Up-regulation Of DR5 Expression Research Articles

Luteolin sensitizes human liver cancer cells to TRAIL‑induced apoptosis via autophagy and JNK‑mediated death receptor 5 upregulation.

The tumor necrosis factor‑related apoptosis‑inducing ligand(TRAIL) is a dynamic cytokine that initiates the apoptosis of cancer cells, but exhibits little or no toxicity in normal cells. Luteolin is a flavonoid compound frequently used in the treatment of cancer. In the current study, we demonstrate that treatment with luteolin and TRAIL exerts a synergistic effect and the mechanisms on TRAIL‑resistant Huh7 cells. The results demonstrated that luteolin induced an autophagic flux in human liver cancer cells. The attenuation of the autophagic flux by applying the specific inhibitor of autophagy, chloroquine, significantly suppressed DR5 expression. Treatment with genetically modified autophagy‑related5 siRNA abrogated the luteolin‑mediated sensitizing effect of TRAIL. Furthermore, pre‑treatment with the c‑Jun N‑terminal kinase(JNK) inhibitor, SP600125, significantly attenuated the luteolin‑induced upregulation of DR5 expression, thereby suggesting that JNK activation promotes DR5 expression. Our findings also revealed that Akt phosphorylation was required for TRAIL sensitization. On the whole, the findings of this study indicated that luteolin effectively enhanced TRAIL‑initiated apoptosis, and that these effects were likely to be mediated by autophagy and JNK‑mediated DR5 expression.

C-27-carboxylated oleanane triterpenoids up-regulate TRAIL DISC assembly via p38 MAPK and CHOP-mediated DR5 expression in human glioblastoma cells

Despite recent tremendous progress, targeting of TNF-related apoptosis-inducing ligand (TRAIL) as a cancer therapy has limited success in many clinical trials, in part due to inactivation of death inducing signaling complex (DISC)-mediated caspase-8 signaling cascade in highly malignant tumors such as glioblastoma. In this study, screening of constituents derived from Astilbe rivularis for TRAIL-sensitizing activity identified C-27-carboxylated oleanolic acid derivatives (C27OAs) including 3β-hydroxyolean-12-en-27-oic acid (C27OA-1), 3β,6β,7α-trihydroxyolean-12-en-27-oic acid (C27OA-2), and 3β-trans-p-coumaroyloxy-olean-12-en-27-oic acid (C27OA-3) as novel TRAIL sensitizers. Interestingly, these C27OAs did not affect apoptotic cell death induced by either ligation of other death receptor (DR) types, such as TNF and Fas or DNA damaging agents, which suggests that C27OAs effectively and selectively sensitize TRAIL-mediated caspase-8 activation. Mechanistically, C27OAs upregulate the expression of cell surface DR5 and DISC formation without affecting downstream intracellular apoptosis-related proteins. The upregulation of DR5 expression by C27OAs strictly depends on transactivation of C/EBP homology protein, which is regulated through the p38 MAPK pathway, rather than p53 and intracellular reactive oxygen species status. Taken together, our results identify the novel C27OAs as TRAIL sensitizers targeting the upstream DISC assembly of DR5, and provide a rationale for further development of C27OAs for facilitating TRAIL-based chemotherapy in glioblastoma patients.

BIX-01294 sensitizes renal cancer Caki cells to TRAIL-induced apoptosis through downregulation of survivin expression and upregulation of DR5 expression

BIX-01294 (BIX), a G9a histone methyltransferase inhibitor, has been reported for its anti-proliferative and anticancer activities against various cancer cell lines. In this study, we investigated whether BIX could sensitize TRAIL-mediated apoptosis in various cancer cells. Combined treatment with BIX and TRAIL markedly induced apoptosis in human renal carcinoma (Caki, ACHN, and A498), breast carcinoma (MCF-7), and lung carcinoma (A549) cells. In contrast, BIX and TRAIL co-treatment did not induce apoptosis in normal cells, specifically mouse kidney cell (TCMK-1) and human skin fibroblast (HSF). BIX downregulated protein expression levels of XIAP and survivin at the post-translational level. Overexpression of survivin markedly blocked combined BIX and TRAIL treatment-induced apoptosis, but XIAP had no effect. Furthermore, BIX induced upregulation of DR5 expression at the transcriptional levels, and knockdown of DR5 expression using small interfering RNAs (siRNAs) markedly attenuated BIX and TRAIL-induced apoptosis. Interestingly, siRNA-mediated G9a histone methyltransferase knockdown also enhanced TRAIL-induced apoptosis in Caki cells. However, knockdown of G9a did not change expression levels of XIAP, survivin, and DR5. Therefore, BIX-mediated TRAIL sensitization was independent of histone methyltransferase G9a activity. Taken together, these results suggest that BIX facilitates TRAIL-mediated apoptosis via downregulation of survivin and upregulation of DR5 expression in renal carcinoma Caki cells.▶ BIX facilitates TRAIL-mediated apoptosis in human renal carcinoma Caki cells.▶ Downregulation of survivin contributes to BIX plus TRAIL-induced apoptosis.▶ Upregulation of DR5 is involved in BIX plus TRAIL-mediated apoptosis.▶ BIX-mediated TRAIL sensitization is independent of ROS production.

