Transcription elongation factors OsSPT4 and OsSPT5 are essential for rice growth and development and act with APO2.

KEY MESSAGE: The transcription elongation factor SPT4/SPT5 complex is essential for rice vegetative and reproductive growth and that OsSPT5-1, with its interactor APO2, is involved in multiple phytohormone pathways. The SPT4/SPT5 complex is a transcription elongation factor that regulates the processivity of transcription elongation. However, our understanding of the role of SPT4/SPT5 complex in developmental regulation remains limited. Here, we identified three SPT4/SPT5 genes (OsSPT4, OsSPT5-1, and OsSPT5-2) in rice, and investigated their roles in vegetative and reproductive growth. These genes are highly conserved with their orthologs in other species. OsSPT4 and OsSPT5-1 are widely expressed in various tissues. By contrast, OsSPT5-2 is expressed at a relatively low level, which could cause osspt5-2 null mutants have no phenotypes. Loss-of-function mutants of OsSPT4 and OsSPT5-1 could not be obtained; their heterozygotes showed severe reproductive growth defects. An incomplete mutant line (osspt5-1#12) displayed gibberellin-related dwarfed defects and a weak root system at an early vegetative phase, and a short life cycle in different planting environments. Furthermore, OsSPT5-1 interacts with the transcription factor ABERRANT PANICLE ORGANIZATION 2 (APO2) and plays a similar role in regulating the growth of rice shoots. RNA sequencing analysis verified that OsSPT5-1 is involved in multiple phytohormone pathways, including gibberellin, auxin, and cytokinin. Therefore, the SPT4/SPT5 complex is essential for both vegetative and reproductive growth in rice.

Glioma progression is suppressed by Naringenin and APO2L combination therapy via the activation of apoptosis in vitro and in vivo.

Naringenin (NG) is a natural antioxidant flavonoid which is isolated from citrus fruits, and has been reported to inhibit colon cancer proliferation. However, the effects of NG treatment on glioma remain to be elucidated. The present study aimed to explore the effects of NG on glioma in vitro and in vivo. Also, the interactions between NG and APO2 ligand (APO2L; also known as tumor necrosis factor-related apoptosis-inducing ligand) were investigated in glioma. A synergistic effect of NG and APO2L combination on apoptotic induction was observed, though glioma cells were insensitive to APO2L alone. After NG treatment, glioma cells resumed the sensitivity to APO2L and cell apoptosis was induced via the activation of caspases, elevation of decoy receptors 4 and 5 (DR4 and DR5) and induction of p53. Coadministration of NG and APO2L decreased levels of anti-apoptotic B cell lymphoma 2 (Bcl-2) family members Bcl-2 and Bcl-extra large (Bcl-xL), while increased levels of proapoptotic factors Bcl-2-associated agonist of cell death (Bad) and Bcl-2 antagonist/killer 1 (Bak). Furthermore, an in vivo mouse xenograft model demonstrated that NG and APO2L cotreatment markedly suppressed glioma growth by activating apoptosis in tumor tissues when compared with NG or APO2L monotherapy. The present study provides a novel therapeutic strategy for glioma by potentiating APO2L-induced apoptosis via the combination with NG in glioma tumor cells.

Quinovic acid purified from medicinal plant Fagonia indica mediates anticancer effects via death receptor 5.

Plants are major source for discovery and development of anticancer drugs. Several plant-based anticancer drugs are currently in clinical use. Fagonia indica is a plant of medicinal value in the South Asian countries. Using mass spectrometry and NMR spectroscopy, several compounds were purified from the F. indica extract. We have used one of the purified compounds quinovic acid (QA) and found that QA strongly suppressed the growth and viability of human breast and lung cancer cells. QA did not inhibit growth and viability of non-tumorigenic breast cells. QA mediated its anticancer effects by inducing cell death. QA-induced cell death was associated with biochemical features of apoptosis such as activation of caspases 3 and 8 as well as PARP cleavage. QA also upregulated mRNA and protein levels of death receptor 5 (DR5). Further investigation revealed that QA did not alter DR5 gene promoter activity, but enhanced DR5 mRNA and protein stabilities. DR5 is one of the major components of the extrinsic pathway of apoptosis. Accordingly, Apo2L/TRAIL, the DR5 ligand, potentiated the anticancer effects of QA. Our results indicate that QA mediates its anticancer effects, at least in part, by engaging DR5-depentent pathway to induce apoptosis. Based on our results, we propose that QA in combination with Apo2L/TRAIL can be further investigated as a novel therapeutic approach for breast and lung cancers.

