Multiple Drug Resistant Cancer Research Articles

Activation of the Anaphase Promoting Complex Restores Impaired Mitotic Progression and Chemosensitivity in Multiple Drug-Resistant Human Breast Cancer.

The development of multiple-drug-resistant (MDR) cancer all too often signals the need for toxic alternative therapy or palliative care. Our recent in vivo and in vitro studies using canine MDR lymphoma cancer cells demonstrate that the Anaphase Promoting Complex (APC) is impaired in MDR cells compared to normal canine control and drug-sensitive cancer cells. Here, we sought to establish whether this phenomena is a generalizable mechanism independent of species, malignancy type, or chemotherapy regime. To test the association of blunted APC activity with MDR cancer behavior, we used matched parental and MDR MCF7 human breast cancer cells, and a patient-derived xenograft (PDX) model of human triple-negative breast cancer. We show that APC activating mechanisms, such as APC subunit 1 (APC1) phosphorylation and CDC27/CDC20 protein associations, are reduced in MCF7 MDR cells when compared to chemo-sensitive matched cell lines. Consistent with impaired APC function in MDR cells, APC substrate proteins failed to be effectively degraded. Similar to our previous observations in canine MDR lymphoma cells, chemical activation of the APC using Mad2 Inhibitor-1 (M2I-1) in MCF7 MDR cells enhanced APC substrate degradation and resensitized MDR cells in vitro to the cytotoxic effects of the alkylating chemotherapeutic agent, doxorubicin (DOX). Using cell cycle arrest/release experiments, we show that mitosis is delayed in MDR cells with elevated substrate levels. When pretreated with M2I-1, MDR cells progress through mitosis at a faster rate that coincides with reduced levels of APC substrates. In our PDX model, mice growing a clinically MDR human triple-negative breast cancer tumor show significantly reduced tumor growth when treated with M2I-1, with evidence of increased DNA damage and apoptosis. Thus, our results strongly support the hypothesis that APC impairment is a driver of aggressive tumor development and that targeting the APC for activation has the potential for meaningful clinical benefits in treating recurrent cases of MDR malignancy.

Abstract B006: Does TFPI1 drive development of multiple drug resistant cancer and can the anaphase promoting complex prevent it

Abstract Chemotherapy is often rendered ineffective by the development of multiple drug resistance (MDR). Given time MDR occurs almost universally and once developed applies in a non-specific manner. The lack of MDR specificity means many drugs, including those a patient has no pervious exposure to, are affected which limits treatment options. The goal of this research is to show nuclear localized tissue factor pathway inhibitor 1 (TFPI1) plays a driving role in development of MDR, and that the Anaphase Promoting Complex (APC) is capable of inhibiting MDR by targeting TFPI1 for degradation. A coagulation regulator, TFPI1 normally inhibits the tissue factor pathway to prevent blood clot formation. However, TFPI1 is highly elevated in many types of MDR cancer where we believe it mediates resistance development. Previous work in our lab has shown plasmid overexpression of TFPI1 induces MDR in MCF7 breast cancer cells, in MDR cells TFPI1 does not localize to the cell surface but instead to the nucleus, and a putative TFPI1 nuclear localization sequence (NLS) was identified. To confirm the putative NLS is required for TFPI1’s localization to nucleus, and subsequent MDR development, NLS mutant or wild type TFPI1-GFP were overexpressed in MCF7 cells. Localization of TFPI1-GFP was observed using widefield and confocal microscopy. Wild type TFPI1-GFP localized to the nucleus while the NLS mutant TFPI1 did not. These results help to confirm the putative NLS and its importance to TFPI1s role in MDR development. The APC is a nuclear E3 ubiquitin ligase and highly conserved regulator of cell cycle progression. Defects in APC function are associated with reduced longevity and cancer. Other work in our lab focuses on the APC’s role in longevity using a yeast model. However, we believe the APC also plays a role in TFPI1’s promotion of MDR. Our observations suggest APC dysfunction exists within MDR cancer cells where APC substrates accumulate, as does TFPI1. We hypothesize that nuclear TFPI1 is an APC substrate, and that APC activation will result in TFPI1 degradation slowing MDR development. Our lab has isolated several small peptides we believe bind and activate the APC, and have used these peptides in conjugation with known APC activators such as Mad2 Inhibitor-1 (M2I-1) to show that APC activation is capable of reducing TFPI1 abundance in MCF7 cells treated with the chemotherapeutic Doxorubicin (Dox). These results suggest TFPI1 is an APC substrate. To summarize, we believe TFPI1 drives development of MDR cancer, for which TFPI1’s accumulation within the nucleus is important. We have shown TFPI1’s NLS is required for localization of the protein to the nucleus, and activation of the APC reduces nuclear accumulation of TFPI1. Moving forward our goal is to demonstrate the effect TFPI1 NLS knockout and APC activation have on the development of MDR in MCF7 cells undergoing drug selection with Dox. By exploring TFPI1’s role in MDR we may elucidate means to slow resistance development and extend chemotherapy effectiveness. Citation Format: Mathew Lubachowski, Troy Harkness. Does TFPI1 drive development of multiple drug resistant cancer and can the anaphase promoting complex prevent it [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B006.

