Papers - Sugimoto, Yoshikazu

Activating mutations in EGFR and PI3K promote ATF4 induction for NSCLC cell survival during amino acid deprivation

Takahashi M., Okamoto Y., Kato Y., Shirahama H., Tsukahara S., Sugimoto Y., Tomida A.

Heliyon (Heliyon)  9 ( 4 )  2023.04

ISSN  24058440

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Some oncoproteins along with stress kinase general control non-derepressible 2 (GCN2) can ensure the induction of activating transcription factor 4 (ATF4) to counteract amino acid deprivation; however, little is known regarding the role of the oncogenic EGFR-PI3K pathway. In this study, we demonstrate that both mutated EGFR and PIK3CA contribute to ATF4 induction following GCN2 activation in NSCLC cells. The inhibition of EGFR or PI3K mutant proteins, pharmacologically or through genetic knockdown, inhibited ATF4 induction without affecting GCN2 activation. A downstream analysis revealed that the oncogenic EGFR-PI3K pathway may utilize mTOR-mediated translation control mechanisms for ATF4 induction. Furthermore, in NSCLC cells harboring co-mutations in EGFR and PIK3CA, the combined inhibition of these oncoproteins markedly suppressed ATF4 induction and the subsequent gene expression program as well as cell viability during amino acid deprivation. Our findings establish a role for the oncogenic EGFR-PI3K pathway in the adaptive stress response and provide a strategy to improve EGFR-targeted NSCLC therapy.

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Kaposi's sarcoma–associated herpesvirus replication and transcription activator protein activates CD274/PD-L1 gene promoter

Miyazawa M., Yamamoto Y., Katayama K., Sugimoto Y., Noguchi K.

Cancer Science (Cancer Science)  114 ( 4 ) 1718 - 1728 2023.04

ISSN  13479032

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Kaposi's sarcoma–associated herpesvirus (KSHV) is responsible for the pathogenesis of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. The expression of immunosuppressive genes, such as IL-10 and CD274/PD-L1 is observed during KSHV-associated pathogenesis, and the modulation of the host immune system by KSHV contributes to establishing viral persistence in the host. Understanding the mechanism that allows the virus to evade host cell immunity would be helpful in order to develop therapeutic strategies for KSHV malignancy. In this study, we show that KSHV replication and transcriptional activator (K-RTA), an essential activator of the viral lytic cycle, transactivates the CD274/PD-L1 gene promoter. Mechanistically, we demonstrate that the binding of K-RTA to the cellular specificity protein 1 (SP1) is critical for K-RTA–mediated CD274/PD-L1 promoter activation. These findings suggest that K-RTA cooperates with intracellular SP1 to activate the expression of CD274/PD-L1, which helps the virus regulate immune checkpoints to escape and survive.

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Inhibitory Effect of Dextran Derivatives on Multidrug Resistance-Related Efflux Transporters in Vitro

Morimoto K., Ishii M., Sugimoto Y., Ogihara T., Tomita M.

Biological and Pharmaceutical Bulletin (Biological and Pharmaceutical Bulletin)  45 ( 8 ) 1036 - 1042 2022.08

ISSN  09186158

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Dextran is a promising candidate as a nanocarrier of chemotherapeutic drugs due to its biocompatibility, biodegradability, and ability to accumulate in tumors. Furthermore, dextran derivatives interact with P-glycoprotein (P-gp), so we hypothesized that they may be available as tumor-specific drug delivery systems with the ability to reverse multidrug resistance. Here, to test this idea, we investigated whether dextran and its derivatives inhibit breast cancer resistance protein (BCRP), multidrug resistance associated protein 1 (MRP1), and P-gp in vitro. First, we examined their effect on the uptake of specific fluorescent substrates by inside-out Sf-9 membrane vesicles overexpressing BCRP, MRP1, and P-gp. BCRP and MRP1 were significantly inhibited by 2-hydroxypropyl-trimethylammonium-dextran of 4 and 70kDa (Q-D4 and Q-D70) at a concentration near the clinically used concentration of dextran; however, P-gp was not inhibited. A structure–activity study showed that Q-D4, Q-D70, and 40kDa diethylaminoethyl-dextran (DEAE-D40) significantly inhibited BCRP, while 4, 40, and 70kDa dextrans (D4, D40, and D70), dextran sulfate (Sul-D40), and the individual saccharide components of dextran did not. These results suggest that the cationic side chains, but not the saccharides, are important for BCRP inhibition. Finally, cell-based efflux assay was conducted. Q-D4, Q-D70, and DEAE-D40 did not specifically increase the retention of Hoechst33342 in BCRP-overexpressing KB cells. Similarly, Q-D4 and Q-D70 did not affect the intracellular retention of specific fluorescent substrates in MRP1- and P-gp-overexpressing KB cells. The ineffectiveness in cellular systems is presumably due to inability of the dextran derivatives to access transporters located on the cytoplasmic side of the cell membrane.

