Breast cancer (BC) is the most common cancer among women worldwide. Due to its complexity in nature, effective BC treatment can encounter many challenges. The human RALBP1 gene encodes a 76-kDa splice variant protein, RLIP (ral-binding protein1, RalBP1), a stress-protective mercapturic acid pathway (MAP) transporter protein, that also plays a key role in regulating clathrin-dependent endocytosis (CDE) as a Ral effector. Growing evidence shows that targeting RLIP may be an effective strategy in cancer therapy, as RLIP is over-expressed in multiple cancers and is known to induce resistance to apoptosis and chemotherapeutic drugs. Recent studies demonstrated that RLIP is expressed in human BC tissues, as well as BC cell lines. Knockdown of RLIP resulted in apoptotic death of BC cells in vitro, and targeted inhibition and depletion of RLIP resulted in regression of BC in xenograft studies of nude mice. Signaling studies showed that RLIP depletion inhibited endocytosis and differentially regulated signaling to Akt, Myc, and ERK1/2. However, the proliferation and multi-specific transport mechanisms that promote RLIP-mediated cell death in BC are not well understood. In this review, we will discuss a missing but an essentially determining and connecting piece of the puzzle on the understanding of proliferation and transport mechanisms by focused analyses of the apoptotic, drug- and radiation-sensitivity regulated by RLIP, a stress-responsive non-ATP-binding cassette (ABC), high capacity MAP transporter, in breast cancer.

Consumption of citrus-fruits is associated with reduced incidence of breast cancer (BC), the most common cancer diagnosed in women across the globe. In this study, we investigated the anticancer potential of 2-Hydroxyflavanone (2HF) in BC. 2HF, a citrus-bioflavonoid, has demonstrated anticancer properties in various cancers, but its anticancer role in BC has not been well studied. We investigated the in vitro and in vivo growth inhibitory effects of 2HF in an array of BC lines and in xenograft mouse models of ER-positive and HER2-positive BC cells. Compared to control, 2HF treatment reduced cell viability and suppressed migratory and invasive potential of BC cells, while, no growth inhibitory effects were observed in non-tumorigenic breast epithelial cells. Further, 2HF inhibited the expression of RLIP76, a stress-defensive and anti-apoptotic protein, which is over-expressed in BC cells and simultaneously reduced proliferation of BC cells. Nude mice bearing MCF7 or SKBR3 BC cells xenografts treated with either 2HF or targeting RLIP76 by RLIP76-antisense or RLIP76-antibody treatment had significantly lower tumor-weight as compared to corresponding controls. In addition, Western-blotting and immunohistochemical analysis of tumor tissue from control and treatment group mice showed that 2HF decreased protein expression levels of RLIP76, and the decrease was similar to those seen following RLIP76-antisense treatment. Furthermore, 2HF decreased expression of Ki67, CD31, vimentin, inhibited phosphorylation of Akt and expression of survivin and Bcl2, and increased levels of Bax, E-cadherin, and cleaved-PARP. Therefore, our results indicate that 2HF may suppress BC growth in vitro and in vivo by targeting RLIP76, and may serve as a potential adjuvant treatment in BC patients.

BACKGROUND: The most frequent type of renal cell carcinoma is called clear-cell renal cell carcinoma (ccRCC) which is associated with a poor prognosis. It has been observed that miR-137 is aberrantly expressed in many different kinds of human malignancies including ccRCC. This research aims to examine the role of miR-137 in ccRCC.
METHODS: Quantitative RT-PCR (qRT-PCR) was applied to measure miR-137 expression in ccRCC and adjacent noncancerous tissue. Gene expression was determined by western blot. Cell Counting Kit-8 (CCK-8) assay, flow cytometry and Transwell assay were used to determine the effects of miR-137 on cell growth, apoptosis and invasion, respectively. Moreover, xenograft and pulmonary metastasis animal models were established to investigate the role of miR-137 in vivo.
RESULTS: Our findings show that there was significant downregulation of miR-137 in ccRCC tissue relative to corresponding non-cancerous tissue. Ectopic miR-137 expression in ccRCC cells led to suppression of cell growth and invasion, as well as apoptosis induction. In contrast, knockdown of miR-137 enhances proliferation and invasion, inhibits apoptosis. It also confirms that miR-137 plays a tumor supressor role in vivo. Mechanically, miR-137 directly targets the 3'-UTR of RLIP76 which is an established oncogene in ccRCC.
CONCLUSION: MiR-137 serves as a tumor suppressor, which can be considered a potential therapeutic target in ccRCC.

RIP1 and RIP3 are necroptosis initiators, but their roles in regulation of glycolysis remain elusive. In this study, we found shikonin activated RIP1 and RIP3 in glioma cells in vitro and in vivo, which was accompanied with glycolysis suppression. Further investigation revealed that shikonin-induced decreases of glucose-6-phosphate and pyruvate and downregulation of HK II and PKM2 were significantly prevented when RIP1 or RIP3 was pharmacologically inhibited or genetically knocked down with SiRNA. Moreover, shikonin also triggered accumulation of intracellular H

Resistance to antiangiogenic drugs limits their applicability in cancer therapy. Here, we show that revascularization and progression of pancreatic neuroendocrine tumors (PNETs) under extended vascular-endothelial growth factor A (VEGFA) blockade are dependent on periostin (POSTN), a matricellular protein expressed by stromal cells. Genetic deletion of Postn in RIP1-Tag2 mice blunted tumor rebounds of M2-like macrophages and αSMA

