Tumor necrosis factor-related apoptosis-inducing ligand activates a lysosomal pathway of apoptosis that is regulated by Bcl-2 proteins

The present studies were performed to determine whether lysosomal permeabilization contributes to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity and to reconcile a role for lysosomes with prior observations that Bcl-2 family members regulate TRAIL-induced apoptosis. In KMCH cholangiocarcinoma cells stably expressing Mcl-1 small interference RNA (siRNA), treatment with TRAIL induced a redistribution of the cathepsin B from lysosomes to the cytosol. Pharmacological and small hairpin RNA-targeted inhibition of cathepsin B attenuated TRAIL-mediated apoptosis as assessed by morphological, biochemical, and clonogenic assays. Neither Bid siRNA nor Bak siRNA prevented cathepsin B release. In contrast, treatment of the cells with Bim siRNA or the JNK inhibitor SP600125 attenuated lysosomal permeabilization and cell death. Moreover, Bim and active Bax co-localized to lysosomes in TRAIL-treated cells in a JNK-dependent manner, and Bax siRNA reduced TRAIL-induced lysosomal permeabilization and cell death. Finally, BH3 domain peptides permeabilized isolated lysosomes in the presence of Bax. Collectively, these data suggest that TRAIL can trigger an apoptotic pathway that involves JNK-dependent activation of Bim, which in turn induces Bax-mediated permeabilization of lysosomes.     

cFLIPL prevents TRAIL-induced apoptosis of hepatocellular carcinoma cells by inhibiting the lysosomal pathway of apoptosis

Sensitivity to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and the lysosomal pathway of cell death are features of cancer cells. However, it is unknown if TRAIL cytotoxic signaling engages the lysosomal pathway of cell death. Our aim, therefore, was to ascertain if TRAIL killing involves lysosomal permeabilization. TRAIL-induced apoptosis of hepatocellular carcinoma cells (HuH-7, Hep3B) was associated with lysosomal permeabilization, as demonstrated by redistribution of the lysosomal protease cathepsin B into the cytosol. Pharmacological and short hairpin RNA-targeted inhibition of cathepsin B reduced apoptosis. Because cellular FLICE-inhibitory protein (cFLIP) inhibits TRAIL-induced cell death and is frequently overexpressed by human cancers, the ability of cFLIP to prevent lysosomal permeabilization during TRAIL treatment was examined. Enforced long-form cFLIP (cFLIP(L)) expression reduced release of cathepsin B from lysosomes and attenuated apoptosis. cFLIP(L) overexpression was also associated with robust p42/44 MAPK activation following exposure to TRAIL. In contrast, cFLIP(L) overexpression attenuated p38 MAPK activation and had no significant effect on JNK and NF-kappaB activation. Inhibition of p42/44 MAPK by PD98059 restored TRAIL-mediated lysosomal permeabilization and apoptosis in cFLIP-overexpressing cells. In conclusion, these results demonstrate that lysosomal permeabilization contributes to TRAIL-induced apoptosis of hepatocellular carcinoma cells and suggest that cFLIP(L) cytoprotection is, in part, due to p42/44 MAPK-dependent inhibition of lysosomal breakdown.     

JNK mediates UVB-induced apoptosis upstream lysosomal membrane permeabilization and Bcl-2 family proteins

UVB irradiation induced phosphorylation of JNK and subsequent apoptosis in human melanocytes. Depletion of both JNK1 and JNK2 expression using siRNA transfection, protected against apoptosis, as detected by decreased nuclear fragmentation and caspase-3 activity, as well as reduced translocation of Bax to mitochondria. Moreover, release of cathepsin B and D from lysosomes to the cytosol was reduced when JNK expression was suppressed by siRNA, demonstrating a JNK dependent regulation of lysosomal membrane permeabilization. In unirradiated control melanocytes, coimmunoprecipitation showed that Bim was sequestered by Mcl-1, which had a pro-survival function. After UVB irradiation, a significant decrease in Mcl-1 protein level was found, which was prevented by addition of a proteasome inhibitor. The interaction between Bim and Mcl-1 was reduced in response to UVB irradiation and Bim was phosphorylated in a JNK dependent manner. In conclusion, these findings suggest JNK to have an important pro-apoptotic function following UVB irradiation in human melanocytes, by acting upstream of lysosomal membrane permeabilization and Bim phosphorylation.     

