as used herein and in the claims , “ comprising ” means including the following elements but not excluding others . nsclc is heterogeneous disease characterized by driver oncogenic alterations that can be targeted through precision tyrosine kinase inhibitors ( tkis ). the most prevalent mutated or rearranged oncogenes identified in nsclcs are kras ( v - ki - ras2 kirsten rat sarcoma viral oncogene homolog ), epidermal growth factor receptor ( egfr ), anaplastic lymphoma kinase ( alk ) and c - ros oncogene 1 ( ros1 ) etc . ros1 encodes a receptor tyrosine kinase of the insulin receptor super family with no known ligand and little known about its normal function . the prevalence of ros1 rearrangement in nsclc is estimated at 1 - 2 %. crizotinib is the only current clinically - available , orally available , atp - competitive small molecule inhibitor of alk / ros1 with potency against ros1 kinase , which displays marked anti - tumor activity both in vitro and in vivo as well as in clinics . although most patients with ros1 fusion - driven nsclc derive remarkable benefit from crizotinib , durable responses to crizotinib therapy lead to the development of eventual drug resistance , a common feature similar to other tki drugs . therefore , development of other novel ros1 kinase inhibitors is essential to treat nsclc with ros1 gene fusion . the inventors applied a nsclc cell line hcc78 harboring the scl34a2 - ros1 fusion gene , and identified mitoxantrone as a new potent ros1 kinase inhibitor . in this study , the inventors found out that mitoxantrone can directly inhibit ros1 kinase activity and suppress the phosphorylation of ros1 as well as its downstream anti - apoptotic and growth signaling . in this study , the inventors also have found the new role and function of mitoxantrone by directly targeting ros1 kinase , which is potentially useful to be developed as new ros1 kinase inhibitor to treat lung cancer , in particular non - small cell lung cancer . the chemical structure of mitoxantrone was shown in fig1 . hcc78 cell line was purchased from the american type culture collection ( atcc ) ( manassas , va ., usa ). cells were cultured in rpmi - 1640 medium containing 10 % fetal bovine serum ( fbs ), 100 units / ml penicillin , and 100 μg / ml streptomycin under a humidified atmosphere of 5 % co 2 at 37 ° c . mitoxantrone was dissolved in dmso . lanthascreen ® eu kinase binding assay was used to determine the changes in kinase activity , which is an assay based on the binding and displacement property of the kinase with substrate . briefly , the assay was performed at room temperature for 1 hour in a total volume of 15 μl , including 5 μl of test compound , 5 μl of kinase / antibody mixture and 5 μl of tracer . the signal of the 384 - well plate was then measured at the end of the reactions . emission ratio was calculated by dividing the acceptor / tracer emission signal ( 665 nm ) by the antibody / donor emission signal ( 615 nm ). 3000 cells were seeded on 96 - well plates , then the plates were cultured overnight for cell adhesion , and treated with dmso or various concentrations of mitoxantrone for 72 h . 10 μl of mtt ( 5 mg / ml ; sigma ) was added in each well , and incubation was continued for another 4 hours , then the dark blue crystals were dissolved in 100 μl resolved solution ( 10 % sds and 0 . 1 mm hcl ). finally , the absorbance was measured at 570 nm by a microplate reader ( tecan , morrisville , n . c ., usa ). the cell viability was calculated relative to untreated controls , and results were presented based on at least 3 independent experiments . hcc78 cells were seeded on a 6 - well plate , and were allowed to attach overnight . the cells were treated with a dilution series of mitoxantrone for 48 h , collected by trypsin and washed twice with ice - cold pbs , and then resuspended in 100 μl 1 × binding buffer . 2 μl annexin - v fitc and 4 μl pi ( 100 μg / ml ) were added to the solution and mixed well . after incubation for 15 min in the dark at room temperature , 400 μl of 1 × binding buffer was added to each tube , where apoptosis level was then quantitatively analyzed using a facsaria iii flow cytometer ( bd biosciences , san jose , calif ., usa ). after cells were plated on 6 - well plates overnight , a wide concentrations of mitoxantrone were administered for 48 h . cells were washed twice with cold pbs and then lysed in ripa lysis buffer containing protease and phosphatase inhibitors , and protein concentration of the cell lysates were measured using the bio - rad protein assay kit ( bio - rad , philadelphia , pa ., usa ). after equalizing the protein concentrations of the samples , 5 × laemmli buffer was added and boiled at 100 ° c . for 5 min . equal amounts of protein samples ( 30 μg ) were subjected to sds - page and separated with a 10 % gel ; then the separated proteins were transferred to a nitrocellulose ( nc ) membrane , which was then exposed to 5 % non - fat dried milk in tbs containing 0 . 1 % tween 20 ( 0 . 