Vincristine (VCR), an important component of childhood acute lymphoblastic leukemia (ALL) therapy, can cause sensory and motor neurotoxicity. This neurotoxicity could lead to dose reduction or treatment discontinuation, which could in turn reduce survival. In this line, several studies associated peripheral neurotoxicity and polymorphisms in genes involved in pharmacokinetics (PK) and pharmacodynamics (PD) of VCR. Nowadays, it is well known that these genes are regulated by microRNAs (miRNAs) and SNPs in miRNAs could modify their levels or function. Therefore, the aim of this study was to determine whether SNPs in miRNAs could be associated with VCR-induced neurotoxicity. To achieve this aim, we analyzed all the SNPs in miRNAs (minor allele frequency (MAF) ≥ 0.01) which could regulate VCR-related genes in a large cohort of Spanish children with B-cell precursor ALL (B-ALL) homogeneously treated with LAL/SHOP protocols. We identified the A allele of rs12402181 in the seed region of miR-3117-3p, that could affect the binding with ABCC1 and RALBP1 gene, and C allele of rs7896283 in pre-mature sequence of miR-4481, which could be involved in peripheral nerve regeneration, significantly associated with VCR-induced neurotoxicity. These findings point out the possible involvement of two SNPs in miRNA associated with VCR-related neurotoxicity.

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) triggers apoptosis by inducing the death-inducing signaling complex (DISC) formation. Recently, TNFα-induced protein 3 (TNFAIP3, A20) was reported to prevent TRAIL-induced caspase 8 cleavage in the DISC by mediating ubiquitination of RIP1 in glioblastoma. However, whether A20 regulates caspase 8 cleavage in the DISC when TRAIL induces apoptosis in gastric cancer cells is unknown. In the present study, A20 interacted with RIP1 and DR4 in MGC803 and SGC7901 gastric cancer cells. Treatment with TRAIL promoted the A20-mediated polyubiquitination of RIP1, caspase 8 translocation into the DISC, and the interaction between caspase 8 and ubiquitinated RIP1. Inhibition of A20 expression prevented the polyubiquitination of RIP1 and promoted caspase 8 cleavage. Moreover, our data clarified that A20 is a target of miR-200a. Overexpression of miR-200a inhibited A20 expression and polyubiquitination of RIP1 and then promoted cleavage of caspase 8 and TRAIL-triggered apoptosis. Taken together, our results indicate that miR-200a enhanced TRAIL-triggered apoptosis in gastric cancer cells by targeting A20.

RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor (GEF) for RalA containing a PH domain and an SH3-binding region and it is involved in several cellular processes, such as cytokinesis, control of cell cycle progression, differentiation, cytoskeleton organization and rearrangement. Up to now, few data have been published regarding RalGPS2 role in cancer cells, and its involvement in bladder cancer is yet to be established. In this paper we demonstrated that RalGPS2 is expressed in urothelial carcinoma-derived 5637 cancer cells and is essential for cellular growth. These cells produces thin membrane protrusions that displayed the characteristics of actin rich tunneling nanotubes (TNTs) and here we show that RalGPS2 is involved in the formation of these cellular protrusions. In fact the overexpression of RalGPS2 or of its PH-domain increased markedly the number and the length of nanotubes, while the knock-down of RalGPS2 caused a strong reduction of these structures. Moreover, using a series of RalA mutants impaired in the interaction with different downstream components (Sec5, Exo84, RalBP1) we demonstrated that the interaction of RalA with Sec5 is required for TNTs formation. Furthermore, we found that RalGPS2 interacts with the transmembrane MHC class III protein leukocyte specific transcript 1 (LST1) and RalA, leading to the formation of a complex which promotes TNTs generation. These findings allow us to add novel elements to molecular models that have been previously proposed regarding TNTs formation.

Chemotherapeutic regimens containing camptothecin (CPT), 5-fluorouracil, and oxaliplatin are used to treat advanced colorectal cancer. We previously reported that an indole derivative, 3-(2-bromoethyl)indole (BEI-9), inhibited the proliferation of colon cancer cells and suppressed NF-κB activation. Here, we show that a combination of BEI-9 with either CPT or tumor necrosis factor alpha (TNFα) enhances cell death. Using colorectal cancer cells, we examined the activation of NF-κB by drugs, the potential of BEI-9 for inhibiting drug-induced NF-κB activation, and the enhancement of cell death by combination treatments. Cells were treated with the chemotherapeutic drugs alone or in combination with BEI-9. NF-κB activation, cell cycle profiles, DNA-damage response, markers of cell death signaling and targets of NF-κB were evaluated to determine the effects of single and co-treatments. The combination of BEI-9 with CPT or TNFα inhibited NF-κB activation and reduced the expression of NF-κB-responsive genes, Bcl-xL and COX2. Compared to CPT or BEI-9 alone, sequential treatment of the cells with CPT and BEI-9 significantly enhanced caspase activation and cell death. Co-treatment with TNFα and BEI-9 also caused more cytotoxicity than TNFα or BEI-9 alone. Combined BEI-9 and TNFα enhanced cell death through caspase activation and cleavage of the switch-protein, RIP1 kinase. BEI-9 reduced the expression of COX2 both alone and in combination with CPT or TNF. We postulate that BEI-9 enhances the effects of these drugs on cancer cells by turning off or redirecting NF-κB signaling. Therefore, the combination of BEI-9 with drugs that activate NF-κB needs to be evaluated for clinical applications.

