Survivin Protein Research Articles

PLK1 and AURKB phosphorylate survivin differentially to affect proliferation in racially distinct triple-negative breast cancer

Protein diversity due to alternative mRNA splicing or post-translational modifications (PTMs) plays a vital role in various cellular functions. The mitotic kinases polo-like kinase 1 (PLK1) and Aurora B (AURKB) phosphorylate survivin, an inhibitor of apoptosis (IAP) family member, thereby regulating cell proliferation. PLK1, AURKB, and survivin are overexpressed in triple-negative breast cancer (TNBC), an aggressive breast cancer subtype. TNBC is associated with high proliferative capacity, high rates of distant metastasis, and treatment resistance. The proliferation-promoting protein survivin and its activating kinases, PLK1 and AURKB, are overexpressed in TNBC. In this study, we investigated the role of survivin phosphorylation in racial disparities in TNBC cell proliferation. Analysis of TCGA TNBC data revealed higher expression levels of PLK1 (P = 0.026) and AURKB (P = 0.045) in African Americans (AAs; n = 41) than in European Americans (EAs; n = 86). In contrast, no significant racial differences in survivin mRNA or protein levels were observed. AA TNBC cells exhibited higher p-survivin levels than EA TNBC cells. Survivin silencing using small interfering RNAs significantly attenuated cell proliferation and cell cycle progression in AA TNBC cells, but not in EA TNBC cells. In addition, PLK1 and AURKB inhibition with volasertib and barasertib significantly inhibited the growth of AA TNBC xenografts, but not of EA TNBC tumors. These data suggest that inhibition of PLK1 and AURKB suppresses cell proliferation and tumor growth, specifically in AA TNBC. These findings suggest that targeting survivin phosphorylation may be a viable therapeutic option for AA patients with TNBC.

PI3K δ inhibitor PI-3065 induces apoptosis in hepatocellular carcinoma cells by targeting survivin

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, and its clinical treatment remains challenging. The development of new treatment regimens is important for effective HCC treatment. Phosphoinositide 3-kinase (PI3K) is a lipid kinase that plays an important role in cell growth and metabolism and is overexpressed in nearly 50% of patients with HCC. Studies have shown that PI-3065, a small-molecule inhibitor of phosphatidylinositol 3-kinase delta, significantly inhibits solid breast cancer. However, its antitumor effects against HCC and the underlying mechanisms remain unclear. In the present study, we found that PI-3065 dose- and time-dependently reduced HCC cell viability and induced apoptosis while posing no obvious apoptotic toxicity in normal liver cells. Further mechanistic analysis showed that PI-3065 induced apoptosis mainly by inhibiting survivin protein expression, decreasing mitochondrial membrane potential, and promoting cytochrome C release. Simultaneously, PI-3065 markedly suppressed the colony formation, migration, and epithelial–mesenchymal transition abilities of HCC cells. Furthermore, transplantation of nude mice with HCC tumors showed that PI-3065 inhibits HCC tumor growth in vivo by targeting survivin. In summary, PI-3065 specifically inhibited survivin expression and exerted anti-HCC activity in vivo and in vitro, suggesting that it may serve as an effective antitumor drug for HCC treatment, which warrants further study.

The prognostic effect of immunohistochemical staining rates in patients with non-muscle-invasive bladder cancer.

Despite the follow-up protocols developed in non-muscle-invasive bladder cancer patients, progression and recurrence could not be prevented. We aimed to investigate whether proteins such as OCT-4, CD47, p53, Ki-67, and Survivin, which increase in bladder cancer cells, can be used as prognostic markers for patients with non-muscle-invasive bladder cancer. The study included a total of 89 patients with newly diagnosed non-muscle-invasive bladder cancer between January 2015 and December 2020. Levels of OCT-4, CD47, p53, Kİ-67, and Survivin proteins in cancer cells were determined with a semi-quantitative immunohistochemical experiment. Pathological data and survival rates were compared according to the staining rates. Data obtained in the study were analyzed statistically with SPSS 22.0 (SPSS, Chicago, IL, USA). The mean age of the patients was 64.25 ± 9.91 years, and the median follow-up period was 55 months. Recurrence rate was determined to be 36% (n = 32), and the rate of progression at 40.4% (n = 36). The staining rates were stronger for each marker in the progression group and advanced-stage tumors (p < 0.001). The findings of the multivariate analysis carried out as part of the study showed that older age and higher tumor stage were independent risk factors for recurrence-free survival (HR = 1.048 and 7.074, respectively; P = 0.02). Also, higher tumor stages, diameters, and grades were associated with reduced progression-free survival (HR = 0.105, 0.395, 0.225, respectively; P < 0.05). Although immunohistochemical staining rates are promising, it is more appropriate to use tumor characteristics when assessing survival rate in patients with non-muscle-invasive bladder cancer.

