Ginsenoside Rh2 enhances the antitumor immunological response of a melanoma mice model.

The treatment of malignant tumors following surgery is important in preventing relapse. Among all the post-surgery treatments, immunomodulators have demonstrated satisfactory effects on preventing recurrence according to recent studies. Ginsenoside is a compound isolated from panax ginseng, which is a famous traditional Chinese medicine. Ginsenoside aids in killing tumor cells through numerous processes, including the antitumor processes of ginsenoside Rh2 and Rg1, and also affects the inflammatory processes of the immune system. However, the role that ginsenoside serves in antitumor immunological activity remains to be elucidated. Therefore, the present study aimed to analyze the effect of ginsenoside Rh2 on the antitumor immunological response. With a melanoma mice model, ginsenoside Rh2 was demonstrated to inhibit tumor growth and improved the survival time of the mice. Ginsenoside Rh2 enhanced T-lymphocyte infiltration in the tumor and triggered cytotoxicity in spleen lymphocytes. In addition, the immunological response triggered by ginsenoside Rh2 could be transferred to other mice. In conclusion, the present study provides evidence that ginsenoside Rh2 treatment enhanced the antitumor immunological response, which may be a potential therapy for melanoma.

Aberrant CXCR4 and ß-catenin expression in osteosarcoma correlates with patient survival.

To determine the clinical significance of C-X-C chemokine receptor type 4 (CXCR4) and ß-catenin in osteosarcoma, their protein expression levels were assessed in 96 osteosarcoma and 20 osteochondroma cases using immunohistochemistry. Additionally, CXCR4 and ß-catenin mRNA expression levels were measured in 16 fresh osteosarcoma and 16 adjacent healthy tissue samples using fluorescent reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In the osteosarcoma samples, the positive CXCR4 protein expression rate was significantly higher than the rate in the osteochondroma samples (68.75 vs. 20.00%; P<0.01). Furthermore, ß-catenin protein expression was detected in 61.46% of osteosarcoma cases and 25.00% of osteochondroma cases. Similarly, the RT-qPCR data identified increased CXCR4 and ß-catenin mRNA expression levels in the osteosarcoma compared with adjacent control tissues. It was determined that CXCR4 (P<0.01) and ß-catenin (P<0.05) expression were significantly associated with the clinical Enneking stage, metastasis and survival of osteosarcoma. Furthermore, multivariate analysis identified CXCR4 and ß-catenin protein expression levels, as well as clinical stage and metastasis, as significant risk factors for survival in patients with osteosarcoma (P<0.05). In conclusion, the present study determined that CXCR4 and ß-catenin are abnormally expressed in osteosarcoma tissues, and, therefore, may be important during osteosarcoma progression.

SDF-1/CXCR4 promotes F5M2 osteosarcoma cell migration by activating the Wnt/ß-catenin signaling pathway.

Osteosarcoma (OS), the most common primary malignant bone tumor in children and adolescents, lacks an effective therapy. Stromal cell-derived factor (SDF-1) and its receptor, CXCR4, play multiple roles in migration, proliferation, and survival of different tumor cells. This study aimed to investigate whether the functional SDF-1/CXCR4 signaling mediates chemotaxis in F5M2 OS cells as well as the underlying mechanisms. Immunohistochemistry and immunofluorescence microscopy were used. RNA expression was detected by real-time quantitative polymerase chain reaction, and protein expression was examined by Western blotting. Migration assays were carried out in F5M2 cells. The results showed that the expression of CXCR4 and ß-catenin mRNA and protein was significantly higher in OS tissues compared to the surrounding non-neoplastic tissues. SDF-1 promoted F5M2 cell migration by activating the AKT and Wnt/ß-catenin signaling pathway, which was abrogated by preincubation with AMD3100 and LY294002. In conclusion, SDF-1/CXCR4 axis-promoted F5M2 cell migration was regulated by the Wnt/ß-catenin signaling pathway.

Proteomic profiling of osteosarcoma cells identifies ALDOA and SULT1A3 as negative survival markers of human osteosarcoma.

