A Chimeric SM5-1 Antibody Inhibits Hepatocellular Carcinoma Cell Growth And Induces Caspase Dependent Apoptosis

8/7/2019 A Chimeric SM5-1 Antibody Inhibits Hepatocellular Carcinoma Cell Growth And Induces Caspase Dependent Apoptosis

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AA ChimericChimeric SM5SM5--11 AntibodyAntibody InhibitsInhibitsHepatocellular Hepatocellular CarcinomaCarcinoma

CellCell GrowthGrowth AndAnd InducesInducesCaspaseCaspase DependentDependent ApoptosisApoptosis

AA ChimericChimeric SM5SM5--11 Antibody

Antibody InhibitsInhibitsHepatocellular Hepatocellular CarcinomaCarcinoma

CellCell GrowthGrowth AndAnd InducesInducesCaspaseCaspase DependentDependent ApoptosisApoptosis


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INTRODUCTIONINTRODUCTIONHepatocellularHepatocellular CarcinomaCarcinoma : : MostMost commoncommon 11rr liverliver malignancymalignancy. .

MostMost HCCHCC 22rr toto HepHep--B/CB/C InfectionInfection && CirrhosisCirrhosis (Alcoholism,(Alcoholism, AD,AD,HemochromatosisHemochromatosis) )..

UsuallyUsually deadlydeadly inin 33--66 months/months/ >> 66monthsmonths. .

SurgicalSurgical resection,resection, LiverLiver transplant,transplant, Chemo/RadiationChemo/Radiation TherapyTherapy etcetc..PrognosisPrognosis poorpoor. . ((1010--3030%% max)max). .

AbsAbs cancan specificallyspecifically bindbind toto targettarget cellscells..

cSMcSM55--11 mousemouse humanhuman chimericchimeric AbAb.. (> 66monthsmonths. .

SurgicalSurgical resection,resection, LiverLiver transplant,transplant, Chemo/RadiationChemo/Radiation TherapyTherapy etcetc..PrognosisPrognosis poorpoor. . ((1010--3030%% max)max). .

AbsAbs cancan specificallyspecifically bindbind toto targettarget cellscells..

cSMcSM55--11 mousemouse humanhuman chimericchimeric AbAb.. (


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Materials & MethodsMaterials & MethodsCellCell LinesLines -- ::

1.1. BreastBreast cancercancer cellcell lineslines (SK (SK--BR BR--33,, MDAMDA--MBMB--231231,MDA,MDA--MBMB--468468,,MCFMCF--77)) && melanomamelanoma cellcell lineslines (A(A375375SS22,, UU1010)) obtainedobtained fromfromATCCATCC ( (Manassas,VAManassas,VA) )..

2.2. HepatocellularHepatocellular carcinomacarcinoma cellcell lineslines (Ch(Ch--HepHep--33,, ChCh--HepHep--11,, ChCh--HepHep--66)) establishedestablished andand maintainedmaintained. .

AntibodiesAntibodies - - ::1.1. cSMcSM55--11 AbAb constructedconstructed andand purifiedpurified. .2.2. FITCFITC- -conjugatedconjugated goatgoat antianti--humanhuman IgGIgG (H+L)(H+L) && HRPHRP--conjugatedconjugated

goatgoat antianti--humanhuman IgGIgG (H+L)(H+L) purchasedpurchased fromfrom ZymedZymed (SF,CA)(SF,CA)3.3. AntiAnti CDCD33 chimericchimeric AbAb cc1212FF66 developeddeveloped. .

FlowFlow CytometryCytometry AnalysisAnalysis - - ::1.1. CancerCancer cellscells incubatedincubated withwith 11QQg/mlg/ml of of cSMcSM55--11/c/c1212FF66 ((11hh atat 44oo C)C)..2.2. CellsCells washedwashed andand incubatedincubated withwith FITCFITC- -conjugatedconjugated goatgoat antianti--

humanhuman IgGIgG (H+L)(H+L) ((3030minmin atat 44oo C)C)..3.3. CellsCells washedwashed andand examinedexamined withwith FACScanFACScan flowflow cytometercytometer. .

CellCell LinesLines -- ::

1.1. BreastBreast cancercancer cellcell lineslines (SK (SK--BR BR--33,, MDAMDA--MBMB--231231,MDA,MDA--MBMB--468468,,MCFMCF--77)) && melanomamelanoma cellcell lineslines (A(A375375SS22,, UU1010)) obtainedobtained fromfromATCCATCC ( (Manassas,VAManassas,VA) )..