Dioscin inhibiting EGFR-mediated Survivin expression promotes apoptosis in oral squamous cell carcinoma cells.

Overexpression of survivin plays a crucial role in tumorigenesis and correlates with poor prognosis in human malignancies, including oral squamous cell carcinoma (OSCC). Thus, survivin has been proposed as an attractive target for new antitumor interventions. In the present study, we found that a natural compound, Dioscin, inhibited OSCC cells by reducing the survivin protein level and activating apoptotic signaling. Dioscin inhibits survivin expression by interrupting EGFR binding to the AT-rich sequences (ATRSs) at the survivin promoter, eventually promoting survivin-mediated cell apoptosis. The in vivo study showed that Dioscin suppressed the tumor development of SCC25 cells. Furthermore, the immunohistochemistry (IHC) results revealed that treated with Dioscin reduced the protein levels of EGFR and survivin in SCC25 xenograft tumors. Overall, our findings indicate that targeting the EGFR-survivin axis might be a promising OSCC treatment strategy.

Immunotoxicity and Anti-Inflammatory Characterizations of Prenylated Flavonoids-The Lipophilic 7-O-Terpenylated Wogonin.

Wogonin, one of the exceptional bioactive flavonoids found abundant in the roots of Huang-Qin (Scutellaria baicalensis Georgi), is a popular health-preserving Chinese medicine. The therapeutic applications can be expanded by improving its bioavailability. The 7-O-terpenylated wogonin consisting one to three prenyl units are chemically synthesized for increasing lipophilic nature for efficient uptake, and also an attempt in mimicry of naturally scarce terpenylated flavonoids found in limited plant families and bee propolis. Wogonin (W) and its lipophilic nature prenyl wogonin (W5), geranyl wogonin (W10), and farnesyl wogonin (W15) were comparatively studied with structure-relationship in immunotoxicity of cell livability on lymphoid, myeloid, and somatic origins cell lines. Anti-inflammatory functions characterized with nitric oxide inhibition and intracellular ROS level of LPS-activated murine macrophage RAW264.7 were assessed. Wogonin and its terpenylated derivatives have selectively influenced livability of lymphoid origin cells but not myeloid and somatic origin cells. The mitotic protein survivin gene expressions analysis further supported the selective suppressions on lymphoid origin YAC-1 cells by wogonin and geranyl wogonin, while oppositely boosted survivin expressions in LPS-activated macrophages. Moreover, wogonin exhibits dose-dependent inhibition on the nitric oxide (NO) production and iNOS gene expressions of LPS-activated RAW264.7 cells. Terpenylated wogonin exhibits profoundly superior control in intracellular ROS level and a sustained action with sound cell integrity than the wogonin. The enhanced cellular uptake with higher lipophilicity to membrane of 7-O-terpenylated wogonin may pose an important biological nature in facilitating better bioavailability and specific immunomodulatory actions of the category of terpenylated flavonoids. The 7-O-terpenylated wogonin having biological merit of fast membrane lipid bilayer integration, lower effective concentration, and better preserving immune cells functions and livability deserved further in-depth investigations and their broadly therapeutic applications.

Bim downregulation by activation of NF-κB p65, Akt, and ERK1/2 is associated with adriamycin and dexamethasone resistance in multiple myeloma cells.

Multiple myeloma (MM) frequently acquires multidrug resistance (MDR), which is due to poor prognosis. Our previous study indicated that high expression of Survivin and multidrug resistance protein 1 (MDR1) and decreased expression of Bim are associated with MDR in adriamycin- and dexamethasone-resistant cells. However, the fundamental mechanism of MDR in adriamycin- and dexamethasone-resistant MM cells is still unidentified. In this study, we examined the MDR mechanism in adriamycin- and dexamethasone-resistant cells. RPMI8226/ADM, ARH-77/ADM, RPMI8226/DEX, and ARH-77/DEX cells exhibited enhanced nuclear factor κB (NF-κB) p65, Akt, and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. Combination treatment with NF-κB p65, phosphoinositide 3-kinase (PI3K), and mitogen-activated protein kinase 1/2 (MEK1/2) inhibitors resensitized to adriamycin and dexamethasone via increased Bim expression. Although treatment with MDR1 or Survivin siRNA did not overcome adriamycin and dexamethasone resistance in RPMI8226/ADM and RPMI8226/DEX cells, administration of Bim siRNA induced adriamycin and dexamethasone resistance in RPMI8226 cells. Moreover, low expression of Bim was related to poor prognosis in MM patients. These results indicate that activation of NF-κB p65, Akt, and ERK1/2 is associated with adriamycin and dexamethasone resistance via decreasing Bim expression, and these signal inhibitor combinations overcome drug resistance in MM. These findings suggest that combination treatment with these inhibitors and adriamycin or dexamethasone may be a promising therapy for adriamycin- and dexamethasone-resistant MM.