Promotion of TRAIL/Apo2L-induced apoptosis by low-dose interferon-ß in human malignant melanoma cells.

Interferon ß (IFN-ß) is considered a signaling molecule with important therapeutic potential in cancer since IFN-ß-induced gene transcription mediates antiproliferation and cell death induction. Whereas, TNF-related apoptosis inducing ligand/Apo2 ligand (TRAIL/Apo2L) has emerged as a promising anticancer agent because it induces apoptosis specifically in cancer cells. In this study, we elucidated that IFN-ß augments TRAIL-induced apoptosis synergistically using five human malignant melanoma cells. All of these cells were induced apoptosis by TRAIL. Whereas, the response against IFN-ß was different in amelanotic cells (A375 and CRL1579) and melanotic cells (G361, SK-MEL-28, and MeWo). The responsibility of amelanotic cells against IFN-ß was higher than those of melanotic cells. The synergism of IFN-ß and TRAIL were correlated with the responsibilities of the cells against IFN-ß. The synergistic interaction was confirmed by a combination index based on the Chou-Talalay method. The upregulation of apoptosis in amelanotic cells was caused by very low doses of IFN-ß (over 0.1 IU/ml). Both of p53-mediated intrinsic pathway and Fas-related extrinsic pathway were activated by IFN-ß alone and combination with TRAIL. Further, TRAIL death receptors (DR4 and DR5) were upregulated by a low-dose IFN-ß (over 0.1 IU/ml) and the expression was more promoted by the combination with TRAIL. It was clarified that the upregulation of DR5 is associated with the declination of viability.

Improved Anticancer Effect of Recombinant Protein izTRAIL Combined with Sorafenib and Peptide iRGD.

One of the main problems in oncology is the development of drugs that cause the death of cancer cells without damaging normal cells. Another key problem to be solved is to suppress the drug resistance of cancer cells. The third important issue is to provide effective penetration of drug molecules to cancer cells. TRAIL (TNFα-related apoptosis inducing ligand)/Apo2L is a highly selective anticancer agent. However, the recombinant TRAIL protein having high efficiency against cancer cells in vitro was not effective in clinical trials. Recently we have discovered an acquisition of TRAIL resistance by cancer cells in confluent cultures, which is apparently a manifestation of the general phenomenon of multicellular resistance. The aim of this study was to evaluate whether the anticancer effect of the recombinant protein TRAIL in vivo can be improved by the suppression of multicellular TRAIL-resistance using sorafenib and a tumor-penetrating peptide iRGD, c(CRGDKGPDC). The results testified a great increase in the resistance of human fibrosarcoma HT-1080 cells to izTRAIL both in confluent cultures and in spheroids. Sorafenib administered at nontoxic concentration effectively suppressed confluent- or spheroid-mediated TRAIL-resistance of HT-1080 cells in vitro. Sorafenib combined with iRGD significantly improved the anticancer effect of the recombinant protein izTRAIL in HT-1080 human fibrosarcoma grafts in BALB/c nude mice. Consistent with this finding, multicellular TRAIL-resistance may be a reason of inefficacy of izTRAIL alone in vivo. The anticancer effect of the recombinant protein izTRAIL in vivo may be improved in combination with sorafenib, an inhibitor of multicellular TRAIL resistance and iRGD, the tumor-penetrating peptide.

Cosmc transfection decreases malignant behavior of Tn+ cells and enhances sensitivity to apoptosis when induced by Apo2L/TRAIL via alteration of O-glycan structure.

