Patent Application: US-83662610-A

Abstract:
the invention is based on the surprising finding that treatment with a chemotherapeutic agent such as 5 - fluorouracil and an autophagy inducer effectively inhibit the continued growth of , and prevent the recovery following drug withdrawal , of cancer cells . in vivo , drug resistance from a failure to adequately engage in apoptotic programmed cell death leads to a recurrence of cancer and tumours can remain dormant for periods of time before re - emerging as drug resistant metastases . it has been hypothesised that autophagy may help cancer cells survive in response to growth limiting conditions , such as nutrient depletion , hypoxia , absence of growth factor , or presence of cytotoxic drug . licl is a known autophagy inducer and accelerates cell survival to autophagic programmed cell death .

Description:
established human esophageal cell lines oe19 , oe21 and oe33 were obtained from the european collection of cell cultures . kyse450 were from die deutsche managementsystem zertifizierungsgesellschaft mbh ( dsmz ). all cell lines were maintained in rpmi 1640 medium , 1 % penicillin / streptomycin and 10 % ( v / v ) foetal calf serum ( gibco , uk ) and grown at 37 ° c ., 5 % co 2 . cell growth / viability was assessed using the real - time cell analyzer . cells were seeded at 2 . 5 × 10 4 cells / cm 2 , in a 96 - well plate and treated for ˜ 80 hours . to assess recovery , at 80 hours post treatment all drugs were removed , culture medium was replaced and cells were cultured for an additional 85 hours , to monitor their ability to recover . viable cells remain adhered to the plate and the relative change in electrical impedance is measured to represent cell status . the cell index ( ci ) is a relative value , representing the impedance change divided by a background value . the greater the cell number adhered , the larger the impedance and as cells are lost , the impedance drops . measurements are taken every fifteen minutes for the first three hours after seeding and treatment , all other measurements of impedance are taken continually , every hour . additionally , a change in a cell status , such as morphology , cell adhesion or cell viability can lead to a change in ci . viability of esophageal cells was assessed using the mtt reduction assay . cells are seeded at 2 × 10 4 ( oe33 / oe19 ) and 1 × 10 4 ( oe21 / kyse450 ) cells per cm 2 , treated for 48 - 96 hours and incubated for an additional 60 minutes at 37 ° c . in 0 . 5 mg / ml mtt dye . viable , metabolizing cells reduce mtt dye , producing a dark formazan product , with absorbance read at 562 nm , reference wavelength 620 nm . to assess recovery , at 48 hours post treatment , in replicate plates ( identical seeding & amp ; treatment times ), all drugs were removed , culture medium was replaced , and these cells were cultured for a further 48 - 96 hours , to monitor their ability to recover and mtt assay was repeated . in analysis of mtt data , values are presented as the mean absorbance +/− standard error of the mean ( s . e . m ) for four independent experiments . statistical analysis was performed with paired student &# 39 ; s t - test . values of p & lt ; 0 . 05 were considered statistically significant . asterisks indicate the level of significance . morphological features of cells treated with 5 - fluorouracil ( 5 - fu )/ cisplatin , without and with 3 - ma , ly294002 , bafilomycin , lithium chloride , rapamycin or ha14 - i were examined by light microscopy . morphologies of treated cells were examined 24 and 48 hours post treatment , treatment times are referred to in figure legends . aliquots of vehicle control and drug treated cells were cytospun onto glass slides and stained with rapi - diff ( braidwood laboratories , uk ). the extent of apoptotic and non - apoptotic cell death was determined by counting the cells in at least three fields of view per slide , with an average of ˜ 100 cells per field . apoptotic cell death is characterized by the presence of two or more of the following morphological features : cell shrinkage , chromatin condensation , dna degradation and fragmentation into ‘ apoptotic bodies ’, within an intact plasma membrane . autophagic cell death was identified by clear elevation of cytoplasmic vesicles , loss of cytoplasmic material , pyknosis of the nuclear material and an intact nuclear membrane . cytospin images are representative of at least three independent experiments . to examine control and 5 - fluorouracil treated cells for evidence of caspase activity cells were processed by trypsinization , 48 hours after treatment . following fixation in 4 % para - formaldehyde , cells were washed in a permeabilisation buffer ( 0 . 