staining) Multiple Comparison Tests. For the long-term therapeutic experiments a two-way repeated measures ANOVA was applied. ☒
Results ☒
Methiothepin inhibits the resistance of yeast expressing Patched to doxorubicin ☒
In a previous study, we reported that the expression of human Patched allowed yeast to grow in the presence of a concentration of doxorubicin (dxr) that inhibits the growth of control
☒ yeast, indicating that Patched confers resistance to dxr6. From these results, we developed a
☒
screening test in 96-well plates to identify compounds capable of inhibiting the resistance to dxr of yeast expressing human Patched16. 1200 drug molecules from the Prestwick Chemical library were tested at a final concentration of 10 uM on yeast expressing Patched in a medium supplemented or not with dxr. We observed that the compound P375, which corresponds to methiothepin maleate, a small molecule known as a non-selective 5-HT receptor antagonist, significantly inhibited the growth of yeast expressing Patched in the presence of dxr in contrast to the majority of the molecules tested such as P298 that had no effect (Fig. 1A). Notably, P375 had no effect on the growth of yeast expressing Patched in the absence of dxr and did not inhibit the growth of control yeast in the presence of smaller amounts of dxr (Fig. 1A) suggesting that this molecule inhibits specifically dxr resistance conferred by Patched activity.
☒
Methiothepin inhibits doxorubicin efflux from yeast expressing Patched
☒
We took advantage of the natural fluorescence properties of dxr27 to carry out dxr efflux measurements, and tested the effect of P375 on dxr efflux as described before17. 2-deoxy-D- glucose was added in buffer during dxr loading and efflux in order to de-energize yeast and inhibit ATP-binding cassette (ABC) transporters which also contribute to dxr efflux in yeast. This enabled us to selectively study ATP independent efflux activity. Dxr fluorescence measured in yeast expressing Patched after efflux in buffer containing DMSO (P375 solvant) was significantly lower than that measured in control yeast, consistently with the dxr efflux activity of Patched already described6 (Fig 1B left panel). Dxr fluorescence in yeast expressing Patched was significantly higher when P375 was present in the efflux buffer while this molecule had no significant effect on the dxr fluorescence of control yeast (Fig. 1B right panel), indicating that P375 specifically inhibited Patched dxr efflux activity. Note that the P- glycoprotein (P-gp) inhibitor PSC833 had no significant effect on the dxr efflux activity of ☒ ☒ ☒ ☒
☒
☒ Adrenocortical carcinoma as a paradigm of Patched-expressing cancers
Adrenocortical carcinoma (ACC) is a rare cancer which presents strong resistance to the best available treatment composed of a mixture of chemotherapeutic agents (etoposide, doxorubicin and cisplatin) combined with the adrenolytic substance mitotane (EDP-M)28,29,30. The expression of Hh signaling components was analyzed in tissue samples of 70 ACC patients. ICH analysis showed that the Hh receptor Patched and the Hh signaling receptor Smoothened were expressed at the protein level in ACC tumor tissues (Fig. 2A), and were positively correlated (Spearman’s R=0.38 p=0.002). Median H-score values were 130 for Patched and 90 for Smoothened. Intensity and distribution was highly heterogeneous among individual tumors (Fig. 2B). As shown in Fig. 2C, Patched is also strongly expressed in the ACC cell line H295R which was initially isolated from a patient diagnosed with ACC31,32. Interestingly, Patched expression was found to be significantly lower in the MUC-1 cell line which had recently been established from an ACC metastasis obtained from a patient with a primary diagnosis of ACC19. ☒
☒
Methiothepin increases the cytotoxicity of doxorubicin on adrenocortical carcinoma cells ☒ expressing Patched
☒
As dxr is used as a standard compound of therapeutic regiments for patients with advanced ACC, we decided to test the effect of P375 on dxr cytotoxicity using the two available human ACC cell lines H295R and MUC-1. Specifically, cells were treated separately with increasing concentrations of P375 in the presence of 2 uM dxr during 48 hours before cell viability measurement. We observed that P375 significantly increased the cytotoxicity of dxr against the H295R cell line in a dose-dependent manner (Fig. 3A left panel), while this molecule had no effect on the cytotoxicity of dxr against the MUC-1 cell line (Fig. 3A right panel).