2.2. HepatocellularHepatocellular carcinomacarcinoma cellcell lineslines (Ch(Ch--HepHep--33,, ChCh--HepHep--11,, ChCh--HepHep--66)) establishedestablished andand maintainedmaintained. .

AntibodiesAntibodies - - ::1.1. cSMcSM55--11 AbAb constructedconstructed andand purifiedpurified. .2.2. FITCFITC- -conjugatedconjugated goatgoat antianti--humanhuman IgGIgG (H+L)(H+L) && HRPHRP--conjugatedconjugated

goatgoat antianti--humanhuman IgGIgG (H+L)(H+L) purchasedpurchased fromfrom ZymedZymed (SF,CA)(SF,CA)3.3. AntiAnti CDCD33 chimericchimeric AbAb cc1212FF66 developeddeveloped. .

FlowFlow Cy

tometryCytometry AnalysisAnalysis - - ::1.1. CancerCancer cellscells incubatedincubated withwith 11QQg/mlg/ml of of cSMcSM55--11/c/c1212FF66 ((11hh atat 44oo C)C)..2.2. CellsCells washedwashed andand incubatedincubated withwith FITCFITC- -conjugatedconjugated goatgoat antianti--

humanhuman IgGIgG (H+L)(H+L) ((3030minmin atat 44oo C)C)..3.3. CellsCells washedwashed andand examinedexamined withwith FACScanFACScan flowflow cytometercytometer. .


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Materials & MethodsMaterials & Methods

Western Blotting AnalysisWestern Blotting Analysis - - ::

1.1. CellsCells lysedlysed inin lysislysis buffer.buffer.2.2. 100100 QQg of total protein separated on 6% SDSg of total protein separated on 6% SDS- -PAGE and blottedPAGE and blottedon PVDF membrane.on PVDF membrane.

3.3. SM5SM5--1 binding protein detected using cSM51 binding protein detected using cSM5- -1 as 11 as 1 oo AbAb and HRPand HRP- -conjugated goat anticonjugated goat anti- -human antibody (H+L) as 2human antibody (H+L) as 2 oo Ab.Ab.

4.4. AgAg--AbAb complexcomplex visualizedvisualized usingusing TMBTMB singlesingle solutionsolution. . cc1212FF66 usedusedasas veve controlcontrol. .

RadioactiveRadioactive LabellingLabelling && ImmunoprecipitationImmunoprecipitation - - ::1.1. CellsCells metabolicallymetabolically labelledlabelled withwith SS3535 methioninemethionine ( (3030minmin atat 3737 oo C)C)..2.2. CellCell lysateslysates preclearedprecleared withwith 22QQgg cc1212FF66 andand 2020QQll A/GA/G AgaroseAgarose

conjugateconjugate proteinprotein overnightovernight atat 44oo CC..

3.3. PostPost centrifugationcentrifugation supernatantsupernatant immunoprecipitatedimmunoprecipitated withwith 22QQggcSMcSM55--11 mAbmAb andand 2020 QQll of of proteinprotein A/GA/G AgaroseAgarose conjugateconjugate proteinprotein. .

4.4. SamplesSamples resolvedresolved byby 66%% SDSSDS--PAGEPAGE andand analyzedanalyzed bybyautoradiographyautoradiography. .

Western Blotting

AnalysisWestern Blotting Analysis - - ::

1.1. CellsCells lysedlysed inin lysislysis buffer.buffer.2.2. 100100 QQg of total protein separated on 6% SDSg of total protein separated on 6% SDS- -PAGE and blottedPAGE and blottedon PVDF membrane.on PVDF membrane.

3.3. SM5SM5--1 binding protein detected using cSM51 binding protein detected using cSM5- -1 as 11 as 1 oo AbAb and HRPand HRP- -conjugated goat anticonjugated goat anti- -human antibody (H+L) as 2human antibody (H+L) as 2 oo Ab.Ab.

4.4. AgAg--AbAb complexcomplex visualizedvisualized usingusing TMBTMB singlesingle solutionsolution. . cc1212FF66 usedusedasas veve controlcontrol. .