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Transcriptomics, molecular docking, and cross-resistance profiling of nobiletin in cancer cells and synergistic interaction with doxorubicin upon SOX5 transfection

Adham A.N., Abdelfatah S., Naqishbandi A., Sugimoto Y., Fleischer E., Efferth T.

Phytomedicine (Phytomedicine)  100 2022.06

ISSN  09447113

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Background: Nobiletin is a polymethoxylated flavone from citrus fruit peels. Among other bioactivities, it acts antioxidative, anti-inflammatory, neuroprotective, and cardiovascular-protective. Nobiletin exerts profound anticancer activity in vitro and in vivo but the underlying mechanisms are not well understood. Purpose: The aim was to unravel the multiple modes of action against cancer cells by bioinformatic and transcriptomic techniques and their verification by molecular pharmacological methods. Methods: The in silico methods used were COMPARE analysis of transcriptomic data, signaling pathway analysis, transcription factor binding motif analysis in promoter sequences of target genes, and molecular docking. The in vitro methods used were resazurin assay, isobologram analysis, generation of stably SOX5-tranfected cells, and Western blotting. Results: Nobiletin was cytotoxic against a wide range of cell lines from different tumor types, including diverse phenotypes to established anticancer drugs (e.g., P-glycoprotein, ABCB5, p53, EGFR). Cross-resistance profiling with 83 standard anticancer drugs revealed a correlation to antihormonal anticancer drugs, which can be explained by the phytoestrogenic features of nobiletin. Transcriptomic analysis showed that the responsiveness of tumor cells was predictable by their specific mRNA expression profile. Nobiletin bound to the transcription factor SOX5 in silico. SOX5 conferred resistance to the control drug doxorubicin but collateral sensitivity to nobiletin in HEK293 cells transfected with a lentiviral GFP-tagged pLOC–ORF-SOX5 vector. The combination of nobiletin and doxorubicin synergistically killed HEK293-SOX5 cells in isobologram analyses, implying attractive new treatment options. Conclusion: Nobiletin represents an interesting candidate for cancer therapy with broad-spectrum activity and multiple modes of action. The identification of novel targets (i.e., SOX5) may allow its use for targeted tumor therapy in individualized treatment protocols.

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Inhibition of BCL2A1 by STAT5 inactivation overcomes resistance to targeted therapies of FLT3-ITD/D835 mutant AML.

Yamatani K, Ai T, Saito K, Suzuki K, Hori A, Kinjo S, Ikeo K, Ruvolo V, Zhang W, Mak PY, Kaczkowski B, Harada H, Katayama K, Sugimoto Y, Myslinski J, Hato T, Miida T, Konopleva M, Hayashizaki Y, Carter BZ, Tabe T, Andreeff M.

