A novel STAT3/ NFκB p50 axis regulates stromal-KDM2A to promote M2 macrophage-mediated chemoresistance in breast cancer.

BACKGROUND: Lysine Demethylase 2A (KDM2A) plays a crucial role in cancer cell growth, differentiation, metastasis, and the maintenance of cancer stemness. Our previous study found that cancer-secreted IL-6 can upregulate the expression of KDM2A to promote further the transition of cells into cancer-associated fibroblasts (CAFs). However, the molecular mechanism by which breast cancer-secreted IL-6 regulates the expression of KDM2A remains unclear. Therefore, this study aimed to elucidate the underlying molecular mechanism of IL-6 in regulating KDM2A expression in CAFs and KDM2A-mediated paclitaxel resistance in breast cancer. METHODS: The ectopic vector expression and biochemical inhibitor were used to analyze the KDM2A expression regulated by HS-578 T conditioned medium or IL-6 in mammary fibroblasts. Immunoprecipitation and chromatin immunoprecipitation assays were conducted to examine the interaction between STAT3 and NFκB p50. M2 macrophage polarization was assessed by analyzing M2 macrophage-specific markers using flow cytometry and RT-PCR. ESTIMATE algorithm was used to analyze the tumor microenvironment-dominant breast cancer samples from the TCGA database. The correlation between stromal KDM2A and CD163 + M2 macrophages was analyzed using the Pearson correlation coefficient. Cell viability was determined using trypan blue exclusion assay. RESULTS: IL-6 regulates gene expression via activation and dimerization of STAT3 or collaboration of STAT3 and NFκB. However, STAT3, a downstream transcription factor of the IL-6 signaling pathway, was directly complexed with NFκB p50, not NFκB p65, to upregulate the expression of KDM2A in CAFs. Enrichment analysis of immune cells/stromal cells using TCGA-breast cancer RNA-seq data unveiled a positive correlation between stromal KDM2A and the abundance of M2 macrophages. CXCR2-associated chemokines secreted by KDM2A-expressing CAFs stimulated M2 macrophage polarization, which in turn secreted CCL2 to increase paclitaxel resistance in breast cancer cells by activating CCR2 signaling. CONCLUSION: This study revealed the non-canonical molecular mechanism of IL-6 secreted by breast cancer upregulated KDM2A expression in CAFs via a novel STAT3/NFκB p50 axis, which STAT3 complexed with NFκB p50 in NFκB p50 binding motif of KDM2A promoter. KDM2A-expressing CAFs dominantly secreted the CXCR2-associated chemokines to promote M2 macrophage polarization and enhance paclitaxel resistance in breast cancer. These findings underscore the therapeutic potential of targeting the CXCR2 or CCR2 pathway as a novel strategy for paclitaxel-resistant breast cancer.

Redefine the role of d-α-Tocopheryl polyethylene glycol 1000 succinate on P-glycoprotein, multidrug resistance protein 1, and breast cancer resistance protein mediated cancer multidrug resistance.

Cancer drug resistance is an ever-changing problem that most patients need to face in their later stages of treatment, especially the multidrug resistant (MDR) type. The drug efflux transporters, including P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP), play the crucial roles in this sophisticated battle. In recent decades, researchers try to find potential inhibitors to impede the drug efflux function of above transporters. d-α-Tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) is a prevalently used excipient in the formulation design. In the present study, the modulatory effects and mechanisms of vitamin E TPGS on the efflux transporters were investigated. And the cancer MDR reversing ability of vitamin E TPGS was evaluated as well. Stable-cloned transporter over-expressed cell lines were used for mechanisms study, while several types of MDR cancer cell lines were adopted as reversing evaluation models. The results exhibited that vitamin E TPGS significantly inhibited the efflux function of P-gp, MRP1, and BCRP under non-cytotoxic concentrations, but not influencing the protein expression levels. Through efflux assay and molecular docking, vitamin E TPGS was found to be an uncompetitive, non-competitive, and competitive inhibitor on chemotherapeutic drug doxorubicin efflux in P-gp, MRP1, and BCRP over-expressing cell lines, respectively. Furthermore, the basal ATPase activity of three transporters were significantly inhibited by vitamin E TPGS at 10 µM. And the cell membrane fluidity of P-gp over-expressing cell line was enhanced by 22.58% with 5 µM vitamin E TPGS treatment, compared to the parental Flp-In™-293 cell line (without P-gp). The resistance reversing ability of vitamin E TPGS was prominent in MCF-7/DOX MDR breast cancer cell line, which over-expressed P-gp, MRP1, and BCRP. These significant results suggested that vitamin E TPGS is a promising modulator on transporters mediated cancer MDR. Vitamin E TPGS is not an inert excipient, but possesses MDR-reversing pharmacological effects, and deserves a re-purposing application on the future combinatorial regimen design for MDR cancer treatment.

