Synthesis of 3-octadecanoxypropyl 6-deoxy-6-sulfo-α-d-glucopyranoside (ODSG) as a lipase-resistant SQAP derivative.

Sulfoquynovosylacyl propanediol (SQAP; 1) has been developed as a radiosensitizer (anti-cancer agent) for solid tumors, but it was easily cleaved in vivo and had a problem of short residence time. We synthesized a novel compound of a SQAP derivative (3-octadecanoxypropyl 6-deoxy-6-sulfo-α-d-glucopyranoside: ODSG; 2) to solve these problems not easily cleaved by lipase. ODSG (2) cytotoxicity was investigated in vitro, resulting in low toxicity like SQAP (1).

Induction of apoptosis, cytotoxicity and radiosensitization by novel 3,4-dihydroquinazolinone derivatives.

Twenty new quinazolinone derivatives bearing a piperonyl moiety were designed and synthesized. The structures of the target compounds were in agreement with the microanalytical and spectral data. Compounds 4-10, 13, 14 and 17-27 were screened for their cytotoxic activity against HepG-2 and MCF-7 cancer cell lines. The target compounds showed IC50 in the range of 2.46-36.85 µM and 3.87-88.93 µM for HepG-2 and MCF-7, respectively. The promising compounds 7, 19, 26 and 27 were selected to measure their EGFR inhibitory activity. The IC50 values of the promising compounds were in the range of 146.9-1032.7 nM for EGFR in reference to Erlotinib (IC50 = 96.6 nM). In further studies on compounds 7, 19, 26 and 27 using HepG-2 cell line, there was significant overexpression of p21 and downregulation of two members of IAPs protein family; Survivin and XIAP, relative to their controls. Annexin V-FITC and caspase-3 analyses have established a significant increase in early apoptosis. Moreover, the four selected compounds have impaired cell proliferation by cell cycle arrest at the G2/M phase compared to their respective control. Considering radiotherapy as the primary treatment for many types of solid tumors, the radiosensitizing abilities of compounds 7, 19, 26 and 27 were measured against HepG-2 and MCF-7 cell lines combined with a single dose of 8 Gy gamma radiation. Measurement of the IC50 of the promising compounds after irradiation revealed their ability to sensitize the cells to the lethal effect of gamma irradiation (IC50 = 1.56-4.32 µM and 3.06-5.93 µM for HepG-2 and MCF-7 cells, respectively). Molecular docking was performed to gain insights into the ligand-binding interactions of 7, 19, 26 and 27 inside the EGFR binding sites and revealed their essential interactions, explaining their good activity towards EGFR.

Biological evaluation, radiosensitizing activity and structural insights of novel halogenated quinazoline-sulfonamide conjugates as selective human carbonic anhydrases IX/XII inhibitors.

A library of iodoquinazolinones endowed with benzenesulfonamide moiety was designed and synthesized as human carbonic anhydrase (hCA) inhibitors. Compounds 4-17 showed generally poor activity against the cytosolic hCA I and hCA II isoforms. Contrarily they were more potent and showed a variable spectrum of selectivity against the tumor-specific isoforms hCA IX and hCA XII. The 4-iodophenyl derivative 12 and the 4-pyridinyl derivative 15 were the most active and selective in this series against hCA IX and hCA XII isoforms with KI of 18 and 9 nM, respectively. Compounds 12 and 15 were further screened for their cytotoxicity against MCF-7, HepG-2 and HCT-116 cancer cell lines besides WI38 and MCF-10A normal cell lines to determine their selectivity towards cancer cells. Compound 12 was selective towards HepG-2 and HCT-116 cell lines but less selective towards MCF-7. While compound 15 showed higher selectivity towards HepG-2 than HCT-116 and MCF-7 cell lines. The ability of compounds 12 and 15 to sensitize the cells against gamma irradiation's effect proved their potential radiosensitizing activity. Molecular docking analysis was carried out to discover the possible binding mode of the compounds within the active site of isoform hCA IX and XII. Compounds 12 and 15 revealed the probable fundamental interactions explaining the good activity and selectivity towards the tumor-specific isoforms.

