Role of nanomedicine in reversing drug resistance mediated by ATP binding cassette transporters and P-glycoprotein in melanoma

Multidrug resistance (MDR) is one of the most common complex phenomenons exhibited by cancer cells. It is a very common property of melanoma postchemotherapy. MDR transporters, ATP binding cassette (ABC) transporters, play a critical role in conferring this property to melanoma cells. miRNA are post-transcriptional regulators that regulate the expression of these ABC transporters. Targeting these miRNA, in turn targeting ABC transporters with the help of nanodelivery systems to overcome drug resistance, is the primary focus for attaining successful treatment methods for drug-resistant melanoma. These delivery systems are endocytosed by the cancer cells and do not require ABC transporters for their delivery, being a promising therapeutic measure for melanoma.

Targeted multimodal liposomes for nano-delivery and imaging: an avenger for drug resistance and cancer.

Understanding the cellular target structure and thereby proposing the best delivery system to achieve sustained release of drugs has always been a significant area of focus in biomedical research for translational benefits. Specific targeting of the receptors expressed on the target cell represents an effective strategy for increasing the pharmacological efficacy of the administered drug. Liposomes offer enhanced conveyance as a potential carrier of biomacromolecules such as anti-cancer proteins, drugs and siRNA for targeting tumour cell death. Commonly used liposomal constructs for various therapies are Doxil, Myocet, DepoCyt and Abraxanes. However, recent strategy of using multifunctional liposomes for the sustained release of drugs with increased plasma residence time and monoclonal antibody-based targeting of tumours coupled with imaging modalities have attracted enormous scientific attention. The ability of liposomes coated with specific ligands such as Apo-E derived RGD R9 and Tat peptide, to reverse the conceptualisation of drug resistance and cross the blood brain barrier, provides promising future for their use as an efficient drug delivery system. By outlining the recent advancements and innovations in the established concept of liposomal drug delivery, this review will focus on the multifunctional liposomes as an emerging novel lipid based drug delivery system.

Multifunctional and multitargeted nanoparticles for drug delivery to overcome barriers of drug resistance in human cancers

The recurrence and metastatic spread of cancer are major drawbacks in cancer treatment. Although chemotherapy is one of the most effective methods for the treatment of metastatic cancers, it is nonspecific and causes significant toxic damage. The development of drug resistance to chemotherapeutic agents through various mechanisms also limits their therapeutic potential. However, as we discuss here, the use of nanodelivery systems that are a combination of diagnostics and therapeutics (theranostics) is as relatively novel concept in the treatment of cancer. Such systems are likely to improve the therapeutic benefits of encapsulated drugs and can transit to the desired site, maintaining their pharmaceutical properties. The specific targeting of malignant cells using multifunctional nanoparticles exploits theranostics as an improved agent for delivering anticancer drugs and as a new solution for overriding drug resistance.

Overcoming acquired drug resistance in colorectal cancer cells by targeted delivery of 5-FU with EGF grafted hollow mesoporous silica nanoparticles

Acquired drug resistance (ADR) can be developed in colorectal cancer cells after 5-fluorouracil (5-FU) treatment and diminish the effectiveness of chemotherapy. In this work, acquired 5-FU resistance in the colorectal cancer cell line SW480 was obtained with the up-regulation of dihydropyrimidine dehydrogenase (DPYD) gene expression which can convert 5-FU to its inactive metabolite. To overcome ADR in colorectal cancer, hollow mesoporous silica nanoparticles (HMSNs) grafted with epidermal growth factor (EGF) were used as nanocarriers to deliver 5-FU to colorectal cancer cells with acquired drug resistance. The effect and mechanism of 5-FU loaded EGF grafted HMSNs (EGF-HMSNs-5-FU) in overcoming acquired drug resistance in SW480/ADR cells were studied. The EGF-HMSNs were demonstrated to be specifically internalized in EGFR overexpressed SW480/ADR cells via a receptor-mediated endocytosis and can escape from endo-lysosomes. The EGF-HMSNs-5-FU exhibited much higher cytotoxicity on SW480/ADR cells than HMSNs-5-FU and free 5-FU while the plain HMSNs did not show significant cytotoxicity. The mechanism of EGF-HMSNs-5-FU in overcoming drug resistance in SW480/ADR cells could be attributed to the specific internalization of EGF-HMSNs-5-FU in EGFR overexpressed cells which can lead to high intracellular drug accumulation and cause cell death through S phase arrest.

Substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development

Human contains 49 ATP-binding cassette (ABC) transporter genes and the multidrug resistance associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP4/ABCC4, MRP5/ABCC5, MRP6/ABCC6, MRP7/ABCC10, MRP8/ABCC11 and MRP9/ABCC12) belong to the ABCC family which contains 13 members. ABCC7 is cystic fibrosis transmembrane conductance regulator; ABCC8 and ABCC9 are the sulfonylurea receptors which constitute the ATP-sensing subunits of a complex potassium channel. MRP10/ABCC13 is clearly a pseudo-gene which encodes a truncated protein that is highly expressed in fetal human liver with the highest similarity to MRP2/ABCC2 but without transporting activity. These transporters are localized to the apical and/or basolateral membrane of the hepatocytes, enterocytes, renal proximal tubule cells and endothelial cells of the blood-brain barrier. MRP/ABCC members transport a structurally diverse array of important endogenous substances and xenobiotics and their metabolites (in particular conjugates) with different substrate specificity and transport kinetics. The human MRP/ABCC transporters except MRP9/ABCC12 are all able to transport organic anions, such as drugs conjugated to glutathione, sulphate or glucuronate. In addition, selected MRP/ABCC members may transport a variety of endogenous compounds, such as leukotriene C(4) (LTC(4) by MRP1/ABCC1), bilirubin glucuronides (MRP2/ABCC2, and MRP3/ABCC3), prostaglandins E1 and E2 (MRP4/ABCC4), cGMP (MRP4/ABCC4, MRP5/ABCC5, and MRP8/ABCC11), and several glucuronosyl-, or sulfatidyl steroids. In vitro, the MRP/ABCC transporters can collectively confer resistance to natural product anticancer drugs and their conjugated metabolites, platinum compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and in concert with alterations in phase II conjugating or biosynthetic enzymes, classical alkylating agents, alkylating agents. Several MRP/ABCC members (MRPs 1-3) are associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. Drug targeting of these transporters to overcome MRP/ABCC-mediated multidrug resistance may play a role in cancer chemotherapy. Most MRP/ABCC transporters are subject to inhibition by a variety of compounds. Based on currently available preclinical and limited clinical data, it can be expected that modulation of MRP members may represent a useful approach in the management of anticancer and antimicrobial drug resistance and possibly of inflammatory diseases and other diseases. A better understanding of their substrates and inhibitors has important implications in development of drugs for treatment of cancer and inflammation.

Role of multidrug resistance associated proteins in drug development

The Multidrug Resistance Associated Proteins (MRPI, MRP2, MRP3, MRp4, MRp5, MRP6, MRP7, MRPS and MRP9) belong to the ATP-binding cassette superfamily (ABCC family) of transporters expressed differentially in the liver, kidney, intestine and blood-brain barrier. MRps transport a structurally diverse array of endo- and xenobiotics and their metabolites (in particular conjugates) and are subject to induction and inhibition by a variety of compounds. An increased efflux of natural product anticancer drugs and other anticancer agents by MRPs in cancer cells is associated with tumor resistance. These transporting proteins play a role in the absorption, distribution and elimination of various compounds in the body. There are increased reports on the clinical impact of genetic mutations of genes encoding MRP1-9. Therefore, MRPs have an important role in drug development, since a better understanding of their function and regulating mechanism can help minimize and avoid drug toxicity, unfavorable drug-drug interactions, and to overcome drug resistance.

Copper-transporting P-Type ATPase, ATP7A, confers multidrug resistance and its expression is related to resistance to SN-38 in clinical colon cancer

We and others have shown that the copper transporters ATP7A and ATP7B play a role in cellular resistance to cisdiaminedichloroplatinum (II) (CDDP).  In this study, we found that ATP7A transfection of Chinese hamster ovary  cells (CHOK1) and fibroblasts isolated from Menkes disease patients  enhanced resistance not only to CDDP but also to various anticancer drugs, such as vincristine, paclitaxel, 7-ethyl-10- hydroxy-camptothecin (SN-38),  etoposide, doxorubicin, mitoxantron, and 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11). ATP7A preferentially localized
doxorubicin fluorescence to the Golgi apparatus in contrast to the more intense nuclear staining of doxorubicin in the parental cells. Brefeldin A   partially and monensin completely altered the distribution of doxorubicin to the nuclei in the ATP7A-expressing cells. ATP7A expression also enhanced the efflux rates of doxorubicin and SN-38 from cells and increased the uptake of SN-38 in membrane vesicles. These findings strongly suggested that   ATP7A confers multidrug resistance to the cells by compartmentalizing drugs in the Golgi apparatus and by enhancing efflux of these drugs, and the trans-Golgi network has an important role of ATP7A-related drug resistance. ATP7A was expressed in 8 of 34 (23.5%) clinical colon cancer specimens but not in the adjacent normal epithelium. Using the histoculture drug response assay that is useful for the prediction of drug sensitivity of clinical cancers, ATP7A-expressing colon cancer cells were significantly more  resistant to SN-38 than ATP7Anegative cells. Thus, ATP7A confers  resistance to various anticancer agents on cancer cells and might be a good index of drug resistance in clinical colon cancers.

