Synthesis, Pharmacokinetic Profile, Anticancer Activity and Toxicity of the New Amides of Betulonic Acid-In Silico and In Vitro Study.

Betulonic acid (B(O)A) is a pentacyclic lupane-type triterpenoid that widely exists in plants. There are scientific reports indicating anticancer activity of B(O)A, as well as the amides and esters of this triterpenoid. In the first step of the study, the synthesis of novel amide derivatives of B(O)A containing an acetylenic moiety was developed. Subsequently, the medium-soluble compounds (EB171 and EB173) and the parent compound, i.e., B(O)A, were investigated for potential cytotoxic activity against breast cancer (MCF-7 and MDA-MB-231) and melanoma (C32, COLO 829 and A375) cell lines, as well as normal human fibroblasts. Screening analysis using the WST-1 test was applied. Moreover, the lipophilicity and ADME parameters of the obtained derivatives were determined using experimental and in silico methods. The toxicity assay using zebrafish embryos and larvae was also performed. The study showed that the compound EB171 exhibited a significant cytotoxic effect on cancer cell lines: MCF-7, A-375 and COLO 829, while it did not affect the survival of normal cells. Moreover, studies on embryos and larvae showed no toxicity of EB171 in an animal model. Compared to EB171, the compound EB173 had a weaker effect on all tested cancer cell lines and produced less desirable effects against normal cells. The results of the WST-1 assay obtained for B(O)A revealed its strong cytotoxic activity on the examined cancer cell lines, but also on normal cells. In conclusion, this article describes new derivatives of betulonic acid-from synthesis to biological properties. The results allowed to indicate a promising direction for the functionalization of B(O)A to obtain derivatives with selective anticancer activity and low toxicity.

Design, Synthesis and Biological Evaluation of Quinoline-8-Sulfonamides as Inhibitors of the Tumor Cell-Specific M2 Isoform of Pyruvate Kinase: Preliminary Study.

Cancer cells need to carefully regulate their metabolism to keep them growing and dividing under the influence of different nutrients and oxygen levels. Muscle isoform 2 of pyruvate kinase (PKM2) is a key glycolytic enzyme involved in the generation of ATP and is critical for cancer metabolism. PKM2 is expressed in many human tumors and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various modulators regulate PKM2, shifting it between highly active and less active states. In the presented work, a series of 8-quinolinesulfonamide derivatives of PKM2 modulators were designed using molecular docking and molecular dynamics techniques. New compounds were synthesized using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Compound 9a was identified in in silico studies as a potent modulator of muscle isoform 2 of pyruvate kinase. The results obtained from in vitro experiments confirmed the ability of compound 9a to reduce the intracellular pyruvate level in A549 lung cancer cells with simultaneous impact on cancer cell viability and cell-cycle phase distribution. Moreover, compound 9a exhibited more cytotoxicity on cancer cells than normal cells, pointing to high selectivity in the mode of action. These findings indicate that the introduction of another quinolinyl fragment to the modulator molecule may have a significant impact on pyruvate levels in cancer cells and provides further directions for future research to find novel analogs suitable for clinical applications in cancer treatment.

The Anticancer Potential of Doxycycline and Minocycline-A Comparative Study on Amelanotic Melanoma Cell Lines.

Malignant melanoma is still a serious medical problem. Relatively high mortality, a still-growing number of newly diagnosed cases, and insufficiently effective methods of therapy necessitate melanoma research. Tetracyclines are compounds with pleiotropic pharmacological properties. Previously published studies on melanotic melanoma cells ascertained that minocycline and doxycycline exerted an anti-melanoma effect. The purpose of the study was to assess the anti-melanoma potential and mechanisms of action of minocycline and doxycycline using A375 and C32 human amelanotic melanoma cell lines. The obtained results indicate that the tested drugs inhibited proliferation, decreased cell viability, and induced apoptosis in amelanotic melanoma cells. The treatment caused changes in the cell cycle profile and decreased the intracellular level of reduced thiols and mitochondrial membrane potential. The exposure of A375 and C32 cells to minocycline and doxycycline triggered the release of cytochrome c and activated initiator and effector caspases. The anti-melanoma effect of analyzed drugs appeared to be related to the up-regulation of ERK1/2 and MITF. Moreover, it was noticed that minocycline and doxycycline increased the level of LC3A/B, an autophagy marker, in A375 cells. In summary, the study showed the pleiotropic anti-cancer action of minocycline and doxycycline against amelanotic melanoma cells. Considering all results, it could be concluded that doxycycline was a more potent drug than minocycline.

