Ultrasound-Guided Delivery of siRNA and a Chemotherapeutic Drug by Using Microbubble Complexes: In Vitro and In Vivo Evaluations in a Prostate Cancer Model

OBJECTIVE: To evaluate the effectiveness of ultrasound and microbubble-liposome complex (MLC)-mediated delivery of siRNA and doxorubicin into prostate cancer cells and its therapeutic capabilities both in vitro and in vivo. MATERIALS AND METHODS: Microbubble-liposome complexes conjugated with anti-human epidermal growth factor receptor type 2 (Her2) antibodies were developed to target human prostate cancer cell lines PC-3 and LNCaP. Intracellular delivery of MLC was observed by confocal microscopy. We loaded MLC with survivin-targeted small interfering RNA (siRNA) and doxorubicin, and delivered it into prostate cancer cells. The release of these agents was facilitated by ultrasound application. Cell viability was analyzed by MTT assay after the delivery of siRNA and doxorubicin. Survivin-targeted siRNA loaded MLC was delivered into the xenograft mouse tumor model. Western blotting was performed to quantify the expression of survivin in vivo. RESULTS: Confocal microscopy demonstrated substantial intracellular uptake of MLCs in LNCaP, which expresses higher levels of Her2 than PC-3. The viability of LNCaP cells was significantly reduced after the delivery of MLCs loaded with siRNA and doxorubicin (85.0 ± 2.9%), which was further potentiated by application of ultrasound (55.0 ± 3.5%, p = 0.009). Survivin expression was suppressed in vivo in LNCaP tumor xenograft model following the ultrasound and MLC-guided delivery of siRNA (77.4 ± 4.90% to 36.7 ± 1.34%, p = 0.027). CONCLUSION: Microbubble-liposome complex can effectively target prostate cancer cells, enabling intracellular delivery of the treatment agents with the use of ultrasound. Ultrasound and MLC-mediated delivery of survivin-targeted siRNA and doxorubicin can induce prostate cell apoptosis and block survivin expression in vitro and in vivo.

siRNA against survivin mediated by adenovirus vector up-regulate estrogen receptor expression in breast cancer MCF-7 cell / 国际外科学杂志

Objective To investigate the effect on ER expression in MCF-7 cell by siRNA against survivin mediated by adenovirus vector.Methods An adenovirus vector of siRNA against survivin was constructed and used to infect MCF-7 cell.The change of expression of survivin and ER was detected by Western Blot.Results The expression strength of survivin are 0.09 ± 0.04、0.86 ± 0.08、0.82 ± 0.17;expression strength of ER are 1.57 ± 0.09、1.16 ± 0.10、1.23 ± 0.01 respectively in the experimental group,negative control group and blank control group.Statistics analysis shows that the adenovirus vector of siRNA against survivin constructed in the study can suppress the expression of survivin significantly,and suppress the expression of survivin can up-regulate the estrogen receptor (ER) expression.Conclusions The results suggest that there may be a certain regulatory mechanism between survivin and ER signal pathway in MCF-7 cell and siRNA against survivin is of important potential value in the endocrine therapy of hormone receptor positive breast cancer.

Mechanism of radiosensitization effect of paeonol on human lung adenocarcinoma cell line A549 / 中华放射医学与防护杂志

Objective To investigate the radiosensitization effect and underlying mechanism of Paeonol on human lung adenocarcinoma cell line A549 in vitro. Methods Cells were assigned to following groups:control,Paeonol alone,irradiation alone,Paeonol combined with irradiation.The effect of Paeonol on cell proliferation was evaluated by the MTT assay. Clonogenic assay was performed to measure the radiosensitization effect of Paeonol under three concentrations around 20％ IC50.Cell apoptosis was determined by TUNEL assay and flow cytometry (FCM).The expression of Survivin protein was analyzed by Western blot.Results Cell growth was inhibited by Paeonol in a dose-dependent manner and the IC50 of Paeonol was (25.2 ± 2.1 ) mg/L. Clonogenic assay showed that Paeonol could markedly enhance cell radiosensitivity and the sensitizing enhancement ratio (SER) was 1.29.After the pretreatment of Paeonol with different concentrations,radiation-induced apoptosis increased with the doses at 24,48,and 72 h post-irradiation ( t =4.95,3.03,3.78,4.59,2.88,3.70,5.54,P ＜ 0.05 ). Moreover,the protein expression of Survivin was obviously down-regulated by 22.6％ - 56.7％ ( t =4.15,7.30,13.47,P ＜0.05 ) due to the treatment of Paeonol.When the Paeonol-treated cells were further irradiated with 6 Gy X-rays,the expression of Survivin was reduced to 22.2％ - 69.4％ ( t =4.30,8.36,16.34,P ＜ 0.05 ).Conclusions Paeonol had radiosensitization effect on the human lung adenocarcinoma cell line A549 in vitro,where the down-regulated Survivin protein might be involved.

