Multidrug-resistant Leukemia Cells Research Articles

Impact of Complex Apoptotic Signaling Pathways on Cancer Cell Sensitivity to Therapy.

Anticancer drugs induce apoptotic and non-apoptotic cell death in various cancer types. The signaling pathways for anticancer drug-induced apoptotic cell death have been shown to differ between drug-sensitive and drug-resistant cells. In atypical multidrug-resistant leukemia cells, the c-Jun/activator protein 1 (AP-1)/p53 signaling pathway leading to apoptotic death is altered. Cancer cells treated with anticancer drugs undergo c-Jun/AP-1-mediated apoptotic death and are involved in c-Jun N-terminal kinase activation and growth arrest- and DNA damage-inducible gene 153 (Gadd153)/CCAAT/enhancer-binding protein homologous protein pathway induction, regardless of the p53 genotype. Gadd153 induction is associated with mitochondrial membrane permeabilization after anticancer drug treatment and involves a coupled endoplasmic reticulum stress response. The induction of apoptosis by anticancer drugs is mediated by the intrinsic pathway (cytochrome c, Cyt c) and subsequent activation of the caspase cascade via proapoptotic genes (e.g., Bax and Bcl-xS) and their interactions. Anticancer drug-induced apoptosis involves caspase-dependent and caspase-independent pathways and occurs via intrinsic and extrinsic pathways. The targeting of antiapoptotic genes such as Bcl-2 enhances anticancer drug efficacy. The modulation of apoptotic signaling by Bcl-xS transduction increases the sensitivity of multidrug resistance-related protein-overexpressing epidermoid carcinoma cells to anticancer drugs. The significance of autophagy in cancer therapy remains to be elucidated. In this review, we summarize current knowledge of cancer cell death-related signaling pathways and their alterations during anticancer drug treatment and discuss potential strategies to enhance treatment efficacy.

Pentagalloyl Glucose-Targeted Inhibition of P-Glycoprotein and Re-Sensitization of Multidrug-Resistant Leukemic Cells (K562/ADR) to Doxorubicin: In Silico and Functional Studies.

Combining phytochemicals with chemotherapeutic drugs has demonstrated the potential to surmount drug resistance. In this paper, we explore the efficacy of pentagalloyl glucose (PGG) in modulating P-gp and reversing multidrug resistance (MDR) in drug-resistant leukemic cells (K562/ADR). The cytotoxicity of PGG was evaluated using a CCK-8 assay, and cell apoptosis was assessed using flow cytometry. Western blotting was used to analyze protein expression levels. P-glycoprotein (P-gp) activity was evaluated by monitoring the kinetics of P-gp-mediated efflux of pirarubicin (THP). Finally, molecular docking, molecular dynamics simulation, and molecular mechanics with generalized Born and surface area solvation (MM-GBSA) calculation were conducted to investigate drug-protein interactions. We found that PGG selectively induced cytotoxicity in K562/ADR cells and enhanced sensitivity to doxorubicin (DOX), indicating its potential as a reversal agent. PGG reduced the expression of P-gp and its gene transcript levels. Additionally, PGG inhibited P-gp-mediated efflux and increased intracellular drug accumulation in drug-resistant cells. Molecular dynamics simulations and MM-GBSA calculation provided insights into the binding affinity of PGG to P-gp, suggesting that PGG binds tightly to both the substrate and the ATP binding sites of P-gp. These findings support the potential of PGG to target P-gp, reverse drug resistance, and enhance the efficacy of anticancer therapies.

Two palladium (II) complexes derived from halogen-substituted Schiff bases and 2-picolylamine induce parthanatos-type cell death in sensitive and multi-drug resistant CCRF-CEM leukemia cells