Activation of the Anaphase Promoting Complex Reverses Multiple Drug Resistant Cancer in a Canine Model of Multiple Drug Resistant Lymphoma.

Simple SummaryMultiple drug resistant cancers develop all too soon in patients who received successful cancer treatment. A lack of treatment options often leaves palliative care as the last resort. We tested whether the insulin sensitizer, metformin, known to have anti-cancer activity, could impact canines with drug resistant lymphoma when added to chemotherapy. All canines in the study expressed protein markers of drug resistance and within weeks of receiving metformin, the markers were decreased. A microarray was performed, and from four canines assessed, a common set of 290 elevated genes were discovered in tumor cells compared to control cells. This cluster was enriched with genes that stall the cell cycle, with a large component representing substrates of the Anaphase Promoting Complex (APC), which degrades proteins. One canine entered partial remission. RNAs from this canine showed that APC substrates were decreased during remission and elevated again during relapse, suggesting that the APC was impaired in drug resistant canines and restored when remission occurred. We validated our results in cell lines using APC inhibitors and activators. We conclude that the APC may be a vital guardian of the genome and could delay the onset of multiple drug resistance when activated.Like humans, canine lymphomas are treated by chemotherapy cocktails and frequently develop multiple drug resistance (MDR). Their shortened clinical timelines and tumor accessibility make canines excellent models to study MDR mechanisms. Insulin-sensitizers have been shown to reduce the incidence of cancer in humans prescribed them, and we previously demonstrated that they also reverse and delay MDR development in vitro. Here, we treated canines with MDR lymphoma with metformin to assess clinical and tumoral responses, including changes in MDR biomarkers, and used mRNA microarrays to determine differential gene expression. Metformin reduced MDR protein markers in all canines in the study. Microarrays performed on mRNAs gathered through longitudinal tumor sampling identified a 290 gene set that was enriched in Anaphase Promoting Complex (APC) substrates and additional mRNAs associated with slowed mitotic progression in MDR samples compared to skin controls. mRNAs from a canine that went into remission showed that APC substrate mRNAs were decreased, indicating that the APC was activated during remission. In vitro validation using canine lymphoma cells selected for resistance to chemotherapeutic drugs confirmed that APC activation restored MDR chemosensitivity, and that APC activity was reduced in MDR cells. This supports the idea that rapidly pushing MDR cells that harbor high loads of chromosome instability through mitosis, by activating the APC, contributes to improved survival and disease-free duration.

Paclitaxel and Lapatinib dual loaded chitosan-coated PLGA nanoparticles enhance cytotoxicity by circumventing MDR1-mediated trastuzumab resistance in HER2 positive breast cancers: In-vitro and in-vivo studies

