The short-chain fatty acid butyrate is a substrate of breast cancer resistance protein

Colorectal cancer is one of the most common cancers worldwide. Butyrate (BT) plays a key role in colonic epithelium homeostasis. The aim of this work was to investigate the possibility of BT being transported by P-glycoprotein (MDR1), multidrug resistance proteins (MRPs), or breast cancer resistance protein (BCRP). Uptake and efflux of (14)C-BT and (3)H-folic acid were measured in Caco-2, IEC-6, and MDA-MB-231 cell lines. mRNA expression of BCRP was detected by RT-PCR. Cell viability, proliferation, and differentiation were quantified with the lactate dehydrogenase, sulforhodamine B, and alkaline phosphatase activity assays, respectively. In both IEC-6 cells and Caco-2 cells, no evidence was found for the involvement of either MDR1 or MRPs in (14)C-BT efflux from the cells. In contrast, several lines of evidence support the conclusion that BT is a substrate of both rat and human BCRP. Indeed, BCRP inhibitors reduced (14)C-BT efflux in IEC-6 cells, both BT and BCRP inhibitors significantly decreased the efflux of the known BCRP substrate (3)H-folic acid in IEC-6 cells, and BCRP inhibitors reduced (14)C-BT efflux in the BCRP-expressing MDA-MB-231 cell line. In IEC-6 cells, combination of BT with a BCRP inhibitor significantly potentiated the effect of BT on cell proliferation. The results of this study, showing for the first time that BT is a BCRP substrate, are very important in the context of the high levels of BCRP expression in the human colon and the anticarcinogenic and anti-inflammatory role of BT at that level. So, interaction of BT with BCRP and with other BCRP substrates/inhibitors is clearly of major importance.     

Reversal of BCRP-mediated multidrug resistance by stable expression of small interfering RNAs

Breast cancer resistance protein (BCRP) is an ATP-binding cassette multidrug transporter that confers resistance to various anticancer drugs like Mitoxantrone. Overexpression of BCRP confers multidrug resistance (MDR) in cancer cells and is a frequent impediment to successful chemotherapy. For stable reversal of BCRP-depending MDR by RNA interference technology, a hU6-RNA gene promoter-driven expression vector encoding anti-BCRP short hairpin RNA (shRNA) molecules was constructed. By treating endogenously and exogenously expresses high levels of BCRP cells with these constructs, expression of the targeted BCRP-encoding mRNA, and transport protein was inhibited completely. Furthermore, the accumulation of mitoxantrone in the anti-BCRP shRNA-treated cells increased. And the sensitivity to mitoxantrone of anti-BCRP shRNA-treated cells is increased 14.6-fold and 2.44-fold respectively compared to their control (P < 0.05). These data indicated that stable shRNA-mediated RNAi could be tremendously effective in reversing BCRP-mediated MDR and showed promises in overcoming MDR by gene therapeutic applications.     

Novel structure–activity relationships and selectivity profiling of cage dimeric 1,4-dihydropyridines as multidrug resistance (MDR) modulators

Synthesized series of cage dimeric 1,4-dihydropyridines have been systematically evaluated as MDR modulators in in vitro assays to investigate structure-dependent selectivity properties of inhibiting most cancer-relevant efflux pump proteins. Structure–activity relationships of each P-glycoprotein (P-gp) and multidrug resistance associated protein (MRP) 1 and MRP2 inhibition are discussed and prove to be mainly determined by certain aromatic substitution patterns. The characterization of breast cancer resistance protein (BCRP) inhibition results in the discovery of benzyloxy substituted derivatives as selective P-gp inhibitors.     

