The survival of Amblyomma sculptum ticks upon blood-feeding depends on the expression of an inhibitor of apoptosis protein.

BACKGROUND: The tick Amblyomma sculptum is the major vector of Rickettsia rickettsii, the causative agent of the highly lethal Brazilian spotted fever. It has been shown that R. rickettsii inhibits apoptosis in both human endothelial cells and tick cells. Apoptosis is regulated by different factors, among which inhibitors of apoptosis proteins (IAPs) play a central role. In the study reported here, we selected an IAP of A. sculptum that has not yet been characterized to assess its role in cell death and to determine the effects of its gene silencing on tick fitness and R. rickettsii infection. METHODS: An A. sculptum cell line (IBU/ASE-16) was treated with specific double-stranded RNA (dsRNA) for either IAP (dsIAP) or green fluorescent protein (dsGFP; as a control). The activity of caspase-3 and the exposure of phosphatidylserine were determined in both groups. In addition, unfed adult ticks, infected or not infected with R. rickettsii, were treated with either dsIAP or dsGFP and allowed to feed on noninfected rabbits. In parallel, noninfected ticks were allowed to feed on an R. rickettsii-infected rabbit. Ticks (infected or not with R. rickettsii) that remained unfed were used as a control. RESULTS: Caspase-3 activity and the externalization of phosphatidylserine were significantly higher in IBU/ASE-16 cells treated with dsIAP than in those treated with dsGFP. The mortality rates of ticks in the dsIAP group were much higher than those in the dsGFP group when they were allowed to feed on rabbits, independent of the presence of R. rickettsii. Conversely, lower mortality rates were recorded in unfed ticks. CONCLUSIONS: Our results show that IAP negatively regulates apoptosis in A. sculptum cells. Moreover, IAP-silenced ticks experienced higher mortality rates following the acquisition of a blood meal, suggesting that feeding may trigger the activation of apoptosis in the absence of this physiological regulator. These findings indicate that IAP is a potential antigen for an anti-tick vaccine.

The LQB-223 Compound Modulates Antiapoptotic Proteins and Impairs Breast Cancer Cell Growth and Migration.

Drug resistance represents a major issue in treating breast cancer, despite the identification of novel therapeutic strategies, biomarkers, and subgroups. We have previously identified the LQB-223, 11a-N-Tosyl-5-deoxi-pterocarpan, as a promising compound in sensitizing doxorubicin-resistant breast cancer cells, with little toxicity to non-neoplastic cells. Here, we investigated the mechanisms underlying LQB-223 antitumor effects in 2D and 3D models of breast cancer. MCF-7 and MDA-MB-231 cells had migration and motility profile assessed by wound-healing and phagokinetic track motility assays, respectively. Cytotoxicity in 3D conformation was evaluated by measuring spheroid size and performing acid phosphatase and gelatin migration assays. Protein expression was analyzed by immunoblotting. Our results show that LQB-223, but not doxorubicin treatment, suppressed the migratory and motility capacity of breast cancer cells. In 3D conformation, LQB-223 remarkably decreased cell viability, as well as reduced 3D culture size and migration. Mechanistically, LQB-223-mediated anticancer effects involved decreased proteins levels of XIAP, c-IAP1, and Mcl-1 chemoresistance-related proteins, but not survivin. Survivin knockdown partially potentiated LQB-223-induced cytotoxicity. Additionally, cell treatment with LQB-223 resulted in changes in the mRNA levels of epithelial-mesenchymal transition markers, suggesting that it might modulate cell plasticity. Our data demonstrate that LQB-223 impairs 3D culture growth and migration in 2D and 3D models of breast cancer exhibiting different phenotypes.

α-lipoic acid reduces postreperfusion syndrome in human liver transplantation - a pilot study.