Treatment options for multidrug-resistant breast cancer are limited and ineffective. Few combinations of chemotherapeutic agents are clinically successful in treating multiple drug-resistant cancers. However, due to their varied pharmacokinetic characteristics, it is challenging to deliver dual drugs to the tumor simultaneously with the best proportion by simple co-administration. This study aimed to generate chitosan-coated PLGA nanoparticles delivering a synergistic combination of Paclitaxel (PTX) and Lapatinib (LPB) to improve efficacy in patients with multidrug-resistant breast cancer. In comparison to sensitive BT-474 cells, the 3:1 M ratio of PTX/LPB demonstrated significant synergism with a reversal index of approximately 13.4 in trastuzumab-resistant BT-474/TR cells. Particle diameters of the prepared chitosan (CS) PLGA Nps ranged from 157.4 ± 9.2 to 263.5 ± 13.5 nm. CS-coated Nps exhibited good EE and DL values of 80.8 ± 3.4% and 1.5 ± 0.12%, respectively. Moreover, CS-coating allowed successful co-delivery of PTX and LPB, resulting in the maximum in-vitro release within 72 h. Cytotoxicity against BT-474/TR cells was significantly increased when CS-coated dual loaded Nps were compared to other Nps, including free PTX, LPB, and their mixture. Quantification of the intracellular drug accumulation revealed that the encapsulation effectively increased drug uptake in BT-474/TR cells. These CS-coated dual loaded Nps exerted an excellent synergistic effect in BT-474/TR cells, leading to significantly improved cell inhibition via circumventing the MDR1 mediated trastuzumab resistance. Additionally, co-delivery of PTX and LPB demonstrated the most effective tumor growth reduction in the BT-474/TR human breast tumor xenograft compared to the saline control, single loaded PTX, and LPB Nps and a mixture of PTX and LPB combinations. The data suggest that CS-coated PLGA Nps loaded with PTX and LPB concurrently may be one of the most promising formulations for treating MDR1 mediated chemoresistance in HER2-positive breast tumors.

Copper-Based Metal-Organic Framework Overcomes Cancer Chemoresistance through Systemically Disrupting Dynamically Balanced Cellular Redox Homeostasis.

Chemodrug resistance is a major reason accounting for tumor recurrence. Given the mechanistic complexity of chemodrug resistance, molecular inhibitors and targeting drugs often fail to eliminate drug-resistant cancer cells, and sometimes even promote chemoresistance by activating alternative pathways. Here, by exploiting biochemical fragility of high-level but dynamically balanced cellular redox homeostasis in drug-resistant cancer cells, we design a nanosized copper/catechol-based metal-organic framework (CuHPT) that effectively disturbs this homeostasis tilting the balance toward oxidative stress. Within drug-resistant cells, CuHPT starts disassembly that is triggered by persistent consumption of cellular glutathione (GSH). CuHPT disassembly simultaneously releases two structural elements: catechol ligands and reductive copper ions (Cu+). Both of them cooperatively function to amplify the production of intracellular radical oxidative species (ROS) via auto-oxidation and Fenton-like reactions through exhausting GSH. By drastically heightening cellular oxidative stress, CuHPT exhibits selective and potent cytotoxicity to multiple drug-resistant cancer cells. Importantly, CuHPT effectively inhibits in vivo drug-resistant tumor growth and doubles the survival time of tumor-bearing mice. Thus, along with CuHPT's good biocompatibility, our biochemical, cell biological, preclinical animal model data provide compelling evidence supporting the notion that this copper-based MOF is a predesigned smart therapeutic against drug-resistant cancers through precisely deconstructing their redox homeostasis.

M6A Modification in Non-Coding RNA: The Role in Cancer Drug Resistance.

Cancer drug resistance has always been a major difficulty in cancer therapy. In the face of drug pressure, resistant cancer cells show complex molecular mechanisms including epigenetic changes to maintain survival. Studies prove that cancer cells exhibit abnormal m6A modification after acquiring drug resistance. m6A modification in the target RNA including non-coding RNA can be a controller to determine the fate and metabolism of RNA by regulating their stability, subcellular localization, or translation. In particular, m6A-modified non-coding RNA plays multiple roles in multiple drug-resistant cancer cells, which can be a target for cancer drug resistance. Here, we provide an overview of the complex regulatory mechanisms of m6A-modified non-coding RNA in cancer drug resistance, and we discuss its potential value and challenges in clinical applications.

Metal-polyphenol-network coated CaCO3 as pH-responsive nanocarriers to enable effective intratumoral penetration and reversal of multidrug resistance for augmented cancer treatments

