Multidrug-resistant Cancer Cell Lines Research Articles

Cytotoxicity of seven naturally occurring phenolic compounds towards multi-factorial drug-resistant cancer cells

IntroductionIn medical oncology, multi-drug resistance (MDR) of cancer cells continues to be a major impediment. We are in quest of novel anti-proliferative agents to overcome drug-resistant tumor cells. MethodsIn the present study, we investigated the cytotoxicity of 7 naturally occurring phenolic compounds including two isoflavonoids alpinumisoflavone (1) and laburnetin (2), one biflavonoid amentoflavone (3), three lignans pycnanthulignene A (4), pycnanthulignene B (5), and syringaresinol (7) and one xanthone, euxanthone (6) against 9 drug-sensitive and MDR cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analyzed via flow cytometry. ResultsThe IC50 values for the investigational phenolics ranged from 5.91 µM (towards leukemia CEM/ADR5000 cells) to 65.65 µM (towards drug-resistant breast adenocarcinoma MDA-MB-231-BCRP cells) for 1, 27.63 µM (towards leukemia CCRF-CEM cells) to 107.57 µM (towards MDA-MB-231-pcDNA cells) for 2, from 5.84 µM (towards CEM/ADR5000 cells) to 65.32 µM (towards colon carcinoma HCT116 (p53−/−) cells) for 4 and 0.20 µM (towards CCRF-CEM cells) to 195.12 µM (towards leukemia CEM/ADR5000) for doxorubicin. Phenolics 3, 5, 6 and 7 displayed selectivity cytotoxic effects on cancer cells lines. Compounds 1 and 4 induced apoptosis in CCRF-CEM cells, mediated by loss of MMP and increase ROS production. ConclusionsThe studied phenolics and mostly isoflavonoid 1 and lignan 4 are potential cytotoxic natural products that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug-resistant cancers.

PH and near-infrared light dual-stimuli responsive drug delivery using DNA-conjugated gold nanorods for effective treatment of multidrug resistant cancer cells

A thiolated pH-responsive DNA conjugated gold nanorod (GNR) was developed as a multifunctional nanocarrier for targeted, pH-and near infrared (NIR) radiation dual-stimuli triggered drug delivery. It was further passivated by a thiolated poly(ethylene glycol)-biotin to improve its cancer targeting ability by specific binding to cancer cell over-expressed biotin receptors. Doxorubicin (DOX), a widely used clinical anticancer drug, was conveniently loaded into nanocarrier by intercalating inside the double-stranded pH-responsive DNAs on the GNR surface to complete the construction of the multifunctional nanomedicine. The nanomedicine can rapidly and effectively release its DOX payload triggered by an acidic pH environment (pH~5) and/or applying an 808nm NIR laser radiation. Compared to free DOX, the biotin-modified nanomedicine displayed greatly increased cell uptake and significantly reduced drug efflux by model multidrug resistant (MDR) breast cancer cell lines (MCF-7/ADR). The application of NIR radiation further increased the DOX release and facilitated its nuclear accumulation. As a result, this new DNA-GNR based multifunctional nanomedicine exerted greatly increased potency (~67 fold) against the MDR cancer cells over free DOX.

Cytotoxicity of 15 Cameroonian medicinal plants against drug sensitive and multi-drug resistant cancer cells

Ethnopharmacological relevanceCameroonian medicinal plants are traditionally used to treat many ailments, including cancer and related diseases. Cancer is characterized as a condition with complex signs and symptoms. It has been recommended that ethnopharmacological usages such as immune and skin disorders, inflammatory, infectious, parasitic and viral diseases should be taken into account when selecting plants for anticancer screenings, since these reflect disease states bearing relevance to cancer or cancer-like symptoms. Aim of the studyThe present study aims at investigating 20 methanol extracts from 15 Cameroonian medicinal plants on a panel of human cancer cell lines, including various drug-resistant phenotypes. Possible modes of action of the of the most active plant were analyzed. Materials and methodsMethanol extracts from different plant parts (leaves, bark, roots, fruits or whole plant) were evaluated for their cytotoxicity using resazurin reduction assay on a panel of nine sensitive and multi-drug resistant (MDR) cancer cell lines. Cell cycle, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were measured by flow cytometry. ResultsPrescreening of extracts at 80µg/mL showed that 6 extracts out of 20 inhibited more than 50% proliferation of leukemia CCRF-CEM cells; these include extracts from Anthocleista schweinfurthii fruits (ASF; 48.28%), Morus mesozygia bark (MMB; 42.76%), Nauclea latifolia bark (NLB; 38.75%), Tridesmostemon omphalocarpoides bark (TOB; 38.53%), Nauclea latifolia leaves (NLL; 35.17%) and Erythrina sigmoidea bark (ESB; 33.77%). Subsequent investigations revealed IC50 values below or around 20µg/mL for extracts from MMB, NLB, NLL and ESB towards sensitive CCRF-CEM cells and its resistant P-glycoprotein over-expressing subline CEM/ADR5000. The best extract, ESB also displayed IC50 values below 20µg/mL colon carcinoma HCT116 (p53+/+) cells with an IC50 value of 19.63µg/mL and it resistant p53 knockout subline HCT116 (p53−/−) with an IC50 value of 16.22µg/mL. ConclusionErythrina sigmoidea, Anthocleista schweinfurthii, Morus mesozygia, Nauclea latifolia, Tridesmostemon omphalocarpoides used in African traditional medicine are good cytotoxic plants that can be exploited to develop phytomedicine to fight cancers including MDR phenotypes.

