Downregulation Of Survivin Research Articles

Periostin Contributes to Cisplatin Resistance in Human Non-Small Cell Lung Cancer A549 Cells via Activation of Stat3 and Akt and Upregulation of Survivin

Background/Aims: Periostin is upregulated in non-small cell lung cancer (NSCLC). This study was done to explore the function of periostin in the development of cisplatin (CDDP) resistance in NSCLC. Methods: The effects of overexpression or knockdown of periostin on CDDP sensitivity was examined in A549 cells. The involvement of signal transducer and activator of transcription 3 (Stat3) and Akt signaling in the action of periostin was checked. The in vivo effect of periostin silencing on CDDP susceptibility was determined in a mouse xenograft model. Results: Periostin was significantly upregulated in CDDP-resistant A549 cells, compared to parental controls. Overexpression of periostin rendered A549 cells more resistant to CDDP-induced apoptosis and enhanced Stat3 and Akt phosphorylation and survivin expression. Periostin-mediated protection against CDDP-induced apoptosis was compromised by downregulation of survivin. Furthermore, knockdown of periostin re-sensitized CDDP-resistant A549 cells to CDDP. After CDDP treatment, greater volume reduction was observed in periostin-silenced xenograft tumors than in control tumors, which was accompanied by reduced levels of phosphorylated Stat3 and survivin in periostin-depleted tumors. Conclusion: In conclusion, periostin promotes CDDP resistance in NSCLC cells largely through activation of Stat3 and Akt and upregulation of survivin and thus represents a promising target for overcoming CDDP resistance.

Survivin activates NF‑κB p65 via the IKKβ promoter in esophageal squamous cell carcinoma.

Survivin and transcription factor p65 (NF‑κB p65) participate in the progression of esophageal squamous cell carcinoma (ESCC). However, the mechanism of NF‑κB p65 activation in ESCC remains to be elucidated. The aim of the present study was to investigate the role of survivin in the activation of NF‑κB p65 in ESCC. The expression levels of survivin, NF‑κB p65, inhibitor of nuclear factor κB kinase subunit α (IKKα) and inhibitor of nuclear factor κB kinase subunit β (IKKβ) were detected in ESCC tissue samples. Eca109 and KYSE150 cells were cultured and survivin activity was modulated via transfection with an overexpression plasmid, a small hairpin RNA plasmid and a specific inhibitor. Quantitative reverse transcription-polymerase chain reaction and western blotting assays were conducted to assess the effects of survivin on the expression levels of IKKα, IKKβ and NF‑κB p65. Cell cycle and apoptosis assays were conducted to detect surviving-dependent cellular behavior changes. In addition, the luciferase reporter gene assay and chromatin immunoprecipitation assay were conducted to determine the genomic sites responsible for surviving-induced activation of NF‑κB p65. The present study demonstrated that the expression of survivin is positively correlated with IKKα and IKKβ in ESCC tissues. Survivin affected the mRNA and protein expression levels of IKKα, IKKβ, and NF‑κB p65 in Eca109 and KYSE150 cells. Furthermore, survivin increased the transcriptional activity of the IKKβ promoter and bound to the IKKβ promoter region in the Eca109 cells. Downregulation of survivin arrested the cell cycle at the G2/M phase and induced apoptosis. Results of the present study suggest that survivin activates NF‑κB p65 in Eca109 cells via binding to the IKKβ promoter region and upregulating IKKβ promoter transcriptional activity. Survivin overexpression activates NF‑κB p65, which is important in the acquisition and maintenance of the oncogenic characteristics of ESCC.

4′-Chlorodiazepam is neuroprotective against amyloid-beta through the modulation of survivin and bax protein expression in vitro

The translocator protein of 18kDa (TSPO) is located in the outer mitochondrial membrane and is involved in the cholesterol transport into the mitochondria and in the regulation of steroidogenesis, mitochondrial permeability transition pore opening and apoptosis. TSPO ligands have been investigated as therapeutic agents that promote neuroprotective effects in experimental models of brain injury and neurodegenerative diseases. The aim of this study was to identify the neuroprotective effects of 4′-chlorodiazepam (4′-CD), a ligand of TSPO, against amyloid-beta (Aβ) in SHSY-5Y neuroblastoma cells and its mechanisms of action. Aβ decreased the viability of SHSY-5Y neuroblastoma cells, while 4′-CD had a neuroprotective effect at the doses of 1nM and 10nM. The neuroprotective effects of 4′-CD against Aβ were associated with the inhibition of Aβ-induced upregulation of Bax and downregulation of survivin. In summary, our findings indicate that 4′-CD is neuroprotective against Aβ-induced neurotoxicity by a mechanism that may involve the regulation of Bax and survivin expression.

Survivin silencing and TRAIL expression using oncolytic adenovirus increase anti-tumorigenic activity in gemcitabine-resistant pancreatic cancer cells.

