Upregulation Of Survivin Research Articles

2-Methoxyestradiol, an Endogenous 17β-Estradiol Metabolite, Induces Antimitogenic and Apoptotic Actions in Oligodendroglial Precursor Cells and Triggers Endoreduplication via the p53 Pathway.

The abnormal growth of oligodendrocyte precursor cells (OPCs) significantly contributes to the progression of glioblastoma tumors. Hence, molecules that block OPC growth may be of therapeutic importance in treating gliomas. 2-Methoxyestradiol (2ME), an endogenous tubulin-interacting metabolite of estradiol, is effective against multiple proliferative disorders. Based on its anti-carcinogenic and anti-angiogenic actions, it is undergoing phase II clinical trials. We hypothesize that 2ME may prevent glioma growth by targeting OPC growth. Here, we tested this hypothesis by assessing the impact of 2ME on the growth of an OPC line, "Oli-neu", and dissected the underlying mechanism(s). Treatment with 2ME inhibited OPC growth in a concentration-dependent manner, accompanied by significant upregulation in the expression of p21 and p27, which are negative cell-cycle regulators. Moreover, treatment with 2ME altered OPC morphology from multi-arm processes to rounded cells. At concentrations of 1uM and greater, 2ME induced apoptosis, with increased expressions of caspase 3, PARP, and caspase-7 fragments, externalized phosphatidylserine staining/APOPercentage, and increased mitochondrial activity. Flow cytometry and microscopic analysis demonstrated that 2ME triggers endoreduplication in a concentration-dependent fashion. Importantly, 2ME induced cyclin E, JNK1/2, and p53 expression, as well as OPC fusion, which are key mechanisms driving endoreduplication and whole-genome duplication. Importantly, the inhibition of p53 with pifithrin-α rescued 2ME-induced endoreduplication. The pro-apoptotic and endoreduplication actions of 2ME were accompanied by the upregulation of survivin, cyclin A, Cyclin B, Cyclin D2, and ppRB. Similar growth inhibitory, apoptotic, and endoreduplication effects of 2ME were observed in CG4 cells. Taken together, our findings provide evidence that 2ME not only inhibits OPC growth and triggers apoptosis, but also activates OPCs into survival (fight or flight) mode, leading to endoreduplication. This inherent survival characteristic of OPCs may, in part, be responsible for drug resistance in gliomas, as observed for many tubulin-interacting drugs. Importantly, the fate of OPCs after 2ME treatment may depend on the cell-cycle status of individual cells. Combining tubulin-interfering molecules with drugs such as pifithrin-α that inhibit endoreduplication may help inhibit OPC/glioma growth and limit drug resistance.

Ethanolic extract of Euphorbia Gypsicola induces differentiation and apoptosis in human myeloid leukemia K562 cells

IntroductionThe Euphorbiaceae family, widely distributed and long utilized in traditional medicine, has been recognized for its potential in developing anti-cancer drugs. The current study investigates the capacity of Euphorbia gypsicola extract to induce apoptosis and differentiation in the human myeloid leukemia K562 cell line. MethodsCell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic activity was visually evaluated through acridine orange/ethidium bromide (AO/EB) dye staining and annexin V/PI double labeling procedures. Induction of differentiation was examined using nitro blue tetrazolium (NBT) reduction and Wright-Giemsa staining assays. The expression of apoptosis-related proteins including Bax, Bcl2 and Survivin was evaluated by western blotting method. ResultsThe E. gypsicola extract exhibited potent medication properties with IC50 values of 207.59, 48.58, and 7.08 ng/mL at 24, 48, and 72 h, respectively. Fluorescence microscopy, DNA fragmentation, annexin V/PI double staining, and sub-G1 cell cycle arrest confirmed apoptosis occurrence in K562 cells treated with the E. gypsicola extract. Our findings demonstrated that differentiated cells reduced NBT yellow solution after endocytosis, forming dark crystals. Wright Giemsa staining observations indicated a decrease in nuclear volume-to-cytoplasm ratio in K562 cells treated with the crude extract, signifying Induction of differentiation. Furthermore, western blot analysis revealed down-regulation of Survivin protein expression and up-regulation of Bax protein level following treatment with the E. gypsicola extract. However, no significant decrease in Bcl2 protein expression level was observed in K562 cells. ConclusionThese findings indicate the growth inhibitory effects of the E. gypsicola extract on leukemia K562 cells, which supports the further study of this plant to discover novel therapeutic compounds for the treatment of leukemia.

Survivin inhibition with YM155 ameliorates experimental pulmonary arterial hypertension.

