The Establishment of Quantitatively Regulating Expression Cassette with sgRNA Targeting BIRC5 to Elucidate the Synergistic Pathway of Survivin with P-Glycoprotein in Cancer Multi-Drug Resistance

Quantitative analysis and regulating gene expression in cancer cells is an innovative method to study key genes in tumors, which conduces to analyze the biological function of the specific gene. In this study, we found the expression levels of Survivin protein (BIRC5) and P-glycoprotein (MDR1) in MCF-7/doxorubicin (DOX) cells (drug-resistant cells) were significantly higher than MCF-7 cells (wild-type cells). In order to explore the specific functions of BIRC5 gene in multi-drug resistance (MDR), a CRISPR/Cas9-mediated knocking-in tetracycline (Tet)-off regulatory system cell line was established, which enabled us to regulate the expression levels of Survivin quantitatively (clone 8 named MCF-7/Survivin was selected for further studies). Subsequently, the determination results of doxycycline-induced DOX efflux in MCF-7/Survivin cells implied that Survivin expression level was opposite to DOX accumulation in the cells. For example, when Survivin expression was down-regulated, DOX accumulation inside the MCF-7/Survivin cells was up-regulated, inducing strong apoptosis of cells (reversal index 118.07) by weakening the release of intracellular drug from MCF-7/Survivin cells. Also, down-regulation of Survivin resulted in reduced phosphorylation of PI3K, Akt, and mTOR in MCF-7/Survivin cells and significantly decreased P-gp expression. Previous studies had shown that PI3K/Akt/mTOR could regulate P-gp expression. Therefore, we speculated that Survivin might affect the expression of P-gp through PI3K/Akt/mTOR pathway. In summary, this quantitative method is not only valuable for studying the gene itself, but also can better analyze the biological phenomena related to it.

Targeting abundant survivin expression in liposarcoma: subtype dependent therapy responses to YM155 treatment

Purpose Liposarcoma (LPS) represent the largest group of malignant soft tissue tumours comprising a heterogeneous group of subtypes in which the degrees of chemoresistance and radiosensitivity strongly vary. Consequently, it is of utmost interest to establish novel therapeutic regimens based on molecular targets. Methods Immunohistochemical staining of survivin was performed in tissue microarrays comprising 49 primary LPS specimens. LPS cell lines were treated with survivin antagonist YM155 and doxorubicin or etoposide alone as well as in combination. Changes in cell viability were investigated and the synergistic effect of a combined therapy analysed. Results Immunohistochemistry revealed an abundant expression of survivin in LPS that significantly concurred with less-differentiated tumour subtypes and grading. In vitro, we demonstrated the impact of the survivin inhibitor YM155 on dedifferentiated LPS (DDLPS) and, even more imposing, pleomorphic LPS (PLS) tumour cell viability with a strong induction of apoptosis. A combined treatment of doxorubicin or etoposide with YM155 augmented the cytotoxic effects on DDLPS and PLS cells. Conclusion These findings support the significant role of survivin in the oncogenesis and progression of LPS subtypes providing a rationale to target survivin in eligible in-vivo models and to pioneer clinical applications of survivin-specific substances unfolding their therapeutic potential in LPS patients prospectively.

Role of Surviving in Bladder Cancer: Issues to Be Overcome When Designing an Efficient Dual Nano-Therapy

Bladder cancer is the 10th most diagnosed cancer, with almost 10 M cancer deaths last year worldwide. Currently, chemotherapy is widely used as adjuvant therapy after surgical transurethral resection. Paclitaxel (PTX) is one of the most promising drugs, but cancer cells acquire resistance, causing failure of this treatment and increasing the recurrence of the disease. This poor chemotherapeutic response has been associated with the overexpression of the protein survivin. In this work, we present a novel dual nano-treatment for bladder cancer based on the hypothesis that the inhibition of survivin in cancer cells, using a siRNA gene therapy strategy, could decrease their resistance to PTX. For this purpose, two different polymeric nanoparticles were developed to encapsulate PTX and survivin siRNA independently. PTX nanoparticles showed sizes around 150 nm, with a paclitaxel loading of around 1.5%, that produced sustained tumor cell death. In parallel, siRNA nanoparticles, with similar sizes and loading efficiency of around 100%, achieved the oligonucleotide transfection and knocking down of survivin expression that also resulted in tumor cell death. However, dual treatment did not show the synergistic effect expected. The root cause of this issue was found to be the cell cycle arrest produced by nuclear survivin silencing, which is incompatible with PTX action. Therefore, we concluded that although the vastly reported role of survivin in bladder cancer, its silencing does not sensitize cells to currently applied chemotherapies.

