Survivin Gene Expression Research Articles

Expression of ErbB-2 (Her-2/neu) and survivin gene (BIRC5) in tumor infiltrating lymphocytes (TILs) in breast cancer

Expression of ErbB-2 (Her-2/neu) and survivin gene (BIRC5) in tumor infiltrating lymphocytes (TILs) in breast cancer

Role of BIRC5 Gene as a Prognostic Marker in Pediatric Acute Lymphoblastic Leukemia

Background and objectives: Acute lymphoblastic leukaemia (ALL) is the most common malignancy diagnosed in children. Survivin, a small inhibitor-of-apoptosis protein (IAP) encoded by the baculoviral inhibitor of apoptosis repeat-Containing 5 (BIRC5) gene, plays critical roles in malignancy by preventing apoptosis through blocking caspase activity. The aim of this study was to assess the prognostic role of BIRC5 gene in paediatric ALL. Subjects and methods: The present study was carried out on 42 children with the’ novo ALL who were followed up for two years and 10 apparently healthy children of matched age and sex (controls). Each child was subjected to complete history taking, clinical examination, laboratory investigations the form of CBC, Leishman-stained peripheral blood smears, Lactate dehydrogenase (LDH), Bone marrow (BM) aspiration and examination of Leishman-stained smears, Immunophenotyping on BM samples for routine panel of ALL determined by flow cytometer and quantitative determination of survivin gene expression by real time PCR on BM samples. Results: Patients with ALL had a significantly higher BIRC5 expression than did the control group (P=0.0004). There was a significant increase in survivin gene expression level in T-ALL when compared to common BALL and pre B-ALL (P=0.001). A significant positive correlation was found between survivin expression and LDH, uric acid and white blood cells (WBCs) (r=0.47, P=0.002; r=0.31, P=0.05, r=0.62, P=0.001), respectively. A significant higher survivin expression levels, LDH and WBCs was found in children with unfavorable outcome. 94.5% of ALL patients with high survivin expression had an unfavorable outcome while, in ALL children with low survivin expression, only 25% had an unfavorable outcome (P=0.001). Conclusion: BIRC5 gene expression was correlated with unfavorable outcome of childhood ALL. So, its measurement at diagnosis may detect a high risk ALL subgroup.

YM155 Reverses Statin Resistance in Renal Cancer by Reducing Expression of Survivin.

The purpose of the present study was to clarify whether treatment with YM155, a novel small-molecule inhibitor of survivin, reverses statin resistance in statin-resistant renal cell cancer (RCC). We induced simvastatin resistance in a renal clear cell carcinoma cell line (Caki-1-staR). In vitro and in vivo models were used to test the efficacy of YM155 and simvastatin. Survivin gene expression was significantly stronger in Caki-1-staR cells than in its parent cells (Caki-1). In Caki-1-staR cells, YM155 significantly reduced expression of survivin gene and cell proliferation in a dose-dependent manner. Treatment with YM155 significantly reversed simvastatin resistance in Caki-1-staR cells. YM155 significantly inhibited the growth of Caki-1-staR tumors in a nude mouse tumor xenograft model. Furthermore, YM155 significantly enhanced the antitumor effects of simvastatin on Caki-1-staR tumors. Our results indicate that inhibition of survivin by YM155 overcomes statin resistance in RCC cells.

Prognostic Value of Gravin Versus Survivin Gene Expression in a Cohort of Egyptian Patients with Adult Acute Myeloid Leukemia

Prognostic Value of Gravin Versus Survivin Gene Expression in a Cohort of Egyptian Patients with Adult Acute Myeloid Leukemia

Drug priming enhances radiosensitivity of adamantinomatous craniopharyngioma via downregulation of survivin.