AGS 인체 위암세포에서 DR5의 발현 및 ROS 생성의 증가를 통한 sanguinarine과 TRAIL 혼합처리의 apoptosis 유도 활성 촉진

혈근초(Sanguinaria canadensis) 뿌리에서 유래된 benzophenanthridine alkaloid의 일종인 sanguinarine은 항균, 항산화 및 항암작용 등 다양한 생리활성을 지니고 있는 것으로 알려져 있다. 비록 TRAIL이 암세포에서는 apoptosis를 유도하지만 정상세포에서는 세포독성을 나타내지 않는다는 큰 장점으로 제 2 임상 단계에서 유의적인 성과를 이루었지만, TRAIL 저항성을 극복해야 하는 큰 어려움이 남아있다. 본 연구실에서는 TRAIL이 세포독성을 나타내지 않는 범위의 sanguinarine과 혼합처리에 의하여 TRAIL 저항성 AGS 위암세포에서 apoptosis를 유발하였음을 보고한 바 있으며, 본 연구에서는 sanguinarine의 TRAIL 저항성 관련 극복에 대한 추가적인 기전 연구를 실시하였다. 본 연구의 결과에 의하면, sanguinarine과 TRAIL의 혼합처리는 각각의 단독 처리에 비하여 AGS 세포의 증식억제 및 apoptosis 유도의 상승 효과가 있었으며, 이를 MTT assay, agarode gel 전기영동, 염색질 응축 현상 및 flow cytometry 분석을 통하여 확인하였다. 또한 sanguinarine과 TRAIL의 혼합처리는 DR5의 발현을 증가시켰으며, ROS의 생성을 촉진시켰다. 그러나 동일 조건에서 MAPKs 신호 전달계에는 큰 영향을 주지 않았다. 아울러 ROS 생성을 인위적으로 차단하였을 경우, sanguinarine과 TRAIL의 혼합처리에 의한 생존도 저하가 유의적으로 회복되었다. 이러한 결과는 sanguinarine과 TRAIL이 DR5의 발현 증가와 ROS의 생성을 촉진시킴으로서 apoptosis 신호를 활성화하였음을 의미하는 결과로서 TRAIL 저항성 극복을 위한 sanguinarine 활용의 유용성을 보여주는 것이다. Sanguinarine, a benzophenanthridine alkaloid originally derived from the root of Sanguinaria canadensis, has been shown to possess antimicrobial, antioxidant, and anti-cancer properties. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to induce apoptosis in cancer cells, but not most normal cells and has shown efficacy in a phase 2 clinical trial, development of resistance to TRAIL by tumor cells is a major roadblock. Our previous study indicated that treatment with TRAIL in combination with subtoxic concentrations of sanguinarine sensitized TRAIL-mediated apoptosis in TRAIL-resistant human gastric carcinoma AGS cells; however, the detailed mechanisms are not fully understood. In this study, we show that sanguinarine sensitizes AGS cells to TRAIL-mediated apoptosis as detected by MTT assay, agarose gel electrophoresis, chromatin condensation and flow cytometry analysis. Combined treatment with sanguinarine and TRAIL effectively induced expression of death receptor (DR) 5 but did not affect expression of DR4 and mitogen activated protein kinases signaling molecules. Moreover, the combined treatment with sanguinarine and TRAIL increased the generation of reactive oxygen species (ROS); however, N-acetylcysteine, ROS scavenger, significantly recovered growth inhibition induced by the combined treatment. Taken together, our results indicate that sanguinarine can potentiate TRAIL-mediated apoptosis through upregulation of DR5 expression and ROS generation.

Abstract 2937: Dual roles of ERK2 to TRAIL sensitivity in SK-N-MC neuroepithelioma cells.

