| Apoptosis is the process of programmed cell death, which is mainly carried outthrough endogenous and exogenous signaling cascade pathways. Many studies havedemonstrated that deficiency in apoptotic cell death induction is one of causes thatlead to failure of chemotherapy in most types of cancers, including HepatocellularCarcinoma (HCC). Bcl-2family proteins are key proteins that play an important rolein regulatiing apoptosis signaling. Bcl-2family proteins share common Bcl-2 homology (BH) domains, and are divided into antiapoptotic and proapoptoticsubfamilies. Two multidomain proapoptotic Bcl-2members, Bax and Bak, are theexecutor of mitochondrial dysfunction. Antiapoptotic Bcl-2family proteins, includingBcl-xL, Bcl-w, Mcl-1, A1and Bcl-2itself, inhibit apoptosis through sequestering Baxand Bak, thus maintaining mitochondrial integrity.Myeloid leukemin gene1(Mcl-1) is a unique Bcl-2family members, cloned andidentified from myeloid leukemia cells in1993. Since Mcl-1had a short half-life (1-4hours), Mcl-1can rapidly response to various stimuli. The expression of Mcl-1can beregulated by a variety of signals at the levels of transcription, posttranscription andprotein. Since overexpression of Mcl-1was found in a variety of malignant tumorsincluding HCC, Mcl-1has been considered as a promising target for the therapy ofthese malignant tumors.ABT-737, ABT-263(Navitoclax) are synthetic small molecule BH3mimetics.Previous studies have shown that ABT-737, ABT-263(Navitoclax) have goodtherapeutic effect for various types hematological malignancies and solid tumors. Inparticularly, ABT-263has shown promising anticancer cancer activity in clinicaltrials. However, both ABT-737and ABT-263(Navitoclax) specifically bind to Bcl-xL and Bcl-2with high affinities, but bind to Mcl-1with very low affinities. Highexpression of Mcl-1in HCC and other malignant tumor cause resistance of thesetumors to ABT-737and ABT-263-induced apoptosis. These situations pose a majorobstacle for using these two drugs to treat cancer patients in clinic. Therefore,combined with other agents targeting Mcl-1might an attractive strategy to promoteABT-737/ABT-263-based anticancer therapy.Norcantharidin (NCTD) is a small molecule anticancer drugs, which is derivedfrom traditional Chinese medicine cantharidin. Previous studies have shown that theantitumor effect of NCTD may be related with suppression of Bcl-2family members. In a previous study, we observed that NCTD have significantly inhibited theexpression of Mcl-1in HCC cells. Aspirin (acetylsalicylic acid) is a common non-steroidal anti-inflammatory drug (NSAID), which has antipyretic, analgesic and anti-inflammatory abilities. Like other NSAIDs, aspirin showed strong anticancer effectsin a variety of tumor cells. Recent studies have shown that aspirin played anti-tumoreffects through downregulating the level of Mcl-1in colorectal cancer, oral squamouscell carcinoma, cervical cancer and leukemia cells. However, it has not been studiedwhether NCTD and aspirin can be used to overcome the resistance of HCC cells toABT-263.ObjectiveTo study the anti-HCC activities and molecular mechanisms of small moleculeBH3mimetic ABT-737/ABT-263alone, or in combination with NCTD in HCC cells.MethodsFour HCC cell lines, SMMC-7721, BEL-7402, HepG2, Hep3B were used ourstudies. MTT, colony formation assay, trypan blue staining, cell transfection, siRNAtechnology, qRT-PCR, flow cytometry, western blotting and other methods were usedin our study.Results1.1NCTD inhibited the expression of Mcl-1in HCC cells. The level of Mcl-1was examined in four HCC cell lines, including HuH-7, HepG2, BEL-7402andSMMC-7721. Western blotting analysis showed that NCTD at15μM partiallyinhibited the expression of Mcl-1, and NCTD at30and60μM completely inhibitedthe expression of Mcl-1in4cell lines. HCC cells treated by NCTD with or withoutpretreated with protease inhibitor MG132in HuH-7and HepG2. The results showedthat MG132treatment resulted in increased expression of Mcl-1in both cell line,MG132had a minimal role on NCTD-mediated Mcl-1inhibition, suggesting that downregulation of Mcl-1level did not caused by the proteasome-dependent proteindegradation. Mcl-1inhibition was observed at6hours after HuH-7and HepG2cellswere treated with NCTD at30μM, showing the effect of NCTD on Mcl-1occurredrapidly. qRT-PCR showed that NCTD also significantly