The use of cisplatin and its derivatives in cancer treatment triggered the interest in metal-containing complexes as potential novel anticancer agents. Palladium (II)-based complexes have been synthesized in recent years with promising antitumor activity. Previously, we described the synthesis and cytotoxicity of palladium (II) complexes containing halogen-substituted Schiff bases and 2-picolylamine. Here, we selected two palladium (II) complexes with double chlorine-substitution or double iodine-substitution that displayed the best cytotoxicity in drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells for further biological investigation. Surprisingly, these compounds did not significantly induce apoptotic cell death. This study aims to reveal the major mode of cell death of these two palladium (II) complexes. We performed annexin V-FITC/PI staining and flow cytometric mitochondrial membrane potential measurement followed by western blotting, immunofluorescence microscopy, and alkaline single cell electrophoresis (comet assay). J4 and J6 still induced neither apoptosis nor necrosis in both leukemia cell lines. They also insufficiently induced autophagy as evidenced by Beclin and p62 detection in western blotting. Interestingly, J4 and J6 induced a novel mode of cell death (parthanatos) as mainly demonstrated in CCRF-CEM cells by hyper-activation of poly(ADP-ribose) polymerase 1 (PARP) and poly(ADP-ribose) (PAR) using western blotting, flow cytometric measurement of mitochondrial membrane potential collapse, nuclear translocation of apoptosis-inducing factor (AIF) by immunofluorescence microscopy, and DNA damage by alkaline single cell electrophoresis (comet assay). AIF translocation was also observed in CEM/ADR5000 cells. Thus, parthanatos was the predominant mode of cell death induced by J4 and J6, which explains the high cytotoxicity in CCRF-CEM and CEM/ADR5000 cells. J4 and J6 may be interesting drug candidates and deserve further investigations to overcome resistance of tumors against apoptosis. This study will promote the design of further novel palladium (II)-based complexes as chemotherapeutic agents.

PP2A modulation overcomes multidrug resistance in chronic lymphocytic leukemia via mPTP-dependent apoptosis.

Targeted therapies such as venetoclax (VEN) (Bcl-2 inhibitor) have revolutionized the treatment of chronic lymphocytic leukemia (CLL). We previously reported that persister CLL cells in treated patients overexpress multiple antiapoptotic proteins and display resistance to proapoptotic agents. Here, we demonstrated that multidrug-resistant CLL cells in vivo exhibited apoptosis restriction at a pre-mitochondrial level due to insufficient activation of the Bax and Bak (Bax/Bak) proteins. Co-immunoprecipitation analyses with selective BH domain antagonists revealed that the pleiotropic proapoptotic protein (Bim) was prevented from activating Bax/Bak by "switching" interactions to other upregulated antiapoptotic proteins (Mcl-1, Bcl-xL, Bcl-2). Hence, treatments that bypass Bax/Bak restriction are required to deplete these resistant cells in patients. Protein phosphatase 2A (PP2A) contributes to oncogenesis and treatment resistance. We observed that small-molecule activator of PP2A (SMAP) induced cytotoxicity in multiple cancer cell lines and CLL samples, including multidrug-resistant leukemia and lymphoma cells. The SMAP (DT-061) activated apoptosis in multidrug-resistant CLL cells through induction of mitochondrial permeability transition pores, independent of Bax/Bak. DT-061 inhibited the growth of wild-type and Bax/Bak double-knockout, multidrug-resistant CLL cells in a xenograft mouse model. Collectively, we discovered multidrug-resistant CLL cells in patients and validated a pharmacologically tractable pathway to deplete this reservoir.

Expression of thimet oligopeptidase (THOP) modulated by oxidative stress in human multidrug resistant (MDR) leukemia cells

Thimet oligopeptidase (THOP) is a cytosolic metallopeptidase known to regulate the fate of post-proteasomal peptides, protein turnover and peptide selection in the antigen presentation machinery (APM) system. Oxidative stress influences THOP expression and regulates its proteolytic activity, generating variable cytosolic peptide levels, possibly affecting the immune evasion of tumor cells. In the present work, we examined the association between THOP expression/activity and stress oxidative resistance in human leukemia cells using the K562 cell line, a chronic myeloid leukemia (CML), and the multidrug-resistant (MDR) Lucena 1 (K562-derived MDR cell line) as model. The Lucena 1 phenotype was validated under vincristine treatment and the relative THOP1 mRNA levels and protein expression compared to K562 cell line. Our data demonstrated increased THOP1 gene and protein levels in K562 cells in contrast to the oxidative-resistant Lucena 1, even after H2O2 treatment, suggesting an oxidative stress dependence in THOP regulation. Further, it was observed higher basal levels of reactive oxygen species (ROS) in K562 compared to Lucena 1 cell line using DHE fluorescent probe. Since THOP activity is dependent on its oligomeric state, we also compared its proteolytic activity under reducing agent treatment, which demonstrated that its function modulation with respect to changes in redox state. Finally, the mRNA expression and FACS analyses demonstrated a reduced expression of MHC I only in K562 cell line. In conclusion, our results highlight THOP redox modulation, which could influence antigen presentation in multidrug resistant leukemia cells.