Functional Assessment of Adipose Stem Cells for Xenotransplantation Using Myocardial Infarction Immunocompetent Models: Comparison with Bone Marrow Stem Cells

Recently, preclinical studies have shown that allogeneic adipose-derived stem cells (ASCs), like bone marrow-derived mesenchymal stem cell (BMSCs) have significant clinical benefits in treating cardiovascular diseases, such as ischemic/infarcted heart. In this study, we tested whether ASCs are also immune tolerant, such that they can be used as universal donor cells for myocardial regenerative therapy. The study also focuses on investigating the potential therapeutic effects of human ASCs (hASCs) for myocardial infarction in xenotransplant model, and compares its effects with that of hBMSCs. The in vitro study confirms the superior proliferation potential and viability of hASCs under normoxic and stressed hypoxic conditions compared with hBMSCs. hASCs also show higher potential in adopting cardiomyocyte phenotype. The major findings of the in vivo study are that (1) both hASCs and hBMSCs implanted into immunocompetent rat hearts with acute myocardial infarction survived the extreme environment of xenogeneic mismatch for 6 weeks; (2) both hASCs and hBMSCs showed significant improvement in myocardial pro/anti-inflammatory cytokine levels with no detectable inflammatory reaction, despite the lack of any immunosuppressive therapy; and (3) hASCs contributed to the remarkable improvement in cardiac function and reduced infarction which was significantly better than that of hBMSC and untreated control groups. Thus, our findings suggest the feasibility of using ASCs, instead of BMSCs, as universal donor cells for xenogeneic or allogeneic cell therapy.     

Long-term in-vivo tumorigenic assessment of human culture-expanded adipose stromal/stem cells

After more than a decade of extensive experimentation, the promise of stem cells to revolutionize the field of medicine has negotiated their entry into clinical trial. Adipose tissue specifically holds potential as an attainable and abundant source of stem cells. Currently undergoing investigation are adipose stem cell (ASC) therapies for diabetes and critical limb ischemia, among others. In the enthusiastic pursuit of regenerative therapies, however, questions remain regarding ASC persistence and migration, and, importantly, their safety and potential for neoplasia. To date, assays of in vivo ASC activity have been limited by early end points. We hypothesized that with time, ASCs injected subcutaneously undergo removal by normal tissue turnover and homeostasis, and by the host's immune system. In this study, a high dose of culture expanded ASCs was formulated and implanted as multicellular aggregates into immunocompromised mice, which were maintained for over one year. Animals were monitored for toxicity, and surviving cells quantified at study endpoint. No difference in growth/weight or lifespan was found between cell-treated and vehicle treated animals, and no malignancies were detected in treated animals. Moreover, real-time PCR for a human specific sequence, ERV-3, detected no persistent ASCs. With the advent of clinical application, clarification of currently enigmatic stem cell properties has become imperative. Our study represents the longest duration determination of stem cell activity in vivo, and contributes strong evidence in support of the safety of adipose derived stem cell applications.     

Lobeline,a piperidine alkaloid from Lobelia can reverse P-gp dependent multidrug resistance in tumor cells

Multidrug resistance (MDR) can limit efficacy of chemotherapy. The best studied mechanism involves P-gp (P-glycoprotein) mediated drug efflux. This study focuses on MDR reversal agents from medicinal plants, which can interfere with P-gp. Rhodamine 123 accumulation assay and flow cytometry analysis were employed to screen for P-gp dependant efflux inhibitors. Lobeline, a piperidine alkaloid from Lobelia inflata and several other Lobelia species, inhibited P-gp activity. MDR reversal potential of lobeline could be demonstrated in cells treated with doxorubicin in that lobeline can sensitize resistant tumor cells at non-toxic concentrations. However, lobeline cannot block BCRP (Breast Cancer Resistance Protein) dependent mitoxantrone efflux. Lobeline could be a good candidate for the development of new MDR reversal agents.     