reduced Mcl-1mRNA levels.1.2Aspirin inhibits the expression of Mcl-1in HCC cells. Aspirin at2.5,5,10mM treatment inhibited the expression of Mcl-1in HepG2and BEL-7402cell lines.In contrast, the expression of Bcl-2, Bcl-xL, XIAP and Survivin did not change in twoHCC cell lines after the treatments. Treatment with5mM aspirin for48h, theexpression of Mcl-1was partially inhibited,10mM aspirin completely inhibited theexpression of Mcl-1.2.1NCTD enhanced ABT-737-mediated cells proliferation and apoptosisinduction in HCC cells. We found that HuH-7and HepG2cells were not sensitive toABT-737. However, ABT-737in combination with NCTD significantly inhibited theproliferation of four HCC cells. In HuH-7and HepG2cells, treatment with NCTDalone or ABT-737for48hours barely induced apoptosis. However, treated by theircombination caused a large number of cells apoptosis.2.2Aspirin potentiated Navitoclax-mediated cell proliferation inhibition inHCC cell lines. Treatment with aspirin alone, Navitoclax alone or their combinationin HCC cells for48hours, cell survival inhibition detected by MTT. There wasminimal cytotoxity treated by aspirin alone or Navitoclax alone in2HCC cell lines.However, their combination tremendously inhibited cell viability in two HCC celllines. It was found that the combination effect was closely related with Mcl-1inhibited by aspirn. Aspirin at5,10mM almost completely inhibited the expressionof Mcl-1, cell proliferation inhibition rate was80-100%after treated by aspirin5,10mM combined with Navitoclax in two HCC cell lines. Aspirin enhanced the anti-HCC activities of Navitoclax mainly through downregulation of Mcl-1. 3.1NCTD potentiates ABT-737-triggered PARP cleavage and caspasesactivation. Western blotting analysis demonstrated that compared with the minimalactivity treated by signal-agents for48hours, treated by ABT-737plus NCTDresulted in obviously increase of cleavage of the caspase substrate PARP. Theexpression of caspase-9activation fragments (37/35kd) was distinctly increased intwo HCC cell lines. Similarly, the expression of caspase-3proteolytic fragments(19/17kd) was significantly increased in the combination treatment. With a pan-caspases inhibitor (zVAD.fmk) at50μM pretreatment of HuH-7and HepG2cells for1hour, then adding30μM NCTD and3μM ABT-737, cell death induction wasvastly reduced the by the combination, indicating anti-cancer activity of two-drugcombination is dependent on caspases pathway.3.2Aspirin potentiates Navitoclax-mediated anticancer activity by inducingapoptosis in HCC cells. Cells apoptosis assayed by Annexin V/propidium iodide (PI)staining and flow cytometry analysis after treatment with5mM aspirin alone,Navitoclax alone or their combined in HCC cell lines for48hours. The resultsshowed that aspirin alone, Navitoclax alone had a minimal effect, however, theircombination induced by89%,81%cells apoptosis in BEL-7402and HepG2respectively. Western blotting analysis showed that treatment with aspirin alone orNavitoclax alone barely induced the cleavage of PARP and the activation of caspase-9,-3, but their combination greatly induced PARP cleavage and caspase-9,-3activation.4.1NCTD potentiates ABT-737-inducing the release of cytochrome c. Therelease of cytochrome C from mitochondria into the cytosol is a major event in Bcl-2family proteins regulating apoptosis. NCTD or ABT-737alone did not inducecytochrome c release into cytoplasm, however, combination treatment induced robustcytochrome c release.4.2Aspirin potentiates the anticancer activity of Navitoclax-mediated dependent on mitochondrial apoptosis signaling pathway. The release ofcytochrome c from mitochondria to cytoplasm in HCC cell was not detected aftertreatment with aspirin alone and Navitoclax alone. The release of cytochrome c wasdetected after their combination, suggesting that the combination activated themitochondrial apoptosis signaling pathways. Z-LEHD-FMK, a pharmacologicalcaspase-9inhibitor, was used to investigate whether the anticancer activity of theircombination is dependent on mitochondrial apoptotic cell death in HCC cells.Pretreatment with Z-LEHD-FMK for1h completely prevented the cell deathinduction by the combination of aspirin at5mM plus Navitoclax at1μM.5.1Knockdown Mcl-1enhances the sensitivity of HCC cells to ABT-737.Western blotting analysis showed that the reduction of Mcl-1by siRNA against Mcl-1enhanced