Biodegradable mesoporous manganese carbonate nanocomposites for LED light-driven cancer therapy via enhancing photodynamic therapy and attenuating survivin expression

Triple-negative breast cancer (TNBC) is one of the most daunting diseases, low toxicity and efficient approaches are in urgent demand. Herein, we developed degradable mesoporous manganese carbonate nanocubes (MnCO3 NCs), incorporated with survivin shRNA-expressing plasmid DNA (iSur-pDNA) and riboflavin (Rf), namely MRp NCs, for synergistic TNBC therapy. The MnCO3, itself, could generate O2 and CO2 under H2O2 and thus relieve the hypoxia and acidic tumor microenvironment (TME). Furthermore, the MnCO3 NCs exhibited high Rf loading capacity and iSur-pDNA delivery ability after polyethyleneimine modification. Specifically, MRp NCs decompose in TME, meanwhile they deprived the endogenous expression of survivin gene and significantly amplified the generation of reactive oxygen species after exposure to LED light, resulting in serious tumor destruction. The multifunctional MRp NCs with LED light-driven characters are able to provide a high efficiency, low toxicity and promising strategy for TNBC therapy.

Chromatin binding of survivin regulates glucose metabolism in the IFN-g producing CD4+ T cells

Interferon-gamma (IFNg) producing T cells develop metabolic adaptation required for their effector functions in tumour biology, autoimmunity and antiviral defence. Using sorted CD4+ cells we demonstrated that glycolytic switch and high glucose uptake in IFNgproducing cells was associated with survivin expression. Inhibition of survivin restored glycolysis by upregulating the transcription of phosphofructokinase PFKFB3 and reducing glucose uptake. Integration of the whole-genome sequencing of the chromatin immunoprecipitated with survivin with transcription changes in CD4+ cells after survivin inhibition revealed co-localization of survivin, IRF1 and SMAD3 in the regulatory elements paired to the differentially expressed genes. Western blot demonstrated direct binding of survivin to IRF1 and SMAD3. Functionally, inhibition of survivin repressed IFNg signalling and activated SMAD3-dependent protein remodelling, which resulted in the effector-to-memory transition of CD4+ cells. These findings demonstrate the key role of survivin in IFNg-dependent metabolic adaptation and identify survivin inhibition as an attractive strategy to counteract these effects.

Biflavonoids from Selaginella doederleinii as Potential Antitumor Agents for Intervention of Non-Small Cell Lung Cancer

Four new biflavonoids (1–4) were isolated from Selaginella doederleinii together with a known biflavonoid derivative (5). Their structures contained a rare linker of individual flavones to each other by direct C-3-O-C-4′′′ bonds, and were elucidated by extensive spectroscopic data, including HRESIMS, NMR and ECD data. All isolates significantly inhibited the proliferation of NSCLC cells (IC50 = 2.3–8.4 μM) with low toxicity to non-cancer MRC-5 cells, superior to the clinically used drug DDP. Furthermore, the most active compound 3 suppressed XIAP and survivin expression, promoted upregulation of caspase-3/cleaved-caspase-3, as well as induced cell apoptosis and cycle arrest in A549 cells. Together, our findings suggest that 3 may be worth studying further for intervention of NSCLC.

