Effect of hyperthermia and chemotherapeutic agents on TRAIL-induced cell death in human colon cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In the previous study [Yoo and Lee, 2007], we have reported that hyperthermia could enhance the cytotoxicity of TRAIL-induced apoptosis. We observed in human colorectal cancer cell line CX-1 that TRAIL-induced apoptotic death and also that mild hyperthermia promoted TRAIL-induced apoptotic death through caspase activation and cytochrome-c release. Although its effects in vivo are not clear, hyperthermia has been used as an adjunctive therapy for cancer. Hyperthermia is often accompanied by chemotherapy to enhance its effect. In this study, CX-1 colorectal adenocarcinoma cells were treated with TRAIL concurrently with hyperthermia and oxaliplatin or melphalan. To evaluate the cell death effects on tumor cells via hyperthermia and TRAIL and chemotherapeutic agents, FACS analysis, DNA fragmentation, and immunoblottings for PARP-1 and several caspases and antiapoptotic proteins were performed. Activities of casapse-8, caspase-9, and caspase-3 were also measured in hyperthermic condition. Interestingly, when analyzed with Western blot, we detected little change in the intracellular levels of proteins related to apoptosis. Clonogenic assay shows, however, that chemotherapeutic agents will trigger cancer cell death, either apoptotic or non-apoptotic, more efficiently. We demonstrate here that CX-1 cells exposed to 42 degrees C and chemotherapeutic agents were sensitized and died by apoptotic and non-apoptotic cell death even in low concentration (10 ng/ml) of TRAIL.     

Effect of hyperthermia in combination with TRAIL on the JNK‐Bim signal transduction pathway and growth of xenograft tumors

Approximately 25% of patients with colorectal cancer develop metastases to the liver, and surgery is currently the best treatment available. But there are several patients who are unresectable, and isolated hepatic perfusion (IHP) offers a different approach in helping to treat these patients. IHP is a method used for isolating the liver and delivering high doses of chemotherapeutic agents. The efficacy of IHP has been improved by combining hyperthermia not only with chemotherapeutics but with other deliverable agents such as tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL). In this study, we used human colorectal cancer CX‐1 cells and treated them with hyperthermia and TRAIL, causing cytotoxicity. We were able to demonstrate that the numbers of live cells were significantly reduced with hyperthermia and 10 ng/ml of TRAIL combined. We also showed that the effect of hyperthermia on TRAIL in our studies was enhancement of the apoptotic pathway by the promotion of JNK and Bim_EL activity as well as PARP cleavage. We have also used our CX‐1 cells to generate tumors in Balb/c nude mice. With intratumoral injections of TRAIL combined with hyperthermia at 42°C, we were able to show a delayed onset of tumor growth in our xenograft model. J. Cell. Biochem. 110: 1073–1081, 2010. Published 2010 Wiley‐Liss, Inc.     

Role of Bcl-xL/Beclin-1 in synergistic apoptotic effects of secretory TRAIL-armed adenovirus in combination with mitomycin C and hyperthermia on colon cancer cells

In this study, we attempted to develop a multimodality approach using chemotherapeutic agent mitomycin C, biologic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L), and mild hyperthermia to treat colon cancer. For this study, human colon cancer LS174T, LS180, HCT116 and CX-1 cells were infected with secretory TRAIL-armed adenovirus (Ad.TRAIL) and treated with chemotherapeutic agent mitomycin C and hyperthermia. The combinatorial treatment caused a synergistic induction of apoptosis which was mediated through an increase in caspase activation. The combinational treatment promoted the JNK-Bcl-xL-Bak pathway which transmitted the synergistic effect through the mitochondria-dependent apoptotic pathway. JNK signaling led to Bcl-xL phosphorylation at serine 62, dissociation of Bak from Bcl-xL, oligomerization of Bak, alteration of mitochondrial membrane potential, and subsequent cytochrome c release. Overexpression of dominant-negative mutant of Bcl-xL (S62A), but not dominant-positive mutant of Bcl-xL (S62D), suppressed the synergistic death effect. Interestingly, Beclin-1 was dissociated from Bcl-xL and overexpression of dominant-negative mutant of Bcl-xL (S62A), but not dominant-positive mutant of Bcl-xL (S62D), suppressed dissociation of Beclin-1 from Bcl-xL. A combinatorial treatment of mitomycin C, Ad.TRAIL and hyperthermia induced Beclin-1 cleavage, but the Beclin-1 cleavage was abolished in Beclin-1 double mutant (D133A/D146A) knock-in HCT116 cells, suppressing the apoptosis induced by the combination therapy. We believe that this study supports the application of the multimodality approach to colon cancer therapy.     