Butein promotes ubiquitination-mediated survivin degradation inhibits tumor growth and overcomes chemoresistance

Overexpression of survivin is frequently observed in human malignancies and is associated with poor prognosis. The present study found that survivin is highly expressed in nasopharyngeal carcinoma (NPC) tumor tissues. Depleting survivin with shRNA inhibited cell viability, colony formation, and in vivo tumorigenesis of NPC cells. With a natural product screening, we identified Butein as a potential anti-tumor compound for NPC by reducing survivin protein level. Butein shortened the half-life of survivin and enhanced ubiquitination-mediated degradation. The mechanism study showed that Butein promoted the interaction between survivin and E3 ligase Fbxl7, and the knockdown of Fbxl7 compromised Butein-induced survivin ubiquitination. Butein suppressed the Akt-Wee1-CDK1 signaling and decreased survivin Thr34 phosphorylation, facilitating E3 ligase Fbxl7-mediated survivin ubiquitination and degradation. Moreover, Butein exhibited a strong in vivo anti-tumor activity, as the tumor volume of Butein-treated xenografts was reduced significantly. Butein alone or combined with cisplatin (CDDP) overcame chemoresistance in NPC xenograft tumors. Overall, our data indicate that Butein is a promising anti-tumor agent for NPC treatment.

Impact of Peptide Sequences on Their Structure and Function: Mimicking of Virus-Like Nanoparticles for Nucleic Acid Delivery.

We rationally designed a series of amphiphilic hepta-peptides enriched with a chemically conjugated guanidiniocarbonylpyrrole (GCP) unit at the lysine side chain. All peptides are composed of polar (GCP) and non-polar (cyclohexyl alanine) residues but differ in their sequence periodicity, resulting in different secondary as well as supramolecular structures. CD spectra revealed the assembly of β-sheet-, α-helical and random structures for peptides 1, 2 and 3, respectively. Consequently, this enabled the formation of distinct supramolecular assemblies such as fibres, nanorod-like or spherical aggregates. Notably, all three cationic peptides are equipped with the anion-binding GCP unit and thus possess a nucleic acid-binding centre. However, only the helical (2) and the unstructured (3) peptide were able to assemble into small virus-like DNA-polyplexes and effectively deliver DNA into cells. Notably, as both peptides (2 and 3) were also capable of siRNA-delivery, they could be utilized to downregulate expression of the caner-relevant protein Survivin.

Formulation of Lipid-Based Nanoparticles for Simultaneous Delivery of Lapatinib and Anti-Survivin siRNA for HER2+ Breast Cancer Treatment.

In this work, lipid-based nanoparticles (LBNP) were designed to combine tyrosine kinase inhibitor (TKI) Lapatinib (LAPA) with siRNA directed against apoptosis inhibitor protein Survivin (siSurvivin) in an injectable form. This nanosystem is based on lipid nanocapsules (LNCs) coated with a cationic polymeric shell composed of chitosan grafted through a transacylation reaction. The hydrophobic LAPA is solubilized in the inner oily core, while hydrophilic siRNA is associated electrostatically onto the nanocarrier's surface. The co-loaded LBNP showed a narrow size distribution (polydispersity index (PDI) &lt; 0.3), a size of 130 nm, and a slightly positive zeta potential (+21 mV). LAPA and siRNA were loaded in LBNP at a high rate of &gt;90% (10.6 mM) and 100% (4.6 µM), respectively. The siRNA-LAPA_LBNP was readily uptaken by the human epidermal growth factor receptor 2 overexpressed (HER2+) breast cancer cell line SK-BR-3. Moreover, the cytotoxicity studies confirmed that the blank chitosan decorated LBNP is not toxic to the cells with the tested concentrations, which correspond to LAPA concentrations from 1 to 10 µM, at different incubation times up to 96 h. Furthermore, siCtrl.-LAPA_LBNP had a more cytotoxic effect than Lapatinib salt, while siSurvivin-LAPA_LBNP had a significant synergistic cytotoxic effect compared to siCtrl.-LAPA_LBNP. All these findings suggested that the developed modified LBNP could potentiate anti-Survivin siRNA and LAPA anti-cancer activity.

Combination of Nanovectorized siRNA Directed against Survivin with Doxorubicin for Efficient Anti-Cancer Activity in HER2+ Breast Cancer Cells.