Expression and Significance of Survivin and Fhit Protein in Colorectal Benign and Malignant Disease / 医学研究杂志

Objective To investigate the expression and significance of Survivin and Fhit protein in colorectal benign and malignant disease.Methods Test the expression of Survivin and Fhit proteins in 20 cases normal colorectal mucosa,30 cases low grade colorectal intraepithelial lesion,30 cases high grade colorectal intraepithelial lesion and 68 cases colorectal adenocarcinoma by immunohistochemical staining S-P method.Results The unexpression of Survivin in normal colorectal mucosa,postive expression rates of Survivin in low grade colorectal intraepithelial lesion,high grade colorectal intraepithelial lision and colorectal adenocarcinoma were 43.3%,76.7% and 91.2%(P

Antisurvivin oligonucleotides inhibit growth and induce apoptosis in human medullary thyroid carcinoma cells

Suvivin is a novel member of the inhibitor of apoptosis protein (IAP) family, which is known to be over-expressed in various carcinomas and associated with their biologically aggressive characteristics. The aim of this study was to investigate survivin expression in human medullary thyroid carcinoma (MTC) and a MTC cell line TT, correlate suvivin expression with clinicopathologic features of MTC, and test effects of antisurvivin oligonucleotides (ASODNs) on growth and apoptosis of TT cells. Survivin expression was immunohistochemically determined in formalin-fixed and paraffin-embedded specimens obtained from 10 cases of normal thyroid (NT) and 10 cases of MTC, and in TT cells. In TT cells, we confirmed survivin expression and its down-regulation by ASODNs using RT-PCR and Western blot analyses, and investigated effects of ASODNs on viability and growth by MTT assay and apoptosis by apoptotic analyses including DNA laddering assay, acridine orange/ethidium bromide staining and flow cytometric cell cycle analysis. Immunohistochemical analysis showed high survivin expression in MTC and TT cells, whereas no immunoreactivity was detectable in NT. Statistical analyses revealed no significant correlation of survivin expression with the clinicopathologic features of MTC. In TT cells, survivin expression at both mRNA and protein levels was confirmed and could be down-regulated by ASODNs concomitant with decrease in viability and growth, and increase in apoptosis. Our results suggest that survivin plays an important role in MTC independent of the conventional clinicopathologic factors, and ASODNs is a promising survivin-targeted gene therapy for MTC.

Expression of Survivin in Non-Small Cell Lung Carcinoma: Relationship to Tumor Biology and Prognosis in Surgically Treated Patients

BACKGROUND: Survivin, a novel member of inhibitor-of-apoptosis, is undetectable in most terminally differentiated nonproliferative adult tissue, but is overexpressed in some human malignancies. The survivin gene expression is repressed by binding of wild-type p53 with the survivin promotor. In this study, we investigated the prevalence of survivin expression, its association with p53 overexpression and proliferative index, and clinicopathological significance in non-small cell lung carcinomas (NSCLC). METHODS: Immunohistochemical stainings were performed in 59 cases of primary NSCLC for survivin, p53 and Ki-67. Correlations between the survivin expression, p53 overexpression and Ki-67 labeling index were analyzed. RESULTS: Survivin expression was detected in 47 carcinomas (80%) with nuclear immunoreactivity (56%). Survivin nuclear immunoreactivity revealed significantly worse prognosis in NSCLC patients (p=0.003), and correlated with lymph node metastasis (p=0.014), lymphovascular invasion (p=0.032), p53 overexpression, and Ki-67 labeling index (KI 24.2 +/- 6.9, p=0.045). Survivin expression was not correlated with histological type and pT status. CONCLUSIONS: High incidence of survivin overexpression in NSCLC suggests that survivin is involved in lung carcinogenesis, and nuclear expression of survivin can be used as a poor prognostic predictor in NSCLC patients. Expression of mutant p53 seems to be a possible mechanism of survivin up-regulation in NSCLC.

Survivin and Fas Ligand Expressions Are Correlated with Angiolymphatic Tumor Spread in Medullary Thyroid Carcinoma

BACKGROUND: Medullary thyroid carcinoma (MTC) that originates from C cells comprises about 10% of all the malignant thyroid tumors. Activating mutations of the RET proto-oncogene have been found to be involved in the anti-apoptotic pathway of MTC that harbors the RET mutation. We investigated the correlation between the clinicopathologic parameters and the expressions of survivin, a novel anti-apoptotic molecule, and the other apoptosis-related proteins, and the known prognostic markers. METHODS: Immunohistochemical staining was performed using antibodies for survivin, Fas, Fas ligand (FasL), bcl-2, calcitonin, CEA and cyclin A in 19 case of MTC; 10 sporadic MTCs, eight multiple endocrine neoplasia (MEN) type 2A MTCs and one familial MTC (FMTC). RESULTS: Survivin protein expression was found in five cases (26%) and this was correlated with the presence of angiolymphatic tumor emboli (p=0.019). FasL was expressed in 14 cases (74%) and it had correlation with the presence of lymph node metastases (p=0.029). The cyclin A-labeling indices were correlated with local invasiveness (p=0.001). CONCLUSIONS: Survivin and FasL might be involved in the lymphatic tumor spread of MTC.