In this study, we demonstrated that survivin downregulation with TRAIL expression greatly enhanced the cytotoxic death of pancreatic cancer cells after gemcitabine treatment. Using real-time RT-PCR, we analyzed five survivin shRNAs to identify the best target sequence for suppression of human survivin, with the goal of treating gemcitabine-resistant pancreatic cancer cells. Survivin shRNA 5, corresponding to target 5, showed the greatest reduction in survivin mRNA levels. Furthermore, combined treatment with survivin shRNA-expressing adenovirus with gemcitabine plus TRAIL decreased uncleaved PARP and increased consequent PARP cleavage, which was correlated with the greatest levels of survivin downregulation and cell death. These results indicate that survivin functions as a common mediator of gemcitabine- and TRAIL-induced cell death. Using a nude mouse model implanted with MiaPaCa-2 pancreatic cancer cells, we observed tumor regression induced by an oncolytic adenovirus expressing survivin shRNA and TRAIL plus gemcitabine. Together, our findings provide a strong rationale for treating pancreatic cancer patients with both gemcitabine and oncolytic adenovirus armed with survivin shRNA and TRAIL.

Effect of survivin knockdown on apoptosis and spatial learning and memory of mice with traumatic brain injury

Objective To examine the effect of survivin downregulation by adenovirus-mediated RNA interference on apoptosis in hippocampal dentate gyrus and spatial learning and memory of mice with traumatic brain injury (TBI). Methods A total of 112 adult male mice were allocated into 4 groups (28 animals each) according to the random number table. Fluid percussion model of TBI was used in the study. In negative control group, animals were stereotactically injected with control recombinant adenovirus immediately after injury. In survivin knockdown group, the animals were stereotactically injected with shRNA adenovirus immediately after injury. In sham group, animals were subjected to identical surgery without fluid percussion injury. In TBI group, animals were subjected to isolated fluid percussion injury. Real-time fluorescent quantitative polymerase chain reaction (RT-PCR), Western blot analysis and immunofluorescence staining were used to detect expression level of survivin in mouse brain tissue. TUNEL assay was used for measuring apoptotic cells in hippocampal denate gyrus. Morris water maze test was adopted to evaluate mouse learning and memory after surviving downregulation. Results Expression levels of survivin mRNA and protein in survivin knockdown group reduced at days 3 postinjury compared to negative control group (1.21±0.11 vs.4.05±0.30; 0.34±0.08 vs.1.11±0.17, respectively) (P<0.01). Immunofluorescence staining showed survivin-positive cells calculated 267.45±38.41 in survivin knockdown group, lower than 896.56±102.23 in negative control group (P<0.01). TUNEL-positive cells in dentate gyrus of the hippocampus in survivin knockdown group increased at days 3 postinjury compared to negative control group (416.1±29.0 vs. 250.2±20.8) (P<0.01). Morris water maze test confirmed that survivin downexpression did lead to a statistically significant changes in escape latency (P<0.05) and target quadrant (P<0.01). Conclusion Decreased expression of survivin may promote apoptotic cell death and result in a negative role in the recovery of learning and memory function of mice after TBI. Key words: Mice, knockout; Brain injuries; Apoptosis; Memory function

Microrna-16 Mediates the Regulation of a Senescence-Apoptosis Switch in Cutaneous T-Cell and Other Non-Hodgkin Lymphomas