OBJECTIVE In this study, the authors investigated the underlying mechanisms responsible for high tumor recurrence rates of adamantinomatous craniopharyngioma (ACP) after radiotherapy and developed new targeted treatment protocols to minimize recurrence. ACPs are characterized by the activation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR), known to mediate radioresistance in various tumor entities. The impact of tyrosine kinase inhibitors (TKIs) gefitinib or CUDC-101 on radiation-induced cell death and associated regulation of survivin gene expression was evaluated. METHODS The hypothesis that activated EGFR promotes radioresistance in ACP was investigated in vitro using human primary cell cultures of ACP (n = 10). The effects of radiation (12 Gy) and combined radiochemotherapy on radiosensitivity were assessed via cell death analysis using flow cytometry. Changes in target gene expression were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Survivin, identified in qRT-PCR to be involved in radioresistance of ACP, was manipulated by small interfering RNA (siRNA), followed by proliferation and vitality assays to further clarify its role in ACP biology. Immunohistochemically, survivin expression was assessed in patient tumors used for primary cell cultures. RESULTS In primary human ACP cultures, activation of EGFR resulted in significantly reduced cell death levels after radiotherapy. Treatment with TKIs alone and in combination with radiotherapy increased cell death response remarkably, assessed by flow cytometry. CUDC-101 was significantly more effective than gefitinib. The authors identified regulation of survivin expression after therapeutic intervention as the underlying molecular mechanism of radioresistance in ACP. EGFR activation promoting ACP cell survival and proliferation in vitro is consistent with enhanced survivin gene expression shown by qRT-PCR. TKI treatment, as well as the combination with radiotherapy, reduced survivin levels in vitro. Accordingly, ACP showed reduced cell viability and proliferation after survivin downregulation by siRNA. CONCLUSIONS These results indicate an impact of EGFR signaling on radioresistance in ACP. Inhibition of EGFR activity by means of TKI treatment acts as a radiosensitizer on ACP tumor cells, leading to increased cell death. Additionally, the results emphasize the antiapoptotic and pro-proliferative role of survivin in ACP biology and its regulation by EGFR signaling. The suppression of survivin by treatment with TKI and combined radiotherapy represents a new promising treatment strategy that will be further assessed in in vivo models of ACP.

72P - Survivin gene expression in the primary tumor and circulating tumor cells – a new biomarker of tumor progression of breast cancer

Breast cancer is a leading cause of morbidity and mortality of the female population from malignant tumors. Distant metastases are the main cause of death of patients, a substrate for the development of which are circulating tumor cells (CTCs). However, the search for these cells alone is not sufficient to provide full information about the nature and course of tumor in a single patient. Determination of the expression of tumor-genes responsible for the different processes of tumor progression allows a more complete picture. Such genes include the gene survivin (BIRC5) family of inhibitors of apoptosis (IAP). Using real-time PCR we investigated the expression of the survivin gene in 36 samples of primary invasive ductal carcinoma of the breast, 10 samples of benign tumors - fibroadenoma of the breast, as well as 36 samples of peripheral blood of patients with breast cancer at various stages of tumor and stage specific treatment, and 10 healthy people as controls. In primary breast carcinoma we determined a high expression of survivin gene in all 36 samples with the average value (M ± m) 1.58 ± 0.31 (min – 1.19; max – 4.41). The highest figures were found in tumors of medium and high grade (G II-III) with lymphovenous invasion (LVSI +). In 2 of 3 samples of benign tumor expression of survivin was not found, and one was 0.015. In CTCs, isolated from peripheral blood of breast cancer patients, all 36 samples as determined by the gene expression of survivin with an average value (M ± m) 1.10 ± 0.19 (min – 0.36; max – 3.79). The level of expression of the control samples did not exceed 0.003. It should be noted that the maximum volume of expression was obtained in samples of tumor patients with stage N +, and especially M1, on TNM classification. Any legitimate expression of survivin, depending on the size of the tumor had been received. In patients, receiving chemotherapy average expression of survivin gene was obswerved, but it never approached the indicators of control. Determination of expression of the survivin gene in primary tumor and in CTCs may be one of the most promising markers of tumor progression and for monitoring of breast cancer therapy.