Upregulation of the Multidrug Resistance P‐glycoprotein ABCB1 by Transcription Factor Pituitary Homeobox 2 (Pitx2) in Human Colon and Kidney Cancers

BackgroundPituitary homeobox 2 (Pitx2) is a master regulator of organ asymmetry during development but is reactivated in neoplastic cells, contributing to cell proliferation, tumor cell invasion and cancer progression. Upregulation of the multidrug resistance P‐glycoprotein ABCB1 confers chemotherapeutic drug resistance to tumor cells, hampering effective treatment. We have previously shown that Pitx2 is upregulated in a multidrug resistant (MDR) kidney cancer cell line and serves as a positive transcriptional regulator of ABCB1 thereby increasing resistance to the anthracycline chemotherapeutic drug, doxorubicin (DOX) (Lee, W.‐K. et al. Int J Cancer 113, 556–67, 2013). The objective of this study was to determine if ABCB1 regulation by Pitx2 is also in engaged by other cancer types that exhibit intrinsic MDR.MethodsExpression of Pitx2 and ABCB1 was determined by immunofluorescence microscopy, immunoblotting and immunohistochemistry. Cell viability was assessed by MTT and clonogenic survival assays. Pitx2 transcriptional activity was measured by luciferase reporter gene assay. Isolation of nuclei was performed by Tenbroeck homogenization in hypoosmotic buffer and a discontinuous sucrose gradient.ResultsIn human colorectal adenocarcinoma Colo320DM cells, which exhibit MDR and harbor augmented ABCB1 expression, nuclear Pitx2 expression and Pitx2 reporter gene activity were significantly increased compared to less drug resistant Colo205 cells. The ABCB1 inhibitor PSC833 (0.1 μM) reversed drug resistance to 1 μM DOX after 24 h in Colo320DM cells. Further, knockdown of Pitx2 by RNAi in Colo320DM cells resulted in decreased ABCB1 protein by almost 3‐fold and diminished resistance to DOX resulting in attenuated cell survival. Moreover, the Pitx2 isoforms that are involved in regulation of ABCB1 in colon and kidney cancer cells were investigated. Both Pitx2A and Pitx2B were found to be present in the nuclear fraction of Colo320DM and MDR A498 kidney carcinoma cells. Analysis of human kidney and colon cancer tissue arrays by co‐immunostaining suggests a causal relationship between nuclear Pitx2 and ABCB1 expression.ConclusionPitx2 is a transcriptional regulator of ABCB1 in cancers of the colon and kidney suggesting a contributory role to the MDR phenotype.Support or Funding InformationFunded by an Internal Research Grant (UW/H) and ZBAF

Design, synthesis and biological evaluation of thiosemicarbazones, hydrazinobenzothiazoles and arylhydrazones as anticancer agents with a potential to overcome multidrug resistance

There is a constant need for new therapies against multidrug resistant (MDR) cancer. An attractive strategy is to develop chelators that display significant antitumor activity in multidrug resistant cancer cell lines overexpressing the drug efflux pump P-glycoprotein. In this study we used a panel of sensitive and MDR cancer cell lines to evaluate the toxicity of picolinylidene and salicylidene thiosemicarbazone, arylhydrazone, as well as picolinylidene and salicylidene hydrazino-benzothiazole derivatives. Our results confirm the collateral sensitivity of MDR cells to isatin-β-thiosemicarbazones, and identify several chelator scaffolds with a potential to overcome multidrug resistance. Analysis of structure-activity-relationships within the investigated compound library indicates that NNS and NNN donor chelators show superior toxicity as compared to ONS derivatives regardless of the resistance status of the cells.