Multiple sequential genetic and epigenetic alterations, including alterations in microRNAs (miRNAs), underlie the development and progression of cancers. For example, overcoming cellular senescence is an early step in cancer pathogenesis. We show here that a tumor-suppressive miRNA, microRNA-16/miR-16 has a potential to induce cellular senescence in cutaneous T-cell lymphoma (CTCL). Firstly, to examine the role of miR-16 in the progression of primary CTCL, we performed q-PCR analysis against early (n=26) and advanced (n=14) Mycosis Fungoides samples. As for control (CD4+ T-cells), we used samples from patients with atopic dermatitis (AD; n=18). We found that expression of miR-16 in both early and advanced stages were significantly lower than AD, with the expression of advanced showing significantly more reduced than early stage. These results suggest that miR-16 is associated with lymphomagenesis from early to advanced stages of CTCL. To examine in vivo tumor-suppressive effects of miR-16, we used NOD/Shi-scid IL2Rgnull (NOG) mouse xenograft model, which would die around day 30-40 after CTCL cells (My-La, MJ, HH or HUT78) injection by invasion and metastasis (Ito et al, Blood 2014). By use of the xenograft model, we found that miR-16 transduced CTCL cells transplanted NOG mice showed significant prolonged survivals than control xenograft mice. We previously demonstrated that miR-16 did not inhibit migration capability of CTCL cells. When we considered that miR-16 successfully contribute to extend survival of mice models despite of lacking migration-inhibition capability, the result suggested that other tumor suppressive mechanisms might exist. Therefore we conducted functional analysis of miR-16, and found that miR-16 could induce cellular senescence against CTCL cells. In CTCL cells that expressed wild-type p53 (My-La and MJ), forced expression of miR-16 enhanced p21, which is a key mediator of senescence, in a p53 dependent manner. Because miR-16 family members are also able to directly suppress the polycomb group protein Bmi1, which is known to negatively regulate p21, we assessed Bmi1 expression in miR-16-transduced CTCL cells and found it to be diminished in all four CTCL cell lines tested. When we knocked down BMI1, expression of p21 was elevated in cells expressing wild-type p53 and the incidence of senescence was increased. We further demonstrated that Bmi1 could directly interact with the promoter lesion of CDKN1A/p21 by cross-linking/chromatin immunoprecipitation assays. From these results, miR-16 has potential to induce cellular senescence via targeting Bmi1-p21 pathway in p53-wild type CTCL cells. Although miR-16 could not induce senescence in p53 non-functional CTCL cells (HH and HUT78), in that case, we found that miR-16 can compensatory induce apoptosis by negative regulation of Bmi1-Survivin pathway. To elucidate this senescence-apoptosis switch mechanism, we examined co-transfection of miR-16 and siCDKN1A/p21 against p53 wild-type CTCL cells. We found that co-transfection of miR-16 and siCDKN1A significantly decreased senescent cells and increased apoptotic cells, with downregulation of Bmi1 and Survivin. In addition, miR-16 negatively regulated cyclinD1-Rb pathway regardless of p53 status, leading to cell growth inhibition against CTCL cells. Together, these results strongly support our idea that presence or absence of functional p53-p21 is an essential factor in miR-16-mediated senescence-to-apoptosis switch in CTCL. Finally, because a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA, vorinostat) has been recently applied and marks good responses in advanced CTCL, we tried to examine whether SAHA might share essential targets with miR-16 in CTCL lymphomagenesis. We found that SAHA restored miR-16 and its essential targets (p21, Bmi1, Survivin), and induced senescence in CTCL cells with wild-type p53 and apoptosis in cells with non-functional p53. Moreover, we found that T or NK/T-cell lymphoma cells (n=8) derived from various subtypes also showed similar tumor-suppressive effects by miR-16 transduction or SAHA treatment. In conclusion, through detailed study of miR-16 function, we have shed light on the mechanism by which lymphoma cells avoid senescence. Furthermore, identification of essential SAHA targets may provide supporting evidence of its clinical application for various non-Hodgkin T/NK-cell lymphomas. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Magnetic Nanoparticles of Fe3O4 Enhance Artesunate-inducing Apoptosis of SKM-1 Cells.

To investigate the inhibitory effect of the copolymer of magnetic nanoparticles of Fe(3)O(4) (MNPs-Fe(3)O(4)) and artesunate (ART) on myelodysplastic syndromes (MDS) cell line SKM-1 cells and the potential mechanisms. The protein expression levels of BCL-2, BAX, Caspase-3, and Survivin in SKM-1 cells treated with or without the co-polymer were measured by Western blot. The co-polymer-induced apoptosis rate of SKM-1 cells was measured by flow cytometry. The apoptosis rate of SKM-1 cells in the copolymer groups was higher than that in both MNPs-Fe(3)O(4) and artesunate groups alone. The MNPs-Fe(3)O(4) may enhance ART-induced cell apoptosis. Western blot assay showed that the expression of survivin and BCL-2 protein were down-regulated in the ART group, and this down-regulation was even more significant in the group of copolymer of ART with MNPs-Fe(3)O(4). The levels of BAX were increased both in ART group and the copolymer of ART with MNPs-Fe(3)O(4) group, as compared with control group and MNPs-Fe(3)O(4) group. The levels of active-caspase-3 were obviously up-regulated when the ART was combined with the MNPs-Fe(3)O(4). The copolymer of ART with MNPs-Fe(3)O(4) could trigger changes in the expression levels of apoptosis-related genes in SKM-1 cells, among which up-regulation of BAX and down-regulation of survivin and BCL-2 are the 2 major alterations. Artesunate can induce the apoptosis of SKM-1 cells, and MNPs-Fe(3)O(4) may enhance the cell apoptosis induced by ART.

BPIQ, a novel synthetic quinoline derivative, inhibits growth and induces mitochondrial apoptosis of lung cancer cells in vitro and in zebrafish xenograft model.