Translational Oncology (Translational Oncology)  18   101354 2022.01

Research paper (scientific journal), Joint Work, Accepted

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Tyrosine kinase inhibitors (TKIs) are established drugs in the therapy of FLT3-ITD mutated acute myeloid leukemia (AML). However, acquired mutations, such as D835 in the tyrosine kinase domain (FLT3-ITD/D835), can induce resistance to TKIs. A cap analysis gene expression (CAGE) technology revealed that the gene expression of BCL2A1 transcription start sites was increased in primary AML cells bearing FLT3-ITD/D835 compared to FLT3-ITD. Overexpression of BCL2A1 attenuated the sensitivity to quizartinib, a type II TKI, and venetoclax, a selective BCL2 inhibitor, in AML cell lines. However, a type I TKI, gilteritinib, inhibited the expression of BCL2A1 through inactivation of STAT5 and alleviated TKI resistance of FLT3-ITD/D835. The combination of gilteritinib and venetoclax showed synergistic effects in the FLT3-ITD/D835 positive AML cells. The promoter region of BCL2A1 contains a BRD4 binding site. Thus, the blockade of BRD4 with a BET inhibitor (CPI-0610) downregulated BCL2A1 in FLT3-mutated AML cells and extended profound suppression of FLT3-ITD/D835 mutant cells. Therefore, we propose that BCL2A1 has the potential to be a novel therapeutic target in treating FLT3-ITD/D835 mutated AML.

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A novel moniliformin derivative as pan-inhibitor of histone deacetylases triggering apoptosis of leukemia cells

Lu X., Yan G., Dawood M., Klauck S.M., Sugimoto Y., Klinger A., Fleischer E., Shan L., Efferth T.

Biochemical Pharmacology (Biochemical Pharmacology)  194   114677 2021.12

Research paper (scientific journal), Joint Work, Accepted,  ISSN  00062952

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New and potent agents that evade multidrug resistance (MDR) and inhibit epigenetic modifications are of great interest in cancer drug development. Here, we describe that a moniliformin derivative (IUPAC name: 3‐(naphthalen‐2‐ylsulfanyl)‐4‐{[(2Z)‐1,3,3‐trimethyl‐2,3‐dihydro‐1H‐indol‐2‐ylidene]methyl}cyclobut‐3‐ene‐1,2‐dione; code: MCC1381) bypasses P-gp-mediated MDR. Using transcriptomics, we identified a large number of genes significantly regulated in response to MCC1381, which affected the cell cycle and disturbed cellular death and survival. The potential targets of MCC1381 might be histone deacetylases (HDACs) as predicted by SwissTargetPrediction. In silico studies confirmed that MCC1381 presented comparable affinity with HDAC1, 2, 3, 6, 8 and 11. Besides, the inhibition activity of HDACs was dose-dependently inhibited by MCC1381. Particularly, a strong binding affinity was observed between MCC1381 and HDAC6 by microscale thermophoresis analysis. MCC1381 decreased the expression of HDAC6, inversely correlated with the increase of acetylated HDAC6 substrates, acetylation p53 and α-tubulin. Furthermore, MCC1381 arrested the cell cycle at the G2/M phase, induced the generation of reactive oxygen species and collapse of the mitochondrial membrane potential. MCC1381 exhibited in vivo anti-cancer activity in xenografted zebrafish. Collectively, MCC1381 extended cytotoxicity towards P-gp-resistant leukemia cancer cells and may act as a pan-HDACs inhibitor, indicating that MCC1381 is a novel candidate for cancer therapy.

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Cytotoxicity of 4-hydroxy-N-(naphthalen-1-yl)-2-oxo-2H-chromene-3-carboxamide in multidrug-resistant cancer cells through activation of PERK/eIF2α/ATF4 pathway

Lu X., Yan G., Klauck S.M., Fleischer E., Klinger A., Sugimoto Y., Shan L., Efferth T.

Biochemical Pharmacology 193   114788 2021.11

Research paper (scientific journal), Joint Work, Accepted,  ISSN  00062952

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2α-Hydroxyalantolactone from Pulicaria undulata: activity against multidrug-resistant tumor cells and modes of action

Hegazy M.E.F., Dawood M., Mahmoud N., Elbadawi M., Sugimoto Y., Klauck S.M., Mohamed N., Efferth T.

Phytomedicine (Phytomedicine)  81   153409 2021.01

Research paper (scientific journal), Joint Work, Accepted,  ISSN  09447113

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GZD824 inhibits GCN2 and sensitizes cancer cells to amino acid starvation stress

Kato Y., Kunimasa K., Takahashi M., Harada A., Nagasawa I., Osawa M., Sugimoto Y., Tomida A.

