Apoptosis Rate Of Tumor Cells Research Articles

Expression of Testis-Specific Gene Antigen 10 (TSGA10) is Associated with Apoptosis and Cell Migration in Bladder Cancer Cells and Tumor Stage and Overall Survival in Patients with Bladder Cancer.

BackgroundTestis-specific gene antigen 10 (TSGA10) is a tumor suppressor in several types of human malignancy. However, there have been few studies that have investigated the role of TSGA10 in bladder cancer. This study aimed to investigate the expression of TSGA10 in human bladder cancer cell lines and bladder cancer tissues and its effects on patient prognosis.Material/MethodsThe expression of TSGA10 in 40 tissue samples of bladder cancer and matched normal adjacent bladder tissue, and five human bladder cancer cell lines was assessed by immunohistochemistry, Western blot, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and flow cytometry. The correlation between the expression level of TSGA10 and the clinicopathological features of patients with bladder cancer was analyzed and overall survival (OS) in patients with bladder cancer was determined by Kaplan-Meier curves.ResultsUpregulation of TSGA10 expression in tissues from patients with bladder cancer was compared with normal adjacent bladder tissue and was significantly correlated with gender, metastasis, lymphovascular invasion, and tumor stage in bladder cancer. In bladder cancer cell lines, down-regulation of TSGA10 reduced cell apoptosis and increased cell migration, and resulted in the formation of an epithelial-mesenchymal transition (EMT) phenotype. Overexpression of TSGA10 resulted in an increased apoptosis rate of tumor cells, reduced cell migration, and contributed to the reversal of the EMT phenotype.ConclusionsThese findings support that TSGA10 deserves further study as a potential novel prognostic biomarker in bladder cancer.

Self‐Delivery Nanoparticles of Amphiphilic Acyclic Enediynes for Efficient Tumor Cell Suppression

Summary of main observation and conclusionFour acyclic maleimide‐based enediyne compounds with different hydrophilicity were synthesized through Sonogashira reaction to reveal a self‐delivery antitumor drug platform. As proved by ESR analysis, the enediyne compounds undergo Bergman‐like cyclization and generate diradical intermediates at physiological temperature, which are able to induce DNA‐cleavage through the abstraction of H atoms from the sugar‐phosphate backbones. When the critical aggregation concentration is reached in water, the amphiphilic enediyne compounds self‐assemble into nanoparticles and possess the self‐delivery ability to be facilely admitted by tumor cells, resulting in greatly improved cytotoxicity (IC50 down to 10 μmol·L–1) and much higher tumor cell apoptosis rate (up to 86.6%) in comparison with either the hydrophilic or the lipophilic enediyne compound. The enhanced endocytosis of the amphiphilic enediyne compounds was further confirmed through confocal laser scanning microscopy analysis. The unveiled relationship between the hydrophilicity of enediyne drugs and their therapeutic efficacy will provide a guideline for the design of new self‐delivery drugs employed in medicinal applications.

Additive antitumor effect of arsenic trioxide combined with intravesical bacillus Calmette–Guerin immunotherapy against bladder cancer through blockade of the IER3/Nrf2 pathway

BackgroundAs an inorganic compound used to treat various cancers and other diseases, arsenic trioxide (As2O3) has been reported to induce cellular apoptosis in certain kinds of cancers including bladder cancer. The aim of the present study was to elucidate the crucial cooperative role of As2O3 and intravesical bacillus Calmette-Guerin (BCG) immunotherapy and its ability to protect against bladder cancer by targeting the IER3/Nrf2 pathway. MethodInitially, an orthotopic bladder cancer model was established in mice by means of intravesical instillation of the human bladder cancer cell line 5637. The expression of IL-6/IL-8 in dendritic cells (DCs) and the proportion of CD4+ cells and ratio of CD4+/CD8+ T cells were subsequently determined. RT-qPCR and Western blot assay methods were employed to determine the expressions of IER3, Nrf2, NQO1, IL-6 and IL-8. Finally, tumor cell apoptosis and the volume and weight of the in vivo tumors were evaluated in an attempt to determine the contributory role of As2O3 in combination with BCG immunotherapy in treating bladder cancer. ResultsThe additive effect of As2O3 and BCG was demonstrated to promote the expressions of IL-6/IL-8 among DCs. Additionally, the proportion of CD4+ cells, ratio of CD4+/CD8+ T cells and rate of tumor cell apoptosis were all elevated, while decreased in vivo tumor volume and weight were detected. Of importance, we determined the role that ad-shNrf2 (adenoviral vectors expressing shRNA against Nrf2) played in inhibiting the effects of As2O3 on bladder cancer. ConclusionTaken together, the key findings of the present study provide evidence defining the effect of As2O3 on inducing the inhibitory effect of BCG on the development of bladder cancer via the IER3/Nrf2 pathway, highlighting the potential of As2O3 as a treatment option for bladder cancer through its enhancement of intravesical BCG.