Chemosensitization of U-87 MG Glioblastoma Cells by Neobavaisoflavone towards Doxorubicin and Etoposide.

Glioblastoma (GB) is the most common type of glioma, which is distinguished by high mortality. Due to the rapid progression of the tumor and drug resistance, the treatment is often ineffective. The development of novel therapies in a big part concerns the application of anti-cancer agents already used in clinical practice, unfortunately often with limited effects. This could be overcome through the use of compounds that possess chemosensitizing properties. In our previous work, it has been shown that neobavaisoflavone (NBIF) enhances the in vitro activity of doxorubicin in GB cells. The aim of this study was a further investigation of the possible chemosensitizing effects of this isoflavone. The experimental panel involving image cytometry techniques, such as count assay, examination of mitochondrial membrane potential, Annexin V assay, and cell cycle analysis, was performed in human glioblastoma U-87 MG cells and normal human astrocytes (NHA) treated with NBIF, doxorubicin, etoposide, and their mixes with NBIF. NBIF in co-treatment with etoposide or doxorubicin caused an increase in the population of apoptotic cells and prompted alterations in the cell cycle. NBIF enhances the pro-apoptotic activity of etoposide and doxorubicin in U-87 MG cells, which could be a sign of the chemosensitizing properties of the isoflavone.

Molecular and Biochemical Basis of Minocycline-Induced Hyperpigmentation-The Study on Normal Human Melanocytes Exposed to UVA and UVB Radiation.

Minocycline is a drug which induces skin hyperpigmentation. Its frequency reaches up to 50% of treated patients. The adverse effect diminishes the great therapeutic potential of minocycline, including antibacterial, neuroprotective, anti-inflammatory and anti-cancer actions. It is supposed that an elevated melanin level and drug accumulation in melanin-containing cells are related to skin hyperpigmentation. This study aimed to evaluate molecular and biochemical mechanism of minocycline-induced hyperpigmentation in human normal melanocytes, as well as the contribution of UV radiation to this side effect. The experiments involved the evaluation of cyto- and phototoxic potential of the drug using cell imaging with light and confocal microscopes as well as biochemical and molecular analysis of melanogenesis. We showed that minocycline induced melanin synthesis in epidermal melanocytes. The action was intensified by UV irradiation, especially with the UVB spectrum. Minocycline stimulated the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase (TYR) gene. Higher levels of melanin and increased activity of tyrosinase were also observed in treated cells. Moreover, minocycline triggered the supranuclear accumulation of tyrosinase, similar to UV radiation. The decreased level of premelanosome protein PMEL17 observed in all minocycline-treated cultures suggests disorder of the formation, maturation or distribution of melanosomes. The study revealed that minocycline itself was able to enhance melanin synthesis. The action was intensified by irradiation, especially with the UVB spectrum. Demonstrated results confirmed the potential role of melanin and UV radiation minocycline-induced skin hyperpigmentation.

Ketoprofen Combined with UVA Irradiation Exerts Higher Selectivity in the Mode of Action against Melanotic Melanoma Cells than against Normal Human Melanocytes.

Malignant melanoma is responsible for the majority of skin cancer-related deaths. The methods of cancer treatment include surgical removal, chemotherapy, immunotherapy, and targeted therapy. However, neither of these methods gives satisfactory results. Therefore, the development of new anticancer therapeutic strategies is very important and may extend the life span of people suffering from melanoma. The aim of this study was to examine the effect of ketoprofen (KTP) and UVA radiation (UVAR) therapy on cell proliferation, apoptosis, and cell cycle distribution in both melanotic melanoma cells (COLO829) and human melanocytes (HEMn-DP) in relation to its supportive effect in the treatment of melanoma. The therapy combining the use of pre-incubation with KTP and UVAR causes a significant increase in the anti-proliferative properties of ketoprofen towards melanoma cells and the co-exposure of melanotic melanoma cells induced apoptosis shown as the mitochondrial membrane breakdown, cell-cycle deregulation, and DNA fragmentation. Moreover, co-treatment led to GSH depletion showing its pro-apoptotic effect dependent on ROS overproduction. The treatment did not show a significant effect on normal cells-melanocytes-which indicates its high selectivity. The results suggest a possible benefit from the use of the ketoprofen and ultraviolet A irradiation as a new concept of melanotic melanoma therapy.