Hypoxia inhibits TRAIL-induced tumor cell apoptosis: Involvement of lysosomal cathepsins

Tumor hypoxia interferes with the efficacy of chemotherapy, radiotherapy, and tumor necrosis factor-α. TRAIL (tumor necrosis factor-related apoptosis inducing ligand) is a potent apoptosis inducer that limits tumor growth without damaging normal cells and tissues in vivo. We present evidence for a central role of lysosomal cathepsins in hypoxia and/or TRAIL-induced cell death in oral squamous cell carcinoma (OSCC) cells. Hypoxia or TRAIL-induced activation of cathepsins (B, D and L), caspases (-3 and -9), Bid cleavage, release of Bax and cytochrome c, and DNA fragmentation were blocked independently by zVAD-fmk, CA074Me or pepstatin A, consistent with the involvement of lysosomal cathepsin B and D in cell death. Lysosome stability and mitochondrial membrane potential were reduced in hypoxia and TRAIL-induced apoptosis. However, TRAIL treatment under hypoxic condition resulted in diminished apoptosis rates compared to treatment under normoxia. This inhibitory effect of hypoxia on TRAIL-induced apoptosis may be based on preventing Bax activation and thus protecting mitochondria stability. Our data show that TRAIL or hypoxia independently triggered activation of cathepsin B and D leading to apoptosis through Bid and Bax, and suggest that hypoxic tissue regions provide a selective environment for highly apoptosis-resistant clonal cells. Molecular therapy approaches based on cathepsin inhibitors need to address this novel tumor-preventing function of cathepsins in OSCC.     

Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors: Evidence for the role of c-Jun N-terminal kinase activation

The discovery of the molecular targets of chemotherapeutic medicines and their chemical footprints can validate and improve the use of such medicines. In the present report, we investigated the effect of mitomycin C (MMC), a classical chemotherapeutic agent on cancer cell apoptosis induced by TRAIL. We found that MMC not only potentiated TRAIL-induced apoptosis in HCT116 (p53−/−) colon cancer cells but also sensitized TRAIL-resistant colon cancer cells HT-29 to the cytokine both in vitro and in vivo. MMC also augmented the pro-apoptotic effects of two TRAIL receptor agonist antibodies, mapatumumab and lexatumumab. At a mechanistic level, MMC downregulated cell survival proteins, including Bcl2, Mcl-1 and Bcl-XL, and upregulated pro-apoptotic proteins including Bax, Bim and the cell surface expression of TRAIL death receptors DR4 and DR5. Gene silencing of DR5 by short hairpin RNA reduced the apoptosis induced by combination treatment of MMC and TRAIL. Induction of DR4 and DR5 was independent of p53, Bax and Bim but was dependent on c-Jun N terminal kinase (JNK) as JNK pharmacological inhibition and siRNA abolished the induction of the TRAIL receptors by MMC.     

Cathepsin D-Bax death pathway in oxidative stressed neuroblastoma cells

Hydrogen peroxide, the major oxidoradical species in the central nervous system, has been involved in neuronal cell death and associated neurodegenerative diseases. In this study, we have investigated the involvement of the lysosomal pathway in the cytotoxic mechanism of hydrogen peroxide in human neuroblastoma cells. Alteration of lysosomal and mitochondrial membrane integrity was shown to be an early event in the lethal cascade triggered by oxidative stress. Desferrioxamine (DFO), an iron chelator that abolishes the formation of reactive oxygen species within lysosomes, prevented lysosome leakage, mitochondrial permeabilization and caspase-dependent apoptosis in hydrogen peroxide-treated cells. Inhibition of cathepsin D, not of cathepsin B, as well as small-interference RNA-mediated silencing of the cathepsin D gene prevented hydrogen peroxide-induced injury of mitochondria, caspase activation, and TUNEL-positive cell death. Cathepsin D activity was shown indispensable for translocation of Bax onto mitochondrial membrane associated with oxidative stress. DFO abolished both the cytosolic relocation of Cathepsin D and the mitochondrial relocation of Bax in hydrogen peroxide-treated cells. siRNA-mediated down-regulation of Bax expression protected the cells from oxidoradical injury. The present study identifies the lysosome as the primary target and the axis cathepsin D-Bax as the effective pathway of hydrogen peroxide lethal activity in neuroblastoma cells.     

Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors

The discovery of the molecular targets of chemotherapeutic medicines and their chemical footprints can validate and improve the use of such medicines. In the present report, we investigated the effect of mitomycin C (MMC), a classical chemotherapeutic agent on cancer cell apoptosis induced by TRAIL. We found that MMC not only potentiated TRAIL-induced apoptosis in HCT116 (p53?/?) colon cancer cells but also sensitized TRAIL-resistant colon cancer cells HT-29 to the cytokine both in vitro and in vivo. MMC also augmented the pro-apoptotic effects of two TRAIL receptor agonist antibodies, mapatumumab and lexatumumab. At a mechanistic level, MMC downregulated cell survival proteins, including Bcl2, Mcl-1 and Bcl-XL, and upregulated pro-apoptotic proteins including Bax, Bim and the cell surface expression of TRAIL death receptors DR4 and DR5. Gene silencing of DR5 by short hairpin RNA reduced the apoptosis induced by combination treatment of MMC and TRAIL. Induction of DR4 and DR5 was independent of p53, Bax and Bim but was dependent on c-Jun N terminal kinase (JNK) as JNK pharmacological inhibition and siRNA abolished the induction of the TRAIL receptors by MMC.     

Phosphatidic acid mediates the targeting of tBid to induce lysosomal membrane permeabilization and apoptosis

Upon apoptotic stimuli, lysosomal proteases, including cathepsins and chymotrypsin, are released into cytosol due to lysosomal membrane permeabilization (LMP), where they trigger apoptosis via the lysosomal-mitochondrial pathway of apoptosis. Herein, the mechanism of LMP was investigated. We found that caspase 8-cleaved Bid (tBid) could result in LMP directly. Although Bax or Bak might modestly enhance tBid-triggered LMP, they are not necessary for LMP. To study this further, large unilamellar vesicles (LUVs), model membranes mimicking the lipid constitution of lysosomes, were used to reconstitute the membrane permeabilization process in vitro. We found that phosphatidic acid (PA), one of the major acidic phospholipids found in lysosome membrane, is essential for tBid-induced LMP. PA facilitates the insertion of tBid deeply into lipid bilayers, where it undergoes homo-oligomerization and triggers the formation of highly curved nonbilayer lipid phases. These events induce LMP via pore formation mechanisms because encapsulated fluorescein-conjugated dextran (FD)-20 was released more significantly than FD-70 or FD-250 from LUVs due to its smaller molecular size. On the basis of these data, we proposed tBid-PA interactions in the lysosomal membranes form lipidic pores and result in LMP. We further noted that chymotrypsin-cleaved Bid is more potent than tBid at binding to PA, inserting into the lipid bilayer, and promoting LMP. This amplification mechanism likely contributes to the culmination of apoptotic signaling.     

Lysosomal membrane permeabilization as apoptogenic factor

Lysosomal membrane labilizing agents (incl. proapoptotic proteins of Bcl-2 family, LAPF, p53), estimation of lysosomal membrane permeabilization in living cells, the new data on differential permeabilization of lysosomal membranes, membrane stabilizing factors (incl. Hsp70), relations between lysosomal membrane damage, and initiation of apoptosis were considered. Signal effect of lysosomal membrane permeabilization is caused preferentially by release of cathepsin B and D in cytosol. Subsequent numerous pathways of apoptogenic signalization include proteolytic attack/activation on signal cytosolic proteins, mitochondria, procaspases, cell nuclei. The mainstream of the cell damage is connected with activation pf proapoptotic Bid and Bax, leading to permeabilization of the outer mitochondrial membrane, release of cytochrome c into cytosol and activation of caspase cascade. Translocation of the lysosoma enzymes in cytosol is capable to induce both the caspase-dependent and caspase-independent paths of apoptosis.     