Study of survivin gene expression in hepatocellular carcinoma patients

Objective The aim of this study was to assess the differential expression of survivin gene in tumorous versus nontumorous liver tissue of hepatocellular carcinoma (HCC) patients and determine the possible influence of survivin gene polymorphism −31G/C and +9809T/C on its expression and its association with the risk for HCC in Egyptian patients. Background Mechanisms of hepatic carcinogenesis are not completely understood, but HCC development and progression could be attributed to accumulated genetic alterations as other malignancies. Survivin has functional involvement in apoptosis and proliferation, and is upregulated in malignancy. It attracts considerable interest as a potential diagnostic, prognostic tumor marker and a new target for cancer treatment. Patients and methods This study was performed on 30 HCC patients and 30 unrelated apparently healthy individuals who served as a control group. Genotyping of survivin gene at −31G/C and +9809T/C was performed for HCC cases and controls using TaqMan allelic discrimination Assay. Real-time PCR was performed for survivin mRNA in paired tumorous and nontumorous liver tissue specimens of the HCC patients. Results No significant difference was found between HCC patients and controls as regards different genotypes and alleles of survivin −31G/C and +9809T/C polymorphism. Moreover, none of these genotypes and alleles was associated with risk for HCC. Survivin mRNA was expressed in all tumorous and nontumorous tissue specimens in HCC cases and its expression was significantly higher in tumorous tissues compared with nontumorous tissues by 13 folds. Survivin expression in tumorous tissue was significantly increased with high α-fetoprotein, larger size of the tumor, vascular invasion, absence of cirrhosis, recurrence within 1 year, and low survival rate. The advanced stage of the tumor was the independent variable for survivin expression (P Conclusion Genetic variations of survivin gene had no effect on the susceptibility to HCC in Egyptian patients. Survivin was significantly overexpressed in HCC cases with advanced tumor stage.

A Study of Gene Expression of Survivin, its Antiapoptotic Variants, and Targeting Survivin In Vitro for Therapy in Retinoblastoma.

Apoptosis is a natural process regulated by apoptotic and antiapoptotic molecules. We investigated mRNA expression of survivin and its splice variants, along with B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax), in a cohort of 20 retinoblastoma (RB) tumors by real-time polymerase chain reaction. We hypothesized a correlation between the Bcl-2/Bax and survivin splice variants and also that expression of these would be associated with clinicopathologic features of tumors. The Bcl-2 expression was significantly higher (P<0.001) in RB, and Bcl-2/Bax ratio was remarkably higher in poorly differentiated tumors. A statistically significant higher expression of Survivin-WT (wild type) compared with its variant Survivin-2β (P<0.05) was observed. Bcl-2 did not exhibit positive correlation with any of the survivin variants except Survivin-2β, whereas Bax exhibited significant (P<0.05) correlation with the variants. Thus, it could be suggested that a superior player out of a likely interaction between the variants and Bcl-2/Bax uses its activity for the progression of RB. Silencing of Survivin-WT in the Y79 cell line was studied by siRNA technology and cell-permeable dominant negative survivin (SurR9-C84A). siRNA showed higher proapoptotic effects and increased caspase 3/7 activity in Y79 cells. Effective internalization of SurR9-C84A in Y79 cells induced cytotoxic effects. Thus, the current study confirms survivin as a promising target for therapy.

Abstract 2791: Hypoxia selectively confers resistance to the survivin-targeting compound YM155 in acute myeloid leukemia and Ewings sarcoma cell lines