Minocycline Impact on Redox Homeostasis of Normal Human Melanocytes HEMn-LP Exposed to UVA Radiation and Hydrogen Peroxide.

Minocycline is a semisynthetic tetracycline antibiotic. In addition to its antibacterial activity, minocycline shows many non-antibiotic, beneficial effects, including antioxidative action. The property is responsible, e.g., for anti-inflammatory, neuroprotective, and cardioprotective effects of the drug. However, long-term pharmacotherapy with minocycline may lead to hyperpigmentation of the skin. The reasons for the pigmentation disorders include the deposition of the drug and its metabolites in melanin-containing cells and the stimulation of melanogenesis. The adverse drug reaction raises a question about the influence of the drug on melanocyte homeostasis. The study aimed to assess the effect of minocycline on redox balance in human normal melanocytes HEMn-LP exposed to hydrogen peroxide and UVA radiation. The obtained results indicate that minocycline induced oxidative stress in epidermal human melanocytes. The drug inhibited cell proliferation, decreased the level of reduced thiols, and stimulated the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). The described changes were accompanied by an increase in the intracellular level of ROS. On the other hand, pretreatment with minocycline at the same concentrations increased cell viability and significantly attenuated the oxidative stress in melanocytes exposed to hydrogen peroxide and UVA radiation. Moreover, the molecular docking analysis revealed that the different influence of minocycline and other tetracyclines on CAT activity can be related to the location of the binding site.

Neobavaisoflavone May Modulate the Activity of Topoisomerase Inhibitors towards U-87 MG Cells: An In Vitro Study.

Despite many advances in therapy, glioblastoma (GB) is still characterized by its poor prognosis. The main reason for this is unsuccessful treatment, which slightly extends the duration of remission; thus, new regimens are needed. One of many types of chemotherapeutics that are being investigated in this field is topoisomerase inhibitors, mainly in combination therapy with other drugs. On the other hand, the search for new anti-cancer substances continues. Neobavaisoflavone (NBIF) is a natural compound isolated from Psoralea corylifolia L., which possesses anti-oxidant, anti-inflammatory, and anti-cancer properties. The aim of this study was to evaluate the effect of NBIF in human U-87 MG glioblastoma cells in comparison to normal human NHA astrocytes, and to examine if it influences the activity of irinotecan, etoposide, and doxorubicin in this in vitro model. We demonstrated that NBIF decreases U-87 MG cells viability in a dose-dependent manner. Furthermore, we found that it inhibits cell growth and causes glutathione (GSH) depletion more intensely in U-87 MG cells than in astrocytes. This study also provides, for the first time, evidence of the potentialization of the doxorubicin effect by NBIF, which was shown by the reduction in the viability in U-87 MG cells.

UVA Radiation Enhances Lomefloxacin-Mediated Cytotoxic, Growth-Inhibitory and Pro-Apoptotic Effect in Human Melanoma Cells through Excessive Reactive Oxygen Species Generation.

Melanoma, the most dangerous type of cutaneous neoplasia, contributes to about 75% of all skin cancer-related deaths. Thus, searching for new melanoma treatment options is an important field of study. The current study was designed to assess whether the condition of mild and low-dose UVA radiation augments the lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effect of the drug in melanoma cancer cells through excessive oxidative stress generation. C32 amelanotic and COLO829 melanotic (BRAF-mutant) melanoma cell lines were used as an experimental model system. The combined exposure of cells to both lomefloxacin and UVA irradiation caused higher alterations of redox signalling pathways, as shown by intracellular reactive oxygen species overproduction and endogenous glutathione depletion when compared to non-irradiated but lomefloxacin-treated melanoma cells. The obtained results also showed that lomefloxacin decreased both C32 and COLO829 cells' viability in a concentration-dependent manner. This effect significantly intensified when melanoma cells were exposed to UVA irradiation and the drug. For melanoma cells exposed to lomefloxacin or lomefloxacin co-treatment with UVA irradiation, the concentrations of the drug that decreased the cells' viability by 50% (EC50) were found to be 0.97, 0.17, 1.01, 0.18 mM, respectively. Moreover, we found that the redox imbalance, mitochondrial membrane potential breakdown, induction of DNA fragmentation, and changes in the melanoma cells' cell cycle distribution (including G2/M, S as well as Sub-G1-phase blockade) were lomefloxacin in a dose-dependent manner and were significantly augmented by UVA radiation. This is the first experimental work that assesses the impact of excessive reactive oxygen species generation upon UVA radiation exposure on lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effects towards human melanoma cells, indicating the possibility of the usage of this drug in the photochemotherapy of malignant melanoma as an innovative medical treatment option which could improve the effectiveness of therapy. The obtained results also revealed that the redox imbalance intensification mediated by the phototoxic potential of fluoroquinolones may be considered as a more efficient treatment model of malignant melanoma and may constitute the basis for the development of new compounds with a high ability to excessive oxidative stress generation upon UVA radiation in cancer cells.