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new promising agent for clinical use since it kills a wide range of tumor cells but not normal cells. It is regulated by 2 death receptors (DR), DR4 (TRAIL-R1) and DR5 (TRAIL-R2), and 2 decoy receptors (DcR), DcR1 (TRAIL-R3) and DcR2 (TRAIL-R4). However, the determining factors of the sensitivity to TRAIL cytotoxicity are not clearly understood. This study aims to elucidate the role of extracellular signal-regulated kinase (ERK) on enhancement of TRAIL cytotoxicity in cultured neuroepithelioma cell line SK-N-MC. When 1 μg/ml cisplatin-pretreated cells were exposed to 100 ng/ml TRAIL for 48 h, apoptotic cell death increased, compared with the cells treated with cisplatin or TRAIL alone (66% versus 27% or 9% apoptotic cell death). In addition, pretreatment with cisplatin in SK-N-MC cells significantly increased ERK1/2 activity and increased expression levels of DR5, but not DR4, DcR1 and DcR2, which correlated with increase of their susceptibility to TRAIL cytotoxicity. Introduction of ERK2 siRNA or ERK2 shRNA in SK-N-MC cells significantly decreased cisplatin-induced DR5 expression, suggesting that cisplatin-induced ERK2 activity is involved in DR5 expression. On the other hand, treatment with 20 μM U0126, an inhibitor of MEK, completely reduced basal ERK1/2 activity and induced cell death in SK-N-MC cells. In addition, pretreatment with U0126 increased TRAIL-mediated apoptosis via up-regulation of DR5 expression in response to the decrease in basal ERK2 activity. Because pretreatment with U0126 completely inhibited not only cisplatin-induced ERK2 activation but also basal ERK2 activity, the following treatment with TRAIL significantly increased cell death, indicating that constitutively activated ERK2 blocks DR5 expression in SK-N-MC cells. Taken together, our results implicate that basal ERK2 activity is involved in TRAIL resistance but induced ERK2 plays a role in TRAIL sensitivity, and suggest that the enhanced sensitivity of SK-N-MC cells to TRAIL is associated with an increase in DR5 expression. Citation Format: Hye Lim Jung, Myoung Woo Lee, Ji Eun Eom, Dea Seong Kim, Hyo-Jung Park, Dong-Kyung Yu, Meong Hi Son, Soo Hyun Lee, Keon Hee Yoo, Ki Woong Sung, Hong Hoe Koo. Dual roles of ERK2 to TRAIL sensitivity in SK-N-MC neuroepithelioma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2937. doi:10.1158/1538-7445.AM2013-2937

Potentiation of bortezomib's Anti-Myeloma Effects in Combination with Anti-DR5 Agonistic Antibody: The Role of ER Stress in DR5 Editing and DR-Mediated Apoptotic Signaling

Abstract Abstract 5104 Bortezomib (BTZ) is widely used in the treatment of myeloma (MM) with marked response rates in both relapsed/refractory as well as newly diagnosed MM. However, significant numbers of patients still remain outside benefit of the BTZ treatment; and various combinatory treatments with BTZ have been implemented to improve BTZ's anti-MM effects. On the other hand, immunotherapies seem attractive for yet incurable malignancies by chemotherapeutic agents such as MM and their clinical application has been studied. One such approach is a TNF-related apoptosis-inducing ligand (TRAIL)-mediated immunotherapy. In the present study, we therefore explored the role of BTZ on TRAIL receptor editing and its downstream signaling with special reference to endoplasmic reticulum (ER) stress and the cytotoxic effects of BTZ and anti-TRAIL receptor agonistic antibody in combination on MM cells. Most MM cells expressed DR4 but weakly DR5, while normal peripheral blood mononuclear cells expressed neither DR4 nor DR5. BTZ at 10 nM markedly up-regulated the surface levels of DR5 and its mRNA expression but not those of DR4 in MM cell lines and primary MM cells. Furthermore, BTZ decreased the levels of c-FLIP, an inhibitor of DISC, along with activation of caspase-8 and caspase-3, suggesting potentiation of the DR-mediated extrinsic apoptotic pathway. Consistently, BTZ and anti-DR5 agonistic antibody cooperatively enhanced the cytotoxicity against MM cells. BTZ induced phosphorylation of eIF2alpha, ATF4 and CHOP, along with disappearance of anti-apoptotic proteins including Mcl-1 in MM cells, suggesting the enhancement of ER stress and subsequent suppression of protein translation by BTZ. However, such induction of ER stress by BTZ was not observed in BTZ-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, with a point mutation in BTZ-binding proteasome beta5 subunit (Ri et al. Leukemia 2010). In KMS-11/BTZ and OPM-2/BTZ, surface protein as well as mRNA levels of DR5 were not up-regulated by BTZ, suggesting a critical role of ER stress in up-regulation of DR5 expression by BTZ. Because DR5 expression has been shown to be transcriptionally up-regulated by CHOP, the up-regulation of DR5 mRNA and protein in MM cells by BTZ is suggested to be at least in part due to CHOP induced by BTZ-mediated ER stress. Although BTZ exerts its anti-MM effects through induction of ER stress, the present study demonstrates that induction of ER stress by BTZ is also able to sensitize MM cells to TRAIL-mediated immunotherapy. Therefore, the combination of BTZ and TRAIL-mediated immunotherapy is warranted for further study. Disclosures: No relevant conflicts of interest to declare.