Impaired Ventrolateral Periaqueductal Gray-Ventral Tegmental area Pathway Contributes to Chronic Pain-Induced Depression-Like Behavior in Mice.

Chronic pain conditions within clinical populations are correlated with a high incidence of depression, and researchers have reported their high rate of comorbidity. Clinically, chronic pain worsens the prevalence of depression, and depression increases the risk of chronic pain. Individuals suffering from chronic pain and depression respond poorly to available medications, and the mechanisms underlying the comorbidity of chronic pain and depression remain unknown. We used spinal nerve ligation (SNL) in a mouse model to induce comorbid pain and depression. We combined behavioral tests, electrophysiological recordings, pharmacological manipulation, and chemogenetic approaches to investigate the neurocircuitry mechanisms of comorbid pain and depression. SNL elicited tactile hypersensitivity and depression-like behavior, accompanied by increased and decreased glutamatergic transmission in dorsal horn neurons and midbrain ventrolateral periaqueductal gray (vlPAG) neurons, respectively. Intrathecal injection of lidocaine, a sodium channel blocker, and gabapentin ameliorated SNL-induced tactile hypersensitivity and neuroplastic changes in the dorsal horn but not depression-like behavior and neuroplastic alterations in the vlPAG. Pharmacological lesion of vlPAG glutamatergic neurons induced tactile hypersensitivity and depression-like behavior. Chemogenetic activation of the vlPAG-rostral ventromedial medulla (RVM) pathway ameliorated SNL-induced tactile hypersensitivity but not SNL-elicited depression-like behavior. However, chemogenetic activation of the vlPAG-ventral tegmental area (VTA) pathway alleviated SNL-produced depression-like behavior but not SNL-induced tactile hypersensitivity. Our study demonstrated that the underlying mechanisms of comorbidity in which the vlPAG acts as a gating hub for transferring pain to depression. Tactile hypersensitivity could be attributed to dysfunction of the vlPAG-RVM pathway, while impairment of the vlPAG-VTA pathway contributed to depression-like behavior.

Reversal of multidrug resistance by Fissistigma latifolium-derived chalconoid 2-hydroxy-4,5,6-trimethoxydihydrochalcone in cancer cell lines overexpressing human P-glycoprotein.

Cancer treatment is an evolving field with various challenges to clinical practice. One unresolved problem in this field is multidrug resistance (MDR) mediated by ABC efflux transporters, particularly P-glycoprotein (P-gp). In this study, by prescreening compounds, we identified the potential of a dihydrochalcone compound, 2-hydroxy-4,5,6-trimethoxydihydrochalcone, for P-gp inhibition. Therefore, we investigated its ability to inhibit P-gp and reverse P-gp-mediated MDR, as well as the underlying mechanisms. The P-gp-inhibitory effects of 2-hydroxy-4,5,6-trimethoxydihydrochalcone were investigated as follows. A P-gp efflux assay and an ATPase assay were used to understand the modulatory mechanisms in the drug-binding and ATP-binding areas, respectively. Prominent reversal effects observed in MDR cancer cell lines; thus, reversal, cytotoxicity, cell cycle, apoptosis, and reactive oxygen species assays were conducted to investigate the underlying mechanism. The results indicated that 2-hydroxy-4,5,6-trimethoxydihydrochalcone functionally inhibited P-gp in a noncompetitive manner, and this inhibition was unrelated to expression. In addition, 2-hydroxy-4,5,6-trimethoxydihydrochalcone served as an ATPase stimulator but not as a P-gp substrate. Moreover, a low binding energy of - 6.85 kcal/mol and one hydrogen bond were obtained, indicating that 2-hydroxy-4,5,6-trimethoxydihydrochalcone has a high affinity for P-gp. P-gp-mediated MDR was reversed by 31.6 µM 2-hydroxy-4,5,6-trimethoxydihydrochalcone in combination with paclitaxel, with a reversal fold value of 379.42. In conclusion, this study provides evidence of the ability of 2-hydroxy-4,5,6-trimethoxydihydrochalcone to inhibit P-gp and reverse MDR.