1 % tbst ) for 1 h at room temperature , followed by overnight incubation at 4 ° c . with primary anti - bodies ( 1 : 1000 dilutions ). after being washed 3 times by tbst , the membranes were incubated with secondary fluorescent antibodies ( 1 : 10000 dilutions ) to rabbit or mouse , the signal intensity of the membranes was detected by an li - cor odessy scanner ( belfast , me ., usa ). antibodies against gapdh , p - akt , p - ros1 , ros1 , p - erk , erk , p - stat3 , stat3 were purchased from cell signaling technology . anti - akt antibody was acquired from santa cruz biotechnology . molecular docking calculation is performed to study the binding mode of mitoxantrone to ros1 kinase by using induced fit docking module in schrodinger software ( schrodinger , inc ., new york , n . y ., 2009 ). the structure of mitoxantrone is prepared and optimized in the ligprep module . the 3d structure of ros1 kinase is derived from the pdb database ( pdb id : 3zbf ) and prepared using the protein preparation wizard . in the molecular docking calculation , crizotinib is used to define the active site . the binding pose of ligand is evaluated with xp docking score . the pose with the best score is selected for further analysis . statistical analysis was conducted using graph prim5 . 0 . difference between datasets was assessed using on - way anvoa . p & lt ; 0 . 05 was considered statistically significant . by performing biochemical enzymatic assays , mitoxantrone was shown in the table 1 to be an atp - competitive inhibitor of ros1 with ic 50 value of 36 . 03 nm , while the ic 50 value of the control drug crizotinib is 52 . 76 nm , indicating that mitoxantrone is even more effective in inhibiting ros1 kinase than the current clinically - applied drug crizotinib . the kinase activity assay of mitoxantrone on hcc78 was shown in fig2 a , and crizotinib was used as positive control drug to demonstrate positive ros kinase inhibition ability as shown in fig2 b . 2 . 2 cytotoxicity effect of mitoxantrone on hcc78 nsclc cells ( with ros1 fusion gene ) and normal lung cells ( ccd19 - lu ) mtt assay showed that mitoxantrone has an inhibition activity against hcc78 cell . the cell viability of hcc78 cells treated by mitoxantrone for 72 h is shown in fig3 a and that that of normal lung cell ccd19 - lu for 72 h treatment by mitoxantrone is shown in fig3 b . as shown in table 2 , the ic 50 of mitoxantrone on hcc78 is 13 . 83 ± 2 . 49 nm , while a lower cytotoxicity on normal lung cells ( ccd19 - lu ) was shown after 72 h treatment . the ic 50 in ccd19 - lu is higher than 160 nm , which is at least 11 . 6 fold than that on hcc78 . when hcc78 cells were treated with mitoxantrone , cells detached and became smaller starting at 10 nm treatment as shown in fig4 , which is a morphological indication of apoptosis . next , by using a more quantitative apoptosis measurement method , the flow cytometry analysis in fig5 a - 5f showed that mitoxantrone exhibits anticancer ability through induction of apoptosis on hcc78 cells in a concentration - dependent manner . treatment on hcc78 cells with mitoxantrone induced significant apoptosis levels when compared with the control group . 2 . 4 mitoxantrone led to a dose - dependent suppression of ros1 phosphorylation as well as its downstream anti - apoptotic and growth signaling as shown in fig6 , treatment of hcc78 cells with mitoxantrone led to a dose - dependent decrease of ros1 phosphorylation as well as its downstream signaling molecules erk1 / 2 , stat3 and akt signaling , further supporting the anti - cancer effect of mitoxantrone and elucidating its treatment mechanism . crizotinib was included as positive control . molecular docking calculations prove that mitoxantrone and crizotinib have similar binding mode with ros1 kinase . the docking scores of mitoxantrone and crizotinib to ros1 kinase are − 13 . 582 and − 9 . 674 kcal / mol , respectively . mitoxantrone is shown to have a better binding affinity to ros1 kinase than crizotinib . this is consistent with the results from both mtt and enzyme inhibition activity . fig7 a shows the n between crizotinib and ros1 kinase . as observed in fig7 a and fig7 b , the anthracenedione scaffold of mitoxantrone overlapped well with the core structure of crizotinib . as further illustrated in fig7 b , residues leu1951 , glu2027 , met2029 , asp2033 , asp2083 , lys2090 of the ros1 kinase had hydrogen bond interaction with mitoxantrone as shown by broken lines in red . residues glu2027 and met2029 in the hinge region formed multiple hydrogen bonds with the polar atoms of anthracenedione part , while residues leu1951 , asp2033 , asp2083 and lys2090 had hydrogen bonds with the two substituted “ tails ”. other residues , such as val1959 , leu2012 , leu2026 , leu2086 , also had hydrophobic interaction with mitoxantrone . the exemplary embodiments of the present invention are thus fully described . although the description referred to particular embodiments , it will be clear to one skilled in the art that the present invention may be practiced with variation of these specific details . hence this invention should not be construed as limited to the embodiments set forth herein .
the present invention discloses that a method of treating cancer , preferably non - small cell lung cancer , comprises administrating mitoxantrone . the present invention also discloses a method of inhibiting ros1 kinase comprising administrating mitoxantrone . a pharmaceutical composition comprising mitoxantrone is also disclosed .