Background: Imbalance between cell proliferation and apoptosis underlies the development of pulmonary arterial hypertension (PAH). Current vasodilator treatment of PAH does not target the uncontrolled proliferative process in pulmonary arteries. Proteins involved in the apoptosis pathway may play a role in PAH and their inhibition might represent a potential therapeutic target. Survivin is a member of the apoptosis inhibitor protein family involved in cell proliferation. Objectives: This study aimed to explore the potential role of survivin in the pathogenesis of PAH and the effects of its inhibition. Methods: In SU5416/hypoxia-induced PAH mice we assessed the expression of survivin by immunohistochemistry, western-blot analysis, and RT-PCR; the expression of proliferation-related genes (Bcl2 and Mki67); and the effects of the survivin inhibitor YM155. In explanted lungs from patients with PAH we assessed the expression of survivin, BCL2 and MKI67. Results: SU5416/hypoxia mice showed increased expression of survivin in pulmonary arteries and lung tissue extract, and upregulation of survivin, Bcl2 and Mki67 genes. Treatment with YM155 reduced right ventricle (RV) systolic pressure, RV thickness, pulmonary vascular remodeling, and the expression of survivin, Bcl2, and Mki67 to values similar to those in control animals. Lungs of patients with PAH also showed increased expression of survivin in pulmonary arteries and lung extract, and also that of BCL2 and MKI67 genes, compared with control lungs. Conclusion: We conclude that survivin might be involved in the pathogenesis of PAH and that its inhibition with YM155 might represent a novel therapeutic approach that warrants further evaluation.

Design, Synthesis, Docking Studies and Biological Evaluation of Novel Benzochromenopyrimidines via Silica Sulfuric Acid Catalyzed Reaction on Apoptosis in Human Cancer Cells

Via one-pot reaction of 2-naphthol, thiobarbituric acid and aldehyde in the presence of silica sulfuric acid (SSA) as a catalyst to synthesis benzochromenopyrimidines derivatives. Then we studied the reaction of benzochromenopyrimidines derivatives with bromoacetic acid to obtain benzo[5,6]chromeno[2,3-d]thiazolo[3,2-a]pyrimidine-11,13(10H,14H)-dione derivatives. Meanwhile, dichloroethane was reacted with 12-(4-fluorophenyl)-9-thioxo-9,10-dihydro-8H-benzo[5,6]chromeno[2,3-d]pyrimidin-11(12H)-one to form 14-(4-fluorophenyl)-10,11-dihydrobenzo[5,6]chromeno[2,3-d]thiazolo[3,2-a]pyrimidin-13(14H)-one. All new products structure was elucidated based on analytical and spectroscopic analyses. Anti-cancer activity of newly synthesized structures was investigated. Cancer cells (MDA-MB-231 and HepG2) were used to evaluate the anti-cancer activity of tested compounds using neutral red uptake assay. Treatment of MDA-MB-231 and HepG2 cells with tested compound 6h revealed more inhibitory influence after 48 h. The expression levels of BCL2, BAX, Caspase3, Survivin and P53 genes were investigated using QRT-PCR. This study exhibited that compound 6h revealed significant pro-apoptotic effect via down regulation of BCL2 and Survivin genes and up-regulation of BAX, Caspase3 and P53 genes when treated with MDA-MB-231 and HepG2 cells as compared to control values. The biological studies of these compounds were proved through molecular docking studies with human cyclin-dependent kinase 2 (PDB code: 2A4L) and compound 6h showed low binding energy and shortage bond length with different amino acids. Noteworthy, the tested compound 6h exhibited the most pronounced effect in this respect.

Survivin knockdown alleviates pathological hydrostatic pressure-induced bladder smooth muscle cell dysfunction and BOO-induced bladder remodeling via autophagy.

Aim: Bladder outlet obstruction (BOO) leads to bladder wall remodeling accompanying the progression from inflammation to fibrosis where pathological hydrostatic pressure (HP)-induced alteration of bladder smooth muscle cells (BSMCs) hypertrophic and excessive extracellular matrix (ECM) deposition play a pivotal role. Recently, we have predicted survivin (BIRC5) as a potential hub gene that might be critical during bladder fibrosis by bioinformatics analyses from rat BOO bladder, but its function during BOO progression remains unknown. Here, we investigated the role of survivin protein on bladder dysfunction of BOO both in vitro and in vivo. Methods: Sprague-Dawley female rats were divided into three groups: control group, BOO group, and BOO followed by the treatment with YM155 group. Bladder morphology and function were evaluated by Masson staining and urodynamic testing. To elucidate the underlying mechanism, hBSMCs were subjected to pathological HP of 200cm H2O and co-cultured with the presence or absence of survivin siRNA and/or autophagy inhibitor 3-MA. Autophagy was evaluated by the detection of Beclin1 and LC3B-II expression, proliferation was conducted by the EdU analysis and PCNA expression, and fibrosis was assessed by the examination of Col 1 and Fn expression. Results: BOO led to a gradual alteration of hypertrophy and fibrosis of the bladder, and subsequently induced bladder dysfunction accompanied by increased survivin expression, while these histological and function changes were attenuated by the treatment with YM155. HP significantly increased survivin expression, upregulated Col1 and Fn expression, enhanced proliferation, and downregulated autophagy markers, but these changes were partially abolished by survivin siRNA treatment, which was consistent with the results of the BOO rat experiment. In addition, the anti-fibrotic and anti-proliferative effects of the survivin siRNA treatment on hBSMCs were diminished after the inhibition of autophagy by the treatment with 3-MA. Conclusion: In summary, the upregulation of survivin increased cell proliferation and fibrotic protein expression of hBSMC and drove the onset of bladder remodeling through autophagy during BOO. Targeting survivin in pathological hBSMCs could be a promising way to anti-fibrotic therapeutic approach in bladder remodeling secondary to BOO.