Background2,9-Bis[2-(pyrrolidin-1-yl)ethoxy]-6-{4-[2-(pyrrolidin-1-yl)ethoxy] phenyl}-11H-indeno[1,2-c]quinolin-11-one (BPIQ) is a derivative from 6-arylindeno[1,2-c]quinoline. Our previous study showed the anti-cancer potential of BPIQ compared to its two analogues topotecan and irinotecan. In the study, the aim is to investigate the potency and the mechanism of BPIQ against lung cancer cells.MethodsBoth in vitro and zebrafish xenograft model were performed to examine the anti-lung cancer effect of BPIQ. Flow cytometer-based assays were performed for detecting apoptosis and cell cycle distribution. Western blot assay was used for detecting the changes of apoptotic and cell cycle-associated proteins. siRNA knockdown assay was performed for confirming the apoptotic role of Bim.ResultsBoth in vitro and zebrafish xenograft model demonstrated the anti-lung cancer effect of BPIQ. BPIQ-induced proliferative inhibition of H1299 cells was achieved through the induction of G2/M-phase arrest and apoptosis. The results of Western blot showed that BPIQ-induced G2/M-phase arrest was associated with a marked decrease in the protein levels of cyclin B and cyclin-dependent kinase 1 (CDK1). The up-regulation of pro-apoptotic Bad, Bim and down-regulation of pro-survival XIAP and survivin was observed following BPIQ treatment.ConclusionsBPIQ-induced anti-lung cancer is involved in mitochondrial apoptosis. BPIQ could be a promising anti-lung cancer drug for further applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1970-x) contains supplementary material, which is available to authorized users.

FOXM1 targets XIAP and Survivin to modulate breast cancer survival and chemoresistance

Drug resistance is a major hurdle for successful treatment of breast cancer, the leading cause of deaths in women throughout the world. The FOXM1 transcription factor is a potent oncogene that transcriptionally regulates a wide range of target genes involved in DNA repair, metastasis, cell invasion, and migration. However, little is known about the role of FOXM1 in cell survival and the gene targets involved. Here, we show that FOXM1-overexpressing breast cancer cells display an apoptosis-resistant phenotype, which associates with the upregulation of expression of XIAP and Survivin antiapoptotic genes. Conversely, FOXM1 knockdown results in XIAP and Survivin downregulation as well as decreased binding of FOXM1 to the promoter regions of XIAP and Survivin. Consistently, FOXM1, XIAP, and Survivin expression levels were higher in taxane and anthracycline-resistant cell lines when compared to their sensitive counterparts and could not be downregulated in response to drug treatment. In agreement with our in vitro findings, we found that FOXM1 expression is significantly associated with Survivin and XIAP expression in samples from patients with IIIa stage breast invasive ductal carcinoma. Importantly, patients co-expressing FOXM1, Survivin, and nuclear XIAP had significantly worst overall survival, further confirming the physiological relevance of the regulation of Survivin and XIAP by FOXM1. Together, these findings suggest that the overexpression of FOXM1, XIAP, and Survivin contributes to the development of drug-resistance and is associated with poor clinical outcome in breast cancer patients.

Therapeutic potential of thalidomide for gemcitabine-resistant bladder cancer.

Controversial effects of thalidomide for solid malignancies have been reported. In the present study, we evaluate the effects of thalidomide for transitional cell carcinoma (TCC), the most common type of bladder cancer. Thalidomide precipitates were observed when its DMSO solution was added to the culture medium. No precipitation was found when thalidomide was dissolved in 45% γ-cyclodextrin, and this concentration of γ-cyclodextrin elicited slight cytotoxicity on TCC BFTC905 and primary human urothelial cells. Thalidomide-γ-cyclodextrin complex exerted a concentration-dependent cytotoxicity in TCC cells, but was relatively less cytotoxic (with IC50 of 200 µM) in BFTC905 cells than the other 3 TCC cell lines, possibly due to upregulation of Bcl-xL and HIF-1α mediated carbonic anhydrase IX, and promotion of quiescence. Gemcitabine-resistant BFTC905 cells were chosen for additional experiments. Thalidomide induced apoptosis through downregulation of survivin and securin. The secretion of VEGF and TNF-α was ameliorated by thalidomide, but they did not affect cell proliferation. Immune-modulating lenalidomide and pomalidomide did not elicit cytotoxicity. In addition, cereblon did not play a role in the thalidomide effect. Oxidative DNA damage was triggered by thalidomide, and anti-oxidants reversed the effect. Thalidomide also inhibited TNF-α induced invasion through inhibition of NF-κB, and downregulation of effectors, ICAM-1 and MMP-9. Thalidomide inhibited the growth of BFTC905 xenograft tumors in SCID mice via induction of DNA damage and suppression of angiogenesis. Higher average body weight, indicating less chachexia, was observed in thalidomide treated group. Sedative effect was observed within one-week of treatment. These pre-clinical results suggest therapeutic potential of thalidomide for gemcitabine-resistant bladder cancer.