TRAIL: a potential agent for cancer therapy

Induction of apoptosis in cancer cells with chemotherapy and radiation treatment is a major strategy in cancer therapy at present. Nevertheless, innate or acquired resistance has been an obstacle for conventional clinical therapy. TNF-related apoptosis inducing ligand (TRAIL/Apo-2L) is a typical member of the TNF ligand family that induces apoptosis through activating the death receptors. In recent years, considerable attention has been focused on the potential benefits of TRAIL in cancer therapy, as the majority of cancer cells are sensitive to TRAIL-induced apoptosis, while most normal cells are TRAIL-resistant. Furthermore, the use of TRAIL in combination with chemotherapeutic agents or irradiation strengthens its apoptotic effects. In this review, we will discuss the regulation mechanism of TRAIL-induced apoptosis and the molecular basis of the synergies created by its use in combination with chemotherapeutic agents and irradiation. We also analyze in detail that TRAIL may be cytotoxic, as this is a potential obstacle to its development for being used in cancer therapy.     

Hyperthermia-enhanced TRAIL- and mapatumumab-induced apoptotic death is mediated through mitochondria in human colon cancer cells

Colorectal cancer is the third leading cause of cancer-related mortality in the world; death usually results from uncontrolled metastatic disease. Previously, we developed a novel strategy of TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) in combination with hyperthermia to treat hepatic colorectal metastases. However, previous studies suggest a potential hepatocyte cytotoxicity with TRAIL. Unlike TRAIL, anti-human TRAIL receptor antibody induces apoptosis without hepatocyte toxicity. In this study, we evaluated the anti-tumor efficacy of humanized anti-death receptor 4 (DR4) antibody mapatumumab (Mapa) by comparing it with TRAIL in combination with hyperthermia. TRAIL, which binds to both DR4 and death receptor 5 (DR5), was approximately tenfold more effective than Mapa in inducing apoptosis. However, hyperthermia enhances apoptosis induced by either agent. We observed that the synergistic effect was mediated through elevation of reactive oxygen species, c-Jun N-terminal kinase activation, Bax oligomerization, and translocalization to the mitochondria, loss of mitochondrial membrane potential, release of cytochrome c to cytosol, activation of caspases, and increase in poly(ADP-ribose) polymerase cleavage. We believe that the successful outcome of this study will support the application of Mapa in combination with hyperthermia to colorectal hepatic metastases.     

Low extracellular pH augments TRAIL-induced apoptotic death through the mitochondria-mediated caspase signal transduction pathway

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/APO-2L), a member of the tumor necrosis factor (TNF) gene family, is considered as one of the most promising cancer therapeutic agents due to its ability to selectively kill tumor cells. Although microenvironments of solid tumors (hypoxia, nutrient deprivation, and low pH) often affect the effectiveness of chemotherapy, few studies have been reported on the relationship between tumor microenvironments and TRAIL. In this study, we investigated whether low extracellular pH affects TRAIL-induced apoptotic death. When human prostate carcinoma DU145 cells were treated with 200 ng/ml His-tagged TRAIL for 4 h, the survival was approximately 10% at pH 6.3-6.6 and 61.3% at pH 7.4. Similar results were observed in human colorectal carcinoma CX-1 cell line. The TRAIL-mediated activation of caspase, cytochrome c release, and poly (ADP-ribose) polymerase (PARP) cleavage was promoted at low extracellular pH. Immunoprecipitation followed by western blot analysis shows that low extracellular pH enhances the association of truncated Bid with Bax during treatment with TRAIL. Western blot analysis also shows that the low extracellular pH-enhanced TRAIL cytotoxicity does not involve modulation of the levels of TRAIL receptors (DR4, DR5, and DcR2), FLIP, inhibitor of apoptosis (IAP), and Bcl-2. Overexpression of Bcl-2 effectively prevented low extracellular pH-augmented TRAIL cytotoxicity. Taken together, we propose that TRAIL-mediated cytotoxicity is greatly enhanced in low pH environments by promoting caspase activation.     