Role of the ATP-binding cassette drug transporters in the intestinal absorption of tanshinone IIB, one of the major active diterpenoids from the root of Salvia miltiorrhiza

There is an increasing use of herbal medicines worldwide, and the extracts from the root of Salvia miltiorrhiza are widely used in the treatment of angina and stroke. In this study, we investigated the mechanism for the intestinal absorption of tanshinone IIB (TSB), a major constituent of S. miltiorrhiza. The oral bioavailability of TSB was about 3% in rats with less proportional increase in its maximum plasma concentration (C_max) and area under the plasma concentration-time curve (AUC) with increasing dosage. The time to C_max (T_max) was prolonged at higher oral dosage. In a single pass rat intestinal perfusion model, the permeability coefficients (P_app) based on TSB disappearance from the lumen (P_lumen) were 6.2- to 7.2-fold higher (p < 0.01) than those based on drug appearance in mesenteric venous blood (P_blood). The uptake and efflux of TSB in Caco-2 cells were also significantly altered in the presence of an inhibitor for P-glycoprotein (PgP) or for multi-drug resistance associated protein (MRP1/2). TSB transport from the apical (AP) to basolateral (BL) side in Caco-2 monolayers was 3.3- to 5.7-fold lower than that from BL to AP side, but this polarized transport was attenuated by co-incubation of PgP or MRP1/2 inhibitors. The P_app values of TSB in the BL-AP direction were significantly higher in MDCKII cells over-expressing MDR1 or MRP1, but not in cells over-expressing MRP2-5, as compared with the wild-type cells. The plasma AUC_0-24hr in mdr1a and mrp1 gene-deficient mice was 10.2- to 1.7-fold higher than that in the wild-type mice. Furthermore, TSB significantly inhibited the uptake of digoxin and vinblastine in membrane vesicles containing PgP or MRP1. TSB also moderately stimulated PgP ATPase activity. Taken collectively, our findings indicate that TSB is a substrate for PgP and MRP1 and that drug resistance to TSB therapy and drug interactions may occur through PgP and MRP1 modulation.

Structural and functional properties of human multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug ABC transporters such as P-glycoprotein (P-gp/MDR1/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. Recently, the mouse P-gp/Abcb1a structure has been determined and this has significantly enhanced our understanding of the structure-activity relationship (SAR) of mammalian ABC transporters. This paper highlights our current knowledge on the structural and functional properties and the SAR of human MRP1/ABCC1. Although the crystal structure of MRP1/ABCC1 has yet to be resolved, the current topological model of MRP1/ABCC1 contains two transmembrane domains (TMD1 and TMD2) each followed by a nucleotide binding domain (NBD) plus a third NH2-terminal TMD0. MRP1/ABCC1 is expressed in the liver, kidney, intestine, brain and other tissues. MRP1/ABCC1 transports a structurally diverse array of important endogenous substances (e.g. leukotrienes and estrogen conjugates) and xenobiotics and their metabolites, including various conjugates, anticancer drugs, heavy metals, organic anions and lipids. Cells that highly express MRP1/ABCC1 confer resistance to a variety of natural product anticancer drugs such as vinca alkaloids (e.g. vincristine), anthracyclines (e.g. etoposide) and epipodophyllotoxins (e.g. doxorubicin and mitoxantrone). MRP1/ABCC1 is associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. However, most compounds that efficiently reverse P-gp/ABCB1-mediated multidrug resistance have only low affinity for MRP1/ABCC1 and there are only a few effective and relatively specific MRP1/ABCC1 inhibitors available. A number of site-directed mutagenesis studies, biophysical and photolabeling studies, SAR and QSAR, molecular docking and homology modeling studies have documented the role of multiple residues in determining the substrate specificity and inhibitor selectivity of MRP1/ABCC1. Most of these residues are located in the TMs of TMD1 and TMD2, in particular TMs 4, 6, 7, 8, 10, 11, 14, 16, and 17, or in close proximity to the membrane/cytosol interface of MRP1/ABCC1. The exact transporting mechanism of MRP1/ABCC1 is unclear. MRP1/ABCC1 and other multidrug transporters are front-line mediators of drug resistance in cancers and represent important therapeutic targets in future chemotherapy. The crystal structure of human MRP1/ABCC1 is expected to be resolved in the near future and this will provide an insight into the SAR of MRP1/ABCC1 and allow for rational design of anticancer drugs and potent and selective MRP1/ABCC1 inhibitors.