Nowadays, lung cancer, as a health problem in worldwide, has high mortality both in men and women. Despite advances in diagnosis and surgical techniques of lung cancer in recent decades, chemotherapy is still a fundamentally and extensively useful strategy. Vinca alkaloids are a class of important and widely used drugs in the treatment of lung cancer, targeting on the Vinca binding site at the exterior of microtubule plus ends. Either intrinsic or acquired resistance to chemotherapy of Vinca alkaloids has been a major obstacle to the treatment of lung cancer, which arose great interests in studies of understanding and overcoming resistance. In this review, we focused on the application and resistance mechanisms of the Vinca alkaloids such as vinblastine, vincristine, vinorelbine and vinflunine in lung cancer. We reviewed characteristic resistance mechanisms in lung cancer including over-expression of ATP-binding cassette (ABC) transporters P-glycoprotein and structural, functional or expression alterations of β-tubulin (βII, βIII, βIV) which may devote to the development of acquired resistance to the Vinca alkaloids; multidrug-resistance proteins (MRP1, MRP2, MRP3) and RLIP76 protein have also been identified that probably play a significant role in intrinsic resistance. Lung resistance-related protein (LRP) is contributed to lung cancer therapy resistance, but is not deal with the Vinca alkaloids resistance in lung cancer. Understanding the principle of the Vinca alkaloids in clinical application and mechanisms of drug resistance will support individualized lung cancer therapy and improve future therapies.

Metastatic breast cancer is still incurable so far; new specifically targeted and more effective therapies for triple-negative breast cancer (TNBC) are required in the clinic. In this study, our clinical data have established that basal and claudin-low subtypes of breast cancer (TNBC types) express significantly higher levels of Annexin A1 (ANXA1) with poor survival outcomes. Using human cancer cell lines that model the TNBC subtype, we observed a strong positive correlation between expression of ANXA1 and PPARγ. A similar correlation between these two markers was also established in our clinical breast cancer patients' specimens. To establish a link between these two markers in TNBC, we show

Since cancer cells often evade apoptosis, induction of necroptosis as another mode of programmed cell death is considered a promising therapeutic alternative. Here, we identify a novel synergistic interaction of Smac mimetics that antagonize x-linked Inhibitor of Apoptosis (XIAP), cellular Inhibitor of Apoptosis (cIAP) 1 and 2 with interferon (IFN)γ to induce necroptosis in apoptosis-resistant cancer cells in which caspase activation is blocked. This synergism is confirmed by calculation of combination indices (CIs) and found in both solid and hematological cancer cell lines as well as for different Smac mimetics (i.e. BV6, Birinapant), pointing to a broader relevance. Importantly, individual genetic knockdown of key components of necroptosis signaling, i.e. receptor-interacting protein (RIP) 1, RIP3 or mixed lineage kinase domain-like pseudokinase (MLKL), significantly protects from BV6/IFNγ-induced cell death. Similarly, pharmacological inhibitors of RIP1 (necrostatin-1(Nec-1)), RIP3 (GSK'872) or MLKL (necrosulfonamide (NSA)) significantly reduce BV6/IFNγ-stimulated cell death. Of note, IFN-regulatory factor (IRF)1 is required for BV6/IFNγ-mediated necroptosis, as IRF1 silencing provides protection from cell death. By comparison, antibodies blocking tumor necrosis factor (TNF)α, TNF-related apoptosis-inducing ligand (TRAIL) or CD95 ligand fail to inhibit BV6/IFNγ-induced cell death, pointing to a mechanism independently of death receptor ligands. This is the first report showing that Smac mimetics synergize with IFNγ to trigger necroptosis in apoptosis-resistant cancer cells with important implications for Smac mimetic-based strategies for the treatment of cancer.

Cancer cells require both migratory and tumorigenic property to establish metastatic tumors outside the primary microenvironment. Identifying the characteristic features of migratory cancer stem cells with tumorigenic property is important to predict patient prognosis and combat metastasis. Here we established one epithelial and two mesenchymal cell lines from ascites of a bladder cancer patient (i.e. cells already migrated outside primary tumor). Analyses of these cell lines demonstrated that the epithelial cells with surface expression of PD-L1, E-cadherin, CD24, and VEGFR2 rapidly formed tumors outside the primary tumor microenvironment in nude mice, exhibited signatures of immune evasion, increased stemness, increased calcium signaling, transformation, and novel E-cadherin-RalBP1 interaction. The mesenchymal cells on the other hand, exhibited constitutive TGF-β signaling and were less tumorigenic. Hence, targeting epithelial cancer stem cells with rapid tumorigenesis signatures in future might help to combat metastasis.

OBJECTIVE: Gastric cancer (GC) is one of the most prevalent types of malignant disease Worldwide. Mounting evidence has demonstrated the involvement of miRNAs in the development of GC. One of these miRNAs, miR-144 has been found aberrantly expressed in a variety of human malignancies.
PATIENTS AND METHODS: GC tissues were collected from patients, and the level of miR-144 was determined by qRT-PCR. GC cell lines SGC7901 and AGS were used as model cell lines and the anti-tumor effect of miR-144 in both cells were examined. The level of miR-144 was restored in GC cells using miR-144 mimic. Moreover, the target gene of miR-144 wad identified.
RESULTS: In this study, our results showed that low miR-144 level significantly correlated with lymph node metastasis stage, TNM stage and differentiation degree. In addition, we found that miR-144 acted as a tumor suppressor in GC. Moreover, our findings showed that miR-144 exerted an anti-tumor effect by directly targeting RLIP76.
CONCLUSIONS:   miR-144 acts as a tumor suppressor in GC and it is a potential therapeutic target for GC treatment.