Tumor-derived biomimetic nanozyme with immune evasion ability for synergistically enhanced low dose radiotherapy.

High doses of radiation can cause serious side effects and efficient radiosensitizers are urgently needed. To overcome this problem, we developed a biomimetic nanozyme system (CF) by coating pyrite (FeS2) into tumor-derived exosomes for enhanced low-dose radiotherapy (RT). CF system give FeS2 with immune escape and homologous targeting abilities. After administration, CF with both glutathione oxidase (GSH-OXD) and peroxidase (POD) activities can significantly lower the content of GSH in tumor tissues and catalyze intracellular hydrogen peroxide (H2O2) to produce a large amount of ·OH for intracellular redox homeostasis disruption and mitochondria destruction, thus reducing RT resistance. Experiments in vivo and in vitro showed that combining CF with RT (2 Gy) can provide a substantial suppression of tumor proliferation. This is the first attempt to use exosomes bionic FeS2 nanozyme for realizing low-dose RT, which broaden the prospects of nanozymes.

Synthesis of some quinazolinones inspired from the natural alkaloid L-norephedrine as EGFR inhibitors and radiosensitizers.

A set of quinazolinones synthesized by the aid of L-norephedrine was assembled to generate novel analogues as potential anticancer and radiosensitizing agents. The new compounds were evaluated for their cytotoxic activity against MDA-MB-231, MCF-7, HepG-2, HCT-116 cancer cell lines and EGFR inhibitory activity. The most active compounds 5 and 6 were screened against MCF-10A normal cell line and displayed lower toxic effects. They proved their relative safety with high selectivity towards MDA-MB-231 breast cancer cell line. Measurement of the radiosensitizing activity for 5 and 6 revealed that they could sensitize the tumour cells after being exposed to a single dose of 8 Gy gamma radiation. Compound 5 was able to induce apoptosis and arrest the cell cycle at the G2-M phase. Molecular docking of 5 and 6 in the active site of EGFR was performed to gain insight into the binding interactions with the key amino acids.

LXR activation radiosensitizes non-small cell lung cancer by restricting myeloid-derived suppressor cells.

Radiotherapy (RT) is an important radical treatment for locally advanced non-small cell lung cancer (NSCLC). However, radioresistance greatly impairs the efficacy of this therapy in the clinic. Radioresistance can be caused by radiation-induced myeloid-derived suppressor cell (MDSC) infiltration. Liver-X nuclear receptor (LXR) agonists have demonstrated potent antitumor activity in preclinic animal models. Here, we report for the first time that LXR agonists, GW3965 and RGX-104, radiosensitized NSCLC in a subcutaneous homograft murine model. LXR activation significantly reduced MDSC abundance in the tumor microenvironment (TME). Treatment with RGX-104 greatly promoted MDSC apoptosis in vitro. Depleting MDSC activated cytotoxic T lymphocyte (CTL) and T-helper 1 (Th1) responses in the TME. In conclusion, the immunosuppressive effects of radiotherapy can be abrogated partly with an LXR agonist by depleting MDSC, which sensitizes NSCLC to RT.

Photofunctional Cyclometalated Iridium(III) Polypyridine Complexes Bearing a Perfluorobiphenyl Moiety for Bioconjugation, Bioimaging, and Phototherapeutic Applications.