Efflux pumps of Mycobacterium tuberculosis play a significant role in antituberculosis activity of potential drug candidates

Active efflux of drugs mediated by efflux pumps that confer drug resistance is one of the mechanisms developed by bacteria to counter the adverse effects of antibiotics and chemicals. To understand these efflux mechanisms in Mycobacterium tuberculosis, we generated knockout (KO) mutants of four efflux pumps of the pathogen belonging to different classes. We measured the MICs and kill values of two different compound classes on the wild type (WT) and the efflux pump (EP) KO mutants in the presence and absence of the efflux inhibitors verapamil and L-phenylalanyl-L-arginyl-β-naphthylamide (PAβN). Among the pumps studied, the efflux pumps belonging to the ABC (ATP-binding cassette) class, encoded by Rv1218c, and the SMR (small multidrug resistance) class, encoded by Rv3065, appear to play important roles in mediating the efflux of different chemical classes and antibiotics. Efflux pumps encoded by Rv0849 and Rv1258c also mediate the efflux of these compounds, but to a lesser extent. Increased killing is observed in WT M. tuberculosis cells by these compounds in the presence of either verapamil or PAβN. The efflux pump KO mutants were more susceptible to these compounds in the presence of efflux inhibitors. We have shown that these four efflux pumps of M. tuberculosis play a vital role in mediating efflux of different chemical scaffolds. Inhibitors of one or several of these efflux pumps could have a significant impact in the treatment of tuberculosis. The identification and characterization of Rv0849, a new efflux pump belonging to the MFS (major facilitator superfamily) class, are reported.

Clinical aspects for survivin: a crucial molecule for targeting drug-resistant cancers

Drug resistance is frequently found in cancer patients who have prolonged chemotherapeutic treatments. Overcoming this phenomenon to make therapy available to these patients is one of the most important features in developing effective cancer therapeutic strategies. Identification of drug resistance causative molecules is one of the most focused areas of cancer research today. Many molecules have been identified in conferring cancer cells the property of drug resistance, and various small molecule inhibitors have been developed to target these molecules to restore the sensitivity of different traditional chemotherapeutic agents, which are frequently found to exhibit reduced potency during prolonged treatment, in cancer patients. Survivin, a member of the inhibitor of apoptosis proteins (IAP) family, has been identified as one of the most crucial biomarkers in the recognition of drug resistance. Survivin is overexpressed in tumor cells, helping in its proliferation and survival, and its overexpression is positively correlated with poor prognosis for cancer patients. Targeted therapeutic measures to inhibit survivin in cancers, particularly drug-resistant tumors, are the recent focus of research for cancer treatment.

Delivery of fluorouracil and siRNA by mesoporous silica nanoparticles to drug resistant colorectal cancer

 Mesoporous silica nanoparticle based drug and gene delivery system was developed to overcome the acquired drug resistance in colorectal cancer by targeted delivery of anti-cancer drug in the cytoplasm of the cancer cells and silencing the gene expression related to drug resistance.

Doxorubicin conjugated to immunomodulatory anticancer lactoferrin displays improved cytotoxicity overcoming prostate cancer chemo resistance and Inhibits tumour development in TRAMP mice

Advanced, metastatic, castration resistant and chemo-resistant prostate cancer has triggered change in the drug development landscape against prostate cancer. Bovine lactoferrin (bLf) is currently attracting attention in clinics for its anti-cancer properties and proven safety profile. bLf internalises into cancer cells via receptor mediated endocytosis, boosts immunity and complements chemotherapy. We employed bLf as an excellent functional carrier protein for delivering doxorubicin (Dox) into DU145 cells, CD44+/EpCAM+ double positive enriched DU145 3D prostaspheres and drug resistant ADR1000-DU145 cells, thus circumventing Dox efflux, to overcome chemo-resistance. Successful bLf-Dox conjugation with iron free or iron saturated bLf forms did not affect the integrity and functionality of bLf and Dox. bLf-Dox internalised into DU145 cells within 6 h, enhanced nuclear Dox retention up to 24 h, and proved significantly effective (p < 0.001) in reducing LC50 value of Dox from 5.3 μM to 1.3 μM (4 fold). Orally fed iron saturated bLf-Dox inhibited tumour development, prolonged survival, reduced Dox induced general toxicity, cardiotoxicity, neurotoxicity in TRAMP mice and upregulated serum levels of anti-cancer molecules TNF-α, IFN-γ, CCL4 and CCL17. The study identifies promising potential of a novel and safer bLf-Dox conjugate containing a conventional cytotoxic drug along with bLf protein to target drug resistance.

