STUDY OF INHIBITION OF B16F10 MELANOMA GROWTH IN MICE BY LANDOMYCIN A IN COMPARISON TO DOXORUBICIN

L. V. Lehka1, R. R. Panchuk1, N. R. Skorokhyd1, Yu. S. Kozak3, J. Rohr2, R. S. Stoika1,3 1Institute of Cell Biology, NAS of Ukraine, 14–16, Drahomanov St., Lviv 79005, Ukraine e-mail: lilyalehka@gmail.com 2Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky 789 S. Limestone St., Lexington, Kentucky, 40536-0596, USA 3Ivan Franko National University of Lviv, 4, Hrushevskyi St., Lviv 79005, Ukraine


INTRODUCTION
A cutaneous melanoma is a highly malignant tumor derived from melanocytes, the pigment-producing cells in skin epidermis [14]. If being early diagnosed and surgically removed while localized in the outermost skin layer, melanoma is potentially curable. When tumor cells have spread to distant lymph nodes or metastasized (stage IV), they become refractory to common chemotherapies and, therefore, incurable. The prognosis for patients with stage IV metastatic melanoma is very poor, with an expected median survival of only 6 to 9 months [7]. The rapid increase in the incidence of malignant melanomas has not been associated with any improved therapeutic options over the years [2].

Рис. 1. Ландоміцин А
LA exhibits a strong cytotoxic effect towards tumor cells of different origin and induces early apoptosis in target cells. Our finding that landomycin A induces oxidative stress in cancer cell lines in vitro provides a possible mechanism of its antineoplastic activity [8]. It is known that cancer cells have increased ROS steady state level, and they are likely to be more vulnerable to damage by further ROS insults induced by exogenous agents [4]. Thus, manipulating ROS levels by redox modulation could be a way to kill cancer cells selectively without causing a significant toxicity to normal cells. Melanoma is a unique type of cancer with melanin biosynthesis that produces high level of ROS and oxidative stress [10,15]. Thus, melanoma cells are expected to be very sensitive to therapeutic strategies whose application leads to induce cancer cell death through ROS production.
In this study, we evaluated the effects of landomycin A in vitro and in vivo on the growth of mouse melanoma cells.