Abstract Background Hypoxia is characteristic of almost all types of solid tumors and is an important component of the bone marrow microenvironment. It is an adverse prognostic indicator in cancer as it is associated with tumor progression and chemoresistance. Cells may adapt to hypoxia in different ways; including growth arrest, stimulation of angiogenic factors, inhibition of apoptosis, induction of the ER stress program, or modification of the cellular energy metabolism. YM155 inhibits survivin gene expression by suppressing promoter activity and subsequent gene expression. The role of hypoxia and its clinical implications in childhood cancer remain largely unknown. Therefore, studying the drug response of cancer cells within a hypoxic environment is critical for the accurate prediction of patients’ response to therapy. The aims of the present study were (1) to perform screening of the effects of 5 molecularly targeted compounds on a large panel of pediatric cancer cell lines including acute myeloid leukemia (AML), Ewings sarcoma (ES), osteosarcoma (OS), and neuroblastoma (NB), under normoxia and hypoxia, and (2) to determine the potential mechanisms of drug resistance under hypoxia. Methods and Results In the present study the IC50 values were determined for the following targeting agents: linsitinib, picropodophylin (insulin-like growth factor-1 receptor), YM155 (survivin), AZD 8055 (mTOR), dovitinib (FLT3, c-kit, FGF4), and Palbociclib (CDK4/CDK6) in a panel of 15 AML, 4 ES, 2 OS and 2 NB cell lines using Cell Titer Glo assay. The effect of hypoxia was differential. It depended on the cell line and the compound used. However, hypoxia induced universal resistance to YM155 in all cell lines tested. YM155 had significant cytotoxicity in AML cell lines (IC50 between 2.5-1775 nM) in normoxia, however, the IC50 values in hypoxia were in the micromolar range, with an increase of &amp;gt; 100 fold. The effect of hypoxia on YM155 response in ES cell lines was less pronounced with (IC50s between 1-10 nM) in normoxia compared to (IC50 between 6 and 350 nM) in hypoxia. We studied the effect of hypoxia on the cell cycle using propidium iodide and flow cytometry in MV4-11 AML cells. Treatment with YM155 for 24 h in normoxia induced a pronounced decrease in the percentage of cells in S phase and G2/M phase and an increase in the sub-G1 phase. However, no change in cell cycle phases was detected under hypoxia. Ongoing studies are focusing on determining the mechanisms of hypoxia-induced YM155 resistance through studying the expression of proteins regulating hypoxia, cell cycle, and apoptosis using western blot. Conclusion These data demonstrate that hypoxia induced different effects in a panel of pediatric cancer cell lines. However, it conferred pronounced resistance to YM155 in AML and ES cell lines. Hypoxia is likely a key factor in programming treatment resistance and, thus, relapse and poor outcomes. Citation Format: Justin Montoya, Kieran Finch, Jan Simper, Robert J. Arceci, Eiman A. Aleem. Hypoxia selectively confers resistance to the survivin-targeting compound YM155 in acute myeloid leukemia and Ewings sarcoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2791.

486 - Expression of survivin gene (BIRC5) and ErbB-2 (Her-2/neu) in lymphocytes in breast cancer

486 - Expression of survivin gene (BIRC5) and ErbB-2 (Her-2/neu) in lymphocytes in breast cancer

Antiproliferative effect of Resveratrol and Oxaliplatin on colon cancer cells

Current cancer treatments include radiation, chemotherapy and surgery. Treatment of primary tumors with radiotherapy and/or chemotherapy has been observed to increase the rate of metastasis. While these treatments contribute to overall well‐being, they are a double‐edged sword and may also cause unintended and painful side effects. Consumption of a diet rich in phytochemicals is efficacious in inhibiting colon cancer. Phytochemicals are plant‐based compounds that are usually nontoxic and have significant effects on cancer cells. They also have been used in a synergistic manner with chemotherapy drugs. This study assessed different plant‐based compounds for effect on colon cancer cells and resveratrol in combination with the known chemotherapy drug, oxaliplatin, displayed interesting results. Resveratrol is found in the skin of red grapes and red wine and has shown promise as an anti‐tumor agent. Our studies focused on assessment of varying concentrations of resveratrol or oxaliplatin used as single compounds and oxaliplatin in combination with resveratrol with resveratrol held constant at the 50uM (IC50) concentration. Results indicated that resveratrol or oxalipatin treatment alone elicited anti‐proliferative activity. Oxaliplatin exhibited greater antiproliferative activity than resveratrol alone. In the double compound treatment analysis, resveratrol (held constant at 50uM) plus oxaliplatin showed significant inhibition at lower concentration (1.56uM) than that of either single compound treatment alone. At higher concentrations, no significant differences were found between oxaliplatin and double‐compound treated cells. Preliminary results suggested that resveratrol plus the chemotherapeutic agent oxaliplatin may inhibit at lower concentrations due to synergistic activities. Studies have shown that both compounds reduce expression of the inhibitor of apoptosis protein known as survivin. Therefore, future experiments will be focused on expression level of micro RNA‐16 and micro RNA‐34a that modulates the survivin gene expression by post transcriptional regulation.