According to Globocan 2020, breast cancer is considered one of the most common cancers affecting women and is one of the leading causes of death in over 100 countries. The available classical treatment options do not always give satisfactory outcomes, and some patients develop resistance to these treatments. This study aims to investigate the combination of nanovectorized siRNA directed against anti-apoptotic protein Survivin (siSurvivin) by targeted stealth magnetic siRNA nanovectors (TS-MSN), designed in our lab, with Doxorubicin (DOX), as an option for HER2+ breast cancer treatment. The hypothesis is that the pretreatment of the HER2+ breast cancer cell line SK-BR-3 with siSurvivin will induce apoptosis in the cancer cells and enhance the therapeutic efficacy of DOX, allowing a dose reduction of DOX and hence a reduction of potential side effects. TS-MSN are based on superparamagnetic iron oxide nanoparticles (SPIONs) covalently coupled with a fluorophore sulfocyanine-5 and polyethylene glycol 5000 (PEG5000) and functionalized with single-chain variable fragments (scFv) of an antibody targeting the HER2 membrane receptor. These covalently functionalized SPIONs are then complexed via electrostatic interactions with therapeutic siRNA and the cationic polymers, chitosan, and poly-L-arginine. TS-MSNsiSurvivin had an average size of 144 ± 30 nm, a PDI of 0.3, and a slightly positive zeta potential value of 10.56 ± 05.70 mV. The agarose gel electrophoresis assay confirmed that the siRNA is well-complexed into TS-MSN without leakage, as no free siRNA was detected. Moreover, siRNA in TS-MSN was protected from RNAse A degradation for up to 6 h at 37 °C. Formulations of TS-MSN with siSurvivin demonstrated in vitro gene knockdown up to 89% in the HER2+ breast cancer cell line SK-BR-3. Furthermore, qRT-PCR confirmed a significant Survivin mRNA relative expression inhibition (about 50%) compared to control siRNA or untreated cells. A combination protocol was evaluated between TS-MSN and Doxorubicin (DOX) for the first time. Therefore, SK-BR-3 cells were pretreated with TS-MSN formulated with siSurvivin at 50 nM for 24 h alone, before a DOX treatment at a concentration of 0.5 µM (corresponding to the IC50) was added for 48 h. The MTT cytotoxicity tests, performed after 72 h of treatment, revealed that the combination had a significant synergistic cytotoxic effect on SK-BR-3 cells compared to monotherapies or untreated cells. We confirmed that pretreatment of cells with siSurvivin potentializes the cytotoxic effect of DOX as an alternative approach for treating HER2+ breast cancer. In conclusion, a combination of anti-Survivin siRNA and DOX would be a good alternative in HER2+ breast cancer therapy.

Safety and Efficacy of the Addition of the Protein Translation Inhibitor Omacetaxine to Induction with Cytarabine and Idarubicin in Newly Diagnosed AML

Background: Omacetaxine mepesuccinate (OM), a semi-synthetic form of homoharringtonine (HHT), mechanistically inhibits aminoacyl-tRNA binding to ribosomal acceptor "A” sites, preventing mRNA translation (Wetzler,Curr Pharm Des 2011, 17:59-64), and causing a rapid decline of short-lived proteins e.g., c-myc, cyclin D, the anti-apoptotic proteins cFLIP, survivin and Mcl-1, an important anti-apoptotic regulator in AML.While HHT added to cytarabine and an anthracycline improved CR and 3-yr EFS in de novo AML patients (pts; Jin et al, Lancet Oncology 2013, 7:599-608), meta-analysis of Chinese studies in elderly pts indicated a CR of 47.5%. Aims: Early analysis of AML-02, a phase Ib trial of escalating doses of OM (an FDA-approved drug) added to a standard 7 + 3 induction regimen was described (NCT02440568; Christian et al., Blood, 2018, 132:5218). Here we report long-term pt outcomes and studies of their correlation with in vitro OM effects on protein translation and apoptosis sensitivity. Methods: From June 2015 to June 2018, 22 pts aged 18-70 yrs with newly diagnosed AML entered AML-02, receiving escalating doses of OM administered SQ q12h d1-7 with cytarabine (100mg/m2 CIV) d1-7 and Idarubicin 12mg/m2 IV d1-3. Four OM dose levels were tested starting with cohort #1 at 0.625mg/m2 sc q12h (also 1.25, 2.0, and 3.0mg/m2 q12h cohorts). The primary endpoint, the optimally safe and active dose (OD) was determined using a Bayesian adaptive design, EFFTOX, which considers trade-offs between efficacy and toxicity to establish the OD (Shah et al, Leukemia, 2015; 29:1945). Secondary endpoints included OS, RFS and EFS. Residual disease at d14 was deemed a study failure and pts were taken off study. Hematological toxicity (HT) was defined as incomplete hematologic recovery at d49 with the BM free of leukemia. Post-induction therapy consisted of standard high dose cytarabine consolidation chemotherapy or allogeneic HSCT, based on cytogenetic risk (ELN 2010). Potential correlations of the effects of OM on protein translation rates, measured using the OP-Puro technique, and apoptosis sensitivity, measured using BH3 profiling techniques, on pretreatment AML samples and subsequent patient outcomes were also examined. Follow-up is complete to August 1st, 2022. Results: Median age was 58 yrs, 12 pts had adverse cytogenetics, 6 intermediate and 3 favorable risk (1 unknown due to fibrosis); overall 73% (16) had adverse cytogenetics or secondary AML. Earlier results (Christian et al) indicated no HTs in 21 evaluable pts, and OD was established as 2.0mg/m2. CR was 45.5%, median OS 22.7 months (OS at 4 yrs 45%), and EFS 80 days. The median OS is now 24.2 months (95% CI 18.3-39.7), median EFS is 80 days (95% CI 280-964), and OS at 6 years remains at 45%. There were no new TEAEs. Notably, of 10 pts achieving CR, 1 pt relapsed at D60, 1 was lost to follow-up and 1 died of opiate overdose (in CR). Five of the remaining 7 underwent HSCT and all 7 remain in remission. In pts achieving CR, median RFS is 62.4 months (95% CI 42.6-82.2; Fig.1), suggesting potential LSC impact. Common adverse effects included neutropenia (82%), nausea (64%), vomiting (59%), diarrhea (45%), hyperglycemia (41%), and a transient desquamating skin reaction (27%) that is dose-related and may be due to OM-mediated downregulation of cFLIP levels in sweat glands (Hwang et al, British Journal of Haematology, 2021; 195:e138-e146). In in vitro studies, 3 pretreatment AML pt BM samples were analyzed after a short-term (non-cytotoxic) OM treatment for 4 hours at a dose (75 nM) equivalent to calculated systemic OM levels achieved in cohort#3 (2mg/m2). Responding pt samples (CR;n=2) had a greater reduction in protein translation than the non-responder (NR). With exposure to OM, CR pts also had greater reductions of proteins with a short T1/2, such as anti-apoptotic protein MCL-1, with a marked increase in cytochrome C loss, indicating high mitochondrial priming and sensitivity to apoptosis (Fig.2). Conclusion: This small phase Ib study of the addition of OM to a standard 7 + 3 regimen in a diverse population with predominantly high-risk AML and frequent co-morbidities demonstrates a manageable safety profile, acceptable efficacy and resulted in a prolonged DoR. Studies of pretreatment pt samples suggest that analysis of blast protein translation inhibition and especially BH3 profiling responses to OM may predict susceptibility of LSC to Omacetaxine, and potentially patient outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Niclosamide Inhibits the Growth of Acute Lymphoblastic Leukemia Cells By Increasing Intracellular ROS and Activation of p53-PUMA/NOXA