OBJECTIVE: Colorectal cancer (CRC) is a common human malignancy and is the second leading cause of cancer deaths worldwide with a dismal prognosis. Previous investigations have shown that miR-340 can modulate the metabolism of CRC cells. The aim of this report is to study the role of miR-340 in the development and progression of CRC.
PATIENTS AND METHODS: The level of miR-340 in CRC cells was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting. CRC cell lines were used as model cell lines and the anti-tumor effect of miR-340 in vitro was examined. The luciferase reporter assay was performed. The level of miR-340 was restored in CRC cells by the usage of the miR-340 mimic. Re-expression of RLIP76 in CRC cells was then constructed. Moreover, the target gene of miR-340 was identified through the experiment of in vivo xenograft model.
RESULTS: The aberrant downregulation of miR-340 is correlated with advanced stage of CRC. Furthermore, the ectopic overexpression of miR-340 in CRC cell lines resulted in growth inhibition, apoptosis and enhanced chemosensitivity in vitro and in vivo, which was mediated by directly targeting RLIP76.
CONCLUSIONS: miR-340 acts as a tumor suppressor in CRC and is involved in the chemoresistance of CRC.

BACKGROUND: Shikonin, a natural naphthoquinone pigment purified from Lithospermum erythrorhizon, induces necroptosis in various cancer types, but the mechanisms underlying the anticancer activity of shikonin in lung cancer are not fully understood. This study was designed to clarify whether shikonin causes necroptosis in non-small cell lung cancer (NSCLC) cells and to investigate the mechanism of action.
METHODS: Multiplex and caspase 8 assays were used to analyze effect of shikonin on A549 cells. Cytometry with annexin V/PI staining and MTT assays were used to analyze the mode of cell death. Western blotting was used to determine the effect of shikonin-induced necroptosis and autophagy. Xenograft and orthotopic models with A549 cells were used to evaluate the anti-tumor effect of shikonin in vivo.
RESULTS: Most of the cell death induced by shikonin could be rescued by the specific necroptosis inhibitor necrostatin-1, but not by the general caspase inhibitor Z-VAD-FMK. Tumor growth was significantly lower in animals treated with shikonin than in the control group. Shikonin also increased RIP1 protein expression in tumor tissues. Autophagy inhibitors, including methyladenine (3-MA), ATG5 siRNA, and bafilomycin A, enhanced shikonin-induced necroptosis, whereas RIP1 siRNA had no effect on the apoptotic potential of shikonin.
CONCLUSIONS: Our data indicated that shikonin treatment induced necroptosis and autophagy in NSCLC cells. In addition, the inhibition of shikonin-induced autophagy enhanced necroptosis, suggesting that shikonin could be a novel therapeutic strategy against NSCLC.

OBJECTIVE: RLIP76 is a stress-responsive membrane protein regulating multiple cellular signaling pathways and is overexpressed in a number of malignant tumor cells. The aim of this study is to test whether the RLIP76 plays a critical role in cellular growth, apoptosis, cell cycle distribution and chemoresistance in melanoma.
MATERIALS AND METHODS: In this study, human melanoma cell line A375 was stably transfected with an RLIP76-targeted shRNA-containing vector to investigate the role of RLIP76 in cellular function. Quantitative RT-PCR and Western blot revealed significant suppression of RLIP76 in A375 cells after transfection with shRNA-containing vector. Cell proliferation was determined by MTT assay. Wound scratch ad transwell assays were used for cell migration and invasion.
RESULTS: The results showed that shRNA decreased the expression of RLIP76 in human melanoma cell line A375, and the knockdown of RLIP76 gene significantly inhibited A375 cell growth, resulted in G1 phase arrest and apoptosis increase of A375. Moreover, by testing the cell migratory and invasive abilities by wound scratch and transwell assays, it determined that the RLIP76 also suppressed cell migration and invasion in A375 cells and P13K/Akt signalling CONCLUSIONS: Overall, this study suggests that RLIP76 is a potential therapeutic target against melanoma.

Necroptosis is a form of programmed necrosis mediated by signaling complexes with receptor-interacting protein 1 (RIP1) and RIP3 kinases as the main mediators. However, the underlying execution pathways of this phenomenon have yet to be elucidated in detail. In this study, a RIP1/RIP3 complex was formed in 2-methoxy-6-acetyl-7-methyljuglone (MAM)-treated HCT116 and HT29 colon cancer cells. With this formation, mitochondrial reactive oxygen species (ROS) levels increased, mitochondrial depolarization occurred, and ATP concentrations decreased. This process was identified as necroptosis. This finding was confirmed by experiments showing that MAM-induced cell death was attenuated by the pharmacological or genetic blockage of necroptosis signaling, including RIP1 inhibitor necrostatin-1s (Nec-1s) and siRNA-mediated gene silencing of RIP1 and RIP3, but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1H-Indol-3-yl)-3-pentylamino-maleimide (IM54). Transmission electron microscopy (TEM) analysis further revealed the ultrastructural features of MAM-induced necroptosis. MAM-induced RIP1/RIP3 complex triggered necroptosis through cytosolic calcium (Ca