A double-blind randomized controlled trial was performed to compare the safety and efficacy of α-lipoic acid (ALA) in liver transplantation (LT). The grafts were randomized to receive ALA or placebo before the cold ischemia time. Furthermore, patients transplanted with the ALA-perfused graft received 600 mg of intravenous ALA, while patients with the nonperfused graft received the placebo just before graft reperfusion. Hepatic biopsy was performed 2 h postreperfusion. Blood samples were collected before, during and 1 and 2 days after reperfusion. Quantitative polymerase chain reaction (qPCR) analysis was performed on biopsies to assess genes involved in the response to hypoxia, apoptosis, cell growth, survival and proliferation, cytokine production and tissue damage protection. Nine of 40 patients developed postreperfusion syndrome (PRS), but seven of them belonged to the control group. There was a decrease in PHD2 and an increase in alpha subunit of hypoxia-inducible factor-1 (HIF-1α) and baculoviral IAP repeat containing 2 (Birc2) transcript levels in the biopsies from the ALA-treated versus the control group of patients. Additionally, plasma levels of alarmins were lower in ALA-treated patients than control patients, which suggests that ALA-treated grafts are less inflammatory than untreated grafts. These results showed that ALA is safe for use in LT, induces gene changes that protect against hypoxia and oxidative stress and reduces the appearance of PRS.

Evaluation of survivin immunoexpression in the differentiation of high- and low-grade breast ductal carcinoma in situ.

Objective To evaluate the expression of survivin protein in low- and high-grade ductal carcinoma in situ. Methods Breast tissue fragments obtained by incisional biopsy and surgical procedures of 37 women with ductal carcinoma in situ of the breast were subdivided into two groups: Group A, composed of women with low-grade ductal carcinoma in situ, and Group B, women with high-grade ductal carcinoma in situ. Survivin protein expression test was performed by immunohistochemistry, using a monoclonal antibody clone I2C4. The criterion to evaluate survivin immunoexpression was based on the percentage of neoplastic cells that presented brown-gold staining. This criterion was positive when the percentage of stained cells was ≥10%. Results The survivin protein was expressed in 22 out of 24 cases of high-grade ductal carcinoma in situ (78%), whereas, in Group A, of low-grade ductal carcinoma in situ (n=13), it was positive in only 6 cases (21.40%; p=0.004). Conclusion The frequency of expression of survivin was significantly higher in the group of patients with high-grade ductal carcinoma in situ compared to those in the low-grade ductal carcinoma in situ group.

p53 expression and subcellular survivin localization improve the diagnosis and prognosis of patients with diffuse astrocytic tumors.

PURPOSE: Diffuse astrocytic tumors are the most frequently occurring primary central nervous system (CNS) tumors. Their histological sub-classification into diffuse astrocytoma (DA), anaplastic astrocytoma (AA) and glioblastoma (GB) is challenging and the available prognostic factors are limited to age and tumor subtype. Biomarkers that may improve the histological sub-classification and/or serve as prognostic factors are, therefore, urgently needed. The relationship between survivin and p53 in diffuse astrocytic tumor progression and survival is currently unclear. Here, we aimed to assess the relevance of these proteins in the accuracy of the histological sub-classification of these tumors and their respective treatment responses. METHODS: One hundred and thirty-three formalin-fixed paraffin-embedded diffuse astrocytic tumor samples were included. The tumor samples were histologically reviewed and subsequently assessed for p53 and survivin expression and the presence of the IDH R132H mutation by immunohistochemistry. p53 expression levels and survivin subcellular localization patterns were correlated with histological classification and clinical outcome. RESULTS: We found that age and histological subtype were the only features with a prognostic impact. In addition, we found that high p53 expression levels and a nuclear survivin localization correlated with the AA subtype, whereas cytoplasmic survivin localization correlated with the GB subtype. We also found that patients carrying tumors with a high cytoplasmic survivin expression, a high nuclear survivin expression or a high p53 expression, and who did not receive radiotherapy, exhibited poorer short-term and long-term overall survival rates. CONCLUSIONS: Our data suggest that subcellular survivin localization and p53 expression may be employed as valuable tools to improve the accuracy of the histological sub-classification of diffuse astrocytic tumors. Patients whose tumors overexpress these proteins may benefit from radiotherapy, irrespective age and/or histological classification.