MATERIALS AND METHODS
Landomycin A (99% purity according to thin-layer chromatography) was obtained in the laboratory of professor Jurgen Rohr (Department of Pharmaceutical Sciences, University of Kentucky, USA). Doxorubicin hydrochloride (Dx, Pfizer, New York, NY) was bought in local pharmacy.
Cell culture. B16F10 cell line (mouse melanoma) used in this study was obtained from the collection of the Institute of Cancer Research at Medical University of Vienna (Austria). Cells were grown in RPMI-1640 medium (Sigma, USA) supplemented with 10% fetal bovine serum (Sigma, USA) and 50 µg/ml gentamicin (Sigma, Missouri, USA) in 5% CO 2 -containing humidified atmosphere at 37 ºC. Cells were re-seeded every two days at the rate of 2.5×10 5 cell/ml of culture medium [1].
Cytotoxicity assay. Cytotoxicity was measured using Trypan Blue exclusion assay. Briefly, exponentially growing B16F10 cells at 1×10 5  with various concentrations of studied drug and cultivated in a 24-well tissue culture plate (Greiner Bio One, Frickenhausen, Germany). After 24 h incubation, the number of cells was calculated in the hemocytometer chamber by counting of the number of dead cells using 0.1% Trypan Blue dye that stains dead cells with damaged membrane in blue, whereas alive cells remain unstained [5].
Animals. Studies of the biological activity of LA were conducted using male C57black/6 mice Kent at animal facility of the Institute of Cell Biology, NAS of Ukraine (Lviv, Ukraine). All in vivo experiments were conducted in accordance with the international principles of the European Convention for protection of vertebrate animals under a control of the Bio-Ethics Committee of the above mentioned institution (Protocol N 9/2015 from 1.09.2015 of the BioEthics committee of the Institute of Cell Biology, NAS of Ukraine). Animals weighing 18-25 g were kept at a temperature of 22±2 °C and with photo cycle of 12-hour light/12-hour dark. Water and food pellets were provided ad libitum.
Testing of LA in vivo toxicity. To define the toxicity of studied compound three experimental groups of male C57black/6 mice were used. Animals were distributed according to the study design: 1 st group -control, intact animals, 2 nd group -animals treated intraperitonealy (IP) with LA (10 mg/kg), 3 rd group -mice treated IP with Dx (cumulative dose 10 mg/kg). There were three mice used in each group. During the period of study, the animals were weighed every day. Clinical symptoms were evaluated in mice daily.
Blood cell formula. For blood sampling amputation of small part of mouse tail was cut with pumping of ~100 µl of blood in a test tube, followed by immediate disinfection of a wound with 70% alcohol. For counting of red blood cells, 5 µl of blood were dissolved in 5 ml of isotonic NaCI solution (1:1000 dilution), while for leukocyte, 5 µl of blood were dissolved in 95 µl of 3% acetic acid solution (1:20 dilution). Erythrocytes and leukocytes were counted under the Evolution 300 Trino microscope (Delta Optical, Mińsk Mazowiecki, Poland) and calculated by standard formulas, described in [11]. For blood smear preparation, 3 µl of blood were put at the edge of a slide, and then spread for 1.5 cm using another narrow polished slide, placed at a 45° angle. The obtained smears were dried at room temperature, then fixed with absolute methanol, and later rehydrated by subsequent washing in ethanol solutions with decreasing concentration (96%, 75%, 50%, 25%, 12.5%). Finally, the smears were washed with distilled water, stained with Giemsa dye and air-dried, after which they were ready for analysis of leukogram. Counting of leukocytes was performed under Evolution 300 Trino microscope (Delta Optical, Mińsk Mazowiecki, Poland) using 90× oil immersion objective. Cell counting was always done using the same system -half of cells were counted in the upper half part of the smear, and the rest 50% of cells were counted on the lower part of the smear. A percentage of certain types of white blood cells in each smear was determined after counting of at least 300 cells.
Tumor implantation. Tumor inoculation was done by a subcutaneous injection of B16F10 cells suspension diluted with sterile 1-x phosphate-buffered saline (PBS) in an amount of 1 mln per one animal. The viability and number of cells stained with 0.1% Trypan Blue were checked by cell counting in the haemocytometric chamber. The vitality of melanoma cells used for transplantation was not less than 98%.
LA and Dx were administered 5 times in dose of 2 mg/kg every 72 hours. Injections of studied compounds were performed on 10 th day after tumor inoculation. The length, width, and height of tumors were measured every three days with calipers. Experiments were terminated on 22 th day, when tumor volumes in the control group reached the clinical endpoint (2500 mm 3 or become necrotic) in accordance with animal ethics guidelines. Tumor volume was calculated as: Vol = ½ L width *L length *L height [12].
Aspartate and alanine aminotransferase activities. For determination of aspartate aminotransferase activity (AST), 10 µl of blood serum were mixed with 100 µl of substrate solution (2 mM α-ketoglutaric acid; 0.2 M D,L-aspartate in 0.1 M phosphate buffer pH 7.4), while in control tube 10 µl of distilled water were added instead of blood serum. The tubes were placed for 60 min at 37 °C, and then 100 µl of 1 mM solution of 2,4-dinitrophenylhydrazine was added to the samples and left for 20 min at RT. Then 1 ml of 0.4 M sodium hydroxide solution was added to each sample for extra 10 min, and optical density of samples was measured using ThermoSpectronic spectrophomometer (Helios, Great Britain) at 540 nm wavelength. For measuring alanine aminotransferase (ALT) activity, the procedure was identical except substrate solution (2 mM α-ketoglutaric acid; 0,2 M D,L-alanine in phosphate buffer pH 7.4).
Statistical analysis. In vitro experiments were performed in triplicate for each variant. For statistical analysis, standard variation data within a group were calculated together with a statistical reliability of differences between two groups of data assessed by t-test. The level of significance was set at 0.05.