Molecular and Biochemical Basis of Fluoroquinolones-Induced Phototoxicity-The Study of Antioxidant System in Human Melanocytes Exposed to UV-A Radiation.

Phototoxicity of fluoroquinolones is connected with oxidative stress induction. Lomefloxacin (8-halogenated derivative) is considered the most phototoxic fluoroquinolone and moxifloxacin (8-methoxy derivative) the least. Melanin pigment may protect cells from oxidative damage. On the other hand, fluoroquinolone-melanin binding may lead to accumulation of drugs and increase their toxicity to skin. The study aimed to examine the antioxidant defense system status in normal melanocytes treated with lomefloxacin and moxifloxacin and exposed to UV-A radiation. The obtained results demonstrated that UV-A radiation enhanced only the lomefloxacin-induced cytotoxic effect in tested cells. It was found that fluoroquinolones alone and with UV-A radiation decreased superoxide dismutase (SOD) activity and SOD1 expression. UV-A radiation enhanced the impact of moxifloxacin on hydrogen peroxide-scavenging enzymes. In turn, lomefloxacin alone increased the activity and the expression of catalase (CAT) and glutathione peroxidase (GPx), whereas UV-A radiation significantly modified the effects of drugs on these enzymes. Taken together, both analyzed fluoroquinolones induced oxidative stress in melanocytes, however, the molecular and biochemical studies indicated the miscellaneous mechanisms for the tested drugs. The variability in phototoxic potential between lomefloxacin and moxifloxacin may result from different effects on the antioxidant enzymes.

Cellular and Molecular Aspects of Anti-Melanoma Effect of Minocycline-A Study of Cytotoxicity and Apoptosis on Human Melanotic Melanoma Cells.

Minocycline is a tetracycline compound with pleiotropic pharmacological properties. In addition to its antibacterial action, it shows many non-antimicrobial effects, including an anti-cancer activity. The anti-cancer action was confirmed in studies on ovarian carcinoma cells, hepatocellular carcinoma cells, glioma cells, or acute myeloid leukemia cells. Malignant melanoma remains a serious medical problem despite the extensive knowledge of the disease. The low effectiveness of the standard treatment, as well as the resistance to therapy, result in high mortality rates. This work aimed to investigate the potential and mechanisms of anti-melanoma action of minocycline. Human skin melanotic melanoma cell line COLO 829 was used in the study. The obtained results showed that minocycline decreased cell viability and inhibited the growth of melanoma cells, proportional to the drug concentration as well as to the time of incubation. The EC50 values were calculated to be 78.6 µM, 31.7 µM, and 13.9 µM for 24 h, 48 h, and 72 h, respectively. It was observed that treated cells had a disturbed cell cycle and significantly changed morphology. Moreover, minocycline caused a decrease in mitochondrial membrane potential and an increase in cells with a low level of reduced thiols. Finally, it was found that the anti-melanoma effect of minocycline was related to the induction of apoptosis. The drug activated caspases 8, 9, and 3/7 as well as increased the number of annexin V-positive cells. The presented results show that minocycline possesses anti-melanoma potential.

Kanamycin induces free radicals formation in melanocytes: An important factor for aminoglycosides ototoxicity.