RadioactiveRadioactive LabellingLabelling && ImmunoprecipitationImmunoprecipitation - - ::1.1. CellsCells metabolicallymetabolically labelledlabelled withwith SS3535 methioninemethionine ( (3030minmin atat 3737 oo C)C)..2.2. CellCell lysateslysates preclearedprecleared withwith 22QQgg cc1212FF66 andand 2020QQll A/GA/G AgaroseAgarose

conjugateconjugate proteinprotein overnightovernight atat 44oo CC..

3.3. PostPost centrifugationcentrifugation supernatantsupernatant immunoprecipitatedimmunoprecipitated withwith 22QQggcSMcSM55--11 mAbmAb andand 2020 QQll of of proteinprotein A/GA/G AgaroseAgarose conjugateconjugate proteinprotein. .

4.4. SamplesSamples resolvedresolved byby 66%% SDSSDS--PAGEPAGE andand analyzedanalyzed bybyautoradiographyautoradiography. .


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Materials & MethodsMaterials & Methods

Cell Growth Inhibition AssayCell Growth Inhibition Assay - - ::

1.1. Cells plated and treated with different conc. of cSM5Cells plated and treated with different conc. of cSM5- -1 (96h).1 (96h).2.2. Cells incubated with MTT (2h at 37Cells incubated with MTT (2h at 37 ooC) and dissolved in DMSO.C) and dissolved in DMSO.

3.3. Reduction of MTT quantified by absorbance measurement atReduction of MTT quantified by absorbance measurement at490nm using490nm using microplatemicroplate reader.reader.

4.4. % Growth Inhibition% Growth Inhibition ==A of A of mAbmAb untreated cellsuntreated cells A of A of mAbmAb treated cellstreated cells

x 100x 100A of A of mAbmAb untreated cellsuntreated cells A of culture mediumA of culture medium

DNA Fragmentation AnalysisDNA Fragmentation Analysis - - ::1.1. Cells treated with cSM5Cells treated with cSM5- -1 then1 then lysedlysed with 200with 200 QQl of l of lysislysis buffer.buffer.2.2. CellCell lysateslysates incubated for 1h at 50incubated for 1h at 50 oo C prior toC prior to RNaseRNase A addition.A addition.

3.3. Genomic DNA extracted with phenol and precipitated withGenomic DNA extracted with phenol and precipitated withethanol.ethanol.

4.4. DNA precipitates dissolved in 50DNA precipitates dissolved in 50 QQl TE buffer and resolved byl TE buffer and resolved byelectrophoresis on 2%electrophoresis on 2% agaroseagarose gel.gel.

Cell Growth Inhibition Assay

Cell Growth Inhibition Assay - - ::

1.1. Cells plated and treated with different conc. of cSM5Cells plated and treated with different conc. of cSM5- -1 (96h).1 (96h).2.2. Cells incubated with MTT (2h at 37Cells incubated with MTT (2h at 37 ooC) and dissolved in DMSO.C) and dissolved in DMSO.

3.3. Reduction of MTT quantified by absorbance measurement atReduction of MTT quantified by absorbance measurement at490nm using490nm using microplatemicroplate reader.reader.

4.4. % Growth Inhibition% Growth Inhibition ==A of A of mAbmAb untreated cellsuntreated cells A of A of mAbmAb treated cellstreated cells

x 100x 100A of A of mAbmAb untreated cellsuntreated cells A of culture mediumA of culture medium

DNA Fragmentation AnalysisDNA Fragmentation Analysis - - ::1.1. Cells treated with cSM5Cells treated with cSM5- -1 then1 then lysedlysed with 200with 200 QQl of l of lysislysis buffer.buffer.2.2. CellCell lysateslysates incubated for 1h at 50incubated for 1h at 50 oo C prior toC prior to RNaseRNase A addition.A addition.

3.3. Genomic DNA extracted with phenol and precipitated withGenomic DNA extracted with phenol and precipitated withethanol.ethanol.

4.4. DNA precipitates dissolved in 50DNA precipitates dissolved in 50 QQl TE buffer and resolved byl TE buffer and resolved byelectrophoresis on 2%electrophoresis on 2% agaroseagarose gel.gel.


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Materials & MethodsMaterials & MethodsRoleRole Of Of CaspasesCaspases inin cSMcSM55--11 InducedInduced ApoptosisApoptosis - - ::

1.1. ChCh--HepHep--33 cellscells incubatedincubated withwith caspasecaspase inhibitorsinhibitors priorprior toto

treatmenttreatment withwith cSMcSM55--11 ((9696h)h)..2.2. CellsCells pellettedpelletted andand suspendedsuspended inin lysislysis bufferbuffer ( (3030minmin atat 44ooC)C)..