RESULTS
To determine the effect of LA on growth of B16F10 mouse melanoma in vitro, tumor cells were treated with studied drug at various concentrations (1, 2, 4, 6 µM) and examined by cell counting using Trypan Blue assay on 24 hour after LA addition to the culture medium. Compared with control group, cell density in groups treated with LA decreased significantly. Thus, LA effectively inhibits the growth of B16F10 cells (Fig. 2, A). It was shown that LA suppressed proliferation of melanoma cells in a dose-dependent manner. LC 50 value (the concentration of compound which cause a death of 50% cells compared to the control) was 2 µM, which is about 5 times lower than for doxorubicin (IC 50 for Dx on B16F10 cells = 10 µM, Fig. 2, B). Doxorubicin is considered to be gold chemotherapy standard and it is widely used for treatment of solid tumors [16]. The next step in our work was studying LA toxicity in male C57black/6 mice.This experiment was designed to investigate potential side effects of one-time IP administration of studied antibiotic in a dose of 10 mg/kg body weight. Fig. 3 demonstrates the results of weighing mice of control group. It was shown that their body weight did not change during the experiment term. We did not reveal any pathological changes in the LA-treated mice, as compared to the control group of animals. No mortality or significant changes were observed in the body weight of mice that received LA (Fig. 4). A similar experiment, as above, was done with Dx. It was revealed (Fig. 5) that already after the first injection of Dx, the weight of experimental animals decreased by 8% in comparison to control group. Starting from the 9 th day after the injection of last Dx dose the further decrease of weight of experimental animals took place. Mice of this group in 3 days lost about 16% of their body weight. Although the weight of animals did not return to the initial value, suggesting general toxic effect of this drug. The hematological profile (number of red and white blood cells as well as a leukogram) in the experimental animals was also studied. After LA administration, no significant changes in the peripheral blood cells were observed in comparing to animals of control group (Table 1). Table 1. In case of mice receiving the same dose of Dx, changes in a blood parameters were more marked and typical for this compound: mielosupressive effect including leukopenia Relative increase in weight ,% Days Doxorubicin, 10 mg/kg (29% decrease in the amount of white blood cells compared to control group), erythropenia (10% reduction in red blood cells number), diminution of SL percentage (from 72.0±1.4% in untreated mice to 58.6±0.9%) and increase in monocytes amount (from 1.3±1.0% to 3.4±0.7% were found (Table 1). These results prove low toxic nature of LA, which is a primary criterion and important step in development of anticancer drugs.

Number of white, red blood cells and peripheral blood leukogram of mice on 7 th day after the last injection of landomycin A and Doxorubicin
B16F10 cells formed large, aggressive tumors in C57black/6 mice. To determine whether LA limits melanoma growth in vivo, the IP injection of LA (cumulative dose 10 mg/kg) was carried out in B16F10 melanoma-bearing mice. As a result (Fig 6), the mean of tumor volume was significantly less (742 mm 3 ) compared with that in control group (3,126 mm 3 ) on 22 th day when control mice were sacrificed. We also used Dx as a positive control in the same dose (cumulative dose 10 mg/kg). Group of mice treated with Dx also did show a decrease in tumor volume (1,878 mm 3 comparing with 3,126 mm 3 in control), however the tumor size in Dx-treated mice was 153% larger compared to LA in the same concentration. The hematological profile was also analyzed in B16F10 melanoma-bearing animals. The growth of B16F10 melanoma was characterized by a significant increase in the level of SN (from 20.0±4.5% to 57.0±16.2%) and marked increase in the level of WBC (from 6.2±0.9% to 13.5±1.7%). Regarding the leukogram in a group treated with LA, it was revealed a restoration of these indicators found in control group, thus, suggesting a therapeutic effect of this drug ( Table 2).
The amount of SL is another important indicator that was decreased in tumor-bearing animals (from 78.0±5.1% in intact mice to 31.9±4.1%). LA partially restored this value to control level -from 31.9±4.1% to 54.0±10.6%.
A therapeutic selectivity and avoiding resistance to drugs are two important issues in the anticancer therapy. Strategies for improving therapeutic selectivity depend significantly on understanding of the biological difference between tumor and normal cells. Tumor cells, compared to normal ones are under big oxidative stress related to the oncogenic transformation and alterations in metabolic activity [13]. Exogenous agents that rapidly increase ROS generation will move the redox equilibrium and induce tumor cells death. In contrast, normal cells are less sensitive to agents that induce an oxidative stress due to low level of ROS production and high antioxidant capacity. Previously we have shown that under LA action in vitro, the level of ROS, mainly H 2 O 2 , had increased several times, comparing to control level already at the 1 st hour after start of LA addition to the culture medium [8]. It is well established that high level of ROS, like H 2 O 2 , induce apoptosis in a wide variety of tumor cells via activating the caspase cascade [9]. We suppose that such early and rapid generation of ROS, accompanied with caspases activation allows LA to inhibit growth of B16F10 melanoma both in vitro and in vivo with much lower negative side effects than such effects of Dx.

CONCLUSION
In this study, we have shown that LA possessed antineoplastic effect towards B16F10 melanoma cells in vitro, and that effect was even stronger than such of Dx used as a positive control. LA's action was not accompanied by cachexy in experimental animals and it didn't cause hematotoxic effects found at Dx's action. Our data on measurement of AST/ALT ratio testify to the lack of cardio-and hepatotoxicity under LA treatment. LA effectively inhibited growth of B16F10 melanoma in vivo without significant myelosuppressive effects.
Taking into consideration these results, investigation of action of LA in the other experimental tumor models that might be especially sensitive to this drug are desired, as well as studying the molecular mechanisms of its therapeutic activity.