Structure-activity relationships of new inhibitors of breast cancer resistance protein (ABCG2)

At the end of the last century tariquidar (XR9576) was synthesized, pharmacologically investigated, and classified as a promising 3rd generation P-glycoprotein (P-gp) modulator. Following the discovery of BCRP in 1998 an increasing number of substances were studied in relation to their potency to interact with this transporter. Recently it has been shown that XR9576 inhibits both P-gp and BCRP transport function similarly to GF120918 (elacridar). This observation prompted us to investigate 5 XR compounds and 25 structurally related derivatives synthesized in our laboratory for their BCRP inhibitory effect. The biological activity data were determined by our new Hoechst 33342 assay that has been transferred from P-gp to BCRP overexpressing cells. 3D-QSAR models (CoMFA and CoMSIA) were generated and validated by the leave-many-out method and the scrambling stability test. The best models yielded an internal predictive squared correlation coefficient higher than 0.8 and contained steric, electrostatic, hydrophobic, and hydrogen bond donor fields. To our knowledge, this is the first 3D-QSAR analysis of BCRP inhibitors. Additionally the biological activity data determined in P-gp overexpressing cells on one side and BCRP overexpressing cells on the other side were compared to identify selective and non-selective inhibitors of P-gp and BCRP. The results may help to get a better insight which structural elements are necessary to direct the interaction of these compounds with P-gp and/or BCRP.     

Small-molecule synthetic compound norcantharidin reverses multi-drug resistance by regulating Sonic hedgehog signaling in human breast cancer cells

Multi-drug resistance (MDR), an unfavorable factor compromising treatment efficacy of anticancer drugs, involves upregulated ATP binding cassette (ABC) transporters and activated Sonic hedgehog (Shh) signaling. By preparing human breast cancer MCF-7 cells resistant to doxorubicin (DOX), we examined the effect and mechanism of norcantharidin (NCTD), a small-molecule synthetic compound, on reversing multidrug resistance. The DOX-prepared MCF-7R cells also possessed resistance to vinorelbine, characteristic of MDR. At suboptimal concentration, NCTD significantly inhibited the viability of DOX-sensitive (MCF-7S) and DOX-resistant (MCF-7R) cells and reversed the resistance to DOX and vinorelbine. NCTD increased the intracellular accumulation of DOX in MCF-7R cells and suppressed the upregulated the mdr-1 mRNA, P-gp and BCRP protein expression, but not the MRP-1. The role of P-gp was strengthened by partial reversal of the DOX and vinorelbine resistance by cyclosporine A. NCTD treatment suppressed the upregulation of Shh expression and nuclear translocation of Gli-1, a hallmark of Shh signaling activation in the resistant clone. Furthermore, the Shh ligand upregulated the expression of P-gp and attenuated the growth inhibitory effect of NCTD. The knockdown of mdr-1 mRNA had not altered the expression of Shh and Smoothened in both MCF-7S and MCF-7R cells. This indicates that the role of Shh signaling in MDR might be upstream to mdr-1/P-gp, and similar effect was shown in breast cancer MDA-MB-231 and BT-474 cells. This study demonstrated that NCTD may overcome multidrug resistance through inhibiting Shh signaling and expression of its downstream mdr-1/P-gp expression in human breast cancer cells.     

The anti-apoptotic effect of IGF-1 on tissue resident stem cells is mediated via PI3-kinase dependent secreted frizzled related protein 2 (Sfrp2) release

Previous studies suggest that IGF-1 may be used as an adjuvant to stem cell transfer in order to improve cell engraftment in ischemic tissue. In the current study, we investigated the effect of IGF-1 on serum deprivation and hypoxia induced stem cell apoptosis and the possible mechanisms involved. Exposure of adipose tissue derived stem cells (ASCs) to serum deprivation and hypoxia resulted in significant apoptosis in ASC which is partially prevented by IGF-1. IGF-1’s anti-apoptotic effect was abolished in ASCs transfected with Sfrp2 siRNA but not by the control siRNA. Using Western blot analysis, we demonstrated that serum deprivation and hypoxia reduced the expression of nuclear β-catenin, which is reversed by IGF-1. IGF-1’s effect on β-catenin expression was abolished by the presence of PI3-kinase inhibitor LY294002 or in ASCs transfected with Sfrp2 siRNA. These results suggest that IGF-1, through the release of the Sfrp2, contributes to cell survival by stabilizing β-catenin.     

