a549 — human neoplastic cells of the lung cancer ; the continuous line obtained from the institute of the immunology and the experimental therapy of polish academy of science in wroclaw . ht - 29 — human neoplastic cells of the large intestine cancer ; the continuous line obtained from the institute of the immunology and the experimental therapy of polish academy of science in wroclaw . c6 — the rat neoplastic cells of the brain cancer ( glioma ); the continuous line obtained from the department of neonatology , humboldt university , berlin , germany . cells of the a549 line were cultured on the basis dmem : f - 12 ham ( 2 : 1 ), ht - 29 and c6 cells on the basis dmem . to the culture basis , 10 % foetal beast serum ( fbs ), penicillin 100 i . u ./ ml and streptomycin 100 . mu . g / ml were added . the basis dmem : f - 12 ham , dmem was produced by sigma company ( sigma , st . louis , mo ., u . s . a .). the foetal beast serum ( fbs ) was produced by life technologies company ( life technologies , karlsruhe , germany ). remaining reagents were produced by sigma company . cells stored in liquid nitrogen in a tissue bank were unfrozen at a temperature of 37 . degree . c ., then poured into the plastic bottles containing the proper basis . they were cultured in a temperature of 37 . degree . c . in the incubator with 5 % co . sub . 2 flow . after the cells &# 39 ; reproduction liquid was poured out , the cells were washed with pbs ( without the calcium and magnesium ions ) and processed with 0 . 25 % trypsin solution + edta to receive the suspension of cells necessary in the experience . prepared earlier , in the culture basis , a suspension of cells with a density 1 . times . 10 . sup . 4 cells / ml ( a549 ), 4 . times . 10 . sup . 4 cells / ml ( ht - 29 ) and 0 . 5 . times . 10 . sup . 4 cells / ml ( c6 ) was poured into the 96 - pits microplate with the flat bottom ( nunc company , roskilde , denmark ) in the volume 100 . mu . 1 / pit . after sticking of cells ( 24 hours ) the liquid was carefully pulled down and then different concentrations of akg and examined cytostatics ( cyclophosphamide , iphosphamid , thiotepa ) in liquid with 10 % fbs ( 100 . parallel . 1 / pit ) were added . the cultures on plates were left for 96 hours incubation at a temperature of 37 . degree . c ., in the atmosphere 95 % air and 5 % co . sub . 2 . the antiproliferative activity of examined substances was assessed with the method mtt . the method mtt ( according to kit “ cell proliferation kit iii ”, boehringer manheim ) this method was worked out to determine the proliferation and vitality of cells in studies on the cytotoxic and antiproliferative substances . in metabolically active cells tetrazolic yellow salt mtt is reduced to formazane blue with the mitochondrial dehydrases . formazane crystals , insoluble in water , accumulate in cells and for their dissolutions the use of organic detergent , breaking the membrane and simultaneously solvent the dye , is necessary . for this purpose the buffer sds - hc1 with ph 7 . 4 is used . the concentration of released dye is evaluated quantitatively in the reader for 96 - pits plates at the wavelength 570 nm . the colour intensity is directly proportional to the quantity of alive cells . mtt solution in pbs condition in concentration 5 mg / ml was added into each pit on a plastic plate in a dose of 15 . mu . 1 / pit . plates were incubated for 3 hours at a temperature of 37 . degree . c . thereafter , buffer sds - hc1 in a does 100 . mu . 1 / pit was added and the plates were left at a temperature of 37 . degree . c . all night . the results were evaluated next day with the use of e - max reader ( molecular devices corporation , menlo park , calif ., u . s . a .). this method is for the estimation of the activity of substances affecting the mobility of cells in vitro . it is used in research on wounds healing , angiogenesis and neoplastic metastases . c6 cells ( 1 . times . 10 . sup . 