Identification and Clinical Significance of Pancreatic Cancer Stem Cells and Their Chemotherapeutic Drug Resistance.

Pancreatic cancer ranks in the 10th-11th position among cancers affecting men in Taiwan, besides being a rather difficult-to-treat disease. The overall 5-year survival rate of pancreatic cancer is only 5-10%, while that of resectable pancreatic cancer is still approximately 15-20%. Cancer stem cells possess intrinsic detoxifying mechanisms that allow them to survive against conventional therapy by developing multidrug resistance. This study was conducted to investigate how to overcome chemoresistance and its mechanisms in pancreatic cancer stem cells (CSCs) using gemcitabine-resistant pancreatic cancer cell lines. Pancreatic CSCs were identified from human pancreatic cancer lines. To determine whether CSCs possess a chemoresistant phenotype, the sensitivity of unselected tumor cells, sorted CSCs, and tumor spheroid cells to fluorouracil (5-FU), gemcitabine (GEM), and cisplatin was analyzed under stem cell conditions or differentiating conditions. Although the mechanisms underlying multidrug resistance in CSCs are poorly understood, ABC transporters such as ABCG2, ABCB1, and ABCC1 are believed to be responsible. Therefore, we measured the mRNA expression levels of ABCG2, ABCB1, and ABCC1 by real-time RT-PCR. Our results showed that no significant differences were found in the effects of different concentrations of gemcitabine on CSCs CD44+/EpCAM+ of various PDAC cell line cultures (BxPC-3, Capan-1, and PANC-1). There was also no difference between CSCs and non-CSCs. Gemcitabine-resistant cells exhibited distinct morphological changes, including a spindle-shaped morphology, the appearance of pseudopodia, and reduced adhesion characteristics of transformed fibroblasts. These cells were found to be more invasive and migratory, and showed increased vimentin expression and decreased E-cadherin expression. Immunofluorescence and immunoblotting experiments demonstrated increased nuclear localization of total ß-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells showed activation of the receptor protein tyrosine kinase c-Met and increased expression of the stem cell marker cluster of differentiation (CD) 24, CD44, and epithelial specific antigen (ESA). We concluded that the expression of the ABCG2 transporter protein was significantly higher in CD44+ and EpCAM+ CSCs of PDAC cell lines. Cancer stem-like cells exhibited chemoresistance. Gemcitabine-resistant pancreatic tumor cells were associated with EMT, a more aggressive and invasive phenotype of numerous solid tumors. Increased phosphorylation of c-Met may also be related to chemoresistance, and EMT and could be used as an attractive adjunctive chemotherapeutic target in pancreatic cancer.

Characterization of Potent ABCG2 Inhibitor Derived from Chromone: From the Mechanism of Inhibition to Human Extracellular Vesicles for Drug Delivery.

Inhibition of ABC transporters is a promising approach to overcome multidrug resistance in cancer. Herein, we report the characterization of a potent ABCG2 inhibitor, namely, chromone 4a (C4a). Molecular docking and in vitro assays using ABCG2 and P-glycoprotein (P-gp) expressing membrane vesicles of insect cells revealed that C4a interacts with both transporters, while showing selectivity toward ABCG2 using cell-based transport assays. C4a inhibited the ABCG2-mediated efflux of different substrates and molecular dynamic simulations demonstrated that C4a binds in the Ko143-binding pocket. Liposomes and extracellular vesicles (EVs) of Giardia intestinalis and human blood were used to successfully bypass the poor water solubility and delivery of C4a as assessed by inhibition of the ABCG2 function. Human blood EVs also promoted delivery of the well-known P-gp inhibitor, elacridar. Here, for the first time, we demonstrated the potential use of plasma circulating EVs for drug delivery of hydrophobic drugs targeting membrane proteins.

MET inhibitor tepotinib antagonizes multidrug resistance mediated by ABCG2 transporter: In vitro and in vivo study.

