A new approach for the characterization of proliferative cells in cestodes.

Cestodes show a remarkable proliferative capability that sustains the constant growth and differentiation of proglottids essential for their lifestyle. It is believed that a separate population of undifferentiated stem cells (the so-called germinative cells) are the only cells capable of proliferation during growth and development. The study of this particular cell subpopulation is hampered by the current lack of methods to isolate it. In this work, we developed a reproducible flow cytometry and cell sorting method to quantify and isolate the proliferating cells in the tetrathyridia larvae of the model cestode Mesocestoides corti, based on the DNA content of the cells. The isolated cells display the typical germinative cell morphology, and can be used for RNA isolation with a yield in the ng to µg range. We expect that this approach may facilitate the characterization of the germinative cells in M. corti and other model tapeworms.

Liver organotropism and biotransformation of a novel platinum-ursodeoxycholate derivative, Bamet-UD2, with enhanced antitumour activity.

BACKGROUND/AIMS: Several members of a novel family of bile acid derivatives with cytostatic and virostatic activity have been synthesized and characterized. The aim of this work was to investigate the liver organotropism and biotransformation of two novel compounds with enhanced DNA-reactivity: Bamet-D3, in which a glycine-polyamine tandem was used as a spacer to separate the glycocholic acid moiety from the platinum(II) atom, and Bamet-UD2, in which cisplatin was directly bound to the carboxylate group of two ursodeoxycholic acid moieties. METHODS: Drug uptake and "in vitro" toxicity were investigated using rat hepatocytes in primary culture. Following i.v. administration of 0.5 mumol cisplatin, Bamet-D3 or Bamet-UD2, bile output, urinary and fecal excretion, organ distribution and pharmacokinetic parameters were determined in short-term (3 h) and long-term (14 days) experiments carried out on anaesthetized and conscious rats, respectively. Liver biotransformation was investigated by HPLC analysis of bile samples. Total platinum was measured by flameless atomic absorption spectroscopy. Using Nude mice, antitumour activity was investigated in subcutaneously implanted Hepa 1-6 mouse hepatoma cells. RESULTS: Uptake by rat hepatocytes was Bamet-UD2 (11.3 nmol/mg protein) > Bamet-D3 (5.6 nmol/mg protein) > cisplatin (2.1 pmol/mg protein). Bamet-UD2 induced "in vitro" cell toxicity, which was not observed for Bamet-D3 or cisplatin. On the contrary, no toxicity "in vivo" for Bamet-UD2 was found which was observed for cisplatin and Bamet-D3. This may be related with the fact that bile output of Bamet-UD2, which occurs with no major biotransformation, was > 10 fold higher than that of cisplatin and 3-fold higher than that of Bamet-D3, which was previously transformed into at least three different metabolites. Fecal excretion was Bamet-UD2 > Bamet-D3 > cisplatin, whereas urinary output was Bamet-D3 > cisplatin > Bamet-UD2. Accordingly, a marked liver- and a reduced kidney-vectoriality for Bamet-UD2, but not for Bamet-D3, was observed. Bamet-UD2 and cisplatin, but not Bamet-D3, were efficient in inhibiting tumour growth whereas, only Bamet-UD2 significantly prolonged survival time. CONCLUSIONS: There results indicate that Bamet-UD2 is a cisplatin-ursodeoxycholate derivative with strong antitumour activity, marked hepatobiliary organotropism, and reduced toxic side-effects as compared to the parent drug cisplatin.

Low in vivo toxicity of a novel cisplatin-ursodeoxycholic derivative (Bamet-UD2) with enhanced cytostatic activity versus liver tumors.

Cisplatin-bile acid derivatives belonging to the Bamet-family maintain both liver organotropism and cytostatic activity. "In vivo" toxicity and usefulness as chemotherapeutic agent versus liver tumors of a novel drug, Bamet-UD2 [cis-diamminechlorocholylglycinate platinum (II)], with enhanced "in vitro" cytostatic activity was investigated. Using orthotopically implanted mouse Hepa 1-6 hepatoma in the liver of Nude mice, the antitumor effect of Bamet-UD2 was compared with that of a previously characterized compound of this family, Bamet-R2 [cis-diamminebis-ursodeoxycholate platinum(II)], and cisplatin. Life span was significantly prolonged in mice treated with both Bamets (Bamet-UD2 > Bamet-R2), compared with animals receiving saline or cisplatin. All these drugs inhibit tumor growth (Bamet-UD2 = cisplatin > Bamet-R2). However, toxicity-related deaths only occurred under cisplatin treatment. Using rats maintained in metabolic cages, organ-specific toxicity and drug accumulation in tissues were investigated. The amount of both Bamets in the liver was severalfold higher than that of cisplatin. By contrast, a significantly higher amount of cisplatin in kidney and nerve was found. In lung, heart, muscle, brain, and bone marrow the amount of drug was small and also significantly lower in animals receiving Bamets. Signs of neurotoxicity (altered nerve conduction velocity), nephrotoxicity (increased serum urea and creatinine concentrations and decreased creatinine clearance), and bone marrow toxicity (decreased platelet and white blood counts) in animals treated with cisplatin but not with the Bamets were found. These results indicate that, owing to strong antitumor activity together with absence of side effects, Bamet-UD2 may be useful in the treatment of liver tumors.