Alisol F 24 Acetate Enhances Chemosensitivity and Apoptosis of MCF-7/DOX Cells by Inhibiting P-Glycoprotein-Mediated Drug Efflux.

Multidrug resistance (MDR) is a prime reason for numerous failed oncotherapy approaches. In the present study, we investigated whether Alisol F 24 acetate (ALI) could reverse the MDR of MCF-7/DOX cells, a multidrug-resistant human breast cancer cell line. We found that ALI was a potent P-glycoprotein (P-gp) inhibitor, in the Caco-2-monolayer cell model. ALI showed a significant and concentration-dependent cytotoxic effect on MCF-7/DOX cells in combination with doxorubicin by increasing intracellular accumulation and inducing nuclear migration of doxorubicin. However, ALI had no such effect on MCF-7 cells. In addition, ALI also promoted doxorubicin-induced early apoptosis of MCF-7/DOX cells in a time-dependent manner. These results suggest that ALI can enhance chemosensitivity of doxorubicin and reinforce its anti-cancer effect by increasing its uptake, especially inducing its nuclear accumulation in MCF-7/DOX cells. Therefore, ALI could be developed as a potential MDR-reversing agent in cancer chemotherapy in further study.

N-alkylated isatins evade P-gp mediated efflux and retain potency in MDR cancer cell lines.

The search for novel anticancer therapeutics with the ability to overcome multi-drug resistance (MDR) mechanisms is of high priority. A class of molecules that show potential in overcoming MDR are the N-alkylated isatins. In particular 5,7-dibromo-N-alkylisatins are potent microtubule destabilizing agents that act to depolymerize microtubules, induce apoptosis and inhibit primary tumor growth in vivo. In this study we evaluated the ability of four dibrominated N-alkylisatin derivatives and the parent compound, 5,7-dibromoisatin, to circumvent MDR. All of the isatin-based compounds examined retained potency against the MDR cell lines; U937VbR and MES-SA/Dx5 and displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell lines. We show that one mechanism by which the isatin-based compounds overcome MDR is by circumventing P-glycoprotein (P-gp) mediated drug efflux. Thus, as the isatin-based compounds are not susceptible to extrusion from P-gp overexpressing tumor cells, they represent a promising alternative strategy as a stand-alone or combination therapy for treating MDR cancer.

Development and evaluation of lipid nanoparticles for paclitaxel delivery: a comparison between solid lipid nanoparticles and nanostructured lipid carriers

Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) were prepared as delivery system for water-insoluble anticancer agent, paclitaxel (PTX). The dispersion of SLNs was consisted of 5 % w/w tristearin, 3.75 % w/w egg phosphatidylcholine (egg PC) and 1 % w/w polysorbate 80 in water. NLCs were prepared using the same composition as SLNs except that 10 % of the solid lipid was replaced by triolein. PTX was incorporated into SLNs and NLCs to attain 0.025 % w/w in the dispersion. The particle size of the prepared SLNs and NLCs were 167.9 ± 21.3 and 121.9 ± 28.3 nm, respectively, and slightly increased to 239.1 ± 32.6 and 183.6 ± 36.2 nm by PTX incorporation. PTX incorporation also increased polydispersity index suggesting broader size distribution compared to that for empty particles. SLNs and NLCs showed sustained release of PTX in cell culture media containing 10 % fetal bovine serum at 37 °C compared to the commercial micellar formulation consisted of Cremophor EL and ethanol. PTX in SLNs and NLCs showed comparable cytotoxicity to the commercial formulation and free PTX against human breast cancer cell line, MCF-7. On the contrary, PTX in SLNs and NLCs showed higher anticancer activity against multidrug resistant (MDR) cancer cell line, MCF-7/ADR, compared to the free PTX delivered in DMSO, which indicates that both SLNs and NLCs would be effective carriers to avoid efflux pump expressed in MDR cancer cells. In conclusion, the SLNs and NLCs prepared in the present study showed similar characteristics each other and both can be used as effective delivery system for PTX.

"Non-Taxifolin" Derived Flavonolignans: Phytochemistry and Biology.

