Patent Application: US-201514799541-A

Abstract:
the present invention provides a compound of formula as a serca inhibitor for treating cancers , a pharmaceutical composition comprising said compound , and methods of using said compound for treating cancers and / or inducing cell death in cells of said cancers . said cancers include but not limited to cervical , lung , liver , breast , and prostate cancer . said cancers also include drug - resistant and / or apoptosis - resistant cancers such as isogenic drug - resistant colon cancer . the subject being administered with said compound or the composition comprising thereof can be human or animal subject . said methods for treating cancers and / or inducing cell death can be a targeting treatment for certain cancers .

Description:
the following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention . they should not be considered as limiting the scope of the invention , but merely as being illustrative and representative thereof . in vitro cytotoxicity test of pomiferin in a panel of human or mouse cancer cells pomiferin is dissolved in dmso at a final concentration of 100 mmol / l and stored at − 20 ° c . cytotoxicity is assessed using the 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide assay . 4000 - 8000 hela ( human cervical cancer ), mcf - 7 ( human breast cancer ), hepg2 and hep3b ( human liver cancer ), h1299 and a549 ( human lung cancer ), lncap ( human prostate cancer ) and llc - 1 ( mouse lewis lung carcinoma ) cells are seeded on 96 - well plates per well . after overnight pre - incubation , the cells are exposed to different concentrations of pomiferin ( 0 . 039 - 100 mol / l ) for 3 days . subsequently , 10 μl of mtt reagents is added to each well and incubated at 37 ° c . for 4 hours followed by the addition of 100 μl solubilization buffer ( 10 % sds in 0 . 01 mol / l hcl ) and overnight incubation . absorbance at 585 nm is determined from each well the next day . the percentage of cell viability is calculated using the following formula : cell viability (%)= cells number treated / cells number dmso control × 100 . data are obtained from three independent experiments . there is significant cell cytotoxicity with mean ic 50 value ranging from 3 . 82 - 20 . 3 μm observed in a panel of human and mouse cancer cells treated with pomiferin for 72 hours , which is revealed by mtt assay ( table 1 ). after pomiferin treatments in the presence or absence of lysosomal inhibitor 50 nm of bafilomycin a1 or autophagy inhibitor 5 mm 3 - ma , hela cancer cells are harvested and lysed in ripa buffer ( cell signaling technologies inc ., beverly , mass .). the cell lysates are then resolved by sds - page . after electrophoresis , the proteins from sds - page are transferred to nitrocellulose membrane which is then blocked with 5 % non - fat dried milk for 60 minutes . the membrane is then incubated with lc3 primary antibodies ( 1 : 1000 ) in tbst overnight at 4 ° c . after that , the membrane is further incubated with hrp - conjugated secondary antibodies for 60 minutes . finally , protein bands are visualized by using the ecl western blotting detection reagents ( invitrogen , paisley , scotland , uk ). gfp - lc3 puncta formation is quantified as previously described 13 . in brief , cells grown on coverslips in a 6 - well plate are treated with or without 10 μm of pomiferin for 24 hours , the cells are then fixed in 4 % paraformaldehyde for 20 minutes at room temperature and then rinsed with pbs . slides are mounted with fluorsave ™ mounting media ( calbiochem , san diego , calif .) and examined by fluorescence microscopy . the number of gfp - positive cells with gfp - lc3 puncta formation is captured and examined under the delta vision fluorescence microscope . to quantify for autophagy , the percentage of cells with punctate gfp - lc3 fluorescence is calculated by counting the number of the cells with punctate gfp - lc3 fluorescence in gfp - positive cells . a minimum of 150 cells from 3 randomly selected fields is scored . western blot analysis showed that the autophagic marker lc3 - ii conversion is induced upon pomiferin treatment as shown in fig1 a . in addition , pomiferin is able to further enhance the lc3 - ii conversion in the presence of lysosomal inhibitor ( bafilomycin a1 ) as illustrated in fig1 b . collectively , these data suggest that pomiferin is able to induce autophagy via increasing of autophagic flux . on the other hand , autophagic inhibitor , 3 - ma is used to confirm and validate the pomiferin - mediated autophagy . obviously , addition of 3 - ma could markedly suppress the pomiferin - mediated lc3 - ii conversion . bar chart represented the quantitation of lc3 - ii protein conversion ( fig1 c ). besides , fig1 d further demonstrated that the pomiferin - mediated gfp - lc3 autophagic puncta formation is significantly inhibited by 3 - ma . collectively , pomiferin is confirmed to induce autophagy in cancer cells via autophagic flux . pomiferin induces endogenous lc3 puncta formation in a panel of cancer and normal cells the detection of endogenous lc3 puncta formation is conducted using immunofluorescence staining method as described below . in brief , pomiferin - treated cancer cells ( hep3b , hepg2 , h1299 , mcf - 7 and a549 ) or normal human hepatocyte ( lo2 ) on cover slips are fixed with 4 % paraformaldehyde ( sigma ) for 20 min at room temperature and then rinsed with pbs . immerse coverslips in methanol at room temperature for 2 min . after ishing with pbs , the cells are then incubated with anti - lc3 ( 1 : 200 ) in tbst ( 100 mm tris hcl , ph 7 . 5 , 150 mm nacl , 0 . 05 % tween 20 and 5 % bsa ) overnight at 4 . after washing with pbs , the cells are incubated with anti - mouse secondary antibody ( tritc ) 1 : 200 in tbst containing 5 % bsa at 37 for 1 hrs in the dark . the coverslips are then mounted with fluorsave ™ mounting media ( calbiochem , san diego , calif ., usa ) for fluorescence imaging and localization of lc3 autophagosomes are captured under the api delta vision live - cell imaging system ( applied precision inc ., ge healthcare company , washington , usa ). to quantify autophagy , guidelines are followed to monitor autophagy 20 , the percentage of cells with punctuate lc3 immunofluorescence staining is calculated by counting the number of the cells showing the punctuate pattern of lc3 fluorescence (& gt ; 10 dots / cell ) in immunofluorescence positive cells over the total number of cells in the same field . a minimum of 1000 cells from randomly selected fields are scored . fig2 indicated that pomiferin induces tritc - lc3 puncta formation in all tested cancer and normal cells , suggesting that the pomiferin - induced autophagy is not cell - type specific . hela cancer cells treated with indicated time and concentrations of pomiferin are harvested and lysed in ripa buffer ( cell signaling ). the cell lysates are then resolved by sds - page . after electrophoresis , the proteins from sds - page are transferred to nitrocellulose membrane which is then blocked with 5 % non - fat dried milk for 60 minutes . the membrane is then incubated with p - p70s6k , p70s6k , p - ampk , ampk and actin primary antibodies ( 1 : 1000 ) in tbst overnight at 4 ° c . respectively . after that , the membrane is further incubated with hrp - conjugated secondary antibodies for 60 minutes . finally , protein bands are visualized by using the ecl western blotting detection reagents ( invitrogen ). after pomiferin treatment in the presence or absence of ampk inhibitor compound c [ 5 μm ] or camkk - β inhibitor sto - 609 [ 25 μm ], hela cancer cells are harvested and lysed in ripa buffer ( cell signaling technologies inc . ( beverly , mass .). the cell lysates are then resolved by sds - page . after electrophoresis , the proteins from sds - page are transferred to nitrocellulose membrane which is then blocked with 5 % non - fat dried milk for 60 minutes . the membrane is then incubated with lc3 primary antibodies ( 1 : 1000 ) in tbst overnight at 4 ° c . after that , the membrane is further incubated with hrp - conjugated secondary antibodies for 60 minutes . finally , protein bands are visualized by using the ecl western blotting detection reagents ( invitrogen , paisley , scotland , uk ). gfp - lc3 puncta formation is quantified as previously described 13 . in brief , cells grown on coverslips in a 6 - well plate are incubated with 10 μm of pomiferin in the presence or absence of ampk inhibitor compound c [ 5 μm ] or camkk - β inhibitor sto - 609 [ 25 μm ] for 24 hours , the cells are then fixed in 4 % paraformaldehyde for 20 minutes at room temperature and then rinsed with pbs . slides are mounted with fluorsave ™ mounting media ( calbiochem , san diego , calif .) and examined by fluorescence microscopy . the number of gfp - positive cells with gfp - lc3 puncta formation is