Optical coherence tomography (OCT) is a high-resolution, nondestructive imaging modality that enables time-serial assessment of adenoma development in the mouse model of colorectal cancer. In this study, OCT was utilized to evaluate the effectiveness of interventions with the experimental antitumor agent α-difluoromethylornithine (DFMO) and a nonsteroidal anti-inflammatory drug sulindac during early [chemoprevention (CP)] and late stages [chemotherapy (CT)] of colon tumorigenesis. Biological endpoints for drug interventions included OCT-generated tumor number and tumor burden. Immunochistochemistry was used to evaluate biochemical endpoints [Ki-67, cleaved caspase-3, cyclooxygenase (COX)-2, β-catenin]. K-Ras codon 12 mutations were studied with polymerase chain reaction-based technique. We demonstrated that OCT imaging significantly correlated with histological analysis of both tumor number and tumor burden for all experimental groups (P < 0.0001), but allows more accurate and full characterization of tumor number and burden growth rate because of its time-serial, nondestructive nature. DFMO alone or in combination with sulindac suppressed both the tumor number and tumor burden growth rate in the CP setting because of DFMO-mediated decrease in cell proliferation (Ki-67, P < 0.001) and K-RAS mutations frequency (P = 0.04). In the CT setting, sulindac alone and DFMO/sulindac combination were effective in reducing tumor number, but not tumor burden growth rate. A decrease in COX-2 staining in DFMO/sulindac CT groups (COX-2, P < 0.01) confirmed the treatment effect. Use of nondestructive OCT enabled repeated, quantitative evaluation of tumor number and burden, allowing changes in these parameters to be measured during CP and as a result of CT. In conclusion, OCT is a robust minimally invasive method for monitoring colorectal cancer disease and effectiveness of therapies in mouse models.

Previous studies suggest the anti-inflammatory drug, sulindac inhibits tumorigenesis by a COX independent mechanism involving cGMP PDE inhibition. Here we report that the cGMP PDE isozymes, PDE5 and 10, are elevated in colon tumor cells compared with normal colonocytes, and that inhibitors and siRNAs can selectively suppress colon tumor cell growth. Combined treatment with inhibitors or dual knockdown suppresses tumor cell growth to a greater extent than inhibition from either isozyme alone. A novel sulindac derivative, ADT-094 was designed to lack COX-1/-2 inhibitory activity but have improved potency to inhibit PDE5 and 10. ADT-094 displayed >500 fold higher potency to inhibit colon tumor cell growth compared with sulindac by activating cGMP/PKG signaling to suppress proliferation and induce apoptosis. Combined inhibition of PDE5 and 10 by treatment with ADT-094, PDE isozyme-selective inhibitors, or by siRNA knockdown also suppresses β-catenin, TCF transcriptional activity, and the levels of downstream targets, cyclin D1 and survivin. These results suggest that dual inhibition of PDE5 and 10 represents novel strategy for developing potent and selective anticancer drugs.

Microsatellite instability (MSI) is caused by DNA mismatch repair deficiency and is an important prognostic and predictive biomarker in colorectal cancer but relatively few studies have exploited mouse models in the study of its clinical utility. Furthermore, most previous studies have looked at MSI in the small intestine rather than the colon of mismatch repair deficient Msh2-knockout (KO) mice. Here we compared Msh2-KO, p53-KO, and wild type (WT) mice that were treated with the carcinogen azoxymethane (AOM) and the nonsteroidal anti-inflammatory drug sulindac or received no treatment. The induced tumors and normal tissue specimens from the colon were analysed with a panel of five mononucleotide repeat markers. MSI was detected throughout the normal colon in untreated Msh2-KO mice and this involved contraction of the repeat sequences compared to WT. The markers with longer mononucleotide repeats (37-59) were the most sensitive for MSI while the markers with shorter repeats (24) showed only minor change. AOM exposure caused further contraction of the Bat37 and Bat59 repeats in the distal colon of Msh2-KO mice which was reversed by sulindac. Thus AOM-induced carcinogenesis is associated with increased instability of mononucleotide repeats in the colon of Msh2-KO mice but not in WT or p53-KO mice. Chemoprevention of these tumors by sulindac treatment reversed or prevented the increased MSI.

Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronectin-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP.

Phospho-ibuprofen (MDC-917) and phospho-sulindac (OXT-328) are highly effective in cancer and arthritis treatment in preclinical models. Here, we investigated their metabolism by major human cytochrome P450s (CYPs) and flavin monooxygenases (FMOs).

Cancer stem cells (CSCs) are shown to be responsible for initiation and progression of tumors in a variety of cancers. We previously showed that anthocyanin-containing baked purple-fleshed potato (PP) extracts (PA) suppressed early and advanced human colon cancer cell proliferation and induced apoptosis, but their effect on colon CSCs is not known. Considering the evidence of bioactive compounds, such as anthocyanins, against cancers, there is a critical need to study anticancer activity of PP, a global food crop, against colon CSCs. Thus, isolated colon CSCs (positive for CD44, CD133 and ALDH1b1 markers) with functioning p53 and shRNA-attenuated p53 were treated with PA at 5.0 μg/ml. Effects of baked PP (20% wt/wt) against colon CSCs were also tested in vivo in mice with azoxymethane-induced colon tumorigenesis. Effects of PA/PP were compared to positive control sulindac. In vitro, PA suppressed proliferation and elevated apoptosis in a p53-independent manner in colon CSCs. PA, but not sulindac, suppressed levels of Wnt pathway effector β-catenin (a critical regulator of CSC proliferation) and its downstream proteins (c-Myc and cyclin D1) and elevated Bax and cytochrome c, proteins-mediating mitochondrial apoptosis. In vivo, PP reduced the number of crypts containing cells with nuclear β-catenin (an indicator of colon CSCs) via induction of apoptosis and suppressed tumor incidence similar to that of sulindac. Combined, our data suggest that PP may contribute to reduced colon CSCs number and tumor incidence in vivo via suppression of Wnt/β-catenin signaling and elevation of mitochondria-mediated apoptosis.

Aim: Autosomal dominant polycystic kidney disease is one of the most common genetic renal diseases. Cyclooxygenase plays an important role in epithelial cell proliferation and may contribute to the mechanisms underlying cyst formation. The aim of the present study was to evaluate the role of cyclooxygenase inhibition in the cyst progression in polycystic kidney disease. Method: Pkd2WS25/- mice, a murine model which harbors a compound cis-heterozygous mutation of the Pkd2 gene were used. Cyclooxygenase expression was assessed in both human and murine kidney specimens. Pkd2WS25/- mice were treated with Sulindac (a nonselective cyclooxygenase inhibitor) or vehicle for 8 months starting at three weeks age, and then renal cyst burden was assessed by kidney weight and volume. Results: Cyclooxygenase-2 expression was up-regulated compared to control kidneys as shown by RNase protection in human polycystic kidneys and immunoblot in mouse Pkd2WS25/- kidneys. Cyclooxygenase-2 expression was up-regulated in the renal interstitium as well as focal areas of the cystic epithelium (p<0.05). Basal Cyclooxygenase-1 levels were unchanged in both immunohistochemistry and real-time PCR. Administration of Sulindac to Pkd2WS25/- mice and to control mice for 8 months resulted in reduced kidney weights and volume in cystic mice. Renal function and electrolytes were not significantly different between groups. Conclusion: Thus treatment of a murine model of polycystic kidney disease with Sulindac results in decreased kidney cyst burden. These findings provide additional implications for the use of Cyclooxygenase inhibition as treatment to slow the progression of cyst burden in patients with polycystic kidney disease.