Overexpression of ABCG2 transporter in cancer cells has been linked to the development of multidrug resistance (MDR), an obstacle to cancer therapy. Our recent study uncovered that the MET inhibitor, tepotinib, is a potent reversal agent for ABCB1-mediated MDR. In the present study, we reported for the first time that the MET inhibitor tepotinib can also reverse ABCG2-mediated MDR in vitro and in vivo by directly binding to the drug-binding site of ABCG2 and reversibly inhibiting ABCG2 drug efflux activity, therefore enhancing the cytotoxicity of substrate drugs in drug-resistant cancer cells. Furthermore, the ABCB1/ABCG2 double-transfected cell model and ABCG2 gene knockout cell model demonstrated that tepotinib specifically inhibits the two MDR transporters. In mice bearing drug-resistant tumors, tepotinib increased the intratumoral accumulation of ABCG2 substrate drug topotecan and enhanced its antitumor effect. Therefore, our study provides a new potential of repositioning tepotinib as an ABCG2 inhibitor and combining tepotinib with substrate drugs to antagonize ABCG2-mediated MDR.

Targeting breast cancer resistance protein (BCRP/ABCG2): Functional inhibitors and expression modulators.

The primary source of failure of cancer therapies is multidrug resistance (MDR), which can be caused by different mechanisms, including the overexpression of ABC transporters in cancer cells. Among the 48 human ABC proteins, the breast cancer resistance protein (BCRP/ABCG2) has been described as a pivotal player in cancer resistance. The use of functional inhibitors and expression modulators is a promising strategy to overcome the MDR caused by ABCG2. Despite the lack of clinical trials using ABCG2 inhibitors, many compounds have already been discovered. This review presents an overview about various ABCG2 inhibitors that have been identified, discussing some chemical aspects and the main experimental methods used to identify and characterize the mechanisms of new inhibitors. In addition, some biological requirements to pursue preclinical tests are described. Finally, we discuss the potential use of ABCG2 inhibitors in cancer stem cells (CSC) for improving the objective response rate and the mechanism of ABCG2 modulators at transcriptional and protein expression levels.

Methods to Measure the Inhibition of ABCG2 Transporter and Ferrochelatase Activity to Enhance Aminolevulinic Acid-Protoporphyrin IX Fluorescence-Guided Tumor Detection and Resection.

Aminolevulinic acid (ALA) has been clinically used as an intraoperative fluorescence probe for protoporphyrin IX (PpIX) fluorescence-guided tumor resection and a PDT agent for cancer treatment. Although tumor tissues often show increased ALA-PpIX fluorescence compared with normal tissues, which enables the use of ALA for tumor imaging and targeting, weak tumor PpIX fluorescence as well as the heterogeneity in tumor fluorescence severely limits its clinical application. Intracellular PpIX in tumor cells is reduced by two major mechanisms, efflux by ATP-binding cassette (ABC) transporters such as ABCG2 and bioconversion to form heme by ferrochelatase (FECH) in the heme biosynthesis pathway. Targeting these two predominant PpIX-reducing mechanisms for the enhancement of ALA-PpIX have yielded a plethora of promising results and stimulated the clinical exploration of these enhancement strategies. Here we describe our methods of evaluating chemicals for the inhibition of ABCG2 transporter and FECH activity. Our goal is to further encourage research and development of novel ABCG2 and FECH inhibitors and promote a rational use of these inhibitors to optimize ALA-based tumor detection and treatment.