Molecular Pharmacology 98 ( 6 ) 669 - 676 2020.12

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0026895X

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Citrus Fruit-Derived Flavanone Glycoside Narirutin is a Novel Potent Inhibitor of Organic Anion-Transporting Polypeptides

Morita T., Akiyoshi T., Sato R., Uekusa Y., Katayama K., Yajima K., Imaoka A., Sugimoto Y., Kiuchi F., Ohtani H.

Journal of Agricultural and Food Chemistry  2020.11

Research paper (scientific journal), Joint Work, Accepted,  ISSN  00218561

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Putative molecular determinants mediating sensitivity or resistance towards carnosic acid tumor cell responses

Mahmoud N., Saeed M.E.M., Sugimoto Y., Klinger A., Fleischer E., Efferth T.

Phytomedicine (Phytomedicine)  77   153271 2020.10

Research paper (scientific journal), Joint Work, Accepted,  ISSN  09447113

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Background: Carnosic acid (CA) is one of the main constituents in rosemary extract. It possesses valuable pharmacological properties, including anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer activities. Numerous in vitro and in vivo studies investigated the anticancer profile of CA and emphasized its potentiality for cancer treatment. Nevertheless, the role of multidrug-resistance (MDR) related mechanisms for CA's anticancer effect is not yet known. Purpose: We investigated the cytotoxicity of CA against known mechanisms of anticancer drug resistance (P-gp, ABCB5, BCRP, EGFR and p53) and determined novel putative molecular factors associated with cellular response towards CA. Study design: Cytotoxicity assays, bioinformatic analysis, flow cytometry and western blotting were performed to identify the mode of action of CA towards cancer cells. Methods: The cytotoxicity to CA was assessed using the resazurin assays in cell lines expressing the mentioned resistance mechanisms. A pharmacogenomic characterization of the NCI 60 cell line panel was applied via COMPARE, hierarchical cluster and network analyses. Flow cytometry was used to detect cellular mode of death and ROS generation. Changes in proteins-related to apoptosis were determined by Western blotting. Results: Cell lines expressing ABC transporters (P-gp, BCRP or ABCB5), mutant EGFR or p53 were not cross-resistant to CA compared to their parental counterparts. By pharmacogenomic approaches, we identified genes that belong to different functional groups (e.g. signal transduction, regulation of cytoskeleton and developmental regulatory system). These genes were predicted as molecular determinants that mediate CA tumor cellular responses. The top affected biofunctions included cellular development, cellular proliferation and cellular death and survival. The effect of CA-mediated apoptosis in leukemia cells, which were recognized as the most sensitive tumor type, was confirmed via flow cytometry and western blot analysis. Conclusion: CA may provide a novel treatment option to target refractory tumors and to effectively cooperate with established chemotherapy. Using pharmacogenomic approaches and network pharmacology, the relationship between cancer complexity and multi-target potentials of CA was analyzed and many putative molecular determinants were identified. They could serve as novel targets for CA and further studies are needed to translate the possible implications to clinical cancer treatment.

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Effect of TNIK upregulation on JQ1-resistant human colorectal cancer HCT116 cells

Takahashi C., Kondo S., Sadaoka K., Ishizuka S., Noguchi K., Kato Y., Sugimoto Y.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications)  530 ( 1 ) 230 - 234 2020.09

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0006291X

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JQ1 disrupts the binding of bromodomain and extra-terminal (BET) family of proteins to acetylated histones, modulates the expression of various genes, and inhibits the proliferation of cancer cells. We established two JQ1-resistant sublines from human colorectal cancer HCT116 cells. These resistant cells showed an 8- to 9-fold higher resistance to JQ1, and a 2- to 4-fold higher resistance to various anti-cancer agents, such as doxorubicin, etoposide, mitoxantrone, SN-38, cisplatin, and methotrexate than the parental HCT116 cells. The JQ1-resistant cells expressed higher levels of TRAF2 and NCK-interacting protein kinase (TNIK), cyclin D1 (CCND1), cyclin E1 (CCNE1), and their corresponding mRNAs than the parental cells. TNIK is a regulator of Wnt/β-catenin signaling and is known to transactivate CCND1. Transient transfection of HCT116 cells with a TNIK expression plasmid resulted in the upregulation of cyclin D1, cyclin E1, and their corresponding mRNAs, as well as an increase in CCNE1 promoter activity. Furthermore, luciferase assay revealed that the JQ1-resistant cells showed high CCNE1 promoter activity. These results suggest that TNIK also transactivates CCNE1. Three stable TNIK transfectant clones of HEK293 cells expressed 1.5- to 2-fold higher levels of TNIK, cyclin D1, and cyclin E1 than the parental cells. The 293/TNIK-6 cells, which expressed the highest level of TNIK among the transfectants, showed a 2.3-fold higher resistance to JQ1 than the parental cells. These results suggest the possible involvement of TNIK in cellular resistance to JQ1.