A variety of commercial analogs and a newer series of Sulindac derivatives were screened for inhibition of M. tuberculosis (Mtb) in vitro and specifically as inhibitors of the essential mycobacterial tubulin homolog, FtsZ. Due to the ease of preparing diverse analogs and a favorable in vivo pharmacokinetic and toxicity profile of a representative analog, the Sulindac scaffold may be useful for further development against Mtb with respect to in vitro bacterial growth inhibition and selective activity for Mtb FtsZ versus mammalian tubulin. Further discovery efforts will require separating reported mammalian cell activity from both antibacterial activity and inhibition of Mtb FtsZ. Modeling studies suggest that these analogs bind in a specific region of the Mtb FtsZ polymer that differs from human tubulin and, in combination with a pharmacophore model presented herein, future hybrid analogs of the reported active molecules that more efficiently bind in this pocket may improve antibacterial activity while improving other drug characteristics.

Tumor associated macrophages (TAMs) have a crucial role in cancer progression, metastasis and drug response. Piroxicam and sulindac sulfide are non-steroidal anti-inflammatory drugs (NSAID) that decrease the incidence and progression of several types of cancer. However, their role in suppressing the interactions between TAMs and cancer cells remain unclear. Herein, we studied the impact of human monocytes conditioned media (CM) on cellular proliferation of ER-dependent MCF-7 and ER-independent MDA-MB-231 cells, and the effects of piroxicam and sulindac sulfide on the expression levels of RAS, COX-2, IL-6, IL-1β and PAR-4 (qRT-PCR), BCL-2 and BAX (western blot), Caspase-3, VEGF-a and PGE2 (ELISA), MMP-2 and -9 (zymography) in the stimulated cells. Our results showed that CM caused a significant increase in cells survival through significant increase in RAS expression which resulted in upregulation of COX-2, PGE2, BCL-2, IL-6, IL-1β, VEGF-A and MMP-9 and down regulation of PAR-4. Treatment with one of the NSAIDs used in this study produced a time and concentration dependent growth inhibition of stimulated cells by inhibiting RAS expression. Suppression of RAS was accompanied by downregulation of its downstream signaling of IL-1β, IL-6, COX-2 and PGE2, activation of apoptotic machinery through upregulation of PAR-4 and caspase-3, as well as, inhibition of BCL-2, VEGF-A, MMP-2 and MMP-9. In conclusion, our data support the role of piroxicam and sulindac sulfide in suppressing inflammation-driven breast cancer progression and identifies promising novel target in RAS and PAR-4 signaling.

Endogenously formed prostacyclin (PGI2) and synthetic PGI2 analogues have recently been shown to regulate cell survival in various cell lines. To elucidate the significance of PGI2 in human breast cancer, we performed immunohistochemistry to analyze expression of prostacyclin-synthase (PGIS) in 248 human breast cancer specimens obtained from surgical pathology files. We examined patients' 10-year survival retrospectively by sending a questionnaire to their general practitioners and performed univariate analysis to determine whether PGIS expression correlated with patient survival. Lastly, the effects of PGI2 and its analogues on cell death were examined in a human breast cancer cell line (MCF-7) and a human T-cell leukemia cell line (CCRF-CEM). PGIS expression was observed in tumor cells in 48.7% of samples and was associated with a statistically significant reduction in 10-year survival (P = 0.038; n = 193). Transient transfection of PGIS into MCF-7 cells exposed to sulindac increased cell viability by 50% and exposure to carbaprostacyclin protected against sulindac sulfone induced apoptosis in CCRF-CEM cells. Expression of PGIS is correlated with a reduced patient survival and protects against cell death in vitro, suggesting that PGIS is a potential therapeutic target in breast cancer.

Macrophage apoptosis coupled with a defective phagocytic clearance of the apoptotic cells promotes plaque necrosis in advanced atherosclerosis, which causes acute atherothrombotic vascular disease. Nonsteroidal anti-inflammatory drug sulindac derivative K-80003 treatment was previously reported to dramatically attenuate atherosclerotic plaque progression and destabilization. However, the underlying mechanisms are not fully understood. This study aimed to determine the role of K-80003 on macrophage apoptosis and elucidate the underlying mechanism.