Remarkably, these results indicate, that this molecule seems to increase dxr efficiency only on cells overexpressing Patched. ☒
Accordingly, we decided to use the H295R cell line to characterize the effect of P375 on dxr cytotoxicity. Cells were treated with increasing concentrations of dxr, with or without P375, during 48 hours before assessment of cell viability. Results showed that P375 induced a significant decrease of the dxr-IC50 on H295R cells (more than 6 times) (Fig. 3B) without affecting Patched protein levels or Hedgehog signaling (Sup fig. 1). We observed the same effect of P375 on cells rendered resistant to dxr (H295R Dxr R) (Fig. 3C). Interestingly, these dxr resistant cells showed an increased level of Patched expression, suggesting that Patched upregulation was the cause for their resistance (Fig. 3D). These results demonstrate that P375 strongly enhanced the efficiency of dxr on ACC cells expressing Patched. The isobologram analysis clearly showed that dxr and P375 exhibit significant synergism as demonstrated by cooperativity indices of << 1 (Sup. Fig. 2). We also observed that P375 increased the efficiency of cisplatin, of ethoposide and of the clinical gold standard EDP-M treatment (Sup. Fig. 3). Dose-responses of P375 on cell viability were performed providing IC50 values of about 3 uM in the presence of 2 uM dxr, a dose which has a very low cytotoxicity alone (about 15%) (Fig. 3E). ☒ ☒
☒
☒
Methiothepin increases the pro-apoptotic, anti-proliferative and anti-clonogenic effects of doxorubicin
☒
☒
In agreement with the results of cytotoxicity, apoptosis experiments based on caspase 3/7 activation measurements indicated that the addition of P375 to dxr treatment significantly increased the percentage of apoptotic cells (Fig. 4A). We also observed that the presence of P375 in the culture medium significantly increased the anti-proliferative effect of dxr on H295R cells, dxr-IC50 on cell proliferation being reduced nearly 10 times with 2 uM of P375 ☒
(Fig. 4B). Moreover, the combination of dxr and P375 inhibited the ability of H295R cells to form clones to a significantly greater extent than dxr alone (P<0.05) (Fig. 4C). This effect was also observed in 3D cultures on the formation of H295R spheroids on which P375 enhanced dxr efficiency by about 2 times (Sup Fig. 4). ☒
☒
Methiothepin inhibits doxorubicin efflux from ACC cells
☒
As in the yeast assay, epifluorescence microscopy was used to measure the effect of P375 on dxr efflux on H295R cells. As exemplified in Fig. 5A, incubation of cells with dxr induced a strong accumulation of dxr in the nuclei. The intracellular dxr amount was drastically reduced after 30 minutes of incubation with the efflux buffer. Quantification of this observation indicated that 60 to 70% of dxr was transported out of cells over this time period. The presence of P375 in the efflux buffer allowed retaining of about 60% of cellular dxr. This finding demonstrated that, in H295R cells as in Patched expressing yeast, P375 strongly inhibits the efflux of dxr. Interestingly, the P-gp antagonist PSC833 had no effect on dxr efflux despite the fact that P-gp is expressed in ACC cells (Fig. 5 D). However, we observed that PSC833 inhibited Hoechst efflux in contrast to P375 (Fig. 5B). These results suggest that P375 inhibits specifically dxr efflux and that Patched strongly contributes to dxr efflux in this model. Moreover, the depletion by 60% of Patched protein in H295R cells using a specific siRNAs equally reduced dxr efflux by 60%, confirming that Patched is the major dxr efflux pump in these cells (Fig. 5C). This is in good agreement with experiments showing that a 70% decrease of P-gp expression using a specific siRNA inhibited only about 10% of the dxr efflux (Fig. 5D), which corroborates that P-gp does not contribute significantly to dxr efflux in these ACC cells.
Methiothepin inhibits doxorubicin efflux and increases doxorubicin cytotoxicity on other cancer cells expressing Patched
The effect P375 was also tested on other human cancer cell lines endogenously overexpressing Patched, such as the colorectal carcinoma cell line HCT116, the breast adenocarcinoma cell line MCF7, and the melanoma cell line A375 (Sup. Fig. 5A). We observed that P375 significantly inhibited dxr efflux (Sup. Fig. 5B), and strongly increased the cytotoxicity of dxr (Sup. Fig. 5C) on these three cell lines. P375 might therefore be of general interest for the development of cancer therapy strategies that rely on the combination of Patched inhibitors with dxr. This will be illustrated below for the example of ACC. ☒
Methiothepin profiling and pharmacokinetics parameters
As summarized in Sup.Table 1, profiling data indicated that P375 is a particularly stable ☒ lipophilic compound. The metabolic stability of P375 was confirmed in vivo in a pharmacokinetic (PK) study, which was conducted on male ICR mice following a single intravenous (IV) or oral (PO) administration of P375 (Sup. Table 2).
Methiothepin enhances doxorubicin efficacy in vivo
☒
Short- and long-term therapeutic efficacy of P375, dxr or a combination of both compounds was investigated in H295R xenograft bearing mice. In this study, liposomal dxr (lipdxr) was preferred over dxr because of its higher effectiveness in this model33.
After administration of one therapeutic cycle, the quantification of apoptotic cells in tumors showed significant anti-tumoral efficacy exclusively for P375 + lipdxr treatment (61.6±19.7 apoptotic cells/mm2 vs. 16.4±4.0 apoptotic cells/mm2 in control mice, p<0.05; Fig. 6A). Moreover, epifluorescence images taken from tumor slides revealed that the amount of dxr in tumors from mice treated with lipdxr and P375 was significantly higher to the amount of dxr in tumors from mice treated with lipdxr alone (Fig. 6B). ☒
For long-term treatment, two drug cycles were administered with a therapy-free interval of six
days in between. Primary endpoint of this study was tumor size (expressed as length x width in cm2). This experiment confirmed the previously indicated higher therapeutic efficacy for the combined administration of P375 + lipdxr (p<0.01 vs. controls on day 39; Fig. 6C), without any obvious signs of undesirable side effects such as weight loss or abnormal ☒ ☒
behavior of the animals. Epifluorescence images taken from tumor slides showed that P375 enhanced significantly (about 3 times) the dxr content in tumors (Fig. 6D left), which corroborated the results obtained from the short-term experiment and was in very good agreement with in vitro studies. Interestingly, epifluorescence images from heart slides showed that P375 did not increase the amounts of dxr in murine hearts (Fig. 6D right).