Soluble factors from ASCs effectively direct control of chondrogenic fate

Background and objectives: Adipose tissue‐derived stem cells (ASCs) have great potential for regenerative medicine. For molecular understanding of specific functional molecules present in ASCs, we analysed 756 proteins including specific chondrogenic functional factors, using high‐throughput nano reverse‐phase liquid chromatography–electrospray ionization–tandem mass spectrometry. Materials, methods and results: Of these proteins, 33 were identified as chondrogenic factors or proteins including type 2 collagen, biglycan, insulin‐like growth factor‐binding protein and transforming growth factor‐beta 1 (TGF‐β1). ASCs are a possible cell source for cartilage regeneration as they are able to secrete a number of functional cytokines including chondrogenesis‐inducing molecules such as TGF‐β1 and bone morphogenetic protein 4 (BMP4). The chondrogenic phenotype of cultured ASCs was effectively induced by ASC‐culture media (CM) containing BMP4 and TGF‐β1, and maintained after pre‐treatment for 14 days in vitro and subcutaneous implantation in vivo. Chondrogenic differentiation efficiency of cultured ASCs and cultured mouse skin‐derived progenitor cells (SPCs) depended absolutely on ASC CM‐fold concentration. Cell density was also a very important factor for chondrogenic behaviour development during differentiation of ASCs and SPCs. Conclusion: ASC CM‐derived TGF‐β1‐induced chondrogenic differentiation of ASCs resulted in significant reduction in chondrogenic activity after inhibition of the p38 pathway, revealing involvement of this MAPK pathway in TGF‐β1 signalling. On the other hand, TGF‐β1 signalling also led to SMAD activation that could directly increase chondrogenic activity of ASCs.     

Investigation of chalcones and benzochalcones as inhibitors of breast cancer resistance protein

Breast cancer resistance protein (BCRP/ABCG2) belongs to the ATP binding cassette family of transport proteins. BCRP has been found to confer multidrug resistance in cancer cells. A strategy to overcome resistance due to BCRP overexpression is the investigation of potent and specific BCRP inhibitors. The aim of the current study was to investigate different multi-substituted chalcones for their BCRP inhibition. We synthesized chalcones and benzochalcones with different substituents (viz. OH, OCH(3), Cl) on ring A and B of the chalcone structure. All synthesized compounds were tested by Hoechst 33342 accumulation assay to determine inhibitory activity in MCF-7 MX and MDCK cells expressing BCRP. The compounds were also screened for their P-glycoprotein (P-gp) and Multidrug resistance-associated protein 1 (MRP1) inhibitory activity in the calcein AM accumulation assay and were found to be selective towards inhibition of BCRP. Substituents at position 2' and 4' on chalcone ring A were found to be essential for activity; additionally there was a great influence of substituents on ring B. Presence of 3,4-dimethoxy substitution on ring B was found to be optimal, while presence of 2- and 4-chloro substitution also showed a positive effect on BCRP inhibition.     

Reduction of infarct size by intravenous injection of uncultured adipose derived stromal cells in a rat model is dependent on the time point of application

Stem cell therapy is a promising tool to improve outcome after acute myocardial infarction (AMI), but needs to be optimized since results from clinical applications remain ambiguous. A potent source of stem cells is the stromal vascular fraction of adipose tissue (SVF), which contains high numbers of adipose derived stem cells (ASC). We hypothesized that: 1) intravenous injection can be used to apply stem cells to the heart. 2) Uncultured SVF cells are easier and safer when cultured ASCs. 3) Transplantation after the acute inflammation period of AMI is favorable over early injection. For this, AMI was induced in rats by 40min of coronary occlusion. One or seven days after AMI, rats were intravenously injected with vehicle, 5×10(6) uncultured rat SVF cells or 1×10(6) rat ASCs. Rats were analyzed 35 days after AMI. Intravenous delivery of both fresh SVF cells and cultured ASCs 7 days after AMI significantly reduced infarct size compared to vehicle. Similar numbers of stem cells were found in the heart, after treatment with fresh SVF cells and cultured ASCs. Importantly, no adverse effects were found after injection of SVF cells. Using cultured ASCs, however, 3 animals had shortness of breath, and one animal died during injection. In contrast to application at 7 days post AMI, injection of SVF cells 1 day post AMI resulted in a small but non-significant infarct reduction (p=0.35). Taken together, intravenous injection of uncultured SVF cells subsequent to the acute inflammation period, is a promising stem cell therapy for AMI.     