Ototoxicity is well-documented but not fully understood undesirable side effect of aminoglycoside antibiotic, kanamycin. Kanamycin is capable of binding to melanin biopolymers-natural pigments of the skin, hair, and eyes. Melanin-producing cells, melanocytes, are also present in the inner ear and are known to be necessary for normal hearing. It was considered that melanin content in the inner ear may influence aminoglycoside-induced ototoxic effect. The impact of kanamycin on melanocytes homeostasis may thus play role in the antibiotic-induced ototoxic effect. Previously, we demonstrated that kanamycin disturbs homeostasis in light-pigmented melanocytes. To investigate if/how melanization contributes to this phenomenon, the study using in vitro model of dark-pigmented melanocytes is required. Spectrophotometric measurements and electron paramagnetic resonance (EPR) spectroscopy analysis were performed. Kanamycin induced a concentration-dependent loss in HEMn-DP melanocytes viability. The value of IC 50 was estimated to be 5.0 mM. Modulation of the activity of analyzed antioxidant enzymes and increased production of free radicals as well as the decrease of the melanin content were observed. Our results confirmed that kanamycin generates oxidative stress in melanocytes. The increased level of free radicals caused by kanamycin may be responsible for the imbalance of antioxidant defense and the reduction of melanin content in melanocytes. The role of melanin in the mechanism of kanamycin-induced hearing impairment was discussed and the obtained results were compared with the previously demonstrated data concerning light-pigmented melanocytes.

Vitamin B12 Deficiency Induces Imbalance in Melanocytes Homeostasis-A Cellular Basis of Hypocobalaminemia Pigmentary Manifestations.

Vitamin B12 deficiency causes significant changes in cellular metabolism leading to various clinical symptoms, such as hematological, psychiatric, and neurological disorders. We hypothesize that skin pigmentation disorders may be a diagnostically important manifestation of vitamin B12 deficiency, however the cellular and molecular mechanisms underlying these effects remain unknown. The aim of this study was to examine the effect of vitamin B12 deficiency on melanocytes homeostasis. Hypocobalaminemia in vitro model was developed by treating epidermal melanocytes with synthesized vitamin B12 antagonist-hydroxycobalamin(c-lactam). The cells were examined using immunoenzymatic, spectrophotometric, and fluorimetric assays as well as image cytometry. Significant melanogenesis stimulation-the increase of relative melanin content and tyrosinase activity up to 131% and 135%, respectively-has been indicated. Cobalamin-deficient cells displayed the elevation (by 120%) in reactive oxygen species level. Moreover, the redox status imbalance was stated. The study provided a scientific evidence for melanocytes homeostasis disturbance under hypocobalaminemia, thus indicating a significant element of the hyperpigmentation mechanism due to vitamin B12 deficiency. Furthermore, the implication between pigmentary and hematological and/or neuropsychiatric symptoms in cobalamin-deficient patients may be an important issue.

Lomefloxacin Induces Oxidative Stress and Apoptosis in COLO829 Melanoma Cells.

Although some fluoroquinolones have been found to exert anti-tumor activity, studies on the effect of these drugs on melanoma cells are relatively rare. The aim of this study was to examine the effect of lomefloxacin on cell viability, reactive oxygen species production, redox balance, cell cycle distribution, DNA fragmentation, and apoptosis in COLO829 melanoma cells. Lomefloxacin decreases the cell viability in a dose- and time-dependent manner. For COLO829 cells treated with the drug for 24, 48, and 72 h, the values of IC50 were found to be 0.51, 0.33, and 0.25 mmol/L, respectively. The analyzed drug also altered the redox signaling pathways, as shown by intracellular reactive oxygen species overproduction and endogeneous glutathione depletion. After lomefloxacin treatment, the cells were arrested in S- and G2/M-phase, suggesting a mechanism related to topoisomerase II inhibition. DNA fragmentation was observed when the cells were exposed to increasing lomefloxacin concentrations and a prolongation of incubation time. Moreover, it was demonstrated that the drug induced mitochondrial membrane breakdown as an early hallmark of apoptosis. The obtained results provide a strong molecular basis for the pharmacologic effect underlying the potential use of lomefloxacin as a valuable agent for the treatment of melanoma in vivo.

Effect of fluoroquinolones on melanogenesis in normal human melanocytes HEMn-DP: a comparative in vitro study.