Methyloxantines are present in many herbs and vegetal foods, among them in tea, coffee and chocolate. Previous studies revealed that theophylline and theobromine have anti-angiogenic properties. Anti-tumor properties of theobromine were also described. Pentoxifylline (3,7-dimethyl-1-(5-oxohexyl)xanthine, PTX) is a synthetic xanthine derivative. It is a phosphodiesterase inhibitor and has various anti-inflammatory abilities. Pentoxifylline is widely used in therapy of inflammatory arterial diseases such as intermittent claudication of upper and lower limbs as well as in coronary heart disease. The aim of our research was to evaluate the effect of pentoxifylline (individually and in combination with non-steroidal anti-inflammatory drug sulindac), on L-1 sarcoma angiogenic activity and tumor formation in syngeneic Balb/c mice. Pre-incubation of tumor cells for 90 min with various PTX concentrations resulted in dose-dependent decrease of their ability to induce newly-formed blood vessels after transplantation into the skin of recipient mice. Administration of PTX to mice, recipients of tumor cells, slows tumor growth and reduces its volume. Synergistic inhibitory effect of PTX and sulindac, expressed as % of tumors sixth and thirteen day after subcutaneous grafting of L-1 sarcoma into syngeneic Balb/c mice, was observed.

Store-operated Ca2+ entry (SOCE) is the most important Ca2+ entry pathway in non-excitable cells. Colorectal cancer (CRC) shows decreased Ca2+ store content and enhanced SOCE that correlate with cancer hallmarks and are associated to remodeling of store-operated channels (SOCs). Normal colonic cells display small, Ca2+-selective currents driven by Orai1 channels. In contrast, CRC cells display larger, non-selective currents driven by Orai1 and transient receptor potential canonical type 1 channels (TRPC1). Difluoromethylornithine (DFMO), a suicide inhibitor of ornithine decarboxylase (ODC), the limiting step in polyamine biosynthesis, strongly prevents CRC, particularly when combined with sulindac. We asked whether DFMO may reverse SOC remodeling in CRC. We found that CRC cells overexpress ODC and treatment with DFMO decreases cancer hallmarks including enhanced cell proliferation and apoptosis resistance. Consistently, DFMO enhances Ca2+ store content and decreases SOCE in CRC cells. Moreover, DFMO abolish selectively the TRPC1-dependent component of SOCs characteristic of CRC cells and this effect is reversed by the polyamine putrescine. Combination of DFMO and sulindac inhibit both SOC components and abolish SOCE in CRC cells. Finally, DFMO treatment inhibits expression of TRPC1 and stromal interaction protein 1 (STIM1) in CRC cells. These results suggest that polyamines contribute to Ca2+ channel remodeling in CRC, and DFMO may prevent CRC by reversing channel remodeling.

The response to a traumatic experience may be rapid recovery or development of psychopathology such as post-traumatic stress disorder (PTSD). Impaired extinction of fear memories is thought to contribute to the development of the persistent trauma memories and avoidance. The Wnt/β-catenin pathway and the endocannabinoid system appear to play significant roles in anxiety and depressive symptoms. Here we examined the involvement of β-catenin in the nucleus accumbens (NAc) in extinction in rats exposed to the shock and reminders model of PTSD. We found that increased β-catenin levels in the NAc were correlated with facilitated extinction kinetics in rats exposed to shock and reminders, suggesting that increased levels of NAc β-catenin are associated with a resilient response to the stressor. Furthermore, downregulating β-catenin expression in the NAc in shocked rats using sulindac (0.0178, 0.178mg/side) impaired extinction whereas upregulating the Wnt/β-catenin pathway using LiCl (2µg/side) facilitated extinction. Exposure to shock and reminders resulted in attenuated levels of the endocannabinoid N-arachidonylethanolamine (AEA) in the NAc; the cannabinoid CB1/2 receptor agonist WIN55,212-2 (5µg/side) microinjected into the NAc facilitated extinction in shocked rats. Importantly, the facilitating effect of WIN55,212-2 on extinction was blocked by co-administration of sulindac in doses that downregulated β-catenin levels. Taken together, the results suggest that β-catenin in the NAc may serve as a protective buffer against the effects of severe stress, and that inhibiting this system in the NAc may prevent the therapeutic effects of cannabinoids against stress-related disorders.