1 % triton , 0 . 1 % sodium azide , 10 mm hepes , 4 % fcs , 150 mm nacl ) and incubated with a primary rabbit polyclonal anti - active caspase - 3 antibody ( bd biosciences uk ) on ice for 1 hr . this was detected with an anti rabbit fitc conjugated secondary antibody , and samples were analyzed by facscan at 530 nm ( fl - 1 ). percentages indicate the proportion of cells with active caspase - 3 , detected as an increase in the number of fitc ( fl - 1 ) labelled cells . similar results were observed in at least three independent experiments . mitochondrial membrane potential was determined using the jc - 1 probe ( molecular probes ). in non - apoptotic cells , jc1 accumulates as aggregates in the mitochondria , which stain red ( fl2 ; 590 nm ). at the onset of apoptosis , a loss of mitochondrial membrane potential ( δψm ) releases the aggregated jc1 , returning it to its monomeric form , which stains the cytosol green ( fl1 ; 530 nm ). therefore , fluorescence of the jc - 1 probe in the fl - 2 channel decreases as mitochondrial membrane integrity is lost , while the fluorescence in fl - 1 channel increases . cells were incubated in jc1 ( 7 . 5 μg / ml ) at 37 ° c . for 15 mins , and washed prior to analysis by flow cytometry ( facscan , becton dickinson ). percentages denote the proportion of cells with depolarised mitochondria following 48 hour incubation with 5 - fluorouracil and represent at least three independent experiments . cells were seeded on semi - porous membranes and incubated in 5 - fluoruracil ( 5 - fu ) for 48 hours . cells were then fixed in a 0 . 165 mm phosphate buffer ( ph 7 . 4 ), containing 2 . 0 % glutaraldehyde , at room temperature ( rt ) for 40 minutes . cells were post - fixed in osmium tetroxide ( oso 4 ) at rt for 60 minutes , dehydrated in ascending grades of ethanol solutions ( 50 %, 70 %, 95 %, 100 % and 100 % dry ), prior to embedding in araldite resin . samples were subjected to a graded infiltration process with araldite ( epoxy resin ) before being set and sectioned . representative areas were chosen for ultra - thin sectioning and samples were examined by electron microscopy . total cellular protein extracts were prepared by scraping the cells into modified ripa buffer ( 50 mm tris hcl ( ph 7 . 4 ), 150 mm nacl , 0 . 25 % sodium deoxycholate , 1 % igepal , protease inhibitors 1 mm edta , 1 × pefabloc , 1 × protease inhibitor cocktail , 1 mm na 3 vo 4 , 1 mm naf ). all protein samples were separated by sds - page ( 10 / 12 %) and electrophoretically transferred onto nitrocellulose membrane . all primary antibodies were incubated overnight at 4 ° c . : anti - phospho mtor ( ser 2448 ) and anti - p70 s6k ( thr 389 ) ( cell signaling technologies , uk ), anti - beclin - 1 ( bd biosciences , uk ) and anti - lc3 ( medical & amp ; biological laboratories , japan ). the membranes were incubated with the relevant horseradish peroxidase conjugated secondary antibodies ( dakocytomation , dublin ) and detected by chemiluminescence ( ecl amersham , uk ). to visualize and quantify the formation of autophagic vesicles , the green fluorescent protein ( gfp )- lc3 ( pegfp - lc3 ) expression vector , kindly supplied by dr . t . yoshimori ( national institute of genetics , japan ) was used . cells were transiently transfected with the amaxa electroporation system according to the supplier &# 39 ; s protocol . twenty - four hours post transfection , cells were treated with 5 - fu and / or cisplatin , fixed in 4 % paraformaldehyde in pbs and transferred onto slides using a non - fluorescent fixative for analysis by fluorescence microscopy . alternatively , western blot analysis was used to assess the expression and processing of lc3 . upon stimulation of autophagy , lc3 is up - regulated