Survivin and cyclin E2 genes expression in a cohort of Egyptian acute leukemia patients: Clinical importance and future prospects.

Abnormalities in the control of apoptosis play an important role in leukemogenesis. Survivin is a member of inhibitor of apoptosis proteins family, it prevents apoptosis by blocking caspase activity and play a role in cell proliferation. While, cyclin E2 is one of the cyclins proteins family that controls progression of cell cycle by activation of cyclin dependant-kinase. Was to assess survivin and cyclin E2 genes expression in acute leukemia (AL) patients, and to define their role in the susceptibility of AL, and their correlation with the clinical presentation, laboratory findings, as well as treatment outcome. This study included 60 de novo AL patients and 40 control subjects to study the expression of survivin and cyclin E2 genes using RT-PCR. Survivin and cyclin E2 genes expression was significantly higher in leukemic patients compared with control subjects (P< 0.001), both genes separately were associated with increased risk of leukemia development and treatment failure (P< 0.01). Moreover, when combining the 2 genes expression, a significant elevation of the risk of leukemia and treatment failure was found (P < 0.01). Survivin and cyclin E2 genes expression may have clinical relevance and can be considered as molecular risk factors for AL. Also they may be useful as predictive markers for treatment outcome in leukemic patients.

Expression of Survivin Gene and Protein in Mycosis Fungoides

Background: Survivin is a member of the inhibitors of apoptosis gene family that has been implicated in cell division and suppression of apoptosis that becomes expressed in human cancers, e.g. colorectal, bladder cancers. But, there is no available data on the role of survivin in cutaneous neoplasms. Aim: To estimate the expression of survivin gene and protein in Mycosis Fungoides (MF). Methods: Seventeen cases of MF have been en-rolled into this study. Skin biopsies from such patient were subjected to RT-PCR technique to detect the level of survivin gene and protein. Results: There was significant increase in expression of surviving gene and protein in cases of MF if compared to normal control. Conclusion: Survivin may have a role in induction of MF and this may influence the therapeutic strategies for the treatment of MF.

Effects of monomethoxypolyethylene glycol-chitosan nanoparticle-mediated dual silencing of livin and survivin genes in prostate cancer PC-3M cells.

Monomethoxypolyethylene glycol-chitosan (mPEG-CS) nanoparticles were used as interfering RNA carriers to transfect human prostate cancer PC-3M cells to evaluate the effects of livin and survivin gene silencing on the proliferation and apoptosis. mPEG-CS nanoparticles with sizes of approximately 60 nm were first synthesized by ionic crosslinking. Through electrostatic adsorption, mPEG-CS-livin short hairpin RNA (shRNA), mPEG-CS-survivin shRNA, and mPEG-CS-(livin shRNA + survivin shRNA) nanoparticles were then prepared to transfect PC-3M cells. The mRNA and protein expression levels of livin and survivin were measured by reverse transcription-PCR and western blotting, respectively. The inhibitory effects of down-regulated livin and survivin gene expression on the cell proliferation were evaluated by MTT assay. Cell apoptosis was assessed visually using Hoechst staining. Livin and survivin expression levels in all shRNA interference groups were effectively down-regulated at both the mRNA and protein levels. Dual silencing of livin and survivin genes markedly inhibited cell proliferation and facilitated apoptosis, with better outcomes than those of individual shRNA treatments. mPEG-CS nanoparticle-mediated dual shRNA interference of livin and survivin genes significantly reduced the expression levels in PC-3M cells, inhibited proliferation, and promoted apoptosis. As these effects were superior to single interference, this method may have synergistic effects.

Targeted Microbubbles for Ultrasound Mediated Short Hairpin RNA Plasmid Transfection to Inhibit Survivin Gene Expression and Induce Apoptosis of Ovarian Cancer A2780/DDP Cells.