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pH-dependent transport kinetics of the human organic anion-transporting polypeptide 1A2

Morita T., Akiyoshi T., Sato R., Katayama K., Yajima K., Kataoka H., Imaoka A., Sugimoto Y., Ohtani H.

Drug Metabolism and Pharmacokinetics (Drug Metabolism and Pharmacokinetics)  35 ( 2 ) 220 - 227 2020.04

Research paper (scientific journal), Joint Work, Accepted,  ISSN  13474367

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Organic anion-transporting polypeptide (OATP) 1A2 is expressed on the apical sides of intestinal and renal epithelial cells and considered to be involved in the intestinal absorption and renal reabsorption of drugs. Although the transport activity of OATP1A2 is considered to be pH-dependent, the effects of pH on its kinetic parameters and on the potency of OATP1A2 inhibitors are yet to be elucidated. Some OATP are known to have multiple binding sites (MBS), but it remains unclear whether OATP1A2 has MBS. In the present study, we evaluated the influence of pH on the OATP1A2-mediated uptake of estrone 3-sulfate using OATP1A2-expressing HEK293 cells. The uptake of 0.3 μM estrone 3-sulfate by HEK293-OATP1A2 cells was pH-dependent. OATP1A2 exhibited bimodal saturation kinetics at pH 6.3 and 7.4. Compared with that seen at pH 6.3 (5.62 μM), the Km value of the high-affinity site was 8-fold higher at pH 7.4 (43.2 μM). In addition, the influence of pH on the potency of inhibitors varied among the examined inhibitors. These results suggest that the transport properties of OATP1A2 under lower pH conditions, such as those found in the microenvironments of the small intestinal mucosa and distal tubules, differ from those seen under neutral pH conditions.

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STAT1 upregulates glutaminase and modulates amino acids and glutathione metabolism

Kondo S., Kato Y., Minagawa S., Sugimoto Y.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications)  523 ( 3 ) 672 - 677 2020.03

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0006291X

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We previously reported the upregulation of cellular Glu and glutathione levels in human ABCB5- and murine Abcb5-transfected cells. Here, we demonstrate the upregulation of STAT1 and glutaminase (GLS) in ABCB5/Abcb5-transfected cells. Among a total of four ABCB5/Abcb5 high-expressing clones with docetaxel resistance, three of the clones expressed STAT1 and GLS highly and showed resistance to docetaxel and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. Neither STAT1 nor GLS upregulation was observed in the remaining ABCB5 high-expressing clone, as well as in another two ABCB5 low-expressing clones; these three clones did not show BSO resistance. The ABCB5/STAT1 high-expressing clones showed higher cellular levels of Ala, Glu, and Asp and lower cellular levels of Phe, Trp, Leu, Ile, Gly, Met, Tyr, Val, and His compared to the ABCB5/STAT1 low-expressing clones. The former clones also showed a higher resistance to Glu. The STAT1-transfected clones expressed high levels of GLS and the corresponding mRNA, suggesting the transactivation of GLS by STAT1. These clones showed resistance to Glu and BSO, similar to the ABCB5/STAT1 high-expressing clones. The cellular glutathione levels of the STAT1-transfected clones were significantly higher than that of the control. The STAT1-transfected clones also showed greater resistance to the effect of BSO on the cellular glutathione depletion compared to the control. These results demonstrate that STAT1 upregulates GLS and modulates amino acids and glutathione metabolism. Although we were unable to directly prove STAT1 upregulation by ABCB5, our results suggest that ABCB5 expression, directly or indirectly, leads to the overexpression of STAT1.