Tumor-Suppressor Role of the α1-Na/K-ATPase Signalosome in NASH Related Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide, with an estimate of 0.84 million cases every year. In Western countries, because of the obesity epidemic, non-alcoholic steatohepatitis (NASH) has become the major cause of HCC. Intriguingly, the molecular mechanisms underlying tumorigenesis of HCC from NASH are largely unknown. We hypothesized that the growing uncoupled metabolism during NASH progression to HCC, manifested by lower cell redox status and an apoptotic ‘switch’ activity, follows a dysregulation of α1-Na/K-ATPase (NKA)/Src signalosome. Our results suggested that in NASH-related malignancy, α1-NKA signaling causes upregulation of the anti-apoptotic protein survivin and downregulation of the pro-apoptotic protein Smac/DIABLO via the activation of the PI3K → Akt pro-survival pathway with concomitant inhibition of the FoxO3 circuit, favoring cell division and primary liver carcinogenesis. Signalosome normalization using an inhibitory peptide resets apoptotic activity in malignant cells, with a significant decrease in tumor burden in vivo. Therefore, α1-NKA signalosome exercises in HCC the characteristic of a tumor suppressor, suggesting α1-NKA as a putative target for clinical therapy.

Tumor‐Suppressor Role of the Caveolar α1‐Na/K‐ATPase Signalosome in NASH related Hepatocellular Carcinoma

BackgroundHepatocellular Carcinoma (HCC) is the second and fastest‐growing cause of cancer‐ related mortality worldwide. In the Western countries, due to the epidemic of obesity, non‐alcoholic steatohepatitis (NASH) has become the major cause of HCC. Intriguingly, the molecular mechanisms that underlie the progression of NASH to HCC is still unclear.HypothesisWe hypothesized that, the growing uncoupled metabolism during NASH progression to ESLD and HCC manifested by lower cell oxi‐redox status and an apoptotic ‘switch’ activity, follows a dysregulation in the wild type Caveolar α1‐Na/K‐ATPase (NKA)/Src signalosome, promoting an amplification of the pSrc → PI3K→ Akt pathway, which mediates survivin overexpression driving local carcinogenesis.MethodsExpression of Cav‐1/Smac‐Diablo/Survivin proteins was performed by confocal‐microscopy on immuno‐stained HCC cell lines (Hep3B and SNU475), and livers in both NASH and NASH‐HCC rodent models, and in humans. Apoptotic activity was assayed via TUNEL. Tumor burden in rodents’ livers were evaluated using ImageJ software on H&E‐stained liver tissues and fibrosis via trichrome staining. Furthermore, signaling pathway studies were explored in‐vitro guided by RNA sequencing. Selective blockage of Src‐p at its kinase domain was performed by administration of a synthetic peptide (pNaKtide). Significant differences among groups were established at p<0.05 using ANOVA/Turkey’s post hoc test.ResultsBlockage of Src‐p at the α1‐NKA promoted apoptosis of Human HCC cell lines and tumors of disease murine models in a dose dependent manner (p< 0.01). pNaKtide at IC50 drove the downregulation and upregulation of Survivin and SMAC expressions (p<0.05), respectively in HCC cell lines. In‐vivo, liver tumor burden and fibrosis were significantly lower in animals treated with pNaKtide vs non‐treated animals (p<0.01). Furthermore, Cav‐1 and Survivin expressions were significantly higher, while SMAC protein expression was significantly lower in livers from rodents with NASH/HCC vs animals treated with pNaKtide (p<0.01). Similar pattern of proteins expressions was noted in tumors from patients with NASH±HCC vs liver tissue from healthy subjects or subjects with liver metastases (p<0.05). In‐vitro, Src‐p at the α1‐NKA activates PI3K/Akt pathway with concomitant inhibition of the FoxO cascade, favoring cell division and primary liver carcinogenesis.ConclusionsCaveolar Src‐phosphorylation at the α1‐Na/K‐ATPase regulated Survivin/SMAC expressions, leading to cellular “switch” from apoptosis to cell division via the activation of the PI3K/Akt pro‐survival pathway with concomitant inhibition of the FoxO cascade. Therefore, point target blockage may be explored as a treatment for tumor regression in HCC.