BACKGROUND: The inhibition of Delta-like 4 (Dll4)/Notch signaling has been shown to result in excessive, nonfunctional vessel proliferation and significant tumor growth suppression. However, safety concerns emerged with the identification of side effects resulting from chronic Dll4/Notch blockade. Alternatively, we explored the endothelial Dll4 overexpression using different mouse tumor models.
METHODS: We used a transgenic mouse model of endothelial-specific Dll4 overexpression, previously produced. Growth kinetics and vascular histopathology of several types of solid tumors was evaluated, namely Lewis Lung Carcinoma xenografts, chemically-induced skin papillomas and RIP1-Tag2 insulinomas.
RESULTS: We found that increased Dll4/Notch signaling reduces tumor growth by reducing vascular endothelial growth factor (VEGF)-induced endothelial proliferation, tumor vessel density and overall tumor blood supply. In addition, Dll4 overexpression consistently improved tumor vascular maturation and functionality, as indicated by increased vessel calibers, enhanced mural cell recruitment and increased network perfusion. Importantly, the tumor vessel normalization is not more effective than restricted vessel proliferation, but was found to prevent metastasis formation and allow for increased delivery to the tumor of concomitant chemotherapy, improving its efficacy.
CONCLUSIONS: By reducing endothelial sensitivity to VEGF, these results imply that Dll4/Notch stimulation in tumor microenvironment could be beneficial to solid cancer patient treatment by reducing primary tumor size, improving tumor drug delivery and reducing metastization. Endothelial specific Dll4 overexpression thus appears as a promising anti-angiogenic modality that might improve cancer control.

BACKGROUND Almost all hepatic cancer cells have resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. c-FLIPL and RIP-1 are apoptotic negative regulatory factors. This study investigated the role of c-FLIPL and RIP-1 in hepatic cancer cell resistance to TRAIL-induced apoptosis. MATERIAL AND METHODS HepG2 cells were treated by TRAIL, RIP-1 siRNA, and/or BY11-7082. Cell viability was detected by MTT assay. Cell apoptosis was tested by flow cytometry. DISC component proteins, RIP-1, and p-p65 were measured by Western blot. Caspase-8 and caspase-3 were determined by spectrophotometry. RESULTS Single TRAIL treatment showed no significant impact on cell proliferation and apoptosis. HepG2 cells expressed high levels of RIP1 and c-FLIPL, while a high concentration of TRAIL upregulated RIP-1 and c-FLIPL expression but not DR4 and DR5. Single TRAIL treatment did not obviously activate caspase-8 and caspase-3. RIP-1 or c-FLIPL siRNA markedly induced cell apoptosis and enhanced caspase-8 and caspase-3 activities. Combined transfection obviously increased apoptotic cells. TRAIL markedly upregulated RIP-1 expression and enhanced p-p65 protein. Downregulating RIP-1 and/or BAY11-7082 significantly reduced NF-kB transcriptional activity, blocked cells in G0/G1 phase, weakened proliferation, elevated caspase-8 and caspase-3 activities, and promoted cell apoptosis. CONCLUSIONS TRAIL can enhance RIP1 and c-FLIPL expression in HepG2 cells. High expression of RIP1 and c-FLIPL is an important reason for TRAIL resistance. Downregulation of RIP1 and c-FLIPL can relieve caspase-8 suppression, activate caspase-3, and promote cell apoptosis. TRAIL mediates apoptosis resistance through upregulating RIP-1 expression, enhancing NF-kB transcriptional activity, and weakening caspase activity.

While reactive oxygen species (ROS) gain their carcinogenic effects by DNA mutations, if generated in the vicinity of genome, lipid peroxidation products, notably 4-hydroxynonenal (HNE), have much more complex modes of activities. Namely, while ROS are short living and have short efficiency distance range (in nm or µm) HNE has strong binding affinity for proteins, thus forming relatively stable adducts. Hence, HNE can diffuse from the site or origin changing structure and function of respective proteins. Consequently HNE can influence proliferation, differentiation and apoptosis of cancer cells on one hand, while on the other it can affect genome functionality, too. Although HNE is considered to be important factor of carcinogenesis due to its ability to covalently bind to DNA, it might also be cytotoxic for cancer cells, as well as it can modulate their growth. In addition to direct cytotoxicity, HNE is also involved in activity mechanisms by which several cytostatic drugs and radiotherapy exhibit their anticancer effects. Complementary to that, the metabolic pathway for HNE detoxification through RLIP76, which is enhanced in cancer, may be a target for anti-cancer treatments. In addition, some cancer cells can undergo apoptosis or necrosis, if exposed to supraphysiological HNE levels in the cancer microenvironment, especially if challenged additionally by pro-oxidative cytostatics and/or inflammation. These findings could explain previously observed disappearance of HNE from invading cancer cells, which is associated with the increase of HNE in non-malignant cells close to invading cancer utilizing cardiolipin as the source of cancer-inhibiting HNE.