3.3. PostPost centrifugationcentrifugation supernatantssupernatants collectedcollected andand apoptosisapoptosis assessedassessedbyby cellcell deathdeath detectiondetection ELISAELISA PLUSPLUS kitkit..

4.4. %% Apoptosis Apoptosis Inhibition Inhibition ==

AA of of caspasecaspase inhibitorinhibitor untreateduntreated cellscells AA of of caspasecaspase inhibitorinhibitor treatedtreated cellscellsxx 100100

AA of of caspasecaspase inhibitorinhibitor untreateduntreated cellscells AA of of cultureculture mediummedium

CaspaseCaspase 1010 ActivityActivity AssayAssay -- ::1.1. ChCh--HepHep--33/Ch/Ch--HepHep--66 cellscells incubatedincubated withwith cSMcSM55--11 forfor differentdifferent

timetime intervalsintervals inin 1010cmcm dishdish..2.2. CellsCells lysedlysed byby lysislysis bufferbuffer andand postpost centrifugationcentrifugation supernatantssupernatants

collectedcollected toto measuremeasure caspasecaspase- -1010 activityactivity byby caspasecaspase- -1010colorimetriccolorimetric assayassay kitkit..

3 .3 . %% CaspaseCaspase- -1010 Activity Activity ==AA of of mAbmAb treatedtreated cellscells AA of of mAbmAb untreateduntreated cellscells

xx 100100AA of of mAbmAb untreateduntreated cellscells AA of of cultureculture mediummedium

RoleRole Of Of Casp

asesCaspases inin cSMcSM55--11 InducedInduced ApoptosisApoptosis - - ::1.1. ChCh--HepHep--33 cellscells incubatedincubated withwith caspasecaspase inhibitorsinhibitors priorprior toto

treatmenttreatment withwith cSMcSM55--11 ((9696h)h)..2.2. CellsCells pellettedpelletted andand suspendedsuspended inin lysislysis bufferbuffer ( (3030minmin atat 44ooC)C)..

3.3. PostPost centrifugationcentrifugation supernatantssupernatants collectedcollected andand apoptosisapoptosis assessedassessedbyby cellcell deathdeath detectiondetection ELISAELISA PLUSPLUS kitkit..

4.4. %% Apoptosis Apoptosis Inhibition Inhibition ==

AA of of caspasecaspase inhibitorinhibitor untreateduntreated cellscells AA of of caspasecaspase inhibitorinhibitor treatedtreated cellscellsxx 100100

AA of of caspasecaspase inhibitorinhibitor untreateduntreated cellscells AA of of cultureculture mediummedium

CaspaseCaspase 1010 ActivityActivity AssayAssay -- ::1.1. ChCh--HepHep--33/Ch/Ch--HepHep--66 cellscells incubatedincubated withwith cSMcSM55--11 forfor differentdifferent

timetime intervalsintervals inin 1010cmcm dishdish..2.2. CellsCells lysedlysed byby lysislysis bufferbuffer andand postpost centrifugationcentrifugation supernatantssupernatants

collectedcollected toto measuremeasure caspasecaspase- -1010 activityactivity byby caspasecaspase- -1010colorimetriccolorimetric assayassay kitkit..

3 .3 . %% CaspaseCaspase- -1010 Activity Activity ==AA of of mAbmAb treatedtreated cellscells AA of of mAbmAb untreateduntreated cellscells

xx 100100AA of of mAbmAb untreateduntreated cellscells AA of of cultureculture mediummedium


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RESULTS & DISCUSSIONRESULTS & DISCUSSION

Fig. 1. The expression level of SM5-1 binding protein in different cancer cell lines. Different cancer cells (a, hepatocellular carcinoma; b,melanoma; c, breast cancer) at 1 106 cells/ml were incubated with 1 lg/ml cSM5-1 or negative control antibody (c12F6) for 1 h at 4 C.

The cells were washed and incubated with FITC-conjugated goat anti-human IgG (H + L) for 30 min at 4 C. Then the cells were washedand analysed by flow cytometry analysis. Open histograms, negative control antibody. Solid histograms, cSM5-1.