Construction of multifunctional stimuli-responsive nanotherapeutics enabling improved intratumoral penetration of therapeutics and reversal of multiple-drug resistance (MDR) is potent to achieve effective cancer treatment. Herein, we report a general method to synthesize pH-dissociable calcium carbonate (CaCO3) hollow nanoparticles with amorphous CaCO3 as the template, gallic acid (GA) as the organic ligand, and ferrous ions as the metallic center via a one-pot coordination reaction. The obtained GA–Fe@CaCO3 exhibits high loading efficiencies to both oxidized cisplatin prodrug and doxorubicin, yielding drug loaded GA–Fe@CaCO3 nanotherapeutics featured in pH-responsive size shrinkage, drug release, and Fenton catalytic activity. Compared to nonresponsive GA–Fe@silica nanoparticles prepared with silica nanoparticles as the template, such GA–Fe@CaCO3 confers significantly improved intratumoral penetration capacity. Moreover, both types of drug-loaded GA–Fe@CaCO3 nanotherapeutics exhibit synergistic therapeutic efficacies to corresponding MDR cancer cells because of the GA–Fe mediated intracellular oxidative stress amplification that could reduce the efflux of engulfed drugs by impairing the mitochondrial-mediated production of adenosine triphosphate (ATP). As a result, it is found that the doxorubicin loaded GA–Fe@CaCO3 exhibits superior therapeutic effect towards doxorubicin-resistant 4T1 breast tumors via combined chemodynamic and chemo-therapies. This work highlights the preparation of pH-dissociable CaCO3-based nanotherapeutics to enable effective tumor penetration for enhanced treatment of drug-resistant tumors.

Synergistic Antitumor Effects on Drug-Resistant Breast Cancer of Paclitaxel/Lapatinib Composite Nanocrystals.

Drug resistance presents serious difficulties for cancer treatment. A combination of paclitaxel (PTX) and lapatinib (LAPA) shows potentials in multiple drug resistant cancers in the clinic, but it is almost impossible to deliver these two drugs to the tumor at the same time with the best proportion by simple co-administration of the respective current formualtions for their different pharmacokinetic profiles. Here composite nanocrystals of PTX and LAPA (cNC) were designed with a ratio of 2:1 (w/w), which was their intracellular ratio at the best synergistic efficacy on a drug-resistant cancer cell line (MCF-7/ADR). Such cNC were prepared using a bottom-up method to achieve a nearly spherical appearance and a narrow size distribution of 95.1 ± 2.1 nm. For nanocrystal stabilization, Polyethylene glycol (PEG) coating was introduced into the cNC via polydopamine (PDA) coating in order to get a PEGylated composite nanocrystal (cNC@PDA-PEG) with nanoscale size (170.5 ± 1.4 nm), considerable drug loading (PTX: 21.33 ± 1.48%, LAPA: 10.95 ± 1.24%) and good stability for at least 4 days in plasma-containing buffers. Differential scanning calorimeter (DSC) and XRD data both indicated the different crystalline states of the cNC as well as the cNC@PDA-PEG in comparison with bulk drugs. In vitro release data showed that PTX and LAPA were gradually and completely released from cNC@PDA-PEG in 3 days, while drug release from bulk drugs or cNC was only 30%. cNC@PDA-PEG also showed negligible hemolysis in vitro. Cellular uptake experiments in the MCF-7/ADR cell line showed that the nanocrystals entered the cells in a complete form through endocytosis and then released the drug in the cell. cNC@PDA-PEG inhibits the growth of this drug-resistant cell more effectively than the unmodified version (cNC). In summary, PEGylated PTX and LAPA composite nanocrystals showed the potential for treament of drug-resistant tumors by simultaneously delivering two drugs to tumor cells with the best proportion.

Microfluidic device for the analysis of MDR cancerous cell-derived exosomes' response to nanotherapy.

Exosomes are membrane-enclosed extracellular vesicles which have been indicated as important biomarkers of cancerous cell functionality, such as multiple drug resistance (MDR). Nanoparticles based chemotherapy is a promising strategy to overcome MDR by interfering the production and composition of exosomes. Therefore, tumor-derived exosomes post-treatment by nanotherapy are implied to play critical roles of biomarkers on cancer MDR analysis. However, the efficient isolation of such exosomes from extracellular environment for their therapeutic response analysis remains challenging. In this study, we presented a microfluidic device featured exosome specific anti-CD63 immobilized ciliated micropillars, which were capable to isolate cancer-derived exosomes from cell culture medium. The captured exosomes can be recovered intact by dissolving the cilia on the micropillars using PBS soaking. Owing to the immobilized antibody in the microfluidic device, nearly 70% of exosome from the biofluid could be isolated. So the secreted exosomes of the MDR and ordinary human breast cancer cells pre-treated by free drug or nanotherapy could be isolated with high purity. The drug contents of the isolated exosomes were measured to analysis of the exosomal pathway response of MDR cells to different chemotherapeutic formulations. Such analyses and further definition of the biomarkers of these exosomes could benefit the future investigations of accurately and reliably determine design principle, functional activity, and mechanisms of nanotherapy for MDR overcoming.