Novel application of rhein and its prodrug diacerein for reversing cancer-related multidrug resistance through the dual inhibition of P-glycoprotein efflux and STAT3-mediated P-glycoprotein expression.

Multidrug resistance (MDR) is a multifactorial issue in cancer treatment. Drug efflux transporters, particularly P-glycoprotein (P-gp), are major contributors to such resistance. In the present study, we evaluated the P-gp-inhibiting and MDR-reversing effects of two compounds, namely rhein, an anthraquinone, and diacerein, the acetylated prodrug of rhein. ABCB1/Flp-In-293 was used as a model for investigating the related molecular mechanisms, and the multi-drug-resistant cancer cell line KB/VIN was used as a platform for evaluating the reversal of MDR0. The results indicated that at a concentration of 2.5 µM, both diacerein and rhein significantly inhibited P-gp efflux function. They also downregulated P-gp expression by interacting with the signal transducer and activator of transcription 3. Further investigation of the inhibitory mechanism of these compounds revealed that both stimulated P-gp ATPase activity dose dependently and engaged in the noncompetitive inhibition of rhodamine 123 efflux. Furthermore, rhein was revealed to be a potent reverser of MDR in cancer, and the combination of 30 µM rhein and 1000 nM vincristine exerted a strong synergistic effect, achieving a high combination index (CI) of 0.092. Diacerein demonstrated potential applications as a selective cytotoxic agent against multi-drug-resistant cancer cells at a concentration of > 18.92 µM and as a mild MDR reverser at doses of < 10 µM. In conclusion, diacerein and rhein are potential candidates for P-gp inhibition and MDR reversal in cancer cells.

Repositioning application of polyoxyethylene (20) sorbitan monooleate on ocular drug resistance and cancer multi-drug resistance by inhibiting the ATPase activity of human multidrug resistance protein 1 and P-glycoprotein.

Drug efflux transporters were highly related to the clinical drug resistance issues, such as cancer multi-drug resistance (MDR) and ocular drug resistance. In the present study, with the focus on human multi-drug resistance protein 1 (MRP1) and P-glycoprotein (P-gp), the inhibitory kinetics of polyoxyethylene (20) sorbitan monooleate (Tween 80) on both drug binding sites and ATPase were in-depth evaluated. We used the stable-cloned ABCB1/Flp-In™-293 and ABCC1/Flp-In™-293 cell lines, and inside-out membrane vesicles for underlying mechanisms investigation while used the drug induced cancer MDR cell line KB/VIN and human retinal pigmented epithelium cell line ARPE-19 for efficacy evaluation. Results showed that Tween 80 exhibited non-competitive inhibition on the doxorubicin efflux of P-gp and MRP1, with the inhibitory affinity 0.00195% (14.89 µM) and 0.00245% (18.7 µM), respectively. Tween 80 inhibited the basal ATPase activity of P-gp and MRP1 in a dose-dependent manner (0.0002-0.02%) and demonstrated significant reversing effects on the doxorubicin, paclitaxel, and vincristine resistance at the concentration of 0.001% (7.63 µM). This was the first thorough study revealing the interactions between Tween 80 and P-gp or MRP1 at a molecular level and these findings suggested that Tween 80 was a potential candidate for future combinatorial regimens applied in the "drug resistance" issue.

Cinnamophilin overcomes cancer multi-drug resistance via allosterically modulating human P-glycoprotein on both drug binding sites and ATPase binding sites.

