Betulinic Acid Induces Bax/Bak-independent Cytochrome C Release In Human Nasopharyngeal Carcinoma Cells

Objective The ralationship of Betulinic acid with the humannasopharyngeal carcinoma CNE2cells be studied. To evaluate whether theapoptosis would be happened and the possible manner would be after BetAincubate with CNE2, that would provide the basis of therapy for NPC usingBetA.Materials and methods After incubate CNE2with BetA at varyingconcentration5μg/mL,10μg/mL,20μg/mL, staining cells with AV/PI, thecell death would be observed by the flow cytometry analysis, and Etoposidewould be positive control. The activity of caspase would be detected byCaspase fluorogenic substrates Ac-DEVD-AFC (caspase-3) andAc-LEHD-AFC (caspase-9), actin would be control; Genomic DNA wasextracted from treated CNE2cells and run on agarose gel to detect whetheror not BetA causes the formation of a DNA ladder; Whether or not BetAinduces the cyt c release would be detected by Western blot, and COX IVwould be positive control. Results1. At varying concentrations of5μg/mL,10μg/mL, and20μg/mL,BetA was able to induce29.7%,41.3%, or80.3%CNE2cell death (AV/PIdouble-positive); death was characterized by AV single-positive cells(87.5%,84.7%, and80.9%, respectively).2. We detected caspase was activated after BetA treatment to confirmthe type of BetA-induced CNE2cell death. Caspase-3was cleaved into its19KDa activated form in a dose-dependent manner;3. BetA caused the formation of a DNA ladder.4. BetA induces the cyt c release in a dose-dependent manner.Conclusion These data indicate that BetA-induced CNE2cell death iscaspase-dependent and a typical apoptosis. Objective To observe the role of Bcl-2/Bcl-xL and Bax/Bak duringBetA induced CNE2cells apoptosis, to provide the basis of of therapy forNPC using BetA.Materials and methods CNE2cells were transfected withpcDNA3.1-Bcl-2or pcDNA3.1-Bcl-xL plasmids, and stable cell lines werescreened with G418. Over-expression of the stable CNE2cell line wasconfirmed with anti-myc antibodies. Different CNE2cells were treated withindicated BetA concentrations, stained with AV/PI, and then subjected toFACS analysis. Dead cells were calculated by PI positive plus AV positiveplus AV/PI double-positive. Data were calculated from three experiments;CNE2cells were treated with20μg/mL of BetAfor48h and then subjectedto immunofluorescent staining. Cells were fixed, permeabilized, and stainedwith a specific anti-Bax antibody that can specifically recognize activatedBax. Etoposide-induced Bax activation served as a positive control; CNE2cells were transfected sequentially with pSuper-Bax and pSuper-Bakplasmid, and stable cell lines were screened with G418. Cells were subjectedto Western blot analysis for knockdown efficiency. Bax and Bak wereprobed with specific antibodies, and actin served as the loading control.Wild-type (WT) and Bax/Bak knockdown CNE2cells were treated with indicated BetA concentrations, stained with AV/PI, and then subjected toFACS analysis. WT and Bax/Bak knockdown CNE2cells were treated withindicated BetA and subjected to Western blot analysis to determine cyt crelease. COX IV was used as the loading control; CNE2cells werepre-treated with or without5μg/mL of CsA for1h and then treated with20μg/mL BetA. After24h of treatment, cells were collected and subjected toFACS analysis to determine the dead cell count. Cyt c release was measuredusing the InnoCyte Flow Cytometric Cytochrome c Release Kit. Data werecalculated from three experiments and are shown as mean±SD. The sametreatment and analysis were applied as previously mentioned,except thatCsA was replaced with BKA. Data were calculated from three experimentsand are shown as mean±SD.Results1. Over-expression of the stable CNE2cell line was confirmed withanti-mycantibodies.At different concentrations(5μg/mL,10μg/mL, and20μg/mL) BetA, Bcl-2/Bcl-xLover-expression cells showed reduced numbersof dead cells compared with WT cells.2. BetA cannot activate Bax. Instead, etoposide, which was used as apositive control to confirm our system, can successfully activate Bax.3. With different concentrations BetA (5μg/mL,10μg/mL, and20μg/mL), Bax/Bak double-knockdown cells showed reduced numbers ofdeadcells compared with WT cells, although the difference was not significant. 4. CsA/BKA partially inhibits BetA-caused CNE2cyt c release..Conclusion Bcl-2or Bcl-xL overexpression can partially preventBetA-induced CNE2cell apoptosis. Bax is not activated duringBetA-induced CNE2apoptosis. that CsA/BKA-sensitive mPTPs areinvolved in Bax/Bak-independent cyt c release of CNE2cells induced byBetA.