and processed from soluble gfp - lc3i ( 45 kda ) to the autophagosome - associated form gfp - lc31i ( 43 kda ). the membrane sequestered , lipid - conjugated form of lc3 - ii remains with the autophagosome membrane after the vesicle has formed , and levels of both isoform are detected by western blot . transfection efficiency was consistent for a given cell line , oe33 and kyse450 cell lines (˜ 60 - 70 %) compared to oe19 and oe21 cell lines (˜ 30 %). mdc is an autofluorescent weak base that accumulates in acidic lysosomal vacuoles , showing high selectivity for autophagosomes , due to the high level of unhydrolyzed membrane lipids from engulfed organelles , which enhance mdc fluorescence . cells were incubated with 0 . 1 mm mdc in pbs at 37 ° c . for 10 minutes ( biederbick et al ., 1995 ), washed and immediately analyzed by fluorescent microscopy . gene silencing with sirna was used to inhibit mammalian beclin1 ( ortholog of atg6 ). cells were transfected with a pre - designed sirna ( 20 - 50 nm ) against beclin1 ( dharmacon on - targetplus smartpool human becn1 , nm — 003766 ) using the transfection reagent lipofectamine 2000 ( invitrogen , ireland ). the transfection efficiency was greater than 60 % ( transfection efficiency was assessed visually using fluorescently tagged rna duplexes , dharmacon ) and the extent of beclin1 silencing was determined by western blot analysis of protein levels . cell death induced by 5 - fluorouracil ( 5 - fu ) and cisplatin in esophageal cancer cells a panel of four esophageal cell lines was evaluated , two squamous ( oe21 / kyse450 ) and two adenocarcinoma ( oe19 / oe33 ), for their sensitivity to the chemotherapeutic drugs 5 - fu and cisplatin . at a range of drug concentrations , oe21 and oe33 cell lines are significantly more sensitive than oe19 and kyse450 cell lines . for example , 10 μm cisplatin induced significant effects on cell viability in both oe21 / oe33 , while the oe19 / kyse450 cell lines were only marginally affected ( fig1 a ). the more drug sensitive esophageal cell lines ( oe21 / oe33 ) induced a predominantly apoptotic cell death morphology ( type i pcd ), in response to both 5 - fu and cisplatin ( arrows , fig1 b ), with low levels of non - apoptotic cell death morphology . at 40 μm cisplatin , oe21 cells display 31 % apoptotic / 3 % non - apoptotic cell death ( fig1 c ). the more drug resistant oe19 / kyse450 cell lines display predominantly non - apoptotic morphology ( arrowheads , fig1 b ) with oe19 cells displaying only 0 . 5 % apoptosis / 38 % non - apoptotic cell death ( fig1 c ). this non - apoptotic morphology includes pyknosis of the nuclear material and vacuolization of the cytoplasm , features resembling those described for autophagic / type ii cell death . typical markers of apoptotic cell death were examined in all cell lines . both drug sensitive ( oe21 / oe33 ) cell lines displayed active caspase - 3 , and mitochondrial depolarization in response to 5 - fu and cisplatin ( fig2 a / b ). in contrast , the more drug resistant ( oe19 / kyse450 ) cell lines do not show caspase - 3 activity or mitochondrial membrane depolarization ( fig2 a / b ). ultra structural features of oe21 cells ( determined by electron microscopy ), incubated with 5 - fu , reveal morphological changes consistent with classical apoptotic cell death including marginalization of the nucleus , with an intact but blebbing plasma membrane ( upper middle fig3 a ). in contrast , oe19 treated cells retain an intact nuclear membrane with a distinct nucleolus , and the nuclei have areas of more electron dense heterochromatin . in addition , numerous cytoplasmic vacuoles are evident , many of which appear to surround cytoplasmic material and components , such as the mitochondria , resembling nascent autophagosomes ( upper / lower right fig3 a ). the expression and processing of gfp - lc3 was examined and western blot analysis showed no induction of a lower autophagosome - associated lc3ii - band in treated oe21 / oe33 cells ( fig3 b ). in contrast , a significant increase in lc3ii levels are evident in oe19 and kyse450 cells , following treatment with 5 - fu at 24 hours ( fig3 b ). redistribution of gfp - lc3 from a diffuse cytosolic to a punctate