FX1, a BCL6 inhibitor, reactivates BCL6 target genes and suppresses HTLV-1-infected T cells.

Human T cell leukemia virus type 1 (HTLV-1) is responsible for adult T cell leukemia (ATL); however, molecular and cellular mechanisms underlying HTLV-1-induced leukemogenesis are unclear. BCL6 oncogene is involved in cancer progression and a preferred target of anti-cancer treatments. Here, we aimed to evaluate BCL6 expression and the effects of BCL6 inhibitor (FX1) on HTLV-1-infected T cell lines. BCL6 expression was higher in HTLV-1-infected T cell lines than that in uninfected T cell lines. BCL6 was localized mostly in the nucleus. The virus oncoprotein Tax induced BCL6 mRNA expression in T cells, whereas BCL6 knockdown reduced HTLV-1-infected T cell proliferation; thus, confirmed the association of BCL6 with cancer progression. Further, FX1 efficiently inhibited the cell growth and survival of HTLV-1-infected T cell lines in a dose- and time-dependent manner. The decreased levels of cell cycle regulatory proteins (phosphorylated retinoblastoma protein, cyclin-dependent kinase 4, cyclin D2 and c-Myc) and the increased levels of BCL6 target proteins (p21, p27 and p53) showed that FX1 arrested cell cycle at the G1 phase. Apoptosis was induced concomitantly with Bak upregulation and downregulation of survivin, Bcl-xL and Mcl-1, as well as with the activation of caspase-3, -8, -9 and poly(ADP-ribose) polymerase. FX1 also inhibited NF-κB and Akt signaling pathways. These events were because of the induction of the activity of cell cycle checkpoint proteins and relief of direct repression of the targets of cell cycle checkpoint proteins. Thus, BCL6 might be considered a novel target for ATL treatment.

Human Bone Mesenchymal Stem Cell-Derived Exosomes Inhibit IL-1β-Induced Inflammation in Osteoarthritis Chondrocytes.

ObjectiveHuman bone marrow mesenchymal stem cell (hBMSC)-derived exosomes exhibit protective effects against inflammatory diseases. This study aimed to explore the effects of hBMSC-derived exosomes on osteoarthritis (OA) in vitro and its related mechanisms.Materials and Methods In this experimental study, we characterised exosomes derived from hBMSCs by transmission electron microscopy, nanoparticle tracking and Western blot analysis. Cellular uptake of exosomes was observed by fluorescent microscopy. Cell viability of chondrocytes exposed to interleukin-1 beta (IL-1β) was determined by the Cell Counting Kit-8 (CCK-8). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine expression levels of genes related to apoptosis, inflammation, cartilage collagen metabolism and mitogen-activated protein kinases. Results Fluorescence microscopy revealed that hBMSC-derived exosomes could be taken up by chondrocytes. hBMSC-derived exosomes could significantly enhance cell viability of chondrocytes in response to IL-1β treatment. RT-qPCR showed significant up-regulation of Survivin, Versican, IL-1β, IL-6, NF-κB, MMP-13, MAPK p38, JNK, ERK, Aggrecan and SOX9 expression levels by IL-1β treatment, while their mRNA expression levels decreased after co- culture with exosomes. The anti-inflammatory gene TGF-β was markedly suppressed by IL-1β treatment; however, we observed its expression after co-culture with exosomes. Additionally, the pro-inflammatory genes IL-1β, IL-6, NF-κB, TNF-α and TNF-β displayed significantly elevated expression levels in the IL-1β group and reduced expression levels after co-culture with exosomes. ConclusionhBMSC-derived exosomes may play a protective role in chondrocytes through inhibiting cell apoptosis and the inflammatory response. These results will provide a novel therapeutic strategy for OA.

Downregulation Of The Wnt Inhibitor CXXC5 Predicts a Better Prognosis In Acute Myeloid Leukemia