Cancer multi-drug resistance (MDR) caused by P-glycoprotein (P-gp) efflux is a critical unresolved clinical concern. The present study analyzed the effect of cinnamophilin on P-gp inhibition and MDR reversion. The effect of cinnamophilin on P-gp was investigated through drug efflux assay, ATPase assay, MDR1 shift assay, and molecular docking. The cancer MDR-reversing ability and mechanisms were analyzed through cytotoxicity and combination index (CI), cell cycle, and apoptosis experiments. P-gp efflux function was significantly inhibited by cinnamophilin without influencing the drug's expression or conformation. Cinnamophilin uncompetitively inhibited the efflux of doxorubicin and rhodamine 123 and exhibited a distinct binding behavior compared with verapamil, the P-gp standard inhibitor. The half maximal inhibitory concentration of cinnamophilin for doxorubicin and rhodamine 123 efflux was 12.47 and 11.59 µM, respectively. In regard to P-gp energy consumption, verapamil-stimulated ATPase activity was further enhanced by cinnamophilin at concentrations of 0.1, 1, 10, and 20 µM. In terms of MDR reversion, cinnamophilin demonstrated synergistic cytotoxic effects when combined with docetaxel, vincristine, or paclitaxel. The CI was < 0.7 in all experimental combination treatments. The present study showed that cinnamophilin possesses P-gp-modulating effects and cancer MDR resensitizing ability.

A novel flavonoid from Fissistigma cupreonitens, 5­hydroxy­7,8­dimethoxyflavanone, competitively inhibited the efflux function of human P-glycoprotein and reversed cancer multi-drug resistance.

BACKGROUND: P-glycoprotein (P-gp) over-expression plays a vital role in not only systemic drug bioavailability but also cancer multi-drug resistance (MDR). Develop functional inhibitors of P-gp can conquer both problems. PURPOSE AND STUDY DESIGN: The aim of the present study was to research the P-gp modulating effects and MDR reversing ability of a novel flavonoid from Fissistigma cupreonitens, the underlying inhibitory mechanisms were further elucidated as well. METHODS: Calcein-AM, rhodamine 123, and doxorubicin were fluorescent substrates for the evaluation of P-gp inhibitory function and detailed drug binding modes. Docking simulation was performed to reveal the in silico molecular bonding. ATPase assay and MDR1 shift assay were adopted to reveal the ATP consumption and conformational change of P-gp. The MDR reversing effects were demonstrated through cytotoxicity, cell cycle, and apoptosis analyses. RESULTS: 5­hydroxy­7,8­dimethoxyflavanone inhibited the efflux of rhodamine 123 and doxorubicin in a competitive manner, and increased the intracellular fluorescence of calcein at a concentration as low as 2.5 µg/ml. 5­hydroxy­7,8­dimethoxyflavanone slightly changed P-gp's conformation and only stimulated ATPase at very high concentration (100 µg/ml). The docking results showed that 5­hydroxy­7,8­dimethoxyflavanone and verapamil exhibited similar binding affinity to P-gp. The MDR reversing effects were prominent in the vincristine group, the reversal folds were 23.01 and 13.03 when combined with 10 µg/ml 5­hydroxy­7,8­dimethoxyflavanone in the P-gp over-expressing cell line (ABCB1/Flp-In™-293) and MDR cancer cell line (KB/VIN), respectively. CONCLUSION: The present study demonstrated that 5­hydroxy­7,8­dimethoxyflavanone was a novel effective flavonoid in the P-gp efflux inhibition and in vitro cancer MDR reversion.

Caffeic Acid Attenuates Multi-Drug Resistance in Cancer Cells by Inhibiting Efflux Function of Human P-glycoprotein.

: Multidrug resistance (MDR) is a complicated ever-changing problem in cancer treatment, and P-glycoprotein (P-gp), a drug efflux pump, is regarded as the major cause. In the way of developing P-gp inhibitors, natural products such as phenolic acids have gotten a lot of attention recently. The aim of the present study was to investigate the modulating effects and mechanisms of caffeic acid on human P-gp, as well as the attenuating ability on cancer MDR. Calcein-AM, rhodamine123, and doxorubicin were used to analyze the interaction between caffeic acid and P-gp, and the ATPase activity of P-gp was evaluated as well. Resistance reversing effects were revealed by SRB and cell cycle assay. The results indicated that caffeic acid uncompetitively inhibited rhodamine123 efflux and competitively inhibited doxorubicin efflux. In terms of P-gp ATPase activity, caffeic acid exhibited stimulation in both basal and verapamil-stimulated activity. The combination of chemo drugs and caffeic acid resulted in decreased IC50 in ABCB1/Flp-InTM-293 and KB/VIN, indicating that the resistance was reversed. Results of molecular docking suggested that caffeic acid bound to P-gp through GLU74 and TRY117 residues. The present study demonstrated that caffeic acid is a promising candidate for P-gp inhibition and cancer MDR attenuation.