Osteosarcoma (OSA) is the most common primary malignancy of bone. Molecular mechanism underlying OSA remains to be fully elucidated. It is critical to identify reliable diagnostic and prognostic markers for OSA at the molecular levels. This study is designed to investigate possible molecular mechanisms behind OSA development and to identify novel prognostic markers related to OSA survival. We conduct a comprehensive proteomic profiling analysis of human OSA cell lines with differential metastatic potential. Through comprehensive combinatorial analyses of the proteomic data and the previously obtained cDNA microarray results, we identify 37 candidate proteins which are differentially expressed in OSA sublines. Among them, ALDOA and SULT1A3 are selected for further investigation. The expressions of protein are confirmed by Western blotting analysis. We further analyze the expression levels of ALDOA and SULT1A3 from 40 clinical cases of OSA. The results demonstrate that the expression of ALDOA and/or SULT1A3 is significantly higher in patients with worse survival time than patients with better survival time. Five-year survival analysis shows there is a statistically significant difference between two patient populations. The data strongly suggest that ALDOA and/or SULT1A3 expression level in biopsy samples may predict the clinical outcomes of OSA patients. Furthermore, the biological functions of ALDOA and SULT1A3 may be implicated in OSA development and/or progression.

A novel recombinant immunocasp-6 fusion gene specifically and efficiently suppresses HER2-overexpressing osteosarcoma.

Osteosarcoma is the most common primary malignant tumor of bone for adolescent or children. The poor prognosis of patients, due to its remote metastasis, has led to the exploration of more effective and less toxic treatments. Immunotherapy is a promising strategy for the treatment of human epidermal growth factor receptor 2 (HER2)-overexpressing tumors. Herein, we describe experiments conducted with a fusion gene, immunocasp-6, which was generated by fusing a HER2-specific single-chain Ab, a single-chain Pseudomonas exotoxin A and an active caspase-6 which can directly cleave lamin A leading to nucleus damage inducing programmed cell death. We demonstrated that immunocasp-6 can specifically and efficiently recognize and induce apoptosis in HER2-overexpressing osteosarcoma cells in vitro. The immunocasp-6 was transferred into BALB/c athymic mice bearing human osteosarcoma by i.m. injection of liposome-encapsulated pCMV-immunocap-6. Expression of immunocasp-6 not only strongly inhibited tumor growth and significantly prolonged animal survival, but also greatly prevented tumor metastasis. Our data showed that the immuno-casp-6 can specifically recognize HER2-overexpressing osteosarcoma cells, can also promptly attack their nucleus and induce apoptotic death, suggesting the potential of this strategy for the treatment of human HER2-overexpressing tumors.

miR-15a and miR-16-1 downregulate CCND1 and induce apoptosis and cell cycle arrest in osteosarcoma.

Osteosarcoma, the most common primary tumor of the bones, causes many deaths due to its rapid proliferation and drug resistance. Recent studies have shown that cyclin D1 plays a key regulatory role during cell proliferation, and non-coding microRNAs (miRNAs) act as crucial modulators of cyclin D1 (CCND1). The aim of the current study was to determine the role of miRNAs in controlling CCND1 expression and inducing cell apoptosis. CCND1 has been found to be a target of miR-15a and miR-16-1 through analysis of complementary sequences between microRNAs and CCND1 mRNA. The upregulation of miR-15a and miR-16-1 in the cell line SOSP-9607 induces apoptosis and cell cycle arrest. Osteosarcoma cells transfected with miR-15a and miR-16-1 show slower proliferation curves. Moreover, the transcription of CCND1 is suppressed by miR-15a and miR-16-1 via direct binding to the CCND1 3'-untranslated region (3'-UTR). The data presented here demonstrate that the CCND1 contributes to osteosarcoma cell proliferation, suggesting that repression of CCND1 by miR-15a and miR-16-1 could be used for osteosarcoma therapy.

[Construction and expression of human anti-HER2 scFv-9R fusion protein and identification of its activity].

AIM: To construct, express and purify a human fusion protein, which is composed of a single-chain antibody fragment scFv that recognizes HER2 protein and an oligo-9-arginine, and to analyze the binding activity of the expressed fusion protein. METHODS: Pairs of oligonucleotide primers were designed and used to amplify the scFv-9R. The fusion protein gene scFv-9R was then cloned into expression vector pQE30 and expressed in E.coli M15.Expressed protein was detected by SDS-PAGE and Western blot and purified by Ni-NTA chelating agarose. Then, the purified protein was refolded by dialysis and concentrated by ultrafiltration. The antigen-binding activity of the scFv-9R fusion protein was confirmed by ELISA, and the siRNA binding ability was confirmed by electrophoretic mobility shift assay (EMSA). RESULTS: HER2 scFv-9R encoding sequence was correctly cloned into the expression vector. The recombinant protein was insolubly expressed in E.coli M15 induced by IPTG. ELISA confirmed that it had specific antigen binding activity; EMSA assured that it had siRNA binding activity. CONCLUSION: The scFv-9R fusion protein can specially bind with both HER2 antigen and siRNA.