Cosmc mutations may cause abnormal O-glycosylation and result in Tn antigen expression. In the current study, it was discovered that proliferation and migration of Tn+ cells (Jurkat T and LS174T-Tn+ cells) with mutant Cosmc decreased after transfected Cosmc, and their sensitivity to apoptosis induced by Apo2L/TRAIL increased. Core 1-, 2-, and 3-derived O-glycans were absent in Tn+ cells. After Cosmc transfection, normal extended core 1-derived O-glycans appeared and were accompanied by increased T-synthase activity. Core 2-derived O-glycans appeared in transfected LS174T-Tn+ cells, and their structural types and levels were lower than those in LS174T-Tn- cells. Core 3-derived O-glycans were present only in LS174T-Tn- cells. The activity of C3GnT in LS174T-Tn+ cells was lower than that in LS174T-Tn- cells, and it was absent in Jurkat T cells. Cosmc transfection did not alter C3GnT activity or core 3-derived O-glycans in Jurkat T and LS174T-Tn+ cells. The results demonstrated that the composition and structure of O-glycans were different among various Tn+ cells, which not only affected cell malignant behavior but also modulated sensitivity to apoptotic stimuli. Thus, Cosmc transfection may effectively decrease the malignant behavior of Tn+ tumor cells and enhance their sensitivity to apoptosis when induced by Apo2L/TRAIL through modification of O-glycans.

Interleukin-6 sensitizes TNF-α and TRAIL/Apo2L dependent cell death through upregulation of death receptors in human cancer cells.

Interleukin-6 (IL-6) enhanced TNF-α and TRAIL/Apo2L induced cell death in various human cancer cells derived from malignant glioma, melanoma, breast cancer and leukemia, although the effect was not detected with IL-6 alone. The effects of IL-6 using SKBR3 cells were associated with the generation of apoptotic cells as analyzed by fluorescence microscopy and flow cytometry. IL-6 activated p53 and upregulated TRAIL death receptors (DR-4 and DR-5) and stimulated the TNF-α and TRAIL dependent extrinsic apoptotic pathway without activation of the p53 mediated intrinsic apoptotic pathway. TNF-α and TRAIL induced cleavage of caspase-8 and caspase-3 was more enhanced by IL-6, although these caspases were not cleaved by IL-6 alone. The dead cell generation elicited by the combination with IL-6 was blocked by anti-human TRAIL R2/TNFRSF10B Fc chimera antibody which can neutralize the DR-5 mediated death signal. These findings indicate that IL-6 could contribute to the enhancement of TNF-α or TRAIL induced apoptosis through p53 dependent upregulation of DR-4 and DR-5. The data suggest that a favorable therapeutic interaction could occur between TNF-α or TRAIL and IL-6, and provide an experimental basis for rational clinical treatments in various cancers.

Expression of tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) in minor salivary glands and saliva.

OBJECTIVE: The aim of this study was to characterize the baseline expression of tumor necrosis factor (tnf)-related apoptosis inducing ligand (TRAIL) in minor salivary glands, gingiva and saliva from healthy individuals. DESIGN: Minor salivary gland and gingival tissues were used in the study for immunohistochemical staining. An enzyme-linked immunosorbent assay was used to measure the levels of TRAIL in unstimulated saliva and parotid saliva collected from non-smoking individuals. The salivary levels of TRAIL are presented as secretory output. RESULTS: Parotid saliva showed higher secretory output (327.8 ±â€¯41.6 pg/min) for TRAIL compared to unstimulated saliva (212.3 ±â€¯32.1 pg/min; p =0.041). For unstimulated saliva, the young female subjects had the lowest secretory output (119 ±â€¯17.2 pg/min) compared to elderly females (275 ±â€¯62.18 pg/min; p =0.046) and young males (294.4 ±â€¯50.2 pg/min; p =0.021). The ductal cells of salivary glands exhibited the strongest positivity for TRAIL, whereas mucous cells showed no staining for TRAIL. Serous cells displayed an intermediate staining. Gingival tissues showed gradually decreasing staining towards the basal layer. CONCLUSIONS: The current study shows that TRAIL is not only expressed by immune cells, but also by the epithelial cells of salivary glands. Saliva contains high concentrations of soluble TRAIL that suggest roles of this protein in the apoptosis of tumor cells.

Oxidative stress-driven DR5 upregulation restores TRAIL/Apo2L sensitivity induced by iron oxide nanoparticles in colorectal cancer.