Discussion
☒
In this study, we show that the Hedgehog receptor Patched is expressed in adrenocortical carcinoma patients, and is a major player of the doxorubicin (drx) efflux and the dxr resistance in the human ACC cell line H295R. We demonstrate that methiothepin (P375) significantly enhances the cytotoxic, pro-apototic, anti-proliferative and anti-clonogenic effects of dxr on ACC cells by inhibiting the dxr efflux activity of Patched. In vivo experiments performed on mice bearing human ACC cells (H295R) xenografts showed that the addition of methiothepin to liposomal dxr treatment inhibited tumor growth more significantly than liposomal dxr alone by enhancing dxr accumulation in tumors. Notably, these effects were achieved without obvious undesirable side effect and specifically, without increasing the amounts of dxr in heart tissues of treated animals. ☒ ☒ ☒
☒
Overall, these findings are especially interesting as dxr is one of the three chemotherapeutic agents included in the gold standard ACC therapy and since Patched was found to be expressed in ACC tumor samples from our cohort. Of note, the standard therapy composed of
☒
dxr in combination with etoposide, cisplatin and mitotane (EDP-M) has many side effects and often fails due to resistance to the treatment causing a poor overall patient survival28,29,34 ☒
Methiothepin is a small molecule known as a non-selective 5-HT receptor antagonist with antipsychotic activity35,36,37. Three chlorinated derivatives Clorotepine, Zotepine and Clotiapine are antipsychotics drugs marketed for the treatment of schizophrenia38. We showed that these drugs also enhanced the doxorubicin cytotoxicity on ACC cells but with lower efficacy than methiothepin (IC50 of 15uM, 17uM and 12uM respectively) (Sup. Fig. 6). This kind of poly-pharmacology profile has been observed for many drugs, probably because of the broad spectrum of molecules capable of being transported by multidrug transporters. For instance, HIV-protease inhibitors like saquinavir or its NO-derivatives have been shown to increase sensitivity to chemotherapy by inhibiting ABC transporters3. However, our results show that methiothepin does not inhibit ABC transporters like P-gp but interacts specifically with Patched. ☒ ☒
☒
Profiling data and pharmacokinetics study indicate that methiothepin possesses excellent drug-like properties, with a particularly favorable metabolic stability. ☒
Altogether, results position methiothepin favorably as a drug-like molecule. Therefore, our study strongly suggests that ACC patients with tumors expressing Patched might benefit from therapies based on the use of methiothepin or one of its analogues in combination with dxr, either alone or as part of the standard therapy formulation EDP-M.
☒ ☒
Dxr has been in use for over five decades as the backbone of chemotherapy treatment regimens for a wide range of cancers. However, due to heart failure, the maximum life time cumulative dose of dxr has been limited decreasing the benefits that patients may receive ☒ ☒
from this potent drug40. Since methiothepin strongly increases dxr efficiency and specifically inhibits Patched dxr efflux in cancer cells, the effective dose of dxr received by patients could
be reduced inducing less impairment in cardiac function. Moreover, our in vivo studies showed that methiothepin enhanced liposomal dxr (commercialized as Caelix or Doxil) which is a new formulation of dxr approved by FDA for cancer therapy. The ability of liposomal dxr to buffer a number of undesirable side effects of dxr, including a major risk reduction in cardiac toxicity, is now well established4 and should strengthen the consideration to further studying the use of a combination of methiothepin and liposomal dxr for Patched expressing cancers treatment. ☒
☒
☒
Interestingly, the present work also brings evidences that dxr efflux in the ACC cell line H295R mainly occurs through Patched and not through ABC transporters such as P-gp. This is in good agreement with previous work suggesting that dxr resistance in human adrenocortical and renal carcinomas is mediated by mechanisms other than P-gp42. Patched is not part of the ABC transporters family. Indeed, we previously showed that Patched uses the proton motive force to efflux drugs similarly to the bacterial efflux pumps from the RND family6. This may seem surprising; however, the alteration of energy metabolism that occurs in hypoxic conditions in cancer cells has been shown to lead to lactate accumulation and intracellular acidification43,44,45. Accordingly, rapidly dividing cancer cells produce and release large amounts of protons into the extracellular compartment due to enhanced glucose utilization. This pattern of acidic extracellular environment and the alkaline cytosol is considered a hallmark of malignant cancers and is referred to as a “reversed pH gradient”46. Therefore, in cancer cells, Patched can function as an efflux pump using the proton gradient. This makes Patched a particularly relevant therapeutic target for recurrent and metastatic cancers such as ACC, and Patched drug efflux inhibitors potentially represent much more cancer specific and less toxic therapeutics than ABC transporters antagonists. ☒ ☒
Moreover, experiments which show that methiothepin is also able to inhibit dxr efflux and increase dxr cytotoxicity on colorectal, breast and melanoma cancer cells endogenously overexpressing Patched suggest that Patched drug efflux inhibition could improve the efficiency of dxr not only on ACC, but also on a panel of Patched-expressing cancers. ☒
In conclusion, our results provide a proof of concept that the combination of dxr with a Patched drug efflux inhibitor could significantly improve the therapeutic treatment of ACC, and most likely also of other Patched-expressing cancers including melanoma, breast and colorectal cancers.