A novel recombinant immuno-tBid with a furin site effectively suppresses the growth of HER2-positive osteosarcoma cells in vitro.

Immunotherapy is a promising strategy for the treatment of human epidermal growth factor receptor 2 (HER2)-positive tumors. Previously, we constructed an immuno-carboxy terminal fragment of Bid (immuno-tBid) and reported its specific and effective destruction of HER2-positive tumor cells. In this study, in order to further reduce the immunogenicity of the previous immuno-proapoptotic protein, we constructed a novel immuno-tBid by replacing domain II of Pseudomonas exotoxin A with a short furin cleavage sequence from the diphtheria toxin. In order to explore the possible application of this novel immuno-tBid in the treatment of osteosarcoma, we first examined the expression of the HER2 protein in a subclone of a human osteosarcoma cell line with relatively high metastatic potential (SOSP-9607-E10), as well as in clinical specimens of osteosarcoma. Quantitative real-time PCR and Western blot analysis revealed that the expression of HER2 was up-regulated in the SOSP-9607-E10 cells, while immunohistochemical analysis revealed that HER2 was overexpressed in 37% of the tissue specimens examined. Both HER2-positive SOSP-9607-E10 and SKBR-3 cells, as well as HER2-negative HeLa cells were transiently transfected with the novel immuno-tBid in order to study its specific pro-apoptotic effect. We demonstrate here that this novel immuno-tBid induces the specific destruction of HER2-overexpressing SOSP-9607-E10 cells through the release of cytochrome C. These results suggest that the novel immuno-tBid with a minimized exogenous fragment could represent a competitive approach for the treatment of HER2-positive osteosarcoma.

Gene expression profiles of human osteosarcoma cell sublines with different pulmonary metastatic potentials.

AIM: to screen the pulmonary metastasis-associated molecules of Osteosarcoma and evaluate their functions concerning prognosis prediction. METHODS: cDNA microarray analysis has been applied to 2 pairs of osteosarcoma cell sublines with differential metastatic potentials to the lung. Immunohistochemistry and survival analysis have been performed to clinical samples of osteosarcoma patients. RESULT: Analysis detected 484 differentially expressed genes between the high metastatic subline, F5M2, and the low metastatic subline, F4. There were 1257 genes differentially expressed between newly established high-metastatic sublines named Saos-2M2 and its parental cell line Saos-2. Furthermore, 16 commonly up-regulated genes and 5 commonly down-regulated genes were identified by clustering analysis. EREG and CHST2, two genes not previously described in osteosarcoma, were finally seen to be differentially expressed in all examined osteosarcoma cell lines and in samples between the different prognosis sample groups. Survival analysis also confirmed these two molecules could be used to predict the outcome of OSA patients. CONCLUSION: This work represents a rationale approach to the evaluation of microarray data and will be useful to identify genes that may be causally associated with metastasis. EREG and CHST2 will be likely considered as clinical molecular markers to predict the outcome of OSA.

[Pro-apoptotic effect on osteosarcoma SOSP-9607 cells by human recombinant caspase-6 fusion protein].