Carbonic Anhydrase IX and Survivin in Colorectal Adenocarcinoma Cells: Slovakian Population Study

The aim of this study was to detect carbonic anhydrase IX (CAIX) and survivin in the colorectal adenocarcinoma cells of the Slovakian population. We used an indirect three-step immunohistochemical method with DAB staining for the localization of the proteins and investigation their expression. We compared their expression with expression in healthy colorectal tissue. In 74 tissues of colorectal adenocarcinomas, 42% showed CAIX positivity and 20% showed survivin positivity. Brown membrane immunostaining was visible in CAIX-positive tumors. Survivin-positive tumors had strong brown cytoplasmic immunostaining. Co-expression of both proteins was present in five specimens (7%). The samples of normal colorectal tissue (without carcinoma) were CAIX-negative and survivin-negative. We also applied the Chi-squared test for evaluation statistically significant association between the expression of proteins and selected clinical and histopathological parameters. We did not find any statistically significant correlations between CAIX or survivin expression and sex of patients, the grade of the tumor, nodal status and presence of metastasis (p > 0.05). The fact that all samples of normal colorectal tissue were CAIX- and survivin-negative could lead to the possibility of using these two proteins as potential tumor diagnostic markers. On the basic of the available publications and data, we suggest that CAIX and survivin could be negative independent prognostic markers of colorectal cancer, which could affect response to therapy.

Prognostic Significance of Survivin and Livin Expression in the Primary Breast Cancer and Their Lymph Node Metastases

Aim: To assess the prognostic significance of Survivin and Livin expression in invasive breast cancer and their lymph node metastases. Materials and Methods: The present series consists of archival samples from 78 women with invasive breast cancer diagnosed and treated during 2010-2014 at National Cancer Institute, Misurata, Libya. Tumor biopsies were analysed for expression of Survivin and Livin by immunohistochemistry, and different grading systems were tested for their expression. Results: In the cancer samples, a significant correlation was established between Survivin expression and site of the tumor (p=0.021), tumor recurrence (p=0.036), and unifocal tumor (p=0.001). Moreover, Her-2 negative tumors had higher Survivin expression than Her-2 positive tumors (p= 0.047). There were no associations between Survivin expression and histological grade, histological type, lymph node status, tumor stage, TNM classification, estrogen and progesterone receptors, distant metastases, chemotherapy, radiotherapy, hormone replacement, vascular invasion, surgical margin, positive family history. Livin expression in primary breast cancer showed a significant correlation (p=0.025) with positive family history, but no significant association with other clinicopathological parameters. In addition, we found that primary tumors showed higher Survivin expression (82%) compared with the lymph node metastases (34%), whereas Livin expression did not differ between the primary (71%) tumors and their metastases (84%). Conclusion: Survivin expression in primary breast cancer is significantly associated with several characteristics of favourable prognosis. Livin expression in primary breast cancer is significantly associated only with a positive family history of breast cancer.

Biodegradable Mesoporous Manganese Carbonate Nanocomposites for LED Light-Driven Cancer Therapy via Enhancing Photodynamic Therapy and Attenuating Survivin Expression

Triple-negative breast cancer (TNBC) is one of the most daunting diseases, and low toxicity and efficient approaches are in urgent demand. Herein, we developed degradable mesoporous manganese carbonate nanocubes (MnCO3 NCs), incorporated with survivin shRNA-expressing plasmid DNA (iSur-pDNA) and riboflavin (Rf), namely MRp NCs, for synergistic TNBC therapy. The MnCO3, itself, could generate O2 and CO2 under H2O2 and thus improve the hypoxia and acidic tumor microenvironment (TME). Furthermore, the MnCO3 NCs exhibited high Rf loading capacity and iSur-pDNA delivery ability after polyethyleneimine modification. Specifically, MRp NCs decompose in TME, meanwhile they deprived the endogenous expression of survivin gene and significantly amplified the generation of reactive oxygen species after exposure to LED light, resulting in serious tumor destruction. The multifunctional MRp NCs with LED light-driven characters are able to provide a high efficiency, low toxicity and promising strategy for TNBC therapy.