Flavonolignans are plant natural products, composed of a flavonoid moiety and a lignan (phenylpropanoid) part. Current literature focuses on flavonolignans formed from taxifolin and coniferyl alcohol as e.g. silybin and its congeners from fruit extract from the purple variety of the milk thistle (Silybum marianum) denoted as "silymarin". This review describes chemistry and biological activity of so far neglected "non-taxifolin" based flavonolignans, derived from apigenin, luteolin, tricin, chrysoeriol, naringenin and eriodictyol, as the flavonoid part. Up-to-date knowledge on hydnocarpin, hydnocarpin-D, pseudotsuganol, hydnowightin, neohydnocarpin, palstatin, salcolins A and B, anastatins A and B, sinaiticin, silyamandin and silandrin is summarized in the present paper. Most of non-taxifolin derived flavonolignans have been shown to exhibit in vitro and/or in vivo anti-hepatotoxic, anti-oxidant, free radical scavenging, anti-inflammatory, anti-proliferative, anti-cancer, chemotherapy potentiating, anti-melanogenic, anti-bacterial, vasorelaxing, anti-platelet aggregation and/or hypotriglyceridemic activity, often stronger than silybin. Many of these compounds inhibited Staphylococcus aureus multidrug resistance pump NorA and sensitized multidrug resistant cancer cell lines showing a potential as adjuvants. Non-taxifolin derived flavonolignans are a relatively unexplored group of compounds with interesting biological activity and great application potential. Their detailed study could provide a new insight into the biomimetic synthesis in order to obtain new compounds with greater activity and identify new lead structures for the biomedicinal research.

Cytotoxicity of compounds from Xylopia aethiopica towards multi-factorial drug-resistant cancer cells

IntroductionMultidrug resistance (MDR) in cancer represent a major hurdle in chemotherapy. Previously, the methanol extract of the medicinal spice Xylopia aethiopica displayed considerable cytotoxicity against multidrug resistant (MDR) cancer cell lines. MethodsThe present study was designed to assess the cytotoxicity of compounds, 16α-hydroxy-ent-kauran-19-oic acid (2), 3,4′,5-trihydroxy-6″,6″-dimethylpyrano[2,3-g]flavone (3), isotetrandrine (5) and trans-tiliroside (6) derived from the methanol crude extract of Xylopia aethiopica against 9 drug-sensitive and -resistant cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analyzed via flow cytometry. ResultsFlavonoid 3 and alkaloid 5 also displayed IC50 values ranging from 2.61 µM (towards leukemia CCRF-CEM cells) to 18.60 µM (towards gliobastoma multiforme U87MG.ΔEGFR cells) and from 1.45 µM (towards HepG2 cells) to 7.28 µM (towards MDA-MB-231-pcDNA cells), respectively. IC50 values ranged from 0.20 µM (against CCRF-CEM cells) to 195.12 µM (against CEM/ADR5000 cells) for doxorubicin. Compound 3 induced apoptosis in leukemia CCRF-CEM cells mediated by the disruption of the MMP, whilst 5 induced apoptosis mediated by ROS production. ConclusionsCompounds 2 and 5 represent potential cytotoxic phytochemicals that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug-resistant cancers.

Expression of ABC drug transporters in human placenta from normal and GDM pregnancies and its relation to cytokines

Multidrug resistance (MDR) of cancer is often associated with the overexpression of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp), multidrug resistance-associated protein-1 (MRP-1) and breast cancer resistance protein (BCRP or ABCG2), in cancer cells, which facilitates the active efflux of a wide variety of chemotherapeutic drugs out of the cells. Marsdenia tenacissima is a traditional Chinese medicinal herb that has long been clinically used for treatment of cancers, particularly in combinational use with anticancer drugs. Polyoxypregnanes (POPs) are identified as main constituents of this herb, and three of them have been reported to exhibit P-gp modulatory effect and thus reverse MDR. Therefore, it is of great necessity to investigate more POPs that have potential to reverse transporters-mediated MDR.We aimed to identify POPs as the chemical basis responsible for circumventing ABC transporters-mediated MDR by M. tenacissima.The MDR reversal effects of M. tenacissima crude extract together with a series of isolated POPs were evaluated on several MDR cancer cell lines that overexpress P-gp, MRP1 or ABCG2. The activities of P-gp, MRP1 and ABCG2 were determined by the flow cytometry-based substrate efflux assay. Molecular docking of POPs to a three-dimensional human P-gp homology structure was also performed.The crude extract of M. tenacissima was firstly found to circumvent P-gp-mediated MDR. Then, 11 polyoxypregnane compounds (POPs) isolated from this herb were found to overcome P-gp-, MRP1- and/or ABCG2-mediated MDR. Further mechanistic study delineated that the reversal of MDR by these POPs was due to significant increase in the intracellular concentrations of the substrate anticancer drugs via their inhibition of different ABC transporter-mediated efflux activities. Furthermore, molecular docking revealed that POPs with P-gp modulatory effect bound to P-gp and fitted well into the cavity between the alpha and beta subunit of P-gp via forming hydrogen bonds. In addition, several key structural determinants for inhibition of P-gp, MRP1 or ABCG2 by POPs were illustrated.Our findings advocated the rational use of M. tenacissima to enhance efficacies of conventional anticancer drugs in tumors with ABC drug transporters-mediated MDR. Furthermore, 11 POPs were found to contribute to MDR reversal effect of M. tenacissima via inhibition of different ABC efflux transporters.

