Cytotoxic effects of haplamine and its major metabolites on human cancer cell lines.

Haplamine, extracted from Haplophyllum perforatum, is widely used in Central Asia for treating various diseases, including testicular cancer. The purpose of the present study was to investigate in vitro the cytotoxic properties of haplamine and its major metabolites (trans/cis-3,4-dihydroxyhaplamine) on human pancreatic cancer, colorectal cancer and hepatic cancer cell lines. The efficacy of haplamine was compared with those of the respective reference drugs for treating digestive cancers (e. g., 5-FU, gemcitabine). Finally, the implication of apoptosis in haplamine-induced cell death was investigated. The IC50 values of of haplamine were 52.5 +/- 2.6, 24.3 +/- 0.7; 41.5 +/- 2.5, 72 +/- 2, 32 +/- 2.2 and 59.7 +/- 2.1 microM in human pancreatic cancer (Capan1 and Capan2), colorectal cancer (LS174T, HT29, and SW620) and hepatic cancer (HepG2) cells, respectively. The IC50 values of trans/cis-3,4-dihydroxyhaplamine were both > 200 microM, thus suggesting that the previously reported cytotoxic efficacy of haplamine was supported by the parent drug only. Besides, our data showed that haplamine leads to cell death through the induction of early/late apoptosis in the target cells. Interestingly, we found that haplamine showed significant antiproliferative efficacy on resistant SW620 colorectal cells, whereas the reference drug 5-FU was ineffective (32 vs. 73 microM, p < 0.01 t- test), thus suggesting that haplamine could be of interest for treating digestive cancers resistant to standard fluoropyrimidines. Similarly, haplamine proved to be significantly more potent in pancreatic cells than gemcitabine, the reference cytotoxic drug for treating pancreatic carcinomas. Overall, these results confirm the anticancer properties of haplamine suggested by its traditional use, and indicate that it could be further considered in various other solid tumours frequently encountered in adults, including those resistant to standard chemotherapy.

Reversal of P-glycoprotein-mediated drug efflux by eudesmin from Haplophyllum perforatum and cytotoxicity pattern versus diphyllin, podophyllotoxin and etoposide.

The present study focuses on eudesmin (bicyclic lignan, 0.15 % of dry leaves) and diphyllin (arylnaphthalene lignan, 0.1 % of dry roots), both isolated from H. perforatum Kar. et Kir, a Rutaceae species endemic to Uzbekistan. We first compared their specificity for cancer cells with those of etoposide and podophyllotoxin by screening their cytotoxicity on 3 healthy cell-lines and 7 sensitive or resistant human solid cancer lines. We then tested their capacity to reverse P-glycoprotein-mediated multidrug resistance (MDR) by assaying dye and drug uptake in MDR1-transfected Madin-Darby canine kidney (MDCK-MDR1) and doxorubicine-resistant human breast carcinoma cells (MCF7/Dox). Eudesmin displays IC (50) values > 100 microM on all tested lines. Our data provide the first demonstration that this non-toxic lignan reverses Pgp-mediated drug efflux and supports the hypothesis that it may inhibit resistance mediated by MDR1 and MRP proteins. Even if its reversal activity is insufficient for clinical application, its capacity to accumulate [(3)H]-vinblastine in MDCK/MDR1 and MCF7/Dox cells suggests that eudesmin may positively affect the bioavailability and, thereby, the therapeutic potency of anticancer drugs in Pgp-overexpressing cells. Diphyllin exhibits IC (50) values ranging from 10 (- 6) to 10 (- 4) M. It is markedly less toxic than podophyllotoxin (IC (50) : 13 - 61 nM), but exhibits tumoricidal effects close to those of etoposide. Unfortunatly, it is 65-fold more toxic than etoposide on human primary fibroblasts. Consequently, it has no value as an anticancer drug. Its value as raw material for the hemisynthesis of anticancer drugs is discussed.

HPLC quantification of alkaloids from Haplophyllum extracts and comparison with their cytotoxic properties.

An efficient system for the analysis of total alkaloids extracted from the aerial parts from different species of genus Haplophyllum (Rutaceae) by HPLC on a reversed-phase column is described. The HPLC method described was validated for its specificity, linearity and precision using external standards (haplopine, skimmianine and haplamine). The chromatographic conditions allowed the separation of alkaloids and the quantification of haplopine, skimmianine and haplamine in different samples of species of Haplophyllum collected in Uzbekistan. The alkaloidal contents of samples were compared with their in vitro cytotoxic properties against two cancer cell lines (HeLa and HCT-116). The cytotoxicity of extracts was correlated with the concentration of haplopine, skimmianine or haplamine in aerial parts of species of Haplophyllum.

Inter-species variability of haplamine metabolism and identification of its phase I metabolites from liver microsomes.

Haplamine, a pyranoquinoline alkaloid, was isolated from the genus Haplophyllum. The inter-species variability of haplamine metabolism was determined by reversed phase high performance liquid chromatography (HPLC) with UV detection. Microsomes from the liver of rats, mice, rabbits, guinea-pigs and humans were incubated with haplamine. After incubation, samples were extracted with a mixture of ethyl acetate and isopropyl alcohol (90 : 10; v/v). Haplamine and its metabolites were separated by HPLC using Nucleosil C18 Nautilus (5 microm) connected with a precolumn of the same type. The HPLC mobile phase consisted of water (A) and a mixture of methanol and acetonitrile (85 : 15; v/v) (B) used in a gradient mode (17 to 27 % B for 10 min, 27 to 90 % B for 37 min, 90 to 17 % B for 3 min, and finally 17 % B for 3 min) at 1 mL/min. Quantitative and qualitative results showed significant inter-species differences in haplamine metabolism. Qualitative similarities were found between guinea-pigs, rabbits, and humans. The metabolites were isolated by HPLC and identified by GC/MS after silylation. The phase I metabolites identified in human liver microsomes were TRANS/CIS-3,4-dihydroxy-9-O-desmethylhaplamine, TRANS/CIS-3,4-dihydroxyhaplamine and 9-O-desmethylhaplamine.

In vitro antileishmanial activity of diphyllin isolated from Haplophyllum bucharicum.

Diphyllin isolated from Haplophyllum bucharicum Litv. (Rutaceae), an endemic plant of Uzbekistan, displayed a moderate antiproliferative activity towards human monocytes (IC50 = 35.2 microM) and Leishmania promastigotes (IC50 = 14.4 microM), by a mechanism of action that involved interaction with macromolecules and resulted in cell cycle arrest in the S-phase and inhibition of protein synthesis. In the intracellular amastigote form of the parasite, diphyllin exerted a strong specific inhibitory activity (IC50 = 0.2 microM) resulting from the inhibition of parasite internalization within macrophages. This property was mainly due to modulation of macrophage phagocytosis and, to a lesser extent, it also involved interference with surface molecules of the promastigote membrane.