Studies on the Expression of Survivin Protein and Its Relationship to the Proliferation of the Tumor Cells and Prognosis in Primary Hepatocellular Carcinoma / 中国普外基础与临床杂志

Objective To study the expression of survivin protein in primary hepatocellular carcinoma(PHC) and its relationship to the proliferation of the tumor cells and prognosis of PHC. Methods The expression of survivin protein and the proliferation of tumor cells marked by proliferating cell nuclear antigen (PCNA) in 48 cases of PHC were determined by immunohistochemical method. Results The survivin protein was expressed in 31 of 48 cases of PHC (64.6%). The expression of PCNA was significantly higher in hepatocellular carcinoma (HCC) with positive survivin expression than in HCC with negative survivin expression. The patients with positive survivin expression had the worse prognosis than those with negative survivin expression. Conclusion The expression of survivin may play an important role in the proliferation of PHC cells and closely associate with the prognosis of PHC, and probably become the prognostic factor and an important target of therapy.

The regulation of As_2O_3 on the expression of apoptosis inhibitory protein:an experimental study in rabbits with implanted liver tumor / 介入放射学杂志

Objective To investigate the relationship between the expression of tumor apoptosis inhibitory protein(survivin) and the apoptosis induced by arsenic trioxide(As2O3) in transcatheter arterial chemoembolization therapy.Methods Sixteen Japanese big-ear white rabbits with implanted hepatic VX2 tumor at both right and left hepatic lobes were randomly and equally divided into two groups.Three weeks after the tumor was inoculated,1 ml lipiodol(UFLP) and 2 mg As2O3 were injected via hepatic arterial cannulation into the rabbits of study group,while only 1 ml UFLP was used for the rabbits in control group.Three weeks later,all the rabbits were sacrificed,and the tumor tissue,the tumor-neighboring tissue and the normal liver were separately collected and sent for TUNEL staining and examinations,which included the observation of apoptosis of tumor cells and the assessment of the expression of survivin protein.Results In study group,a large number of yellow apoptosis cells was seen in the tumor tissue but no apoptosis cell was found in the tumor-neighboring tissue or in the normal liver tissue.In the control group,no yellow apoptosis cell was observed in the tumor tissue,tumor-neighboring tissue or normal liver tissue.The survivin protein expression rate of the tumor tissue was 100%(16 / 16) in the control group,including strongly-positive in 12 and weakly-positive in 4 rabbits.In contrast,the survivin protein expression rate of both the tumor-neighboring tissue and the normal tissue was 0%.In study group,the survivin protein expression rate of the tumor tissue was 37.5%(6 / 16),including strongly-positive in 2 and weakly-positive in 4 cases,and the survivin protein expression rate of both the tumor-neighboring tissue and the normal tissue was 0%.Significant difference in survivin protein expression rate of the tumor tissue existed between two groups(P

Expression and correlation between Survivin and PTEN proteins in astrocytoma / 临床神经病学杂志

Objective To investigate the expressions and correlation between Survivin and PTEN proteins in astrocytoma.Methods The expressions of Survivin and PTEN proteins were examined by immunohistrochemistry in astrocytoma specimens from 65 patients with astrocytoma.Results The positive expression rate of Survivin in astrocytoma grade Ⅱ was significantly lower than that in grade Ⅲ and grade Ⅳ(all P

Construction of short hairpin RNA expression plasmid and its inhibition of survivin expression / 中国普通外科杂志

Objective To study the inhibit effect of survivin short hairpin RNA on the survivin mRNA and protein expression of gallbladder carcinoma cells.Methods Human gallbladder carcinoma cells(GBC-SD) were transfected with recombinant plasmid.RT-PCR and Western blot were used to analyze the changes in(expression) levels of survivinmRNA and protein.Results The size of the PCR product was 350bp.DNA(sequencing) showed that the sequence of recombinant vector pshRNA-survivin was successfully constructed and suppressed the expression of(GBC-SD) survivinmRNA.Conclusions The recombinant plasmid constructed can inhibit the expression of survivinmRNA in transfected cells.This provides a new method and material for the biological therapy of cancer.