Non-steroidal anti-inflammatory drugs (NSAIDs) display anti-inflammatory, antipyretic and analgesic properties by inhibiting cyclooxygenases and blocking prostaglandin production. Previous studies, however, suggested that some NSAIDs also modulate peroxisome proliferator activated receptors (PPARs), raising the possibility that such off target effects contribute to the spectrum of clinically relevant NSAID actions. In this study, we set out to understand how peroxisome proliferator activated receptor-γ (PPARγ/PPARG) interacts with NSAIDs using X-ray crystallography and to relate ligand binding modes to effects on receptor activity. We find that several NSAIDs (sulindac sulfide, diclofenac, indomethacin and ibuprofen) bind PPARγ and modulate PPARγ activity at pharmacologically relevant concentrations. Diclofenac acts as a partial agonist and binds to the PPARγ ligand binding pocket (LBP) in typical partial agonist mode, near the β-sheets and helix 3. By contrast, two copies of indomethacin and sulindac sulfide bind the LBP and, in aggregate, these ligands engage in LBP contacts that resemble agonists. Accordingly, both compounds, and ibuprofen, act as strong partial agonists. Assessment of NSAID activities in PPARγ-dependent 3T3-L1 cells reveals that NSAIDs display adipogenic activities and exclusively regulate PPARγ-dependent target genes in a manner that is consistent with their observed binding modes. Further, PPARγ knockdown eliminates indomethacin activities at selected endogenous genes, confirming receptor-dependence of observed effects. We propose that it is important to consider how individual NSAIDs interact with PPARγ to understand their activities, and that it will be interesting to determine whether high dose NSAID therapies result in PPAR activation.

Cyclodextrins (CDs) as a pseudophase in pseudophase-to-pseudophase microextraction (P2ME) in capillary zone electrophoresis (CZE) are proposed. In this P2ME mode called CD to admicelle ME, a long plug of dilute analyte solution prepared in cetyltrimethylammonium bromide (CTAB) at the critical micellar concentration was injected into the capillary. This formed CTAB admicelles at the interface between the solution and the negatively charged capillary surface, where the analytes were trapped. The injection of CD solution released the admicelles and the analytes from the capillary surface due to the formation of stable CD/CTAB inclusion complexes. The analytes are concentrated at the CD front during injection and voltage separation. Various neutral CDs were found to be effective for CD to admicelle ME. To implement this in-line sample concentration technique in CZE, CD concentration, sample injection time, and sample:CD solution injection ratio were optimized. The optimized conditions for five model anionic analytes, namely, 4-bromophenol, sulindac, sulfamethizole, 4-vinylbenzoic acid, and succinylsulfathiazole, were 20 mM α-CD in 20 mM sodium tetraborate (pH 9.2) solution, sample injection time of 370 s, and CD:sample injection ratio of 1:2. The sensitivity enhancement factors (SEFs) were between 112 and 168. The SEFs of sulindac and sulfamethizole in particular were similar to previously published off-line microextraction techniques, which are typically time-consuming. The calculated values of LOQ, intra-/inter-day (n = 6/n = 10, 3 days) repeatability, and linearity (R2) of CD to admicelle ME were 0.0125-0.05 µg/mL, 1.5-4.6%, 1.8-4.8%, and ≥0.999, respectively. Finally, the potential of CD to admicelle ME to the analysis of artificial urine samples was demonstrated.

