Galloflavin, a new lactate dehydrogenase inhibitor, induces the death of human breast cancer cells with different glycolytic attitude by affecting distinct signaling pathways.

Galloflavin (GF), a recently identified lactate dehydrogenase inhibitor, hinders the proliferation of cancer cells by blocking glycolysis and ATP production. The aim of the present experiments was to study the effect of this compound on breast cancer cell lines reproducing different pathological subtypes of this tumor: MCF-7 (the well differentiated form), MDA-MB-231 (the aggressive triple negative tumor) and MCF-Tam (a sub-line of MCF-7 with acquired tamoxifen resistance). We observed marked differences in the energetic metabolism of these cell lines. Compared to MCF-7 cells, both MDA-MB-231 and MCF-Tam cells exhibited higher LDH levels and glucose uptake and showed lower capacity of oxygen consumption. In spite of these differences, GF exerted similar growth inhibitory effects. This result was explained by the finding of a constitutively activated stress response in MDA-MB-231 and MCF-Tam cells, which reproduce the poor prognosis tumor forms. As a further proof, different signaling pathways were found to be involved in the antiproliferative action of GF. In MCF-7 cells we observed a down regulation of the ERα-mediated signaling needed for cell survival. On the contrary, in MCF-Tam and MDA-MB-231 cells growth inhibition appeared to be contributed by an oxidative stress condition. The prevalent mechanism of cell death was found to be apoptosis induction. Because of the clinical relevance of breast cancer forms having the triple negative and/or chemoresistant phenotype, our results showing comparable effects of GF even on aggressively growing cells encourage further studies to verify the potential of this compound in improving the chemotherapy of breast cancer.

The enhancement of interstitial transport of a doxorubicin-lactosaminated albumin conjugate by imatinib: in rat hepatocellular carcinoma it is not preferentially higher than that in liver and bone marrow.

The finding that imatinib enhances the drug transport from bloodstream to neoplastic cells suggested a possible role of this drug as an adjuvant to the chemotherapeutics given in the treatment of solid malignancies.The present experiments aimed to verify whether imatinib can selectively increase the penetration of a doxorubicin-lactosaminated human albumin conjugate (L-HSA-DOXO) in chemically induced rat hepatocellular carcinomas (HCCs). We observed that imatinib increased the uptake of L-HSA-DOXOby HCCs but at the same time caused a similar enhanced penetration of the conjugate in liver and bone marrow. To our knowledge, this is the first demonstration that the enhancing effect of imatinib on interstitial drug transport is not restricted to the tumors, but can be also displayed in normal tissues. This observation casts some doubts about the possibility that the value of anticancer agents with toxic side effects on liver and bone marrow can be improved by imatinib.

Efficacy of doxorubicin coupled to lactosaminated albumin on rat hepatocellular carcinomas evaluated by ultrasound imaging.

BACKGROUND/AIMS: Doxorubicin was conjugated with lactosaminated human albumin, a hepatotropic drug carrier, in order to increase its efficacy in the treatment of hepatocellular carcinoma. In rats bearing hepatocellular carcinomas induced by diethylnitrosamine, lactosaminated human albumin coupled doxorubicin enhanced the drug concentrations in the tumours and lowered those in extrahepatic tissues. The aim of the present study was to investigate the effects of lactosaminated human albumin coupled doxorubicin on the growth of established rat hepatocellular carcinomas induced by diethylnitrosamine. METHODS: Lactosaminated human albumin coupled doxorubicin and the free drug were i.v. administered to rats twice a week for 4 weeks at the single dose of 1 microg/g. Growth of individual tumours was followed through time by ultrasonography. RESULTS: In the control animals injected with saline the mean area of the tracked tumours significantly increased during the whole period of treatment. In the group of rats treated with lactosaminated human albumin coupled doxorubicin the mean area of the followed hepatocellular carcinomas remained practically unchanged. The free drug inhibited tumour growth only in the first period of drug administration. Lactosaminated human albumin coupled doxorubicin also hindered the development of new neoplastic nodules, which was unaffected by the free drug. CONCLUSIONS: The results support lactosaminated human albumin coupled doxorubicin as a promising agent for a systemic chemotherapy of hepatocellular carcinomas to treat noncurable patients.

Doxorubicin coupled to lactosaminated albumin: Effects on rats with liver fibrosis and cirrhosis.

BACKGROUND: The conjugate of doxorubicin with lactosaminated human albumin has the potential of increasing the doxorubicin efficacy in the treatment of hepatocellular carcinomas expressing the asialoglycoprotein receptor. However, coupled doxorubicin also accumulates in the liver, which might damage hepatocytes. AIMS: To verify whether coupled doxorubicin impairs liver function in rats with liver fibrosis and cirrhosis. METHODS: Coupled doxorubicin was administered using the same schedule which exerted an antineoplastic effect on rat hepatocellular carcinomas (4-weekly injections of doxorubicin at 1 microg/g). Liver fibrosis/cirrhosis was produced by carbon tetrachloride (CCl4) poisoning. Liver samples were studied histologically. Serum parameters of liver function and viability were determined. RESULTS: In normal rats, administration of coupled doxorubicin neither caused microscopic changes of hepatocytes nor modified serum liver parameters. In rats with fibrosis/cirrhosis, although a selective doxorubicin accumulation within the liver followed coupled doxorubicin administration, the drug did not have a detrimental effect on the histology of the liver and, among serum liver tests, only alanine aminotransferase and aspartate aminotransferase levels were moderately modified. CONCLUSIONS: Coupled doxorubicin can be administered to rats with liver fibrosis/cirrhosis without inducing a severe liver damage. If further studies will confirm the efficacy and safety of this compound, coupled doxorubicin therapy may open a new perspective in the treatment of hepatocellular carcinoma.

Detection of Legionella pneumophila in water samples by species-specific real-time and nested PCR assays.

AIMS: Legionella pneumophila is a contaminant of man-made water systems, including potable water, cooling towers, water systems of large buildings, etc. It is the most common causative agent of legionellosis, a respiratory infection, which may give rise to restricted outbreaks. To survey environmental water samples from hospitals and private habitations in Bologna, we developed a species-specific nested and a TaqMan real-time PCR for the detection of L. pneumophila. We compared the two assays and both to cultural isolation. METHODS AND RESULTS: The targeted gene was macrophage infectivity potentiator (mip), conserved in L. pneumophila, and divergent in other legionellae. One assay was based on a nested PCR and the other on a TaqMan real-time PCR protocol. Their sensitivities were 14 % or 5% higher than that of cultural isolation respectively. The detection limits were 1-2 genome equivalents per 50-microl reaction. Specificity was assessed using DNA from nine target and 20 nontarget organisms. CONCLUSIONS: When applied to water samples, both assays detected L. pneumophila at 80% or higher frequency. SIGNIFICANCE AND IMPACT OF THE STUDY: The species-specific molecular diagnosis of L. pneumophila by means of nested PCR does not require a specific instrumentation, exhibits a high sensitivity, and is advantageous over the cultural isolation and real-time PCR detection. It allows to quickly monitor water samples for the risk assessment of environmental contaminations.

Doxorubicin coupled to lactosaminated human albumin remains confined within mouse liver cells after the intracellular release from the carrier.

BACKGROUND: The hepatocyte receptor for asialoglycoproteins, which binds and internalises galactosyl terminating peptides, was found to be expressed also on the cells of the majority of hepatocarcinomas. AIMS: To verify whether doxorubicin coupling to lactosaminated albumin, a galactosyl terminating neoglycoprotein, produces selective drug accumulation in hepatocytes with reduced concentrations in extra-hepatic tissues, thus facilitating the use of the drug in hepatocarcinoma treatment. METHODS: Doxorubicin concentrations were measured in organs of mice injected with the free or coupled drug. RESULTS: In mice injected with the coupled drug, the ratios between doxorubicin concentrations in liver and those in heart, intestine, spleen and kidney were 8-14 times higher than in animals that received the same dose of the free drug. CONCLUSIONS: Due to the very efficient liver targeting of doxorubicin, the lactosaminated human albumin-doxorubicin conjugate appears to have the potential of improving the chemotherapy of hepatocellular carcinomas through the asialoglycoprotein receptor.

Floxuridine coupling with lactosaminated human albumin to increase drug efficacy on liver micrometastases.

BACKGROUND: Conjugates of nucleoside analogues with galactosyl terminating peptides selectively enter hepatocytes through the asialoglycoprotein receptor. After intracellular release from the carrier, the drugs partly exit from hepatic cells into hepatic blood. AIMS: To establish whether administration of a conjugate of floxuridine with lactosaminated human albumin selectively enhances drug concentrations in hepatic blood. Floxuridine is a fluoropyrimidine active on human colorectal cancer, a tumour which metastasises first to the liver. METHODS: In rats injected with free or conjugated floxuridine, plasma levels of the drug were determined in hepatic veins and in inferior vena cava, in order to measure drug concentrations in hepatic blood and in the systemic circulation, respectively. RESULTS: Ratios between floxuridine levels in hepatic veins and those in systemic circulation were found to be seven times higher in rats injected with the conjugate (p=0.000). CONCLUSIONS: The present results suggest that coupling to lactosaminated albumin might improve the effect of floxuridine in adjuvant chemotherapy of colorectal cancer by exposing the cells of liver micrometastases (nourished by hepatic sinusoids) to enhanced drug concentrations.

Coupling of 5-fluoro 2'-deoxyuridine to lactosaminated poly-l-lysine: an approach to a regional, non-invasive chemotherapy of liver micrometastases.

Nucleoside analogs conjugated with galactosyl-terminating peptides selectively enter liver cells and after intracellular release from the carrier partly exit into bloodstream, resulting in higher concentrations in liver blood than in systemic circulation. The aim of the present experiments was to ascertain whether, in mice injected with non-toxic doses of a 5-fluoro 2'-deoxyuridine (FUdR) conjugate with lactosaminated poly-L-lysine (L-poly(LYS)), the drug was released by hepatic cells in high enough amounts to be pharmacologically active on neoplastic cells infiltrating the liver. We observed that L-poly(LYS)-FUdR inhibited the growth of hepatic metastases induced by intrasplenic administration of murine colon carcinoma C-26 cells. L-poly(LYS)-FUdR was not toxic for C-26 cells in vitro, was selectively taken up by mouse liver, and was stable in mouse blood, indicating that the effect on the metastases was due to FUdR (and/or its active metabolites) released in liver blood after the conjugate was taken up by the hepatic cells. These results suggest that L-poly(LYS)-FUdR might be useful in adjuvant chemotherapy of tumors giving liver metastases. The drug released from hepatic cells into liver blood following conjugate administration via the peripheral venous route might accomplish a locoregional, non-invasive treatment of micrometastases nourished by liver sinusoids.