Oral delivery of shRNA based on amino acid modified chitosan for improved antitumor efficacy

In this investigation, chitosan–histidine–cysteine (CHC) was engineered for oral delivery of Survivin short hairpin RNA (shRNA)-expressing plasmid DNA (shSur-pDNA) to promote hepatoma regression through integrating the advantages of histidine and cysteine to conquer serial cellular and systemic barriers. CHC could effectively encapsulate shSur-pDNA to form compact nanocomplexes (NC) at adequate weight ratios. Sequential modification with histidine and cysteine conferred CHC NC with the beneficial attributes for shRNA delivery including improved stability, facilitated internalization, promoted endosomal escape, increased nuclear localization, and GSH-responsive release, which contributed to their superior performance in terms of apoptosis promotion, proliferation inhibition, and Survivin down-regulation of tumor cells. More importantly, in hepatoma-bearing mice, orally delivered CHC NC overweighed chitosan counterparts with respect to suppressed Survivin expression, retarded tumor growth, and prolonged surviving time, owing to their above-mentioned merits in combination with enhanced intestinal permeation. Especially, rapid intracellular release of CHC NC with lower molecular weight of 30 kDa (CHC30 NC) might be responsible for the most satisfactory antitumor efficacy with tumor inhibition ratio (TIR) of 92.5%, which rendered CHC30 NC a promising vehicle for oral delivery of shRNA. This investigation would shed light on the deliberate design of oral shRNA delivery vehicles to mediate effective antitumor efficacy.

Abstract 20: Honokiol radiosensitizes squamous cells carcinoma of head and neck by down-regulation of survivin

Abstract Background: Overexpression of survivin is associated with resistance to radiotherapy and poor survival in many cancer types. However, in squamous cells carcinoma of head and neck (SCCHN), previous studies have revealed diverging results regarding survivin's role in clinical outcome. In this study, we aim to 1) evaluate the clinical significance of survivin expression in SCCHN; 2) identify the function of survivin on DNA damage repair following ionizing radiation (IR) in SCCHN cells; 3) investigate the potential of using honokiol, a nature compound, to enhance radiation therapy through down regulation of survivin. Methods: Protein expression of survivin in SCCHN patient primary tissues (n = 99) was analyzed by immunohistochemistry and correlated with clinical parameters. IR-sensitive (JHU-022), IR -insensitive (PCI-15A, Tu212), and IR acquired resistance (PCI-15A-R) SCCHN cell lines were used to evaluate the role of survivin in DNA double strain break repairer following RT. The in vitro effects of and potential molecular targets of honokiol were also evaluated using those cell lines. Results: Overexpression of survivin is significantly associated with lymph node metastasis, worse overall survival and worse disease free survival in patients receiving radiation therapy (n = 65) and in all patients with SCCHN (n = 99). In SCCHN cells, depletion of survivin by siRNA inhibited, whereas overexpression of survivin increased, clonogenic survival following IR. IR induces nuclear accumulation of survivin and complexation with γ-H2X, and DNA-PKCs, suggesting survivin is involved in DNA damage response and repair induced by IR. Further, honokiol significantly reduces survivin expression and increases DNA damage induced by IR, as demonstrated by higher amount of DNA breaks and an increased amount of γ-H2X foci post IR. Conclusions: Survivin is a negative prognostic factor in SCCHN, and is involved in DNA damage response and repair induced by IR in SCCHN cells. Down regulation of survivin by honokiol enhances the efficacy of IR, and may provide a novel therapeutic approach to improve the efficacy of radiotherapy in SCCHN. (This research was supported by the National Cancer Institute award P50 CA128613, and GCC Distinguished Cancer Scholar to Dong M. Shin, Zhuo (Georgia) Chen, and Jonathan J Beitler) Citation Format: Xu Wang, Jonathan J. Beitler, Wen Huang, Guoqing Qian, Kelly Magliocca, Jun Zhang, Sreenivas Nannapaneni, Sungjin Kim, Zhengjia Chen, Saba F. Nabil, Zhuo G. Chen, Jack L. Arbiser, Dong M. Shin. Honokiol radiosensitizes squamous cells carcinoma of head and neck by down-regulation of survivin. [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 20. doi:10.1158/1538-7445.AM2015-20

Abstract 2532: Influence of survivin-targeted therapy on chemosensitivity in the treatment of acute myeloid leukemia