autophagosome - associated pattern , is observed in oe19 ( fig3 c ) and kyse450 ( data not shown ) cells following treatment with 5 - fu & amp ; cisplatin . diffuse cytoplasmic localization of gfp - lc3 was observed in oe21 and oe33 cell lines , in response to both chemotherapeutic drugs ( images not shown ). monodansylcadaverine ( mdc ) dye was also employed to assess levels of mature autophagic vesicle formation in all esophageal cell lines following drug treatment . oe21 / oe33 cells failed to develop a punctate staining pattern , in contrast , the more drug resistant , autophagic , oe19 and kyse450 cell lines demonstrate bright blue punctate staining in response to both chemotherapeutic drugs , consistent with accumulation of mdc in acidic vesicles ( fig4 ). these results collectively suggest that the cytoplasmic vesicles that develop following incubation with either 5 - fu or cisplatin , in the oe19 and kyse450 cells are predominantly autophagosomes . the induction of autophagy in esophageal cancer cell lines is associated with the ability of the oe19 and kyse450 cell lines to recover , following the removal of drugs as the autophagic process is associated with survival , we examined whether the cell population had the ability to recover , following drug removal . cells were treated with 5 - fu / cisplatin and viability was determined . when recovery was assessed , both oe19 and kyse450 cell lines demonstrate a remarkable ability to recover and cell cultures are re - populated following even high dose ( 40 μm ) treatment ( fig5 a / b ). the apoptotic competent cell line oe21 fails to recover from low drug doses , and the oe33 cells show minimal recovery . oe19 and kyse450 cells display morphological features of autophagy when recovering ( data not shown ). this is the first demonstration that the induction of autophagy , in response to chemotherapeutic agents , has been associated with recovery from drug treatment . while type ii cell death is present ( fig1 c ), sufficient cells seem capable of recovery when the cytotoxic insult is removed . to assess whether autophagy contributed to this recovery , autophagy was inhibited by depleting the key regulator , beclin1 with short interfering rna ( sirna ). a complex containing beclin1 , a class iii pi3 - kinase ( hvps34 ) and other cofactors initiates the formation of the autophagosome ( liang et al ., 1999 , levine et al ., 2008 , pattingre et al ., 2008 ), and is critical for autophagic survival ( qu et al ., 2003 ). beclin1 silencing ( maintained for 72 hours , fig6 a ) attenuated the ability of kyse450 cells to recover ( 96 hours recovery ) from a 48 hr treatment with 5 - fu ( fig6 b ). beclin1 silencing clearly reduced autophagic survival , yet autophagic / type ii cell death was unaffected . there was no elevation of necrosis or apoptosis in 5 - fu treated cells with beclin1 silencing ( fig6 c middle right ) compared to cells treated with 5 - fu alone ( middle left ). both display a highly vesicular cytoplasm ( arrowheads ), typical of type ii cell death . morphology of populations following drug withdrawal ( lower right ) also show that transfected cells fail to recover , compared to cells treated with 5 - fu alone ( lower left — many cells regaining cytoplasmic material ). these data suggest that the induction of beclin1 - dependent autophagy , in response to chemotherapeutic drugs , contributes to recovery , when drugs are withdrawn . however , beclin1 is dispensable for autophagic / type ii cell death ( arrowheads ). modulation of chemo - sensitivity and recovery in oe21 and kyse450 cells by pharmacological inhibitors 3 - ma and ly294002 was examined ( fig7 a & amp ; b ). pi - 3 kinase inhibitors do not inhibit autophagy in these cells . in kyse450 cells , autophagy and type ii morphologies are enhanced in combination treatments with 5 - fu and pi - 3 kinase inhibitors ( 3 - ma and ly294002 ). recovery is also reduced at higher concentrations of inhibitors . addition of bafilomycin does not significantly enhance cytotoxicity ( fig8 ), however the inhibition of autophagosome / lysosome fusion elevates vesicular content in all cells and provides striking evidence of autophagy in apoptotic cells . as specific inhibitors of autophagy