Delisheng, a Chinese medicinal compound, exerts anti-proliferative and pro-apoptotic effects on HepG2 cells through extrinsic and intrinsic pathways

The anti-proliferative, cytotoxic and apoptogenic activities of delisheng, a Chinese medicinal compound, has been investigated. In this study, the hepatocarcinoma cell line (HepG2) and the liver cell line (L-02) were exposed to delisheng (6.25, 50 and 100 μl/ml). Delisheng suppressed the proliferation and viability of normal liver L-02 cells slightly, but strongly inhibited the proliferation and viability of hepatocarcinoma HepG2 cells. The flow cytometric analysis of HepG2 cells demonstrated that delisheng primarily arrested the HepG2 cells at the G1 phase of the cell cycle. Annexin V-FITC/PI staining corroborates the apoptogenic nature of delisheng on HepG2 cells. The anti-proliferative and pro-apoptotic effect of delisheng in HepG2 cells was associated with changes in the Bcl-2/Bax ratio and the induction of caspase-mediated apoptosis. Upregulation of DR5 expression was observed in HepG2 cells after treatment with delisheng. The findings from the present study suggest that delisheng has selective cytotoxic activities against HepG2 cells. Delisheng triggered time- and dose-dependent apoptosis in HepG2 cells by activating the mitochondria-mediated and death receptor-mediated apoptotic pathways.

Molecular Mechanism by Which Rituximab Sensitizes B-NHL Cells to TRAIL Apoptosis: Induction of RKIP and DR5 Expression Via Inhibition of NF-Kb, Snail, and YY1.

Abstract There have been significant advances in the treatment of patients with B-NHL using combination of rituximab and CHOP. However, a subset of patients does not initially respond or develop resistance to further treatments; hence, the need for alternative therapies to overcome resistance. TRAIL and agonist DR4/DR5 monoclonal antibodies have been examined clinically against a variety of tumors in Phase I/II. However, the majority of B-NHL derived from patients and cell lines are resistant to TRAIL-induced apoptosis. Recent findings demonstrated that treatment of TRAIL-resistant-B-NHL with rituximab sensitizes the tumor cells to TRAIL apoptosis. The underlying mechanism of rituximab-induced sensitization to TRAIL, however, is not clear. We have recently reported that treatment of tumor cells with sensitizing agents (example CDDP, proteasome inhibitors) resulted in the reversal of resistance to TRAIL via induction of Raf-1 kinase inhibitor protein (RKIP) and demonstrated the pivotal role of RKIP in the regulation of tumor cell sensitivity to TRAIL. Hence, since rituximab induces the expression of RKIP in B-NHL, we determined the role of RKIP induction by rituximab in the sensitization of B-NHL to TRAIL apoptosis. Various B-NHL cell lines were used as models for study. Treatment of B-NHL cells with rituximab (20 ng/ml) and TRAIL (5–10 ng/ml) resulted in significant potentiation of apoptosis and synergy was achieved. Rituximab induced the expression of RKIP as determined by RT-PCR and western concomitantly with inhibition of NF-kB. The inhibition of NF-kB resulted in upregulation of RKIP expression and was mediated, in large part, by inhibition of the transcription repressor Snail (downstream of NF-kB). Further, RKIP-induced inhibition of NF-kB by rituximab resulted in downstream inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) and concomitantly with the upregulation of DR5 expression. The role of RKIP induction by rituximab in the upregulation of DR5 and sensitization to TRAIL apoptosis was corroborated by the use of cells over expressing RKIP which were sensitive to TRAIL apoptosis in the absence of rituximab. Our findings reveal a novel mechanism of rituximab-induced sensitization of B-NHL to TRAIL apoptosis via inhibition of NF-kB and Snail and upregulation of RKIP and DR-5. The combination of rituximab and TRAIL may be effective in the treatment of B-NHL. Further, our studies suggest that agents other than rituximab that can induce RKIP can reverse resistance to TRAIL in B-NHL that are unresponsive to rituximab treatment.

Nitric oxide sensitizes tumor cells to TRAIL-induced apoptosis via inhibition of the DR5 transcription repressor Yin Yang 1

Treatment of TRAIL-resistant tumor cells with the nitric oxide donor DETANONOate sensitizes the tumor cells to TRAIL-induced apoptosis concomitantly with DR5 upregulation. The mechanism of sensitization was examined based on the hypothesis that DETANONOate inhibits a transcription repressor Yin Yang 1 (YY1) that negatively regulates DR5 transcription. Treatment of the prostate carcinoma cell lines with DETANONOate inhibited both NF-kappaB and YY1 DNA-binding activities concomitantly with upregulation of DR5 expression. The direct role of YY1 in the regulation of TRAIL resistance was demonstrated in cells treated with YY1 siRNA resulting in TRAIL-induced apoptosis. The role of YY1 in the transcriptional regulation of DR5 was examined in cells treated with a DR5 luciferase reporter system (pDR5) and two constructs, namely, the pDR5/-605 construct with a deletion of the putative YY1 DNA-binding region (-1224 to -605) and a construct pDR5-YY1 with a mutation of the YY1 DNA-binding site. A significant (3-fold) augmentation of luciferase activity over baseline transfection with pDR5 was observed in cells transfected with the modified constructs. ChIP analysis corroborated the YY1 binding to the DR5 promoter. In vivo, tissues from nude mice bearing the PC-3 xenograft and treated with DETANONOate showed inhibition of YY1 and upregulation of DR5. The present findings demonstrate that YY1 negatively regulates DR5 transcription and expression and these correlated with resistance to TRAIL-induced apoptosis. DETANONOate inhibits both NF-kappaB and YY1 and in combination with TRAIL reverses tumor cell resistance to TRAIL apoptosis.