Abstract Background: Survivin is a dual functional protein. It acts as a critical inhibitor of apoptosis and a key cell cycle regulator. In general, survivin expresses in embryonic tissues during development and is undetectable in most terminally differentiated tissues. It plays a pivotal role in normal hematopoiesis; however, overexpression of survivin has been reported in various hematological malignancies, including acute myeloid leukemia (AML). Studies with AML patients indicate that elevated expression of survivin correlates with a worse clinical outcome. To date, no study initiated to test the efficacy of survivin-targeted therapy in AML patients; and it remains unclear whether downregulation/inactivation of survivin may alter chemosensitivity against AML. Thus, we evaluate the effects of specific knockdown of survivin expression on AML cells and the AML cells’ responsiveness to chemotherapy. We have also investigated the therapeutic potential of the survivin inhibitor YM155, which is under clinical trials in a variety of human cancers, either alone or in combination with the commonly used chemotherapeutic agents in the treatment of AML. Material and Methods: Traditional PCR and real-time PCR were used to measure mRNA levels in AML cell lines. Western blot analyses were performed to determine protein expression and activation. Cell death was quantified by a specific apoptotic ELISA (enzyme linked immunosorbent assay). Flow cytometric analyses were used to explore cell cycle progression. Cell Titer AQueous Non-Radioactive Cell Proliferation Assays (MTS) were carried out to evaluate changes of cell proliferation. Results: Kasumi-1 and HL-60 cells showed relatively higher expression levels of survivin among all AML cell lines tested. Specific knockdown of survivin expression in Kasumi-1 and HL-60 cells resulted in: inhibition of cell proliferation; cell cycle G2/M arrest; upregulation of DNA damage response and apoptotic-related proteins; and increased histone-associated DNA fragmentation. Downregulation of survivin enhanced AML cells’ sensitivity to etoposide- or doxorubicin-induced anti-proliferative/anti-survival activity. The small inhibitor YM155 trigged dose- and time-dependent reduction of survivin and apoptosis in both Kasumi-1 and HL-60 cells. The combinatorial effects of YM155 and chemotherapeutics were either synergetic or antagonistic, depending on the drugs used for combination and the type of cells being treated. Conclusions: These data demonstrate that survivin is important for the maintenance and proliferation of AML cells. Downregulation of survivin in AML cells enhances chemosensitivity. The combinatorial effects of YM155 and chemotherapeutics can be synergetic or antagonistic, depending on the drugs and the AML cell types. Our findings provide a rationale for further evaluation of survivin-targeted therapy in the treatment of patients with AML. Citation Format: Jingcao Huang, Hui Lyu, Ying Wu, Bolin Liu. Influence of survivin-targeted therapy on chemosensitivity in the treatment of acute myeloid leukemia. [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 2532. doi:10.1158/1538-7445.AM2015-2532

Abstract 4139: Role of Akt2 in cell survival and metastasis of colorectal cancer (CRC) cells

Abstract Background: Colorectal Cancer (CRC) is the second highest cause of cancer related deaths in United States. Metastatic dissemination of CRC cells to distant organs (primarily to the liver and lungs) makes the disease more lethal and incurable. Inappropriate cell survival signaling mechanisms contribute to successful colonization of cells at metastatic sites. Akt is a major cell survival-signaling node, which undergoes aberrant activation in various types of cancer. The Akt family has three members: Akt1, Akt2 and Akt3 which share structural homology and have similar mechanism of activation; however, differences in the phenotype of Akt1, Akt2 and Akt3 knock out mice suggest that they have non-redundant functional characteristics. Recently Akt2 has been identified as a significant contributor to colon cancer malignancy in addition to Akt1. Earlier reports have shown that knockdown of Akt2 results in reduced liver metastasis of CRC cells, however there are no studies related to the mechanism responsible for the reduction in metastasis. We observed a significant increase in the expression of Akt2 (microarray analysis) in liver metastatic samples formed by highly metastatic IGF1R-dependent CRC cells in comparison to the primary tumor. This was further confirmed by western blot and immunohistochemistry (IHC) analysis. Additionally, correlative studies using human primary colon carcinoma and corresponding liver metastatic samples showed a significant (p&amp;lt;0.001) increase in the expression of Akt2. These preliminary data led us to hypothesize that Akt2 may be playing a role in regulating cell survival and metastasis of CRC cells. Materials and Methods: IGF1R-dependent CRC cells (GEO and CBS) were used for the study. Two doxycycline inducible shRNAs for Akt2 were introduced by lentiviral transfection. Non-Targeting shRNA was used as control. Results: shRNA-mediated knockdown of Akt2 led to reduction in the expression of XIAP and survivin. We and others have shown that these IAPs (XIAP and survivin) are implicated to play an important role in cancer cell survival and metastasis. Furthermore, loss of Akt2 resulted in loss of pBad (S136), which is an essential cell survival marker. Loss of pBad results in increased cell death by PARP activation. Akt2 knockdown also affects cell survival by increasing p21 and decreasing the expression of cyclin E. PCR array analysis for metastatic genes revealed that a metastatic suppressor gene MTSS1 was 9 fold increased on knockdown of Akt2. It has been shown that MTSS1 inhibits metastasis by affecting cell junction assembly and stability. Conclusion: We have shown that loss of Akt2 promotes cell death and inhibits cell proliferation. This is the first study, which suggests the mechanism of Akt2 mediated cell death by XIAP and survivin downregulation. These results suggest that Akt2 inhibits metastasis by increasing the expression of MTSS1. Citation Format: Ekta Agarwal. Role of Akt2 in cell survival and metastasis of colorectal cancer (CRC) cells. [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 4139. doi:10.1158/1538-7445.AM2015-4139

EpCAM Aptamer-siRNA Chimera Targets and Regress Epithelial Cancer.