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SERCA and P-glycoprotein inhibition and ATP depletion are necessary for celastrol-induced autophagic cell death and collateral sensitivity in multidrug-resistant tumor cells

Xu S.W., Law B.Y.K., Qu S.L.Q., Hamdoun S., Chen J., Zhang W., Guo J.R., Wu A.G., Mok S.W.F., Zhang D.W., Xia C., Sugimoto Y., Efferth T., Liu L., Wong V.K.W.

Pharmacological Research (Pharmacological Research)  153   104660 2020.03

Research paper (scientific journal), Joint Work, Accepted,  ISSN  10436618

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Multidrug resistance (MDR) represents an obstacle in anti-cancer therapy. MDR is caused by multiple mechanisms, involving ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp), which reduces intracellular drug levels to sub-therapeutic concentrations. Therefore, sensitizing agents retaining effectiveness against apoptosis- or drug-resistant cancers are desired for the treatment of MDR cancers. The sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pump is an emerging target to overcome MDR, because of its continuous expression and because the calcium transport function is crucial to the survival of tumor cells. Previous studies showed that SERCA inhibitors exhibit anti-cancer effects in Bax-Bak-deficient, apoptosis-resistant and MDR cancers, whereas specific P-gp inhibitors reverse the MDR phenotype of cancer cells by blocking efflux of chemotherapeutic agents. Here, we unraveled SERCA and P-gp as double targets of the triterpenoid, celastrol to reverse MDR. Celastrol inhibited both SERCA and P-gp to stimulate calcium-mediated autophagy and ATP depletion, thereby induced collateral sensitivity in MDR cancer cells. In vivo studies further confirmed that celastrol suppressed tumor growth and metastasis by SERCA-mediated calcium mobilization. To the best of our knowledge, our findings demonstrate collateral sensitivity in MDR cancer cells by simultaneous inhibition of SERCA and P-gp for the first time.

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Cytotoxicity of cucurbitacin E from Citrullus colocynthis against multidrug-resistant cancer cells

Saeed M., Boulos J., Elhaboub G., Rigano D., Saab A., Loizzo M., Hassan L., Sugimoto Y., Piacente S., Tundis R., Yagi S., Khalid H., Efferth T.

Phytomedicine (Phytomedicine)  62   152945 2019.09

Research paper (scientific journal), Joint Work, Accepted,  ISSN  09447113

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Background: Cucurbitacin E (CuE) is an oxygenated tetracyclic triterpenoid isolated from the fruits of Citrullus colocynthis (L.) Schrad. Purpose: This study outlines CuE's cytotoxic activity against drug-resistant tumor cell lines. Three members of ABC transporters superfamily, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and ABCB5 were investigated, whose overexpression in tumors is tightly linked to multidrug resistance. Further factors of drug resistance studied were the tumor suppressor TP53 and the epidermal growth factor receptor (EGFR). Methods: Cytotoxicity assays (resazurin assays) were used to investigate the activity of Citrullus colocynthis and CuE towards multidrug resistant cancer cells. Molecular docking (In silico) has been carried out to explore the CuE's mode of binding to ABC transporters (P-gp, BCRP and ABCB5). The visualization of doxorubicin uptake was done by a Spinning Disc Confocal Microscope. The assessment of proteins expression was done by western blotting analysis. COMPARE and hierarchical cluster analyses were applied to identify, which genes correlate with sensitivity or resistance to cucurbitacins (CuA, CuB, CuE, CuD, CuI, and CuK). Results: Multidrug-resistant cells overexpressing P-gp or BCRP were cross-resistant to CuE. By contrast, TP53 knock-out cells were sensitive to CuE. Remarkably, resistant cells transfected with oncogenic ΔEGFR or ABCB5 were hypersensitive (collateral sensitive) to CuE. In silico analyses demonstrated that CuE is a substrate for P-gp and BCRP. Immunoblot analyses highlighted that CuE targeted EGFR and silenced its downstream signaling cascades. The most striking result that emerged from the doxorubicin uptake by ABCB5 overexpressing cells is that CuE is an effective inhibitor for ABCB5 transporter when compared with verapamil. The COMPARE analyses of transcriptome-wide expression profiles of tumor cell lines of the NCI identified common genes involved in cell cycle regulation, cellular adhesion and intracellular communication for different cucurbitacins. Conclusion: CuE represents a potential therapeutic candidate for the treatment of certain types of refractory tumors. To best of our knowledge, this is the first time to identify CuE and verapamil as inhibitors for ABCB5 transporter.