Triterpenoid pyrazines and pyridines – Synthesis, cytotoxicity, mechanism of action, preparation of prodrugs

A set of fifteen triterpenoid pyrazines and pyridines was prepared from parent triterpenoid 3-oxoderivatives (betulonic acid, dihydrobetulonic acid, oleanonic acid, moronic acid, ursonic acid, heterobetulonic acid, and allobetulone). Cytotoxicity of all compounds was tested in eight cancer and two non-cancer cell lines. Evaluation of the structure-activity relationships revealed that the triterpenoid core determined whether the final molecule is active or not, while the heterocycle is able to increase the activity and modulate the specificity. Five compounds (1b, 1c, 2b, 2c, and 8) were found to be preferentially and highly cytotoxic (IC50 ≈ 1 μM) against leukemic cancer cell lines (CCRF-CEM, K562, CEM-DNR, or K562-TAX). Surprisingly, compounds 1c, 2b, and 2c are 10-fold more active in multidrug-resistant leukemia cells (CEM-DNR and K562-TAX) than in their non-resistant analogs (CCRF-CEM and K562). Pharmacological parameters were measured for the most promising candidates and two types of prodrugs were synthesized: 1) Sugar-containing conjugates, most of which had improved cell penetration and retained high cytotoxicity in the CCRF-CEM cell line, unfortunately, they lost the selectivity against resistant cells. 2) Medoxomil derivatives, among which compounds 26–28 gained activities of IC50 0.026–0.043 μM against K562 cells. Compounds 1b, 8, 21, 22, 23, and 24 were selected for the evaluation of the mechanism of action based on their highest cytotoxicity against CCRF–CEM cell line. Several experiments showed that the majority of them cause apoptosis via the mitochondrial pathway. Compounds 1b, 8, and 21 inhibit growth and disintegrate spheroid cultures of HCT116 and HeLa cells, which would be important for the treatment of solid tumors. In summary, compounds 1b, 1c, 2b, 2c, 24, and 26–28 are highly and selectively cytotoxic against cancer cell lines and were selected for future in vivo tests and further development of anticancer drugs.

Small molecule MMRi62 targets MDM4 for degradation and induces leukemic cell apoptosis regardless of p53 status.

MDM2 and MDM4 proteins are key negative regulators of tumor suppressor p53. MDM2 and MDM4 interact via their RING domains and form a heterodimer polyubiquitin E3 ligase essential for p53 degradation. MDM4 also forms heterodimer E3 ligases with MDM2 isoforms that lack p53-binding domains, which regulate p53 and MDM4 stability. We are working to identify small-molecule inhibitors targeting the RING domain of MDM2-MDM4 (MMRi) that can inactivate the total oncogenic activity of MDM2-MDM4 heterodimers. Here, we describe the identification and characterization of MMRi62 as an MDM4-degrader and apoptosis inducer in leukemia cells. Biochemically, in our experiments, MMRi62 bound to preformed RING domain heterodimers altered the substrate preference toward MDM4 ubiquitination and promoted MDM2-dependent MDM4 degradation in cells. This MDM4-degrader activity of MMRi62 was found to be associated with potent apoptosis induction in leukemia cells. Interestingly, MMRi62 effectively induced apoptosis in p53 mutant, multidrug-resistant leukemia cells and patient samples in addition to p53 wild-type cells. In contrast, MMRi67 as a RING heterodimer disruptor and an enzymatic inhibitor of the MDM2-MDM4 E3 complex lacked MDM4-degrader activity and failed to induce apoptosis in these cells. In summary, this study identifies MMRi62 as a novel MDM2-MDM4-targeting agent and suggests that small molecules capable of promoting MDM4 degradation may be a viable new approach to killing leukemia cells bearing non-functional p53 by apoptosis.