In this article, we report the design, synthesis, and characterization of a series of cyclometalated iridium(III) polypyridine complexes containing a perfluorobiphenyl (PFBP) moiety [Ir(N^C)2(bpy-PFBP)](PF6) (bpy-PFBP = 4-(S-(perfluoro-(1,1'-biphenyl)-4-yl)-N-mercaptoethylaminocarbonyloxymethyl)-4'-methyl-2,2'-bipyridine; HN^C = 2-phenylpyridine (Hppy) (1a), 2-(4-hydroxymethylphenyl)pyridine (Hppy-CH2OH) (2a), 2-((1,1'-biphenyl)-4-yl)pyridine (Hpppy) (3a), 2-((4'-hydroxymethyl-1,1'-biphenyl)-4-yl)pyridine (Hpppy-CH2OH) (4a), 2-phenylquinoline (Hpq) (5a), 2-(4-hydroxymethylphenyl)quinoline (Hpq-CH2OH) (6a)). Their PFBP-free counterparts [Ir(N^C)2(bpy-C4)](PF6) (bpy-C4 = 4-(N-n-butylaminocarbonyloxymethyl)-4'-methyl-2,2'-bipyridine; HN^C = Hppy (1b), Hppy-CH2OH (2b), Hpppy (3b), Hpppy-CH2OH (4b), Hpq (5b), Hpq-CH2OH (6b)) were also prepared for comparison studies. Upon irradiation, all the complexes displayed intense and long-lived greenish-yellow to orange luminescence in solutions under ambient conditions and in low-temperature alcohol glass. Reactions of the PFBP complexes with peptides containing the FCPF sequence via the π-clamp-mediated cysteine conjugation afforded luminescent peptide conjugates that exhibited rich photophysical properties. Using complex 3a as an example, we demonstrated that the conjugation of complexes to organelle-targeting peptides is an effective means to modulate their intracellular localization behavior, which was further shown to be important to their performance in photodynamic therapy. The results of this work will contribute to the development of photofunctional transition metal complexes as theranostic agents.

MicroRNA-621 Acts as a Tumor Radiosensitizer by Directly Targeting SETDB1 in Hepatocellular Carcinoma.

Radiotherapy is one of the most important treatment methods of tumors. However, the application of radiotherapy in hepatocellular carcinoma (HCC) is limited due to the low tolerance of normal liver cells for radiation and inherent radiation resistance in HCC. With the in-depth study of microRNAs (miRNAs) in tumor therapy, the regulation of tumor radiosensitivity by miRNAs has been a research hotspot in recent years. In the present study, the expression of miR-621 was lower in HCC tissues and cells, and such low expression of miR-621 was associated with poor prognosis in HCC patients. In addition, in vivo and in vitro assays confirmed that the high expression of miR-621 could significantly enhance the radiosensitivity of HCC. Moreover, the expressions of miR-621 and SETDB1 in HCC tissues were negatively correlated. Dual-luciferase reporter assays indicated that miR-621 could directly target the 3' UTR of SETDB1. In addition, miR-621 enhanced the radiosensitivity of HCC cells via directly inhibiting SETDB1. Besides, the miR-621 and/or SETDB1 axis improved the radiosensitivity of HCC cells via activating the p53-signaling pathway. Taken together, miR-621 and/or SETDB1 might be used as a novel therapeutic target for the treatment of HCC.

DNA Strand Break Properties of Protoporphyrin IX by X-Ray Irradiation against Melanoma.

Recent reports have suggested that 5-aminolevulinic acid (5-ALA), which is a precursor to protoporphyrin IX (PpIX), leads to selective accumulation of PpIX in tumor cells and acts as a radiation sensitizer in vitro and in vivo in mouse models of melanoma, glioma, and colon cancer. In this study, we investigated the effect of PpIX under X-ray irradiation through ROS generation and DNA damage. ROS generation by the interaction between PpIX and X-ray was evaluated by two kinds of probes, 3'-(p-aminophenyl) fluorescein (APF) for hydroxyl radical (•OH) detection and dihydroethidium (DHE) for superoxide (O2•-). •OH showed an increase, regardless of the dissolved oxygen. Meanwhile, the increase in O2•- was proportional to the dissolved oxygen. Strand breaks (SBs) of DNA molecule were evaluated by gel electrophoresis, and the enhancement of SBs was observed by PpIX treatment. We also studied the effect of PpIX for DNA damage in cells by X-ray irradiation using a B16 melanoma culture. X-ray irradiation induced γH2AX, DNA double-strand breaks (DSBs) in the context of chromatin, and affected cell survival. Since PpIX can enhance ROS generation even in a hypoxic state and induce DNA damage, combined radiotherapy treatment with 5-ALA is expected to improve therapeutic efficacy for radioresistant tumors.

MiR-365 enhances the radiosensitivity of non-small cell lung cancer cells through targeting CDC25A.

