Moonlighting Proteins: Diverse Functions Found in Fungi.

Moonlighting proteins combine multiple functions in one polypeptide chain. An increasing number of moonlighting proteins are being found in diverse fungal taxa that vary in morphology, life cycle, and ecological niche. In this mini-review we discuss examples of moonlighting proteins in fungi that illustrate their roles in transcription and DNA metabolism, translation and RNA metabolism, protein folding, and regulation of protein function, and their interaction with other cell types and host proteins.

Use of the proton pump inhibitor pantoprazole to modify the distribution and activity of doxorubicin: a potential strategy to improve the therapy of solid tumors.

PURPOSE: Limited drug distribution within solid tumors is an important cause of drug resistance. Basic drugs (e.g., doxorubicin) may be sequestered in acidic organelles, thereby limiting drug distribution to distal cells and diverting drugs from their target DNA. Here we investigate the effects of pantoprazole, a proton pump inhibitor, on doxorubicin uptake, and doxorubicin distribution and activity using in vitro and murine models. EXPERIMENTAL DESIGN: Murine EMT-6 and human MCF-7 cells were treated with pantoprazole to evaluate changes in endosomal pH using fluorescence spectroscopy, and uptake of doxorubicin using flow cytometry. Effects of pantoprazole on tissue penetration of doxorubicin were evaluated in multilayered cell cultures (MCC), and in solid tumors using immunohistochemistry. Effects of pantoprazole to influence tumor growth delay and toxicity because of doxorubicin were evaluated in mice. RESULTS: Pantoprazole (>200 µmol/L) increased endosomal pH in cells, and also increased nuclear uptake of doxorubicin. Pretreatment with pantoprazole increased tissue penetration of doxorubicin in MCCs. Pantoprazole improved doxorubicin distribution from blood vessels in solid tumors. Pantoprazole given before doxorubicin led to increased growth delay when given as single or multiple doses to mice bearing MCF7 xenografts. CONCLUSIONS: Use of pantoprazole to enhance the distribution and cytotoxicity of anticancer drugs in solid tumors might be a novel treatment strategy to improve their therapeutic index.

Distribution of the anticancer drugs doxorubicin, mitoxantrone and topotecan in tumors and normal tissues.

PURPOSE: Pharmacokinetic analyses estimate the mean concentration of drug within a given tissue as a function of time, but do not give information about the spatial distribution of drugs within that tissue. Here, we compare the time-dependent spatial distribution of three anticancer drugs within tumors, heart, kidney, liver and brain. METHODS: Mice bearing various xenografts were treated with doxorubicin, mitoxantrone or topotecan. At various times after injection, tumors and samples of heart, kidney, liver and brain were excised. RESULTS: Within solid tumors, the distribution of doxorubicin, mitoxantrone and topotecan was limited to perivascular regions at 10 min after administration and the distance from blood vessels at which drug intensity fell to half was ~25-75 µm. Although drug distribution improved after 3 and 24 h, there remained a significant decrease in drug fluorescence with increasing distance from tumor blood vessels. Drug distribution was relatively uniform in the heart, kidney and liver with substantially greater perivascular drug uptake than in tumors. There was significantly higher total drug fluorescence in the liver than in tumors after 10 min, 3 and 24 h. Little to no drug fluorescence was observed in the brain. CONCLUSIONS: There are marked differences in the spatial distributions of three anticancer drugs within tumor tissue and normal tissues over time, with greater exposure to most normal tissues and limited drug distribution to many cells in tumors. Studies of the spatial distribution of drugs are required to complement pharmacokinetic data in order to better understand and predict drug effects and toxicities.

Use of molecular biomarkers to quantify the spatial distribution of effects of anticancer drugs in solid tumors.

Poor distribution of anticancer drugs within solid tumors may limit their effectiveness. Here, we characterize the distribution within solid tumors of biomarkers of drug effect. γ-H2AX, cleaved-caspase-3 or -6, and Ki67 were quantified in tumor sections in relation to blood vessels (recognized by CD31) using monoclonal antibodies and immunohistochemistry. To validate their use, we compared their time-dependent distribution with that of (i) fluorescent doxorubicin and (ii) a monoclonal antibody that detects melphalan-induced DNA adducts. The biomarkers were then used to quantify the distribution of docetaxel in relation to tumor blood vessels. Activation of γ-H2AX was evaluated following in vitro exposure of tumor cells to multiple drugs. Distributions of doxorubicin in MDA-MB-231 and MCF-7 xenografts and of melphalan-induced DNA adducts in MCF-7 and EMT-6 tumors decreased with distance from blood vessels, similar to the distributions of (i) γ-H2AX at 10 minutes, (ii) cleaved caspase-3 or -6, and (iii) change in Ki67 at 24 hours following treatment. The distribution of these biomarkers following treatment with docetaxel also decreased with increasing distance from tumor blood vessels. Activation of γ-H2AX occurred within 1 hour after exposure to several drugs in culture. Multiple anticancer drugs show a decrease in activity with increasing distance from tumor blood vessels; poor drug distribution is an important cause of drug resistance. The above biomarkers may be used in designing strategies to overcome therapeutic resistance by modifying or complementing the limited spatial distribution of drug activity in solid tumors.

The influence of P-glycoprotein expression and its inhibitors on the distribution of doxorubicin in breast tumors.

BACKGROUND: Anti-cancer drugs access solid tumors via blood vessels, and must penetrate tumor tissue to reach all cancer cells. Previous studies have demonstrated steep gradients of decreasing doxorubicin fluorescence with increasing distance from blood vessels, such that many tumor cells are not exposed to drug. Studies using multilayered cell cultures show that increased P-glycoprotein (PgP) is associated with better penetration of doxorubicin, while PgP inhibitors decrease drug penetration in tumor tissue. Here we evaluate the effect of PgP expression on doxorubicin distribution in vivo. METHODS: Mice bearing tumor sublines with either high or low expression of PgP were treated with doxorubicin, with or without pre-treatment with the PgP inhibitors verapamil or PSC 833. The distribution of doxorubicin in relation to tumor blood vessels was quantified using immunofluorescence. RESULTS: Our results indicate greater uptake of doxorubicin by cells near blood vessels in wild type as compared to PgP-overexpressing tumors, and pre-treatment with verapamil or PSC 833 increased uptake in PgP-overexpressing tumors. However, there were steeper gradients of decreasing doxorubicin fluorescence in wild-type tumors compared to PgP overexpressing tumors, and treatment of PgP overexpressing tumors with PgP inhibitors led to steeper gradients and greater heterogeneity in the distribution of doxorubicin. CONCLUSION: PgP inhibitors increase uptake of doxorubicin in cells close to blood vessels, have little effect on drug uptake into cells at intermediate distances, and might have a paradoxical effect to decrease doxorubicin uptake into distal cells. This effect probably contributes to the limited success of PgP inhibitors in clinical trials.