Design, synthesis and biological evaluation of quinoline-2-carbonitrile-based hydroxamic acids as dual tubulin polymerization and histone deacetylases inhibitors

A series of quinoline and quinazoline analogs were designed and synthesized as new tubulin polymerization (TP) and histone deacetylases (HDAC) inhibitors. Compounds 12a and 12d showed the best cytotoxicity activities against a panel of human cancer cell lines with an averaged IC50 value of 0.6 and 0.7 nM, respectively. Furthermore, these lead compounds showed good activities against CA-4-resistant colon-carcinoma and multidrug-resistant leukemia cells. In addition, compounds 12a and 12d induced HT29 cell cycle arrest in the G2/M phase and produced caspase-induced apoptosis of HT29 cells through mitochondrial dysfunction. Also, 12a and 12d inhibited HDAC8, 6, and 11 activities. Furthermore, lead compound 12a exhibited higher metabolic stability than isoCA-4 and was highly potent in suppressing tumor growth in the fibrosarcoma MCA205 tumor model. Collectively, these studies suggest that 12a represents a new dual inhibitor of TP and HDAC activities, which makes it a suitable candidate for further investigations in clinical development.

Dihydroartemisinin Triggers Ferroptosis in Multidrug-Resistant Leukemia Cells.

The molecular mechanisms and role of ferroptosis in tumor drug resistance remain unclear. In this study, we found that multidrug-resistant (MDR) K562/adriamycin (ADM) leukemia cells possessed higher glutathione (GSH) levels and iron-regulatory protein 2 (IRP2), transferrin receptor, ferritin heavy chain 1 (FTH1), and peroxidase-4 (GPX4) expression than parental drug-sensitive K562 leukemia cells. These elevations might have increased the antioxidant ability of K562/ADM cells and granted them increased buffering capacity against iron disorder, protecting them from ferroptosis and favoring drug resistance. However, dihydroartemisinin (DHA) restrained MDR K562/ADM cell viability and enhanced the sensitivity to ADM by strengthening ferroptosis induced by downregulation of GSH levels and GPX4, IRP2, and FTH expression, upregulation of reactive oxygen species (ROS) levels, and the consequent suppression of total serine/threonine kinase (AKT), total mammalian target of rapamycin (t-mTOR), phosphorylated mTOR (p-mTOR), and p-mTOR/t-mTOR levels. Moreover, compared with K562 cells, MDR K562/ADM cells exhibited greater ROS increases, GSH decreases, and viability rescue after ferroptosis inhibitor treatment owing to further suppression of FTH1, GPX4, p-mTOR, and p-mTOR/t-mTOR. Collectively, the increase in oxidative damage and the blockade of antioxidant defence shaped DHA-induced ferroptosis, which was responsible for the sensitivity of MDR leukemia cells to DHA. Regulating iron homeostasis/ROS/AKT/mTOR might be a potential chemotherapeutic strategy for sensitizing drug-resistant leukemia.

Reversal of Drug Resistance in K562/ADM Cells Caused by RA and the Related Mechanisms

To investigate the effect of ursane triterpenoids 3β,19α-dihydroxyursu-12-ene-23,28-dicarboxylic acid (Rotundioic acid, RA) on the sensitivity of adriamycin-resistant K562 cells (K562/ADM Cell) anti-tumor drug, and to explore the effect and mechanism of RA on the multidrug resistance of K562/ADM cells. CCK-8 method was used to detect the effect of RA on the sensitivity of K562 cells and K562/ADM cells to anti-tumor drug. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression level of mRNA and the protein in K562 and K562/ADM cells, and the effect of RA on the expression of MDR1 mRNA and P-gp in K562/ADM cells was also detected; Western blot was used to detect the expression of p-JNK, p-p38 and p-ERK1/2 in K562/ADM cells. RA could increased the sensitivity of K562/ADM cells to adriamycin(the reversal factor was 1.61 times), the difference showed statistically significantly (P<0.05); the resistance factor of K562/ADM to ADM was 41.76 times. The expression of MDR1 mRNA in K562 cells was extremely low, and the protein product P-glycoprotein (P-gp) was almost not expressed; MDR1 mRNA and P-gp in K562/ADM cells were highly expressed; RA could down-regulate the expression levels of MDR1 and P-gp in K562/ADM cells. In addition, RA could upregulate the phosphorylation levels of p38 and ERK1/2 in K562/ADM cells, but it has no effect on the expression of p-JNK. RA may participate in the regulation of MAPK signaling pathway by upregulating the expression levels of p-p38 and p-ERK1/2 in K562/ADM cells, and thus inhibit the transcription and translation levels of MDR1, and finally reverse the multidrug resistance of leukemia cells.