MET inhibitor tepotinib antagonizes multidrug resistance mediated by ABCG2 transporter: In vitro and in vivo study

Overexpression of ABCG2 transporter in cancer cells has been linked to the development of multidrug resistance (MDR), an obstacle to cancer therapy. Our recent study uncovered that the MET inhibitor, tepotinib, is a potent reversal agent for ABCB1-mediated MDR. In the present study, we reported for the first time that the MET inhibitor tepotinib can also reverse ABCG2-mediated MDR in vitro and in vivo by directly binding to the drug-binding site of ABCG2 and reversibly inhibiting ABCG2 drug efflux activity, therefore enhancing the cytotoxicity of substrate drugs in drug-resistant cancer cells. Furthermore, the ABCB1/ABCG2 double-transfected cell model and ABCG2 gene knockout cell model demonstrated that tepotinib specifically inhibits the two MDR transporters. In mice bearing drug-resistant tumors, tepotinib increased the intratumoral accumulation of ABCG2 substrate drug topotecan and enhanced its antitumor effect. Therefore, our study provides a new potential of repositioning tepotinib as an ABCG2 inhibitor and combining tepotinib with substrate drugs to antagonize ABCG2-mediated MDR.

Predictors of therapeutic efficacy of 5-aminolevulinic acid-based photodynamic therapy in human prostate cancer.

BACKGROUND: Photodynamic therapy (PDT) is a minimally invasive cancer therapy. However, its therapeutic efficacy for prostate cancer is not yet fully understood. In this study, the predictors of therapeutic efficacy of 5-aminolevulinic acid-based PDT (ALA-PDT) on prostate cancer cells are investigated. MATERIALS AND METHODS: The human prostate cancer cell lines, PC-3, 22Rv1, DU145, and LNCap were used to investigate the effects of ALA-PDT on protoporphyrin IX (PpIX) intracellular accumulation, which was measured by flow cytometry. The cytotoxicity of ALA-PDT was evaluated by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. The levels of porphyrin metabolism-related enzyme and transporter mRNA were comprehensively evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was evaluated by Western blot. A xenograft model was created using PC-3 and 22Rv1, and then, pathological analysis was performed to determine the therapeutic effect of ALA-PDT RESULTS: PC-3 and LNCap cells showed high accumulation of PpIX and high sensitivity to ALA-PDT, while 22Rv1 and DU145 showed low accumulation of PpIX and low sensitivity to ALA-PDT. ALA-PDT-induced cytotoxicity correlated negatively with PpIX accumulation. The in vitro assays identified the ATP-binding cassette transporter subfamily G2 (ABCG2) transporter dimer as a predictor of treatment response. In vivo immunohistochemical staining of ABCG2 transporter showed low expression in PC-3 cells and high expression in 22Rv1 cells, and ALA-PDT-induced tumor tissue degeneration was greater in PC-3 cells than in 22Rv1 cells. CONCLUSION: The ABCG2 transporter is a useful predictor of the therapeutic effect of ALA-PDT on human prostate cancer cells.

Water-soluble inhibitors of ABCG2 (BCRP) - A fragment-based and computational approach.

A good balance between hydrophilicity and lipophilicity is a prerequisite for all bioactive compounds. If the hydrophilicity of a compound is low, its solubility in water will be meager. Many drug development failures have been attributed to poor aqueous solubility. ABCG2 inhibitors are especially prone to be insoluble since they have to address the extremely large and hydrophobic multidrug binding site in ABCG2. For instance, our previous, tariquidar-related ABCG2 inhibitor UR-MB108 (1) showed high potency (79 nM), but very low aqueous solubility (78 nM). To discover novel potent ABCG2 inhibitors with improved solubility we pursued a fragment-based approach. Substructures of 1 were optimized and the fragments 'enlarged' to obtain inhibitors, supported by molecular docking studies. Synthesis was achieved, i.a., via Sonogashira coupling, click chemistry and amide coupling. A kinetic solubility assay revealed that 1 and most novel inhibitors did not precipitate during the short time period of the applied biological assays. The solubility of the compounds in aqueous media at equilibrium was investigated in a thermodynamic solubility assay, where UR-Ant116 (40), UR-Ant121 (41), UR-Ant131 (48) and UR-Ant132 (49) excelled with solubilities between 1 µM and 1.5 µM - an up to 19-fold improvement compared to 1. Moreover, these novel N-phenyl-chromone-2-carboxamides inhibited ABCG2 in a Hoechst 33342 transport assay with potencies in the low three-digit nanomolar range, reversed MDR in cancer cells, were non-toxic and proved stable in blood plasma. All properties make them attractive candidates for in vitro assays requiring long-term incubation and in vivo studies, both needing sufficient solubility at equilibrium. 41 and 49 were highly ABCG2-selective, a precondition for developing PET tracers. The triple ABCB1/C1/G2 inhibitor 40 qualifies for potential therapeutic applications, given the concerted role of the three transporter subtypes at many tissue barriers, e.g. the BBB.