ABT-737-triggered PARP cleavage and caspase-3activation.Downregulation of Mcl-1also enhanced ABT-737-induced HCC cells death.5.2Knockdown of Mcl-1by siRNA phenotypes aspirin in potentiatingNavitoclax-mediated anti-HCC activities. Knockdown Mcl-1by siRNA obtainedsimilar anti-HCC cytotoxicity as the combination of5mM aspirin in combinationwith Navitoclax in the two HCC cell lines.6Bax and Bak play an essential role in the enhancement of ABT-737-mediated HCC cells apoptosis by NCTD. Knockdown Bax and Bak by siRNA andinvestigated the suppression of Bax/Bak on PARP cleavage and cell death by NCTDin combination with ABT-737in HepG2and HuH-7HCC cell lines. The resultsshowed that downregulation of Bax/Bak significantly reduced the combinationinduced PARP cleavage and cells death in HCC cells.ConclusionsNCTD and aspirin can suppress the expression of Mcl-1in HCC cells. NCTDand aspirin can significantly potentiate HCC cells to small molecule BH3mimetic ABT-737/ABT-263-mediated apoptosis. Moreover, the potentiation of ABT-737/ABT-263-triggered apoptosis was dependent upon mitochondrial apoptosis pathway inHCC cells. Inhibitor of Apoptosis Proteins (IAPs) play an important role in negativelyregulating apoptosis by inhibition of caspases activation. Overexpression of IAPs wasfound in many cancers, including hepatocellular carcinoma (HCC). Moreover,previous studies showed that overexpression of IAPs leads to HCC cells resistance tochemotherapy and high recurrence rate. The function of IAPs can be antagonized by amitochondrial protein, named second mitochondria-derived activator of caspase(Smac). Small molecule mimicking Smac (Smac mimetics) is being developed as aclass of novel anticancer drugs. Previous studies have shown that Smac mimetics caninduce apoptosis in certain tumor cells by targeting IAPs. A few Small moleculeSmac mimetics have entered clinical studies. However, whether small molecule Smacmimetic could sensitize HCC cells to chemotherapy has not been reported. In thisstudy, we have studied effect and molecular mechanism of small molecule Smacmimetic SM-164in potentiating APO2L/TRAIL-and doxorubicin-mediatedanticancer activity in HCC cell lines.ObjectiveTo study effect and molecular mechanism of small molecule Smac mimetic SM-164in potentiating APO2L/TRAIL-and doxorubicin-mediated anticancer activity inHCC cell lines.MethodsFour HCC cell lines, BEL-7402, SMMC-7721, Hep3B, HepG2, a normal liver cell line, L02, and12primary HCC cells freshly collected from tumor samples ofpatients with HCC were used in this study. Cell viability MTT assays, clonalformation assays and cell death induction were used to investigate the anticanceractivity. Western blotting was used to explore the mechanisms.Results1. SM-164induced rapid cIAP-1degradation but had a modest effect inHCC cells. Treatment with0.1nM of SM-164within1h induced cIAP-1degradation in these HCC cells, however, same treatment even for24h did not inhibitthe expression of XIAP. MTT assays showed that treatment with SM-164for4d hadminimal effect on inhibition of cell viability in these4HCC cell lines. Hep3B cellswere the most sensitive to SM-164, with an IC50of21μM. HepG2was the mostresistant, SM-164at100μM did not achive a50%cell survival inhibition. Inaddition, the IC50value of SM-164was45and63μM in BEL-7402, SMMC-7721cell lines, respectively.2. SM-164enhance APO2L/TRAIL anti-HCC activity. The sensitivity toAPO2L/TRAIL in the four HCC cell lines, BEL-7402, SMMC-7721, HepG2andHep3B, was assayed by MTT. The results demonstrated that BEL-7402was the mostsensitivity to APO2L/TRAIL. The cells was treated with10,30,100ng/ml ofAPO2L/TRAIL for4days, the viability inhibition was27%,54%and74%,respectively. SMMC-7721, HepG2and Hep3B cell lines were resistant toAPO2L/TRAIL. Treatment with APO2L/TRAIL at1000ng/ml for4days obtained asmall survival inhibition rate (<10%) in three cell lines. SM-164at100nMsignificantly enhanced APO2L/TRAIL-mediated cell survival inhibition (p<0.05) inBEL-7402cells. SM-164also drastically potentiated the anticancer activity ofAPO2L/TRAIL on SMMC-7721, HepG2and Hep3B cell lines. APO2L/TRAIL at1000ng/ml as a single had barely effect in SMMC-7721, however, APO2L/TRAIL at 100,300ng/ml combined with SM-164at100nM inhibited cell viability by80%and100%, respectively.3SM-164enhanced APO2L/TRAIL-mediated hepatocellular carcinoma cellcolony