Smoking cessation leads to changes in survivin expression in oral mucosa.

BACKGROUND: Survivin is an inhibitor protein of apoptosis and plays a role in oral carcinogenesis mechanism. METHODS: The aim of this study was to evaluate the effect of smoking in survivin expression of oral mucosa of chronic smokers with and without oral squamous cell carcinoma (OSCC). The study was composed of three groups: Group 1-26 patients smoking more than 20 cigarettes/day/10 years without either history of oral malignant neoplasia or visible clinical signs in the examined site; Group 2-26 patients with OSCC; Group 3-22 patients surgically treated for OSCC for at least 1 month. The immunohistochemistry was performed with 1 smear for each group and analyzed by microscopy regarding extension, intensity of positive cells for survivin, and intracellular location. RESULTS: The survivin expression was observed in 100% of the cases in Group 1, 88.5% in Group 2, and 100% in Group 3. Concerning to Groups 1 and 3, the survivin expression with cytoplasmic location occurred in 100%, while in Group 2 occurred in 87.5%. The cytoplasmic and nuclear expression was observed only in Group 2, with 7.69%. The results were correlated with clinical-pathological data by Fischer's exact test with significant relation between smoking cessation and intensity (P = .015) for Group 2. CONCLUSIONS: The extension and intensity of survivin expression in the cytological smears were related to the smoking cessation in the group with OSCC. However, the smoking history (packs/years) did not influence the survivin expression.

Evaluation of survivin immunoexpression in the differentiation of high- and low-grade breast ductal carcinoma in situ / Expressão da proteína survivina em carcinoma ductal in situ de mama de alto e de baixo grau

ABSTRACT Objective To evaluate the expression of survivin protein in low- and high-grade ductal carcinoma in situ. Methods Breast tissue fragments obtained by incisional biopsy and surgical procedures of 37 women with ductal carcinoma in situ of the breast were subdivided into two groups: Group A, composed of women with low-grade ductal carcinoma in situ, and Group B, women with high-grade ductal carcinoma in situ. Survivin protein expression test was performed by immunohistochemistry, using a monoclonal antibody clone I2C4. The criterion to evaluate survivin immunoexpression was based on the percentage of neoplastic cells that presented brown-gold staining. This criterion was positive when the percentage of stained cells was ≥10%. Results The survivin protein was expressed in 22 out of 24 cases of high-grade ductal carcinoma in situ (78%), whereas, in Group A, of low-grade ductal carcinoma in situ (n=13), it was positive in only 6 cases (21.40%; p=0.004). Conclusion The frequency of expression of survivin was significantly higher in the group of patients with high-grade ductal carcinoma in situ compared to those in the low-grade ductal carcinoma in situ group.

RESUMO Objetivo Avaliar a imunoexpressão da proteína survivina nos carcinomas ductais in situ de mama de baixo e de alto graus. Métodos Fragmentos de tecido mamários obtidos por biópsia incisional e procedimentos cirúrgicos de 37 mulheres acometidas por carcinoma ductal in situ de mama foram subdivididos em dois grupos: Grupo A, formado por mulheres com carcinoma ductal in situ de baixo grau; e Grupo B, por mulheres com carcinoma ductal in situ de alto grau. A pesquisa de expressão da proteína survivina foi realizada pela técnica de imuno-histoquímica, utilizando-se anticorpo monoclonal clone I2C4. O critério de avaliação da imunoexpressão da survivina baseou-se na percentagem de células neoplásicas que apresentava coloração castanho-dourada. Considerouse tal critério positivo quando a percentagem de células apresentasse marcação ≥10%. Resultados A proteína survivina apresentou-se expressa em 22 dos 24 casos de carcinoma ductal in situ de alto grau (78%), enquanto no Grupo A, de carcinoma ductal in situ de baixo grau (n=13), apresentou-se positiva em apenas 6 casos (21,40%; p=0,004). Conclusão O índice de frequência de expressão da survivina foi significativamente mais elevado no grupo de pacientes com carcinoma ductal in situ de alto grau, quando comparado às do grupo com carcinoma ductal in situ de baixo grau.