Acute lymphoblastic leukemia (ALL) is a heterogeneous clonal malignancy originated from B- or T-cell lymphoid precursor cells. The prognosis is extremely poor in refractory or relapsed cases. Niclosamide is an FDA-approved oral anthelmintic drug with anti-cancer property. In the present study, we investigated the anti-leukemic effect of niclosamide in ALL cells in vitro and in vivo. ALL cell lines REH and NALM-6, and primary cells of patients with ALL were treated with niclosamide according to the concentration gradient, and then detected by trypan blue, MTS, and soft agar colony assays. The transcriptional and translational levels of apoptosis/anti-apoptosis-related genes were assessed by immunoblotting and quantitative real-time PCR. Flow cytometry was used to detect the effect of niclosamide on apoptosis of ALL cells, mitochondrial membrane potential, and intracellular specific reactive oxygen species (ROS) levels. For animal studies, we generated a mouse xenograft model with NOD prkdc-/-IL-2Rg-/- mice intravenously engrafted with NALM-6-luc cells, the NALM-6 cells infected with retrovirus expressing luciferase. Mice were treated with 25 mg/kg/day niclosamide or solvent twice a day intraperitoneally. The effect of niclosamide against leukemia cells in mice was dynamically evaluated by an in vivo imaging system. Flow cytometry was used to analyze the percentage of leukemia cells in spleens and bone marrows of sacrificed mice. The overall survival of mice was evaluated by Kaplan-Meier survival curve. In the cell viability assay, we found that niclosamide effectively inhibited REH, NALM-6 and primary ALL cells with IC50 values of 1.72, 3.58 and 1.36 μmol/L, respectively. Immunoblotting showed increased levels of pro-apoptotic proteins p53, PUMA, NOXA, cyto C and PARP, but decreased level of anti-apoptotic protein survivin in ALL cells treated with niclosamide in a dose- and time-dependent manner. Flow cytometry showed that niclosamide induced apoptosis and mitochondrial membrane potential in ALL cells. We also found that the level of ROS began to increase in cells treated by niclosamide for 2 hours, which reached the maximum at 8 hours. The treatment of ROS inhibitor N-acetylcysteine (NAC) attenuated niclosamide-induced apoptosis of REH and NALM-6 cells, which reduced by 18.12% and 8.02%, respectively. NAC also inhibited niclosamide-induced accumulation of intracellular p53/PUMA/NOXA proteins in these cells. In the animal study, in vivo imaging system showed significantly reduced tumor burdens in mice treated with niclosamide compared with controls, with bioluminescence signals of 15.53 and 4.04 on day 21, respectively. Mice treated with niclosamide survived longer than controls (32.5 days vs 26.5 days, p < 0.01), which had lower percentage of CD45+ CD19+ leukemia cells in their bone marrows and spleens, compared with the control group (61.10% vs 34.76% and 54.88% vs 26.40%, respectively). In the present study, we found that niclosamide potently inhibited the growth of ALL cells and induced apoptosis via increased levels of ROS and the activation of p53 signaling. Our findings suggest that niclosamide may be a promisingly potent agent for ALL therapy.