Encorafenib enhances TRAIL-induced apoptosis of colorectal cancer cells dependent on p53/PUMA signaling

TRAIL has been demonstrated to play a critical role in the apoptosis of colorectal cancer (CRC) cells, but drug resistance markedly restricts its therapeutic effects. Objectives: This study aims to investigate whether encorafenib can enhance TRAIL-induced apoptosis of colorectal cancer cells and the underlying mechanism. TRAIL was first used to induce CRC cells. CCK-8 assays were conducted for detecting cell viability of TRAIL-induced CRC cells with encorafenib treatment. Flow cytometry was used to detect the cell apoptosis of CRC cells and western blot was used to measure the expressions of apoptosis-related proteins. The expressions of DR4, DR5, p53, and PUMA were then evaluated by qPCR and western blot. After transfecting the interference plasmid of p53 into CRC cells, the expressions of PUMA and DR5 were further explored. TRAIL reduced the cell viability of CRC cells, and the inhibition was further reinforced under co-treatment of TRAIL and encorafenib. Encorafenib also triggered the promotion of CRC cell apoptosis induced by TRAIL. It was also found that encorafenib exerted its promoting effects on cell apoptosis of CRC cells via the elevation of DR5. Besides, encorafenib administration promoted the expression levels of p53 and PUMA in TRAIL-induced CRC cells. Furthermore, p53 knockdown attenuated the expression of PUMA and DR5 in TRAIL-induced CRC cells treated with encorafenib. This study indicates that encorafenib stimulates TRAIL-induced apoptosis of CRC cells dependent on p53/PUMA signaling, which may provide instructions for the treatment of CRC.     

Codium fragile F2 sensitize colorectal cancer cells to TRAIL-induced apoptosis via c-FLIP ubiquitination

This study demonstrates that combined treatment with subtoxic doses of Codium extracts (CE), a flavonoid found in many fruits and vegetables, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), induces apoptosis in TRAIL-resistant colorectal cancer (CRC) cells. Effective induction of apoptosis by combined treatment with CE and TRAIL was not blocked by Bcl-xL overexpression, which is known to confer resistance to various chemotherapeutic agents. While TRAIL-mediated proteolytic processing of procaspase-3 was partially blocked in various CRC cells treated with TRAIL alone, co-treatment with CE efficiently recovered TRAIL-induced caspase activation. We observed that CE treatment of CRC cells did not change the expression of anti-apoptotic proteins and pro-apoptotic proteins, including death receptors (DR4 and DR5). However, CE treatment markedly reduced the protein level of the short form of the cellular FLICE-inhibitory protein (c-FLIPS), an inhibitor of caspase-8, via proteasome-mediated degradation. Collectively, these observations show that CE recovers TRAIL sensitivity in various CRC cells via down-regulation of c-FLIPS.     