Author contributions
Conception and design, I.M-V. and E.L .; Development of methodology, M.V., C.H. and I.M- V .; Acquisition of data, A.H., C.R., S.J., I.R., M.T., L.S. and M.B.H .; Analysis and ☒
interpretation of data, A.H., C.R., M.T., M.V., C.H., E.L. and I.M-V .; Writing - Original ☒ Draft, I.M-V; Writing -Review & Editing, C.H., E.L. and I.M-V .; Funding Acquisition, I.M-
☒ V., F.B., C.H., and E.L .; Resources, I.M-V., E.L., M.V.,C.H., and F.B .; Supervision, I.M-V., M.V., and C.H.
ba
☒
Acknowledgments
This work was supported by grants from Centre National de la Recherche Scientifique (CNRS) through the “Soutien au transfert” program, Association France Cancer, Region Provence Alpes Côte d’Azur through the APRF program (2013-17362), Canceropôle PACA, ☒
and French National Research Agency (ANR) through the Investments for the Future UCAJEDI (ANR-15-IDEX-01) to I.M-V., from French National Research Agency (ANR)
through the «Investments for the Future »> LABEX SIGNALIFE program (ANR-11-LABX- 0028-01) to E.L. and A.H., from Fondation de France “Recherche fondamentale et clinique sur le cancer 2015” (#00057927) to E.L. and C.R., and from the Wilhelm-Sander-Stiftung to C.H. and F.B. (2014.033.1). The authors are indebted to Igor Shapiro and Claudia Siebert for their excellent assistance, and to Emilia Pinto for H295R cell line DNA STR analysis.
References
1. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat. Rev. Cancer 2002; 2: 48-58.
2. Szakács G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM. Targeting multidrug resistance in cancer. Nat. Rev. Drug Discov. 2006; 5: 219-234.
3. Wang YJ, Zhang YK, Kathawala RJ, Chen ZS. Repositioning of Tyrosine Kinase Inhibitors as Antagonists of ATP-Binding Cassette Transporters in Anticancer Drug Resistance. Cancers (Basel). 2014; 6: 1925-1952.
4. Kathawala RJ, Wang YJ, Shukla S, Zhang YK, Alqahtani S, Kaddoumi A, Ambudkar SV, Ashby CR Jr., Chen ZS. ATP-binding cassette subfamily B member 1 (ABCB1) and subfamily C member 10 (ABCC10) are not primary resistance factors for cabazitaxel. Chin. J. Cancer. 2015; 34: 115-20.
5. Luqmani YA. Mechanisms of drug resistance in cancer chemotherapy. Med. Princ. Pract. 2005; 14: 35-48.
☒
6. Bidet M, Tomico A, Martin P, Guizouarn H, Mollat P, Mus-Veteau I. The Hedgehog receptor patched functions in multidrug transport and chemotherapy resistance. Mol. Cancer Res. 2012; 10: 1496-508.
☒
7. Varjosalo M, Taipale J. Hedgehog: functions and mechanisms. Genes Dev. 2008; 22: 2454- 2472.
8. Scales SJ, de Sauvage FJ. Mechanisms of Hedgehog pathway activation in cancer and implications for therapy. Trends Pharmacol. Sci. 2009; 30: 303-312.
9. Cochrane CR, Szczepny A, Watkins DN, Cain JE. Hedgehog Signaling in the Maintenance of Cancer Stem Cells. Cancers (Basel). 2015; 7: 1554-1585.
10. Blotta S, Jakubikova J, Calimeri T, Roccaro AM, Amodio N, Azab AK, Foresta U, Mitsiades CS, Rossi M, Todoerti K, Molica S, Morabito F, Neri A, Tagliaferri P, Tassone P, Anderson KC, Munshi NC. Canonical and noncanonical Hedgehog pathway in the pathogenesis of multiple myeloma. Blood. 2012; 120: 5002-5013.
☒
11. Jeng KS, Sheen IS, Jeng WJ, Yu MC, Hsiau H, Chang FY. High expression of Sonic Hedgehog signaling pathway genes indicates a risk of recurrence of breast carcinoma. Onco Targets Ther. 2013; 7: 79-86.
☒ 12. Zhao C, Chen A, Jamieson CH, Fereshteh M, Abrahamsson A, Blum J, Kwon HY, Kim J, Chute JP, Rizzieri D, Munchhof M, VanArsdale T, Beachy PA, Reya T. Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia. Nature. 2009 ; 458: ☒ 776-779. Erratum in: Nature. 2009; 460 : 652.
13. Queiroz KC, Ruela-de-Sousa RR, Fuhler GM, Aberson HL, Ferreira CV,
Peppelenbosch MP, Spek CA. Hedgehog signaling maintains chemoresistance in myeloid leukemic cells. Oncogene. 2010; 29: 6314-6322. ☒
☒
14. Saze Z, Terashima M, Kogure M, Ohsuka F, Suzuki H, Gotoh M. Activation of the sonic hedgehog pathway and its prognostic impact in patients with gastric cancer. Dig Surg. 2012; 29: 115-123.