Radioresistance is a major challenge in lung cancer radiotherapy (RT), and consequently, new radiosensitizers are urgently needed. MicroRNAs (miRNAs) have been demonstrated to participate in many important cellular processes including radiosensitization. MiR-365 is dysregulated in non-small cell lung cancer (NSCLC) and is able to restrain the development of NSCLC. However, the relationship between miR-365 and radiosensitivities of NSCLC cells remains largely unknown. Here we reveal that overexpression of miR-365 is able to enhance the radiosensitivity of NSCLC cells through targeting CDC25A. We found that the expression level of miR-365 was positively correlated with the radiosensitivity of NSCLC cell lines. Furthermore, our results showed that overexpression of miR-365 could sensitize A549 cells to the irradiation. However, knockdown of miR-365 in H460 cells could act the converse manner. Mechanically, miR-365 was able to directly target 3'UTR of cell division cycle 25A (CDC25A) mRNA and reduce the expression of CDC25A at the levels of mRNA and protein. And we confirmed that miR-365 could increase the radiosensitivity of NSCLC cells by targeting CDC25A using in vitro and in vivo assays. Taken together, restoration of miR-365 expression enhances the radiosensitivity of NSCLC cells by suppressing CDC25A, and miR-365 could be used as a radiosensitizer for NSCLC therapy.

Combined Cancer Chemo-Photodynamic and Photothermal Therapy Based on ICG/PDA/TPZ-Loaded Nanoparticles.

Although photodynamic therapy (PDT) has been an attractive strategy for several cancer treatments in the clinical setting, PDT efficacy is attenuated by consumption of oxygen. To address this photodynamic issue, we adopted a phototherapy-chemotherapy combination strategy based on targeted delivery of the near-infrared photosensitizer indocyanine green (ICG), photothermal conversion agent polydopamine (PDA), and tirapazamine (TPZ), a hypoxia-activated prodrug. Under laser irradiation, ICG consumption of oxygen and aggravated hypoxia in tumor sites can activate TPZ to damage DNA. In parallel, ICG produces reactive oxygen species which work in synergy with PDA to enhance phototherapeutic efficiency. Herein, hybrid CaCO3/TPGS nanoparticles delivering ICG, PDA, and TPZ (ICG-PDA-TPZ NPs) were designed for effective and safe cancer therapy. ICG-PDA-TPZ NPs showed significantly improved cellular uptake and accumulation in tumors. Furthermore, we demonstrated that ICG-PDA-TPZ NPs showed intensive photodynamic and photothermal effects in vitro and in vivo, which synergized with TPZ in subcutaneous U87 malignant glioma growth and orthotopic B16F10 tumor inhibition, with negligible side effects. Thus, ICG-PDA-TPZ NPs could be an effective strategy for improvement of PDT.

Inhibiting DNA-PKCS radiosensitizes human osteosarcoma cells.

Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PKCS), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PKCS in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PKCS inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PKCS was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PKCS inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma.

Breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (P-gp/ABCB1) transport afatinib and restrict its oral availability and brain accumulation.

Afatinib is a highly selective, irreversible inhibitor of EGFR and HER-2. It is orally administered for the treatment of patients with EGFR mutation-positive types of metastatic NSCLC. We investigated whether afatinib is a substrate for the multidrug efflux transporters ABCB1 and ABCG2 and whether these transporters influence oral availability and brain and other tissue accumulation of afatinib. We used in vitro transport assays to assess human (h)ABCB1-, hABCG2- or murine (m)Abcg2-mediated transport of afatinib. To study the single and combined roles of Abcg2 and Abcb1a/1b in oral afatinib disposition, we used appropriate knockout mouse strains. Afatinib was transported well by hABCB1, hABCG2 and mAbcg2 in vitro. Upon oral administration of afatinib, Abcg2-/-, Abcb1a/1b-/- and Abcb1a/1b-/-;Abcg2-/- mice displayed a 4.2-, 2.4- and 7-fold increased afatinib plasma AUC0-24 compared with wild-type mice. Abcg2-deficient strains also displayed decreased afatinib plasma clearance. At 2h, relative brain accumulation of afatinib was not significantly altered in the single knockout strains, but 23.8-fold increased in Abcb1a/1b-/-;Abcg2-/- mice compared to wild-type mice. Abcg2 and Abcb1a/1b restrict oral availability and brain accumulation of afatinib. Inhibition of these transporters may therefore be of clinical importance for patients with brain (micro)metastases positioned behind an intact blood-brain barrier.