OBJECTIVE: To investigate the pro-apoptotic effect of Her-2 targeted recombinant caspase-6 fusion protein on osteosarcoma SOSP-9607 cells. METHODS: Recombinant immunocasp-6 was generated by sequential fusion of the genes of a signal peptide, a single-chain Her-2 antibody (e23sFv), a PEA translocation domain (PEA aa253-364) and an active caspase-6. The immunocasp-6 gene was cloned into pCMV plasmid to construct a kind of eukaryotic expression vector, i.e. pCMV-e23sfv-PE II-caspase-6 (abbr. pCMV-6) and transfected into SOSP-9607 cells. Murine xenograft models were randomly divided into two groups that received i.m. injections of liposome encapsulated pCMV-6 or pCMV alone. The tumor volume and weight of the nude mice and the tumor weight of the cured mice were observed and statistically analyzed. The morphological changes of the tumors were examined with HE staining, apoptotic morphology of the tumor was observed by TUNEL staining and the gene expression was analyzed by immunohistochemical staining. RESULTS: The tumor growth of the mice in the treatment group was significantly slower than that of the control group (P = 0.001). The weight of the nude mice in the treatment group was significantly higher than that of the control group (P = 0.0002). The tumor weight of the mice in the treatment group was significantly lower than that of the control group (P = 0.0006). HE and TUNEL staining of the tumor of nude mice in the treatment groups showed typical characteristics of apoptosis, while normal structure was found in the control group. Furthermore, caspase-6 was not found in the tumor and muscle tissues in the control group, but only in the treatment group by immunohistochemistry. CONCLUSION: Immunocasp-6 can selectively recognize and bind to and kill HER-2 positive osteosarcoma cells, therefore, to offer some foundation for the clinical treatment of osteosarcoma.

Hydroxysafflor Yellow A protects spinal cords from ischemia/reperfusion injury in rabbits.

BACKGROUND: Hydroxysafflor Yellow A (HSYA), which is one of the most important active ingredients of the Chinese herb Carthamus tinctorius L, is widely used in the treatment of cerebrovascular and cardiovascular diseases. However, the potential protective effect of HSYA in spinal cord ischemia/reperfusion (I/R) injury is still unknown. METHODS: Thirty-nine rabbits were randomly divided into three groups: sham group, I/R group and HSYA group. All animals were sacrificed after neurological evaluation with modified Tarlov criteria at the 48th hour after reperfusion, and the spinal cord segments (L4-6) were harvested for histopathological examination, biochemical analysis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. RESULTS: Neurological outcomes in HSYA group were slightly improved compared with those in I/R group. Histopathological analysis revealed that HSYA treatment attenuated I/R induced necrosis in spinal cords. Similarly, alleviated oxidative stress was indicated by decreased malondialdehyde (MDA) level and increased superoxide dismutase (SOD) activity after HSYA treatment. Moreover, as seen from TUNEL results, HSYA also protected neurons from I/R-induced apoptosis in rabbits. CONCLUSIONS: These findings suggest that HSYA may protect spinal cords from I/R injury by alleviating oxidative stress and reducing neuronal apoptosis in rabbits.

Selective cytotoxicity to HER2-positive tumor cells by a recombinant e23sFv-TD-tBID protein containing a furin cleavage sequence.

PURPOSE: The HER2 antigen is a recognized target on breast cancer cells for immunotherapy. To overcome the immunogenicity and systemic toxicity of traditional immunotoxins, a novel human immunoproapoptotic molecule was developed and its antitumor activity was investigated. EXPERIMENTAL DESIGN: Recombinant e23sFv-TD-tBID, consisting of a single-chain anti-HER2 antibody fragment linked to a human active truncated Bid by a 10-amino acid residue furin cleavage sequence, was bacterially expressed. Purified e23sFv-TD-tBID was tested for binding, internalization, and cytotoxic activity in cell and for tumor localization and antitumor activity in athymic nude mice bearing established human tumor xenografts. RESULTS: e23sFv-TD-tBID selectively binds to HER2-positive cells and induces apoptotic cell death in vitro and in vivo. An investigation of its mechanism of action has revealed that e23sFv-TD-tBID was internalized on binding to the surface of HER2-positive tumor cells, proteolytically cleaved and transported directly to cytosol. The antitumor activity of e23sFv-TD-tBID was shown in a dose-dependent manner when injected i.p. into immunodeficient mice bearing human breast carcinomas. Moreover, this immunoproapoptotic protein, either given as a single dose or in combination with chemotherapy agents, significantly inhibited tumor growth without any observed toxic side effects on mice. Magnetic resonance imaging further showed the specific targeting and good penetration of tumors by e23sFv-TD-tBID in vivo. The therapeutic value of e23sFv-TD-tBID to human was shown by its cytotoxic effects on primary patient-derived breast tumor cells but not on endothelial cells. CONCLUSION: These data suggest that recombinant e23sFv-TD-tBID has therapeutic potential for HER2-positive tumors and warrant further testing for clinical applications.

A caspase-6 and anti-HER2 antibody chimeric tumor-targeted proapoptotic molecule decreased metastasis of human osteosarcoma.