captured and examined under the delta vision fluorescence microscope . to quantify for autophagy , the percentage of cells with punctate gfp - lc3 fluorescence is calculated by counting the number of the cells with punctate gfp - lc3 fluorescence in gfp - positive cells . a minimum of 150 cells from 3 randomly selected fields is scored . as shown in fig3 a , pomiferin is found to activate the phosphorylation of ampk in a time dependent manner and this activation is also accompanied by a concomitant reduction in its mtor downstream p70s6k phosphorylation . in order to demonstrate whether the upstream of ampk signaling is involved in pomiferin - induced autophagy , specific inhibitors such as ampk inhibitor , compound c and camkk - β inhibitor , sto - 609 are used in the study . results showed that there is a significant reduction in pomiferin - induced lc3 - ii conversion and gfp - lc3 puncta formation in hela cells treated with the presence of ampk inhibitor ( compound c ) ( fig3 b & amp ; c ) and camkk - β inhibitor , sto - 609 ( fig3 d & amp ; e ). these findings further suggested that pomiferin induces autophagy via activation of ampk - mtor signaling pathways . pomiferin mobilizes cytosolic calcium for induction of autophagy in hela cancer cells changes in intracellular free calcium are measured by a fluorescent dye , fluo - 3 , as described previously 21 . briefly , hela cells are washed twice with mem medium after pomiferin treatment ( 10 μm ) for various times ( 10 min , 0 . 5 h , 1 h , 2 h , 4 h ). then cell suspensions are incubated with 5 μm fluo - 3 at 37 ° c . for 30 min . then the cells are washed twice with hbss . after re - suspended cell samples are subjected to facs analysis , at least 10 , 000 events are analyzed . after pomiferin treatment in the presence or absence of calcium chelator , bapta / am [ 10m ], hela cancer cells are harvested and lysed in ripa buffer ( cell signaling technologies inc . ( beverly , mass .). the cell lysates are then resolved by sds - page . after electrophoresis , the proteins from sds - page are transferred to nitrocellulose membrane which is then blocked with 5 % non - fat dried milk for 60 minutes . the membrane is then incubated with lc3 primary antibodies ( 1 : 1000 ) in tbst overnight at 4 ° c . after that , the membrane is further incubated with hrp - conjugated secondary antibodies for 60 minutes . finally , protein bands are visualized by using the ecl western blotting detection reagents ( invitrogen , paisley , scotland , uk ). quantification of autophagy gfp - lc3 puncta : gfp - lc3 puncta formation is quantified as previously described 13 . in brief , cells grown on coverslips in a 6 - well plate are incubated with 10 μm of pomiferin in the presence or absence of calcium chelator , bapta / am [ 10m ] for 24 hours , the cells are then fixed in 4 % paraformaldehyde for 20 minutes at room temperature and then rinsed with pbs . slides are mounted with fluorsave ™ mounting media ( calbiochem , san diego , calif .) and examined by fluorescence microscopy . the number of gfp - positive cells with gfp - lc3 puncta formation is captured and examined under the delta vision fluorescence microscope . to quantify for autophagy , the percentage of cells with punctate gfp - lc3 fluorescence is calculated by counting the number of the cells with punctate gfp - lc3 fluorescence in gfp - positive cells . a minimum of 150 cells from 3 randomly selected fields is scored . given that calcium mobilization in cells will contribute to autophagy induction , and fig3 d & amp ; e indicated that camkk - β is involved in pomiferin - mediated autophagy induction , suggesting that calcium may involve for pomiferin - mediated cellular functions . this example further demonstrated that pomiferin is able to increase the cytosolic calcium level in a time dependent manner as shown in fig4 a . to examine whether the release of cytosolic calcium would contribute autophagy , a calcium chelator , bapta / am , is adopted to validate the pomiferin - induced autophagic effect . as expected , addition of bapta / am could markedly suppress the pomiferin - mediated lc3 - ii conversion ( fig4 b ). fig4 c also indicated that bapta / am suppressed pomiferin - mediated lc3 - ii conversion as compared to the same which was treated with pomiferin only . concomitantly , the pomiferin - induced autophagic puncta formation was also inhibited in the presence of bapta / am . collectively , pomiferin induces autophagy via the mobilization of cytosolic calcium in cancer cells . the 3d structure of pomiferin is obtained from the pubchem ( http :// pubchem . ncbi . nlm . nih . gov ). then , the compound is preprocessed by the ligprep 22 which uses opls - 2005 force field 23 to obtain the corresponding low energy 3d conformers . the ionized state is assigned by using epik3 at a target ph value of 7 . 