Hydrogen peroxide enhances TRAIL-induced cell death through up-regulation of DR5 in human astrocytic cells

The central nervous system (CNS) is particularly vulnerable to reactive oxygen species (ROS), which have been implicated in the pathogenesis of various neurological disorders. The TNF superfamily of cytokines, especially tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), induces caspase-dependent cell death and is also implicated in various neurodegenerative diseases. In this study, we investigated the relationship between ROS and TRAIL-induced cell death. Exposure to hydrogen peroxide (H(2)O(2)) (100 microM) sensitized human astrocytic cells to TRAIL-induced cell death (up to 7-fold induction). To delineate the molecular mechanisms responsible for H(2)O(2)-induced sensitization, we examined expression of various genes (Caspase-8, Fas, FasL, DR4, DR5, DcR1, DcR2, TRAIL, TNFRp55) related to TRAIL-induced cell death. Treatment with H(2)O(2) significantly increased DR5 mRNA and protein expression in a time- and dose-dependent manner. H(2)O(2)-mediated cell death was blocked upon treatment with DR5:Fc protein, a TRAIL-specific antagonistic protein. These findings collectively suggest that oxidative stress sensitizes human astroglial cells to TRAIL-induced cell death through up-regulation of DR5 expression.

Rituximab Sensitizes TRAIL-Resistant B-NHL Lines to Apoptosis by Both TRAIL and Fully Humanized Antibodies Targeting TRAIL-R1 (Mapatumumab) and TRAIL-R2 (Lexatumumab).

Abstract Clinical trials are currently in progress to evaluate the therapeutic efficacy of recombinant human TRAIL and fully humanized monoclonal antibodies that target TRAIL-R1 (DR4, mapatumumab) and TRAIL-R2 (DR5, lexatumumab). Although these proteins are cytotoxic to sensitive tumor cells, many tumors are resistant and require a sensitization stimulus. We have reported that rituximab inhibits anti-apoptotic survival pathways such as NF-κB activity and sensitizes tumor cells to both chemotherapeutic drugs and Fas-L- induced apoptosis (Vega et al., J. Immunol175(4):2174–83, 2005). Sensitization to TRAIL is also regulated by NF-κB. Thus, we hypothesize that rituximab-induced inhibition of NF-κB also sensitizes B-NHL cells to TRAIL apoptosis. Treatment of Ramos, Daudi, 2F7 and Raji with rituximab sensitized the cells to TRAIL apoptosis in a concentration dependant feature. Sensitization to TRAIL by Rituximab involved the type II mitochondrial apoptosis pathway. Examination of the mechanism of rituximab sensitization to TRAIL revealed that inhibition of NF-κB was associated with inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) and up regulation of DR5 (Vega et al., Blood 108(11):2387, ASH Annual Meeting Abstract 2006). There was no up regulation of DR4 by rituximab. The direct role of YY1 in the rituximab induced sensitization to TRAIL was corroborated by YY1siRNA. These findings suggested that one mechanism of Rituximab sensitization to TRAIL involved up regulation of DR5 expression. However, since TRAIL binds to both DR4 and DR5, the role of DR4 in rituximab induced sensitization to TRAIL needed further clarification. This was addressed by the use of the specific fully humanized monoclonal antibodies directed against DR4 (TRAIL-R1) (HGS-ETR1, mapatumumab) and DR5 (TRAIL-R2) (HGS-ETR2, lexatumumab) provided by Human Genome Sciences. Treatment of Ramos with rituximab sensitized the cells to both HGS-ETR1 and HGS-ETR2 induced apoptosis in a concentration dependant fashion. The sensitization by these antibodies was comparable to that achieved with TRAIL. These findings demonstrate that rituximab sensitizes B-NHL cells to both TRAIL and DR4/DR5 agonist-antibodies. The studies also suggest that, while the up regulation of DR5 expression by rituximab via inhibition of YY1 was critical for TRAIL apoptosis, up regulation of DR4 by rituximab was not required for sensitization with HGS-ETR1 monoclonal antibody. Further, these results suggest that rituximab may affect cell signaling induced by both DR4 and DR5. The molecular mechanism by which rituximab sensitizes B-NHL cells to TRAIL and both HGS-ETR1 and HGS-ETR2 will be presented.

Induction of Raf-1 Kinase Inhibitory Protein (RKIP) by the Proteasome Inhibitor NPI-0052 and Reversal of B-NHL Resistance to Apoptosis.