Sensitizing multidrug-resistant leukemia cells to common cytostatics by an aluminium-salen complex that has high-apoptotic effects in leukemia, lymphoma and mamma carcinoma cells.

We investigated the aluminium-salen complex MBR-8 as a potential anti-cancer agent. To see apoptotic effects induced by MBR-8, alone and in combination with common cytostatic drugs, DNA-fragmentations were studied using the flow cytometric analysis. Western blot analysis and measurement of the mitochondrial membrane potential with a JC-1 dye were employed to identify the pathway of apoptosis. An impressive overcoming of multidrug-resistance in leukemia (Nalm6) cells was observed. Additionally, solid tumor cells including Burkitt-like lymphoma (BJAB) and mamma carcinoma cells (MCF-7) are affected by MBR-8 in the same way. Western blot analysis revealed activation of caspase-3. MBR-8 showed very pronounced selectivity with regard to tumor cells and high synergistic effects in Nalm6 and daunorubicin-resistant Nalm6 cells when administered in combination with vincristine, daunorubicin and doxorubicin. The aluminium-salen complex MBR-8 showed very promising anti-cancer properties which warrant further development towards a cytostatic agent for future chemotherapy. Studies on aluminium compounds for cancer therapy are rare, and our report adds to this important body of knowledge.

Plant extracts and betulin from Ligaria cuneifolia inhibit P-glycoprotein function in leukemia cells

Overexpression of P-glycoprotein (P-gp), which is linked to multidrug resistance (MDR), is one of the underlying obstacles to the success of chemotherapy as it reduces the efficacy of anticancer drugs and the side effects of these increase as a result of any increased dose to achieve the therapeutic effect. To identify agents with P-gp inhibitory properties, ethanol extracts from 80 plants were screened for their ability to increase intracellular doxorubicin-associated fluorescence, and the extract of Ligaria cuneifolia was found to be the most effective. Its bioassay-guided isolation yielded the pentacyclic triterpene betulin as active agent. This efficiently inhibited P-gp mediated efflux, as demonstrated by the enhancement of the intracellular accumulation of doxorubicin and rhodamine 123 from 1.56 μM in the P-gp overexpressing MDR leukemia cell, Lucena 1. Betulin was also able to render Lucena 1 sensitive to Dox from 0.39 μM. The docking studies revealed that betulin tightly binds to a key region of the TMDs, with a binding mode overlapping one main site of doxorubicin and, more interestingly, emulating the same contacts as tariquidar, as revealed by the per-residue energetic analysis from molecular dynamics simulations. MTT assay using peripheral blood mononuclear cells and hemolysis assay showed that betulin is devoid of toxicity.These findings provide important evidence that betulin may be a safe and promising entity to be further investigated to develop agents able to overcome P-gp-mediated MDR, resulting in a more effective and less toxic chemotherapy.

Phytochemical characterization and biological activities of green tea (Camellia sinensis) produced in the Azores, Portugal