0 ± 2 . 0 . the 3d crystal structure of the sarco ( endo ) plasmic reticulum ca2 + atpase ( serca ) for molecular docking is retrieved from the protein data bank ( pdb id code 2agv ) 24 . the protein preparation wizard is used to remove crystallographic water molecules , add hydrogen atoms , and assign partial charges based on opls - 2005 force field 25 . energy minimization is also performed and terminated when the root - mean - square deviation ( rmsd ) reached a maximum value of 0 . 3 a . pomiferin is docked into the thapsigargin ( tg ) binding site of the serca using glide program 26 with the extra precision ( xp ) scoring mode . the docking grid box is defined by centering on tg in the serca . purified ca 2 + atpase ( serca1a ) is prepared from female rabbit hind leg muscle 27 . atpase activity is determined using the enzyme - coupled method utilizing pyruvate kinase and lactate dehydrogenase as previously described in michelangeli et al . ( 1990 ). all serca inhibition data are fitted to the allosteric dose versus effect equation using fig p ( biosoft ): in order to explore the possible binding poses of pomiferin in serca , molecular docking method was applied . the performance of molecular docking is usually evaluated by re - docking the crystal structure pose . herein , tg in the x - ray co - crystallized complex 2agv 24 is re - docked into the binding sites and the xp docking score was − 7 . 23 kcal / mol . the rmsd of the atomic positions between the ligand and the docked pose was 1 . 78 å , which means the atoms of the tg in the docked pose is coincided with the ligand atoms in the crystal structure . the calculated interaction energy ( xp docking score ) for pomiferin was − 5 . 86 kcal / mol . fig5 illustrated the structure of pomiferin docked into the serca tg binding site . in the predicted binding pose of the pomiferin ( fig5 a ), the hydrophobic groups of pomiferin made favorable hydrophobic effects and van der waals interactions with residues phe256 , leu260 , val263 , ile267 , ile264 , leu302 , val772 , val773 , ile765 , val769 , pro827 , leu828 , ile829 , ser830 , and phe834 . additionally , pomiferin was found to form hydrogen bond with residue glu255 . to ascertain whether the serca pump is suppressed by pomiferin , the serca inhibitory effect was quantified using purified rabbit skeletal muscle sarcoplasmic reticulum ( sr ) membranes , which measured the activity from the serca1a isoform in the sr membrances 28 . most of existing serca inhibitors show similar inhibitory effect in serca isoform [ 15 , 16 ] . the serca1a pump ( from rabbit skeletal muscle sr ) is inhibited by pomiferin in a dose - dependent manner ( fig5 b ), which is fitted to an allosteric dose versus effect equation . taken together , pomiferin targets on serca for calcium mobilization in cancer cells . pomiferin requires autophagy - related gene 7 ( atg7 ) for autophagy induction and induces autophagic cell death quantification of endogenous autophagic lc3 puncta in atg7 wild type and deficient mefs : endogenous lc3 puncta formation is quantified as previously described 2 . in brief , both atg7 wild - type and deficient mouse embryonic fibroblasts ( mefs ) grown on coverslips in a 6 - well plate are treated with indicated concentrations of pomiferin . both atg7 wild - type and deficient mouse embryonic fibroblasts are then fixed in 4 % paraformaldehyde for 20 minutes at room temperature and then rinsed with pbs . after washing with pbs , the cells are then incubated with anti - lc3 ( 1 : 200 ) in tbst ( 100 mm tris hcl , ph 7 . 5 , 150 mm nacl , 0 . 