☒
15. Xu X, Ding H, Rao G, Arora S, Saclarides CP, Esparaz J, Gattuso P, Solorzano CC, Prinz RA. Activation of the Sonic Hedgehog pathway in thyroid neoplasms and its potential role in tumor cell proliferation. Endocr. Relat. Cancer. 2012; 19: 167-179.
☒
16. Fiorini L, Mus-Veteau I. Method to Screen Multidrug Transport Inhibitors Using Yeast Overexpressing a Human MDR Transporter. Methods Mol. Biol. 2016; 1432: 303-318.
☒
17. Fiorini L, Tribalat MA, Sauvard L, Cazareth J, Lalli E, Broutin I, Thomas OP, Mus- Veteau I. Natural paniceins from mediterranean sponge inhibit the multidrug resistance activity of Patched and increase chemotherapy efficiency on melanoma cells. Oncotarget. 2015; 6: 22282-97.
18. Bidet M, Joubert O, Lacombe B, Ciantar M, Nehmé R, Mollat P, Brétillon L, Faure H, Bittman R, Ruat M, Mus-Veteau I. The hedgehog receptor patched is involved in cholesterol transport. PLOS One 2011; 6: e23834.
☒
☒
19. Hantel C, Shapiro I, Poli G, Chiapponi C, Bidlingmaier M, Reincke M, Luconi M, Jung S, Beuschlein F. Targeting heterogeneity of adrenocortical carcinoma: Evaluation and extension ☒ of preclinical tumor models to improve clinical translation. Oncotarget. 2016; 7: 79292- 79304.
20. Donia M, Maksimovic-Ivanic D, Mijatovic S, Mojic M, Miljkovic D, Timotijevic
G, Fagone P, Caponnetto S, Al-Abed Y, McCubrey J, Stosic-Grujicic S, Nicoletti F. In vitro and in vivo anticancer action of Saquinavir-NO, a novel nitric oxide-derivative of the protease inhibitor saquinavir, on hormone resistant prostate cancer cells. Cell Cycle. 2011; 10: 492.
☒
☒ 21. Kroiss M, Sbiera S, Kendl S, Kurlbaum M, Fassnacht M. Drug Synergism of Proteasome Inhibitors and Mitotane by Complementary Activation of ER Stress in Adrenocortical Carcinoma Cells. Horm Cancer. 2016; 7: 345.
22. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev. 2006; 58: 621.
23. Xu D, Kang H, Fisher M, Juliano RL. Strategies for inhibition of MDR1 gene expression. Mol. Pharmacol. 2004; 66: 268-275.
24. Doghman-Bouguerra M, Granatiero V, Sbiera S, Sbiera I, Lacas-Gervais S, Brau F, ☐ Fassnacht M, Rizzuto R, Lalli E. FATE1 antagonizes calcium- and drug-induced apoptosis by uncoupling ER and mitochondria. EMBO Rep. 2016; 17: 1264-1280.
25. Jung S, Nagy Z, Fassnacht M, Zambetti G, Weiss M, Reincke M, Igaz P, Beuschlein F, Hantel C. Preclinical progress and first translational steps for a liposomal chemotherapy ☐ protocol against adrenocortical carcinoma. Endocr. Relat. Cancer. 2016; 23: 825-37.
☒ 26. Hantel C, Jung S, Mussack T, Reincke M, Beuschlein F. Liposomal polychemotherapy improves adrenocortical carcinoma treatment in a preclinical rodent model. Endocr. Relat. Cancer. 2014; 21: 383-94. ☒ ☒
27. Mohan P, Rapoport N. Doxorubicin as a molecular nanotheranostic agent: effect of doxorubicin encapsulation in micelles or nanoemulsions on the ultrasound-mediated intracellular delivery and nuclear trafficking. Mol. Pharm. 2010; 7: 1959-1973. ☒
28. Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, Welin S, Schade- Brittinger C, Lacroix A, Jarzab B, Sorbye H, Torpy DJ, Stepan V, Schteingart DE, Arlt W, Kroiss M et al. Combination chemotherapy in advanced adrenocortical carcinoma. N. Engl. J. Med. 2012; 366: 2189- 2197.
29. Else T, Kim AC, Sabolch A, Raymond VM, Kandathil A, Caoili EM, Jolly S, Miller BS, Giordano TJ, Hammer GD. Adrenocortical carcinoma. Endocr. Rev. 2014; 35: 282-326.
30. Lalli E, Sasano H. 5th International ACC Symposium: An Outlook to Current and Future Research on the Biology of Adrenocortical Carcinoma: Diagnostic and Therapeutic Applications. Horm. Cancer. 2016; 7: 44-48.
31. Gazdar AF, Oie HK, Shackleton CH, Chen TR, Triche TJ, Myers CE, Chrousos GP, Brennan MF, Stein CA, La Rocca RV. Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis. Cancer Res. 1990; 50: 5488-96.