▪Different CXXC zinc finger proteins like MLL, DNMT1 and TET1 have been identified to play a role in leukemia development. The CXXC family member CXXC5 is located on 5q31, a region recurrently deleted in acute myeloid leukemia (AML), suggesting that inactivation of CXXC5 might be involved in leukemogenesis. CXXC5 has previously been shown to exert tumor suppressor functions in solid tumor cells and to negatively regulate Wnt signaling, a known pro-survival pathway in leukemia. Here, we investigated prognostic and biological implications of CXXC5expression in AML.CXXC5 mRNA expression was determined by real time quantitative PCR in pretreatment bone marrow samples of 186 AML patients enrolled on the Medical Research Council (MRC) AML15 and UK National Cancer Research Institute (NCRI) AML17 trials. We found downregulation of CXXC5 in AML compared to healthy controls, particularly in cases with del(5q)/-5, MLL rearrangements and t(8;21). To investigate CXXC5-associated gene expression, microarray data from 351 normal karyotype AML derived from the Microarray Innovations in LEukemia study and 529 unselected AML patients treated in Dutch-Belgian-Swiss Hemato-Oncology Cooperative Group (HOVON/SAKK) trials were analyzed. CXXC5 was associated with expression of several genes implicated in leukemogenesis (WT1, GATA2, MLL, DNMT3B, RUNX1), indicating that alteration of CXXC5 expression is a downstream effect of different leukemic pathways. Data from the HOVON/SAKK cohort (median age 46 years (range 15-77) was further used to perform outcome analyses according to microarray-based CXXC5 expression. Patients with CXXC5 expression below the median level had a lower relapse rate (45% vs 59%; P=0.007) and a better overall survival (OS: 46% vs 28%; P<0.001) and event-free survival (EFS: 36% vs 21%; P<0.001) at 5 years. This was of independent significance in multivariate analyses after adjustment for age, white blood cell count, cytogenetic risk group, FLT3-ITD status, biallelic CEBPA mutations, as well as mutations of NPM1, DNMT3A and ASXL1.DNA sequencing of CXXC5 exons 1-3 on 84 AML cases revealed no mutations, suggesting that CXXC5 is predominantly inactivated by gene regulatory mechanisms in AML. Pyrosequencing analysis showed that lower expression of CXXC5 in AML samples was correlated with hypermethylation within the promoter region. Treatment of NB4 and U937 cell lines with 5-Aza-2´-deoxycytidine led to demethylation of this region and induction of CXXC5 at the mRNA and protein level.Functional analyses confirmed Wnt inhibitor function of CXXC5 in leukemic cells. K562 cells overexpressing pEGFP-C2-CXXC5 showed impaired upregulation of active β-catenin protein, decreased upregulation of Wnt target genes Axin2 and Survivin, as well as decreased Wnt reporter activity after Wnt3a stimulation. In addition, we found evidence for involvement of CXXC5 in the DNA damage-induced p53 response in leukemia. Knockdown of CXXC5in OCI-AML3 cells harboring wildtype p53 led to attenuated p53 upregulation and cell cycle arrest after γ-irradiation.In summary, we have identified CXXC5 as a novel independent prognostic factor in AML. Our data suggest a tumor suppressor function of CXXC5 in AML and association of the gene with different leukemic pathways. These functional and prognostic implications build the framework for further investigating the role of CXXC5 in leukemia. AML patients with downregulation of CXXC5 might be candidates for evaluation of Wnt inhibition and cases with epigenetically silenced CXXC5 expression may particularly benefit from demethylating agents. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.

Hypoxia induces inflammatory microenvironment by priming specific macrophage polarization and modifies LSC behaviour via VEGF-HIF1α signalling.

BackgroundLeukemia stem cells (LSCs) play pivotal roles in leukemogenesis, and are closely implicated in leukemia relapse and chemoresistance. LSCs are tightly modulated by hypoxic exposure and macrophage-conditioned microenvironment. Nevertheless, the impacts on the biology of LSCs imposed by the interaction of hypoxia and macrophage polarization remain elusive.MethodsIn the study, LSCs characterized by CD34+CD38− immunophenotype sorted from KG1α and primary AML cells were employed as in vitro and ex vivo cell models. mRNA and protein expressions of cytokine/chemokine of cells under normoxic and hypoxic conditions were determined by RT-PCR and western blot. Macrophage polarization, cell cycle and apoptosis were determined by flowcytometry. Cell viability was assayed by CCK-8.ResultsMacrophages preferentially presented with M2 polarization phenotype characterized by upregulated VEGF and CCL17 cytokine/chemokine profile, when stimulated by specific set of cytokines under hypoxic exposure, and induced an anti-inflammatory microenvironment. LSCs exhibited significantly increased cell viability, colony-forming capacity and chemoresistance when co-incubated in hypoxic conditioned medium (H-CM) primed by polarized M1 macrophages. VEGF expression was upregulated in LSCs which in turn activated survivin expression. VEGF-mediated upregulation of survivin could be abolished by inhibition of VEGF receptor, but not blocked by survivin-targeting siRNA. In addition, survivin upregulation exerted antiapoptotic effects and was associated with increased chemoresistance. Finally, VEGF mediated transcriptional induction of HIF-1α of LSCs coincubated in H-CM, and HIF-1α induction in turn enhanced chemoresistance and reduced cell apoptosis.ConclusionsTo our best knowledge, this is the first study that focus to explore molecule players and interacting signal pathways regulating LSC biology under hypoxic exposure. It reveals that hypoxia preferentially skew macrophage M2 polarization with specific cytokine profile and proinflammatory microenvironment, which impacts malignant behavior of LSCs. VEGF-HIF-1α interaction is closely implicated in sustaining LSCs survival under hypoxic exposure and might be of potential target of novel therapy.