BackgroundGreen tea is not only one of the most widely consumed beverages worldwide, but is also known for its health promoting and therapeutic effects. Green tea is cultivated in areas with high humidity and acidic soils in China, Indonesia and Japan. Those places have well-marked dry and rainy seasons. In opposite, Azores have a climate with constant average annual rainfall and, unlike eastern regions, relatively constant air humidity throughout the year. While a brand implemented on the Portuguese market, the quality of green tea produced in Azores must be guaranteed. Quality control measures based on phytochemical determination of the chemical composition and biological activities are needed in order to address whenever climate changes interferes significantly with composition and biological effects. PurposeMake the phytochemical characterization of various extracts of green tea leaves coming from Azores and evaluate the anti-cancer activities of the extracts in order to compare the obtained results with those of teas coming from eastern regions. MethodsPhytochemical characterization (catechins, oxyaromatic acids, flavonols, alkaloids and theanine) and total catechins contents (TCC) was performed by using HPLC-DAD analysis, in infusions (5–7 min and 30 min), maceration and methanolic extracts of Camellia sinensis samples coming from Azores, Portugal. The antioxidant activity of extracts was measured by the DPPH assay and the total phenolics contents (TPC) were estimated using the Folin-Ciocalteu colorimetric method. The cytotoxic activity towards drug sensitive and multidrug-resistant leukemia cell lines was determined by the resazurin assay. ResultsThe TCC was higher in methanolic extracts and lower in maceration, as epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) concentrations were significantly higher in methanolic extracts and were only residual in maceration extracts. Maceration extracts showed the highest content of gallic acid, indicating that methanol extracts contained more flavonols of higher molecular weight and/or that maceration may lead to the degalloylation of catechins. The amount of o-caffeoylquinic acid extracted was significantly higher in methanolic samples. Short-term extraction at high temperatures resulted in high amounts of neochlorogenic acid. The contents of glycosylated quercitin-3-d-galactoside and kaempferol-3-glucoside were small in maceration samples and high in methanolic samples. Caffeine was easily extracted by methanol (99%) compared with water, while extraction of the amino-acid l-theanine was unattainable with methanol. TPC values correlated linearly with DPPH•IC50, with infusion samples showing the best antioxidant capacities. The aqueous and the methanol/water extracts were active in multidrug-resistant and drug sensitive cancer cells. ConclusionDespite these climatic differences, the phytochemical characterization revealed that tea coming from the Azores compares well with those from China and Japan. In addition, tea can be produced without fungicides and pesticides to control pests that appear during rainy seasons. This allows the production of high-quality bioorganic tea. Aqueous and methanol/water extracts of the Azorean tea showed to be useful targeting sensitive and drug-resistant tumor cells.

Chemopreventive Property of Sencha Tea Extracts towards Sensitive and Multidrug-Resistant Leukemia and Multiple Myeloma Cells.

The popular beverage green tea possesses chemopreventive activity against various types of tumors. However, the effects of its chemopreventive effect on hematological malignancies have not been defined. In the present study, we evaluated antitumor efficacies of a specific green tea, sencha tea, on sensitive and multidrug-resistant leukemia and a panel of nine multiple myelomas (MM) cell lines. We found that sencha extracts induced cytotoxicity in leukemic cells and MM cells to different extents, yet its effect on normal cells was limited. Furthermore, sencha extracts caused G2/M and G0/G1 phase arrest during cell cycle progression in CCRF/CEM and KMS-12-BM cells, respectively. Specifically, sencha-MeOH/H2O extracts induced apoptosis, ROS, and MMP collapse on both CCRF/CEM and KMS-12-BM cells. The analysis with microarray and COMPARE in 53 cell lines of the NCI panel revealed diverse functional groups, including cell morphology, cellular growth and proliferation, cell cycle, cell death, and survival, which were closely associated with anti-tumor effects of sencha tea. It is important to note that PI3K/Akt and NF-κB pathways were the top two dominant networks by ingenuity pathway analysis. We demonstrate here the multifactorial modes of action of sencha tea leading to chemopreventive effects of sencha tea against cancer.

Vertically aligned multi-walled carbon nanotubes based flexible immunosensor for extreme low level detection of multidrug resistant leukemia cells

We report an advanced flexible immunosensor for extreme low level detection of multidrug resistant myeloid leukemia cells. The detection mechanism is analyzed in terms of novel nanoscale interactions of target molecules with vertically aligned multi-walled carbon nanotubes (VA-MWNT). The immunosensor is fabricated by transferring VA-MWNTs on a polyethylene terephthalate substrate by hot press technique without losing CNTs’ pristine character. Sensing is performed using doxorubicin treated leukemia K562 cells in varying concentrations from 1.5 × 102 cells mL–1 to 1.5 × 107 cells mL–1 and sensor showed detection limit of 10 cells mL–1. The calibration plot of peak current versus logarithmic concentration of DOX/leukemia K562 cells exhibited good linearity with a regression coefficient of ˜0.98. Sensing mechanism is explained in terms of charge transfer induced Fermi level shift of sub μeV order, causing band bending at the interface of CNT-molecular species; and it is reflected in lowering detection limit of the fabricated sensor. Theoretical analysis is done to correlate Fermi energy shift with sensitivity of the device on cancer cell immobilization. Additionally, the developed immunosensor shows good stability, reproducibility and fast detection vis-à-vis the devices reported so far. The notable advantages of proposed flexible sensor are its durability, chemical and moisture resistance, making it a potentially competitive device for point-of-care diagnostics.