Effects of a novel porphyrin-based photosensitizer on sensitive and multidrug-resistant human gastric cancer cell lines

Photodynamic therapy (PDT) has been considered to be a possible candidate approach in combating multidrug resistance (MDR) phenomenon during the treatment of cancer. To investigate the photocytotoxicity of a novel porphyrin-based photosensitizer, meso-5-[ρ-DTPA-aminophenyl]-10, 15, 20-triphenyl-porhyrin (DTP) (Fig. 1A), on MDR cells, the intracellular DTP uptake, phototoxicity and subcellular DTP localization were studied by using a human gastric cancer MGC803 cell line and its paclitaxel selected subline MGC803/PA expressing MDR phenotype. No significant difference was observed in intracellular DTP accumulation between sensitive and resistant cell lines after exposure to 1.56μM concentration for 6h. DTP-PDT induced significant photocytotoxicity on both MGC803 and MGC803/PA cell lines and the photokilling was greater in MGC803 cell line in comparison to MGC803/PA. The fluence that caused 50% cell death was 4.42 and 6.29J/cm2 in MGC803 and MGC803/PA cell lines, respectively. The presence of Pgp inhibitors verapamil and cyclosporin A could not modify the intracellular DTP level in MGC803/PA cell line and the phototoxic effects. DTP was localized at lysosomes of MGC803 cell line but at lysosomes and mitochondria of MGC803/PA. Our results indicated that DTP-mediated PDT could eradicate gastric cancer cells whether or not they express MDR although the efficacy is slightly reduced in the MDR cells. The photokilling in MDR cells could not be altered by MDR inhibitor verapamil. The slightly different photocytotoxicity between sensitive and resistant cell lines could not explained by classical Pgp MDR and might be attributed to the differential intracellular DTP localization sites.

Cytotoxicity of three naturally occurring flavonoid derived compounds (artocarpesin, cycloartocarpesin and isobavachalcone) towards multi-factorial drug-resistant cancer cells

IntroductionCancer remains an aggressive deadly disease, if drug resistance develops. This problem is aggravated by the fact that multiple rather than single mechanisms are involved in resistance and that multidrug resistance (MDR) phenomena cause inefficacy of many clinical established anticancer drugs. We are seeking for novel cytotoxic phytochemicals to combat drug-resistant tumour cells. MethodsIn the present study, we investigated the cytotoxicity of three naturally occurring flavonoids including two flavones artocarpesin (1) and cycloartocarpesin (2) and one chalcone, isobavachalcone (3) against 9 drug-sensitive and MDR cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analysed via flow cytometry. ResultsFlavones 1 and 2 as well as chalcone 3 displayed cytotoxic effects at various extent on all the 9 tested cancer cell lines with IC50 values respectively below 106 µM, 50 µM and 25 µM. The IC50 values for the three investigational flavonoids ranged from 23.95 µM (towards hepatocarcinoma HepG2 cells) to 105 µM [towards colon carcinoma HCT116 (p53−/−) cells] for 1, from 15.51 µM (towards leukemia CCRF-CEM cells) to 49.83 µM [towards glioblastoma U87MG.ΔEGFR cells] for 2 and from 2.30 µM (towards CCRF-CEM cells) to 23.80 µM [towards colon carcinoma HCT116 (p53+/+) cells] for 3 and from 0.20 µM (towards CCRF-CEM cells) to 195.12 µM (towards leukemia CEM/ADR5000 cells) for doxorubicin. Compounds 2 and 3 induced apoptosis in CCRF-CEM leukemia cells, mediated by caspase activation and the disruption of MMP. ConclusionsThe three tested flavonoids and mostly chalcone 3 are potential cytotoxic natural products that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug-resistant cancers.