PURPOSE: Fluoroquinolones are one of the most commonly prescribed classes of antibiotics. However, their use is often connected with high risk of phototoxic reactions that lead to various skin or eye disorders. The aim of this study was to examine the effect of ciprofloxacin, lomefloxacin, moxifloxacin and fluoroquinolone derivatives with different phototoxic potential, on the viability and melanogenesis in melanocytes. MATERIALS AND METHODS: Normal human epidermal melanocytes, dark pigmented (HEMn-DP) were used as an in vitro model system. The effect of the tested antibiotics on cell viability and melanization in pigmented cells was investigated using a spectrophotometric method. The WST-1 assay was used to detect the cytotoxic effect of antibiotics. RESULTS: Ciprofloxacin, lomefloxacin and moxifloxacin induced the concentration-dependent loss in melanocytes viability. The values of EC50 for the tested fluoroquinolone derivatives were found to be 2.0 mM for ciprofloxacin, 0.51 mM for lomefloxacin and 0.27 mM for moxifloxacin. The exposure of cells to different concentrations of the analyzed drugs resulted in decrease in melanin content and tyrosinase activity. The highest decrease was observed for lomefloxacin which may explain its high phototoxic potential in vivo. The role of melanin in the mechanism of the toxicity of fluoroquinolones was discussed and the obtained results were compared with the previously obtained data concerning light-pigmented melanocytes (HEMa-LP). CONCLUSIONS: The results obtained in vitro suggest that the phototoxic potential of fluoroquinolones in vivo depends on specific drug-melanin interaction, the ability of drugs to affect melanogenesis as well as on the degree of melanocytes pigmentation.

The effect of simultaneous exposure of HEMn-DP and HEMn-LP melanocytes to nicotine and UV-radiation on the cell viability and melanogenesis.

Nicotine is a main compound of tobacco plants and may affect more than a billion people all over the world that are permanently exposed to nicotine from cigarettes, various forms of smoking cessation therapies, electronic cigarettes or second-hand smoke. It is known that nicotine forms complexes with melanin what may lead to accumulation of this alkaloid in tissues of living organisms containing the pigment. This may affect the viability of cells and process of melanin biosynthesis that takes place in melanocytes. Although UV radiation is known to be a particular inductor of melanin biosynthesis, its simultaneous effect with nicotine on this process as well as the viability of human cells containing melanin have not been assessed so far. The aim of this study was to examine the simultaneous impact of nicotine and UV radiation on viability and melanogenesis in cultured normal human melanocytes dark (HEMn-DP) and light (HEMn-LP) pigmented. Nicotine together with UV radiation induced concentration-dependent loss in melanocytes viability. The higher cell loss was observed in dark pigmented melanocytes in comparison to light pigmented cells. Simultaneous exposure of cells to nicotine and UV radiation also caused changes in melanization process in both tested cell lines. The data suggest that simultaneous exposure of melanocytes to nicotine and UV radiation up-regulates melanogenesis and affects cell viability. Observed processes are more pronounced in dark pigmented cells.

From tyrosine to melanin: Signaling pathways and factors regulating melanogenesis.

Melanins are natural pigments of skin, hair and eyes and can be classified into two main types: brown to black eumelanin and yellow to reddish-brown pheomelanin. Biosynthesis of melanins takes place in melanosomes, which are specialized cytoplasmic organelles of melanocytes - dendritic cells located in the basal layer of the epidermis, uveal tract of the eye, hair follicles, as well as in the inner ear, central nervous system and heart. Melanogenesis is a multistep process and begins with the conversion of amino acid L-tyrosine to DOPAquinone. The addition of cysteine or glutathione to DOPAquinone leads to the intermediates formation, followed by subsequent transformations and polymerization to the final product, pheomelanin. In the absence of thiol compounds DOPAquinone undergoes an intramolecular cyclization and oxidation to form DOPAchrome, which is then converted to 5,6-dihydroksyindole (DHI) or 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Eumelanin is formed by polymerization of DHI and DHICA and their quinones. Regulation of melanogenesis is achieved by physical and biochemical factors. The article presents the intracellular signaling pathways: cAMP/PKA/CREB/MITF cascade, MAP kinases cascade, PLC/DAG/PKCß cascade and NO/cGMP/PKG cascade, which are involved in the regulation of expression and activity of the melanogenesis-related proteins by ultraviolet radiation and endogenous agents (cytokines, hormones). Activity of the key melanogenic enzyme, tyrosinase, is also affected by pH and temperature. Many pharmacologically active substances are able to inhibit or stimulate melanin biosynthesis, as evidenced by in vitro studies on cultured pigment cells.

FLUPHENAZINE AND PERPHENAZINE IMPACT ON MELANOGENESIS AND ANTIOXIDANT ENZYMES ACTIVITY IN NORMAL HUMAN MELANOCYTES.