Bone Marrow Mesenchymal Stromal Cells Can Render Multiple Myeloma Cells Resistant to Cytotoxic Machinery of CAR T Cells through Inhibition of Apoptosis.

The microenvironment of multiple myeloma (MM) can critically impair therapy outcome, including immunotherapies. In this context, we have earlier demonstrated that bone marrow mesenchymal stromal cells (BMMSC) protect MM cells against the lytic machinery of MM-reactive cytotoxic T cells (CTL) and daratumumab-redirected natural killer (NK) cells through the upregulation of antiapoptotic proteins Survivin and Mcl-1 in MM cells. Here, we investigated the significance of this mode of immune escape on T cells engineered to express chimeric antigen receptors (CAR T cells). We tested the cytolytic ability of a panel of 10 BCMA-, CD38-, and CD138-specific CAR T cells with different affinities against a model MM cell line and against patient-derived MM cells in the presence versus absence of BMMSCs. Although BMMSCs hardly protected MM cells from lysis by high-affinity, strongly lytic BCMA- and CD38-CAR T cells, they significantly protected against lower affinity, moderately lytic BCMA-, CD38-, and CD138-specific CAR T cells in a cell-cell contact-dependent manner. Overall, there was a remarkable inverse correlation between the protective ability of BMMSCs and the lytic activity of all CAR T cells, which was dependent on CAR affinity and type of costimulation. Furthermore, BMMSC-mediated resistance against CAR T cells was effectively modulated by FL118, an inhibitor of antiapoptotic proteins Survivin, Mcl-1, and XIAP. These results extend our findings on the negative impact of the microenvironment against immunotherapies and suggest that outcome of CAR T cell or conventional CTL therapies could benefit from inhibition of antiapoptotic proteins upregulated in MM cells through BMMSC interactions.

Chemotherapy-Induced Survivin Regulation in Acute Myeloid Leukemia Cells

Survivin is a 16.5 kDa protein highly expressed in centrosomes, where it controls proper sister chromatid separation. In addition to its function in mitosis, survivin is also involved in apoptosis. Overexpression of survivin in many cancer types makes it a suitable target for cancer therapy. Western blotting and confocal microscopy were used to characterize the effect of chemotherapy on acute myeloid leukemia (AML) cells. We found enhanced survivin expression in a panel of AML cell lines treated with cytarabine (Ara-C), which is part of a first-line induction regimen for AML therapy. Simultaneously, Ara-C caused growth arrest and depletion of the mitotic cell fraction. Subsequently, the effect of a second component of standard therapy protocol, idarubicin, and of a known survivin inhibitor, YM-155, on cell viability and survivin expression and localization in AML cells was investigated. Idarubicin reversed Ara-C-induced survivin upregulation in the majority of AML cell lines. YM-155 caused survivin deregulation together with a viability decrease in cells resistant to idarubicin treatment, suggesting that YM-155 might be efficient in a specific subset of AML patients. Expression levels of other apoptosis-related proteins, in particular X-linked inhibitor of apoptosis (XIAP), Mcl-1, and p53, and of the cell-cycle inhibitor p21 considerably changed in almost all cases, confirming the off-target effects of YM-155.

Targeting Neuregulin 1 (NRG1): A Novel Biomarker for Non-Small-Cell Lung Cancer.

The aim of this study was to identify the roles of neuregulin 1 (NRG1) during the tumor progression in non-small-cell lung cancer (NSCLC). NSCLC patients with lung squamous cell carcinoma and lung adenocarci-noma were enrolled in this study. The expression of NRG1, vascular endothelial growth factor (VEGF) and surviving in clinical specimens was examined using immunohistochemistry analysis. The cytokine production in plasma was evaluated by ELISA. The levels of NRG1-associated molecules were determined using western blotting. The proliferation and apoptosis of cells with NRG1 knockdown were accessed by CCK-8 assay and flow cytometry. Upregulation of NRG1 as well as tumor-associated angiogenesis markers VEGF and survivin was detected in tissue and serum samples of NSCLC patients compared with the control. Furthermore, positive correlation with NSCLC levels and VEGF/survivin was also found in NSCLC specimens. In addition, upregulation of NRG1, VEGF and survivin was associated with poor overall survival in NSCLC patients. Moreover, enhanced production of NRG1 was detected in serum samples from NSCLC patients compared with healthy donors, and ROC analysis revealed the importance of NRG1 levels on distinguishing NSCLC samples and the controls. These findings suggested the novel diagnostic value of NRG1 in NSCLC. Additionally, upregulated protein levels of NRG1 and its target genes were also found in tissues samples of NSCLC patients compared with normal controls. These data indicated that NRG1 was a promising marker NSCLC, and it could be involved in tumor progression by targeting its downstream target including ErbB-Akt axis. Furthermore, the growth of lung cancer cells was suppressed by the knockdown of NRG1. Our findings could provide guidance for more accurate diagnosis for NSCLC, and future therapeutic approaches might be developed by better understanding of NRG-1-modulated molecular mechanisms during the tumor development in NSCLC.