Abstract 3048: Study of mitochondria in multi-drug resistance and Polyphyllin D anti-cancer effect in hepatocellular carcinoma

Abstract Liver cancer, being the sixth common cancer, causes approximately 700,000 deaths worldwide annually. Major treatment relies on chemotherapies because of the late diagnosis in many cases of liver cancer. However, the results are not satisfactory since the cancer cells develop multi-drug resistance (MDR), which greatly hampers the efficacy of multiple kinds of structurally different drugs, during the course of treatment. Several mechanisms of MDR were identified, the most notable one is the over-expression of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp) and multi-drug resistance protein 1 (MRP-1), on plasma membrane. These transporters act as efflux pump of various types of chemo-drugs to accentuate the MDR phenotype. Nevertheless, the complete picture of MDR formation is yet to be explored. As mitochondria contribute vitally in apoptosis and energy production, in which both criteria are substantially altered in cancer context, we hypothesized that the organelle also takes part in the development of MDR in liver cancer. In our study, with the support of conventional assays such as western blotting and confocal imaging analysis, we adopted a novel approach by making use of the tunable resistive pulse sensing (TRPS) technique to characterize isolated mitochondria of HepG2 and R-HepG2, which corresponds to the native and multi-drug resistant liver cancer cell lines respectively. We identified a difference in volume and amount of mitochondria present in HepG2 and R-HepG2, plus the expression of P-gp and MRP-1 on mitochondria of R-HepG2. These suggest a potential role of mitochondria in MDR. On the other hand, a drug candidate Polyphyllin D (PD) was found to exert selective potency against R-HepG2. However, the mechanism is not lucid. We investigated the possible participation of mitochondria in this phenomenon and identified PD causes stronger swelling and membrane potential depolarization to isolated mitochondria of R-HepG2. Future attempt in elucidating pathways involved in PD toxicity will bring insight on designing therapeutics against cancer cells with MDR phenotype. This study also suggested future research in mitochondria can aid the discovery of more mechanisms in MDR development. Citation Format: Brandon Ho Ngai, Jacky Fong Chuen Loo, Siu Kai Kong. Study of mitochondria in multi-drug resistance and Polyphyllin D anti-cancer effect in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3048. doi:10.1158/1538-7445.AM2015-3048

Abstract 4433: P-gp-overexpressing MDR cells shed larger extracellular vesicles than their drug-sensitive counterparts

Abstract P-glycoprotein (P-gp) overexpression is a major cause of multidrug resistance (MDR) and presents a serious challenge to the efficient treatment of cancer1. In spite of being a major clinical challenge, to date there are no non-invasive means to diagnose the MDR phenotype, using non-tumour biological samples. Recently, the non-genetic acquisition of functional P-gp has been described as being possibly mediated by extracellular vesicles (EVs). Indeed, P-gp may be transferred by EVs from MDR cells to drug-sensitive (DS) recipient cells3. Collectively, EVs may refer to exosomes (with endosomal origin and sizes ranging from 30-100nm), microvesicles (with plasma membrane origin and sizes ranging from 50-2000nm) or even apoptotic bodies (ranging from 50-5000nm)4. This study aimed at characterizing and comparing EVs from P-gp-overexpressing MDR cancer cells and their respective DS counterparts, as an attempt to identify MDR biomarkers. Two pairs of MDR cancer cell lines and their DS counterparts were used: a chronic myeloid leukaemia pair (K562Dox, a kind gift from Dr. Jean-Pierre Marie and K562, from EACC) and a non-small cell lung cancer pair (RH460 and NCI-H460, a kind gift from Dr. Milica Pesic). EVs shed by these cells were characterized in terms of size and molecular markers. In addition, their protein content was investigated by proteomic analysis and by Western blot. This study describes, for the first time, that P-gp overexpressing MDR cells produce larger EVs than their DS counterparts. In addition, a distinct protein signature was found between the EVs shed by DS cells and their MDR counterparts, consisting mainly in differences in the relative abundances of particular proteins. As predicted, P-gp was found present only in the EVs shed by MDR cells. Interestingly, proteins involved in the biogenesis of exosomes, such as Syntenin-1, TSG-101, CD63, Clathrin LCB and CHMP4, were found decreased in EVs shed by MDR cells. These results were in agreement with the identified size differences between the EVs from MDR and DS cells mentioned above, which may suggest that MDR cells produce more microvesicles and that DS cells produce more exosomes. In conclusion, it is proposed that EVs shed by the P-gp overexpressing MDR cells may have a specific size and protein signature, consisting in P-gp presence and a lower abundance of proteins involved in the biogenesis of exosomes. This may allow the development of a non-invasive way of detecting and predicting MDR, by analysing EVs shed into the circulation of cancer patients. 1. Lopes-Rodrigues, V. et al. The network of P-glycoprotein and microRNAs interactions. Int J Cancer 135, 253-263, (2014). 2. Bebawy, M. et al. Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells. Leukemia 23, 1643-1649, (2009). 3. Akers, J. C. et al. Biogenesis of extracellular vesicles (EV). J Neurooncol 113, 1-11, (2013). Citation Format: Vanessa Lopes-Rodrigues, Alessio Di Luca, Diana Sousa, Hugo Seca, Paula Meleady, Michael Henry, Raquel T. Lima, Robert O'Connor, M. Helena Vasconcelos. P-gp-overexpressing MDR cells shed larger extracellular vesicles than their drug-sensitive counterparts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4433. doi:10.1158/1538-7445.AM2015-4433