Tuberous sclerosis complex (TSC) is a genetic multiorgan disorder characterized by the development of neoplastic lesions in kidney, lung, brain, heart, and skin. It is caused by an inactivating mutation in tumor suppressor genes coding the TSC1/TSC2 complex, resulting in the hyperactivation of mTOR- and Raf/MEK/MAPK-dependent signaling that stimulates tumor cell proliferation and metastasis. Despite its oncogenic effect, cells with TSC deficiency were more sensitive to oxidative stress and dependent on mitochondrial metabolism, providing a rationale for a new therapeutic approach. The current study shows that simultaneous inhibition of two major pathways regulating redox homeostasis using l-buthionine-sulfoximine (BSO, glutathione synthesis inhibitor) and auranofin (thioredoxin reductase inhibitor) induces oxidative burst, mitochondrial damage, and necrotic cell death in TSC-deficient cells in a highly synergistic and cell context-specific manner. Furthermore, blocking RIP1/RIP3/MLKL-dependent signaling using chemical inhibitors necrostatin-1 (Nec-1) and necrosulfonamide (NSA) synergizes with BSO and auranofin in killing TSC-deficient cells. Expression analysis demonstrated that RIP1, RIP3, and MLKL protein levels are elevated in cells with TSC2 deficiency, and their inactivation enhances mitochondrial dysfunction in a glutaminolysis-dependent and autophagy-independent manner. Finally, supplementation with the mitochondrial metabolite α-ketoglutarate, whose synthesis is regulated by RIP1/RIP3/MLKL, rescues cells from the sensitizing effect of Nec-1 and NSA. Together, this study identifies a previously unrecognized novel regulated necrotic death pathway that involves mitochondrial homeostasis, is suppressed by the RIP1/RIP3/MLKL signaling in TSC-deficient cells, and could be a promising therapeutic target for TSC-associated tumors. Cancer Res; 76(24); 7130-9. ©2016 AACR.

Medulloblastoma (MDB) represents a major form of malignant brain tumors in the pediatric population. A vast spectrum of research on MDB has advanced our understanding of the underlying mechanism, however, a significant need still exists to develop novel therapeutics on the basis of gaining new knowledge about the characteristics of cell signaling networks involved. The Ras signaling pathway, one of the most important proto-oncogenic pathways involved in human cancers, has been shown to be involved in the development of neurological malignancies. We have studied an important effector down-stream of Ras, namely RalA (Ras-Like), for the first time and revealed overactivation of RalA in MDB. Affinity precipitation analysis of active RalA (RalA-GTP) in eight MDB cell lines (DAOY, RES256, RES262, UW228-1, UW426, UW473, D283 and D425) revealed that the majority contained elevated levels of active RalA (RalA-GTP) as compared with fetal cerebellar tissue as a normal control. Additionally, total RalA levels were shown to be elevated in 20 MDB patient samples as compared to normal brain tissue. The overall expression of RalA, however, was comparable in cancerous and normal samples. Other important effectors of RalA pathway including RalA binding protein-1 (RalBP1) and protein phosphatase A (PP2A) down-stream of Ral and Aurora kinase A (AKA) as an upstream RalA activator were also investigated in MDB. Considering the lack of specific inhibitors for RalA, we used gene specific silencing in order to inhibit RalA expression. Using a lentivirus expressing anti-RalA shRNA we successfully inhibited RalA expression in MDB and observed a significant reduction in proliferation and invasiveness. Similar results were observed using inhibitors of AKA and geranyl-geranyl transferase (non-specific inhibitors of RalA signaling) in terms of loss of in vivo tumorigenicity in heterotopic nude mouse model. Finally, once tested in cells expressing CD133 (a marker for MDB cancer stem cells), higher levels of RalA activation was observed. These data not only bring RalA to light as an important contributor to the malignant phenotype of MDB but introduces this pathway as a novel target in the treatment of this malignancy.

The objective of the present study was to identify the association between mir-143-3p and RalA-binding protein 1 (RALBP1), and their roles in regulating the development of ovarian cancer. Overexpression of RALBP1 induced apoptosis of the ovarian cancer cells, and developed ovarian cancer. In silico analysis and luciferase assay were used to identify whether RALBP1 was the target of mir-143-3p. Subsequently, real‑time PCR and western blotting were used to determine the expression level of mir-143-3p, RALBP1 mRNA and protein in different groups, furthermore, MTT assay and flow cytometry were used to detect the viability and apoptosis of cells in different treatment groups. We identified RALBP1 as a target gene of miR-143-3p using computational analysis, and the luciferase activity of cells transfected with wild-type RALBP1 and RALBP1 siRNA were much lower than the scramble control, however, the luciferase activity of cells transfected with mutant RALBP1 was similar with scramble control. The real-time PCR and western blot results suggested that the miR‑143-3p level was markedly lower in participants with ovarian cancer compared with normal control, while the expression of RALBP1 mRNA and protein were evidently overexpressed in participants with ovarian cancer compared with normal control. Furthermore, the RALBP1 mRNA and protein level in cells transfected with miR-143-3p mimics and RALBP1 siRNA were downregulated, while notably upregulated subsequent to transfection with miR-143-3p inhibitor, when compared with scramble control. Additionally, the viability of cells were inhibited following transfection with miR-143-3p mimics and RALBP1 siRNA, while notably promoted subsequent to transfection with miR-143-3p inhibitor. Apoptosis of cells were promoted following transfection with miR-143-3p mimics and RALBP1 siRNA, while notably inhibited subsequent to transfection with miR-143-3p inhibitor. These findings provide support that downregulation of the miR-143-3p is associated with a decreased risk of ovarian cancer.