Codelivery of survivin inhibitor and chemotherapeutics by tumor-derived microparticles to reverse multidrug resistance in osteosarcoma.

Reportedly, the elevated expression of survivin has been observed in several tumor types, strictly involved in tumor development. In the present study, we detected elevated survivin expression in tumor tissues derived from patients with chemoresistant osteosarcoma when compared with those from chemosensitive patients. Importantly, knockdown of survivin in osteosarcoma cells significantly suppressed cell proliferation and chemoresistance both in vitro and in vivo. Simultaneously, chemotherapy mediates the upregulation of survivin in osteosarcoma cells through a survivin-based selective killing effect, resulting in the development of multidrug resistance. The utilization of tumor-derived microparticles to coencapsulate the survivin inhibitor YM155 and chemotherapeutic agents could effectively reverse multidrug resistance, leading to improved anticancer effects, as well as reduced systemic toxicity. In summary, the expression of survivin contributes to resistance toward osteosarcoma drugs, whereas employing survivin inhibiting combination therapy, based on a microparticle codelivery system, could efficiently reverse resistance and avoid potential systemic toxicity.

Long Noncoding RNA KCNQ1OT1 Confers Gliomas Resistance to Temozolomide and Enhances Cell Growth by Retrieving PIM1 From miR-761.

Many studies have found that the dysregulation of long noncoding RNA (lncRNA) contributed to cancer initiation, progression, and recurrence via multiple signaling pathways. However, the underlying mechanisms of lncRNA in temozolomide (TMZ)-resistant gliomas were not well understood, hindering the improvement of TMZ-based therapies. The present study demonstrated that the lncRNA KCNQ1OT1 increased in TMZ-resistant glioma cells compared to the TMZ-sensitive cells. The introduction of KCNQ1OT1 promoted cell viability, clonogenicity, and rhodamine 123 efflux while hampering TMZ-induced apoptosis. Moreover, KCNQ1OT1 directly sponged miR-761, which decreased in TMZ-resistant sublines. The overexpression of miR-761 attenuated cell viability and clonogenicity, while triggering apoptosis and rhodamine 123 accumulation post-TMZ exposure, leading to a response to TMZ. The interaction between miR-761 and 3'-untranslated region of PIM1 attenuated PIM1-mediated signaling cascades. Furthermore, the knockdown of KCNQ1OT1 augmented the TMZ-induced tumor regression in TMZ-resistant U251 mouse models. Briefly, the present study evaluated that KCNQ1OT1 conferred TMZ resistance by releasing PIM1 expression from miR-761, resulting in the upregulation of PIM-mediated MDR1, c-Myc, and Survivin. The present findings demonstrated that the interplay of KCNQ1OT1: miR-761: PIM1 regulated chemoresistance in gliomas and provided a promising therapeutic target for TMZ-resistant glioma patients.

Targeting 14-3-3ε activates apoptotic signaling to prevent cutaneous squamous cell carcinoma.

More than a million cases of cutaneous squamous cell carcinoma are diagnosed in the USA each year, and its incidence is increasing. Most of these malignancies arise from premalignant lesions, providing an opportunity for intervention before malignant progression. We previously documented how cytoplasmic mislocalization of CDC25A in premalignant and malignant skin cancers confers resistance to apoptotic cell death via a mechanism that depends on its interaction with 14-3-3ε. From these data, we hypothesized that 14-3-3ε overexpression drives skin tumor development and progression, such that targeting 14-3-3ε may be a useful strategy for skin cancer treatment. Like CDC25A, 14-3-3ε was overexpressed and mislocalized to the cytoplasm of both benign and malignant human skin cancer. Skin-targeted deletion of the 14-3-3ε gene reduced skin tumor development by 75% and blocked malignant progression. 14-3-3ε suppressed apoptosis through activation of Akt, leading to inhibition of BCL2 associated agonist of cell death and upregulation of Survivin. Using virtual tetrapeptide libraries, we developed a novel peptide that specifically blocked 14-3-3ε heterodimerization and thereby prevented its interaction with CDC25A. The peptide reduced prosurvival signaling, killed skin cancer cells and reduced skin tumor growth in xenograft. Normal skin keratinocytes were unaffected by inhibition or deletion of 14-3-3ε. Thus, targeting of 14-3-3ε dimerization is a promising strategy for the treatment of premalignant skin lesions.