Increased expression of sorcin is associated with multidrug resistance in leukemia cells via up-regulation of MDR1 expression through cAMP response element-binding protein

Sorcin, a 22 kDa Ca²⁺ binding protein, was first identified in a vincristine-resistant Chinese hamster lung cell line, and was later demonstrated to be involved in the development of multidrug-resistance (MDR) phenotypes in a variety of human cancer cell lines. However, the exact role of sorcin in MDR cells is yet to be fully elucidated. Here we explored the role of sorcin in the development of MDR in leukemia cells, and revealed that the expression level of sorcin was directly correlated to the expression of MDR1/P-glycoprotein (P-gp). In addition, it was shown that sorcin induced the expression of MDR1/P-gp through a cAMP response element (CRE) between −716 and −709 bp of the mdr1/p-gp gene. Furthermore, overexpression of sorcin increased the phosphorylation of CREB1 and the binding of CREB1 to the CRE sequence of mdr1/p-gp promoter, and induced the expression of MDR1/P-gp. These findings suggested that sorcin induces MDR1/P-gp expression markedly through activation of the CREB pathway and is associated with the MDR phenotype. The new findings may be helpful for understanding the mechanisms of MDR in human cancer cells, prompting its further investigation as a molecular target to overcome MDR.     

Proinflammatory interleukins' production by adipose tissue‐derived mesenchymal stromal cells: the impact of cell culture conditions and cell‐to‐cell interaction

The impact of culture conditions and interaction with activated peripheral blood mononuclear cells on the interleukin (IL) gene expression profile and proinflammatory IL‐6 and IL‐8 production by adipose‐derived stromal cells (ASCs) was investigated. A microarray analysis revealed a wide range of IL genes either under standard (20%) or hypoxic (5%) O₂ concentrations, some highly up‐regulated at hypoxia. IL‐6 and IL‐8 production was inversely dependent on cell culture density. In early (first–third) passages, IL‐6 and IL‐8 concentration was higher at 20% O₂ and in late (8th‐12th) passages under 5% O₂. Interaction between ASCs and mononuclear cells in indirect setting was accompanied with a significant decrease of IL‐6 and did not result in the elevation of IL‐8 concentration. Thereby, the production of proinflammatory interleukins (IL‐6 and IL‐8) may be affected by the ASC intrinsic features (density in culture, and duration of expansion), as well as by microenvironmental factors, such as hypoxia and the presence of blood‐borne cells. These data are important for elucidating ASC paracrine activity regulation in vitro. They would also be on demand for optimisation of the cell therapy protocols, based on the application of ASC biologically active substances. Copyright © 2015 John Wiley & Sons, Ltd. SIGNIFICANCE PARAGRAPH Ex vivo expansion is widely used for increasing the number of adipose‐derived stromal cells (ASCs) and improving of their quality. The present study was designed to elucidate the particular factors influencing the interleukin production in ASCs. The presented data specified the parameters (i.e. cell density, duration of cultivation, hypoxia, etc.) that should be taken in mind when ASCs are intended to be used in protocols implying their paracrine activity. These data would be of considerable interest for researchers and clinicians working in the biomedical science.     

Discovery of alkoxyl biphenyl derivatives bearing dibenzo[c,e]azepine scaffold as potential dual inhibitors of P-glycoprotein and breast cancer resistance protein

We recently reported alkoxyl biphenyl derivatives bearing dibenzo[c,e]azepine scaffold as novel P-glycoprotein (P-gp, ABCB1) inhibitors. In this study, their ability to reverse breast cancer resistance protein (BCRP, ABCG2)-mediated multidrug resistance was tested in HEK293/BCRP cells which was BCRP-transfected stable HEK293 cells. It was observed that compounds 4d, 4h, 4i increased mitoxantrone accumulation in HEK293/BCRP cells via inhibiting BCRP efflux function. Notably, the inhibitory activity of 4i was comparable to that of the classical BCRP inhibitor Ko143 at an equimolar concentration. Interestingly, 4i had little inhibitory effect on multidrug resistance-associated protein 1 (MRP1, ABCC1), another drug efflux transporter. These results, together with the previous findings, suggest that 4i may be a dual inhibitor of P-gp and BCRP to warrant further investigation.     