BACKGROUND: LCL161, a novel Smac mimetic, is known to have anti-tumor activity and improve chemosensitivity in various cancers. However, the function and mechanisms of the combination of LCL161 and paclitaxel in non-small cell lung cancer (NSCLC) remain unknown.
METHODS: Cellular inhibitor of apoptotic protein 1 and 2 (cIAP1&2) expression in NSCLC tissues and adjacent non-tumor tissues were assessed by immunohistochemistry. The correlations between cIAP1&2 expression and clinicopathological characteristics, prognosis were analyzed. Cell viability and apoptosis were measured by MTT assays and Flow cytometry. Western blot and co-immunoprecipitation assay were performed to measure the protein expression and interaction in NF-kB pathway. siRNA-mediated gene silencing and caspases activity assays were applied to demonstrate the role and mechanisms of cIAP1&2 and RIP1 in lung cancer cell apoptosis. Mouse xenograft NSCLC models were used in vivo to determine the therapeutic efficacy of LCL161 alone or in combination with paclitaxel.
RESULTS: The expression of cIAP1 and cIAP2 in Non-small cell lung cancer (NSCLC) tumors was significantly higher than that in adjacent normal tissues. cIAP1 was highly expressed in patients with late TNM stage NSCLC and a poor prognosis. Positivity for both cIAP1 and cIAP2 was an independent prognostic factor that indicated a poorer prognosis in NSCLC patients. LCL161, an IAP inhibitor, cooperated with paclitaxel to reduce cell viability and induce apoptosis in NSCLC cells. Molecular studies revealed that paclitaxel increased TNFα expression, thereby leading to the recruitment of various factors and the formation of the TRADD-TRAF2-RIP1-cIAP complex. LCL161 degraded cIAP1&2 and released RIP1 from the complex. Subsequently, RIP1 was stabilized and bound to caspase-8 and FADD, thereby forming the caspase-8/RIP1/FADD complex, which activated caspase-8, caspase-3 and ultimately lead to apoptosis. In nude mouse xenograft experiments, the combination of LCL161 and paclitaxel degraded cIAP1,2, activated caspase-3 and inhibited tumor growth with few toxic effects.
CONCLUSION: Thus, LCL161 could be a useful agent for the treatment of NSCLC in combination with paclitaxel.

BACKGROUND: Hsp90 proteins are important therapeutic targets for many anti-cancer drugs in clinical trials. Geldanamycin (GA) was identified as the first natural inhibitor of Hsp90, increasing evidence suggests that GA was not a good choice for clinical trials. In this study, we investigated two new non-benzoquinone geldanamycin analogs of Hsp90 inhibitors, DHQ3 and 17-demethoxy-reblastatin (17-DR), to explore the molecular mechanisms of their anti-cancer activity in vivo and vitro.
METHODS: MTT and colony formation assays were used to measure cell viability. Flow cytometry, DAPI staining, ATP assay, electron microscopy, western blots, siRNAs transfection and immunofluorescence were used to determine the molecular mechanism of DHQ3- or 17-DR-induced different forms of death in human breast cancer MDA-MB-231 cells. Malachite green reagent was used to measure ATPase activity of the analogs.
RESULTS: DHQ3 and 17-DR presented efficiently inhibitory effect in MDA-MB-231 cell lines, and DHQ3 induced necroptosis by activation of the RIP1-RIP3-MLKL necroptosis cascade. And DHQ3-induced cell death was inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), but not by a caspase inhibitor z-VAD-fmk. On the other hand, 17-DR induced apoptosis in MDA-MB-231 cells, indicating a caspase-dependent killing mechanism. We further demonstrated that down-regulation of RIP1 and RIP3 by siRNA protected against DHQ3 but not 17-DR induced cell death. These results were confirmed by electron microscopy. DHQ3 and 17-DR induced the degradation of Hsp90 client proteins, and they showed strong antitumor effects in MDA-MB-231 cell-xenografted nude mice.
CONCLUSIONS: These findings supported that DHQ3 and 17-DR induce different forms of death in some cancer cell line via activation of different pathways. All of the results provided evidence for its anti-tumorigentic action with low hepatotoxicity in vivo, making them promising anti-breast cancer agents.

RLIP76 is a stress-responsive multifunctional protein and is usually overexpressed in malignant carcinomas. It plays a significant role in multiple cellular biological behaviors, including cell growth, motility, division and apoptosis, in many types of malignant cells. However, functions of RLIP76 in gastric cancer (GC) remain unknown. In the present study, RLIP76 was overexpressed in GC tissues by immunohistochemistry. RLIP76-targeted shRNA-containing lentivirus (KD) and the scrambled shRNA (NC) were used to explore the knockout of RLIP76 on cellular functions of human GC SGC-7901 and MGC-803 cells. Quantitative RT-PCR and western blotting were used to confirm that the RLIP76 was suppressed both on mRNA and protein levels after transfection. The mRNA level in SGC-7901 and MGC-803 after transfection of RLIP76-targeted shRNA was 0.245722±0.021077 (p<0.05) and 0.225389±0.00974 (p<0.05), respectively. Our results showed that the konckdown of RLIP76 downregulated cell growth after 24 h in Cell Counting Kit-8 (CCK-8) assay, reduced migration from 486.7±128.8 to 219.7±43.6 in SGC-7901 (p<0.05) and from 630±95 to 333.7±46.5 in MGC-803 (p<0.05), decreased invasion from 306±33.5 to 97.7±24.3 in SGC-7901 (p<0.05) and from 350±50.9 to 163.3±87.5 in MGC-803 (p<0.05). Length of vascular endothelial growth factor (VEGF)-induced tube formation also decreased from 202.8±83.3 to 44.5±3.69 in SGC-7901 and from 193±3.5 to 71.8±8.83 in MGC-803 (p<0.05). Phosphorylation level of Akt declined from 138.45±13.8 to 69.9±29.7% in SGC-7901, and from 115.5±26.6 to 49.07±27% in MGC-803 (p<0.05) and phosphorylation level of mTOR also significantly decreased (p<0.05). While apoptosis of GC cells increased which we verified with apoptosis proteins and staining analysis. Our data showed that RLIP76 plays a significant oncogenic role in GC and it maybe a potential target in GC treatment.