Desmocollin-2 inhibits cell proliferation and promotes apoptosis in hepatocellular carcinoma via the ERK/c-MYC signaling pathway.

Hepatocellular carcinoma (HCC) is one of the most common cancers around the world with a poor prognosis. The main reason for poor prognosis is early stage HCC is inconspicuous so it is difficult to detect and effective treatment strategies are lacking for advanced HCC. In this context, novel molecular targets are urgently needed for the diagnosis and therapy of HCC. In this study, we investigated the expression level, biological function, and relative mechanism of Desmocollin-2(DSC2) in HCC. DSC2 expression levels were decreased significantly in HCC cell lines SMMC-7721(7721), Huh7, HCC-LM3(LM3), and MHCC-97H(97H), especially in LM3 cells, compared with human liver cell line L02(L02). DSC2 overexpression in LM3 cells could inhibit the proliferation (in vitro and in vivo), colony formation, migration, and invasion abilities of HCC cells, and promote cell apoptosis, while DSC2 inhibition in 7721 cells performed the opposite effect. Consistent with these results, regulating DSC2 expression in 7721 and LM3 cells could affect the expression levels of apoptosis-related proteins (Bax, Bcl-2, c-Caspase-3, Caspase-3, Caspase-8, and Survivin) and cell cycle-related proteins (Cyclin D1, Cyclin B1, CDK1, and CDK2). Furthermore, DSC2 expression was significantly negatively correlated with the levels of p-ERK and c-MYC in both LM3 and 7721 cell lines. These findings confirmed that DSC2 overexpression could inhibit the proliferation, migration, and invasion abilities while promoting apoptosis of HCC cells via the ERK/c-MYC signaling pathway. In a conclusion, DSC2 was a tumor suppressor with low expression in liver cancer.

Mechanism of HSP90 Inhibitor in the Treatment of DSS-induced Colitis in Mice by Inhibiting MAPK Pathway and Synergistic Effect of Compound Sophorae Decoction.

The mechanism of Heat Shock Protein 90 (HSP90) in Ulcerative Colitis (UC) has been studied, and mitogenic-activated protein kinases (MAPK) also contribute to the pathogenesis of UC. However, the effect of the HSP90/MAPK pathway in UC is still unclear. Therefore, the mainstay of this research is to explore the mechanism of action of this pathway in UC. Compound sophorae decoction (CSD), as a Chinese herbal decoction, can synergistically affect the above process. This study aimed to uncover the synergistic effects of HSP90 inhibitors regulating the MAPK pathway for treating DSS-induced colitis in mice and the synergistic effects of CSD. This experiment used oral administration of standard diets containing 3% dextran sodium sulfate (DSS) to establish an experimental colitis model in mice. The model was treated with HSP90 inhibitor, CSD, or dexamethasone. Mouse feces, mobility, body weight, colon length, and colon histopathology scores were recorded daily to assess the degree of colitis inflammation. Expression levels of HSP90 and MAPK pathway-related genes and proteins were evaluated by Western blot and qPCR. The evaluation of intestinal mucosal permeability was measured by enzyme-linked immunosorbent assay (ELISA), which could detect the protein level of D-Amino Acid Oxidase (DAO) and D-lactic acid (D-LA). The same went for downstream molecules AFT-2, p53, and apoptosis-related proteins BAX, BCL-2, Caspase3, and survivin in the MAPK pathway. Immunohistochemical measured p-38, p-JNK, and p-ERK expressions. JAM-A and claudin-1 connexin were tested by immunofluorescence staining. The TUNEL method was for measuring the apoptosis rate of colonic epithelial cells. CBA kit determined the level of inflammatory factors of colons. HSP90 inhibitor can improve the degree of pathological damage in the colon of mice treated with DSS, increase the mice's weight and the length of the colon, and significantly reduce the disease activity index (DAI) score. Intraperitoneal injection of HSP90 inhibitor can reduce the expression of MAPK pathway markers P38, JNK, ERK, and their phosphorylation and decrease the content of AFT-2 and p53, which is downstream of the MAPK pathway. In addition, treatment of the HSP90 inhibitor up-regulated the expression of anti-apoptotic proteins BCL-2 and survivin, as well as down-regulated apoptotic protein caspase3, BAX in the colon of mice with colitis. Lower levels of inflammatory factors such as IL-6, MCP-1, IFN-γ, TNF, IL-12p70, and increased IL-10 were observed after HSP90 inhibitor therapy. Furthermore, the combination treatment of CSD can enhance the effect of the single HSP90 inhibitor treatment and play a synergistic effect. These data suggest that an HSP90 inhibitor is available to treat UC by inhibiting the MAPK signaling pathway. This axis can restore the intestinal mucosa barrier's function by reducing intestinal mucosa's permeability and inhibiting apoptosis of intestinal epithelial cells. The specific mechanism is that HSP90 inhibitor can reduce the pathological damage and inflammation levels of colitis mice, and reduce the apoptosis rate of colonic epithelial cells and the mucosal permeability, thereby restoring the mucosal barrier function. During this process, CSD works synergistically to improve the therapeutic effect of the HSP90 inhibitor.