Correlation between adenosine triphosphate (ATP)-binding cassette transporter G2 (ABCG2) and drug resistance of esophageal cancer and reversal of drug resistance by artesunate

The present study investigated the correlation between the abnormal expression of adenosine triphosphate (ATP)-binding cassette transporter G2 (ABCG2) in esophageal cancer and the drug resistance of esophageal cancer, the reversal effect in drug resistance of artesunate (Art), and the mechanism underlying esophageal cancer using nude mice with subcutaneous xenograft as an animal model. The gene and protein expression of ABCG2 was detected in 80 cases of esophageal cancer, atypical dysplasia, and normal mucosa. A subcutaneous xenograft tumor mouse model was established by subcutaneous inoculation of esophageal cancer cell line Eca109/ABCG2 into BALB/c nu/nu nude mice. The reversal of drug resistance by Art in esophageal cancer was studied in vivo. The mice model was injected intraperitoneally with Art and/or doxorubicin (ADM). The animals were divided into six groups: high dose Art (50 mg/kg), low dose Art (25 mg/kg), ADM (1 mg/kg), ADM and high-dose Art, ADM and low-dose Art, and physiological saline as a control. ABCG2 protein expression and cellular ADM concentration were detected by flow cytometry. The mRNA and protein expressions of ABCG2 in esophageal cancer were significantly higher than that in the normal esophagus (P < 0.01). The volume and mass of the subcutaneously implanted tumors in the ADM+Art high- and low-dose groups were significantly decreased than that in the control group (P < 0.05), while the apoptosis rate of tumor cells and the cellular concentration of ADM were increased significantly (P < 0.05), and the ABCG2 protein expression in the tumor cells decreased significantly (P < 0.05). Abnormally high expression of ABCG2 in esophageal cancer tissues is involved in the multidrug resistance of esophageal cancer. In vivo studies showed that Art could reverse the esophageal cancer drug resistance by regulating the ABCG2 expression in tumor cells.

Inhibiting the proliferation and metastasis of hilar cholangiocarcinoma cells by blocking the expression of vascular endothelial growth factor with small interfering RNA.

The aim of the present study was to investigate whether the proliferation and metastasis of hilar cholangiocarcinoma cells can be suppressed and whether apoptosis can be induced by small interfering RNA (siRNA) repression of vascular endothelial growth factor (VEGF). siRNA sequences targeting the VEGF gene were designed and the human hilar cholangiocarcinoma QBC939, HCCC-9810 and RBE cell lines were transfected with VEGF-siRNA plasmids for 48 h. Reverse transcription-quantitative polymerase chain reaction and western blotting measured the levels of VEGF-A, VEGF-C and matrix metalloproteinase 2 (MMP2) mRNA expression and protein content. The cell invasion potential was evaluated using the Transwell invasion and migration assay and the MTT assay was employed to detect the proliferation of hilar cholangiocarcinoma cells. Flow cytometry was used to quantify cell apoptosis and necrosis. Following the transfection of VEGF-siRNA, a significant reduction of mRNA and protein levels of VEGF-A, VEGF-C and MMP2 was observed in the hilar cholangiocarcinoma cells. The invasion, migration and proliferation of tumor cells were also notably decreased. The rate of tumor cell apoptosis was increased in the VEGF-siRNA group (15.42%) compared with the non-siRNA control (2.22%) and the negative control (2.71%) groups. It was concluded that blocking the expression of VEGF via VEGF-siRNA effectively inhibited the invasion, migration and proliferation, and induced apoptosis in hilar cholangiocarcinoma cells. These observations suggested that targeting VEGF with RNAi may be an effective therapeutic strategy for treating hilar cholangiocarcinoma.

Invivo antitumor activity of liposome‑plasmid DNA encoding mutant survivin‑T34A in cervical cancer.

The aim of the present study was to investigate the influence of liposome-plasmid encoding mutant survivin-T34A (PST34A) on tumor growth in cervical cancer in vivo. Liposome-plasmid DNA encoding mutant survivin-T34A was constructed and administered via an intraperitoneal injection in mice inoculated with cervical cancer cells. Following the establishment of the tumor model, the animals were randomly divided into four groups: i) The normal saline group (NS; 100 µl sterile saline once/3 days for 15 days); ii) the 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) control (100 µg DOTAP once/3 days for 15 days); iii) the plasmid PST34A (10 µg PST34A once/3 days for 15 days); and iv) the PST34A+DOTAP (10 µg PST34A+100 µg DOTAP once/3 days for 15 days). All treatments were administered via intraperitoneal injections. Tumor growth was evaluated following injection with liposome-plasmid DNA encoding mutant survivin-T34A. Apoptosis of cells in ascitic fluid was detected by flow cytometry. The expression of Ki67 and CD34 was detected by immunohistochemical staining. Administration of liposome-plasmid complexes encoding mutant survivin-T34A inhibited tumor growth, reduced the number of tumor nodules and the volume of ascitic fluid, and decreased abdomen circumference and tumor weight. The number of Ki67-positive cells was markedly reduced in the DOTAP+PST34A group compared with the remaining groups. Flow cytometry demonstrated that the number of cells in the sub-G1 phase (apoptosis) increased in the DOTAP+PST34A group compared with all other groups. In addition, tumors in the DOTAP+PST34A group exhibited lower microvessel density compared with all other groups. In the present study, liposome-plasmid DNA encoding mutant survivin-T34A could inhibit tumor growth of cervical cancer. This inhibition may be associated with an increase in the apoptosis rate of tumor cells and a reduction in angiogenesis.