There exists an emergency clinical demand to overcome TRAIL/Apo2L (tumor necrosis factor-related apoptosis-inducing ligand) resistance, which is a major obstacle attributed to insufficient level or mutation of TRAIL receptors. Here, we developed an iron oxide cluster-based nanoplatform for both sensitization and MR image-guided evaluation to improve TRAIL/Apo2L efficacy in colorectal cancer, which has an inadequate response to TRAIL/Apo2L or chemotherapy. Specifically, NanoTRAIL (TRAIL/Apo2L-iron oxide nanoparticles) generated ROS (reactive oxygen species)-triggered JNK (c-Jun N-terminal kinase) activation and induced subsequent autophagy-assisted DR5 upregulation, resulting in a significant enhanced antitumor efficacy of TRAIL/Apo2L, which confirmed in both TRAIL-resistant HT-29, intermediately resistant SW-480 and sensitive HCT-116 cells. Furthermore, in a subcutaneous colorectal cancer mouse model, the in vivo tumor retention of NanoTRAIL can be demonstrated by MR T2 weighted contrast imaging, and NanoTRAIL significantly suppressed tumor growth and prolonged the survival time without observable adverse effects compared with control and TRAIL/Apo2L monotherapy. Importantly, in the study of colorectal cancer patient-derived xenograft models, we found that the NanoTRAIL treatment could significantly improve the survival outcome with consistent ROS-dependent autophagy-assisted DR5 upregulation and tumor apoptosis. Our results describe a transformative design that can be applied clinically to sensitize Apo2L/TRAIL-resistant patients using FDA-approved iron oxide nanoparticles.

Nitric Oxide Inhibits NF-κB-mediated Survival Signaling: Possible Role in Overcoming TRAIL Resistance.

BACKGROUND/AIM: Chemoresistance is a major consequence of multicycle chemotherapy and can be attributed to constitutive activation of pro-survival signaling pathways. Nitric oxide is a ubiquitous signaling molecule which has been shown to inhibit several pathways involved with survival signaling in cancer cells. We have previously demonstrated the anti-tumor activity of a nitric oxide-donor, nitrosylcobalamin (NO-Cbl), mediated by increased expression of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and its receptors in human tumors. We also demonstrated that a functional Apo2L/TRAIL receptor is necessary for the induction of cell death by NO-Cbl and the Apo2L/TRAIL death receptor DR4 (TRAIL R1) is S-nitrosylated. The aim of the study was to examine the effects of nitric oxide (NO) on nuclear factor kappa B (NF-κB) and determine whether nitric oxide could sensitize drug-resistant melanomas to Apo2L/TRAIL via inhibition of NF-κB or inhibitor kappa B kinase (IKK). MATERIALS AND METHODS: Antiproliferative effects of NO-Cbl and Apo2L/TRAIL were assessed in malignant melanomas and non-tumorigenic melanocyte and fibroblast cell lines. Athymic nude mice bearing human melanoma A375 xenografts were treated with NO-Cbl and Apo2L/TRAIL. Apoptosis was measured by the TUNEL assay. The activation status of NF-κB was established by assaying luciferase reporter activity, the phosphorylation status of IκBα, and in vitro IKK activity. RESULTS: NO-Cbl sensitized Apo2L/TRAIL-resistant melanoma cell lines to growth inhibition by Apo2L/TRAIL, but had minimal effect on normal cell lines. NO-Cbl and Apo2L/TRAIL exerted synergistic anti-tumor activity against A375 xenografts. NO-Cbl suppressed Apo2L/TRAIL- and TNF-α-mediated activation of a transfected NF-κB-driven luciferase reporter. NO-Cbl inhibited IKK activation, characterized by decreased phosphorylation of IκBα. CONCLUSION: NO-Cbl treatment rendered Apo2L/TRAIL-resistant malignancies sensitive to the anti-tumor effects of Apo2L/TRAIL in vitro and in vivo. The use of nitric oxide to inhibit NF-κB and potentiate the effects of chemotherapeutic agents, such as Apo2L/TRAIL, represents a promising anti-cancer combination based on recent clinical investigations of anti-TRAIL antibodies for cancer treatment strategies.

Repositioning Quinacrine Toward Treatment of Ovarian Cancer by Rational Combination With TRAIL.