32. Wang T, Rainey WE. Human Adrenocortical Carcinoma Cell Lines. Mol. Cell. Endocrinol. 2012; 351: 58-65.
33. Hantel C, Lewrick F, Reincke M, Süss R, Beuschlein F. Liposomal doxorubicin-based treatment in a preclinical model of adrenocortical carcinoma. J. Endocrinol. 2012; 213: 155- 161.
34. Hammer GD, Fassnacht M, Lalli E. Adrenal cancer: scientific advances. Mol. Cell. Endocrinol. 2011; 351: 1.
35. Monachon MA, Burkard WP, Jalfre M, Haefely W. Blockade of central
5-hydroxytryptamine receptors by methiothepin. Naunyn Schmiedebergs Arch Pharmacol. 1972; 274: 192-197. ☒
☒
36. Dall’Olio R, Vaccheri A, Montanaro N. Reduced head-twitch response to quipazine of rats previously treated with methiothepin: possible involvement of dopaminergic system. Pharmacol. Biochem. Behav. 1985; 23: 43-8.
37. Mao QQ, Huang Z, Ip SP, Xian YF, Che CT. Role of 5-HT(1A) and 5-HT(1B) receptors in the antidepressant-like effect of piperine in the forced swim test. Neurosci. Lett. 2011; 504: 181-184.
38. Hrbek J, Macáková J, Komenda S, Siroká A, Rypka M. On acute effects of some drugs on the higher nervous activity in man (the acoustic analyser). Clorotepin (0.5 mg and 1.0 mg), pemoline (100 mg). Part LI. Acta Univ Palacki Olomuc Fac. Med. 1985; 111: 139-153.
39. Maksimovic-Ivanic D, Fagone P, McCubrey J, Bendtzen K, Mijatovic S, Nicoletti F. HIV-protease inhibitors for the treatment of cancer: Repositioning HIV protease inhibitors while developing more potent NO-hybridized derivatives? Int J Cancer. 2017; 140: 1713- 1726.
☒
40. Magdy T, Burmeister BT, Burridge PW. Validating the pharmacogenomics of chemotherapy-induced cardiotoxicity: What is missing? Pharmacol. Ther. 2016; 168: 113- 125.
41. Gabizon AA, Patil Y, La-Beck NM. New insights and evolving role of pegylated liposomal doxorubicin in cancer therapy. Drug Resist Updat. 2016; 29: 90-106.
42. Fridborg H, Larsson R, Juhlin C, Rastad J, Akerström G, Backlin K, Nygren, P. P- glycoprotein expression and activity of resistance modifying agents in primary cultures of human renal and adrenocortical carcinoma cells. Anticancer Res. 1994; 14: 1009-16.
☒
43. Chen Z, Lu W, Garcia-Prieto C, Huang P. The Warburg effect and its cancer therapeutic implications. J. Bioenerg. Biomembr. 2007; 39: 267-74.
44. Taylor S, Spugnini EP, Assaraf YG, Azzarito T, Rauch C, Fais S. Microenvironment acidity as a major determinant of tumor chemoresistance: Proton pump inhibitors (PPIs) as a novel therapeutic approach. Drug Resist. Updat. 2015; 23: 69-78.
45. Kondapalli KC, Llongueras JP, Capilla-González V, Prasad H, Hack A, Smith C, Guerrero-Cázares H, Quiñones-Hinojosa A, Rao R. A leak pathway for luminal protons in endosomes drives oncogenic signalling in glioblastoma. Nat. Commun. 2015; 6: 6289.
46. Damaghi M, Wojtkowiak JW, Gillies RJ. pH sensing and regulation in cancer. Front. Physiol. 2013; 17: 370.
☒ t
Figure legends
☒
Figure 1. P375 inhibits yeast expressing Patched resistance to doxorubicin. (A) Yeast expressing Patched or control yeast were grown in the presence of 10 uM of each molecule to be tested, and in the presence or the absence of doxorubicin (dxr) (10 uM for yeast expressing Patched and 5 uM for control yeast). DMSO was used as control. The growth of yeast was measured by absorbance at 600 nm. (B) Yeast expressing wild-type Patched and control yeast were incubated with dxr for 2 hours and fixed for dxr loading control, or resuspended in buffer supplemented with DMSO, 10 uM P375 or 2.5 uM PSC833 for 10 min. and fixed.
Histograms represent the intracellular dxr fluorescence quantification which was carried out using Image J software on more than 100 yeast from 3 different fields for each condition on 3 independent experiments. Data are represented as mean ± SEM and were analyzed using Anova multiple comparison test and Bonferroni correction. Significance is attained at P < 0.05 (*) ( ***: P < 0.0005).
Figure 2. Patched is expressed in Adrenocortical carcinoma. (A) Patched and Smoothened proteins are expressed in ACC tumor tissues at a variable extent of intensity and distribution. For each protein, an example of weak and intense expression is shown. (B) Patched and Smoothened protein expression (H-score) greatly varied among individual cases. (C) Western blotting showing Patched protein expression in total extracts from ACC cell lines H295R and MUC-1. B-tubulin was used as loading control. Histogram reports the quantification of Patched protein obtained from 3 western blots performed on 3 extracts of each cell line. Patched and ß-tubulin signals were quantified using ImageJ software. Student’s t-test performed showed a significant difference with a p value of 0.003.