Differential inhibition of TRAIL-mediated DR5-DISC formation by decoy receptors 1 and 2

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that induces cancer cell death by apoptosis with some selectivity. TRAIL-induced apoptosis is mediated by the transmembrane receptors death receptor 4 (DR4) (also known as TRAIL-R1) and DR5 (TRAIL-R2). TRAIL can also bind decoy receptor 1 (DcR1) (TRAIL-R3) and DcR2 (TRAIL-R4) that fail to induce apoptosis since they lack and have a truncated cytoplasmic death domain, respectively. In addition, DcR1 and DcR2 inhibit DR4- and DR5-mediated, TRAIL-induced apoptosis and we demonstrate here that this occurs through distinct mechanisms. While DcR1 prevents the assembly of the death-inducing signaling complex (DISC) by titrating TRAIL within lipid rafts, DcR2 is corecruited with DR5 within the DISC, where it inhibits initiator caspase activation. In addition, DcR2 prevents DR4 recruitment within the DR5 DISC. The specificity of DcR1- and DcR2-mediated TRAIL inhibition reveals an additional level of complexity for the regulation of TRAIL signaling.     

PS-341 (bortezomib) induces lysosomal cathepsin B release and a caspase-2-dependent mitochondrial permeabilization and apoptosis in human pancreatic cancer cells

PS-341 (bortezomib) is a potent and reversible proteosome inhibitor that functions to degrade intracellular polyubiquitinated proteins. PS-341 induces apoptosis and has shown broad antitumor activity with selectivity for transformed cells. We studied the effect of PS-341 on lysosomal and mitochondrial permeabilization, including the role of caspase-2 activation in apoptosis induction in the BxPC-3 human pancreatic carcinoma cell line. PS-341 induced a dose-dependent apoptosis in association with reactive oxygen species generation and cleavage of caspase-2 to its 33- and 14-kDa fragments. PS-341 disrupted lysosomes with redistribution of cathepsin B to the cytosol, as shown using fluorescence confocal microscopy, that was blocked by the free radical scavenger tiron but not by a caspase-2 inhibitor (benzyloxycarbonyl (Z)-VDVAD-fluoromethyl ketone (FMK)). PS-341-induced caspase-2 activation was attenuated by a selective pharmacological inhibitor of cathepsin B (R-3032), suggesting that cathepsin B release occurs upstream of caspase-2. PS-341-induced mitochondrial depolarization was attenuated by Z-VDVAD-FMK, tiron, and an inhibitor of the mitochondrial permeability transition pore (bongkrekic acid). Regulation of mitochondrial permeability by caspase-2 was confirmed using caspase-2 small interfering RNA. PS-341-induced cytochrome c release and phosphatidylserine externalization were attenuated by Z-VDVAD-FMK and partially by R-3032. PS-341 activated the BH3-only proteins Bik and Bim and down-regulated Bcl-2 and Bcl-xL mRNA and protein expression. Taken together, PS-341 induces lysosomal cathepsin B redistribution upstream of caspase-2. Caspase-2 activation regulates PS-341-induced mitochondrial depolarization and apoptosis, suggesting that caspase-2 can serve as a link between lysosomal and mitochondrial permeabilization.     