Quinacrine has been identified as a potent DR5-inducing agent that sensitizes cancer cells to TRAIL-induced apoptosis. In the current study, we found that quinacrine increased DR5 mRNA levels significantly in ovarian cancer cell lines regardless of p53 status. Further study showed the half-life of DR5 in quinacrine-treated cells was significantly prolonged, indicating that DR5 protein degradation was inhibited by quinacrine. We tested if the combination of TRAIL and quinacrine could be effective in ovarian cancer treatment in vitro and in ovarian cancer xenograft mouse models. We found that quinacrine enhanced TRAIL sensitivity or reversed TRAIL resistance in all the ovarian cancer cell lines tested. Mice treated with quinacrine and TRAIL remained disease-free for up to 20 weeks, however, mice treated with TRAIL or quinacrine alone and in control group died within ~8 weeks after treatment. Intraperitoneal delivery of quinacrine and TRAIL is rational and practical with extraordinary synergistic anti-cancer effects in preclinical models of ovarian cancer. Clinical investigation of combining quinacrine with TRAIL for ovarian cancer treatment is warranted.

Autophagy as a mechanism of Apo2L/TRAIL resistance.

Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is unique to selectively induce apoptosis in tumor cells while sparing normal cells. Thus there is tremendous interest in Apo2L/TRAIL therapy; however, drug resistance is a serious limitation. Autophagy is a cellular housekeeping process that controls protein and organelle turnover, and is almost consistently activated in response to apoptosis-inducing stimuli, including Apo2L/TRAIL. Unlike apoptosis, autophagy leads to cell death or survival depending on the context. Various molecular mechanisms by which autophagy regulates Apo2L/TRAIL-induced apoptosis have been identified. Further, whether autophagy is completed (intact autophagic flux) or not could determine the fate of cancer cells, either cell survival or death. Thus, targeting autophagy is an attractive strategy to overcome Apo2L/TRAIL resistance. We present the current view of how these regulatory mechanisms of this interplay between autophagy and apoptosis may dictate cancer cell response to Apo2L/TRAIL therapy.

Betulinic acid promotes TRAIL function on liver cancer progression inhibition through p53/Caspase-3 signaling activation.

Betulinic acid (BA), isolated from the tree bark, is a pentacyclic triterpenoid, showing inhibitory role in cancer cells. However, the effects of BA treatment on liver cancer have little to be known. Thus, the study is conducted to explore the in vitro and in vivo role of BA in liver cancer. And the interactions between BA and tumor necrosis factor-related apoptosis-inducing ligand of APO2, also known as TRAIL, were investigated in liver cancer cells. A synergistic effect of BA and APO2 combination on apoptosis induction in liver cancer cells was observed. The cancer cells were insensitive to APO2 single therapy. However, liver cancer cells receiving BA were sensitive to APO2-triggered apoptotic response by enhancing Caspases cleavage, due to elevation of decoy receptor 1 and 2 (DcR1 and DcR2) dependent on p53. Bcl-2 family members of Bcl-2 and Mcl-1, belonging to anti-apoptosis, were decreased, whereas Bad and Bak, as pro-apoptotic members, were increased for BA and APO2 combined treatment. Additionally, the mouse xenograft model suggested that BA and APO2 in combination markedly inhibited liver cancer growth in comparison to BA or APO2 monotherapy without toxicity. The present study revealed a dramatically therapeutic strategy for promoting APO2-induced anti-cancer effects on liver cancer cells via BA combination.

Caspase-6 Induces 7A6 Antigen Localization to Mitochondria During FAS-induced Apoptosis of Jurkat Cells.

BACKGROUND: Mitochondria are central to apoptosis. However, apoptosis progression involving mitochondria is not fully understood. A factor involved in mitochondria-mediated apoptosis is 7A6 antigen. 7A6 localizes to mitochondria from the cytosol during apoptosis, which seems to involve 'effector' caspases. In this study, we investigated the precise role of effector caspases in 7A6 localization to mitochondria during apoptosis. MATERIALS AND METHODS: Human T-cell lymphoma Jurkat cells were treated with an antibody against FAS. 7A6 localization was analyzed by confocal laser scanning microscopy and flow cytometry. Caspases activation was determined by western blot analysis. RESULTS: 7A6 localization to mitochondria during anti-FAS-induced apoptosis was significantly reduced by the caspase-6 inhibitor, N-acetyl-Val-Glu-Ile-Asp-aldehyde, but not by the caspase-3 inhibitor, N-acetyl-Asp-Asn-Leu-Asp-aldehyde, nor caspase-7/3 inhibitor, N-acetyl-Asp-Gln-Thr-Asp-aldehyde. Moreover, caspase-6 down-regulation suppressed 7A6 localization to mitochondria. CONCLUSION: Caspase-6 regulates 7A6 localization to mitochondria during anti-FAS-induced apoptosis of Jurkat cells.