Nonviral gene transfer by ultrasound-targeted microbubble destruction (UTMD) is an promising technique for RNA interference (RNAi) therapy. Targeting silence survivin gene may provide an important therapeutic option for patients with ovarian cancer. However, UTMD mediated RNAi therapy typically uses nontargeted microbubbles with suboptimal gene transfection efficiency. In this work, a LHRHa targeted microbubble agent and recombinant expression plasmid of shRNA targeting survivin gene (pshRNA survivin) were constructed for UTMD mediated pshRNA survivin therapy in ovarian cancer A2780/DDP cells that express LHRH receptors. The targeted microbubbles (TMBs) mixed with the pshRNA survivin were added to cultured ovarian cancer cells followed by ultrasound exposure (1 MHz, 0.5 W/cm(2)) for 30 s. After transfection for 48 h, the expression of survivin mRNA and protein were (0.36 ± 0.036) and (0.05 ± 0.02), respectively. The cell proliferation inhibitory rates at 24, 48, and 72 h after treatment are (42.08 ± 3.20)%, (54.60 ± 1.02)%, and (74.25 ± 2.14)%, respectively, and the apoptosis rate was (28.99 ± 2.70)%. The expression of apoptosis related protein caspase-9 and caspase-3 were (0.95 ± 0.09) and (2.6 ± 0.21). In comparison with the other treatment groups, ultrasound mediation of targeted microbubbles yielded higher RNAi efficiency and higher cell apoptosis rate and cell proliferation inhibitory rate (p < 0.05). Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance RNAi efficiency in ovarian cancer cells. This novel method for RNA interference represents a powerful, promising no viral technology that can be used in the tumor gene therapy and research.

Sphingosine 1-phosphate interacts with Survivin pathway to enhance tumorigenesis in cancer cells.

Degradation of sphingosine 1-phosphate (S1P), as a bioactive lipid, or deregulation of its production involves in tumor progression, metastasis and chemoresistance. Since the tumor progression effects of S1P and its mechanism in chronic lymphoblastic leukemia and non-small cell lung cancer is not fully understood, we investigated the role and one of the mechanisms of S1P in tumor progression of SKW3 and H1299 cells. The effects of S1P on proliferation, invasion and migration was studied using MTT assay, soft-agar colony forming assay and trans-well migration assay, respectively. In order to find out the mechanisms of S1P action, the role of S1P on expression of Survivin gene was assessed by real-time RT-PCR. Our results demonstrated that although invasion was shown only in H1299 cells, low concentration of S1P, especially at 1 μM, mediated proliferation and migration in both cell lines. In addition, these effects of S1P in tumor progression are S1P receptor-dependent, and Survivin plays a key role in S1P tumorigenesis. Our results confirmed the involvement of S1P and its receptors in tumor progression of SKW3 and H1299. We also investigated another mechanism of S1P involved in cell survival, tumor progression, and Survivin signaling. In conclusion, data demonstrated the importance of this molecule as a target for designing new anticancer drugs such as anti-S1P monoclonal antibody for inhibiting major downstream signaling, which plays significant role in tumorigenesis.

Abstract 5407: T-type Ca2+ channel inhibitors sensitize ovarian cancer to carboplatin through downregulation of survivin gene expression