Structural characterization, kinetic studies, and in vitro biological activity of new cis-diamminebis-cholylglycinate(O,O') Pt(II) and cis-diamminebis-ursodeoxycholate(O,O') Pt(II) complexes.

The complexes cis-diamminebis-cholylglycinate (O,O') [Pt(II) C(52)H(90)N(4)O(12)Pt, for convenience referred to as Bamet-R1] and cis-diamminebis-ursodeoxycholate (O,O') Pt(II) (C(48)H(84)N(2)O(8)Pt, Bamet-UD2) were prepared. The structural integrity of the compounds was confirmed by elemental analysis, FT-IR, NMR, FAB-MS, and UV spectroscopies. The kinetic study of both compounds was accomplished by combining the conductivity measurement and those of the analysis of the electronic spectra in aqueous solution for NaCl concentrations of 4 mM (similar to cytoplasmatic concentration), 150 mM (similar to plasmatic concentration), and 500 mM. In water, the compound Bamet-R1 showed a half-life, t(1/2), of 3.0 h. This compound forms the chelate species through loss of a ligand, and the other one acts as a bidentate ligand. Ring opening in the presence of chloride ion was produced with a k(Cl)()-of 0.25 M(-)(1) h(-)(1). The half-life of Bamet-UD2 in aqueous solution was 3.2 h. However, since this species is not able to chelate and has a lower degree of solubility in the presence of chloride ion, its kinetic behavior was very different from that of the other compound. We consider this to be of great interest with regards to its cytostatic activity. All kinetic measurements were performed under pseudo-first-order conditions, and a pseudo-first-order behavior was found. The antitumoral effect of Bamet-UD2 on several cell lines derived from rat hepatoma, human hepatoma, mouse leukemia, and human colon carcinoma was found to be, in general, similar to that of cisplatin, but higher than that observed for Bamet-R1.

In vitro test to determine the effect of cytostatic drugs on co-cultured rat hepatocytes and hepatoma cells.

Using uptake of the fluorescent bile acid derivative cholylglycylamido-fluorescein (FITC-GC) as a measurement of liver cell population size and function, the antiproliferative and toxic effects of the well known cytostatic drug, cisplatin was evaluated on rapidly growing rat hepatoma McA-RH7777 cells and rat hepatocytes in primary culture under non-proliferating conditions. Co-culture set up to mimic the in vivo situation of tumour and extratumoural liver tissue exposed to cytostatic chemotherapy does not markedly affect the survival or the growth dynamics of both cell types. FITC-GC uptake as corrected for DNA and protein contents in the dish was significantly lower in hepatoma cells than in rat hepatocytes throughout the experimental period (96 h). Effect of 0.1-100 microM cisplatin exposure from 24 to 96 h of culture on cell population size, as measured by protein and DNA contents in the culture dishes, were consistent with changes observed in total FITC-GC uptake. Cisplatin concentrations lower than 50 microM did not affect FITC-GC uptake by rat hepatocytes. By contrast, a progressively increasing effect on hepatoma cells as from 2 microM cisplatin was observed. Two phases in the decay of FITC-GC uptake versus cisplatin concentrations were found in co-cultures exposed to this drug. The first segment, between 2 microM and 50 microM, was characterized by a slow decay that matched the response of hepatoma cells to cisplatin exposure. This was considered to be due to the antiproliferative effect of cisplatin. The second segment, with a steeper decay, matched the effect of cisplatin on hepatocytes. This was interpreted as being due to non-specific toxicity. These results suggest that FITC-GC uptake by co-culture of hepatocytes and tumour cells provides a useful experimental model to explore the mechanism of action and the size of beneficial effect window for new drugs in vitro.