Development of a bioassay as a measure of drozitumab-mediated apoptosis induced by soluble Fc gamma receptors.

Drozitumab is an agonistic therapeutic monoclonal antibody (mAb) against the pro-apoptotic death receptor 5 (DR5). In vitro cell killing assays using drozitumab have traditionally required cross-linking with anti-Fc antibody to amplify the pro-apoptotic signal, although drozitumab shows activity in in vivo tumor models without artificial cross-linking. Recently it has been shown that FcγR expressing cells play an important role in the activity of drozitumab by mediating cross-linking in vivo (Wilson et al., 2011). To provide a more biologically relevant alternative to cross-linking with anti-Fc antibody in in vitro bioassays, methods for cross-linking with soluble FcγR extracellular domain (ECD) were developed in this work. FcγR cross-linking methods developed in this work were assessed in solution, bead-bound, and plate-bound assay formats, as well as a cell-based assay format. The assays showed reproducible drozitumab dose-response curves in the concentration range of 5-20,000ng/mL and had acceptable precision and accuracy. The assays are also able to detect degradative changes in drozitumab samples subjected to thermal stress. The data suggest that FcγR cross-linking of drozitumab is a viable alternative to anti-Fc cross-linking of drozitumab to measure effector mediated apoptosis of drozitumab in vitro.

Pancreatic cancer stem cells in patient pancreatic xenografts are sensitive to drozitumab, an agonistic antibody against DR5.

BACKGROUND: Therapeutic resistance and tumor recurrence are two major hurdles in the treatment of pancreatic ductal adenocarcinoma. Recent findings suggest that both of these attributes are associated with a small subset of pancreatic tumor initiating cancer stem cells (CSCs). Here, we demonstrate that drozitumab, a human agonistic monoclonal antibody which binds the death receptor DR5, selectively eliminates CSCs, resulting in tumor growth inhibition and even regression of pancreatic tumors. METHODS: To examine the efficacy of drozitumab against pancreatic CSCs, we treated patient-derived pancreatic tumor xenografts (PDX) in immunocompromised SCID mice and evaluated tumor control. To assess apoptosis following drozitumab treatment, we identified the CSCs as CD24+, CD44+, and EpCAM+ by FACS analysis, and measured in vivo and in vitro levels of cleaved caspase-3. Lastly, in vitro evaluation of DR5 re-expression was performed using isolated patient pancreatic cancer xenograft cells along with the cell line, Panc-1. After treatment with drozitumab, the remaining DR5- cells were assessed by FACS analysis for DR5 expression at the cell surface at 8, 24 and 48 h post-treatment. All in vivo growth data was analyzed by 2-way Anova, incidence data was analyzed using Mantel-Cox, and in vitro studies statistics were performed with a t-test. RESULTS: We find that while 75-100 % of CSCs express DR5, only 25 % of bulk tumor cells express the death receptors at any one time. Consequently, drozitumab treatment of SCID mice bearing PDX kills higher percentages of CSCs than bulk tumor cells. Additionally, SCID mice implanted with isolated CSCs and then immediately treated with drozitumab fail to ever develop tumors. In vitro studies demonstrate that while drozitumab treatment reduces the DR5+ cell population, the remaining tumor cells begin to express DR5, suggesting a mechanism by which continuous administration of drozitumab can ultimately result in tumor regression despite the initially low percentage of DR5+ cells. CONCLUSIONS: Overall, our work reveals that treatment of pancreatic tumors with the drozitumab can lead to long-term tumor control by targeting both bulk cells and CSCs.

Enhancing tumor-targeting monoclonal antibodies therapy by PARP inhibitors.