Bile acids stimulate cFLIP phosphorylation enhancing TRAIL-mediated apoptosis

Bile acids induce hepatocyte injury by enhancing death receptor-mediated apoptosis. In this study, bile acid effects on TRAIL-mediated apoptosis were examined to gain insight into bile acid potentiation of death receptor signaling. TRAIL-induced apoptosis of HuH-7 cells, stably transfected with a bile acid transporter, was enhanced by bile acids. Caspase 8 and 10 activation, bid cleavage, cytosolic cytochrome c, and caspase 3 activation by TRAIL were all increased by the bile acid glycochenodeoxycholate (GCDCA). GCDCA (100 microm) did not alter expression of TRAIL-R1/DR4, TRAIL-R2/DR5, procaspase 8, cFLIP-L, cFLIP-s, Bax, Bcl-xL, or Bax. However, both caspase 8 and caspase 10 recruitment and processing within the TRAIL death-inducing signaling complex (DISC) were greater in GCDCA-treated cells whereas recruitment of cFLIP long and short was reduced. GCDCA stimulated phosphorylation of both cFLIP isoforms, which was associated with decreased binding to GST-FADD. The protein kinase C antagonist chelerythrine prevented bile acid-stimulated cFLIP-L and -s phosphorylation, restored cFLIP binding to GST-FADD, and attenuated bile acid potentiation of TRAIL-induced apoptosis. These results provide new insights into the mechanisms of bile acid cytotoxicity and the proapoptotic effects of cFLIP phosphorylation in TRAIL signaling.     

Nimbolide sensitizes human colon cancer cells to TRAIL through reactive oxygen species- and ERK-dependent up-regulation of death receptors, p53, and Bax

TNF-related apoptosis-inducing ligand (TRAIL) shows promise as a cancer treatment, but acquired tumor resistance to TRAIL is a roadblock. Here we investigated whether nimbolide, a limonoid, could sensitize human colon cancer cells to TRAIL. As indicated by assays that measure esterase activity, sub-G(1) fractions, mitochondrial activity, and activation of caspases, nimbolide potentiated the effect of TRAIL. This limonoid also enhanced expression of death receptors (DRs) DR5 and DR4 in cancer cells. Gene silencing of the receptors reduced the effect of limonoid on TRAIL-induced apoptosis. Using pharmacological inhibitors, we found that activation of ERK and p38 MAPK was required for DR up-regulation by nimbolide. Gene silencing of ERK abolished the enhancement of TRAIL-induced apoptosis. Moreover, our studies indicate that the limonoid induced reactive oxygen species production, which was required for ERK activation, up-regulation of DRs, and sensitization to TRAIL; these effects were mimicked by H(2)O(2). In addition, nimbolide down-regulated cell survival proteins, including I-FLICE, cIAP-1, cIAP-2, Bcl-2, Bcl-xL, survivin, and X-linked inhibitor of apoptosis protein, and up-regulated the pro-apoptotic proteins p53 and Bax. Interestingly, p53 and Bax up-regulation by nimbolide was required for sensitization to TRAIL but not for DR up-regulation. Overall, our results indicate that nimbolide can sensitize colon cancer cells to TRAIL-induced apoptosis through three distinct mechanisms: reactive oxygen species- and ERK-mediated up-regulation of DR5 and DR4, down-regulation of cell survival proteins, and up-regulation of p53 and Bax.     

IFN-gamma inhibition of TRAIL-induced IAP-2 upregulation, a possible mechanism of IFN-gamma-enhanced TRAIL-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine molecule of TNF family and a potent inducer of apoptosis. The anticancer activities of TNF family members are often modulated by interferon (IFN)-gamma. Thus, we investigated whether IFN-gamma enhances TRAIL-induced apoptosis. We exposed HeLa cells to IFN-gamma for 12 h and then treated with recombinant TRAIL protein. No apoptosis was induced in cells pretreated with IFN-gamma, and TRAIL induced 25% cell death after 3 h treatment. In HeLa cells pretreated with IFN-gamma, TRAIL induced cell death to more than 70% at 3 h, indicating that IFN-gamma pretreatment sensitized HeLa cells to TRAIL-induced apoptosis. We investigated molecules that might be regulated by IFN-gamma pretreatment that would affect TRAIL-induced apoptosis. Western blotting analyses demonstrated that TRAIL treatment increased the level of IAP-2 protein and IFN-gamma pretreatment inhibited the upregulation of IAP-2 protein by TRAIL protein. Our data indicate that TRAIL can signal to activate both apoptosis induction and antiapoptotic mechanism, at least, through IAP-2 simultaneously. IFN-gamma or TRAIL treatment alone did not change expression of other pro- or antiapoptotic proteins such as DR4, DR5, FADD, Bax, IAP-1, XIAP, Bcl-2, and Bcl-XL. Our findings suggest that IFN-gamma may sensitize HeLa cells to TRAIL-induced apoptosis by preventing TRAIL-induced IAP-2 upregulation, and IFN-gamma may play a role in anticancer therapy of TRAIL protein through such mechanism.     