Abstract Patients with B-cell malignancies initially respond to conventional cytotoxic therapies. However, many patients experience relapses and recurrences. Novel therapeutics are being explored in the reversal of resistance such as TRAIL. TRAIL has been shown to be selectively cytotoxic to few tumors and both TRAIL and agonist TRAIL-R1 (DR4) and TRAIL-R2 (DR5) antibodies are currently being explored clinically in unresponsive cancer patients. We have recently found that treatment of TRAIL resistant B-NHL cell lines such as Ramos with the novel proteasome inhibitor NPI-0052 (Nereus Pharmaceuticals) sensitizes tumor cells to TRAIL-induced apoptosis. The concentration of NPI-0052 achieving optimal sensitization to TRAIL was in the range of 1–5 nM. In contrast 400 fold more VelcadeTM (bortezomib) was necessary to achieve a similar level of apoptosis. The mechanism of NPI-0052- mediated sensitization to TRAIL was examined. Treatment of Ramos with NPI-0052 inhibited significantly the constitutively activated NF-κB concomitantly with inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) resulting in up-regulation of DR5 expression. The role of YY1 in NPI-0052-induced sensitization to TRAIL was corroborated by the use of YY1siRNA as treatment with the YY1siRNA mimicked NPI-0052 induced DR5 up-regulation and sensitization to TRAIL. Studies examining the inhibition of the NF-κB pathway by NPI-0052 revealed a novel finding demonstrating the induced expression of the metastatic suppressor gene, Raf-1 kinase inhibitory protein (RKIP). RKIP has been reported to inhibit NF-κB activity and, thus, its induction by NPI-0052 contributed to the inhibition of NF-κB activity. The role of RKIP in NPI-0052 induced sensitization to TRAIL was corroborated by the use of RKIPsiRNA and transfectants mimicked NPI-0052 mediated inhibition of YY1, DR5 upregulation and sensitization to TRAIL mediated apoptosis. The above findings suggest that NPI-0052-induced inhibition of NF-κB results at least from two distinct mechanisms, namely, inhibition of p-IκBα degradation by the proteasome and by the induction of RKIP expression. The apoptosis induced by combination of NPI-0052 and TRAIL was the result of the activation of the type II mitochondrial pathway. It has been reported that the RKIP levels are reduced in many tumors (e.g. breast, prostate, ovarian) and the expression levels are of prognostic significance. Hence, the present findings demonstrating NPI-0052 induced RKIP expression may be therapeutically relevant in the prevention of metastasis. In addition, NPI-0052 should facilitate the reversal of tumor resistance when used in combination with subtoxic levels of cytotoxics.

Chemotherapeutic drugs sensitize cancer cells to TRAIL-mediated apoptosis: up-regulation of DR5 and inhibition of Yin Yang 1

Several chemotherapeutic drugs in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) result in reversal of resistance to TRAIL-mediated apoptosis through up-regulation of DR5 expression. The promoter of DR5 has one putative binding site for the transcription repressor Yin Yang 1 (YY1), and thus, we hypothesized that the sensitizing drugs may inhibit YY1. We have found that treatment of tumor cells with various chemotherapeutic drugs inhibited nuclear factor-kappaB. We examined whether drugs also inhibit YY1 activity and whether YY1 inhibition correlates with up-regulation of DR5 expression and sensitization of cells to TRAIL-induced apoptosis. The TRAIL- and drug-resistant prostate carcinoma PC-3 cell line was treated with CDDP, VP-16, ADR, and vincristine. DR5 luciferase reporter constructs and small interfering RNA against YY1 were used to determine the role of YY1 in DR5 transcription. Pretreatment of PC-3 cells and other tumor cell lines with various chemotherapeutic drugs sensitized the cells to TRAIL-induced apoptosis concurrently with up-regulation of DR5 expression and inhibition of YY1 expression and its DNA-binding activity. The baseline luciferase activity in PC-3 cells transfected with the wild-type DR5 reporter was significantly augmented in cells transfected with DR5 constructs carrying deletions or mutation in the YY1-binding site. Treatment with drug enhanced DR5 wild-type luciferase activity, with no increase in cells transfected with the YY1-deleted or YY1-mutated constructs. Cells transfected with YY1 small interfering RNA showed up-regulation of DR5 expression and sensitization to TRAIL-mediated apoptosis. The findings provide evidence that drug-induced sensitization of tumor cells to TRAIL is mediated, in part, by inhibition of the transcription repressor YY1 and up-regulation of DR5 expression. Hence, YY1 may be a potential therapeutic target to reverse resistance to TRAIL-induced apoptosis.

Induction of RKIP and Inhibition of the Transcription Repressor YY1 in B-NHL by the Novel Proteasome Inhibitor NPI-0052 and Sensitization to TRAIL-Induced Apoptosis.