Modulation of the spacer in N,N-bis(alkanol)amine aryl ester heterodimers led to the discovery of a series of highly potent P-glycoprotein-based multidrug resistance (MDR) modulators

In this study, a new series of N,N-bis(alkanol)amine aryl ester heterodimers was synthesized and studied. The new compounds were designed based on the structures of our previous arylamine ester derivatives endowed with high P-gp-dependent multidrug resistance reversing activity on a multidrug-resistant leukemia cell line. All new compounds were active in the pirarubicin uptake assay on the doxorubicin–resistant erythroleukemia K562 cells (K562/DOX). Compounds bearing a linker made up of 10 methylenes showed unprecedented high reversal activities regardless of the combination of aromatic moieties. Docking results obtained by an in silico study supported the data obtained by the biological tests and a study devoted to establish the chemical stability in phosphate buffer solution (PBS) and human plasma showed that only a few compounds exhibited a significant degradation in the human plasma matrix. Ten selected non-hydrolysable derivatives were able to inhibit the P-gp-mediated rhodamine-123 efflux on K562/DOX cells, and the evaluation of their apparent permeability and ATP consumption on other cell lines suggested that the compounds can behave as unambiguous or not transported substrates. The activity of these the compounds on the transport proteins breast cancer resistance protein (BCRP) and multidrug resistance associated protein 1 (MRP1) was also analyzed. All tested derivatives displayed a moderate potency on the BCRP overexpressing cells; while only four molecules showed to be effective on MRP1 overexpressing cells, highlighting a clear structural requirement for selectivity. In conclusion, we have identified a new very powerful series of compounds which represent interesting leads for the development of new potent and efficacious P-gp-dependent MDR modulators.

Cytotoxicity of fagaramide derivative and canthin-6-one from Zanthoxylum (Rutaceae) species against multidrug resistant leukemia cells

In our continuous search for cytotoxic compounds from the genus Zanthoxylum, chromatographic separation of the MeOH/CH2Cl2 (1:1) extract of Z. chalybeum yielded one new alkamide; 4-(isoprenyloxy)-3-methoxy-3,4-deoxymethylenedioxyfagaramide (1) and a known one; fagaramide (2). Similarly, from the MeOH/CH2Cl2 (1:1) extract of the stem bark of Z. parachanthum four known compounds; canthin-6-one (3), dihydrochelerythrine (4), lupeol (5) and sesamin (6) were isolated. Characterization of the structures of these compounds was achieved using spectroscopic techniques (NMR and MS). Using resazurin reduction assay 1, 3 and 6 displayed moderate cytotoxicity with IC50 values below 50 μM against the drug sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cell lines. It is interesting to note that 3 was more active than the standard drug, doxorubicin against CEM/ADR5000 leukemia cells. Compounds 3 and 6 showed good selectivity on leukemia cells than normal cells. In future studies 3 should be tested against a panel of drug resistant human cells.

1,1-Diheterocyclic Ethylenes Derived from Quinaldine and Carbazole as New Tubulin-Polymerization Inhibitors: Synthesis, Metabolism, and Biological Evaluation.

We report the synthesis and metabolic and biological evaluation of a series of 17 novel heterocyclic derivatives of isocombretastatin-A4 (iso-CA-4) and their structure-activity relationships. Among these derivatives, the most active compound, 4f, inhibited the growth of a panel of seven cancer cell lines with an IC50 in the low nanomolar range. In addition, 4f showed interesting activity against CA-4-resistant colon-carcinoma cells and multidrug-resistant leukemia cells. It also induced G2/M cell-cycle arrest. Structural data indicated binding of 4f to the colchicine site of tubulin, likely preventing the curved-to-straight tubulin structural changes that occur during microtubule assembly. Also, 4f disrupted the blood-vessel-like assembly formed by human umbilical-vein endothelial cells in vitro, suggesting its function as a vascular-disrupting agent. An in vitro metabolism study of 4f showed its high human-microsomal stability in comparison with that of iso-CA-4. The physicochemical properties of 4f may be conducive to CNS permeability, suggesting that this compound may be a possible candidate for the treatment of glioblastoma.