Human growth factor receptor-2 (HER2), overexpressed as a result of gene amplification, is detected in 20-40% of patients with breast, ovarian, endometrial, gastric, bladder, prostate, or lung cancers, correlated to metastasis of many tumors, and considered to be a poor prognostic indicator for these tumors. However, the data was controversial for HER2 overexpression and the prognosis of osteosarcoma, which is the most common primary malignant bone tumor, presents a therapeutic challenge in medical oncology due to its metastasis and poor response to current treatments. Previously, we reported that the immunocasp-6 gene fused by a HER2-specific single-chain antibody with domain II of Pseudomonas exotoxin A (PEA) and the 5' end of the truncated active caspase-6 could selectively suppress the HER2-positive tumor growth. In this study, we extend its application. We first confirmed the higher HER2 expression on the surface of metastatic osteosarcoma SOSP-9607(E10) cells, which then be proved specifically addicted to immunocasp-6-induced cells killing in vitro. Thereafter, the efficacy of immunocasp-6 was tested in an osteosarcoma lung metastasis mouse model using intramuscular (i.m.) injections of liposome-encapsulated vectors. Our results showed that the expression of the immunocasp-6 gene not only significantly prolonged animal's survival, but also greatly inhibited tumor metastasis. Thereby, our strategy suggests an alternative approach to treating HER2/neu-positive osteosarcoma.

scFv-mediated delivery of truncated BID suppresses HER2-positive osteosarcoma growth and metastasis.

Osteosarcoma is the most common primary malignant bone tumor, with high rates of metastasis. Here, we examined the expression of human epidermal growth factor receptor-2 (HER-2) in osteosarcoma cell lines with different metastatic potential, finding that the expression was correlated with metastasis of implanted tumors. We then introduced an expression vector encoding the e23sFv-PEA II-Bid Delta1-60 gene, composed of a HER2-specific single-chain antibody fused with domain II of Pseudomonas exotoxin A (PEA) and the carboxy end of truncated active Bid. We demonstrated that the e23sFv-PEA II-Bid Delta1-60 molecule selectively recognized and killed HER2-overexpressing osteosarcoma cells in vitro. Subsequently, we introduced the e23sFv-PEA II-bid Delta1-60 gene into BALB/c athymic mice bearing HER2-positive osteosarcomas using i.m. injections of liposome-encapsulated vector. Expression of the e23sFv-PEA II-Bid Delta1-60 gene suppressed tumor growth, significantly prolonged animal survival and inhibited metastasis, thereby suggesting it may represent a competitive approach to treating HER2/neu-positive osteosarcoma.

Single-chain antibody/activated BID chimeric protein effectively suppresses HER2-positive tumor growth.

BH3-interacting domain death agonist (BID) is a crucial element in death signaling pathways and is recognized as an intracellular link connecting the intrinsic mitochondrial apoptotic and extrinsic death receptor-mediated apoptotic pathways. Herein, we describe experiments conducted with a fusion protein, which was generated by fusing a human epidermal growth factor receptor-2 (HER2)-specific single-chain antibody with domain II of Pseudomonas exotoxin A and the truncated active BID (tBID). These experiments extend our previous work on several other immuno-proapoptotic proteins. Specifically, by excluding cells with undetectable HER2, we showed that the secreted immuno-tBID molecule selectively recognized and killed HER2-overexpressing tumor cells in vitro by attacking their mitochondria and inducing their apoptotic death. This apoptosis could only be inhibited partially by caspase pan-inhibitor zVAD and mitochondrial protector TAT-BH4. Subsequently, we transferred the immuno-tbid gene into BALB/c athymic mice bearing HER2-positive tumors together with other immuno-proapoptotic proteins using i.m. injections of liposome-encapsulated vectors. The expression of the immuno-tbid gene suppressed tumor growth and prolonged animal survival significantly. We also shortened the translocation domain of Pseudomonas exotoxin A II to only 10-amino acid sequence, which were crucial for furin cleavage. The new recombinant molecule retained the translocation efficiency and the ability of specific killing HER2-positive tumor cells. Our data showed that, compared with the toxins employed before, the chimeric immuno-tBID molecule can not only specifically recognize HER2-positive tumor cells but also certainly induce apoptosis even in the presence of zVAD and TAT-BH4, thereby suggesting an alternative approach to treating HER2/neu-positive tumors.