Down-regulation of cFLIP following reovirus infection sensitizes human ovarian cancer cells to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) shows promise as a chemotherapeutic agent. However, many human cancer cells are resistant to killing by TRAIL. We have previously demonstrated that reovirus infection increases the susceptibility of human lung (H157) and breast (ZR75-1) cancer cell lines to TRAIL-induced apoptosis. We now show that reovirus also increases the susceptibility of human ovarian cancer cell lines (OVCAR3, PA-1 and SKOV-3) to TRAIL-induced apoptosis. Reovirus-induced increases in susceptibility of OVCAR3 cells to TRAIL require virus uncoating and involve increased activation of caspases 3 and 8. Reovirus infection results in the down-regulation of cFLIP (cellular FLICE inhibitory protein) in OVCAR3 cells. Down-regulation of cFLIP following treatment of OVCAR3 cells with antisense cFLIP oligonucleotides or PI3 kinase inhibition also increases the susceptibility of OVCAR3 cells to TRAIL-induced apoptosis. Finally, over-expression of cFLIP blocks reovirus-induced sensitization of OVCAR3 cells to TRAIL-induced apoptosis. The combination of reovirus and TRAIL thus represents a promising new therapeutic approach for the treatment of ovarian cancer.     

TNFα cooperates with IFN-γ to repress Bcl-xL expression to sensitize metastatic colon carcinoma cells to TRAIL-mediated apoptosis

Background

TNF-related apoptosis-inducing ligand (TRAIL) is an immune effector molecule that functions as a selective anti-tumor agent. However, tumor cells, especially metastatic tumor cells often exhibit a TRAIL-resistant phenotype, which is currently a major impediment in TRAIL therapy. The aim of this study is to investigate the synergistic effect of TNFα and IFN-γ in sensitizing metastatic colon carcinoma cells to TRAIL-mediated apoptosis.

Methodology/Principal Findings

The efficacy and underlying molecular mechanism of cooperation between TNFα and IFN-γ in sensitizing metastatic colon carcinoma cells to TRAIL-mediated apoptosis were examined. The functional significance of TNFα- and IFN-γ-producing T lymphocyte immunotherapy in combination with TRAIL therapy in suppression of colon carcinoma metastasis was determined in an experimental metastasis mouse model. We observed that TNFα or IFN-γ alone exhibits minimal sensitization effects, but effectively sensitized metastatic colon carcinoma cells to TRAIL-induced apoptosis when used in combination. TNFα and IFN-γ cooperate to repress Bcl-xL expression, whereas TNFα represses Survivin expression in the metastatic colon carcinoma cells. Silencing Bcl-xL expression significantly increased the metastatic colon carcinoma cell sensitivity to TRAIL-induced apoptosis. Conversely, overexpression of Bcl-xL significantly decreased the tumor cell sensitivity to TRAIL-induced apoptosis. Furthermore, TNFα and IFN-γ also synergistically enhanced TRAIL-induced caspase-8 activation. TNFα and IFN-γ was up-regulated in activated primary and tumor-specific T cells. TRAIL was expressed in tumor-infiltrating immune cells in vivo, and in tumor-specific cytotoxic T lymphocytes (CTL) ex vivo. Consequently, TRAIL therapy in combination with TNFα/IFN-γ-producing CTL adoptive transfer immunotherapy effectively suppressed colon carcinoma metastasis in vivo.

Conclusions/Significance

TNFα and IFN-γ cooperate to overcome TRAIL resistance at least partially through enhancing caspase 8 activation and repressing Bcl-xL expression. Combined CTL immunotherapy and TRAIL therapy hold great promise for further development for the treatment of metastatic colorectal cancer.     

Cellular inhibitor of apoptosis 1 (cIAP-1) degradation by caspase 8 during TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis

TNF-related apoptosis-inducing ligand (TRAIL) is a potential chemotherapeutic agent with high selectivity for malignant cells. Many tumors, however, are resistant to TRAIL cytotoxicity. Although cellular inhibitors of apoptosis 1 and 2 (cIAP-1 and -2) are often over-expressed in cancers, their role in mediating TRAIL resistance remains unclear. Here, we demonstrate that TRAIL-induced apoptosis of liver cancer cells is associated with degradation of cIAP-1 and X-linked IAP (XIAP), whereas cIAP-2 remains unchanged. Lower concentrations of TRAIL causing minimal or no apoptosis do not alter cIAP-1 or XIAP protein levels. Silencing of cIAP-1 expression, but not XIAP or cIAP-2, as well as co-treatment with a second mitochondrial activator of caspases (SMAC) mimetic (which results in rapid depletion of cIAP-1), sensitizes the cells to TRAIL. TRAIL-induced loss of cIAP-1 and XIAP requires caspase activity. In particular, caspase 8 knockdown stabilizes both cIAP-1 and XIAP, while caspase 9 knockdown prevents XIAP, but not cIAP-1 degradation. Cell-free experiments confirmed cIAP-1 is a substrate for caspase 8, with likely multiple cleavage sites. These results suggest that TRAIL-mediated apoptosis proceeds through caspase 8-dependent degradation of cIAP-1. Targeted depletion of cIAP-1 by SMAC mimetics in conjunction with TRAIL may be beneficial for the treatment of human hepatobiliary malignancies.     

Chemotherapy overcomes TRAIL-R4-mediated TRAIL resistance at the DISC level

TNF-related apoptosis-inducing ligand or Apo2L (Apo2L/TRAIL) is a promising anti-cancer drug owing to its ability to trigger apoptosis by binding to TRAIL-R1 or TRAIL-R2, two membrane-bound receptors that are often expressed by tumor cells. TRAIL can also bind non-functional receptors such as TRAIL-R4, but controversies still exist regarding their potential to inhibit TRAIL-induced apoptosis. We show here that TRAIL-R4, expressed either endogenously or ectopically, inhibits TRAIL-induced apoptosis. Interestingly, the combination of chemotherapeutic drugs with TRAIL restores tumor cell sensitivity to apoptosis in TRAIL-R4-expressing cells. This sensitization, which mainly occurs at the death-inducing signaling complex (DISC) level, through enhanced caspase-8 recruitment and activation, is compromised by c-FLIP expression and is independent of the mitochondria. Importantly, TRAIL-R4 expression prevents TRAIL-induced tumor regression in nude mice, but tumor regression induced by TRAIL can be restored with chemotherapy. Our results clearly support a negative regulatory function for TRAIL-R4 in controlling TRAIL signaling, and unveil the ability of TRAIL-R4 to cooperate with c-FLIP to inhibit TRAIL-induced cell death.     

TRAIL apoptosis is enhanced by quercetin through Akt dephosphorylation

TNF-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapy that preferentially induces apoptosis in cancer cells. However, many neoplasms are resistant to TRAIL by mechanisms that are poorly understood. Here we demonstrated that human prostate cancer cells, but not normal prostate cells, are dramatically sensitized to TRAIL-induced apoptosis and caspase activation by quercetin. Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells. We have shown that quercetin can potentiate TRAIL-induced apoptotic death. Human prostate adenocarcinoma DU-145 and LNCaP cells were treated with various concentrations of TRAIL (10-200 ng/ml) and/or quercetin (10-200 microM) for 4 h. Quercetin, which caused no cytotoxicity by itself, promoted TRAIL-induced apoptosis. The TRAIL-mediated activation of caspase, and PARP (poly(ADP-ribose) polymerase) cleavage were both enhanced by quercetin. Western blot analysis showed that combined treatment with TRAIL and quercetin did not change the levels of TRAIL receptors (death receptors DR4 and DR5, and DcR2 (decoy receptor 2)) or anti-apoptotic proteins (FLICE-inhibitory protein (FLIP), inhibitor of apoptosis (IAP), and Bcl-2). However, quercetin promoted the dephosphorylation of Akt. Quercetin-induced potent inhibition of Akt phosphorylation. Taken together, the present studies suggest that quercetin enhances TRAIL-induced cytotoxicity by activating caspases and inhibiting phosphorylation of Akt.     