Zhankuic Acids A, B and C from Taiwanofungus Camphoratus Act as Cytotoxicity Enhancers by Regulating P-Glycoprotein in Multi-Drug Resistant Cancer Cells.

: Since P-glycoprotein (P-gp)-related multidrug resistance (MDR) remains the most important unsolved problem in cancer treatment, scientists are attempting to find potential structures from natural resources. The aim of the present study was to elucidate whether the triterpenoids from Taiwanofungus camphoratus could reverse cancer MDR by influencing P-gp efflux pump. Substrates efflux assay and P-gp ATPase activity assay were conducted to reveal the molecular mechanisms of P-gp inhibition, while SRB assay, cell cycle analyses and apoptosis analyses were performed to confirm the cancer MDR modulating effects. The results indicated that Zhankuic acids A, B and C (ZA-A, ZA-B and ZA-C) impacted P-gp efflux function in competitive, noncompetitive and competitive manners, respectively. Furthermore, these triterpenoids all demonstrated inhibitory patterns on both basal P-gp ATPase activity and verapamil-stimulated ATPase activity. In terms of MDR reversal effects, ZA-A sensitized the P-gp over-expressing cell line (ABCB1/Flp-InTM-293) and MDR cancer cell line (KB/VIN) toward clinically used chemotherapeutic drugs, including doxorubicin, paclitaxel and vincristine, exhibiting the best cytotoxicity enhancing ability among investigated triterpenoids. The present study demonstrated that ZA-A, ZA-B and ZA-C, popular triterpenoids from T. camphoratus, effectively modulated the drug efflux transporter P-gp and reversed the cancer MDR issue.

Danazol mediates collateral sensitivity via STAT3/Myc related pathway in multidrug-resistant cancer cells.

Multidrug resistance presents an obstacle in cancer treatment. Among numerous combative strategies, collateral sensitivity (CS) drugs have opened a new avenue to defeat cancer by exploiting selective toxicity against multidrug-resistant (MDR) cancer. In the present study, a clinically used synthetic steroid hormone, danazol, was investigated for its CS properties and cytotoxic mechanisms. Compared with natural hormones, danazol possessed a stronger selective cytotoxicity against MDR cancer cells. Danazol induced the arrest of MDR cancer cells at the G2/M phase and caspase-8-related early apoptosis. Furthermore, in MDR cancer cells, danazol reduced STAT3 phosphorylation as well as the expression of STAT3-regulated genes involved in cell survival, such as c-Myc, CDC25, and CDK1. Danazol also upregulated the cell cycle inhibitor p21 in MDR cancer cells. Supporting the experimental results, docking studies have revealed that danazol can likely bind favourably with STAT3. Taken together, our results suggest that danazol exerts a CS effect by inhibiting the STAT3 pathway in MDR cancer cells and thus provides a possible solution for MDR cancers.

Development of novel amino-quinoline-5,8-dione derivatives as NAD(P)H:quinone oxidoreductase 1 (NQO1) inhibitors with potent antiproliferative activities.

Fourteen novel amino-quinoline-5,8-dione derivatives (6a-h and 7a-h) were designed and synthesized by coupling different alkyl- or aryl-amino fragments at the C6- or C7-position of quinoline-5,8-dione. All target compounds showed antiproliferative potency in the low micromolar range in both drug sensitive HeLaS3 and multidrug resistant KB-vin cell lines. Compounds 6h, 6d, 7a, and 7d exhibited more potent antiproliferative effects than the other compounds. Especially, compounds 6d and 7d displayed NQO1-dependent cytotoxicity and competitive NQO1 inhibitory effects in both drug sensitive HeLaS3 and multidrug resistant KB-vin cell lines. Furthermore, compounds 6h, 6d, 7a, and 7d induced a dose-dependent lethal mitochondrial dysfunction in both drug sensitive HeLaS3 and multidrug resistant KB-vin cells by increasing intracellular reactive oxygen species (ROS) levels. Notably, compound 7d selectively inhibited cancer cells, but not non-tumor liver cell proliferation in vitro, and significantly triggered HeLaS3 cell apoptosis by regulating apoptotic proteins of Bcl-2, Bax, and cleaved caspase-3 in a dose-dependent manner. Our findings suggest that these novel C6- or C7-substituted amino-quinoline-5,8-dione derivatives, such as 7d, could be further developed in the future as potent and selective antitumor agents to potentially circumvent multi-drug resistance (MDR).