are not currently available , the acceleration of autophagy beyond a survival process , into autophagic cell death was examined as potential therapeutic approach . the effects of two known autophagy inducers rapamycin and lithium chloride ( licl ) were assessed for drug sensitivity and recovery of esophageal cancer cells ( rapamycin data is included as fig9 ). evidence suggests that licl inhibits inositol monophosphatase , with a reduction in inositol - 1 , 4 , 5 - triphosphate ( ip 3 ) levels ( sarkar and rubinsztein , 2006 ), therefore representing an mtor - independent mechanism of action . apoptosis competent - oe21 cells respond to treatment with 10 and 30 mm licl alone . recovery from 10 mm is complete , indicating that mtt reduction is primarily due to a drop in metabolism . at 30 mm , recovery is reduced , but evident ( fig1 a ). a limited recovery in 5 - fu treated oe21 cells is apparent at this 96 hours recovery period , possibly due to the presence of autophagic cells as a minor population ( 7 %) ( fig1 c ), which have the potential to recover . a combined licl and 5 - fu treatment eliminated these recovering cells . licl ( fig1 b upper left ) induced mixed morphologies - autophagic and apoptotic in oe21 cells , present also in recovering populations with the appearance of some large cells displaying morphology previously described as mitotic catastrophy ( lower left , red arrow head ). combination treatments resulted in predominantly apoptotic morphology , with many shrunken nuclei ( upper right ). similar responses were observed in the oe33 cell line ( data not shown ). kyse450 cells respond to licl treatment alone ( 48 hour ), followed by extensive recovery at 96 hours . however , when 30 mm licl is combined with 5 - fu , all cells fail to recover , even after 96 hours ( fig1 c ). licl alone induced an autophagic morphology ( fig1 b arrowheads ), which was greatly enhanced by the addition of 5 - fu ( upper right ). morphology confirms that kyse450 cells recover from licl alone ( lower left ), however cells treated with both licl and 5 - fu fail to recover ( lower right ). this is the first drug combination to which these resistant cells were completely susceptible . the bh3 mimetic , ha14 - i enhanced the cytotoxicity of 5 - fu in kyse450 oesophageal cells . the sensitivity of the kyse450 oesophageal cells was evaluated in response to the bh3 mimetic , ha14 - i ( 5 - 30 μm ) alone and in combination with 5 - fu ( 50 μm ). morphological analysis demonstrated that 24 hours post treatment , at the lowest concentration of ha14 - i ( 5 μm ) without and with 5 - fu ( fig1 ( 3 ) and ( 4 ) respectively ), the induction of apoptosis is observed , but at very low levels . likewise , with 10 μm ha14 - i , without and with 5 - fu ( fig1 ( 1 ) and ( 2 )) and with 20 μm ha14 - i ( fig1 ( 3 )), the induction of apoptosis is apparent , but again at low levels . at 48 hours post treatment , the lower concentrations of ha14 in combination with 5 - fu ( fig1 ( 1 )) induces a non - apoptotic / type ii morphology ( with many of the cells exhibiting loss of cytoplasmic material , no discernable plasma membrane , and pyknosis of the nuclear maerial ), with little or no evidence of apoptosis . the higher concentration of ha14 - i ( 30 μm ) alone , however induced clear levels of apoptotic cell death , at both 24 and 48 hours post treatment ( fig1 and fig1 ). initial data from the real - time cell analyzer , monitoring cell growth and viability , suggests that the combination of 10 and 20 μm ha14 - i & amp ; 5 - fu ( 50 μm ), enhanced the cytotoxic effect of 5 - fu alone . the corresponding morphology for combination treatments ( fig1 ( 1 )), would indicate that this enhanced cytotoxicity is due to the induction of type ii cell death . combination treatments with 5 - fu and lithium chloride significantly reduced llc - luc derived tumours in a murine model , when compared to treatment ( via localised and systemic delivery ) with either 5 - fu or lithium alone , while also reducing the spread / metastatic burden from llc ( wt ) derived tumours ( fig1 - 19 ). to further assess the antitumour effect of systemically delivered treatment on metastatic disease , a spontaneously metastasizing mammary adenocarcinoma model ( 4t1 ) in balb / c mice was utilized . combination treatments with 5 - fu and lithium chloride significantly reduced the spread or burden of metastatic disease in this spontaneously metastasizing breast carcinoma model , when compared to treatment with either 5 - fu or lithium alone , confirmed by both lung weights and the quantifiable 6 - thio guanine assay ( p = 0 . 