Monoclonal antibodies (mAbs) have become a successful therapeutic approach in cancer. However, some patients do not achieve long-term clinical benefit and most mAbs only exert modest effects as monotherapies. Therefore, combinations with chemotherapy are currently being investigated. Emerging studies have shown a synergistic therapeutic effect of PARP inhibitors and mAbs in cancer. PARP enzymes catalytically cleave ß-NAD+ and transfer the ADP-ribose moiety to acceptor proteins, modifying their function. In here, we update recent data about the therapeutic effect of the combination of PARP inhibitors with mAbs in cancer treatment and discuss the molecular mechanisms involved in this synergy.

An optimized antibody-single-chain TRAIL fusion protein for cancer therapy.

Fusion proteins combining oligomeric assemblies of a genetically obtained single-chain (sc) variant of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with antibodies directed against tumor-associated antigens represent a promising strategy to overcome the limited therapeutic activity of conventional soluble TRAIL. To further improve the scTRAIL module in order to obtain a robust, thermostable molecule of high activity, we performed a comprehensive analysis of the minimal TNF homology domain (THD) and optimized linkers between the 3 TRAIL subunits constituting a scTRAIL. Through a stepwise mutagenesis of the N- and C-terminal region and the joining linker sequences, we generated bioactive scTRAIL molecules comprising a covalent linkage of the C-terminal Val280 and the N-terminal position 122 by only 2 amino acid residues in combination with conservative exchanges at positions 122 and 279. The increased thermal stability and solubility of such optimized scTRAIL molecules translated into increased bioactivity in the diabody-scTRAIL (Db-scTRAIL) format, exemplified here for an epidermal growth factor receptor-specific Db-scTRAIL. Additional modifications within the diabody linkers resulted in a fusion protein exerting high, target-dependent apoptosis induction in tumor cell lines in vitro and potent antitumor activity in vivo. Our results illustrate that protein engineering of scTRAIL and associated peptide linkers provides a promising strategy to develop antibody-scTRAIL fusion proteins as effective antitumor therapeutics.

TRAIL causes deletions at the HPRT and TK1 loci of clonogenically competent cells.

When chemotherapy and radiotherapy are effective, they function by inducing DNA damage in cancerous cells, which respond by undergoing apoptosis. Some adverse effects can result from collateral destruction of non-cancerous cells, via the same mechanism. Therapy-related cancers, a particularly serious adverse effect of anti-cancer treatments, develop due to oncogenic mutations created in non-cancerous cells by the DNA damaging therapies used to eliminate the original cancer. Physiologically achievable concentrations of direct apoptosis inducing anti-cancer drugs that target Bcl-2 and IAP proteins possess negligible mutagenic activity, however death receptor agonists like TRAIL/Apo2L can provoke mutations in surviving cells, probably via caspase-mediated activation of the nuclease CAD. In this study we compared the types of mutations sustained in the HPRT and TK1 loci of clonogenically competent cells following treatment with TRAIL or the alkylating agent ethyl methanesulfonate (EMS). As expected, the loss-of-function mutations in the HPRT or TK1 loci triggered by exposure to EMS were almost all transitions. In contrast, only a minority of the mutations identified in TRAIL-treated clones lacking HPRT or TK1 activity were substitutions. Almost three quarters of the TRAIL-induced mutations were partial or complete deletions of the HPRT or TK1 genes, consistent with sub-lethal TRAIL treatment provoking double strand breaks, which may be mis-repaired by non-homologous end joining (NHEJ). Mis-repair of double-strand breaks following exposure to chemotherapy drugs has been implicated in the pathogenesis of therapy-related cancers. These data suggest that TRAIL too may provoke oncogenic damage to the genomes of surviving cells.

Death ligands and granulysin: mechanisms of tumor cell death induction and therapeutic opportunities.

The immune system plays a key role in cancer immune surveillance to control tumor development. The final goal is recognizing and killing transformed cells and consequently the elimination of the tumor. The main effector cell types exerting cytotoxicity against tumors are natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Although the mechanism of activation of NK cells and CTLs are quite different, both cell types share common antitumor effector mechanisms of cytotoxicity which lead to induction of cell death of tumor cells by apoptosis. Among these mechanisms are the death ligand- and granulysin-mediated cell deaths. In this review, we summarize the main concepts of these effector cytotoxic mechanisms against cancer cells, how NK cells and CTLs use them to control tumor development and the therapeutic approaches currently developed based on these molecules.