LKB1 inactivation leads to centromere defects and genome instability via p53-dependent upregulation of survivin.

Inactivating mutations in the liver kinase B1 (LKB1) tumor suppressor gene underlie Peutz-Jeghers syndrome (PJS) and occur frequently in various human cancers. We previously showed that LKB1 regulates centrosome duplication via PLK1. Here, we report that LKB1 further helps to maintain genomic stability through negative regulation of survivin, a member of the chromosomal passenger complex (CPC) that mediates CPC targeting to the centromere. We found that loss of LKB1 led to accumulation of misaligned and lagging chromosomes at metaphase and anaphase and increased the appearance of multi- and micro-nucleated cells. Ectopic LKB1 expression reduced these features and improved mitotic fidelity in LKB1-deficient cells. Through pharmacological and genetic manipulations, we showed that LKB1-mediated repression of survivin is independent of AMPK, but requires p53. Consistent with the key influence of LKB1 on survivin expression, immunohistochemical analysis indicated that survivin is highly expressed in intestinal polyps from a PJS patient. Lastly, we reaffirm a potential therapeutic avenue to treat LKB1-mutated tumors by demonstrating the increased sensitivity to survivin inhibitors of LKB1-deficient cells.

The Role of Opioids and Their Receptors in Urological Malignancy: A Review.

We reviewed the literature surrounding the role of opioids and their receptors in urological malignancy. Recent studies have suggested clinically significant effects of agonism or antagonism of opioid receptors on cancer related outcomes and tumorigenesis. The focus of these efforts has centered on nonurological malignancies. However, a compelling body of evidence is growing in the fields of prostate, bladder and kidney cancer. A systematic review of English language articles published through 2020 was conducted with key phrases related to kidney, bladder or prostate cancer, and opioids or narcotics. A total of 837 unique records were identified, of which 49 were selected for full text review and 33 were included in the qualitative analysis. Eight records were identified via citation review and 1 study was recently presented at a national meeting. Retrospective reviews suggest poorer disease specific and recurrence-free survival with increased perioperative opioid administration in patients undergoing prostate or bladder cancer surgery. However, the data are controversial. Kappa opioid receptors are implicated in both proliferation and inhibition of prostate cancer cell growth across in vitro studies, with a proposed interaction with the androgen cascade. Similarly opioid growth factor receptor is highly expressed in prostate cancer cells and repressed by androgens. Prostate cancer tissue stains more intensely for the mu opioid receptor, and patients with higher expression have poorer oncologic outcomes. Opioid agonism in vitro induces urothelial cell carcinoma proliferation, migration and invasion, with possible additional influence from interactions with the bradykinin b2 receptor. Agonism of the mu, kappa and delta opioid receptors induces renal cell carcinoma tumorigenesis, possibly via upregulation of survivin. Meanwhile, opioid growth factor receptor agonism has the opposite effect in renal cell carcinoma. Evidence surrounding the role of opioids and their receptors in urological malignancy is provocative and should serve as an impetus for further investigation.

Dasatinib reverses drug resistance by downregulating MDR1 and Survivin in Burkitt lymphoma cells

BackgroundCurrent chemotherapies for Burkitt lymphoma (BL) have dramatically improved its clinical outcome. However, chemoresistance can lead to chemotherapy failure and very poor prognosis; thus, novel strategies are urgently required for patients with drug-resistant BL. To investigate the mechanisms underlying drug resistance in BL, we established drug-resistant BL cell lines: HS-Sultan/ADM (adriamycin-resistant), HS-Sultan/VCR (vincristine-resistant), HS-Sultan/DEX (dexamethasone-resistant), and HS-Sultan/L-PAM (melphalan-resistant).MethodsDrug transporter and survival factor expression were investigated the using western blotting and real time polymerase chain reaction. Cell survival was analyzed by trypan blue dye exclusion method.ResultsThe established cell lines acquired cross-resistance to adriamycin, vincristine, dexamethasone, and melphalan and exhibited 50% inhibitory concentration values 106-, 40-, 81-, and 45-fold higher than the parental cell lines, respectively. We found that protein and mRNA expression of MDR1 and Survivin were higher in drug-resistant BL cells than in the parent cells. Treatment with verapamil, an MDR1 inhibitor, or Survivin siRNA alongside each anti-cancer drug suppressed the proliferation of all drug-resistant BL cells. Src kinase activity was higher in all resistant cell lines than the parental cells; suppressing Src with dasatinib restored drug sensitivity by reducing MDR1 and Survivin expression.ConclusionsMDR1 and Survivin upregulation are responsible for resistance to conventional drugs and dasatinib can restore drug sensitivity by reducing MDR1 and Survivin expression in drug-resistant BL cells. Src inhibitors could therefore be a novel treatment strategy for patients with drug resistant BL.