ABC drug transporter expression and functional activity in trophoblast-like cell lines and differentiating primary trophoblast

ATP-binding cassette (ABC) efflux transporters are expressed in the human placenta where they are thought to help protect the fetus from xenobiotics. To evaluate models for analysis of ABC transporter function and regulation in the placenta, we have characterized the expression and activity of multidrug resistance (MDR) 1/P glycoprotein (Pgp), MDR3/Pgp, breast cancer resistance protein (BCRP), and multidrug resistance proteins 1 and 2 (MRPs 1, 2) in differentiating primary trophoblast cells and BeWo and Jar cell lines. Real-time PCR and immunoblotting were used for analysis of mRNA and protein expression, respectively. Functional activity was measured using selective inhibitors of efflux of fluorescent substrates, calcein-AM (Pgp and MRPs) and Hoechst 33342 (BCRP). The levels of MDR1 mRNA and protein expression were much higher in trophoblast than in Jar and especially BeWo cells. Expression of MDR3 protein was also lower in BeWo cells. Levels of MDR3 expression were markedly higher than MDR1 levels in all tested cell types. Levels of both MDR1 and MDR3 expression decreased during trophoblast differentiation/syncytialization. BCRP was highly expressed in all cell types and increased with trophoblast differentiation. MRP1 expression was much lower in trophoblasts compared with both cell lines. In contrast to its abundant mRNA expression, MRP2 protein was practically undetectable in BeWo and Jar cells and was present only at very low levels in trophoblast. Functional studies confirmed the presence of active Pgp and BCRP in all studied cell types, whereas MRP functional activity was detected only in BeWo and Jar cells. Both cell lines may be useful models for studying various aspects of placental ABC transporter expression and function, but also have significant limitations. With respect to their ABC protein expression profile, Jar cells are more similar to nondifferentiated cytotrophoblast, whereas BeWo appear to more closely reflect differentiated syncytiotrophoblast.     

A multiple-targets alkaloid nuciferine overcomes paclitaxel-induced drug resistance in vitro and in vivo

ObjectiveMultidrug resistance (MDR) is the major barrier to the successful treatment of chemotherapy. Compounds from nature products working as MDR sensitizers provided new treatment strategies for chemo-resistant cancers patients.MethodsWe investigated the reversal effects of nuciferine (NF), an alkaloid from Nelumbo nucifera and Nymphaea caerulea, on the paclitaxel (PTX) resistance ABCB1-overexpressing cancer in vitro and in vivo, and explored the underlying mechanism by evaluating drug sensitivity, cell cycle perturbations, intracellular accumulation, function and protein expression of efflux transporters as well as molecular signaling involved in governing transporters expression and development of MDR in cancer.ResultsNF overcomes the resistance of chemotherapeutic agents included PTX, doxorubicin (DOX), docetaxel, and daunorubicin to HCT-8/T and A549/T cancer cells. Notably, NF suppressed the colony formation of MDR cells in vitro and the tumor growth in A549/T xenograft mice in vivo, which demonstrated a very strong synergetic cytotoxic effect between NF and PTX as combination index (CI) (CI<0.1) indicated. Furthermore, NF increased the intracellular accumulation of P-gp substrates included DOX and Rho123 in the MDR cells and inhibited verapamil-stimulated ATPase activity. Mechanistically, inhibition of PI3K/AKT/ERK pathways by NF suppressed the activation of Nrf2 and HIF-1α, and further reduced the expression of P-gp and BCRP, contributing to the sensitizing effects of NF against MDR in cancer.ConclusionThis novel finding provides a promising treatment strategy for overcoming MDR and improving the efficiency of chemotherapy by using a multiple-targets MDR sensitizer NF.