Trail-induced apoptosis in Type I leukemic cells is not enhanced by overexpression of bax

We have previously shown that Bax translocation was crucial in TNFalpha or etoposide-induced apoptosis. Overexpression of Bax sensitized chronic myeloid leukemic K562 cells to etoposide-induced apoptosis. Treatment with TNF-related apoptosis-inducing ligand (TRAIL) induces a loss of mitochondrial membrane potential (DeltaPsim), cytochrome c release from mitochondria, activation of caspases-8, -9, and -3, and cleavage of Bid in the K562 cell line. Bax failed to sensitize K562 cells to TRAIL-induced apoptosis. TRAIL did not induce Bax expression and/or translocation from cytosol to mitochondria in the K562 cell line. However, 100 microM Z-VAD.fmk, a pan caspase inhibitor, completely blocked TRAIL-initiated mitochondrial alterations and cleavages of caspases and Bid. We propose that TRAIL-induced apoptosis in K562 cells is via Type I apoptotic signal pathway. Bax translocation is not essential for TRAIL-induced cytochrome c release and DeltaPsim collapse in the Type I cells.     

Activation of protein kinase C inhibits TRAIL-induced caspases activation, mitochondrial events and apoptosis in a human leukemic T cell line

TRAIL causes apoptosis in numerous types of tumor cells. However, the mechanisms regulating TRAIL-induced apoptosis remain to be elucidated. We have investigated the role of PKC in regulating TRAIL-induced mitochondrial events and apoptosis in the Jurkat T cell line. We found a caspase-dependent decline in mitochondrial membrane potential and translocation of cytochrome c from mitochondria into the cytosol in response to TRAIL. Both these events were prevented by PKC activation. Moreover, PKC activation considerably reduced the activation of caspases, PARP cleavage and apoptosis when induced upon TRAIL treatment. MAPK activation was involved in the mechanism of PKC-mediated inhibition of TRAIL-induced cytochrome c release from mitochondria. Furthermore, inhibition of the MAPK pathway partially reversed the PKC-mediated inhibition of TRAIL-induced apoptosis. Besides, PKC activation may also inhibit the TRAIL-induced apoptosis through a MAPK-independent mechanism. Altogether, these results indicate a negative role of PKC in the regulation of apoptotic signals generated upon TRAIL receptor activation.     

TNF-related apoptosis-inducing ligand-induced apoptosis of melanoma is associated with changes in mitochondrial membrane potential and perinuclear clustering of mitochondria

Past studies have shown that TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis in a high proportion of cultured melanoma by caspase-dependent mechanisms. In the present studies we have examined whether TRAIL-induced apoptosis of melanoma was mediated by direct activation of effector caspases or whether apoptosis was dependent on changes in mitochondrial membrane potential (MMP) and mitochondrial-dependent pathways of apoptosis. Changes in MMP were measured by fluorescent emission from rhodamine 123 in mitochondria. TRAIL, but not TNF-alpha or Fas ligand, was shown to induce marked changes in MMP in melanoma, which showed a high correlation with TRAIL-induced apoptosis. This was associated with activation of proapoptotic protein Bid and release of cytochrome c into the cytosol. Overexpression of B cell lymphoma gene 2 (Bcl-2) inhibited TRAIL-induced release of cytochrome c, changes in MMP, and apoptosis. The pan caspase inhibitor z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and the inhibitor of caspase-8 (z-Ile-Glu-Thr-Asp-fluoromethylketone; zIETD-fmk) blocked changes in MMP and apoptosis, suggesting that the changes in MMP were dependent on activation of caspase-8. Activation of caspase-9 also appeared necessary for TRAIL-induced apoptosis of melanoma. In addition, TRAIL, but not TNF-alpha or Fas ligand, was shown to induce clustering of mitochondria around the nucleus. This process was not essential for apoptosis but appeared to increase the rate of apoptosis. Taken together, these results suggest that TRAIL induces apoptosis of melanoma cells by recruitment of mitochondrial pathways to apoptosis that are dependent on activation of caspase-8. Therefore, factors that regulate the mitochondrial pathway may be important determinants of TRAIL-induced apoptosis of melanoma.     