Epithelial cell adhesion molecule (EpCAM), a cancer stem cell (CSC) marker is over expressed in epithelial cancers and in retinoblastoma (RB). We fabricated an EpCAM targeting aptamer-siRNA chimera and investigated its anti-tumor property and EpCAM intracellular domain (EpICD) mediated signaling in epithelial cancer. The anti-tumor efficacy of EpCAM aptamer-siEpCAM chimera (EpApt-siEp) was evaluated by qPCR, northern and Western blotting in WERI-Rb1- RB cell line, primary RB tumor cells and in MCF7- breast cancer cell line. Anti-tumor activity of EpApt-siEp was studied in vivo using epithelial cancer (MCF7) mice xenograft model. The mechanism and pathways involved in the anti-tumor activity was further studied using protein arrays and qPCR. EpApt-siEp chimera was processed in vitro by dicer enzyme. Treatment of the WERI-Rb1 and MCF7 cells with EpApt-siEp revealed statistically significant down regulation of EpCAM expression (P<0.005) and concomitant reduction in cellular proliferation. In primary RB cells cultured from RB tumors, EpApt-siEp silenced EpCAM, significantly inhibited (P<0.01) cell proliferation and induced cytotoxicity. Knockdown of EpICD expressed in RB primary tumors led to repression of pluripotency markers, SOX2, OCT4, NANOG, and CD133. In vivo studies showed complete tumor growth regression without any toxicity in animals (P<0.001) and tumor tissues showed significant downregulation (P<0.05) of EpCAM, MRP1, ABCG2, stathmin, survivin and upregulation of ATM (P<0.05) leading to apoptosis by intrinsic pathway with minor alteration in cytokines. Our results revealed that EpApt-siEp potentially eradicated EpCAM positive cancer cells through CSC marker suppression and apoptosis, while sparing normal EpCAM negative adjacent cells.

Tanshinone IIA enhances the effects of TRAIL by downregulating survivin in human ovarian carcinoma cells

BackgroundTanshinone IIA (TIIA), a diterpene quinone from the medicinal plant Salvia miltiorrhiza Bunge (Lamiaceae) was shown to possess apoptotic and TRAIL-sensitizing effects. Still, the molecular mechanisms whereby TIIA induces apoptosis remain largely unknown. PurposeThe role of survivin, an inhibitor of apoptosis protein, in TIIA-induced apoptosis has never been addressed before and hence was the primary goal of this study. MethodsIn this study, we explored the anticancer effect of TIIA in TOV-21G, SKOV3, and OVCAR3 ovarian carcinoma cells. Cytotoxicity was determined by MTS assay. Real-time RT-PCR and Western blotting were used to assess the mRNA and protein expression of related signaling proteins. ResultsOur results illustrated that TIIA's cytotoxic effect was caused by apoptosis with the involvement of caspases activity. Moreover, TIIA downregulated survivin in a concentration-dependent manner without affecting the expression of Bcl-2, Bcl-xL, and Bax. TIIA-induced survivin downregulation is regulated by both transcriptional processes and proteasomal degradation. Using TOV-21G cells as our cellular model, we demonstrated that TIIA-induced survivin downregulation requires p38 MAPK activation. Importantly, genetic overexpression of survivin rendered cells more resistant to TIIA, indicating an essential role of survivin downregulation in TIIA-induced apoptosis. This TRAIL sensitization effect of TIIA is ascribed to survivin downregulation because the effect was abrogated in cells that overexpressed survivin. ConclusionOur findings provide new insights into the action modes of TIIA-mediated anticancer effects and further implicate a rational design for cancer therapeutic regimens by combining TIIA-sensitized TRAIL via downregulating survivin to elicit ovarian cancer cell death.

Influence of survivin-targeted therapy on chemosensitivity in the treatment of acute myeloid leukemia

Overexpression of survivin is observed in various hematological malignancies, including acute myeloid leukemia (AML). Studies show that elevated expression of survivin correlates with a worse clinic outcome in AML patients. It remains unclear whether inhibition of survivin may alter the efficacy of chemotherapy against AML. Here, we evaluate the effects of specific knockdown of survivin on AML cells' sensitivity to chemotherapy, and investigate the therapeutic potential of the transcription inhibitor of survivin YM155 either alone or in combination with chemotherapeutic agents. We found Kasumi-1 and HL-60 cells had relatively higher expression levels of survivin among all AML cell lines tested. Specific knockdown of survivin in Kasumi-1 and HL-60 cells resulted in: inhibition of cell proliferation; cell cycle G2/M arrest; induction of DNA damage response and apoptosis. Downregulation of survivin enhanced etoposide- or doxorubicin-induced anti-proliferative/anti-survival activity in AML cells. The small molecule inhibitor YM155 reduced survivin in a dose- and time-dependent manner and trigged apoptosis in Kasumi-1 and HL-60 cells. The combinatorial effects of YM155 and chemotherapeutics were either synergetic or antagonistic, depending upon the drugs used for combination and the type of AML cells being treated. Collectively, our data demonstrate that survivin plays an important role in the maintenance and proliferation of AML cells. While specific knockdown of survivin enhances chemosensitivity, the combinations of YM155 and chemotherapeutic agents exhibit synergetic or antagonistic effects on AML cells. Our findings provide a rationale for further assessment of survivin-targeted therapy in the treatment of patients with AML.