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Fig. 2. Characterization of SM5-1 binding protein by Western blotting and immunoprecipitation. (a) Western blotting assay of thelysates of Ch-Hep-3 cells with cSM5-1 or c12F6. (b) Immunoprecipitation of lysates from 35S-methionine metabolically labeled

Ch- Hep-3 cells with cSM5-1 or c12F6. (c) Immunoprecipitation of lysates from 125 I surface-labeled Ch-Hep-3 cells with cSM5-1 orc12F6. c12F6 is a negative control antibody.


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Fig. 3. The inhibitory effect of cSM5-1 on the growth of different hepatocellular carcinoma cell lines. Cells (Ch-Hep-3, Ch-Hep-1or Ch-Hep-6) were treated with 0.2, 1 or 5 lg/ml of cSM5-1 (a) or c12F6 (b) for 96 h. Then the antiproliferative activity was

determined by MTT assay as described in Section 2. The percentage of cell growth inhibition was presented. Data arepresented as means SD (n = 6).


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Fig. 4. DNA fragmentation induced by cSM5-1 in hepatocellular carcinoma cells. Hepatocellular carcinoma cells Ch-Hep-3, Ch-Hep-1, Ch-Hep-6 were treated with 5 lg/mL of cSM5-1 (a) or c12F6 (b) for 96 h. Then the genomic DNA of the cells

were extracted and subjected to 2% agarose gel electrophoresis


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Fig. 5. The role of caspases in cSM5-1-induced apoptosis. (a) Ch-Hep-3 cells were treated with the various caspase inhibitors for 2h and then incubated with 5 lg/ml of cSM5-1 for an additional 96 h. Then the cells were lysed and the cell lysates were collected forapoptosis assessment as described in Section 2. Percentage of the apoptosis inhibition was presented. Data are presented as means

SD (n = 6). (b)Ch-Hep-3 or Ch-Hep-6 cells were incubated with 5 lg/ml of cSM5-1 for 48, 72 or 96 h. Then the cells were lysedand the cell lysates werecollected for caspase-10 activity assay as described in Section 2. Percentage of caspase-10 activity increase

was presented. Data are presented as means SD (n = 6).


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CONCLUSIONCONCLUSIONcSMcSM55--11 activityactivity EE SMSM55--11 bindingbinding proteinprotein expressionexpression. .

CaspasesCaspases (Caspase(Caspase- -1010//PancaspasePancaspase) ) criticalcritical forfor cSMcSM55--11 inducedinducedapoptosisapoptosis. .

AlthoughAlthough effectiveeffective cSMcSM55--11ss effectivenesseffectiveness cancan bebe increasedincreased bybycombiningcombining withwith otherother treatmenttreatment methodsmethods. .

NewlyNewly arisingarising cryocryo--surgerysurgery coupledcoupled liverliver resectionresection cancan bebe followedfollowedbyby AbAb TherapyTherapy usingusing cSMcSM55--11 mAbmAb.. (Hypothetical)(Hypothetical). .

cSMcSM55--11 isis chimericchimeric andand hencehence immunogenicityimmunogenicity is is minimizedminimized. .

ResultsResults ultimatelyultimately showshow thatthat cSMcSM55--11 inhibitsinhibits cellcell growthgrowth andand inducesinducesapoptosisapoptosis. .

cSMcSM55--11 activityactivity EE SMSM55--11 bindingbinding proteinprotein expressionexpression. .

CaspasesCaspases (Caspase(Caspase- -1010//PancaspasePancaspase) ) criticalcritical forfor cSMcSM55--11 inducedinducedapoptosisapoptosis. .

AlthoughAlthough effectiveeffective cSMcSM55--11ss effectivenesseffectiveness cancan bebe increasedincreased bybycombiningcombining withwith otherother treatmenttreatment methodsmethods. .

NewlyNewly arisingarising cryocryo--surgerysurgery coupledcoupled liverliver resectionresection cancan bebe followedfollowedbyby AbAb TherapyTherapy usingusing cSMcSM55--11 mAbmAb.. (Hypothetical)(Hypothetical). .

cSMcSM55--11 isis chimericchimeric andand hencehence immunogenicityimmunogenicity is is minimizedminimized. .

ResultsResults ultimatelyultimately showshow thatthat cSMcSM55--11 inhibitsinhibits cellcell growthgrowth andand inducesinducesapoptosisapoptosis. .


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A Chimeric SM5-1 Antibody Inhibits Hepatocellular Carcinoma Cell Growth And Induces Caspase Dependent Apoptosis