Dietary flavonoid fisetin binds to β-tubulin and disrupts microtubule dynamics in prostate cancer cells

Microtubule targeting based therapies have revolutionized cancer treatment; however, resistance and side effects remain a major limitation. Therefore, novel strategies that can overcome these limitations are urgently needed. We made a novel discovery that fisetin, a hydroxyflavone, is a microtubule stabilizing agent. Fisetin binds to tubulin and stabilizes microtubules with binding characteristics far superior than paclitaxel. Surface plasmon resonance and computational docking studies suggested that fisetin binds to β-tubulin with superior affinity compared to paclitaxel. Fisetin treatment of human prostate cancer cells resulted in robust up-regulation of microtubule associated proteins (MAP)-2 and -4. In addition, fisetin treated cells were enriched in α-tubulin acetylation, an indication of stabilization of microtubules. Fisetin significantly inhibited PCa cell proliferation, migration, and invasion. Nudc, a protein associated with microtubule motor dynein/dynactin complex that regulates microtubule dynamics, was inhibited with fisetin treatment. Further, fisetin treatment of a P-glycoprotein overexpressing multidrug-resistant cancer cell line NCI/ADR-RES inhibited the viability and colony formation. Our results offer in vitro proof-of-concept for fisetin as a microtubule targeting agent. We suggest that fisetin could be developed as an adjuvant for treatment of prostate and other cancer types.

Lower antioxidative capacity of multidrug-resistant cancer cells confers collateral sensitivity to protoflavone derivatives.

Multidrug resistance (MDR) may develop due to a series of adaptive responses under a new stress conditions, such as chemotherapy. Novel strategies are urgently needed to fight MDR in cancer, and chemotherapeutics that are selective for MDR cancer cells offer a promising approach. Certain protoflavones were previously found to have potential in this regard. Cytotoxicity of six protoflavones was assessed in different P-glycoprotein overexpressing MDR cancer cell lines and in their non-MDR counterparts. The impacts of compound 5, 6-methylprotoflavone previously published and a new derivative, 6-bromoprotoflavone (compound 6), on the cell cycle distribution were evaluated, and 6 was also studied for its potential to regulate the intracellular antioxidative capacity. Protoflavones showed a significant cytotoxicity against all cancer cell lines and selectivity toward MDR cancer cells adapted to conventional chemotherapeutics. Inverse sensitivity versus MDR selectivity pattern was observed. Treatment with H₂O₂ showed that MDR cancer cells are more vulnerable to oxidative stress. Compounds 5 and 6 significantly decreased the portion of MDR cells in the G1 phase. The levels of reactive oxygen and nitrogen species (ROS/RNS) between MDR and non-MDR cells significantly differed upon exposure to 6, accompanied by changes in the glutathione (GSH) levels and in the expression of manganese superoxide dismutase (MnSOD), glutathione-S-transferase π (GST π) and hypoxia-inducible factor-1α (HIF-1α). Our results suggest that MDR cancer cells can be more vulnerable to the pro-oxidative activity of protoflavones due to an impaired antioxidative defense that might arise during the adaptation processes provoked by chemotherapy.