Audit of the use of antimicrobial prophylaxis in nasal surgery at a speciali

Background: In 2002−03 a retrospective audit of the use of surgical antimicrobial prophylaxis (AMP) for elective nasal surgery was undertaken at the Royal Victorian Eye and Ear Hospital (RVEEH). The RVEEH is a publicly funded teaching hospital that provides specialist eye, nose and throat medicine in Victoria, Australia. The aim of the audit was to determine the extent to which the use of antimicrobial prophylaxis in the hospital was consistent with Australian and international evidence-based guidelines and if there was a need for the hospital to develop internal guidelines for the use of AMP.

Methods: The histories of 500 consecutive patients who had undergone nasal surgery within the study period of August 2001 and July 2002 were examined. The data collected from these histories included information such as the patients' age, gender, diagnosis, surgical procedure performed, antimicrobial agents used, and the length of follow up and a range of factors shown in previous studies to increase the risk of surgical site infection.

Results: A total of 306 (72.86%) patients were found to have received antimicrobial agents either prior to admission, during admission or on discharge. Only 24 patients (5.71%) were administered antimicrobials immediately prior to surgery and at no other time.

Conclusions: The findings of this study support the need for further research to examine the appropriateness of the use of AMP at the RVEEH and the need for internal guidelines for its use.

Role of P-glycoprotein in limiting the brain penetration of glabridin, an active isoflavan from the root of Glycyrrhiza glabra.

Purpose. Glabridin is a major active constituent of Glycyrrhiza glabra which is commonly used in the treatment of cardiovascular and central nervous system (CNS) diseases. Recently, we have found that glabridin is a substrate of P-glycoprotein (PgP/MDR1). This study aimed to investigate the role of PgP in glabridin penetration across the blood–brain barrier (BBB) using several in vitro and in vivo models.
Materials and Methods. Cultured primary rat brain microvascular endothelial cells (RBMVECs) were used in the uptake, efflux and transcellular transport studies. A rat bilateral in situ brain perfusion model was used to investigate the brain distribution of glabridin. The brain and tissue distribution of glabridin in rats with or without coadministered verapamil or quinidine were examined with correction for the tissue residual blood. In addition, the brain distribution of glabridin in mdr1a(-/-) mice was compared with the wild-type mice. Glabridin in various biological matrices was determined by a validated liquid chromatography mass spectrometric method.
Results. The uptake and efflux of glabridin in cultured RBMVECs were ATP-dependent and significantly altered in the presence of a PgP or multi-drug resistance protein (Mrp1/2) inhibitor (e.g. verapamil or MK-571). A polarized transport of glabridin was found in RBMVEC monolayers with
facilitated efflux from the abluminal (BL) to luminal (AP) side. Addition of a PgP or Mrp1/2 inhibitor in both luminal and abluminal sides attenuated the polarized transport across RBMVECs. In a bilateral in situ brain perfusion model, the uptake of glabridin into the cerebrum increased from 0.42 T 0.09% at 1 min to 9.27 T 1.69% (ml/100 g tissue) at 30 min and was significantly greater than that for sucrose. Coperfusion of a PgP or Mrp1/2 inhibitor significantly increased the brain distribution of glabridin by 33.6j142.9%. The rat brain levels of glabridin were only about 27% of plasma levels when corrected by tissue residual blood and it was increased to up to 44% when verapamil or quinidine was coadministered. The area under the brain concentration-time curve (AUC) of glabridin in mdr1a(-/-) mice was 6.0-fold higher than the wild-type mice.
Conclusions. These findings indicate that PgP limits the brain penetration of glabridin through the BBB and PgP may cause drug resistance to glabridin (licorice) therapy for CNS diseases and potential drugglabridin interactions. However, further studies are needed to explore the role of other drug transporters (e.g. Mrp1-4) in restricting the brain penetration of glabridin.