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| Description | Chloroquine(diphosphate)isanantimalarialdrugandautophagy/lysosomeinhibitor,inhibitingToll-likereceptor(TLR)signalling. |
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| InVitro | Chloroquine(CHQ,20μM)inhibitsIL-12p70releaseandreducesTh1-primingcapacityofactivatedhumanmonocyte-derivedLangerhans-likecells(MoLC).Chloroquine(CHQ,20μM)enhancesIL-1–inducedIL-23secretioninMoLCandsubsequentlyincreasesIL-17AreleasebyprimedCD4+Tcells[1].Chloroquine(25μM)suppressesMMP-9mRNAexpressioninnormoxiaandhypoxiainparentalMDA-MB-231cells.Chloroquinehascell-,dose-andhypoxia-dependenteffectsonMMP-2,MMP-9andMMP-13mRNAexpression[2].TLR7andTLR9inhibitionusingIRS-954orchloroquinesignificantlyreducesHuH7cellproliferationinvitro[3]. |
| InVivo | Chloroquine(80mg/kg,i.p.)doesnotpreventthegrowthofthetriple-negativeMDA-MB-231cellswithhighorlowTLR9expressionlevelsintheorthotopicmousemodel[2].TLR7andTLR9inhibitionusingIRS-954orchloroquinesignificantlyinhibitstumourgrowthinthemousexenograftmodel.HCCdevelopmentintheDEN/NMORratmodelisalsosignificantlyinhibitedbychloroquine[3]. |
| ClinicalTrial | ViewMoreCollapse |
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| CellAssay [2] | Thecellsareculturedin6-wellplateswithnormalculturemediuminthepresenceofvehicleor25or50μMchloroquine,untilnearconfluency,afterwhichtheyarerinsedwithsterilephosphate-bufferedsaline(PBS)andculturedfurtherfortheindicatedtimesinserum-freeculturemedium.Atthedesiredtime-points,theculturemediumisdiscardedandthecellsarequicklyharvestedinlysisbufferandclarifiedbycentrifugation.Subsequenttoboilingthesupernatantsinreducingsodiumdodecylsulphate(SDS)samplebuffer,equalamountsofprotein(100μg)areloadedperlaneandthesamplesareelectrophoresedinto10or4-20%grADIentpolyacrylamideSDSgels,thentransferredtoanitrocellulosemembrane.TodetectTLR9,theblotsareincubatedovernightat4°Cwithanti-TLR9antibodies,diluted1:500inTris-bufferedsalinewith0.1%(v/v)Tween-20(TBST).Equalloadingisconfirmedwithpolyclonalrabbitanti-actin.Secondarydetectionisperformedwithhorseradishperoxidase-linkedsecondaryantibodies.TheproteinbandsarevisualizedbychemiluminescenceusinganECLkit.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly. AnimalAdmiNISTration | [2] ChloroquinediphosphateisdissolvedinPBS. ControlandTLR9siRNAMDA-MB-231cells(5×105 cellsin100μL)areinoculatedintothemammaryfatpadsoffour-week-old,immune-deficientmice(athymicnude/nuFoxn1).Treatmentsarestartedsevendaysaftertumorcellinoculation.Themicearetreateddailyeitherwithintraperitoneal(i.p.)chloroquine(80mg/kg)orvehicle(PBS).Theanimalsaremonitoreddailyforclinicalsigns.TumormeasurementsareperformedtwiceaweekandtumorvolumeiscalculatedaccordingtotheformulaV=(π/6)(d1×d2)3/2,whered1 andd2 areperpendiculartumordiameters.Thetumorsareallowedtogrowfor22days,atwhichpointthemicearesacrificedandthetumorsaredissectedforafinalmeasurement.Throughouttheexperiments,theanimalsaremaintainedundercontrolledpathogen-freeenvironmentalconditions(20-21ºC,30-60%relativehumidityanda12-hlightingcycle).Themicearefedwithsmall-animalfoodpelletsandsuppliedwithsterilewater adlibitum. MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly. References |
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