formation. After2weeks, inhibition of colony formation was observed whenAPO2L/TRAIL resistant SMMC-7721and HepG2cell lines were treated with thecombination. In SMMC-7721cells, whereas neither APO2L/TRAIL nor SM-164as asingle agent showed an obvious efficacy, the combination therapy achieved acomplete inhibition of colony formation (p <0.001). Further, in HepG2cell line,although the combination demonstrated a minimal effect on cell viability inhibitionand apoptosis induction. However, in colony formation assay, as opposed totreatment with either single drugs, the same concentration of two drugs combinationcaused great inhibition (p<0.001). The results showed that SM-164combined withAPO2L/TRAIL displayed a clear long-term effect on inhibition of HCC cell survivaland proliferation.4SM-164did not enhance the cytotoxicity of APO2L/TRAIL to humannormal liver cells. Human liver cell line L02was used to detect whether SM-164canenhance the cell toxicity of APO2L/TRAIL to normal human liver. The resultsshowed that treatment with300and3000ng/ml of APO2L/TRAIL for4days, cellsurvival inhibition rate was7%and23%. SM-164at100nM did not increaseAPO2L/TRAIL mediated cell survival inhibition.5SM-164enhances APO2L/TRAIL-induced apoptosis in HCC cell lines.Annexin-V staining and flow cytometry assays and western blotting assays were usedto study the apoptosis induction. Treatment with SM-164alone or APO2L/TRAILalone for24h, cell apoptosis rate was7%and14%, respectively. However, theircombination induced apoptosis in77%of HCC cells. Western blotting showed thatSM-164significantly enhanced APO2L/TRAIL-triggered apoptotic signaling pathway and led to activation of caspase-8, caspase-3and cleavage of PARP. Pan-caspases inhibitor z-VAD-fmk can weaken the combination activity of SM-164andAPO2L/TRAIL. Similarly, SM-164also enhanced APO2L/TRAIL-mediated cellsapoptosis in BEL-7402and HepG2cell lines.6SM-164combined with APO2L/TRAIL inhibited AKT activity in HCCcells. SMMC-7721, BEL-7402and HepG2cells were treated with SM-164alone,APO2L/TRAIL alone or their combination, AKT protein was examined by westernblotting. The results showed that the activation of AKT protein was observed in allthree HCC cell lines. APO2L/TRAIL alone or SM-164did not significantly inhibitthe activation of AKT, APO2L/TRAIL in combination with SM-164drasticallyreduced the level of phosphorylated AKT.7SM-164enhances APO2L/TRAIL-mediated cell death induction inprimary HCC cells. Primary HCC cells were obtained from12patients withsurgically resected fresh specimens. Cells treated with SM-164at100nM alone,APO2L/TRAIL at100ng/ml alone and their combination for24h, cell deathinduction was analyzed with trypan blue staining. In12HCC cell, only2primaryHCC cells were sensitive to APO2L/TRAI. However, SM-164and APO2L/TRAILcombination therapy induced robust cell death in11primary cells. Results of7primary cells show a statistically significant difference (p <0.05). Cell death inducedby SM-164+APO2L/TRAIL on primary cells was accompanied by the activation ofPARP.8SM-164enhanced anti-HCC activity of doxorubicin. HCC Cells weretreated with1μM of doxorubicin (Dox) alone,100nM of SM-164alone and theircombination for48h, the results showed that Hep3B cells were most sensitive to Dox.Dox at10μM induced32%,31%cell death in SMMC-7721, BEL-7402cell linesrespectively. SM-164at100nM significantly enhanced Dox-mediated inducing cells death in all three HCC cell lines. Western blotting assay showed that treatment withSM-164in combination with Dox induced activation of caspase-3and cleavage ofPARP.9SM-164in combination with Dox inhibited the activation of AKT in HCCcells. Cells treatment with1μM of Dox in Hep3B cell lines and10μM of Dox inBEL-7402, SMMC-7721cells alone,100nM of SM-164alone and their combinationfor24h, the activiation of AKT was examined. SM-164or Doxorubicin alone hadminimal effect on AKT phosphorylation. However, their combination treatmentsignificantly reduced the expression level of phosphorylated AKT in all3cell lines.ConclusionSmall molecule Smac mimetics can effectively enhance the anti-HCC activitiesof APO2L/TRAIL and Dox. The study showed that the anticancer mechanism by thecombination was dependent on enhancement of apoptosis. Meanwhile, this study alsodemonstrated that Smac mimetics is a promising drug for HCC. |
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