Abstract Proteasome inhibitors have been shown to exert multiple effects including direct cytotoxic activity in sensitive cancer cells. In addition, proteasome inhibitors have recently been used therapeutically for certain cancers such as multiple myeloma and mantle cell lymphoma. We have recently reported that the novel proteasome inhibitor, NPI-0052 (Nereus Pharmaceuticals), can sensitize drug-resistant B-NHL tumor cells to apoptosis by various chemotherapeutic drugs. Also, NPI-0052 inhibits the NF-κB survival pathway and inhibition of NF-κB sensitizes tumor cells to TRAIL-induced apoptosis. We hypothesized that inhibition of NF-κB by NPI-0052 may also lead to sensitization of TRAIL-resistant B-NHL cells to TRAIL-mediated apoptosis. Human Burkitt’s lymphoma,Ramos cells, were treated with various concentrations of NPI-0052 (0–10ng/ml for 24h) and were then treated with recombinant human TRAIL (0–100ng/ml for 24h). The cells were examined for apoptosis by Annexin V/PI. The combination treatment resulted in significant potentiation of cytotoxicity and synergy in apoptosis was achieved. We have then examined a potential underlying mechanism of NPI-0052-mediated sensitization. The transcription repressor YY1 was recently shown to negatively regulate the transcription of the TRAIL death receptor DR5 and thus overexpression of YY1 is a resistant factor for TRAIL-induced apoptosis. Hence, treatment of Ramos cells with NPI-0052 resulted in significant inhibition of YY1 expression and upregulation of surface DR5 expression. We have also examined the effect of NPI-0052 on RKIP (Raf-kinase inhibitor protein) (Yeung et al., Molecular Cellular Biology; 21:7207, 2001) expression. Since both NPI-0052 and RKIP inhibit the NF-κB pathway, we hypothesized that treatment of Ramos cells with NPI-0052 may result in significant upregulation of RKIP expression. The findings, indeed, demonstrate that NPI-0052 significantly induced RKIP expression in Ramos cells. Overall, the findings show, for the first time, that NPI-0052 sensitizes B-NHL tumor cells to TRAIL-mediated apoptosis. Sensitization by NPI-0052 was correlated with both the induction of RKIP expression and inhibition of YY1 and upregulation of DR5 expression. Currently, TRAIL or agonist monoclonal antibodies against DR4 or DR5 are being examined clinically for anti-tumor activity. Therefore, our findings suggest the potential application of the combination of NPI-0052 and TRAIL or agonistic monoclonal antibodies against DR4 or DR5 in the treatment of tumor cells that are resistant to drugs and TRAIL.

Curcumin sensitizes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis through CHOP-independent DR5 upregulation

Death receptor DR5 (DR5/TRAIL-R2) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we showed that curcumin, a plant product containing the phenolic phytochemical, is a potent enhancer of TRAIL-induced apoptosis through upregulation of DR5 expression. Both treatment with DR5/Fc chimeric protein and silencing of DR5 expression using small interfering RNA (siRNA) attenuated curcumin plus TRAIL-induced apoptosis, showing that the critical role of DR5 in this cell death. Curcumin also induced the expression of a potential pro-apoptotic gene, C/EBP homologous protein (CHOP), both at its mRNA and protein levels. However, suppression of CHOP expression by small interfering RNA did not abrogate the curcumin-mediated induction of DR5 and the cell death induced by curcumin plus TRAIL, demonstrating that CHOP is not involved in curcumin-induced DR5 upregulation. Taken together, the present study demonstrates that curcumin enhances TRAIL-induced apoptosis by CHOP-independent upregulation of DR5.

The Induction of Apoptosis by Histone Deacetylase Inhibition in Chronic Lymphocytic Leukemia (CLL) Cells Is Mediated through the TNF-Related Apoptosis-Inducing Ligand (TRAIL) Pathway.