Mechanism of reversal of multidrug resistance by curcumin in human colorectal cancer cell line HCT-8/5-FU.

Since there are no studies on the reversal of multidrug resistance by curcumin in the human colorectal cancer cell line HCT-8/5-FU, our aim was to search for highly efficient reversal agents and investigate the underlying mechanisms of this reversal. The cytotoxic effects of curcumin and 5-FU on HCT-8 and HCT-8/5-FU cells and the reversal effects of 5-FU in combination with curcumin on HCT-8/5-FU cells were measured using cell counting kit-8. Apoptosis and the cell cycle were analyzed by flow cytometry. Protein and mRNA expression levels of BCL-2, survivin, P-gp, and HSP-27 were detected by western blotting and quantitative real-time reverse transcription polymerase chain reaction, respectively. Curcumin inhibited the growth of HCT-8 and HCT-8/5-FU cells. It significantly reduced the IC50 of 5-FU for HCT-8/5-FU cells (P < 0.01) and the expression of BCL-2, survivin, P-gp, and HSP-27 in the cells. Curcumin can effectively reverse multidrug resistance in human colorectal cancer drug-resistant HCT-8/5-FU cells. The mechanism through which this occurs may be associated with decreased expression of BCL-2, survivin, P-gp, and HSP-27. Curcumin may therefore have clinical implications as a new agent for colorectal cancer.

Predictive value of p63, ki-67, and survivin expression in oral leukoplakia: A tissue microarray study.

The aim of this study was to analyze the immunohistochemical expression of survivin, ki-67, and p63 in oral leukoplakic lesions, histopathologically differentiated into dysplastic and nondysplastic. A tissue microarray containing 57 samples of biopsies from clinically classified lesions, such as leukoplakia, was immunolabeled for survivin, ki-67, and p63. Samples were scored for percentage of positively stained. Scores were designated as follows: low = less than 25% of positive cells; and high = more than 25% of positive cells. On performing histopathological diagnosis, 20 dysplastic lesions and 37 nondysplastic lesions were seen, in which female patients (56.1%) were predominant with an average age of 58.27 years. The study showed a high expression of 37.5% for survivin, 43.7% for ki-67, and 88.2% for p63 in dysplastic lesions. However, there was a high expression of 16.7% for survivin, 16.7% for ki-67, and 92% for p63 in nondysplastic lesions. There is a positive correlation of expression among the three antibodies. In the association of immunoreactivity, in both dysplastic and nondysplastic lesions, increased expression of survivin reflects on the increased expression of ki-67, and there is an overexpression of p63. In leukoplakia, the expression of survivin associated with that of ki-67 reinforces the assumption that all these lesions are potentially malignant, regardless of histopathology; and the overexpression of p63 may indicate carcinogenic potential. These findings may help in the treatment of patients with this type of lesion.

Oral delivery of double-stranded RNAs induces mortality in nymphs and adults of the Asian citrus psyllid, Diaphorina citri.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is one of the most important citrus pests. ACP is the vector of the phloem-limited bacteria Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, the causal agents of the devastating citrus disease huanglongbing (HLB). The management of HLB is based on the use of healthy young plants, eradication of infected plants and chemical control of the vector. RNA interference (RNAi) has proven to be a promising tool to control pests and explore gene functions. Recently, studies have reported that target mRNA knockdown in many insects can be induced through feeding with double-stranded RNA (dsRNA). In the current study, we targeted the cathepsin D, chitin synthase and inhibitor of apoptosis genes of adult and nymph ACP by feeding artificial diets mixed with dsRNAs and Murraya paniculata leaves placed in dsRNAs solutions, respectively. Adult ACP mortality was positively correlated with the amount of dsRNA used. Both nymphs and adult ACP fed dsRNAs exhibited significantly increased mortality over time compared with that of the controls. Moreover, qRT-PCR analysis confirmed the dsRNA-mediated RNAi effects on target mRNAs. These results showed that RNAi can be a powerful tool for gene function studies in ACP and perhaps for HLB control.