Lipidomic analysis reveals a radiosensitizing role of gamma-linolenic acid in glioma cells.

Previous studies have demonstrated that gamma-linolenic acid (GLA) is effective against glioma cells under both in vitro and in vivo conditions. In the present study we determined how GLA alone or in combination with irradiation alters the fatty acid (FA) and lipid profiles, the lipid droplet (LD) content, the lipid biosynthetic gene expression and the apoptosis of glioma cells. In GLA-treated cells direct correlations were found between the levels of various FAs and the expression of the corresponding FA biosynthetic genes. The total levels of saturated and monosaturated FAs decreased in concert with the down-regulation of FASN and SCD1 gene expression. Similarly, decreased FADS1 gene expression was paralleled by lowered arachidonic acid (20:4 n-6) and eicosapentaenoic acid (20:5 n-3) contents, while the down-regulation of FADS2 expression was accompanied by a diminished docosahexaenoic acid (22:6 n-3) content. Detailed mass spectrometric analyses revealed that individual treatments gave rise to distinct lipidomic fingerprints. Following uptake, GLA was subjected to elongation, resulting in dihomo-gamma-linolenic acid (20:3 n-6, DGLA), which was used for the synthesis of the LD constituent triacylglycerols and cholesteryl esters. Accordingly, an increased number of LDs were observed in response to GLA administration after irradiation. GLA increased the radioresponsiveness of U87 MG cells, as demonstrated by an increase in the number of apoptotic cells determined by FACS analysis. In conclusion, treatment with GLA increased the apoptosis of irradiated glioma cells, and GLA might therefore increase the therapeutic efficacy of irradiation in the treatment of gliomas.

Therapeutic delivery of miR-200c enhances radiosensitivity in lung cancer.

The microRNA (miR)-200s and their negative regulator ZEB1 have been extensively studied in the context of the epithelial-mesenchymal transition. Loss of miR-200s has been shown to enhance cancer aggressiveness and metastasis, whereas replacement of miR-200 miRNAs has been shown to inhibit cell growth in several types of tumors, including lung cancer. Here, we reveal a novel function of miR-200c, a member of the miR-200 family, in regulating intracellular reactive oxygen species signaling and explore a potential application for its use in combination with therapies known to increase oxidative stress such as radiation. We found that miR-200c overexpression increased cellular radiosensitivity by direct regulation of the oxidative stress response genes PRDX2, GAPB/Nrf2, and SESN1 in ways that inhibits DNA double-strand breaks repair, increase levels of reactive oxygen species, and upregulate p21. We used a lung cancer xenograft model to further demonstrate the therapeutic potential of systemic delivery of miR-200c to enhance radiosensitivity in lung cancer. Our findings suggest that the antitumor effects of miR-200c result partially from its regulation of the oxidative stress response; they further suggest that miR-200c, in combination with radiation, could represent a therapeutic strategy in the future.

Electron induced single strand break and cyclization: a DFT study on the radiosensitization mechanism of the nucleotide of 8-bromoguanine.

Cleavage of the O-P bond in 8-bromo-2'-deoxyguanosine-3',5'-diphosphate (BrdGDP), considered as a model of single strand break (SSB) in labelled double-stranded DNA (ds DNA), is investigated at the B3LYP/6-31++G(d,p) level. The thermodynamic and kinetic characteristics of the formation of SSB are compared to those related to the 5',8-cycloguanosine lesion. The first reaction step, common to both damage types, which is the formation of the reactive guanyl radical, proceeds with a barrier-free or low-barrier release of the bromide anion. The guanyl radical is then stabilized by hydrogen atom transfer from the C3' or C5' sites of the 2'-deoxyribose moiety to its C8 center. The C3' path, via the O-P bond cleavage, leads to a ketone derivative (the SSB model), while the C5' path is more likely to yield 5',8-cycloguanosine.