Amiloride potentiates TRAIL-induced tumor cell apoptosis by intracellular acidification-dependent Akt inactivation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor gene family, is considered as one of the most promising cancer therapeutic agents due to its ability to selectively induce tumor cell apoptosis. In this study, we investigated whether the Na(+)/H(+) exchanger inhibitor, amiloride, promotes TRAIL-induced apoptotic death both in sensitive and resistant tumor cells, HeLa and LNCaP cells, respectively, and its underlying molecular mechanism. Amiloride enhanced TRAIL-induced apoptosis and activation of caspase-3 and -8 in both cells. This compound increased TRAIL-induced mitochondrial cytochrome c release and poly(ADP-ribose) polymerase cleavage. Moreover, amiloride-induced intracellular acidification, and inhibited the phosphorylated activation of the serine/threonine kinase Akt, which is known to promote cell survival, in both tumor cells. These data suggest that amiloride sensitizes both tumor cells to TRAIL-induced apoptosis by promoting Akt dephosphorylation and caspase-8 activation via the intracellular acidification and that Na(+)/H(+) exchanger inhibitors may play an important role in the anti-cancer activity of TRAIL, especially, in TRAIL-resistant tumors with highly active and expressed Akt.     

Down-regulation of FoxO-dependent c-FLIP expression mediates TRAIL-induced apoptosis in activated hepatic stellate cells

Activated hepatic stellate cells which contribute to liver fibrosis have represented an important target for antifibrotic therapy. In this study, we found that TRAIL inhibited PI3K/Akt-dependent FoxO phosphorylation and relocated FoxO proteins into the nucleus from the cytosol in activated human hepatic stellate LX-2 cells. The accumulated FoxO proteins in the nucleus led to down-regulation of c-FLIP_L/S expression, resulting in the activation of apoptosis-related signaling molecules including the activation of caspase-8, -3, and Bid, as well as mitochondrial cytochrome c release. These results were supported by showing that siRNA-mediated knockdown of FoxO led to restoration of c-FLIP_L/S expression and resistance to TRAIL-induced apoptosis after treatment of LX-2 cells with TRAIL. Furthermore, c-FLIP_L/S-transfected LX-2 cells showed the decreased sensitivity to TRAIL-induced apoptosis. Collectively, our data suggest that sequential activation of FoxO proteins under conditions of suppressed PI3K/Akt signaling by TRAIL can down-regulate c-FLIP_L/S, consequently promoting TRAIL-induced apoptosis in LX-2 cells. Therefore, the present study suggests TRAIL may be an effective strategy for antifibrotic therapy in liver fibrosis.     

Polyphenol acertannin prevents TRAIL-induced apoptosis in human keratinocytes by suppressing apoptosis-related protein activation

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been implicated in the inflammatory and immune responses, and apoptosis in skin diseases, such as atopic dermatitis. Dysregulated apoptosis is associated with various pathologic conditions, including inflammation and cancer in skin. Polyphenols, including flavonoids and tannins, have been shown to have anti-oxidant, anti-inflammatory and anti-tumor effects. However, the effect of acertannin on TRAIL-induced apoptosis in keratinocytes has not been determined. To assess the preventive effect of acertannin on apoptosis-mediated skin inflammation, we investigated the effect of acertannin on TRAIL-induced apoptosis in human keratinocytes. TRAIL induced nuclear damage, decreased Bid, Bcl-2, Bcl-xL and survivin protein levels, increased Bax levels, induced cytochrome c release, activated caspases (-8, -9 and -3) and increased tumor suppressor p53 levels. Acertannin prevented the TRAIL-induced formation of reactive oxygen/nitrogen species, apoptosis-related protein activation and cell death. The results suggest that acertannin may reduce apoptotic effect of TRAIL on human keratinocytes by suppressing the activation of the caspase-8- and Bid-pathways and the mitochondria-mediated apoptotic pathway, leading to caspase-3 activation. The preventive effect of acertannin on TRAIL-induced apoptosis may be associated with the inhibitory effect on formation of reactive oxygen/nitrogen species. Acertannin may prevent the TRAIL-induced apoptosis-mediated skin inflammation.