Abstract 4851: Potent cell cycle inhibitors suitable for treatment of multidrug-resistant tumors

Abstract We have developed a class of small-molecule cytotoxic compounds that are highly potent in an in vitro tubulin polymerization assay. Preclinical studies show that these drug candidates are potent against a range of cancer cell lines and may be well suited for the treatment of hematologic or solid tumors resistant to existing chemotherapy agents. We will present data on representative members of this class that inhibit cancer cell growth at nanomolar concentrations and are potent against a variety of cancer cell lines, including liver, breast, ovarian, and lung. The compounds induce cell cycle arrest in the G2M state within 24 hours with cell death occurring over multiple days. These tubulin inhibitors also behave as potent antiangiogenesis agents and inhibit endothelial tube formation in HUVEC cells at nanomolar concentrations. Functional assays using efflux pump inhibitors show that, compared to many major chemotherapy agents, our class of drug candidates is significantly less affected by the efflux pumps MDR1 and MRP1, which are commonly overexpressed as a mechanism of multidrug resistance. Our compounds show comparable potency against wild-type and cell lines developed to be resistant to other cancer drugs, while the potency of clinically relevant compounds such as paclitaxel or doxorubicin is reduced between 80x and about 3000x in the same assay. This feature suggests that our tubulin inhibitors may be active against tumors that are resistant to common cancer drugs. Overexpression of the β-III isotype of tubulin, which is known to occur in many aggressive and metastatic tumors, is another clinically relevant mechanism of resistance to microtubule-targeting anticancer agents. It has been correlated with significantly lower response to docetaxel-based chemotherapy in a number of cancers and is considered an indicator of resistance to paclitaxel and vinorelbine. We will present preclinical data showing that our compounds have similar activity against cells showing normal levels and those highly overexpressing β-III tubulin. The compounds display good in vitro physicochemical properties as well as favorable in vivo pharmacokinetics. In addition, we will present in vivo tolerability and efficacy data in mice. The ability of this class of tubulin inhibitors to maintain their efficacy across multiple drug-resistant cancer cell lines makes them attractive candidates for development as chemotherapy agents. In particular, a new anticancer agent that is less susceptible to major transporters and retains its potency when β-III tubulin is overexpressed could lead to more effective precision second-line therapy. Citation Format: Mohan Sivaraja, Sivan Sizikov, Nilantha Sirisoma, Tamari Kirtadze, Madhuri Chattopadhyay, Makena Ewald, Subhadra Dash, Anne Wong, Georg Neckermann, Elaine To, Stephanie Chang, Timothy P. Shiau, David C. Williams, Kevin M. Short, Angels Estiarte, Anirban Datta, David B. Kita. Potent cell cycle inhibitors suitable for treatment of multidrug-resistant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4851.

Affinity‐Driven Covalent Modulator of the Glyceraldehyde‐3‐Phosphate Dehydrogenase (GAPDH) Cascade

AbstractTraditional medicines provide a fertile ground to explore potent lead compounds, yet their transformation into modern drugs is fraught with challenges in deciphering the target that is mechanistically valid for its biological activity. Herein we reveal that (Z)‐(+)‐isochaihulactone (1) exhibited significant inhibition against multiple‐drug‐resistant (MDR) cancer cell lines and mice xenografts. NMR spectroscopy showed that 1 resisted an off‐target thiolate, thus indicating that 1 was a target covalent inhibitor (TCI). By identifying the pharmacophore of 1 (α,β‐unsaturated moiety), a probe derived from 1 was designed and synthesized for TCI‐oriented activity‐based proteome profiling. By MS/MS and computer‐guided molecular biology approaches, an affinity‐driven Michael addition of the noncatalytic C247 residue of GAPDH was found to control the “ON/OFF” switch of apoptosis through non‐canonically nuclear GAPDH translocation, which bypasses the common apoptosis‐resistant route of MDR cancers.

Affinity-Driven Covalent Modulator of the Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Cascade.