Effect of miRNA-203 on cervical cancer cells and its underlying mechanism.

miRNA-203 is involved in the development and progression of various types of cancer. However, its role in cervical cancer remains unclear. The aim of this study was to investigate the effect of miRNA-203 on the proliferation and migration of HeLa cervical cancer cells, as well as survivin expression in these cells. A miRNA-203 primer probe was designed according to a sequence obtained from NCBI. The expression of miRNA-203 in cervical epithelial cells and cervical cancer cells was detected by quantitative reverse transcriptase-polymerase chain reaction. The miRNA-203 expression pattern was compared between these two cell lines. The cervical cancer cells were transfected with miRNA-203 mimic or inhibitor to determine their effects on proliferation and migration. The expression of the miRNA-203 target protein (survivin) was analyzed by western blot. Cervical cancer cells showed reduced miRNA-203 expression compared to cervical epithelial cells. Transfection of miRNA-203 mimic upregulated the expression of miRNA-203, suppressed cell proliferation and migration, and downregulated survivin expression (P < 0.05). However, downregulation of miRNA-203 expression did not affect proliferation, migration, and survivin expression in cervical cancer cells (P > 0.05). In conclusion, upregulation of miRNA-203 in cervical cancer cells inhibits the proliferative and migratory capacities of these cells by downregulating the expression of survivin.

Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors.

We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown in syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy.

Increased survivin expression and its association with clinical parameters of congenital choledochal cysts.

The aim of the current study was to investigate survivin expression in congenital choledochal cysts (CCCs), and its associations with clinical parameters of CCCs. In total, 121 children with CCCs were included in this study as the case group, and their cysts were staged according to the Todani classification system. Additionally, 49 normal gallbladder specimens from healthy children were included as the control group. Survivin detection was conducted using immunohistochemical staining. Associations between positive survivin expression and clinical parameters of CCCs were then analyzed. Positive survivin expression was observed in the cytoplasm, and was seen as granular with yellow or dark brown staining. In the case group, positive survivin expression was detected in most tissues. Specifically, compared to that of normal tissues, the cystic-shaped and fusiform-shaped CCC tissues had significantly higher positive survivin expression rates (all P < 0.05). Importantly, positive survivin expression was also shown to be significantly associated with gender and histological type (both P < 0.05). In conclusion, increased survivin expression was observed in CCC tissues, and was correlated with certain clinical parameters of CCCs, suggesting a possible prognostic value of survivin for CCC progression.

Investigation of cellular mechanisms involved in apoptosis induced by a synthetic naphthylchalcone in acute leukemia cell lines.

We have previously reported the cytotoxic effects of chalcone A1, derived from 1-naphthaldehyde, in leukemia cell lines. On the basis of these findings, the main aim of this study was to elucidate some of the molecular mechanisms involved in apoptosis induced by chalcone A1 toward K562 and Jurkat cells. In both cell lines, chalcone A1 decreased the mitochondrial membrane potential, increased the expression of Bax proapoptotic protein, and decreased the expression of Bcl-2 antiapoptotic protein (resulting in the inversion of the Bcl-2/Bax ratio), which indicates the involvement of the intrinsic pathway. In addition, chalcone A1 increased the expression of FasR in Jurkat cells, which also indicates the involvement of the extrinsic pathway in this cell line. The results also showed an increased expression of effector caspase-3 and cleaved PARP-1 and a decreased expression of IAP protein survivin, which are consistent with apoptotic cell death. The decreased expression of Ki67 suggests that the mechanism involved in cell death induced by chalcone A1 also involves a decrease in cell proliferation. In ex-vivo experiments, chalcone A1 reduced the cell viability of blast cells collected from eight patients with different types of acute leukemia, confirming the cytotoxicity results found in vitro. The results obtained so far are very promising and further studies need to be carried out so that chalcone A1 can be used as a prototype for the development of new antileukemia agents.