05 % tween 20 and 5 % bsa ) overnight at 4 . after washing with pbs , the cells are incubated with anti - mouse secondary antibody ( tritc ) 1 : 200 in tbst containing 5 % bsa at 37 for 1 hrs in the dark . the coverslips are then mounted with fluorsave ™ mounting media ( calbiochem , san diego , calif ., usa ) for fluorescence imaging and localization of lc3 autophagosomes are captured under the api delta vision live - cell imaging system ( applied precision inc ., ge healthcare company , washington , usa ). to quantify for autophagy , the percentage of cells with punctate tritc - lc3 fluorescence is calculated by counting the number of the cells with punctate tritc - lc3 fluorescence in tritc - positive cells . a minimum of 150 cells from 3 randomly selected fields is scored . cell viability is measured using an annexin v staining kit ( bd biosciences , san jose , calif ., usa ). briefly , atg7 wild - type ( atg7 +/+ or atg7 - wt ) and atg7 deficient ( atg7 −/− or atg7 - ko ) mouse embryonic fibroblasts ( mefs ) are treated with the 2 μm pomiferin for 24 h . cells are then harvested and analysed by multiparametric flow cytometry using fitc - annexin v and propidium iodide staining ( bd biosciences , san jose , calif ., usa ) according to the manufacturer &# 39 ; s instructions . flow cytometry is then carried out using a facscalibur flow cytometer ( bd biosciences , san jose , calif ., usa ). data acquisition and analysis is performed with cellquest ( bd biosciences , san jose , calif ., usa ). data are obtained from three independent experiments . pomiferin is found to induce tritc - lc3 puncta formation in wild type atg7 cells ( atg7 +/+) but not in atg7 - knockout ( atg7 −/−) mouse embryonic fibroblasts as shown in fig6 a . thus , pomiferin works as a novel autophagy enhancer which depends on autophagy related gene , atg7 , for the induction of autophagy . to determine the role of pomiferin - induced autophagy in cell death , we adopted the atg7 wild - type and deficient mefs to investigate the pomiferin - mediated autophagic effect . as shown in fig6 b , pomiferin was found to markedly induce cell death in atg7 +/+ cells , but not in autophagy deficient cells ( atg7 −/−). these findings suggest that pomiferin - mediated cell death is autophagy dependent ; in other words , pomiferin is able to induce autophagic cell death . pomiferin exhibits potent cytotoxic effect and induces autophagy in a panel of apoptosis - resistant cells the test compound of pomiferin is dissolved in dmso at a final concentration of 100 mmol / l and stored at − 20 ° c . cytotoxicity is assessed using the 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide assay as previously described 29 . 2500 of caspase wild - type ( caspase wt ), caspase - 3 deficient ( caspase 3ko ), caspase - 7 deficient ( caspase 7ko ), caspase - 3 /- 7 deficient ( caspase 3 / 7 dko ), caspase - 8 deficient ( caspase 8ko ), bax - bak wild - type ( bak - bak wt ) and bax - bak double knock out ( bak - bak dko ) mouse embryonic fibroblasts ( mefs ) are seeded on 96 - well plates per well . after overnight pre - incubation , the cells are exposed to different concentrations of pomiferin ( namely 100 , 50 , 25 , 12 . 5 , 6 . 25 , 3 . 125 , 1 . 5625 , 0 . 78 , 0 . 39 , 0 . 195 , 0 . 079 , 0 . 039 μmol / l ) for 3 days . subsequently , 10 μl of mtt reagents is added to each well and incubated at 37 ° c . for 4 hours , followed by the addition of 100 μl solubilization buffer ( 10 % sds in 0 . 01 mol / l hcl ) and overnight incubation . absorbance at 585 nm is determined from each well on the following day . the percentage of cell viability is calculated using the following formula : cell viability (%)= cells number treated / cells number dmso control × 100 . data is obtained from three independent experiments . endogenous lc3 puncta formation is quantified as previously described 2 . in brief , caspase wild - type ( caspase wt ), caspase - 3 deficient ( caspase 3ko ), caspase - 7 deficient ( caspase 7ko ), caspase - 3 /- 7 deficient ( caspase 3 / 7 dko ), caspase - 8 deficient ( caspase 8ko ), bax - bak wild - type ( bak - bak wt ) and bax - bak double knock out ( bak - bak dko ) mefs are grown on coverslips in a 6 - well plate are treated with 5 m of pomiferin . both wild - type and deficient mefs are then fixed in 4 % paraformaldehyde for 20 minutes at room temperature and then rinsed with pbs . after washing with pbs , the cells are then incubated with anti - lc3 ( 1 : 200 ) in tbst ( 100 mm tris hcl , ph 7 . 5 , 150 mm nacl , 0 . 