Integrins have been suggested as possible targets in anticancer therapy. Here we show that knockdown of integrins αVβ3, αVβ5, α3β1 and α4β1 and pharmacological inhibition using a cyclo-RGD integrin αVβ3/αVβ5 antagonist sensitized multiple high-grade glioma cell lines to temozolomide (TMZ)-induced cytotoxicity. The greatest effect was observed in LN229 cells upon integrin β3 silencing, which led to inhibition of the FAK/Src/Akt/NFκB signaling pathway and increased formation of γH2AX foci. The integrin β3 knockdown led to the proteasomal degradation of Rad51, reduction of Rad51 foci and reduced repair of TMZ-induced DNA double-strand breaks by impairing homologous recombination efficiency. The down-regulation of β3 in Rad51 knockdown (LN229-Rad51kd) cells neither further sensitized them to TMZ nor increased the number of γH2AX foci, confirming causality between β3 silencing and Rad51 reduction. RIP1 was found cleaved and IκBα significantly less degraded in β3-silenced/TMZ-exposed cells, indicating inactivation of NFκB signaling. The anti-apoptotic proteins Bcl-xL, survivin and XIAP were proteasomally degraded and caspase-3/-2 cleaved. Increased H2AX phosphorylation, caspase-3 cleavage, reduced Rad51 and RIP1 expression, as well as sustained IκBα expression were also observed in mouse glioma xenografts treated with the cyclo-RGD inhibitor and TMZ, confirming the molecular mechanism in vivo. Our data indicates that β3 silencing in glioma cells represents a promising strategy to sensitize high-grade gliomas to TMZ therapy.

Overexpression of microRNA-182 (miR-182) is found in multiple cancers, but the association of miR-182 expression with the sensitivity of triple-negative breast cancer (TNBC) cells to tumor necrosis factor-alpha (TNF-α) remains unknown. In this study, up-regulation of miR-182 was validated in TNBC patients and cell lines. Knockdown of miR-182 was observed to hinder the proliferation of BT-549 cells. More importantly, knockdown of miR-182 significantly promoted the apoptosis induced by TNF-α treatment in BT-549. JC-1 staining and western blot assays revealed that the K63-linked ubiquitin chains on receptor-interacting protein 1 (RIP1) were removed and the outer mitochondrial membrane potential (MMP) and permeability was altered upon combination of TNF-α with anti-miR-182. We then demonstrated that knockdown of miR-182 up-regulated the expression of cylindromatosis (CYLD) deubiquitinase, which promoted the formation of death-inducing signaling complex (DISC) and subsequent caspase-8 activation in TNF-α-treated BT-549 cells. Collectively, the results of the present study improve our understanding of the role of miR-182 in TNBC, knockdown of which facilitates the degradation of ubiquitin chains on RIP1, leading to the caspase-8 activation and apoptosis in TNF-α-treated TNBC cells. This may be valuable for the development of cancer therapy.

Our previous study showed that RalA-binding protein 1 (RLIP76) is overexpressed in gliomas and is associated with higher tumour grade and decreased patient survival. Furthermore, RLIP76 downregulation increases chemosensitivity of glioma cells to temozolomide by inducing apoptosis. However, other mechanisms underlying RLIP76-associated chemoresistance are unknown. In this study, we investigated the effect of RLIP76 depletion on autophagy. RLIP76 was knocked down in U251 glioma cells using shRNA and autophagy-related proteins, and PI3K/Akt signalling components were evaluated. RLIP76 depletion significantly increased cell autophagy as demonstrated by a significant increase in LC3 II, autophagy protein 5 (ATG-5), and Beclin1, and a decrease in p62 expression levels. Furthermore, RLIP76 knockdown increased autophagic flux in U251 cells as autolysosome numbers increased relative to autophagosome numbers. Autophagy induced by RLIP76 knockdown resulted in increased apoptosis that was independent of temozolomide treatment. Moreover, RLIP76 knockdown decreased PI3K and Akt activation. RLIP76 depletion also resulted in decreased levels of the anti-apoptotic protein Bcl2. LY294002, a PI3K/Akt pathway inhibitor, led to increased autophagy and apoptosis in U251 RLIP76-depleted cells. Therefore, RLIP76 knockdown increased autophagic flux and apoptosis in U251 glioma cells, possibly through inhibition of the PI3K/Akt pathway. Thus, this study provides a novel mechanism for the role of RLIP76 in glioma pathogenesis and chemoresistance.