[Cloning and identification of microRNA from human osteosarcoma cell line SOSP-9607].

BACKGROUND & OBJECTIVE: MicroRNA (miRNA), a group of non-coding small RNA (20-25 nt) involved in post-transcriptional regulation, regulate gene expression and closely relate to cancer pathogenesis. This study was to clone miRNA from osteosarcoma cell line SOSP-9607, and identify the expression of some functional genes. METHODS: Low molecular weight RNA fraction (< or =200 nt) was extracted from SOSP-9607 cells, and polyadenylated by poly(A) polymerase. Then a 5' RNA adapter was ligated to poly(A)-tailed RNA using T4 RNA ligase. RNAs were reversely transcribed and amplified by reverse transcription-polymerase chain reaction (RT-PCR). The PCR product of 109 bp was recovered and cloned into pCR 4-TOPO vector. After sequencing, database searching, and expression profiling, the expression of miRNA in SOSP-9607 cells was sieved. The expression of novel and some known miRNAs was examined by Northern blot with small RNAs (< or =200 nt) isolated from SOSP-9607 cells, osteosarcoma tissue, and HeLa cells. RESULTS: A total of 182 clones were subsequently characterized through DNA sequencing and database searching; 47 clones (correspond to 25 species) out of the 182 clones from SOSP-9607 cells were identified as miRNAs. Two novel miRNAs (miR-165 and miR-166) were discovered among other 23 known miRNAs, which were predicted in Nature. Northern blot confirmed that the 2 novel miRNAs and 3 solid cancer miRNAs (miR-21, miR-20a,miR-17-5p) were stably expressed in SOSP-9607 cells and osteosarcoma tissue, but miR-166 was not expressed in HeLa cells. CONCLUSION: We have cloned miRNAs for SOSP-9607 cells and identified parts of the functional ones, which imply that miRNAs may closely relate to the cancer pathogenesis.

A caspase-6 and anti-human epidermal growth factor receptor-2 (HER2) antibody chimeric molecule suppresses the growth of HER2-overexpressing tumors.

Clinical studies have suggested that human epidermal growth factor receptor-2 (HER2) provide a useful target for antitumor therapy. We previously described the generation of a chimeric HER2-targeted immunocasp-3 protein. In this study, we extend the repertoire of chimeric proapoptotic proteins with immunocasp-6, a construct that comprises a HER2-specific single-chain Ab, a single-chain Pseudomonas exotoxin A, and an active caspase-6, which can directly cleave lamin A leading to nucleus damage and inducing programmed cell death. We demonstrate that the secreted immunocasp-6 molecule selectively recognizes and induces apoptosis in HER2-overexpressing tumor cells in vitro, but not in cells with undetectable HER2. The immunocasp-6 gene was next transferred into BALB/c athymic mice bearing human breast SK-BR-3 tumors by i.m. injection of liposome-encapsulated vectors, by intratumor injection of adenoviral vectors, or by i.v. injection of PBMC modified by retroviral infection. Regardless of the method used, expression of immunocasp-6 suppressed tumor growth and prolonged animal survival significantly. Our data show that the chimeric immunocasp-6 molecule can recognize HER2-positive tumor cells, promptly attack their nucleus, and induce their apoptotic death, suggesting the potential of this strategy for the treatment of human cancers that overexpress HER2.

[The cloning and expression of the truncated human bid gene and its pro-apoptotic effect on Hela cells].

AIM: To explore the expression of the truncated bid gene and its pro-apoptotic effect on Hela cells. METHODS: A full-length human bid gene was cloned by RT-PCR and confirmed by sequence analysis. By deleting the 60 amino acids of N-terminal the truncated bid (tbid) gene was obtained and then inserted into the eukaryotic expression vector pIRES2-EGFP. After the tbid gene was transfected into Hela cells under lipofectamine mediation, the effect of target gene expression on morphology and growth of Hela cells were observed under fluorescence and electron microscopes and analysed by TUNEL staining. RESULTS: pIRES2-EGFP containing tbid gene was constructed successfully. After Hela cells were transfected with GFP expression vector of tbid gene, tbid was expressed which was followed by decreased cell fluorescence intensity, poor cell growth, and cell death. Cell shrinkage and nuclear condensation, typical apoptotic characteristics, were observed by electron microscope observation and Tunel staining. CONCLUSION: The expression of tbid can effectively induce apoptosis of Hela cells.