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.

Dehydroleucodine inhibits tumor growth in a preclinical melanoma model by inducing cell cycle arrest, senescence and apoptosis.

Malignant melanoma represents the fastest growing public health risk of all cancer types worldwide. Several strategies and anti-cancer drugs have been used in an effort to improve treatments, but the development of resistance to anti-neoplastic drugs remains the major cause of chemotherapy failure in melanomas. Previously, we showed that the sesquiterpene lactone, dehydroleucodine (DhL), promotes the accumulation of DNA damage markers, such as H2AX and 53BP1, in human tumor cells. Also DhL was shown to trigger either cell senescence or apoptosis in a concentration-dependent manner in HeLa and MCF7 cells. Here, we evaluated the effects of DhL on B16F0 mouse melanoma cells in vitro and in a pre-clinical melanoma model. DhL inhibited the proliferation of B16F0 cells by inducing senescence or apoptosis in a concentration-dependent manner. Also, DhL reduced the expression of the cell cycle proteins cyclin D1 and B1 and the inhibitor of apoptosis protein, survivin. In melanomas generated by subcutaneous injection of B16F0 cells into C57/BL6 mice, the treatment with 20 mg DhL /Kg/day in preventive, simultaneous and therapeutic protocols reduced tumor volumes by 70%, 60% and 50%, respectively. DhL treatments reduced the number of proliferating, while increasing the number of senescent and apoptotic tumor cells. To estimate the long-term effects of DhL, a mathematical model was applied to fit experimental data. Extrapolation beyond experimental time points revealed that DhL administration following preventive and therapeutic protocols is predicted to be more effective than simultaneous treatments with DhL in restricting tumor growth.

Protection From Apoptotic Cell Death During Cold Storage Followed by Rewarming in 13-Lined Ground Squirrel Tubular Cells: The Role of Prosurvival Factors X-Linked Inhibitor of Apoptosis and PhosphoAkt.

BACKGROUND: Hibernators, such as the 13-lined ground squirrel, endure severe hypothermia during torpor followed by periodic rewarming (REW) during interbout arousal (IBA), proapoptotic conditions that are lethal to nonhibernating mammals. We have previously shown that 13-lined ground squirrel tubular cells are protected from apoptotic cell death during IBA. To understand the mechanism of protection, we developed an in vitro model of prolonged cold storage (CS) followed by REW, which is akin to the in vivo changes of hypothermia followed by REW observed during IBA. We hypothesized that renal tubular epithelial cells (RTECs) isolated from hibernating ground squirrels would be protected against apoptosis during CS/REW versus nonhibernating mouse RTECs. METHODS: Isolated hibernating ground squirrel and mouse RTECs were subjected to CS at 4°C for 24 hours followed by REW to 37°C for 24 hours (CS/REW). RESULTS: Ground squirrel RTECs had significantly less apoptosis compared to mouse RTECs when subjected to CS/REW. Next, we hypothesized that the mechanism of protection was related to the antiapoptotic proteins X-linked inhibitor of apoptosis (XIAP), phospho-Akt (pAkt), and phospho-BAD. There was a significantly increased pAkt and pBAD expression in ground squirrel versus mouse RTECs subjected to CS/REW. The XIAP expression was maintained in ground squirrel RTECs but was significantly decreased in mouse RTECs after CS/REW. Ground squirrel RTECs in which gene expression of Akt1 and XIAP was silenced lost their protection and demonstrated increased apoptosis and cleaved caspase-3 expression after CS/REW. CONCLUSIONS: Our findings suggest that ground squirrel RTECs are protected against apoptosis during prolonged CS/REW by the "prosurvival" factors XIAP and pAkt.

Role of survivin in the pathogenesis of papillary thyroid carcinoma.

