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Therapeutics

Nemorosone blocks proliferation and induces apoptosis in leukemia cells

D. Díaz-Carballo, S. Malak, M. Freistühler, A. Elmaagacli, W. Bardenheuer and H.P. Reusch

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Volume 46 p. 428 - 439

Abstract

D. Díaz-Carballo¹, S. Malak², M. Freistühler³, A. Elmaagacli⁴, W. Bardenheuer² and H.P. Reusch¹

¹Abteilung für Klinische Pharmakologie, Ruhr-Universität Bochum, ²Innere Klinik und Poliklinik (Tumorforschung), Zentrum für konservative Onkologie, ³Klinik für Augenheilkunde and ⁴Klinik für Knochenmarktransplantation, Universitätsklinikum Essen, Germany
Objective: This work is aimed at characterizing nemorosone, isolated from Clusia rosea, as a potential antileukemic agent. In addition, we analyzed its influence on hematopoiesis in a mouse model. Materials and methods: The isolation of nemorosone was carried out employing the RP-HPLC (reversed phase high-performance liquid chromatography) technique. Cytotoxicity was assessed in human leukemia cell lines including parental and chemotherapy-refractory sublines based on the MTT compound. Its effects on the cell cycle were analyzed using FACS (fluorescence-activated cell sorting) and Western blot techniques. Studies on the drug-induced early apoptotic process were carried out by means of fluorescence microscopy. Major signal transducers and the enzymatic inhibition of immunoprecipitated Akt/PKB were detected by Western blot. Hematopoiesis was analyzed in NMRI nu/nu mice after chronic nemorosone treatment, measuring hematological parameters by conventional laboratory techniques. Results: Nemorosone proved cytotoxic in both parental and chemoresistant leukemia cell lines with IC₅₀ values between 2.10 and 3.10 mg/ml. No cross-resistances could be detected. Cell cycle studies showed apoptosis induction accompanied by an increase in the G0/G1 population in both cell lines studied, whereas a significant decrease in the S-phase was found in Jurkat cells. Nemorosone induced a down-regulation of cyclins A, B1, D1, and E as well as a dephosphorylation of cdc2. Major signal transduction elements such as ERK1/2 and p38 MAPK, as well as important oncoproteins such as c-Myb and BCR/ABL were also found down-regulated. The enzymatic activity of immunoprecipitated Akt/PKB was substantially inhibited in vitro. Moreover, subchronic nemorosone treatment induced reversible monocytosis and thrombocytosis in the mouse model examined. Conclusions: Here, we demonstrate for the first time that nemorosone exerts cytotoxicity in leukemia cells, partly by targeting the Akt/PKB signal transducer, affecting protein levels and cell cycle progression. Finally, in vivo studies suggest that nemorosone significantly affects hematopoiesis in mice.Correspondence to:
D. Díaz-Carballo, PhD
Abteilung Klinische Pharmakologie, Ruhr-Universität Bochum, Universitätsstraße 150, 44801 Bochum, Germany
Email: [email protected]

Pharmacodynamics

Mucronulatol from Caribbean propolis exerts cytotoxic effects on human tumor cell lines

D. Díaz-Carballo, M. Freistühler, S. Malak, W. Bardenheuer and H.P. Reusch

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Volume 46 p. 226 - 235

Abstract

D. Díaz-Carballo, M. Freistühler, S. Malak, W. Bardenheuer and H.P. Reusch

¹Abteilung Klinische Pharmakologie, Ruhr-Universität Bochum, ²Universitätsklinikum Essen, Klinik für Augenheilkunde, Essen and ³Innere Klinik und Poliklinik, Tumorforschung, Universitätsklinikum Essen, Germany
Objective: Mucronulatol is one of the most cytotoxic substances present in Caribbean propolis. This work aimed at initially characterizing the biological effects of mucronulatol in cancer cell lines comprehending both wildtype and resistant sublines. Materials and methods: An RP-HPLC technique was employed to separate and purify mucronulatol. IC50 values were determined using the sulforhodamine B (SRB) proliferation assay. FACS-based cell cycle studies were carried out combining propidium iodide staining and 5-bromo-2’-deoxyuridine incorporation. Cell cycle regulator proteins were detected by Western blotting. The transcription of genes of interest was analyzed using RT-PCR. Results: In MDR1–/MDR3+ cells, mucronulatol exhibited cytotoxicity in the range of 2.7 – 10.2 µg/ml, while no cytotoxic effects were observed in MDR1+ systems at up to 100 µg/ml. Cytometric studies revealed that mucronulatol promoted a global reduction in all cell cycle phases, with a remarkable increase of the apoptotic sub-G1 population. Immunoblotting showed that mucronulatol induced an up-regulation of p21Cip1 and p27Kip1 while down-regulating cyclin E and CDK4 in a drug concentration-dependent manner. No effect on topoisomerase I was observed, while we detected an altered expression of topoisomerases II-α/β. RT-PCR studies showed that 2-fold the IC50 in HCT8 colon carcinoma cells was sufficient for altering the expression pattern of genes in this cell line, including topoisomerase I, thymidilate synthase, EGF receptor and c-myc, amongst others. Conclusion: Here, we demonstrate for the first time that mucronulatol exerts cytotoxicity in cancer cell lines by targeting the control of cell cycle progression, indicating that the mechanism of action of this compound involves interference with the cell cycle machinery.Correspondence to:
Dr. D. Díaz-Carballo; Abteilung Klinische Pharmakologie, Ruhr-Universität, Bochum, Universitätsstraße 150, 44801 Bochum, Germany
Email: [email protected]