Traditional medicines provide a fertile ground to explore potent lead compounds, yet their transformation into modern drugs is fraught with challenges in deciphering the target that is mechanistically valid for its biological activity. Herein we reveal that (Z)-(+)-isochaihulactone (1) exhibited significant inhibition against multiple-drug-resistant (MDR) cancer cell lines and mice xenografts. NMR spectroscopy showed that 1 resisted an off-target thiolate, thus indicating that 1 was a target covalent inhibitor (TCI). By identifying the pharmacophore of 1 (α,β-unsaturated moiety), a probe derived from 1 was designed and synthesized for TCI-oriented activity-based proteome profiling. By MS/MS and computer-guided molecular biology approaches, an affinity-driven Michael addition of the noncatalytic C247 residue of GAPDH was found to control the "ON/OFF" switch of apoptosis through non-canonically nuclear GAPDH translocation, which bypasses the common apoptosis-resistant route of MDR cancers.

Metformin inhibits the development, and promotes the resensitization, of treatment-resistant breast cancer

Multiple drug resistant (MDR) malignancy remains a predictable and often terminal event in cancer therapy, and affects individuals with many cancer types, regardless of the stage at which they were originally diagnosed or the interval from last treatment. Protein biomarkers of MDR are not globally used for clinical decision-making, but include the overexpression of drug-efflux pumps (ABC transporter family) such as MDR-1 and BCRP, as well as HIF1α, a stress responsive transcription factor found elevated within many MDR tumors. Here, we present the important in vitro discovery that the development of MDR (in breast cancer cells) can be prevented, and that established MDR could be resensitized to therapy, by adjunct treatment with metformin. Metformin is prescribed globally to improve insulin sensitivity, including in those individuals with Type 2 Diabetes Mellitus (DM2). We demonstrate the effectiveness of metformin in resensitizing MDR breast cancer cell lines to their original treatment, and provide evidence that metformin may function through a mechanism involving post-translational histone modifications via an indirect histone deacetylase inhibitor (HDACi) activity. We find that metformin, at low physiological concentrations, reduces the expression of multiple classic protein markers of MDR in vitro and in preliminary in vivo models. Our demonstration that metformin can prevent MDR development and resensitize MDR cells to chemotherapy in vitro, provides important medical relevance towards metformin’s potential clinical use against MDR cancers.

Aminoglucose-functionalized, redox-responsive polymer nanomicelles for overcoming chemoresistance in lung cancer cells

BackgroundChemotherapeutic drugs used for cancer therapy frequently encounter multiple-drug resistance (MDR). Nanoscale carriers that can target tumors to accumulate and release drugs intracellularly have the greatest potential for overcoming MDR. Glucose transporter-1 (GLUT-1) and glutathione (GSH) overexpression in cancer cells was exploited to assemble aminoglucose (AG)-conjugated, redox-responsive nanomicelles from a single disulfide bond-bridged block polymer of polyethylene glycol and polylactic acid (AG-PEG-SS-PLA). However, whether this dual functional vector can overcome MDR in lung cancer is unknown.ResultsIn this experiment, AG-PEG-SS-PLA was synthetized successfully, and paclitaxel (PTX)-loaded AG-PEG-SS-PLA (AG-PEG-SS-PLA/PTX) nanomicelles exhibited excellent physical properties. These nanomicelles show enhanced tumor targeting as well as drug accumulation and retention in MDR cancer cells. Caveolin-dependent endocytosis is mainly responsible for nanomicelle internalization. After internalization, the disulfide bond of AG-PEG-SS-PLA is cleaved in the presence of high intracellular glutathione levels, causing the hydrophobic core to become a polar aqueous solution, which subsequently results in nanomicelle disassembly and the rapid release of encapsulated PTX. Reduced drug resistance was observed in cancer cells in vitro. The caspase-9 and caspase-3 cascade was activated by the AG-PEG-SS-PLA/PTX nanomicelles through upregulation of the pro-apoptotic proteins Bax and Bid and suppression of the anti-apoptotic protein Bcl-2, thereby increasing apoptosis. Furthermore, significantly enhanced tumor growth inhibition was observed in nude mice bearing A549/ADR xenograft tumors after the administration of AG-PEG-SS-PLA/PTX nanomicelles via tail injection.ConclusionsThese promising results indicate that AG-PEG-SS-PLA/PTX nanomicelles could provide the foundation for a paradigm shift in MDR cancer therapy.

Development, Maintenance, and Reversal of Multiple Drug Resistance: At the Crossroads of TFPI1, ABC Transporters, and HIF1.