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Collateral sensitivity of drug-resistant ABCB5- and mutation-activated EGFR overexpressing cells towards resveratrol due to modulation of SIRT1 expression

Saeed M., Rahama M., Kuete V., Dawood M., Elbadawi M., Sugimoto Y., Efferth T.

Phytomedicine (Phytomedicine)  59   152890 2019.06

Research paper (scientific journal), Joint Work, Accepted,  ISSN  09447113

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Background: In the drug discovery field, natural products deemed a precious source of novel lead compounds. They have the ability to bypass or overcome multidrug resistance (MDR) in cancer cells. Purpose: In this study, the natural polyphenolic stilbene resveratrol (RES) has been studied for its cytotoxic activity toward MDR cancer cells. Methods: Resazurin assay was used to investigate the cytotoxicity of RES not only against a panel of drug-resistant cancer cells overexpressing P-glycoprotein/ABCB1, BCRP/ABCG2, ABCB5 (ATP-binding cassette transporters), but also mutation-activated EGFR. The assessment of proteins expression was done by Western blot analysis. COMPARE and hierarchical cluster analyses were applied to identify, which genes correlate with sensitivity or resistance to RES. The NF-κB activation was evaluated using NF-kB reporter cells assay. Results: Interestingly, MDR cells overexpressing ABCB5 and mutation-activated EGFR were collateral sensitive (CS) to RES. Our immunoblotting analysis highlighted that CS may be attributed to RES-induced sirtuin 1 (SIRT1) overexpression. Indeed, the SIRT1 inhibitor, sirtinol completely abolished CS to RES, indicating a causative role of SIRT1 for CS to RES. In addition, COMPARE and hierarchical cluster analyses of transcriptomic data indicated genes associated with diverse cellular mechanisms ranging from the immune response, inflammation signaling, and microtubule formation to cell migration. Searching for transcription factor binding motifs in the promoters of these genes pointed to NF-κB as one of the master regulators related to RES activity. Conclusion: The findings demonstrate that RES alone or in combination with established chemotherapeutic agents might overcome the refractory tumors. This information may be immensely useful for the development of personalized treatment.

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Cytotoxicity of 40 Egyptian plant extracts targeting mechanisms of drug-resistant cancer cells

Hegazy M., Abdelfatah S., Hamed A., Mohamed T., Elshamy A., Saleh I., Reda E., Abdel-Azim N., Shams K., Sakr M., Sugimoto Y., Paré P., Efferth T.

Phytomedicine (Phytomedicine)  59   152771 2019.06

Research paper (scientific journal), Joint Work, Accepted,  ISSN  09447113

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Background: The multidrug resistance (MDR) phenotype encounters a major challenge to the success of established chemotherapy in cancer patients. We hypothesized that cytotoxic medicinal plants with novel phytochemicals can overcome MDR and kill MDR-cells with similar efficacy as drug sensitive cells. Purpose: We evaluated plant extracts from an unexplored ecosystem in Egypt with unusual climate and nutrient conditions for their activity against sensitive and multidrug-resistant cancer cell lines. Material and methods/study design: Methylene chloride: methanol (1:1) and methanol: H 2 O (7:3) extracts of 40 plants were prepared resulting in a sum of 76 fraction containing compounds with varying polarity. The resazurin reduction assay was employed to evaluate the cytotoxicity of these extracts on five matched pairs of drug-sensitive and their drug-resistant cell lines. Flow cytometry and Western blotting was used to determine cell cycle analyses, apoptosis, and autophagy. Reactive oxygen species (ROS) were measured spectrophotometrically. Results: Extracts derived from Withania obtusifolia (WO), Jasonia candicans (JC), Centaurea lippii (CL), and Pulicaria undulata (PU) were the most active ones among 76 extracts from 40 Egyptian medicinal plants. They showed a significant reduction of cell viability on drug-sensitive CCRF-CEM leukemia cell line with IC 50 values less than 7 µg/ml. Low cross-resistance degrees were observed in multidrug-resistant CEM/ADR5000 cells towards CL (1.82-fold) and JC (6.09-fold). All other drug-resistant cell lines did not reveal cross-resistance to the four extracts. Further mechanistic assessment have been studied for these four extracts. Conclusion: The methylene chloride: methanol (1:1) fractions of WO, JC, CL, and PU are promising cytotoxic extracts that could be used to combat MDR cancer cells through different cell death pathways.