The purpose of this study was to assess the correlation between the survivin gene and the occurrence and pathogenesis of papillary thyroid carcinoma (PTC). Sixty patients with PTC and no preoperative chemotherapy were recruited for the study and 30 thyrophyma patients receiving operative treatment in Drum Tower Hospital (Nanjing, China) were included as the control group. The protein expression levels of survivin were assessed by immunoblotting and immunohistochemical analysis of tissues from both patient groups. For survivin gene knockdown experiments, two target sequences were selected based on the mRNA sequence of survivin and two pairs of siRNA interference sequences were designed and synthesized accordingly. The siRNAs were shown to be successfully transfected into SW579 carcinoma cells and the resulting survivin knockdown was assessed by RT-PCR and immunofluorescence. Survivin was shown by immunohistochemistry to be distributed in the cytoplasm of PTC and thyrophyma cells, with the signal being significantly stronger in PTC cells than in thyrophyma cells and statistical analysis of immunostaining data further showed survivin to be more highly expressed  (P < 0.05) in the PTC tissue than in the thyrophyma tissue. Transfection of SW579 cells with siRNA was found to be effective in knocking down the expression levels of survivin: 87.3 and 76.2% knockdown was achieved with sh-Survivin-1 and sh-Survivin-2, respectively. The findings reported here show that survivin is highly expressed in PTC and may therefore play a role in the occurrence, lymph node metastasis, and clinical staging of PTC.

Expression levels of survivin, Bcl-2, and KAI1 proteins in cervical cancer and their correlation with metastasis.

Cervical cancer is associated with abnormal expression of multiple genes. Survivin and Bcl-2 proteins are apoptosis inhibitors. The tumor suppressor gene CD82, which encodes the protein KAI1, is downregulated in cervical cancer, and is associated with differentiation degree. We investigated the expression levels of three proteins and their correlation with metastasis in cervical cancer by comparing them in different cervical lesions. Immunohistochemistry was used to detect their three protein expression levels in the normal cervix, chronic cervicitis, cervical intraepithelial neoplasia (CIN) lesions, and cervical cancer. The relationships between the protein expression levels and tumor type, clinical stage, tissue differentiation, invasion, and metastasis were analyzed. Survivin and Bcl-2 expression levels in cervical cancer were significantly higher than in the normal cervix, chronic cervicitis, or CIN (P < 0.05). KAI1 expression was markedly lower in cervical cancer than in the normal cervix, chronic cervicitis, or CIN (P < 0.05). There was no statistical difference between the expression levels of the three proteins in CIN and chronic cervicitis, but there were differences in expression between CIN and normal cervical tissues (P < 0.05). Bcl-2 and survivin levels were positively correlated while KAI1 expression was negatively correlated with clinical stage. Survivin and KAI1 expression levels were associated with lymph node metastasis (P < 0.05), and KAI1 expression was positively related with differentiation degree (P < 0.05). Survivin, Bcl-2, and KAI1 are metastasis-related factors in cervical cancer. Overexpression of survivin and Bcl-2, and low expression of KAI1 promotes cervical cancer progress and metastasis.

The twisted survivin connection to angiogenesis.

Survivin, a member of the inhibitor of apoptosis family of proteins (IAPs) that controls cell division, apoptosis, metastasis and angiogenesis, is overexpressed in essentially all human cancers. As a consequence, the gene/protein is considered an attractive target for cancer treatment. Here, we discuss recent findings related to the regulation of survivin expression and its role in angiogenesis, particularly in the context of hypoxia. We propose a novel role for survivin in cancer, whereby expression of the protein in tumor cells promotes VEGF synthesis, secretion and angiogenesis. Mechanistically, we propose the existence of a positive feed-back loop involving PI3-kinase/Akt activation and enhanced ß-Catenin-TCF/LEF-dependent VEGF expression followed by secretion. Finally, we elaborate on the possibility that this mechanism operating in cancer cells may contribute to enhanced tumor vascularization by vasculogenic mimicry together with conventional angiogenesis.