Guggulsterone inhibits human cholangiocarcinoma Sk-ChA-1 and Mz-ChA-1 cell growth by inducing caspase-dependent apoptosis and downregulation of survivin and Bcl-2 expression.

Guggulsterone has recently been reported to demonstrate anti-tumor effects in a variety of cancers. The present study aims to investigate the biological roles and underlying mechanism of the action of guggulsterone in cholangiocarcinoma. The immortalized human cholangiocarcinoma Sk-ChA-1 and Mz-ChA-1 cell lines were treated with various concentrations of the trans isomer of guggulsterone, Z-guggulsterone. Cellular proliferation was determined using the XTT assay. The apoptotic status of cholangiocarcinoma cells was assessed by Hoechst 33258 staining, DNA fragmentation assay and flow cytometry. Specific caspase inhibitor was used to explore the role of caspase in guggulsterone-induced apoptosis. A colorimetric assay was performed to measure the alterations of the activities of caspase-3, -8 and -9. Western blot analysis was used to detect the protein expression of survivin, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein and cleaved poly (adenosine diphosphate-ribose) polymerase (PARP). As revealed by the present data, guggulsterone significantly inhibited the growth of the two human cholangiocarcinoma cell lines by inducing cellular apoptosis. In addition, guggulsterone-induced apoptosis of cholangiocarcinoma cells was demonstrated to be partially inhibited by the caspase inhibitors z-VAD-fmk, z-LEHD-fmk and z-IETD-fmk, accompanied by the activation of caspases-3, -8 and -9, accumulation of cleaved PARP and decreased expression of survivin and Bcl-2. In conclusion, the present study demonstrated that guggulsterone was able to suppress the proliferation of cholangiocarcinoma by inducing caspase-dependent apoptosis and downregulating survivin and Bcl-2.

S6K1 inhibition enhances the apoptotic cell death of breast cancer cells in response to Bcl-2/Bcl-xL inhibition by the downregulation of survivin.

Breast cancer cells possess a deregulated apoptotic pathway with increased expression levels of anti-apoptotic B-cell lymphoma-2 (Bcl-2) family proteins and ribosomal S6 kinase 1 (S6K1) protein activity. Therefore, combined interference of anti-apoptotic Bcl-2 family and S6K1 protein expression may be a reasonable therapeutic strategy for the treatment of patients with breast cancer. In the present study, it was identified that the administration of a combination of ABT263 [navitoclax; a Bcl-2/Bcl-extra large (Bcl-xL) inhibitor] and PF4708671 (an S6K1 inhibitor) markedly increased apoptotic cell death in the BT474 breast cancer cells compared with the administration of either agent alone. Furthermore, the downregulation of Bcl-2/Bcl-xL and S6K1 with small interfering RNA induced a significant increase in cell death compared with RNA interference of either agent alone. Notably, combination treatment with ABT263 and PF4708671 decreased the expression level of survivin protein, with this ectopic expression of survivin attenuating cell death. Thus, the present study determined that the combined inhibition of Bcl-2/Bcl-xL and S6K1 may be a good strategy for treating patients with breast cancer.

IRF-1 inhibits NF-κB activity, suppresses TRAF2 and cIAP1 and induces breast cancer cell specific growth inhibition

Interferon Regulatory Factor (IRF)-1, originally identified as a transcription factor of the human interferon (IFN)-β gene, mediates tumor suppression and may inhibit oncogenesis. We have shown that IRF-1 in human breast cancer cells results in the down-regulation of survivin, tumor cell death, and the inhibition of tumor growth in vivo in xenogeneic mouse models. In this current report, we initiate studies comparing the effect of IRF-1 in human nonmalignant breast cell and breast cancer cell lines. While IRF-1 in breast cancer cells results in growth inhibition and cell death, profound growth inhibition and cell death are not observed in nonmalignant human breast cells. We show that TNF-α or IFN-γ induces IRF-1 in breast cancer cells and results in enhanced cell death. Abrogation of IRF-1 diminishes TNF-α and IFN-γ-induced apoptosis. We test the hypothesis that IRF-1 augments TNF-α-induced apoptosis in breast cancer cells. Potential signaling networks elicited by IRF-1 are investigated by evaluating the NF-κB pathway. TNF-α and/or IFN-γ results in decreased presence of NF-κB p65 in the nucleus of breast cancer cells. While TNF-α and/or IFN-γ can induce IRF-1 in nonmalignant breast cells, a marked change in NF-κB p65 is not observed. Moreover, the ectopic expression of IRF-1 in breast cancer cells results in caspase-3, -7, -8 cleavage, inhibits NF-κB activity, and suppresses the expression of molecules involved in the NF-κB pathway. These data show that IRF-1 in human breast cancer cells elicits multiple signaling networks including intrinsic and extrinsic cell death and down-regulates molecules involved in the NF-κB pathway.