Abstract Recent studies by our group and others have shown that T-type Ca2+ channels play a reinforcing role in cancer cell proliferation, cell cycle progression and apoptosis evasion. We have demonstrated drug cooperativity between T-type Ca2+ channel inhibitor, mibefradil and carboplatin in vivo in ovarian cancer xenografts. To investigate the molecular mechanism by which T-type Ca2+ channels affect tumor growth, we used a panel of ovarian cancer cell lines together with mibefradil or small interfering RNA (siRNA) to silence the T-channels gene expression. Inhibition of T-type Ca2+ channels with mibefradil or by silencing expression resulted in growth suppression in ovarian cancer with simultaneous increase in apoptosis and decrease in expression of the anti-apoptotic gene survivin (BIRC5). Combining carboplatin with mibefradil resulted in increased apoptosis. Analysis of intracellular signaling revealed T-type Ca2+ channel inhibitors induced changes in the levels of FOXO proteins, which regulate BIRC5 gene expression. Mibefradil treatment caused greater nuclear retention of FoxO1 and FoxO3a, two transcription factors that repress BIRC5 expression, while protein expression of the transcription factor that activates BIRC5 expression, FoxM1, decreased. Chromatin immunoprecipitation of FOXO proteins from mibefradil treated ovarian cancer cells demonstrated binding of FoxM1 and FoxO1 within the BIRC5 (survivin) promoter. Together, the data show that T-type Ca2+ channels support ovarian cancer cell cycle, proliferation and regulate pro-survival pathways through the FOXO-survivin signaling axis, which has been reported to be deregulated in ovarian cancer. Moreover, our results provide the rationale for future use of T-type antagonists as sensitizing agents in combination with standard chemotherapeutics. Citation Format: Barbara Dziegielewska, Eli V. Casarez, Wesley Z. Yang, Jaroslaw Dziegielewski, Jill K. Slack-Davis. T-type Ca2+ channel inhibitors sensitize ovarian cancer to carboplatin through downregulation of survivin gene expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5407. doi:10.1158/1538-7445.AM2015-5407

Effect of lactoferrin protein on red blood cells and macrophages: mechanism of parasite-host interaction.

BackgroundLactoferrin is a natural multifunctional protein known to have antitumor, antimicrobial, and anti-inflammatory activity. Apart from its antimicrobial effects, lactoferrin is known to boost the immune response by enhancing antioxidants. Lactoferrin exists in various forms depending on its iron saturation. The present study was done to observe the effect of lactoferrin, isolated from bovine and buffalo colostrum, on red blood cells (RBCs) and macrophages (human monocytic cell line-derived macrophages THP1 cells).MethodsLactoferrin obtained from both species and in different iron saturation forms were used in the present study, and treatment of host cells were given with different forms of lactoferrin at different concentrations. These treated host cells were used for various studies, including morphometric analysis, viability by MTT assay, survivin gene expression, production of reactive oxygen species, phagocytic properties, invasion assay, and Toll-like receptor-4, Toll-like receptor-9, and MDR1 expression, to investigate the interaction between lactoferrin and host cells and the possible mechanism of action with regard to parasitic infections.ResultsThe mechanism of interaction between host cells and lactoferrin have shown various aspects of gene expression and cellular activity depending on the degree of iron saturation of lactoferrin. A significant increase (P<0.05) in production of reactive oxygen species, phagocytic activity, and Toll-like receptor expression was observed in host cells incubated with iron-saturated lactoferrin when compared with an untreated control group. However, there was no significant (P>0.05) change in percentage viability in the different groups of host cells treated, and no downregulation of survivin gene expression was found at 48 hours post-incubation. Upregulation of the Toll-like receptor and downregulation of the P-gp gene confirmed the immunomodulatory potential of lactoferrin protein.ConclusionThe present study details the interaction between lactoferrin and parasite host cells, ie, RBCs and macrophages, using various cellular processes and expression studies. The study reveals the possible mechanism of action against various intracellular pathogens such as Toxoplasma, Plasmodium, Leishmania, Trypanosoma, and Mycobacterium. The presence of iron in lactoferrin plays an important role in enhancing the various activities taking place inside these cells. This work provides a lot of information about targeting lactoferrin against many parasitic infections which can rule out the exact pathways for inhibition of diseases caused by intracellular microbes mainly targeting RBCs and macrophages for their survival. Therefore, this initial study can serve as a baseline for further evaluation of the mechanism of action of lactoferrin against parasitic diseases, which is not fully understood to date.

Inhibition of skin squamous cell carcinoma proliferation and promote apoptosis by dual silencing of NET-1 and survivin.