05 % tween 20 and 5 % bsa ) overnight at 4 . after washing with pbs , the cells are incubated with anti - mouse secondary antibody ( tritc ) 1 : 200 in tbst containing 5 % bsa at 37 for 1 hrs in the dark . the coverslips are then mounted with fluorsave ™ mounting media ( calbiochem , san diego , calif ., usa ) for fluorescence imaging and localization of lc3 autophagosomes are captured under the api delta vision live - cell imaging system ( applied precision inc ., ge healthcare company , washington , usa ). pomiferin was found to exhibit similar cytotoxic effect on both wild - type and apoptosis - resistant cells , i . e . caspase - 3 /- 7 /- 8 as compared to the caspase wild - type mefs as shown in table 2 . in addition , it also showed similar cytotoxicity in bax - bak dko apoptosis - resistant cells as compared to bax - bak wild - type mefs ( table 2 ), indicating that pomiferin is able to induce cell death in apoptosis - resistant cells . in addition , pomiferin was able to induce autophagy in all these wild - type and apoptosis - resistant cells ( fig7 ). cell death is measured using an annexin v staining kit ( bd biosciences , san jose , calif ., usa ). briefly , bax - bak wild - type and bax - bak dko mefs are treated with the 5 μm pomiferin for 24 h . cells are then harvested and analysed by multiparametric flow cytometry using fitc - annexin v and propidium iodide staining ( bd biosciences , san jose , calif ., usa ) according to the manufacturer &# 39 ; s instructions . flow cytometry is then carried out using a facscalibur flow cytometer ( bd biosciences , san jose , calif ., usa ). data acquisition and analysis is performed with cellquest ( bd biosciences , san jose , calif ., usa ). data are obtained from three independent experiments . as shown in fig8 a , 5 μm of pomiferin only showed ˜ 40 % of cell death in bax - bak wild - type mefs , however , it demonstrated around ˜ 80 % of cell death in bax - bak dko mefs . these findings suggested that pomiferin shows collateral sensitivity toward the apoptosis - resistant cells . cell death is measured using an annexin v staining kit ( bd biosciences , san jose , calif ., usa ). briefly , bax - bak dko mefs are treated with the 5 μm pomiferin in the presence or absence of 5 mm autophagic inhibitor 3 - ma for 24 h . cells are then harvested and analysed by multiparametric flow cytometry using fitc - annexin v and propidium iodide staining ( bd biosciences , san jose , calif ., usa ) according to the manufacturer &# 39 ; s instructions . flow cytometry is then carried out using a facscalibur flow cytometer ( bd biosciences , san jose , calif ., usa ). data acquisition and analysis is performed with cellquest ( bd biosciences , san jose , calif ., usa ). data are obtained from three independent experiments . as shown in fig8 b , pomiferin alone significantly induced cell death in bax - bak dko apoptosis - resistant cells , whereas addition of autophagic inhibitor 3 - ma markedly suppressed the pomiferin - mediated cytotoxicity , suggesting that pomiferin induce cell death in apoptosis - resistant cells via autophagy induction . pomiferin induces cell death in drug - resistant hct - 116 p 53 deficient isogenic colon cancer cells via calcium mobilization and autophagy induction the test compound of pomiferin is dissolved in dmso at a final concentration of 100 mmol / l and stored at − 20 ° c . cytotoxicity is assessed using the 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide assay as previously described 29 . 4000 of drug - resistant hct - 116 p53 +/+, hct - 116 p53 −/− and hct - 116 p53 −/− isogenic colon cancer cells are seeded on 96 - well plates per well . after overnight pre - incubation , the cells are exposed to different concentrations of pomiferin ( namely 100 , 50 , 25 , 12 . 5 , 6 . 25 , 3 . 125 , 1 . 5625 , 0 . 78 , 0 . 39 , 0 . 195 , 0 . 079 , 0 . 039 mol / l ) for 3 days . subsequently , 10 μl of mtt reagents is added to each well and incubated at 37 ° c . for 4 hours , followed by the addition of 100 μl solubilization buffer ( 10 % sds in 0 . 01 mol / l hcl ) and overnight incubation . absorbance at 585 nm is determined from each well on the following day . the percentage of cell viability is calculated using the following formula : cell viability (%)= cells number treated / cells number dmso control × 100 . data is obtained from three independent experiments . cell death is measured using an annexin v staining kit ( bd biosciences , san jose , calif ., usa ). briefly , drug - resistant hct - 116 p53 −/− deficient colon cancer cells are treated with the 10 μm pomiferin in the presence or absence of 5 mm autophagic inhibitor 3 - ma or 10 m calcium chelator bapta / am for 24 h . cells are then harvested and analysed