6 ) suspended in the culture basis with the addition of 10 % serum ( fbs ) were poured onto the culture plates ( nunc , roskilde , denmark ) with 4 cm diameter . next day in the equal layer of cells the flaw ( wound ) was done with the ending of automatic pipette and unstuck cells were removed by twice - rinsing the plates with a pbs solution . then , akg ( 10 and 20 mm ) dissolved in the culture basis was added to the prepared culture . plates were incubated for 24 hours in a temperature of 37 . degree . c . and in a humid atmosphere 95 % air and 5 % co . sub . 2 . thereafter the cultures were coloured with the may - grunwald - giemza method . then the microscopic analysis was performed with the microscope olympus bx51 ( olympus optical co ., ltd , tokyo , japan ) with the use of the software analysis . rtm . ( soft imaging system gmbh , munster , germany ). the degree of cells migration was assessed in cytometry as the number of cells which populated the wound done earlier in the layer of cells . at least 50 chosen fields on 8 photographs were assessed . antiproliferative activity of akg in the cultures was estimated in differen kinds of neoplastic cells : the lung cancer cells ( a549 ), the large intestine cancer cells ( ht - 29 ) and glioma cells ( c6 ). cells were processed with akg in concentrations 0 . 5 , 1 , 2 . 5 , 5 , 10 and 20 mm , for 96 hours . the examined substance has had antiproliferative activity with relation to all neoplastic cells types ( see the diagram — fig1 fig2 fig3 ). statistically significant ( 4 . 5 %) inhibition of cells &# 39 ; growth was observed at akg concentration 2 . 5 mm in a549 cell line in the comparison to the control group . that effect was correlated with a dose of akg and was 7 . 8 %, 12 . 4 %, 17 . 5 % with doses 5 mm , 10 mm and 20 mm respectively ( see the diagram on fig1 ). the growth inhibition in glioma cells ( c6 ) was 12 . 6 % with akg dose 2 . 5 mm , 7 . 9 %— 5 mm , 16 %— 10 mm and 19 . 8 %— 20 mm , respectively . the growth inhibition of large intestine cancer cells ( ht - 29 ) hadn &# 39 ; t had the lineal character with akg concentrations between 1 and 10 mm . the dose 1 mm inhibited the cells growth by 11 . 8 %. the similar effect was obtained with a dose 5 mm ( 11 . 8 %) and 10 mm ( 11 . 5 %). only a dose 20 mm caused the significant ( 25 %) inhibition of these cells &# 39 ; growth ( the diagram — fig3 ). the research on the interaction between akg and popular cytostatics used in cancer chemotherapy was performed in the culture of lung cancer cells ( a549 ). for that purpose cells were treated with the following cytostatics : cyclophosphamide ( 1 . 5 mm ), iphosphamid ( 1 . 5 mm ) and thiotepa ( 5 . mu . m ), alone and in combination with akg ( 5 , 10 and 20 mm ). the additive effect of akg on cytostatic activity of used chemotherapeutics was observed ( results are presented on fig4 fig5 and fig6 ). cyclophosphamide in a concentration 1 . 5 mm inhibited the growth of a549 cells by 21 . 4 %. the addition of akg increased its cytostatic activity by 6 . 4 %, 9 . 8 % and 14 . 4 % respectively ( the diagram — fig4 ). iphosphamid ( 1 . 5 mm ) inhibited the cells growth by 7 . 3 %. after akg addition ( 5 , 10 and 20 mm ) that effect increased by 5 %, 5 . 3 % and 8 . 8 % respectively ( the diagram — fig5 ). akg intensified also cytostatic activity of thiotepa ( 5 . mu . m — 25 . 9 %) by 4 . 2 % ( 5 mm ), 8 % ( 10 mm ) and 11 . 2 % ( 20 mm ) ( results are presented on fig6 ). the research on the mobility of neoplastic cells was passed in the “ wound assay ” model . at the photograph 1 the wound in the layer of c6 cells ( a ), the wound population with cells after 24 hours of incubation without akg ( b ) and significant inhibition of cells migration in the presence of akg 20 mm ( c ) are presented . on the diagram — fig7 the average number of cells migrating to one field of flaw done in the uniform layer of cells is presented . statistically significant inhibition of cells migration in the presence of 10 mm and 20 mm akg was shown . the statistical analysis was performed with the use of t - student test . * p & lt ; 0 . 05 , ** p & lt ; 0 . 01 , *** p & lt ; 0 . 001 .
a method for treatment or prophylaxis of neoplastic diseases includes administering an antineoplastic preparation to a subject in need thereof . the active component of the antineoplastic preparation is alpha - ketoglutarate and / or a salt of alpha - ketoglutarate .