Fluphenazine and perphenazine as a phenothiazine-class antipsychotic drugs are widely used to treat psychoses and schizophrenia, however their use is associated with significant side effects such as extrapyramidal symptoms as well as ocular and skin disorders. The aim of this study was to examine the effect of fluphenazine and perphenazine on cell viability, melanogenesis and antioxidant defense system in normal human melanocytes. It has been shown that both phenothiazines induce concentration-dependent loss in cell viability. The value of EC50. was calculated to be 1.24 and 2.76 µM for fluphenazine and perphenazine, respectively. Fluphenazine in concentration of 1.0 µM and perphenazine in concentrations of 1.0 and 3.0 µM inhibied melanogenesis and decreased microphthalmia-associated transcription factor content. To study the effect of both analyzed drugs on antioxidant defense system in melanocytes, the level of hydrogen peroxide and the activities of antioxidant enzymes: superoxide dismutase, catalase and glutathione peroxidase were determined. Fluphenazine and perphenazine in higher analyzed concentrations caused depletion of melanocytes antioxidant status, what indicated the induction of oxidative stress. The observed changes in melanization process and antioxidant defense system in pigmented cells exposed to fluphenazine and perphenazine in vibo suggest a significant role of melanin and melanocytes in the mechanisms of undesirable side effects of these drugs in vivo, especially directed to pigmented tissues. Moreover, the presented differences in modulation of biochemical processes in melanocytes may be an explanation for various toxic activity of the analyzed phenothiazine derivatives in vivo.

EFFECT OF PARACETAMOL ON MELANIZATION PROCESS IN HUMAN EPIDERMAL MELANOCYTES.

Paracetamol (acetaminophen) is commonly used as a drug of choice for treatment of pain and fever. Unlike non-steroidal anti-inflammatory drugs (NSAIDs) it does not cause gastrointestinal damage or untoward cardiorenal effects, however cutaneous adverse effects have been reported. It is known that paracetamol binds to melanin biopolymers, but the relation between the affinity of this drug to melanin and its toxicity is not documented. The aim of this work was to examine the impact of paracetamol on melanogenesis in cultured human normal epidermal melanocytes (HEMn-DP). The effect of paracetamol on cell viability was determined by WST-1 assay, melanin content and tyrosinase activity were measured spectrophotometrically. It has been demonstrated that paracetamol induced concentration-dependent loss in melanocytes viability. The value of EC50 was found to be - 20.0 mM. The analyzed drug inhibited melanin biosynthesis in a concentration-dependent manner by decreasing the melanin content as well as the tyrosinase activity. The demonstrated inhibitory effect of paracetamol on melanization process in normal epidermal melanocytes in vitro may explain the potential role of melanin biopolymer in the mechanisms of undesirable side effects of this drug in vivo, as a result of its accumulation in pigmented tissues.

Effect of norfloxacin and moxifloxacin on melanin synthesis and antioxidant enzymes activity in normal human melanocytes.

Fluoroquinolone antibiotics provide broad-spectrum coverage for a number of infectious diseases, including respiratory as well as urinary tract infections. One of the important adverse effects of these drugs is phototoxicity which introduces a serious limitation to their use. To gain insight the molecular mechanisms underlying the fluoroquinolones-induced phototoxic side effects, the impact of two fluoroquinolone derivatives with different phototoxic potential, norfloxacin and moxifloxacin, on melanogenesis and antioxidant enzymes activity in normal human melanocytes HEMa-LP was determined. Both drugs induced concentration-dependent loss in melanocytes viability. The value of EC50 for these drugs was found to be 0.5 mM. Norfloxacin and moxifloxacin suppressed melanin biosynthesis; antibiotics were shown to inhibit cellular tyrosinase activity and to reduce melanin content in melanocytes. When comparing the both analyzed fluoroquinolones, it was observed that norfloxacin possesses greater inhibitory effect on tyrosinase activity in melanocytes than moxifloxacin. The extent of oxidative stress in cells was assessed by measuring the activity of antioxidant enzymes: SOD, CAT, and GPx. It was observed that norfloxacin caused higher depletion of antioxidant status in melanocytes when compared with moxifloxacin. The obtained results give a new insight into the mechanisms of fluoroquinolones toxicity directed to pigmented tissues. Moreover, the presented differences in modulation of biochemical processes in melanocytes may be an explanation for various phototoxic activities of the analyzed fluoroquinolone derivatives in vivo.