Receptor-selective mutants of apoptosis-inducing ligand 2/tumor necrosis factor-related apoptosis-inducing ligand reveal a greater contribution of death receptor (DR) 5 than DR4 to apoptosis signaling

Apoptosis-inducing ligand 2 (Apo2L), also called tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), triggers programmed cell death in various types of cancer cells but not in most normal cells. Apo2L/TRAIL is a homotrimeric protein that interacts with five receptors: death receptor 4 (DR4) and DR5 mediate apoptosis activation, whereas decoy receptor 1 (DcR1), DcR2, and osteoprotegerin counteract this function. Many cancer cell lines express both DR4 and DR5, and each of these receptors can initiate apoptosis independently of the other. However, the relative contribution of DR4 and DR5 to ligand-induced apoptosis is unknown. To investigate this question, we generated death receptor-selective Apo2L/TRAIL variants using a novel approach that enables phage display of mutated trimeric proteins. Selective binding to DR4 or DR5 was achieved with three to six-ligand amino acid substitutions. The DR4-selective Apo2L/TRAIL variants examined in this study showed a markedly reduced ability to trigger apoptosis, whereas the DR5-selective variants had minimally decreased or slightly increased apoptosis-inducing activity. These results suggest that DR5 may contribute more than DR4 to Apo2L/TRAIL-induced apoptosis in cancer cells that express both death receptors.     

Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells

Although resveratrol, an active ingredient derived from grapes and red wine, possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. Here, we examined the molecular mechanisms of resveratrol and its interactive effects with TRAIL on apoptosis in prostate cancer PC-3 and DU-145 cells. Resveratrol inhibited cell viability and colony formation, and induced apoptosis in prostate cancer cells. Resveratrol downregulated the expression of Bcl-2, Bcl-X_L and survivin and upregulated the expression of Bax, Bak, PUMA, Noxa, and Bim, and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Treatment of prostate cancer cells with resveratrol resulted in generation of reactive oxygen species (ROS), translocation of Bax to mitochondria and subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, Smac/DIABLO, and AIF) to cytosol, activation of effector caspase-3 and caspase-9, and induction of apoptosis. Resveratrol-induced ROS production, caspase-3 activity and apoptosis were inhibited by N-acetylcysteine. Bax was a major proapoptotic gene mediating the effects of resveratrol as Bax siRNA inhibited resveratrol-induced apoptosis. Resveratrol enhanced the apoptosis-inducing potential of TRAIL, and these effects were inhibited by either dominant negative FADD or caspase-8 siRNA. The combination of resveratrol and TRAIL enhanced the mitochondrial dysfunctions during apoptosis. These properties of resveratrol strongly suggest that it could be used either alone or in combination with TRAIL for the prevention and/or treatment of prostate cancer.     

Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells

Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3.     

Caveolin-1 negatively regulates TRAIL-induced apoptosis in human hepatocarcinoma cells

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) offers promising therapeutic potential based on its ability to induce apoptosis in various cancer cell lines without obvious adverse effect to normal cells. However, the mechanism of the differential sensitivity towards TRAIL-induced apoptosis remains unclear. Here, we demonstrate that caveolin-1 directly regulated TRAIL-induced apoptosis in HepG2 cells. ShRNA-mediated caveolin knockdown sensitized TRAIL-induced apoptosis and disruption of caveolae structure by the cholesterol-extracting reagent, methyl-β-cyclodextrin (MCD), enhanced TRAIL-induced apoptosis. Over-expression of caveolin-1 partially blocked TRAIL-induced apoptosis. The engagement of TRAIL with its receptor DR4 reduced the localization of DR4 in caveolae and resulted in its internalization. Blockade of caveolae-mediated internalization of DR4 by filipin III effectively enhanced TRAIL-induced apoptosis. Collectively, our results reveal a new mechanism by which caveolin-1 negatively regulates TRAIL-induced apoptosis in human hepatocarcinoma cells.