Original Research

Antiretroviral activity of two polyisoprenylated acylphloroglucinols, 7-epi-nemorosone and plukenetione A, isolated from Caribbean propolis

D. Díaz-Carballo, K.Ueberla, V. Kleff, S. Ergun, S. Malak, M.Freistuehler, S. Somogyi, C. Kücherer, W. Bardenheuer and D. Strumberg

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Volume 48 p. 670 - 677

Abstract

International Journal of Clinical Pharmacology and Therapeutics, Vol. 48 – No. 10/2010 (670-677)

Antiretroviral activity of two polyisoprenylated acylphloroglucinols, 7-epi-nemorosone and plukenetione A, isolated from Caribbean propolis

D. Díaz-Carballo¹*, K. Ueberla²*, V. Kleff³, S. Ergun³, S. Malak⁴, M. Freistuehler⁵, S. Somogyi⁶, C. Kücherer⁶, W. Bardenheuer¹ and D. Strumberg¹

¹Institute for Molecular Oncology and Experimental Therapeutics, Marienhospital Herne, Ruhr University of Bochum, Herne, ²Department of Molecular and Medical Virology, Ruhr University of Bochum, Bochum, ³Institute of Anatomy, ⁴West German Cancer Center, ⁵Department of Ophthalmology, University of Duisburg-Essen, School of Medicine, Essen and ⁶Project HIV Variability and Molecular Epidemiology/P¹¹, Robert Koch Institute, Berlin, Germany

*The two first authors contributed equally to this work.
Objectives: Polyisoprenylated acylphloroglucinols have recently emerged as antitumoral agents. This study aims at elucidating the antiretroviral activity of two such compounds which were isolated from Caribbean propolis: 7-epi-nemorosone and plukenetione A, the structure of which is based on an adamantane moiety. Plukenetione A is for the first time shown to have antiretroviral activity. Material and methods: The isolation of both small molecules was carried out using RP-HPLC. Their antiretroviral activity was studied based on lentiviral particles produced in HEK293T cells from the SIV-based vector VLDBH; their cytotoxicity was monitored by MTT proliferation assay. The antiviral activity of 7-epi-nemorosone was studied in CEMx174-SEAP infected with the HIV-1-strain pNL4.3wt. Reverse transcriptase inhibition was determined by a standard two-step RT-PCR using MMLV RT. Results: 7-epi-nemorosone and plukenetione A were found to be potent antilentiviral agents in the employed system, inhibiting viral infection at concentrations below 1 µM/2 µM, respectively. Whereas 7-epi-nemorosone was not able to inhibit the reverse transcriptase in vitro (IC₅₀ > 25 µM), plukenetione A effectively inhibited its enzymatic activity at an IC₅₀ of 1.75 µM. Conclusions: Despite 7-epi-nemorosone and plukenetione A sharing some structural core elements, the mechanism of action involved in their antiretroviral activity seems to be different. We propose that 7-epi-nemorosone inhibits the viral replication by interrupting the Akt/PKB signaling cascade, as was demonstrated previously in various cell lines. Since plukenetione A effectively inhibits the enzymatic activity of MMLV reverse transcriptase at concentrations that show antilentiviral activity, we suggest that this small molecule acts by interfering with the enzyme’s catalytic site.Correspondence to:
Dr. D. Diaz-Carballo
Institut für Molekulare Onkologie und Substanzentwicklung
Marienhospital Herne
Katholische Stiftung
Ruhr-Universität Bochum
Düngelstraße 33, 44625 Herne, Germany
Email: [email protected]

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