☒
Figure 3. P375 enhances the cytotoxic effect of doxorubicin on ACC cells over- expressing Patched. (A) Treatment-dependent quantification of cell viability of H295R (left) and MUC-1 (right) cells upon addition of 2 uM doxorubicin (dxr) and different concentrations of P375. Stars represent statistical significance over cells incubated with 2 uM dxr. (B) Cell viability was measured after 48 hours treatment with serial dilutions of dxr with or without P375 on H295R cells. (C) dxr IC50 values calculated on H295R cells and H295R cells rendered resistant to dxr (H295RdxrR). IC50 of dxr of H295R and H295RdxrR cells are statistically different with a p value of 0.013. The difference between IC50 of dxr of H295R and H295RdxrR cells in the presence of P375 is not significant. (D) Western blotting showing Patched protein expression in total extracts from H295R and H295RdoxR cells. ß-tubulin was
used as loading control. Patched and ß-tubulin signals were quantified using ImageJ software. Student’s t-test performed showed a significant difference with a p value of 0.03. (E) P375 IC50 on H295R cell viability was measured after 48 hours treatment with serial dilutions of P375 with or without 2 uM dxr.
IC50 values were calculated from the mean of at least three experiments using Prism6 software.
Figure 4. P375 enhances the pro-apoptotic, anti-proliferative and anti-clonogenic effect of doxorubicin. (A) Cell apoptosis was evaluated using the luminescent assay Caspase-Glo 3/7 after 48 hours treatment with medium alone or with medium containing dxr alone, P375 alone, or dxr and P375 together. Histograms represent the mean ± SEM of 3 independent experiments and data were analyzed using Anova multiple comparison test and Bonferroni correction. Significance is attained at P < 0.05 (*) ( : P < 0.005, *: P < 0.0005). (B) For proliferation tests, cells were seeded in 96 well-plates and treated with medium containing serial dilutions of dxr in the presence or the absence of P375. After 7 days, Neutral Red was used for quantification of living cells. Data are represented as mean ± SEM. IC50 were calculated from the mean of at least three independent experiments using Prism6 software. (C) For clone formation tests, cells were seeded in 24 well-plates and treated with DMSO as control, dxr alone, P375 alone or a combination of dxr and P375. After 14 days, clones were revealed with crystal violet solution, pictures were taken and absorbance was read at 550 nm after solubilisation. Histograms represent the mean ± SEM of at least three independent experiments for each cell line and were analyzed using Anova multiple comparison test and Bonferroni correction. Significance is attained at P < 0.05 (*) ( ***: P < 0.0005).
Figure 5. P375 inhibits the doxorubicin efflux activity of Patched. (A) Cells were seeded on coverslips and incubated with dxr. After 2 hours, three coverslips were fixed for dxr loading control. The other coverslips (a triplicate per condition) were incubated with DMSO, P375 or the P-gp antagonist PSC833 for 30 min. and fixed. (B) Cells were seeded on coverslips and incubated with Hoechst. After 2 hours, three coverslips were fixed for Hoechst loading control. The other coverslips (a triplicate per condition) were incubated with DMSO, P375 or PSC833 for 30 min. and fixed. (C) H295R cells were transfected with Patched siRNA or negative control siRNA. Patched protein expression (right panel) and dxr efflux (left panel) were analyzed 16 hours after transfection. (D) H295R cells were transfected with P-gp siRNA or negative control siRNA. P-gp protein expression (right panel) and dxr efflux (left panel) were analyzed. ☒
Images were acquired with a fluorescence microscope using a 40X objective. Dxr or Hoechst fluorescence was quantified using ImageJ software for about 100 cells per condition per experiment. Patched, P-gp and ß-tubulin signals were also quantified using ImageJ software. All data presented are the mean ± SEM of 3 independent experiments and were analyzed using Anova multiple comparison test and Bonferroni correction. Significance is attained at P < 0.05 (*).