Tariquidar-related triazoles as potent, selective and stable inhibitors of ABCG2 (BCRP).

Tariquidar derivatives have been described as potent and selective ABCG2 inhibitors. However, their susceptibility to hydrolysis limits their applicability. The current study comprises the synthesis and characterization of novel tariquidar-related inhibitors, obtained by bioisosteric replacement of the labile moieties in our previous tariquidar analog UR-ME22-1 (9). CuAAC ("click" reaction) gave convenient access to a triazole core as a substitute for the labile amide group and the labile ester moiety was replaced by different acyl groups in a Sugasawa reaction. A stability assay proved the enhancement of the stability in blood plasma. Compounds UR-MB108 (57) and UR-MB136 (59) inhibited ABCG2 in a Hoechst 33342 transport assay with an IC50 value of about 80 nM and belong to the most potent ABCG2 inhibitors described so far. Compound 57 was highly selective, whereas its PEGylated analog 59 showed some potency at ABCB1. Both 57 and 59 produced an ABCG2 ATPase-depressing effect which is in agreement with our precedent cryo-EM study identifying 59 as an ATPase inhibitor that exerts its effect via locking the inward-facing conformation. Thermostabilization of ABCG2 by 57 and 59 can be taken as a hint to comparable binding to ABCG2. As reference substances, compounds 57 and 59 allow additional mechanistic studies on ABCG2 inhibition. Due to their stability in blood plasma, they are also applicable in vivo. The highly specific inhibitor 57 is suited for PET labeling, helping to further elucidate the (patho)physiological role of ABCG2, e.g. at the BBB.

Reversal effect of FW-04-806, a macrolide dilactone compound, on multidrug resistance mediated by ABCB1 and ABCG2 in vitro and in vivo.

BACKGROUND: Overexpression of ATP-binding cassette (ABC) transporters, such as ABCB1 and ABCG2, has been proved to be a major trigger for multidrug resistance (MDR) in certain types of cancer. A promising approach to reverse MDR is the combined use of nontoxic and potent ABC transporters inhibitor with conventional anticancer drugs. We previously reported that FW-04-806 (conglobatin) as a novel Hsp90 inhibitor with low toxicity, capable of attenuating Hsp90/Cdc37 /clients interactions and producing antitumor action in vitro and in vivo. Our early activity screening found that FW-04-806 at non-cytotoxic concentration was able to enhance the cytotoxicity of chemotherapeutic agents on the ABCB1 overexpressing cells. Therefore, we speculated that FW-04-806 might be a promising MDR reversal agent. In the present study we further investigated its reversal effect of MDR induced by ABC transporters in vitro and in vivo. METHODS: MTT assay in vitro and xenograftes in vivo were used to investigate reversal effect of FW-04-806 on MDR in ABCB1 or ABCG2 overexpressing cancer cells. To understand the mechanisms for the MDR reversal, we examined the effects of FW-04-806 on intracellular accumulation of doxorubicin (DOX, adriamycin, adr)/Rhodamine 123 (Rho 123), efflux of doxorubicin, expression levels of gene and protein of ABCB1 or ABCG2 and ATPase activity of ABCB1, and carried out molecular docking between FW-04-806 and human ABCB1. RESULTS: The results indicated that FW-04-806 significantly enhanced the cytotoxicity of substrate chemotherapeutic agents on the ABCB1 or ABCG2 overexpressing cells in vitro and in vivo suggesting its reversal MDR effects. FW-04-806 increased the intracellular accumulation of DOX or Rho123 by inhibiting the efflux function of ABC transporters in MDR cells rather than in their parental sensitive cells. However, unlike other ABC transporter inhibitors, FW-04-806 had no effect on the ATPase activity nor on the expression of ABCB1 or ABCG2 on either mRNA or protein level. Molecular docking suggested that FW-04-806 may have lower affinity to the ATPase site, which was consistent with its no significant effect on the ATPase activity of ABCB1; However FW-04-806 may bind to substrate binding site in TMDs more stably than substrate anticancer drugs therefore obstruct the anticancer drugs pumped out of the cell. CONCLUSIONS: FW-04-806 is a compound that has both anti-tumor and reversal MDR effects, and its antitumor clinical application is worth further study.