by multiparametric flow cytometry using fitc - annexin v and propidium iodide staining ( bd biosciences , san jose , calif ., usa ) according to the manufacturer &# 39 ; s instructions . flow cytometry is then carried out using a facscalibur flow cytometer ( bd biosciences , san jose , calif ., usa ). data acquisition and analysis is performed with cellquest ( bd biosciences , san jose , calif ., usa ). data are obtained from three independent experiments . pomiferin was found to exhibit similar cytotoxic effect on hct - 116 isogenic colon cancer cells with different p53 status ( table 3 ). in addition , pomiferin alone significantly induced cell death in drug - resistant hct - 116 p53 −/− colon cancer cells , whereas addition of autophagic inhibitor 3 - ma or calcium chelator bapta / am could markedly suppress the pomiferin - induced cell death in this drug - resistant cancer ( fig9 a & amp ; b ). taken together , these findings suggested that pomiferin is able to kill drug - resistant cancer cells via calcium mobilization and autophagy induction . the present invention provides the potential use of pomiferin in developing drug for treating drug - resistant or apoptosis - resistant cancer via specific inhibition of serca in order to induce autophagy in the drug - resistant or apoptosis - resistant cancer cells . the following references are incorporated herein by reference in their entirety : 1 . denmeade , s . r . & amp ; 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tobin , d . j . in vitro and ex vivo examination of topical pomiferin treatments . fitoterapia 94 , 164 - 171 . 15 . vesela , d ., kubinova , r ., muselik , j ., zemlicka , m . & amp ; suchy , v . antioxidative and erod activities of osajin and pomiferin . fitoterapia 75 , 209 - 211 ( 2004 ). 16 . svasti , j ., et al . proteomic profiling of cholangiocarcinoma cell line treated with pomiferin from derris malaccensis . proteomics 5 , 4504 - 4509 ( 2005 ). 17 . son , i . h ., chung , i . m ., lee , s . i ., yang , h . d . & amp ; moon , h . i . pomiferin , histone deacetylase inhibitor isolated from the fruits of maclura pomifera . bioorg med chem lett 17 , 4753 - 4755 ( 2007 ). 18 . yang , r ., hanwell , h ., zhang , j ., tsao , r . & amp ; meckling , k . a . antiproliferative activity of pomiferin in normal ( mcf - 10a ) and transformed ( mcf - 7 ) breast epithelial cells . j agric food chem 59 , 13328 - 13336 ( 2011 ). 19 . bartosikova , l ., et al . [ examination of the antioxidative and antidiabetic effect of pomiferin in alloxan - induced diabetes mellitus in an experiment ( a pilot study )]. ceska slov farm 56 , 135 - 140 ( 2007 ). 20 . klionsky , d . j ., et al . guidelines for the use and interpretation of assays for monitoring autophagy . autophagy 8 , 445 - 544 . 21 . liu , m . j ., wang , z ., ju , y ., wong , r . n . & amp ; wu , q . y . diosgenin induces cell cycle arrest and apoptosis in human leukemia k562 cells with the disruption of ca2 + homeostasis . cancer chemother pharmacol 55 , 79 - 90 ( 2005 ). 22 . schrodinger , l . new york , n . y . ligprep , version 2 . 3 . ( 2009 ). 23 . kaminski , g . a . f ., r . a . ; tirado - rives , j . ; jorgensen , w . l . evaluation and reparametrization of the opls - aa force field for proteins via comparison with accurate quantum chemical calculations on peptides . j . phys . chem . b 105 , 6474 - 6487 ( 2001 ). 24 . obara , k ., et al . structural role of countertransport revealed in ca ( 2 +) pump crystal structure in the absence of ca ( 2 +). proc natl acad sci usa 102 , 14489 - 14496 ( 2005 ). 25 . epik . version 2 . 0 , schrodinger , llc , new york , n . y ., ( 2009 ). 26 . glide . version 5 . 5 , schrodinger , llc , new york , n . y . ( 2009 ). 27 . michelangeli , f . & amp ; munkonge , f . m . methods of reconstitution of the purified sarcoplasmic reticulum ( ca ( 2 +)- mg2 +)- atpase using bile salt detergents to form membranes of defined lipid to protein ratios or sealed vesicles . anal biochem 194 , 231 - 236 ( 1991 ). 28 . wu , k . d ., lee , w . s ., wey , j ., bungard , d . & amp ; lytton , j . localization and quantification of endoplasmic reticulum ca ( 2 +)- atpase isoform transcripts . am j physiol 269 , c775 - 784 ( 1995 ). 29 . wong , v . k ., zhou , h ., cheung , s . s ., li , t . & amp ; liu , l . mechanistic study of saikosaponin - d ( ssd ) on suppression of murine t lymphocyte activation . j cell biochem 107 , 303 - 315 ( 2009 ).