The simultaneous silencing of multiple upregulated genes is an attractive and viable strategy to treat many incurable diseases including cancer. In the present study, skin squamous cell carcinoma (SSCC) tissue microarray was constructed and the expression of NET-1 and survivin was identified. The high expression of NET-1 and survivin gene in SSCC was confirmed as an important event for the formation and development of the cancer. A total of 100 primary SSCC patients were included in the present study. Expression of NET-1 and survivin in cancer cells was evaluated immunohistochemically in tissue microarrays. The interaction between NET-1 and survivin in SSCC by co-immunoprecipitation was subsequently verified by producing the siRNA sequence targeting the single gene (siRNA-NET-1 and siRNA-survivin) as well as NET-1 and survivin gene (one-chain-double-target siRNA). The levels of NET-1 and survivin mRNA and protein expression in A431 cells were detected by RT-qPCR and western blotting, and the expression of related genes including vascular endothelial growth factor (VEGF), cortactin, Bcl-2, caspase-3 and -8 was identified using RT-qPCR. The protein localization and expression of NET-1 and survivin in A431 cells were documented by immunohistochemistry and immuno-fluorescence staining. The proliferation and apoptosis of A431 cells were detected by CCK-8 assay and flow cytometry (FCM). The tissue microarray showed that NET-1 and survivin were highly expressed in SSCC, while the correlation analysis showed NET-1 expression was positively associated with survivin. In addition, we reported that using the one-chain-double-target siRNA conjugate composed of NET-1 and survivin siRNA sequences in the same backbone inhibited proliferation and promoted apoptosis of SSCC. The one-chain-double-target siRNA showed further downregulation on NET-1 and survivin mRNA and protein levels compared with NET-1 siRNA or survivin siRNA. It also exhibited greater suppression on proliferation and triggering of apoptosis in A431 cells than NET-1 siRNA or survivin siRNA. This result may be explained by the significant downregulation of VEGF, cortactin and Bcl-2, and upregulation of caspase-3 and -8. NET-1 and survivin were overexpressed in SSCC and an interaction between NET-1 and survivin was identified. The one-chain-double-target siRNA appears to be superior in inhibiting cell proliferation and promoting apoptosis compared with the single target siRNA. NET-1 and survivin may have correlative signaling pathways with VEGF, cortactin, Bcl-2, caspase-3 and -8. Simultaneous silencing of NET-1 and survivin using one-chain-double-target siRNA thus provides an advantageous alternative in the development of therapeutics for SSCC.

Effect of lentivirus-mediated survivin transfection on the morphology and apoptosis of nucleus pulposus cells derived from degenerative human disc in vitro

Lower back pain is a common concern, and 40% of all cases involve the degeneration of the intervertebral disc (IVD). However, the excessive apoptosis of disc cells plays an important role in IVD degeneration, particularly in the nucleus pulposus (NP). Thus, anti-apoptotic gene therapy to attenuate or reverse the degenerative process within the NP is being developed. Survivin is a unique inhibitor of apoptosis (IAP) and has been extensively investigated in cancer cells. However, little is known of the effects of survivin transfection on NP cells derived from degenerative human disc. In this study, we aimed to investigate the effects of lentivirus (LV)-mediated survivin transfection on the morphology and apoptosis of NP cells derived from degenerative human disc in vitro. NP cells were transfected with LV-mediated survivin. Subsequently, cell morphology was observed and the survivin mRNA expression levels were measured by RT-qPCR. Apoptosis was analyzed by flow cytometry and by measuring caspase-3 activity. The results revealed that the morphology of the NP cells derived from degenerative human disc transfected with LV-mediated survivin was significantly altered as evidenced by cytomorphosis, the reduction of the cytoplasm and cell shrinkage. Following transfection, survivin gene expression significantly increased in the transfected cells and subsequent generation cells; however, no significant differences in the cell apoptotic rate and caspase-3 activity were observed. We found that transfection of the survivin gene into NP cells led to the stable expression of survivin and induced marked changes in cell morphology. Furthermore, no significant anti-apoptotic effects were observed following LV-mediated survivin transfection. Overall, our findings demonstrate that LV carrying surviving may be used to successfully enforce the expression of survivin in NP cells. However, cell morphology was evidently altered, whereas the apoptotic rate did not decrease. Comprehensive studies on the feasibility of using survivin in gene therapy in an aim to attenuate disc degeneration are warranted. Further research on the mechanisms responsible for the changes in cell morphology and cell function are also required.