0002 ; fig2 ). oesophageal kyse450 derived tumours deveopled in mf1 nu / nu mice from treated animals were surgically removed , dissected , and processed by routine histology for immunohistochemistry analysis . sections were stained for lc3 ( bio - marker of autophagy ) and active caspase3 ( bio - marker of apoptosis ). tumours treated with combination therapy ( 20 mg / kg 5 - fu & amp ; 200 mg / kg lithium chloride ) display enhanced lc3 staining ( as a result of augmented autophagy ) in the absence of any detectable apoptosis ( fig2 ). as such , these findings demonstrate that enhanced autophagy therapy enhances bio - markers of autophagy , in the absence of apoptosis . to investigate autophagy therapy in an in vivo pre - clinical colorectal carcinoma model , ct26 cells ( colorectal carcinoma cells ) were injected subcutaneously into the right flank of adult female balb / c mice , after anaesthesia . all treatments were delivered directly into the tumour , thrice weekly . following drug treatment , tumours were monitored by alternate day measurements in two dimensions . there were no control or lithium treated animals alive beyond day 28 , and the final 5 - fu treated animal was euthanized on day 33 . at this point , all tumours on the combination treated animals were reduced to scabs . treatment of these animals was maintained until day 58 , following which all treatments ceased . 5 months after cessation of treatment these animals remain tumour free ( fig2 ). in an identical model system , oxaliplatin ( 10 mg / kg ) was used in place of 5 - fu and a combination of both lithium and oxaliplatin ( 200 mg / kg , 10 mg / kg ). the combination treatment had a significant effect on tumour volume , following systemic delivery when compared to both single agent treatments oxaliplatin and lithium (* p = 0 . 013 and * p = 0 . 004 respectively ). this combination treatment also enhanced survival when compared to either agent alone ( fig2 ). in vivo implementation in pre - clinical colorectal carcinoma model : enhanced autophagy therapy enhances survival . for routine tumour induction , 1 × 10 6 ct26 ( colorectal carcinoma cells ) suspended in 200 μl of serum free dmem are injected subcutaneously into the right flank of adult female balb / c mice , after anaesthesia . mice are randomly divided into experimental groups . mice are treated at a tumour volume of approximately 60 mm 3 in volume ( 5 - 7 mm major diameter ). all treatments are delivered in 50 μl volumes , administered directly into the tumour , thrice weekly with pbs ( control ), rapamycin ( 0 . 6 - 2 mg / kg ), 5 - fu ( 20 mg / kg ) and a combination of both rapamycin and 5 - fu ( 0 . 6 - 2 mg / kg , 20 mg / kg ). following drug treatment , tumours are monitored by alternate day measurements in two dimensions , using verniers calipers . tumor volumes are calculated according to the formula v = ab 2 / 6 , where ‘ a ’ is the longest diameter of the tumor and ‘ b ’ is the longest diameter perpendicular to diameter ‘ a ’. animals are culled when tumour volumes exceed ˜ 500 mm 3 ( no greater than 15 mm in diameter ). there are no control or rapamycin - treated animals alive beyond day 28 , and the final 5 - fu treated animal are euthanized on day 33 . at this point , all tumours on the combination treated animals are reduced to scabs . treatment of these animals is maintained until day 58 , following which all treatments cease . the invention is not limited to the embodiments heretofore described which may be varied in construction and detail without departing from the spirit of the invention . the references cited below and throughout the specification are incorporated herein by reference . amaravadi , r . k ., yu , d ., lum , j . j ., bui , t ., christophorou , m . a ., evan , g . i ., thomas - 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