Figure 6. P375 enhances the efficiency of doxorubicin on H295R tumor bearing mice. ☒
☒
(A) Quantification of TUNEL positive cells in tumor slides after one therapeutic cycle. Stars denote significant differences compared with controls. (B) Dxr quantification in mice tumors after one therapeutic cycle was performed using fluorescence microscopy analysis of tissue ☒
slides and ImageJ software. Data presented are mean ± SEM and were analyzed using Student’s t-test. Significance is attained at P < 0.05 (*). (C) Evolution of the tumor size for the 4 groups of mice treated with two therapeutic cycles with different treatment modalities. The
☒
two-way repeated measures ANOVA analysis showed a significant difference between the group control and the group treated with P375 and lipdxr. (D) Dxr in mice tumors and hearts after two therapeutic cycles was quantified using fluorescence microscopy analysis of tissue slides and ImageJ software. Data presented are mean ± SEM and were analyzed using Student’s t-test. Significance is attained at P < 0.05 (*) ( **: P < 0.005). ☒
Accepted Arti
1.6 1.2 0.8 0.4 Yeast growth (Abs 600 nm) 0.0 Article Article Article
Patched expressing yeast
Control yeast
- Doxorubicin
+ Doxorubicin
+ Doxorubicin
DMSO P298
P375
DMSO
P375
20
40
60
80
0
20
40
60
80
0
20
40
60
Time (h)
Time (h)
Time (h)
*
100
ns
ns
60 Accepted 20 Accepted After cept Figure 1
Dxr fluorescence (%)
80
Loading
Efflux
Efflux +P375
Efflux +PSC833
Loading
Efflux
Efflux +P375
Patched expressing yeast
Control yeast
Article Article Figure 2 Accepted Article Accepted
Smoothened
650 micron
100 micron
foo micron
Normal adrenal
ACC, weak
ACC, intense
Patched
650 micron
100 micron
100 micron
Normal adrenal
ACC, weak
ACC, intense
300
H-score
200-
100-
0
Patched
Smoothened
H295R MUC-1
130 kDa-
Patched
55 kDa
ß tubulin
Patched protein (%)
100
I
80
60
40
**
20
I
0
A
NCI-H295R cell viability [% of 100% basal] Article Article
150
150
MUC-1 cell viability [% of 100% basal]
100
100
£
*
E
王
王
50
50
0
=
0
DXR [[M]
0
2
2
2
2
2
DXR [M]
0
2
2
2
2
2
P375 [uM]
0
0
1.25
5
12.5
50
P375 [uM]
0
0
1.25
5
12.5
50
C
Dxr alone
Dxr + 2.5 µM P375
6
Cell viability ( Abs)
F
IT
11
4
T
I
D
H295R
H295RdxrR
Accepted Accepted Accepted 0.8 0.4 Accepted Accepted Accepted Cell viability (Abs) 0.0 Figure 3
130 kDa
Patched
55 kDa
B-Tubulin
0.07
0.15
0.3
0.6
1.25
2.5
5
10
20
30
300
*
Patched (%)
Dxr (M)
200
100
0
1
0.0
0.5
1.0
2.0
2.5
3.7
5.0
7.5
10
15
20
P375 (UM)
| Dxr IC50 (M) | ||
|---|---|---|
| -P375 | + P375 | |
| H295R | 17.3 ± 3.6 | 2.6±0.2 |
| H295R Dxr R | 36 ± 6.5 | 4.2 ±0.7 |
| Dxr (μM) | P375IC50 (μM) |
|---|---|
| 0 | 18.5 ±3 |
| ☒ 2 | 3.4 ±0.8 |
*
Article Article Article Dxr: Article Article Article Article Article Article Article Article Article Article Caspase 3/7 activation
Cell viability (Abs 550nm) Accepted Article Clone formation (%)
12
+ DMSO
10
+ P375 1µM
+ P375 2uM
AAk
+ P375 3uM
Ak
AAck:
I
T
7
0
1 µM
2uM
- P375
+ 2uM P375
0.8
T
I
T
I
L
I
I
I
Accepted Accepted Accepted Accepted Accepted 100 80 60 40 20 Accepted Figure 4
0.000
0.005
0.010
0.020
0.040
0.060
0.080
0.120
0.250
Dxr (UM)
*
Y
T
I
DMSO
P375
Dxr
Dxr + P375
| P375 (μM) | IC50 Dxr (μM) |
|---|---|
| 0 | 0.1 |
| 1 | 0.05 |
| 1.5 | 0.03 |
| 2 | 0.015 |
After: 100 60 40 20 xr fluorescence % After: Article Article Article Article
B
Loading
Efflux
Efflux+P375
After: Loading
Efflux
Efflux +P375
Efflux +PSC833
Accepted Artis
100
ns
Hoechst fluorescence %
80
ns
T
60
40
T
T
T
T
20
0
Loading
Efflux
Efflux +P375
Efflux +PSC833 2.5UM
After:
Loading
Efflux
Efflux +P375
Efflux +PSC833
5uM
siCtrl
130 kDa
Patched
siPatched
55 kDa
ß-tubulin
100
Dxr fluorescence (%) Accepted Accepted Dxr fluorescence (%)
Patched protein (%)
**
ns
ns
50
100
80
0
sictrl
siPatched
60
40
20
After:
Loading
Efflux
Efflux + P375
siCtrl
130 kDa
P-gp
55 kDa
siP-gp
₿ tubulin
P-gp protein (%)
100
100
siCtrl
80
80
siP-gp
60
60
*
40
40
20
20
0
siCtl
siP-gp
0
After:
Loading
Dxr efflux
Figure 5
C Short term study Long term study Figure 6 Accept Art itemshoy Accepted
A
B
Number of apoptotic cells / mm2
100
*
*
Drx fluorescence (AU)
300
75
200
50
25
100
0
0
Ctrls
LipDxr
P375
LipDxr
LipDxr
P375
+ P375
+ LipDxr
1.00
Control
LipDxr
Tumor length width [cm2]
P375
0.75
P375+LipDxr
0.50
0.25
**
0.00
19
21
23
25
27
29
31
33
35
37
39
Time (Days after tumor induction)
400
In tumors
In hearts
600
Drx fluorescence (AU)
**
Drx fluorescence (AU)
ns
300
400
200
100
200
0
0
LipDxr
P375
LipDxr
P375
+ LipDxr
+ LipDxr