Radiation-sensitising effects of antennapedia proteins (ANTP)-SmacN7 on tumour cells.

The objective of this study was to investigate the underlying mechanisms behind the radiation-sensitising effects of the antennapedia proteins (ANTP)-smacN7 fusion protein on tumour cells. ANTP-SmacN7 fusion proteins were synthesised, and the ability of this fusion protein to penetrate cells was observed. Effects of radiation on the expression of X-linked inhibitor of apoptosis protein (XIAP) were detected by western blotting. The radiation-sensitising effects of ANTP-SmacN7 fusion proteins were observed by a clonogenic assay. The effects of drugs and radiation on tumour cell apoptosis were determined using Annexin V/FITC double staining. Changes in caspase-8, caspase-9 and caspase-3 were detected by western blot before and after ANTP-SmacN7 inhibition of XIAP. The ANTP-SmacN7 fusion protein could enter and accumulate in cells; in vitro XIAP expression of radiation-induced tumour cells was negatively correlated with tumour radiosensitivity. The ANTP-SmacN7 fusion protein promoted tumour cell apoptosis through the activation of caspase3. ANTP-SmacN7 fusion protein may reduce tumour cell radioresistance by inducing caspase3 activation.

Evaluation of novel combined carbogen USPIO (CUSPIO) imaging biomarkers in assessing the antiangiogenic effects of cediranib (AZD2171) in rat C6 gliomas.

The recently described combined carbogen USPIO (CUSPIO) magnetic resonance imaging (MRI) method uses spatial correlations in independent imaging biomarkers to assess specific components of tumor vascular structure and function. Our study aimed to evaluate CUSPIO biomarkers for the assessment of tumor response to antiangiogenic therapy. CUSPIO imaging was performed in subcutaneous rat C6 gliomas before and 2 days after treatment with the potent VEGF-signaling inhibitor cediranib (n = 12), or vehicle (n = 12). Histological validation of Hoechst 33342 uptake (perfusion), smooth muscle actin staining (maturation), pimonidazole adduct formation (hypoxia) and necrosis were sought. Following treatment, there was a significant decrease in fractional blood volume (-43%, p < 0.01) and a significant increase in hemodynamic vascular functionality (treatment altered ΔR(2) *(carbogen) from 1.2 to -0.2 s(-1) , p < 0.05). CUSPIO imaging revealed an overall significant decrease in plasma perfusion (-27%, p < 0.05) following cediranib treatment, that was associated with selective effects on immature blood vessels. The CUSPIO responses were associated with a significant 15% reduction in Hoechst 33342 uptake (p < 0.05), but no significant difference in vascular maturation or necrosis. Additionally, treatment with cediranib resulted in a significant 40% increase in tumor hypoxia (p < 0.05). The CUSPIO imaging method provides novel and more specific biomarkers of tumor vessel maturity and vascular hemodynamics, and their response to VEGF-signaling inhibition, compared to current MR imaging biomarkers utilized in the clinic. Such biomarkers may prove effective in longitudinally monitoring tumor vascular remodeling and/or evasive resistance in response to antiangiogenic therapy.

Superparamagnetic iron oxide nanoparticles as radiosensitizer via enhanced reactive oxygen species formation.

Internalization of citrate-coated and uncoated superparamagnetic iron oxide nanoparticles by human breast cancer (MCF-7) cells was verified by transmission electron microscopy imaging. Cytotoxicity studies employing metabolic and trypan blue assays manifested their excellent biocompatibility. The production of reactive oxygen species in iron oxide nanoparticle loaded MCF-7 cells was explained to originate from both, the release of iron ions and their catalytically active surfaces. Both initiate the Fenton and Haber-Weiss reaction. Additional oxidative stress caused by X-ray irradiation of MCF-7 cells was attributed to the increase of catalytically active iron oxide nanoparticle surfaces.

Use of baculovirus BacMam vectors for expression of ABC drug transporters in mammalian cells.

ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters.