Comparative Analysis of miRNA and EMT Markers in Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is the fourth most commonly diagnosed malignant condition in the world. Micro RNAs (miRNAs) as well as epithelial to mesenchymal transition (EMT) play an important role in the pathogenesis of CRC. We performed a comparative analysis of the expression of selected miRNA genes and EMT markers in bioptic samples from patients (n = 45) with primary CRC or metastatic (m)CRC to the regional lymph node using reverse transcription-quantitative PCR and IHC staining. Results: Out of all miRNA analyzed, the miR-17 expression was most significantly different and associated with lower risk of CRC spread to the lymph node. In addition, significant relationships were found between the tumor side localization and several miRNAs expressions (miR-9, miR-29b, miR-19a, miR-19b, miR-21, miR-106a, miR-20a and miR-17). In addition, of the examined EMT markers, only VEGFA expression correlated with tumor progression (tumor grade G2). In the examined set of patient samples and their matched healthy tissue, several specific molecular markers (miRNAs associated with EMT and tumor progression) were identified with a promising prognostic potential. Their further examination in larger patient cohorts is planned to validate the present data.

Inositol hexaphosphate limits the migration and the invasiveness of colorectal carcinoma cells in vitro.

Inositol hexaphosphate (IP6), also known as phytic acid, has been shown to exhibit anticancer effects in a number of preclinical tumor models. IP6 decreases proliferation by arresting cells in the G0/G1 phase, inhibits iron-mediated oxidative reactions, enhances differentiation and stimulates apoptosis. The present study attempted to characterize the effect of IP6 on the migration and adhesion of colon cancer SW620 cells. IP6 was assessed at concentrations of 0.2 and 1 mM during 12, 24 and 48 h of exposure. Migration ability was measured with the real-time xCELLigence Real-Time Cell Analyzer Dual Purpose system. The expression of mRNA and proteins involved in migration and cancer progression [epithelial cell adhesion molecule, intercellular adhesion molecule-1, ß-catenin, N-cadherin, E-cadherin, matrix metalloproteinase (MMP)-2 and MMP-9] was determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis. The changes in the expression and subcellular localization of E-cadherin were determined by indirect immunofluorescence. IP6 induced a decrease in the migration ability of the tested SW620 cell line. IP6-treated cells also showed decreased expression of N-cadherin, increased levels of E-cadherin and decreased expression of MMP-2 and MMP-9. These results indicated that IP6 has potential to modulate the migration ability and expression of markers associated with invasion in SW620 cells; however, further analysis is necessary to obtain a detailed understanding of the mechanism of action.

Flubendazole and mebendazole impair migration and epithelial to mesenchymal transition in oral cell lines.

Benzimidazole anthelmintics flubendazole and mebendazole are microtubule-targeting drugs that showed considerable anti-cancer activity in different preclinical models. In this study, the effects of flubendazole and mebendazole on proliferation, migration and cadherin switching were studied in a panel of oral cell lines in vitro. Both compounds reduced the viability of the PE/CA-PJ15 and H376 oral squamous carcinoma cells and of the premalignant oral keratinocytes DOK with the IC50 values in the range of 0.19-0.26 µM. Normal oral keratinocytes and normal gingival fibroblasts were less sensitive to the treatment. Flubendazole and mebendazole also reduced the migration of the PE/CA-PJ15 cell in concentrations that had no anti-migratory effects on the normal gingival fibroblasts. Levels of the focal adhesion kinase FAK, Rho-A and Rac1 GTPases and the Rho guanine nucleotide exchange factor GEF-H1 were decreased in both PE/CA-PJ15 cells and gingival fibroblasts following treatment. Both drugs also interfered with cadherin switching in the model of TGF-ß-induced epithelial to mesenchymal transition (EMT) in the DOK cell line. Levels of N-cadherin were reduced in the TGF-ß induced cells co-treated with flubendazol and mebendazole in very low concentration (50 nM). These results suggest direct effects of both benzimidazoles on selected processes of EMT in oral cell lines such as cadherin switching as well as cellular migration.

The metabolism of flubendazole in human liver and cancer cell lines.

Flubendazole (FLU), a benzimidazole anthelmintic drug widely used in veterinary medicine, has been approved for the treatment of gut-residing nematodes in humans. In addition, FLU is now considered a promising anti-cancer agent. Despite this, information about biotransformation of this compound in human is lacking. Moreover, there is no information regarding whether cancer cells are able to metabolize FLU in order to deactivate it. For these reasons, the present study was designed to identify all metabolites of Phase I and Phase II of FLU in human liver and in various cancer cells using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. Precision-cut human liver slices and 9 cell lines of different origin (breast, colon, oral cavity) were used as in vitro model systems. Our study showed that FLU with a reduced carbonyl group (FLUR) is the only FLU metabolite formed in the human liver. All human cancer cell lines were able to form FLUR. In addition, methylated FLUR was detected in breast cells MCF7 and intestinal SW480 cells. The accumulation of FLU and its reduction to FLUR markedly differed among cells. The extent of FLU reduction was in a good correlation with the detected expression level of carbonyl reductase 1. In most cases, FLU entered in a higher amount and was reduced to a lesser extent in proliferating (metastatic) cells than in differentiated (non-cancerous, non-metastatic) ones. These results support the promising potential of FLU in anti-cancer therapy.

The Effect of Flubendazole on Adhesion and Migration in SW480 and SW620 Colon Cancer Cells.

BACKGROUND: Colon cancer is the most common type of gastrointestinal cancer. Despite advances during the last two decades, the efficacy of colorectal cancer treatment is still insufficient and new anticancer agents are necessary. METHODS: In our study, colon cancer cells derived from a primary tumor (SW480) and lymph node metastasis (SW620) from the same patient were used and compared. The effect of flubendazole (FLU) on cell adhesion and migration was monitored using the x-CELLigence Real-Time Cell Analysis system. Expressions of molecules involved in adhesion and migration were analyzed using RT-PCR and western blot. Furthermore, RNA silencing of nuclear factor-κB in SW620 cells was used to determine the involvement of the NF-κB p65 regulation pathway in FLU action. RESULTS: FLU significantly suppressed the adhesion of SW480 cells and reduced the expression of adhesion markers (ICAM-1, αE-catenin; ß-catenin; integrin α5 and ß1). Moreover, a significant anti-migratory potential of FLU was manifested in the SW620 cells. In addition, FLU suppressed the phosphorylation of NF-κB p65 and potentiated the suppression of several metastatic markers (ICAM-1, EpCAM, integrin α5, ß1, α-tubulin) caused by NF-κB p65 silencing. CONCLUSION: FLU has a significant anti-migratory effect in intestinal cancer cell SW480 and its lymph node metastatic cells SW620. FLU decreases the expression of some proteins involved in metastatic processes and inhibits activation of NF-κB p65.

Derivation and basic characterization of colorectal carcinoma primary cell lines.

BACKGROUND: Colorectal carcinoma is one of the most common malignancies in western countries. Among different approaches to its research, primary cancer cell lines can play an important role. AIM: The main purposes of this study were: 1) to establish an effective and reproducible method of colorectal cancer cell isolation and cultivation from primary tumours and lymph node metastases and 2) to elucidate the biological features of the tumours favouring successful cell cultivation. MATERIALS AND METHODS: The tumour cells were obtained from colectomy specimens. Primary tumour and lymph node metastasis tissue was used for establishing the tissue cultures. Colectomy samples were further processed for routine histopathological assessment: tumour grade, stage, angioinvasion and perineural spread were evaluated. Features of tissue culture cells were assessed using phase contrast microscopy and immune-histochemical techniques. WST-1 assay and X-CELLigence real time analysis were carried out for viability and proliferation testing before and after treatment with irinotecan and oxaliplatin. Molecular features of the tumour including K-RAS/B-RAF/N-RAS mutations were tested using allele-specific PCR. Results of the cultivation process were compared to the histopathological and molecular features of the tumours. RESULTS: In total, we obtained 33 samples from the primary site of tumours and 20 samples from lymph node metastases; in total, 27 cell lines were successfully isolated. Morphologic features characteristic of tumour cells in primary cell lines and epithelial differentiation (positive for AE1/AE3 cytokeratin) were evaluated. Higher tumour stage, angioinvasion and presence of perineural spread in primary tumour correlated positively with successful cell isolation from lymph node metastasis. All samples tested were NRAS wild-type. No correlation was found between molecular phenotype and the cell culture features. A higher proliferation potential was observed in the primary tumour cells, whereas higher sensitivity to irinotecan was found in the lymph node metastatic cells. CONCLUSIONS: Using mechanical dissociation, we successfully derived and cultivated CRC cells from primary tumours and lymph node metastases with success rate 3 % and 70% respectively. Primary tumour features favouring successful establishment of cell cultures were identified.

The effects of ß-caryophyllene oxide and trans-nerolidol on the efficacy of doxorubicin in breast cancer cells and breast tumor-bearing mice.

BACKGROUND: One approach to improve effect of chemotherapy is combination of classical cytostatic drugs with natural compounds, e. g. sesquiterpenes. In our previous study, sesquiterpenes ß-caryophyllene oxide (CAO) and trans-nerolidol (NER) improved the anti-proliferative effect of doxorubicin (DOX) in intestinal cancer cell lines. PURPOSE: The present study was designed to evaluate the effect of CAO and NER on DOX efficacy, focusing on cell proliferation, migration, apoptosis and DOX accumulation in breast cancer cells MDA-MB-231 and MCF7 in vitro and in mice bearing solid Ehrlich tumors (EST) in vivo. METHODS: The impact of cytotoxic effect was assessed by the neutral red uptake test. The ability to migrate was tested using real-time measurement in x-CELLigence system. Expressions of molecules were examined using western blot analysis. The accumulation of DOX inside the cells using time lapse microscopy was observed. The mice with inoculated EST cells were treated repeatedly with DOX and DOX+CAO or DOX+NER and the growth of tumors were monitored. DOX concentrations in plasma and tumor were assayed using HPLC. RESULTS: In MDA-MB-231, combination of DOX with CAO enhanced anti-proliferative effect and acted strongly synergistic. NER increased accumulation of DOX inside the cells; moreover combination DOX with NER suppressed migration ability in vitro. In vivo, apoptosis was activated especially in group treated with DOX and CAO. However, none of tested sesquiterpenes was able to improve DOX accumulation in tumors and DOX-mediated inhibition of tumor growth. CONCLUSION: In conclusion, sesquiterpenes CAO and NER increased the efficacy of DOX in breast cancer cells in vitro, but did not improve its effect in vivo, in Ehrlich solid tumor bearing mice.

Flubendazole induces mitotic catastrophe and senescence in colon cancer cells in vitro.

OBJECTIVES: Flubendazole (FLU), a member of benzimidazole family of anthelmintic drugs, is able to inhibit proliferation of various cancer cells. The aim of present study was to elucidate the mechanisms of antiproliferative effect of FLU on colorectal cancer cells in vitro. METHODS: The effect of FLU on proliferation, microtubular network, DNA content, caspase activation and senescence induction was studied in SW480 and SW620 cell lines. KEY FINDINGS: Flubendazole significantly affected cell proliferation in a pattern typical for mitotic inhibitor. This was accompanied by decrease in cyclin D1 levels, increase in cyclin B1 levels, activation of caspase 2 and caspase 3/7 and PARP cleavage. Morphological observations revealed disruption of microtubular network, irregular mitotic spindles, formation of giant multinucleated cells and increase in nuclear area and DNA content. In SW620 cell line, 37.5% giant multinucleated cells induced by FLU treatment showed positivity for SA-ß-galactosidase staining. Cell lines were able to recover from the treatment and this process was faster in SW480 cells. CONCLUSION: Flubendazole in low concentration temporarily inhibits cell proliferation and induces mitotic catastrophe and premature senescence in human colon cancer cells in vitro.

Contribution of in vitro comparison of colorectal carcinoma cells from primary and metastatic lesions to elucidation of mechanisms of tumor progression and response to anticancer therapy.

Colorectal cancer has been a leading cause of cancer-related morbidity and mortality. For the research and individualization of therapy, primary cell lines of the colorectal cancer appear to be still an invaluable tool. We evaluated the differences in metastatic potential between four isolated primary colon cancer cells and cells derived from their lymph node metastasis. These results were compared with correspond immortalized cells-SW480 and SW620, respectively. The ability to migrate was tested using real-time measurement in xCELLigence system. Expressions of molecules involved in adhesion and invasion processes were examined using RT-PCR and western blot analysis. Furthermore, impact of cytotoxic effect of selected chemotherapeutics (irinotecan, oxaliplatin) and biological therapy (bevacizumab, cetuximab, panitumumab) was assessed by the WST assay. As expected, cell lines derived from lymph node migrated more aggressively and higher expression of adhesion molecules ICAM-1, EpCAM, and N-cadherin was detected. The expression of MMP-2 and -9 was elevated, on the other hand, in cell lines derived from primary tumor cancer cells as well as the expression of miR-21, miR-29a, and miR-200a. The most pronounced cytotoxic effect has been recorded with oxaliplatin and irinotecan (IC50 = 48.23 resp. 0.11 µg/ml), especially in cells originating from lymph node metastases. In total, comparison of isolated cell lines and immortalized cell lines has shown many similarities, as well as several differences. Adhesion/invasion molecules and several miRNAs, which play an important role in tumor development and the invasive and migratory behavior, could be a useful therapeutic target in malignant colorectal cancer.

Essential Oil from Myrica rubra Leaves Potentiated Antiproliferative and Prooxidative Effect of Doxorubicin and its Accumulation in Intestinal Cancer Cells.

Essential oil from the leaves of Myrica rubra, a subtropical Asian fruit tree traditionally used in folk medicines, has a significant antiproliferative effect in several intestinal cancer cell lines. Doxorubicin belongs to the most important cytostatics used in cancer therapy. The present study was designed to evaluate the effects of defined essential oil from M. rubra leaves on efficacy, prooxidative effect, and accumulation of doxorubicin in cancer cell lines and in non-cancerous cells. For this purpose, intestinal adenocarcinoma CaCo2 cells were used. Human fibroblasts (periodontal ligament) and a primary culture of rat hepatocytes served as models of non-cancerous cells. The results showed that the sole essential oil from M. rubra has a strong prooxidative effect in cancer cells while it acts as a mild antioxidant in hepatocytes. Combined with doxorubicin, the essential oil enhanced the antiproliferative and prooxidative effects of doxorubicin in cancer cells. At higher concentrations, synergism of doxorubicin and essential oil from M. rubra was proved. In non-cancerous cells, the essential oil did not affect the toxicity of doxorubicin and the doxorubicin-mediated reactive oxygen species formation. The essential oil increased the intracellular concentration of doxorubicin and enhanced selectively the doxorubicin accumulation in nuclei of cancer cells. Taken together, essential oil from M. rubra leaves could be able to improve the doxorubicin efficacy in cancer cells due to an increased reactive oxygen species production, and the doxorubicin accumulation in nuclei of cancer cells.

Influence of diet supplementation with green tea extract on drug-metabolizing enzymes in a mouse model of monosodium glutamate-induced obesity.

PURPOSE: Consumption of dietary supplements with green tea extract (GTE) is popular for weight management, but it may be accompanied by various side effects, including interactions with drugs. The aim of the present in vivo study was to evaluate the effect of defined GTE (Polyphenon 60) in three dosage schemes on insulin, leptin and drug-metabolizing enzymes in obese mice. METHODS: Experimental obesity was induced by repeated s.c. application of monosodium glutamate to newborn mice. Green tea extract was administered in three dosage schemes in chow diet. The plasmatic levels of insulin and leptin were assayed using enzyme-linked immunosorbent assay. Enzyme activities and mRNA expressions of drug-metabolizing enzymes (totally 13) were analyzed in liver and small intestine using spectrophotometric and HPLC assays and RT-PCR, respectively. RESULTS: GTE-treatment decreased insulin and leptin levels. Eleven enzymes were significantly affected by GTE-treatment. Long-term administration of 0.01% GTE caused increase in the activity and mRNA level of cytochrome P450 3A4 (CYP3A4) ortholog in the liver as well as in the small intestine. Interestingly, short-term overdose by GTE (0.1%) had more pronounced effects on enzyme activities and mRNA expressions than long-term overdose. CONCLUSIONS: GTE-mediated induction of CYP3A4 ortholog, the main drug-metabolizing enzyme, could result in decreased efficacy of simultaneously or subsequently administered drug in obese individuals.

The Influence of Sesquiterpenes from Myrica rubra on the Antiproliferative and Pro-Oxidative Effects of Doxorubicin and Its Accumulation in Cancer Cells.

The sesquiterpenes ß-caryophyllene, ß-caryophyllene oxide (CAO), α-humulene (HUM), trans-nerolidol (NER), and valencene (VAL) are substantial components of the essential oil from Myrica rubra leaves which has exhibited significant antiproliferative effects in several intestinal cancer cell lines, with CaCo-2 cells being the most sensitive. The present study was designed to evaluate the effects of these sesquiterpenes on the efficacy and toxicity of the anticancer drug doxorubicin (DOX) in CaCo-2 cancer cells and in primary culture of rat hepatocytes. Our results showed that HUM, NER, VAL and CAO inhibited proliferation of CaCo-2 cancer cells but they did not affect the viability of hepatocytes. CAO, NER and VAL synergistically potentiated the efficacy of DOX in cancer cells killing. All sesquiterpenes exhibited the ability to selectively increase DOX accumulation in cancer cells and did not affect DOX concentration in hepatocytes. Additionally, CAO and VAL were able to increase the pro-oxidative effect of DOX in CaCo-2 cells. Moreover, CAO mildly ameliorated DOX toxicity in hepatocytes. Based on all results, CAO seems to be the most promising compound for further testing.

Potential anti-cancer drugs commonly used for other indications.

An increasing resistance of mammalian tumor cells to chemotherapy along with the severe side effects of commonly used cytostatics has raised the urgency in the search for new anti-cancer agents. Several drugs originally approved for indications other than cancer treatment have recently been found to have a cytostatic effect on cancer cells. These drugs could be expediently repurposed as anti-cancer agents, since they have already been tested for toxicity in humans and animals. The groups of newly recognized potential cytostatics discussed in this review include benzimidazole anthelmintics (albendazole, mebendazole, flubendazole), anti-hypertensive drugs (doxazosin, propranolol), psychopharmaceuticals (chlorpromazine, clomipramine) and antidiabetic drugs (metformin, pioglitazone). All these drugs have a definite potential to be used especially in combinations with other cytostatics; the chemotherapy targeting of multiple sites now represents a promising approach in cancer treatment. The present review summarizes recent information about the anti-cancer effects of selected drugs commonly used for other medical indications. Our aim is not to collect all the reported results, but to present an overview of various possibilities. Advantages, disadvantages and further perspectives regarding individual drugs are discussed and evaluated.

Antioxidant, pro-oxidant and other biological activities of sesquiterpenes.

Sesquiterpenes, 15-carbon compounds formed from 3 isoprenoid units, are secondary metabolites produced mainly in higher plants but also in fungi and invertebrates. Sesquiterpenes occur in human food, but they are principally taken as components of many folk medicines and dietary supplements. Moreover, sesquiterpenes could become a rich reservoir of candidate compounds for drug discovery as several sesquiterpenes and their derivatives possess interesting biological activities. Recent efforts in the research and development of new drugs derived from natural products have led to the identification of a variety of sesquiterpenes that possess promising anti-inflammatory, antiparasitic and anti-carcinogenic activities. On the other hand, some sesquiterpenes can cause serious toxicity and other adverse effects. Therefore, more and more attention has been paid to the investigation of the mechanisms of biological activities of sesquiterpenes in vitro as well as in vivo. The data collected in this review show that many of sesquiterpenes biological activities are based on antioxidant or pro-oxidant actions of sesquiterpenes. Structure, concentration, metabolism as well as type of cells determine if sesquiterpene acts as anti-oxidant or pro-oxidant. Therefore, detailed research of sesquiterpenes is very important for evaluation of their efficacy and for their safe use.

Effect of selected catechins on doxorubicin antiproliferative efficacy and hepatotoxicity in vitro.

Catechins may influence both desirable and undesirable effects of many drugs. In this study, the in vitro effect of (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate, and (-)-epigallocatechin gallate (EGCG) on the efficacy of anticancer drug doxorubicin (DOX) was studied in HCT-8 cancer cells. Rat hepatocytes were used to study the influence of EGCG on DOX hepatotoxicity. Cell proliferation and viability were studied by 3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyl tetrazolium bromide and neutral red uptake test assays. Formation of reactive oxygen species (ROS) was determined using the dichlorofluorescein assay. All of the studied catechins (1-25 µmol L(-1)) had no effect on the proliferation of intestinal cancer cells and did not affect the antiproliferative effect of DOX (1-8 µmol L(-1)) in these cells. Moreover, EGCG at 25 µmol L(-1) increased the viability of isolated hepatocytes and significantly protected these cells against DOX-induced toxicity and ROS production. Consumption of EGCG during DOX therapy seems to be safe and beneficial, since EGCG does not decrease DOX anticancer efficacy and could ameliorate DOX hepatotoxicity.

Reference genes for real-time PCR quantification of messenger RNAs and microRNAs in mouse model of obesity.

Obesity and metabolic syndrome is increasing health problem worldwide. Among other ways, nutritional intervention using phytochemicals is important method for treatment and prevention of this disease. Recent studies have shown that certain phytochemicals could alter the expression of specific genes and microRNAs (miRNAs) that play a fundamental role in the pathogenesis of obesity. For study of the obesity and its treatment, monosodium glutamate (MSG)-injected mice with developed central obesity, insulin resistance and liver lipid accumulation are frequently used animal models. To understand the mechanism of phytochemicals action in obese animals, the study of selected genes expression together with miRNA quantification is extremely important. For this purpose, real-time quantitative PCR is a sensitive and reproducible method, but it depends on proper normalization entirely. The aim of present study was to identify the appropriate reference genes for mRNA and miRNA quantification in MSG mice treated with green tea catechins, potential anti-obesity phytochemicals. Two sets of reference genes were tested: first set contained seven commonly used genes for normalization of messenger RNA, the second set of candidate reference genes included ten small RNAs for normalization of miRNA. The expression stability of these reference genes were tested upon treatment of mice with catechins using geNorm, NormFinder and BestKeeper algorithms. Selected normalizers for mRNA quantification were tested and validated on expression of NAD(P)H: quinone oxidoreductase, biotransformation enzyme known to be modified by catechins. The effect of selected normalizers for miRNA quantification was tested on two obesity- and diabetes- related miRNAs, miR-221 and miR-29b, respectively. Finally, the combinations of B2M/18S/HPRT1 and miR-16/sno234 were validated as optimal reference genes for mRNA and miRNA quantification in liver and 18S/RPlP0/HPRT1 and sno234/miR-186 in small intestine of MSG mice. These reference genes will be used for mRNA and miRNA normalization in further study of green tea catechins action in obese mice.

Antiproliferative effect of benzimidazole anthelmintics albendazole, ricobendazole, and flubendazole in intestinal cancer cell lines.

This study aimed to test the antiproliferative effect of three benzimidazole anthelmintics in intestinal cancer cells and to investigate whether these drugs, which inhibit tubulin polymerization, can potentiate the efficacy of the microtubule-stabilizing drug paclitaxel (PTX). Four intestinal cancer cell lines, SW480, SW620, HCT8, and Caco2, with different origins and growth characteristics were used. The antiproliferative effect of albendazole (ABZ), ricobendazole (RBZ), flubendazole (FLU), and their combinations with PTX was tested using three different end-point viability assays, cell cycle distribution analysis, and the x-CELLigence System for real-time cell analysis. ABZ and FLU inhibited cell proliferation significantly in a concentration-dependent and time-dependent manner through cell arrest in the G2/M phase. RBZ was not effective at any concentration tested. The cell lines differed in sensitivity to FLU and ABZ, with HCT8 being the most sensitive, showing IC50 values for ABZ and FLU that reached 0.3 and 0.9 µmol/l, respectively. Combinations of PTX+ABZ and PTX+FLU decreased cell viability more effectively when compared with treatment with individual drugs alone. The anthelmintic benzimidazole drugs ABZ and FLU show a significant cytostatic effect and potentiate the efficacy of PTX in intestinal cancer cells.

In vivo effect of oracin on doxorubicin reduction, biodistribution and efficacy in Ehrlich tumor bearing mice.

BACKGROUND: The limitation of carbonyl reduction represents one possible way to increase the effectiveness of anthracycline doxorubicin (DOX) in cancer cells and decrease its toxicity in normal cells. In vitro, isoquinoline derivative oracin (ORC) inhibited DOX reduction and increased the antiproliferative effect of DOX in MCF-7 breast cancer cells. Moreover, ORC significantly decreases DOX toxicity in non-cancerous MCF-10A breast cells and in hepatocytes. The present study was designed to test in mice the in vivo effect of ORC on plasma and tissue concentrations of DOX and its main metabolite DOXOL. The effect of ORC on DOX efficacy in mice bearing solid Ehrlich tumors (EST) was also studied. METHODS: DOX and DOX + ORC combinations were iv administered to healthy mice. Blood samples, livers and hearts were collected during the following 48 h. DOX and DOXOL concentrations were assayed using HPLC. The mice with inoculated EST cells were treated repeatedly iv with DOX and DOX + ORC combinations, and the growth of tumors was monitored. RESULTS: ORC in combination with DOX significantly decreased DOXOL plasma concentrations during four hours after administration, but this significantly affected neither DOX plasma concentrations nor DOX or DOXOL concentrations in the liver and heart at any of intervals tested. In EST bearing mice, ORC did not significantly affect DOX efficacy on tumor growth. However, EST was shown to be an improper model for the testing of ORC efficacy in vivo, as ORC did not inhibit DOXOL formation in EST. CONCLUSIONS: In vivo, ORC was able to retard DOXOL formation but was not able to improve DOX efficacy in EST-bearing mice.

Possibilities to increase the effectiveness of doxorubicin in cancer cells killing.

Anthracycline antibiotic doxorubicin (DOX) belongs among the most important antineoplastics used in cancer therapy. Unfortunately, its cytostatic effect in therapeutic doses is frequently insufficient; but the use of higher DOX doses is limited by the development of systemic toxicity, especially cardiotoxicity. Therefore, a searching for some possibilities of how to increase DOX efficacy in cancer cells, and minimizing associated toxicities to noncancerous tissues, is in the forefront of scientific research. Many approaches are based on altered DOX metabolism. The classical strategies include an enhancing of DOX uptake by cancer cells and/or an activation of DOX prodrug within cancer cells via liposomal encapsulation or conjugation with antibodies, peptides, or synthetic polymers. The diminishing of DOX deactivation, restriction of DOX efflux from cancer cells, decreased antioxidant defense of cancer cells, changes in cell cycle, or modulation of signaling pathways represent newer approaches in increasing DOX toxicity in tumors. Each way has certain advantages and limitations. The aim of this review was not to collect all reported results, but to bring an overview of various approaches and a summary of their principles. Possible advantages, disadvantages, and further perspectives are discussed and evaluated.

Paclitaxel conjugation with the analog of the gonadotropin-releasing hormone as a targeting moiety.

A new targeted conjugates in which paclitaxel was used as a cytostatic compound and an analog of the gonadotropin-releasing hormone (GnRH) as a targeting moiety were synthesized. The molecule of the peptide hormone GnRH was modified to allow its connection to paclitaxel via spacer. The conjugates were prepared as prodrugs using 2'-hydroxyl group of paclitaxel. 4-Maleimidobutyric acid and chloroacetic acid served as spacers. The structures of the prepared derivatives were analysed by NMR and HR-MS. The conjugates MP264 and MP265 were chosen and their antiproliferative effect was tested in the breast cancer cell line MCF-7 using the MTT test of cell viability and neutral red uptake test. In MCF-7 cells, conjugate MP265 showed higher antiproliferative effect than paclitaxel alone. Receptor saturation tests showed that the unconjugated peptide analog of GnRH decreased efficacy of conjugate MP265 in concentration- and time-dependent manner. In conclusion, the paclitaxel conjugate with the analog of GnRH exhibited targeted antiproliferative effect for which its further testing will be implemented.