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

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.

Nemorosone blocks proliferation and induces apoptosis in leukemia cells.

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.

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

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. IC(50) 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 microg/ml, while no cytotoxic effects were observed in MDR1+ systems at up to 100 microg/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 p21(Cip1) and p27(Kip1) 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-I+/-/I(2). RT-PCR studies showed that 2-fold the IC(50) 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.

Cytotoxic activity of nemorosone in neuroblastoma cells.

Neuroblastoma is the second most common solid tumour during childhood, characterized by rapid disease progression. Most children with metastasized neuroblastoma die despite intensive chemotherapy due to an intrinsic or acquired chemotherapy resistance. Thus, new therapeutic strategies are urgently needed. Here, we demonstrate that the novel compound nemorosone isolated from alcoholic extracts of Clusia rosea resins by reverse phase high pressure liquid chromatography (RP-HPLC) exerts cytotoxic activity in neuroblas-toma cell lines both parental and their clones selected for resistance against adriamycin, cisplatin, etoposide or 5-fluorouracil. Cell cycle studies revealed that nemorosone induces an accumulation in G0/G1- with a reduction in S-phase population combined with a robust up-regulation of p21Cip1. Furthermore, a dose-dependent apoptotic DNA laddering accompanied by an activation of caspase-3 activity was detected. Nemorosone induced a significant dephosphorylation of ERK1/2 in LAN-1 parental cells probably by the inhibition of its upstream kinase MEK1/2. No significant modulation of signal transducers JNK, p38 MAPK and Akt/PKB was detected. The enzymatic activity of immunoprecipitated Akt/PKB was strongly inhibited in vitro, suggesting that nemorosone exerts its anti-proliferative activity at least in part by targeting Akt/PKB in the cell lines studied. In addition, a synergistic effect with Raf-1 inhibitor BAY 43-9006 was found. Finally, nemorosone induced a considerable down-regulation of N-myc protein levels in parental LAN-1 and an etoposide resistant sub-line at the same drug-concentrations.

Evaluación de la influencia del subcitrato de bismuto coloidal sobre la actividad antitumoral del CP en ratones transplantados con leucemia P-388 / Assessment of the influence of colloid bismuth subcitrate upon the antitumoral activity of cis-platinum in mice transplanted with P-388

El Cis-Platino (CP), antitumoral ampliamente utilizado en oncología, posee una marcada nefrotoxicidad. En el presente trabajo se determina la influencia del pretratamiento, con Subcitrato de Bismuto Coloidal (SBC), a dosis de 100, 500 y 1000 mg/kg, sobre la nefrotoxicidad y actividad antitumoral del CP (5 y 10 mg/kg) en ratones B602F-1 previamente transplantados con leucemia p 388. La actividad antitumoral se evaluó mediante la sobrevida promedio y el índice de aumento de sobrevida. Como indicador del daño renal se midieron los niveles plasmáticos de urea y se analizaron histológicamente los riñones. Los resultados evidenciaron que el pretratamiento con SBC no afectó la actividad antitumoral del CP disminuyendo la nefrotoxicidad inherente al mismo al revertir a la normalidad los valores plasmáticos de urea y atenuando el daño a nivel histológico (AU)