Early detection and improved therapies for many cancers are enhancing survival rates. Although many cytotoxic therapies are approved for aggressive or metastatic cancer; response rates are low and acquisition of de novo resistance is virtually universal. For decades; chemotherapeutic treatments for cancer have included anthracyclines such as Doxorubicin (DOX); and its use in aggressive tumors appears to remain a viable option; but drug resistance arises against DOX; as for all other classes of compounds. Our recent work suggests the anticoagulant protein Tissue Factor Pathway Inhibitor 1α (TFPI1α) plays a role in driving the development of multiple drug resistance (MDR); but not maintenance; of the MDR state. Other factors; such as the ABC transporter drug efflux pumps MDR-1/P-gp (ABCB1) and BCRP (ABCG2); are required for MDR maintenance; as well as development. The patient population struggling with therapeutic resistance specifically requires novel treatment options to resensitize these tumor cells to therapy. In this review we discuss the development, maintenance, and reversal of MDR as three distinct phases of cancer biology. Possible means to exploit these stages to reverse MDR will be explored. Early molecular detection of MDR cancers before clinical failure has the potential to offer new approaches to fighting MDR cancer.

Restoration of chemosensitivity by multifunctional micelles mediated by P-gp siRNA to reverse MDR

One of the main obstacles in tumor therapy is multiple drug resistance (MDR) and an underlying mechanism of MDR is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins, especially P-glycoprotein (P-gp). In the synergistic treatment of siRNA and anti-cancer drug doxorubicin, it is crucial that both the siRNA and doxorubicin are simultaneously delivered to the tumor cells and the siRNA can fleetly down-regulate P-g before doxorubicin inactivates the P-gp and is pumped out. Herein, a type of micelles comprising a polycationic PEI-CyD shell to condense the siRNA and hydrophobic core to package doxorubicin is reported. The structure of the polymer is determined by 1H NMR, FT-IR, DSC, and XRD and the micelles are characterized by DLS, 2D-NOESY NMR, and TEM to study the self-assembly of the micelles with siRNA and drugs. In vitro studies demonstrate controlled release and temporal enhancement of the therapeutic efficacy of P-gp siRNA and doxorubicin. Release of siRNA down-regulates the mRNA and protein levels of P-gp in the MCF-7/ADR cell lines effectively and the accumulated doxorubicin facilitates apoptosis of the cells to reverse MDR. Moreover, in vivo research reveals that the siRNA and doxorubicin loaded micelles induce tumor cell apoptosis and inhibit the growth of MDR tumor. The western blotting and RT-PCR results illustrate that the synergistic treatment of siRNA and doxorubicin leads to efficient reduction of the P-gp expression as well as cell apoptotic induction in MDR tumors at a small dosage of 0.5 mg/kg. The micelles have large clinical potential in drug/RNAi synergistic treatment via restoration of the chemosensitivity in MDR cancer therapy.

Abstract 1908: MDR1 gene overexpression confers resistance to C-MET inhibitor in multidrug resistant cancer cell line

Abstract c-MET signaling is implicated in a wide variety of human malignancies. Hepatocyte growth factor (HGF) binding to the proto-oncogenic c-MET receptor lead to activate multiple tyrosine kinase signaling pathway. Inappropriate c-MET signaling in cancer can enhance tumor cell proliferation, survival, motility, and invasion. Recently, several reports have indicated that inhibition of c-MET signaling induced apoptosis in a variety of cancer cells and recognized as a novel anticancer therapy approach. Previous study from our laboratory also showed that the higher mRNA expression level of HGF, and its downstream genes including HGF receptor (c-Met) and urokinase (uPA) were detected in drug resistant Dx5-C5 cell compared with parental human uterus sarcoma MES-SA cell line. Furthermore, report indicated that the constitutive expression of P-glycoprotein (P-gp) is involved in the HGF/MET related pathway of MDR-positive human hepatocellular carcinoma cell line. Moreover, MDR1 gene overexpressed leukemia cell line also shown more resistance to tyrosine kinase inhibitor imatinib mesylate. These finding suggested that chemoresistant cancer cells may also develop a similar mechanism to against c-MET inhibitor. Our results shown: when treated both cell cancer lines with c-MET inhibitor PHA665752, we found that compared to parental MES-SA cells, the Dx5-C5 cells with highly expressed P-gp was more resistant to PHA665752, the resistance was also reversed by co-treatment of MDR inhibitor verapamil and PHA665752. In addition, when transfection of c-MET shRNA into Dx5-C5 and MES-SA cell line for 48 hrs, we found that the viability was decreased in the c-MET shRNA transfected Dx5-C5 cell line in a dose dependent manner but not in the MES-SA cell line. In conclusion, our study demonstrated that existence of P-glycoprotein in MDR cell line would attenuate c-MET inhibitor induced cell death and may help us to develop a new strategy for treatment of multiple drug resistance cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1908. doi:1538-7445.AM2012-1908