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.

Survivin: A Potential Marker of Resistance to Somatostatin Receptor Ligands.

Invasive and somatostatin receptor ligand (SRL)-resistant pituitary tumors represent a challenge in the clinical practice of endocrinologists. Efforts have been made to elucidate reliable makers for both. Survivin and eukaryotic translation initiation factor-binding protein 1 (4EBP1) are upregulated in several cancers and involved in apoptosis and cell proliferation. We explored the role of these markers in somatotropinomas. Immunostains for survivin and 4EBP1, and also for somatostatin receptor type 2 (SSTR2), Ki-67, and cytokeratin 18, were analyzed in tissue microarrays containing 52 somatotropinoma samples. Tumor invasiveness was evaluated in all samples while drug resistance was evaluated in 34 patients who received SRL treatment. All these parameters were correlated with first-generation SRL (fg-SRL) responsiveness and tumor invasiveness. Low survivin expression (P = 0.04), hyperintense signal on T2 weighted image (T2WI) (P = 0.01), younger age (P = 0.01), sparsely granular adenomas (SGA) (P = 0.04), high postoperative growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels (P = 0.049 and P < 0.001, respectively), and large postoperative tumor size (P = 0.02) were associated with resistance to fg-SRL. Low survivin and SSTR2 expression and high 4EBP1 expression were associated with SGA (P = 0.04, P = 0.01, and P = 0.001, respectively). Younger age (P = 0.03), large tumor pre- and postoperative (P = 0.04 and P = 0.006, respectively), low SSTR2 expression (P = 0.03), and high baseline GH and IGF-1 (P = 0.01 and P = 0.02, respectively) were associated with tumor invasiveness. However, survivin, 4EBP1, Ki-67, and granulation patterns were not associated with tumor invasion. This study suggests that low survivin expression is predictive of resistance to fg-SRL in somatotropinomas, but not of tumor invasiveness.

Co-inhibition of Aurora A and Haspin kinases enhances survivin blockage and p53 induction for mitotic catastrophe and apoptosis in human colorectal cancer

Colorectal cancer (CRC) is a leading cause and mortality worldwide. Aurora A and haspin kinases act pivotal roles in mitotic progression. However, the blockage of Aurora A and Haspin for CRC therapy is still unclear. Here we show that the Haspin and p-H3T3 protein levels were highly expressed in CRC tumor tissues of clinical patients. Overexpression of Haspin increased the protein levels of p-H3T3 and survivin in human CRC cells; conversely, the protein levels of p-H3T3 and survivin were decreased by the Haspin gene knockdown. Moreover, the gene knockdown of Aurora A induced abnormal chromosome segregation, mitotic catastrophe, and cell growth inhibition. Combined targeted by co-treatment of CHR6494, a Haspin inhibitor, and MLN8237, an Aurora A inhibitor, enhanced apoptosis and CRC tumor inhibition. MLN8237 and CHR6494 induced abnormal chromosome segregation and mitotic catastrophe. Meanwhile, MLN8237 and CHR6494 inhibited survivin protein levels but conversely induced p53 protein expression. Ectopic survivin expression by transfection with a survivin-expressed vector resisted the cell death in the MLN8237- and CHR6494-treated cells. In contrast, the existence of functional p53 increased the apoptotic levels by treatment with MLN8237 and CHR6494. Co-treatment of CHR6494 and MLN8237 enhanced the blockage of human CRC xenograft tumors in nude mice. Taken together, co-inhibition of Aurora A and Haspin enhances survivin inhibition, p53 pathway induction, mitotic catastrophe, apoptosis and tumor inhibition that may provide a potential strategy for CRC therapy.

Regulation of survivin and caspase/Bcl-2/Cyto-C signaling by TDB-6 induces apoptosis of colorectal carcinoma LoVo cells.