Abstract Background and Objectives: Histone Deacetylase Inhibitors (HDIs) have been shown to induce cell cycle arrest, differentiation and apoptosis in a variety of myeloid and lymphoid malignancies while being relatively nontoxic to normal cells. HDIs have also been shown to sensitize CLL cells to TRAIL-induced apoptosis. In this study we investigated the effects of two HDIs, trichostatin (TSA) and valproic acid (VPA), alone and in combination with fludarabine (FLU) on apoptosis in the BJAB lymphoblastoma B-cell line and in primary CLL cells. Materials and Methods: Cell viability was assessed by the MTT (3-(3,5-dimethylthiazol-2yl)- 2,5 diphenyl tetrazolium bromide) colorimetric assay, following 4 days of continuous drug treatment. Apoptosis was assessed by staining cell suspensions with acridine orange and quantitating apoptotic cell populations using fluorescence microscopy. Protein expression for the TRAIL receptors, DR4 and DR5, was determined by western blotting. Results: As determined by the MTT assay, the mean concentrations of TSA and VPA to reduce cell viability by 50% (IC50) in CLL cells were 0.23 uM and 650uM, respectively. TSA at 0.5uM and VPA at 2mM induced 60% apoptosis following 48 hours of drug exposure in both BJAB and CLL cells, as measured by acridine orange staining. Addition of DR4:FC, which sequesters TRAIL away from its receptors, decreased TSA- and VPA-induced apoptosis in BJAB and CLL cells by 9 and 20% respectively, indicating activation of the TRAIL apoptotic pathway by these HDIs. In addition, exposure to TSA at a concentration of 1uM for 24 hours, up regulated the expression of DR5 in BJAB cells, but this was not observed with VPA at a concentration of 1.5mM, after up to 48 hours of treatment. Neither HDI influenced DR4 protein expression in these cells. Preliminary results show that VPA potentiated FLU-induced apoptosis by 20% in both BJAB and CLL cells. Conclusions: Both TSA and VPA induce apoptosis in BJAB and CLL cells by activation of the TRAIL apoptotic pathway. For TSA, this effect may be potentiated by up regulation of DR5 expression. VPA potentiates FLU-induced apoptosis in BJAB and CLL cells, suggesting a therapeutic role for the combination of HDIs and nucleoside analogues in the management of CLL. Ongoing studies are determining whether this effect is specific for CLL cells, as compared to normal lymphocytes, and the optimum scheduling for synergy.

Tunicamycin Enhances Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis in Human Prostate Cancer Cells

Death receptor 5 (DR5/TRAIL-R2) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L). In this study, we showed that tunicamycin, a naturally occurring antibiotic, is a potent enhancer of TRAIL-induced apoptosis through up-regulation of DR5 expression. Tunicamycin significantly sensitized PC-3, androgen-independent human prostate cancer cells, to TRAIL-induced apoptosis. The tunicamycin-mediated enhancement of TRAIL-induced apoptosis was markedly blocked by a recombinant human DR5/Fc chimeric protein. Tunicamycin and TRAIL cooperatively activated caspase-8, -10, -9, and -3 and Bid cleavage and this activation was also blocked in the presence of the DR5/Fc chimera. Tunicamycin up-regulated DR5 expression at the mRNA and protein levels in a dose-dependent manner. Furthermore, the tunicamycin-mediated sensitization to TRAIL was efficiently reduced by DR5 small interfering RNA, suggesting that the sensitization was mediated through induction of DR5 expression. Tunicamycin increased DR5 promoter activity and this enhanced activity was diminished by mutation of a CHOP-binding site. In addition, suppression of CHOP expression by small interfering RNA reduced the tunicamycin-mediated induction of DR5. Of note, tunicamycin-mediated induction of CHOP and DR5 protein expression was not observed in normal human peripheral blood mononuclear cells. Moreover, tunicamycin did not sensitize the cells to TRAIL-induced apoptosis. Thus, combined treatment with tunicamycin and TRAIL may be a promising candidate for prostate cancer therapy.

Sensitization of prostate cancer cells to TRAIL-induced apoptosis by CDDP: Involvement of NF-κB, YY1 and RKIP in upregulation of DR5 expression

3189 Background: Treatment of the androgen-independent, p53- PC-3 prostate cancer cells with CDDP or the NO donor, DETANONOate, resulted in inhibition of NF-κB activity, upregulation of DR5 expression and sensitization to TRAIL-induced apoptosis. CDDP-dependent mechanisms of regulation of DR5 were examined. Methods: Apoptosis was assessed by PI staining and by activated caspase-3; DNA-binding activity by EMSA; transcription by luciferase reporter systems; and protein expression by flow cytometry and western analysis. Results: Preliminary findings demonstrated that inhibition of NF-κB activity by CDDP and DETANANOate, as measured by NF-κB reporters, was corroborated in PC-3 cells. DHMEQ, an inhibitor of NF-κB nuclear translocation, paralleled CDDP and DETANONOate effects and resulted in upregulation of DR5 expression and sensitization to TRAIL-induced apoptosis. DR5 promoter activity was augmented by deletion of the -1224 to -605 region (Yoshida et al., FEBS Lett., 2001, 507: 381–5), or direct mutation of YY1 binding motif at -800 to -796. DR5 upregulation and sensitization to TRAIL was further demonstrated by YY1 siRNA. RKIP has been shown to regulate the level of activity of NF-κB, MAPK and GPCR-dependent pathways. Forced RKIP overexpression inhibited CDDP-dependent DR5 induction, suggesting that CDDP-induced response is the outcome of positive and negative regulatory mechanisms. Conclusions: The findings reveal for the first time that CDDP- mediated sensitization of tumor cells to TRAIL-induced apoptosis results in inhibition of NF-κB-dependent transcription of YY1. Inhibition of YY1 upregulates DR5 expression and sensitizes the tumor cells to TRAIL-induced apoptosis. The findings identify NF-κB, YY1 and RKIP as new targets for therapeutic intervention for reversal of tumor cells resistance to TRAIL. No significant financial relationships to disclose.