Benzoyl indoles with metabolic stability as reversal agents for ABCG2-mediated multidrug resistance.

Ko143, a potent ABCG2 inhibitor that reverses multidrug resistance in cancer, cannot be used clinically due to its unsuitable metabolic stability. We identified benzoyl indoles as reversal agents that reversed ABCG2-mediated multidrug resistance (MDR), with synthetic tractability and enhanced metabolic stability compared to Ko143. Bisbenzoyl indole 2 and monobenzoyl indole 8 significantly increased the accumulation of mitoxantrone (MX) in ABCG2-overexpressing NCI-H460/MX20 cells, and sensitized NCI-H460/MX20 cells to mitoxantrone. Mechanistic studies were conducted by [3H]-MX accumulation assay, Western blot analysis, immunofluorescence analysis and ABCG2 ATPase assay. The results revealed that the reversal efficacies of compounds 2 and 8 were not due to an alteration in the expression level or localization of ABCG2 in ABCG2-overexpressing cell lines. Instead, compounds 2 and 8 significantly stimulated the ATP hydrolysis of ABCG2 transporter, suggesting that these compounds could be competitive substrates of ABCG2 transporter. Overall, the results of our study indicated that compounds 2 and 8 significantly reversed ABCG2-mediated MDR by blocking the efflux of anticancer drugs.

Effect of levofloxacin, pazufloxacin, enrofloxacin, and meloxicam on the immunolocalization of ABCG-2 transporter protein in rabbit retina.

Adenosine triphosphate-binding cassette (ABC) sub-family G member-2 (ABCG-2) is a transporter protein, implicated for multi-drug efflux from tissues. This study evaluated the effect of fluoroquinolones; levofloxacin, pazufloxacin and enrofloxacin, and non-steroidal anti-inflammatory drug, meloxicam; on the immunolocalization of ABCG-2 transporter protein of rabbit retinas. Thirty-two male rabbits were randomly divided in to eight groups. Control group was gavaged, 2% benzyl alcohol in 5% dextrose since these chemicals are excipients of the drug preparations used in the treatment groups of this study. Four groups were exclusively gavaged, levofloxacin hemihydrate (10 mg/kg body weight b.i.d 12 h), pazufloxacin mesylate (10 mg/kg body weight b.i.d 12 h), enrofloxacin (20 mg/kg body weight o.d.), and meloxicam (0.2 mg/kg body weight o.d.), respectively. Three other groups were co-gavaged meloxicam with above fluoroquinolones, respectively. These drugs were administered for 21 days. ABCG-2 immunolocalization was mild in the retinas of control and levofloxacin-alone-treated groups. The immunolocalization intensity was significantly higher in meloxicam-alone-treated group when compared to control and levofloxacin-alone-treated groups. Immunolocalization of this transporter increased in the levofloxacin-meloxicam co-treated group when compared to the levofloxacin-alone-treated group. Highest immunolocalization was observed in the enrofloxacin-meloxicam co-treated group although the immunolocalization of all treatment groups, except the levofloxacin-alone-treated group, was significantly higher than the control and levofloxacin-alone-treated groups.

Fumitremorgins and Relatives - From Tremorgenic Compounds to Valuable Anti-Cancer Drugs.