Colorectal carcinoma (CRC) treatment remains severe. Survivin is aberrantly overexpressed in CRC tissues and might be a potential target for CRC treatment. TDB-6 is a new taspine derivative. The purpose of this study is to investigate the inhibitory effect of TDB-6 on CRC and its underlying mechanism. The MTT assay and xenograft model were utilized to investigate the inhibitory effect of TDB-6 on LoVo cells in vitro and in vivo. Hoechst staining and Annexin-V FITC/PI analysis were conducted to study the effect of TDB-6 on LoVo cell apoptosis. Mitochondrial membrane potential (Δψm) assay was conducted to demonstrated whether TDB-6 could induce mitochondrial-mediated apoptosis of LoVo cells. Western blotting was conducted to investigate the effect of TDB-6 on survivin protein and caspase/Bcl-2/Cyto-C signaling. The results indicated that TDB-6 induced mitochondria-mediated apoptosis and inhibited the proliferation and growth of LoVo cells in vitro and in vivo. Mechanistic investigation utilizing western blotting indicated that TDB-6 inhibited survivin protein expression, and the inhibitory effect was augmented by TDB-6 and YM-155 co-administration, which revealed that TDB-6 might induce apoptosis of LoVo cells by targeted regulation of survivin. TDB-6 also regulated survivin downstream signaling. It significantly increased the protein level of cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, cleaved-PARP, and Cyto-C, and decreased the protein level of Bcl-2. TDB-6 might be a promising survivin inhibitor with great potential for CRC treatment.

Inhibition of USP1 enhances anticancer drugs-induced cancer cell death through downregulation of survivin and miR-216a-5p-mediated upregulation of DR5

Ubiquitin-specific protease 1 (USP1) is a deubiquitinase involved in DNA damage repair by modulating the ubiquitination of major regulators, such as PCNA and FANCD2. Because USP1 is highly expressed in many cancers, dysregulation of USP1 contributes to cancer therapy. However, the role of USP1 and the mechanisms underlying chemotherapy remain unclear. In this study, we found high USP1 expression in tumor tissues and that it correlated with poor prognosis in RCC. Mechanistically, USP1 enhanced survivin stabilization by removing ubiquitin. Pharmacological inhibitors (ML23 and pimozide) and siRNA targeting USP1 induced downregulation of survivin expression. In addition, ML323 upregulated DR5 expression by decreasing miR-216a-5p expression at the post-transcriptional level, and miR-216a-5p mimics suppressed the upregulation of DR5 by ML323. Inhibition of USP1 sensitized cancer cells. Overexpression of survivin or knockdown of DR5 markedly prevented the co-treatment with ML323 and TRAIL-induced apoptosis. These results of in vitro were proved in a mouse xenograft model, in which combined treatment significantly reduced tumor size and induced survivin downregulation and DR5 upregulation. Furthermore, USP1 and survivin protein expression showed a positive correlation, whereas miR-216a-5p and DR5 were inversely correlated in RCC tumor tissues. Taken together, our results suggest two target substrates of USP1 and demonstrate the involvement of survivin and DR5 in USP1-targeted chemotherapy.

Cellular Concentration of Survivin and Caspase 3 in Habitual Tobacco Chewers with and without Oral Squamous Cell Carcinoma in South Indian Rural Population-A Case Control Study.

Background: There is paucity of data on tissue levels of Survivin and Caspase 3 in south Indian tobacco chewers with oral Squamous cell carcinoma (OSCC). Oral cancer is a rapidly growing, highly prevalent head and neck malignancy; it involves a mucosal epithelium of a buccal cavity exposed to tobacco and other carcinogens. The basis of the survival of a tumor cell or transformed normal cell into a neoplastic cell is by the suppression of apoptosis regulation. Recently, researchers have focused on Survivin, an inhibitor of apoptosis family of proteins (IAP), involved in apoptosis regulation in cancer cells targeting the executioner Caspase 3. The current study aims to quantify the cellular levels of Survivin and Caspase 3 in tobacco chewers with OSCC and in habitual tobacco chewers without OSCC, in comparison to controls. Methods: A single centric case control study included 186 study subjects, categorized into: Group I (n = 63), habitual tobacco chewers with OSCC; Group 2 (n = 63), habitual tobacco chewers without OSCC; and Group 3 (n = 63), the controls. Resected tumor tissue from Group 1 and buccal cell samples from Groups 2 and 3 were collected into phosphate buffer saline (PBS) and assayed for Survivin and Caspase 3 levels by the ELISA sandwich method. Results: The mean ± SD of the Survivin protein in Group 1 was (1670.9 ± 796.21 pg/mL); in Group 2, it was (1096.02 ± 346.17 pg/mL); and in Group 3, it was (397.5 ± 96.1 pg/mL) with a significance of p < 0.001. Similarly, the level of Caspase 3 in Group 1 was (7.48 ± 2.67 ng/mL); in Group 2, it was (8.85 ± 2.41 ng/mL); and in Group 3, it was (2.27 ± 2.24 ng/mL) with a significance of p < 0.001. Conclusion: The progressive transformation of buccal cells to neoplastic cells is evident; in the case of OSCC, this indicates that the over-expression of Survivin compared to Caspase 3 confirms the suppression and dysregulation of apoptosis.