Cytotoxicity of a naturally occurring furoquinoline alkaloid and four acridone alkaloids towards multi-factorial drug-resistant cancer cells

IntroductionChemotherapy is one of the preferred mode of treatment of malignancies, but is complicated by the expression of diverse resistance mechanisms of cancer cells. MethodsIn the present study, we investigated the cytotoxicity of five alkaloids including a furoquinoline montrofoline (1) and four acridones namely 1-hydroxy-4-methoxy-10-methylacridone (2), norevoxanthine (3), evoxanthine (4), 1,3-dimethoxy-10-methylacridone (5) against 9 drug-sensitive and multidrug-resistant (MDR) cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analyzed via flow cytometry. ResultsFuroquinoline 1 as well as the acridone alkaloids 2–5 displayed cytotoxic effects with IC50 values below 138 µM on all the 9 tested cancer cell lines. The IC50 values ranged from 41.56 µM (towards hepatocarinoma HepG2 cells) to 90.66 µM [towards colon carcinoma HCT116 (p53−/−) cells] for 1, from 6.78 µM [towards HCT116 (p53−/−) cells) to 106.47 µM [towards breast adenocarcinoma MDA-MB-231-pcDNA cells] for 2, from 5.72 µM (towards gliobastoma U87MG.ΔEGFR cells) to 137.62 µM (towards leukemia CCRF-CEM cells] for 3, from 6.11 µM [towards HCT116 (p53+/+) cells] to 80.99 µM (towards HepG2 cells] for 4, from 3.38 µM (towards MDA-MB-231-BCRP cells) to 58.10 µM (towards leukemia CEM/ADR5000 cells] for 5 and from 0.20 µM (against CCRF-CEM cells) to 195.12 µM (against CEM/ADR5000 cells) for doxorubicin. Acridone alkaloid 5 induced apoptosis in CCRF-CEM leukemia cells, mediated by increased ROS production. ConclusionsThe five tested alkaloids and mostly acridone 5 are potential cytotoxic natural products that deserve more investigations to develop novel cytotoxic compounds against multifactorial drug-resistant cancers.

Abstract B20: Design, synthesis and SAR studies of novel survivin inhibitors with potent antiproliferative properties

Abstract A series of novel survivin inhibitors were designed and synthesized based our recently reported compound UC-112. All the new analogs were tested against several cancer cell lines including multidrug-resistant melanoma cancer cell lines. The in vitro results showed that several compounds had excellent antiproliferative activity with IC50 values within nanomolar range. Mechanism of action of those new analogs was investigated by Western blot assay. The study confirmed that the new analogs acted by selectively suppressing survivin with the similar mechanism as the parent compound UC-112. Those new analogs represent promising agents that can be further developed for selective anticancer treatment. Citation Format: Min Xiao, Jin Wang, Yan Lu, Duane D. Miller, Wei Li. Design, synthesis and SAR studies of novel survivin inhibitors with potent antiproliferative properties. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B20.

Cytotoxicity of anthraquinones from the roots of Pentas schimperi towards multi-factorial drug-resistant cancer cells.

Multidrug resistance in cancer represents a major problem in chemotherapy. The present study was designed to assess the cytotoxicity of anthraquinones from Pentas schimperi, namely damnacanthal (1), damnacanthol (2), 3-hydroxy-2-hydroxymethyl anthraquinone (3) and schimperiquinone B (4) against nine drug-sensitive and multidrug resistant (MDR) cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of the above compounds, whilst caspase-Glo assay was used to detect the activation of caspases enzymes by compounds 1 and 2. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species were all analyzed via flow cytometry. Anthraquinones 1 and 2 displayed cytotoxic effects with IC50 values below 81 μM on all the nine tested cancer cell lines whilst 3 and 4 displayed selective activities. The recorded IC50 values for compounds 1 and 2 ranged from 3.12 μM and 12.18 μM (towards leukemia CCRF-CEM cells) and from 30.32 μM and 80.11 μM (towards gliobastoma U87MG.ΔEGFR cells) respectively, and from 0.20 μM (against CCRF-CEM cells) to 195.12 μM (against CEM/ADR5000 cells) for doxorubicin. Compounds 1 and 2 induced apoptosis in CCRF-CEM leukemia cells, mediated by the disruption of the MMP and increase in ROS production. Anthraquinones from Pentas schimperi and mostly 1 and 2 are potential cytotoxic natural products that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug resistant cancers.