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Novel pharmacological effects of poly (ADP-ribose) polymerase inhibitor rucaparib on the lactate dehydrogenase pathway

Nonomiya Y., Noguchi K., Katayama K., Sugimoto Y.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications)  510 ( 4 ) 501 - 507 2019.03

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0006291X

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Poly (ADP-ribose) polymerases (PARPs) are involved in various cellular events, including DNA repair. PARP inhibitors including olaparib and rucaparib, have been specially developed against breast and ovarian cancers deficient in DNA repair systems. In this study, we found that PARP1-defective olaparib-resistant A2780 cells (ola-R cells) cells were still sensitive to two PARP inhibitors, rucaparib and veliparib. Metabolomic analysis revealed that rucaparib suppressed the lactate dehydrogenase (LDH)-mediated conversion of pyruvic acid to lactic acid in A2780 cells, although olaparib did not. The inhibition of LDH by siRNA-mediated knockdown or by LDH inhibitors suppressed the growth of ovarian cancer cells. Our results suggested that the suppression of the LDH-associated pathway contributed to the pharmacological effects of rucaparib.

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Synthesis of 5-Hydroxy-3′,4′,7-trimethoxyflavone and Related Compounds and Elucidation of Their Reversal Effects on BCRP/ABCG2-Mediated Anticancer Drug Resistance

Tsunekawa R., Katayama K., Hanaya K., Higashibayashi S., Sugimoto Y., Sugai T.

ChemBioChem (ChemBioChem)  20 ( 2 ) 210 - 220 2019.01

Research paper (scientific journal), Joint Work, Accepted,  ISSN  14394227

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3′,4′,7-Trimethoxyflavone (TMF) has been reported to show a potent reversal effect on drug resistance mediated by breast cancer resistance protein (BCRP)/ATP-binding cassette subfamily G member 2 (ABCG2). In this study, we designed and synthesized five derivatives with either a hydroxy group or a fluorine atom at C-5 and several kinds of capping moiety at the C-7 hydroxy group, on the same 3′,4′-dimethoxy-substituted flavone skeleton. We subsequently evaluated the efficacies of these compounds against BCRP-expressing human leukaemia K562/BCRP cells. Reversal of drug resistance was expressed as the concentration of compound causing a twofold reduction in drug sensitivity (RI 50 ). Of the synthesized compounds, the reversal effect of 5-hydroxy-3′,4′,7-trimethoxyflavone (HTMF, RI 50 7.2 nm) towards 7-ethyl-10-hydroxycamptothecin (SN-38) was stronger than that of TMF (RI 50 18 nm). Fluoro-substituted 5-fluoro-3′,4′,7-trimethoxyflavone (FTMF, RI 50 25 nm) and monoglycosylated 7-(β-glucosyloxy)-5-hydroxy-3′,4′-dimethoxyflavone (GOHDMF, 91 nm) also exhibited reversal effects, whereas the di- and triglycoside derivatives did not. TMF, HTMF and FTMF at 0.01–10 μm upregulated the K562/BCRP cellular accumulation of Hoechst 33342 nuclear staining dye. In addition, western blotting revealed that treatment of K562/BCRP cells with 0.1 μm TMF, HTMF or FTMT suppressed the expression of BCRP. HTMF showed the strongest inhibition of BCRP-mediated efflux and suppression of BCRP expression of the three effective synthesized flavones.

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