Mechanism of action of cytotoxic compounds from the seeds of Euphorbia lathyris.

BACKGROUND: The seeds of Euphorbia lathyris are used in traditional Chinese medicines for the treatment of various medical conditions. E. lathyris contains many natural diterpenes with a lathyrane skeleton. PURPOSE AND STUDY DESIGN: Five lathyrane-type diterpenoids named Euphorbia factors L1, L2, L3, L8, and L9 (1-5), were investigated for cytotoxicity against A549, MDA-MB-231, KB, and MCF-7 cancer cell lines and the KB-VIN multidrug resistant (MDR) cancer cell line. Also, a tetraol derivative (6) of Euphorbia factor L2 (2) was synthesized to assess the effect of hydroxy moieties. METHODS: An ethanolic extract of seeds of Euphorbia lathyris was prepared and separated into petroleum ether, EtOAc, n-butanol, and n-hexane extracts. The natural diterpenes were isolated by using silica gel and Sephadex LH-20 column chromatography as well as preparative thin-layer chromatography. Saponification of 2 gave tetraol derivative 6. Cytotoxic activity was determined by the sulforhodamine B (SRB) colorimetric assay. Mechanism of action studies focused on the impact of compounds on the cell cycle progression as well as cell morphology. RESULTS: Compound 5 exhibited the strongest cytotoxicity against all cell lines, while compound 2 showed selectivity against KB-VIN. In cells treated with 3 and 5, accumulation of G1 to early S phase cells was obvious, while no effect was seen on G2/M phase. CONCLUSION: Analysis of the screening data compared with compound structures suggested that the substitutions at C-3, C-5, C-7, and C-15 are critical for cytotoxicity, as well as cell type-selectivity. Furthermore, results of cytotoxic mechanism analysis demonstrated for the first time that compounds 3 and 5 disrupted normal cell cycle progression, whereas compounds 2‒5 induced obvious actin filament aggregation, as well as partial interference of the microtubule network.

ß-carotene reverses multidrug resistant cancer cells by selectively modulating human P-glycoprotein function.

BACKGROUND: The issue of multidrug resistance (MDR) cancer is one of the major barriers to successful chemotherapy treatment. The ATP-binding cassette (ABC) efflux transporters play an important role in the chemotherapeutic failure. Several generations of ABC efflux transporter inhibitors have been developed, however, none of them could provide better clinical outcome due to systemic toxicities and significant drug-drug interactions. Therefore, the present study focused on identifying the effect of the natural carotenoid on ABC transporters and may provide a safer choice to defeat MDR cancer. PURPOSE: The aim of the present study was to evaluate the inhibitory potency of ß-carotene on the ABC efflux transporters, as well as the reversal effect of ß-carotene toward MDR cancers. The underlying molecular mechanisms and inhibitory kinetics of ß-carotene on the major ABC efflux transporter, P-glycoprotein, were further investigated. METHODS: The human P-gp (ABCB1/Flp-In(TM)-293), MRP1 (ABCC1/Flp-In(TM)-293) and BCRP (ABCG2/Flp-In(TM)-293) stable expression cells were established by using the Flp-In(TM) system. The cytotoxicity of ß-carotene was evaluated by MTT assay in the established cell lines, sensitive cancer cell lines (HeLaS3 and NCI-H460) and resistant cancer cell lines (KB-vin and NCI-H460/MX20). Surface protein detection assay and eFluxx-ID Green Dye assay were applied for confirmation of surface expression and function of the transporters. The transporter inhibition potency of ß-carotene was evaluated by calcein-AM uptake assay and mitoxantrone accumulation assay. Further interaction kinetics between ß-carotene and P-gp were analyzed by rhodamine123 and doxorubicin efflux assay. The influence of ß-carotene on ATPase activity was evaluated by Pgp-Glo(TM) Assay System. RESULTS: Among the tested ABC efflux transporters, ß-carotene significantly inhibited human P-gp efflux function without altering ABCB1 mRNA expression. Furthermore, ß-carotene stimulated both P-gp basal ATPase activity and the verapamil-stimulated P-gp ATPase activity. In addition, ß-carotene exerted partially inhibitory effect on BCRP efflux function. The combination of ß-carotene and chemotherapeutic agents significantly potentiated their cytotoxicity in both cell stably expressed human P-gp (ABCB1/Flp-In(TM)-293) and MDR cancer cells (KB-vin and NCI-H460/MX20). CONCLUSION: The present study indicated that ß-carotene may be considered as a chemo-sensitizer and regarded as an adjuvant therapy in MDR cancer treatment.