BACKGROUND: Fumitremorgins are mycotoxins but can also inhibit cancer cells and reverse their drug resistance. OBJECTIVE: The bioactivity of prenylated cyclo-Trp-Pro dipeptides and their derivatives concerning their application in anti-cancer therapies will be discussed. METHODS: Reports on the discovery and assessment of this class of fungal compounds are compiled from literature using Google Scholar and PubMed. The bioactivities of the natural compounds are discussed with the aim of their improvement for cancer therapy. RESULTS: Although a number of compounds of this class have been found, only a minority of them showed bioactivity in the applied bioassays. Fumitremorgins and related compounds are active against various cancer cells but they are also mycotoxins. Some of these natural compounds can arrest cancer cells in their cell cycle and some can block ABC-transporters and reverse resistance in chemotherapy. Structure activity relationships have been deduced leading to the prediction of highly active compounds. Several easily accessible derivatives of these natural products have been discovered being highly selective and non-toxic. CONCLUSION: Sophisticated screening methods, high throughput screening, metabolic engineering, and synthetic biology are novel and promising technologies for the search for highly active drugs. Rapid gene sequencing in combination with engineered biosynthetic pathways should contribute substantially to novel pharmaceutics.

Pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinolines: Novel compounds that reverse ABCG2-mediated resistance in cancer cells.

Overexpression of ATP-binding cassette transporter (ABC) subfamily G2 in cancer cells is known to elicit a MDR phenotype, ultimately resulting in cancer chemotherapy failure. Here, we report, for the first time, the effect of eight novel pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline (IND) derivatives that inhibit ABCG2 transporter restoring cancer cell chemosensitivity. IND -4, -5, -6, -7, and -8, at 10 µM, and nilotinib at 5 µM, significantly potentiated (8-10 fold) the cytotoxicity of the ABCG2 substrates mitoxantrone (MX) and doxorubicin in HEK293 cells overexpressing ABCG2 transporter, MX (~14 fold) in MX-resistant NCI-H460/MX-20 small cell lung cancer, and of topotecan (~7 fold) in S1-M1-80 colon cancer cells which all stably expressing ABCG2. In contrast, cytotoxicity of cisplatin, which is not an ABCG2 substrate, was not altered. IND-5,-6,-7, and -8 significantly increased the accumulation of rhodamine-123 in multidrug resistant NCI-H460/MX-20 cells overexpressing ABCG2. Both IND-7 and -8, the most potent ABCG2 inhibitors, had the highest affinities for the binding sites of ABCG2 in modeling studies. In conclusion, the beneficial actions of new class of agents warrant further development as potential MDR reversal agents for clinical anticancer agents that suffer from ABCG2-mediated MDR insensitivity.

A combination of curcumin with either gramicidin or ouabain selectively kills cells that express the multidrug resistance-linked ABCG2 transporter.

This paper introduces a strategy to kill selectively multidrug-resistant cells that express the ABCG2 transporter (also called breast cancer resistance protein, or BCRP). The approach is based on specific stimulation of ATP hydrolysis by ABCG2 transporters with subtoxic doses of curcumin combined with stimulation of ATP hydrolysis by Na(+),K(+)-ATPase with subtoxic doses of gramicidin A or ouabain. After 72 h of incubation with the drug combinations, the resulting overconsumption of ATP by both pathways inhibits the efflux activity of ABCG2 transporters, leads to depletion of intracellular ATP levels below the viability threshold, and kills resistant cells selectively over cells that lack ABCG2 transporters. This strategy, which was also tested on a clinically relevant human breast adenocarcinoma cell line (MCF-7/FLV1), exploits the overexpression of ABCG2 transporters and induces caspase-dependent apoptotic cell death selectively in resistant cells. This work thus introduces a novel strategy to exploit collateral sensitivity (CS) with a combination of two clinically used compounds that individually do not exert CS. Collectively, this work expands the current knowledge on ABCG2-mediated CS and provides a potential strategy for discovery of CS drugs against drug-resistant cancer cells.