Aldo-keto reductase 1C3 (AKR1C3) catalyses the NADPH dependent reduction of carbonyl groups in a number of important steroid and prostanoid molecules. The enzyme is also over-expressed in prostate and breast cancer and its expression is correlated with the aggressiveness of the disease. The steroid products of AKR1C3 catalysis are important in proliferative signalling of hormone-responsive cells, while the prostanoid products promote prostaglandin-dependent proliferative pathways. In these ways, AKR1C3 contributes to tumour development and maintenance, and suggest that inhibition of AKR1C3 activity is an attractive target for the development of new anti-cancer therapies. Non-steroidal anti-inflammatory drugs (NSAIDs) are one well-known class of compounds that inhibits AKR1C3, yet crystal structures have only been determined for this enzyme with flufenamic acid, indomethacin, and closely related analogues bound. While the flufenamic acid and indomethacin structures have been used to design novel inhibitors, they provide only limited coverage of the NSAIDs that inhibit AKR1C3 and that may be used for the development of new AKR1C3 targeted drugs. To understand how other NSAIDs bind to AKR1C3, we have determined ten crystal structures of AKR1C3 complexes that cover three different classes of NSAID, N-phenylanthranilic acids (meclofenamic acid, mefenamic acid), arylpropionic acids (flurbiprofen, ibuprofen, naproxen), and indomethacin analogues (indomethacin, sulindac, zomepirac). The N-phenylanthranilic and arylpropionic acids bind to common sites including the enzyme catalytic centre and a constitutive active site pocket, with the arylpropionic acids probing the constitutive pocket more effectively. By contrast, indomethacin and the indomethacin analogues sulindac and zomepirac, display three distinctly different binding modes that explain their relative inhibition of the AKR1C family members. This new data from ten crystal structures greatly broadens the base of structures available for future structure-guided drug discovery efforts.

Desmoid tumors are a rare group of locally aggressive, non malignant tumors of fibroblastic origin that can lead to significant morbidity due to local invasion. Despite advances in the understanding of these tumors, their natural history is incompletely understood and the optimal treatment is still a matter of debate. Local control is the main goal of treatment and there has been a change in philosophy regarding the management of these tumors from aggressive surgical resection to function preservation. A multidisciplinary approach is essential to plan local control with acceptable morbidity. The current Mayo Clinic algorithm for the treatment of these tumors is based on institutional experience and the available evidence in the literature: asymptomatic/non progressive lesions away from vital structures are managed with observation and regular imaging; primary or recurrent desmoid tumors which are symptomatic or progressive or near vital structures are managed with wide surgical resection when wide surgical margins are possible with minimal functional and cosmetic loss. When positive or close surgical margins are likely, surgical resection with adjuvant radiotherapy or definitive radiotherapy is preferred. If likely functional or cosmetic deficit is unacceptable, radiotherapy is the treatment of choice. Unresectable lesions are considered for radiotherapy, chemotherapy or newer modalities however an unresectable lesion associated with a painful, functionless, infected extremity is managed with an amputation.

Since colorectal cancer is one of the world's most common cancers, studies on its prevention and early diagnosis are an emerging area of clinical oncology these days. For this study, a review of randomized controlled, double-blind clinical trials of selected NSAIDs (aspirin, sulindac and celecoxib) in chemoprevention of colorectal cancer was conducted. The main molecular anticancer activity of NSAIDs is thought to be a suppression of prostaglandin E2 synthesis via cyclooxygenase-2 inhibition, which causes a decrease in tumor cell proliferation, angiogenesis, and increases apoptosis. The lower incidence of colorectal cancer in the NSAID patients suggests the long-lasting chemopreventive effect of drugs studied. This new approach to therapy of colorectal cancer may transform the disease from a terminal to a chronic one that can be taken under control.

Human multidrug resistance protein 4 (hMRP4, also known as ABCC4), with a representative topology of the MRP subfamily, translocates various substrates across the membrane and contributes to the development of multidrug resistance. However, the underlying transport mechanism of hMRP4 remains unclear due to a lack of high-resolution structures. Here, we use cryogenic electron microscopy (cryo-EM) to resolve its near-atomic structures in the apo inward-open and the ATP-bound outward-open states. We also capture the PGE1 substrate-bound structure and, importantly, the inhibitor-bound structure of hMRP4 in complex with sulindac, revealing that substrate and inhibitor compete for the same hydrophobic binding pocket although with different binding modes. Moreover, our cryo-EM structures, together with molecular dynamics simulations and biochemical assay, shed light on the structural basis of the substrate transport and inhibition mechanism, with implications for the development of hMRP4-targeted drugs.