A Pharmacological Strategy Using Stemofoline for more Efficacious Chemotherapeutic Treatments Against Human Multidrug Resistant Leukemic Cells

Our previous study reported that stemofoline (STF) exhibited a synergistic effect with chemotherapeutic drugs in human multidrug-resistant (MDR) leukemic cells (K526/Adr) by inhibiting the function of P-glycoprotein, which is a membrane transporter that is overexpressed in several types of MDR cancers. This study further investigated the effects of a combination treatment of STF and doxorubicin (DOX) in vitro and in vivo. The combination treatment of 50 mg/kg of STF strongly enhanced the anti-tumor activity of DOX in SCID-beige mice bearing K562/Adr xenografts without additional toxicity when compared to the single treatment groups. Additionally, an examination of the proliferation markers (Ki67) and the apoptotic marker (TUNEL) in tumor tissues in each group revealed that the combination therapy significantly reduced Ki67 positive cells and increased apoptotic cells. From the in vitro experiments we also found that this combination treatment dramatically induced G1 and G2M arrest in K562/Adr when compared to a single treatment of DOX. STF treatment alone did not show any cytotoxic effect to the cells. These results suggest that the accumulation of DOX enhanced by STF was sufficient to induce cell cycle arrest in K562/Adr. These findings support our previous in vitro data and indicate the possibility of developing STF as an adjuvant therapy in cancer treatments.

Reversal of Multidrug Resistance in Human Colon Cancer and Human Leukemia Cells by Three Plant Extracts and Their Major Secondary Metabolites.

Background: We studied the effect of three plant extracts (Glycyrrhiza glabra, Paeonia lactiflora, Eriobotrya japonica) and six of their major secondary metabolites (glycyrrhizic acid, 18β glycyrrhetinic acid, liquiritigenin, isoliquiritigenin, paeoniflorin, ursolic acid) on the multidrug resistant human colon cancer cell line Caco-2 and human leukemia cell line CEM/ADR 5000 as compared to the corresponding sensitive cell line CCRF-CEM, and human colon cancer cells HCT-116, which do not over-express ATP-binding cassette (ABC) transporters. Methods: The cytotoxicity of single substances in sensitive and resistant cells was investigated by MTT assay. We also applied combinations of extracts or single compounds with the chemotherapeutic agent doxorubicin or doxorubicin plus the saponin digitonin. The intracellular retention of the ABC transporter substrates rhodamine 123 and calcein was examined by flow cytometry to explore the effect of the substances on the activity of ABC transporters P-glycoprotein and MRP1. Real-time PCR was applied to analyse the gene expression changes of ABCB1, ABCC1, caspase 3, caspase 8, AhR, CYP1A1, and GSTP1 in resistant cells under the treatment of the substances. Results: All the substances moderately inhibited cell growth in sensitive and resistant cells to some degree. Whereas ursolic acid showed IC50 of 14 and 22 µM in CEM/ADR 5000 and Caco-2 cells, respectively, glycyrrhizic acid and paeoniflorin were inactive with IC50 values above 400 μM. Except for liquiritigenin and isoliquiritigenin, all the other substances reversed MDR in CEM/ADR 5000 and Caco-2 cells to doxorubicin. Ue, ga, 18ga, and urs were powerful reversal agents. In CEM/ADR 5000 cells, high concentrations of all the substances, except Paeonia lactiflora extract, increased calcein or rhodamine 123 retention in a dose-dependent manner. In Caco-2 cells, all the substances, except liquiritigenin, retained rhodamine 123 in a dose-dependent manner. We also examined the effect of the plant secondary metabolite (PSM) panel on the expression of ABCB1, ABCC1, caspase 3, caspase 8, AhR, CYP1A1, and GSTP1 genes in MDR cells. Conclusions: The extracts and individual PSM could reverse MDR in CEM/ADR 5000 and Caco-2 cells, which overexpress ABC transporters, in two- and three-drug combinations. Most of the PSM also inhibited the activity of ABC transporters to some degree, albeit at high concentrations. Ue, ga, 18ga, and urs were identified as potential multidrug resistance (MDR) modulator candidates, which need to be characterized and validated in further studies.