Demethoxycurcumin modulates human P-glycoprotein function via uncompetitive inhibition of ATPase hydrolysis activity.

Curcuminoids are major components of Curcuma longa L., which is widely used as spice in food. This study aimed at identifying whether curcumin, demethoxycurcumin, and bisdemethoxycurcumin could modulate efflux function of human P-glycoprotein and be used as chemosensitizers in cancer treatments. Without altering P-glycoprotein expression levels and conformation, the purified curcuminoids significantly inhibited P-glycoprotein efflux function. In rhodamine 123 efflux and calcein-AM accumulation assays, demethoxycurcumin demonstrated the highest inhibition potency (inhibitory IC50 = 1.56 ± 0.13 µM) among the purified curcuminoids, as well as in the fold of reversal assays. Demethoxycurcumin inhibited P-glycoprotein-mediated ATP hydrolysis under concentrations of <1 µM and efficiently inhibited 200 µM verapamil-stimulated ATPase activity, indicating a high affinity of demethoxycurcumin for P-glycoprotein. These results suggested that demethoxycurcumin may be a potential additive natural product in combination with chemotherapeutic agents in drug-resistant cancers.

The functional influences of common ABCB1 genetic variants on the inhibition of P-glycoprotein by Antrodia cinnamomea extracts.

Antrodia cinnamomea is a traditional healthy food that has been demonstrated to possess anti-inflammatory, antioxidative, and anticacer effects. The purpose of this study was to evaluate whether the ethanolic extract of A. cinnamomea (EEAC) can affect the efflux function of P-glycoprotein (P-gp) and the effect of ABCB1 genetic variants on the interaction between EEAC and P-gp. To investigate the mechanism of this interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established and the expression of P-gp was confirmed by Western blot. The results of the rhodamine 123 efflux assay demonstrated that EEAC efficiently inhibited wild-type P-gp function at an IC50 concentration of 1.51 ± 0.08 µg/mL through non-competitive inhibition. The IC50 concentrations for variant-type 1236T-2677T-3435T P-gp and variant-type 1236T-2677A-3435T P-gp were 5.56 ± 0.49 µg/mL and 3.33±0.67 µg/mL, respectively. In addition, the inhibition kinetics of EEAC also changed to uncompetitive inhibition in variant-type 1236T-2677A-3435T P-gp. The ATPase assay revealed that EEAC was an ATPase stimulator and was capable of reducing verapamil-induced ATPase levels. These results indicate that EEAC may be a potent P-gp inhibitor and higher dosages may be required in subjects carrying variant-types P-gp. Further studies are required to translate this basic knowledge into clinical applications.

Functional impact of ABCB1 variants on interactions between P-glycoprotein and methadone.

Methadone is a widely used substitution therapy for opioid addiction. Large inter-individual variability has been observed in methadone maintenance dosages and P-glycoprotein (P-gp) was considered to be one of the major contributors. To investigate the mechanism of P-gp's interaction with methadone, as well as the effect of genetic variants on the interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established in the present study. The RNA and protein expression levels of human P-gp were confirmed by real-time quantitative RT-PCR and western blot, respectively. Utilizing rhodamine 123 efflux assay and calcein-AM uptake study, methadone was demonstrated to be an inhibitor of wild-type human P-gp via non-competitive kinetic (IC50 = 2.17±0.10 µM), while the variant-type human P-gp, P-gp with 1236T-2677T-3435T genotype and P-gp with 1236T-2677A-3435T genotype, showed less inhibition potency (IC50 = 2.97±0.09 µM and 4.43±1.10 µM, respectively) via uncompetitive kinetics. Methadone also stimulated P-gp ATPase and inhibited verapamil-stimulated P-gp ATPase activity under therapeutic concentrations. These results may provide a possible explanation for higher methadone dosage requirements in patients carrying variant-type of P-gp and revealed the possible drug-drug interactions in patients who receive concomitant drugs which are also P-gp substrates.