Signal Transduction Pathways and Transcriptional Mechanisms of ABCB1/Pgp-mediated Multiple Drug Resistance in Human Cancer Cells

Multiple drug resistance (MDR), defined as the ability of tumour cells to survive exposure to many chemotherapeutic agents, is a major cause of treatment failure in human cancers. The membrane transporter P-glycoprotein (Pgp, encoded by the ABCB1 [adenosine triphosphate-binding cassette, subfamily B, member 1] gene) is the main mechanism for decreased intracellular drug accumulation in human MDR cancer. ABCB1/Pgp-mediated MDR involves several signal transduction pathways and transcription factors. Activation of these signal transduction pathways influences the prognosis of MDR human cancer. Signalling pathways involved in ABCB1/Pgp-mediated MDR include the mitogen-activated protein kinase (MAPK), c-Jun NH(2)-terminal kinase (JNK), p38, cyclic adenosine monophosphate-dependent protein kinase, phosphatidylino sitol 3-kinase and protein kinase C signalling pathways. This review summarizes the biological characteristics, target points and signalling cascade mediators of these pathways. Drugs targeted against these pathways may provide new therapies for treatment of ABCB1/Pgp-mediated MDR.

Abstract 4914: Methylation profiling of CpG islands in multiple drug resistant cell line

Abstract Multiple drug resistance (MDR) is a major issue to attenuate the effectiveness of chemotherapy to many human malignancies. Despite extensively investigation, MDR inhibitors have been discovered serve side effect. Recently, scientists have reported that proteasome inhibitor, Bertezomib (PS-341), induced apoptosis in a variety of cancer cells and recognized as a novel anticancer therapy approach. Previous study revealed that bertezomib inhibits DNA methyltransferase and led to DNA hypomethylation in myeloid leukemia. Despite its success, some patients are resistant to ongoing proteasome inhibitor treatment. These reports suggested that chemoresistant cell line may develop a novel mechanism to against proteasome inhibitor. To verify the hypothesis, we used human CpG island microarray to indentify epigenetic change and genes expression profile in parental human uterus sarcoma cell line (MES-SA) and MES-SA variant drug resistant cell line (Dx5-C5) Our data has shown that the global genes were hypomethylated in both PS-341 treated MES-SA and Dx5-C5 cell line. Furthermore, the Wnt signalling pathway and cell cycle related gene have significant altered in DNA methylation pattern of Dx5-C5. In summary, our study demonstrated that the cell cycle related gene(s) and upstream gene(s) of Wnt pathway altered its DNA methylation pattern and mRNA expression level in MDR cell line. These data may help us to develop a new strategy for treatment of multiple drug resistance cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4914.

A novel copper chelate modulates tumor associated macrophages to promote anti-tumor response of T cells.

BackgroundAt the early stages of carcinogenesis, the induction of tumor specific T cell mediated immunity seems to block the tumor growth and give protective anti-tumor immune response. However, tumor associated macrophages (TAMs) might play an immunosuppressive role and subvert this anti tumor immunity leading to tumor progression and metastasis.Methodology/Principal FindingsThe Cu (II) complex, (chelate), copper N-(2-hydroxy acetophenone) glycinate (CuNG), synthesized by us, has previously been shown to have a potential usefulness in immunotherapy of multiple drug resistant cancers. The current study demonstrates that CuNG treatment of TAMs modulates their status from immunosuppressive to proimmunogenic nature. Interestingly, these activated TAMs produced high levels of IL-12 along with low levels of IL-10 that not only allowed strong Th1 response marked by generation of high levels of IFN-γ but also reduced activation induced T cell death. Similarly, CuNG treatment of peripheral blood monocytes from chemotherapy and/or radiotherapy refractory cancer patients also modulated their cytokine status. Most intriguingly, CuNG treated TAMs could influence reprogramming of TGF-β producing CD4+CD25+ T cells toward IFN-γ producing T cells.Conclusion/SignificanceOur results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancers through reprogramming of TAMs that in turn reprogram the T cells and reeducate the T helper function to elicit proper anti-tumorogenic Th1 response leading to effective reduction in tumor growth.