Specific light-up pullulan-based nanoparticles with reduction-triggered emission and activatable photoactivity for the imaging and photodynamic killing of cancer cells.

Activatable photosensitizers that can be activated by cancer-associated stimuli have drawn increasing attention for simultaneous fluorescence imaging and photodynamic ablation of cancer cells. Here, we developed a cancer-cell specific photosensitizer nano-delivery system by synthesizing protoporphyrin IX (PpIX)-conjugated pullulan (P) with reducible disulfide bonds. The amphiphilic P-s-s-PpIX conjugate self-assembled in aqueous condition to form core-shell structured nanoparticles (P-s-s-PpIX NPs) with average size of 166nm, showing reduction-controllable stability. In in vitro, the photoactivity of P-s-s-PpIX NPs in an aqueous environment was significantly suppressed by the self-quenching effect, which kept P-s-s-PpIX NPs in a photo-inactive and quenched state. But in the presence of GSH, P-s-s-PpIX NPs quickly dissociated by reductive breakage of disulfide linkers, followed by the significant recovery of fluorescent emission and singlet oxygen generation. In MCF-7 cells, compared to non-reducible P-PpIX NPs with stable amide linkages, P-s-s-PpIX NPs displayed higher cytotoxicity and induced higher apoptosis rate of tumor cells with light irradiation treatment. As a result, the P-s-s-PpIX NPs may serve as an effective smart nanomedicine platform for specific light-up and reduction-triggered cancer imaging and photodynamic therapy with the prominently reduced damage to normal tissues and cells.

Effects of high-intensity focused ultrasound on apoptosis-associated gene expression in xenografts with human pancreatic cancer

Objective: To identify the expression of apoptosis-associated genes of high- intensity focused ultrasound(HIFU) in xenograft with human pancreatic cancer. Methods: Mice implated with human pancreatic cancer cells (YY-1) were divided into HIFU group or control group. Tumor cell apoptosis was verified by TUNEL. The expression of the apoptosis-associated genes was analyzed by Agilent Human Gene Expression. Selected genes was validated by quantitative real-time PCR(RT-PCR)and Western blot. Results: The rate of tumor cell apoptosis in HIFU group was higher than that of control group at 7, 14 days after HIFU treatment ((63.6%±15.2%)vs (19.0%±2.4%), P<0.01)and ((41.4%±7.3%)vs(18.0%±2.4%), P<0.01). Gene expression profiling revealed a total of 69 differentially expressed genes in related to apoptosis pathway, among which 44 genes were up-regulated, and 25 genes down-regulated. The RT-PCR results of selected 4 genes were consistent with those of gene expression profiling. The results of Western blot analysis at 7, 14 days after HIFU treatment showed that the expressions level of Bax protein in HIFU group was greater that of in control group ((0.39±0.11)vs (0.20±0.09), P<0.05)and ((0.46±0.12)vs(0.24±0.10), P<0.05), while the expressions level of Bcl-2 protein in HIFU group was lower than that of in control group ((0.68±0.14)vs(1.56±0.21), P<0.05)and((0.51±0.16)vs(1.57±0.22), P<0.05). Conclusions: HIFU could induce apoptosis and results in dramatic changes in gene expression, indicating that multiple pathways are involved. Although intrinsic pathway might be predominantly involved in HIFU-elicited apoptosis, further research is needed to clarify the detailed mechanisms.

Biocompatible cationic pullulan-g-desoxycholic acid-g-PEI micelles used to co-deliver drug and gene for cancer therapy

The greatest crux in the combination of chemotherapy and gene therapy is the construction of a feasible and biocompatible carrier for loading the therapeutic drug and gene simultaneously. Here, a new amphiphilic bifunctional pullulan derivative (named as PDP) synthesized by grafting lipophilic desoxycholic acid and low-molecular weight (1kDa) branched polyethylenimine onto the backbone of pullulan was evaluated as a nano-carrier for the co-delivery of drug and gene for potential cancer therapy. PDP exhibited good blood compatibility and low cytotoxicity in the hemolysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. By self-assembly process, the amphiphilic PDP polymer formed cationic core-shell nanomicelles in aqueous solution with an average diameter of 160.8nm and a zeta potential of approximate 28mV. The PDP micelles had relative higher drug encapsulation efficiency (84.05%) and loading capacity (7.64%) for doxorubicin (DOX), an effective anti-tumor drug, demonstrating sustained drug release profile and good DNA-binding ability. The flow cytometry and confocal laser scanning microscopy showed that PDP/DOX micelles could be successfully internalized by MCF-7 cells, and presenting higher cytotoxicity against MCF-7 cells than that of free DOX. Furthermore, PDP micelles could efficiently transport tumor suppressor gene p53 into MCF-7 cells, and the expressed exogenous p53 protein induced MCF-7 cells to die. More excitedly, in comparison with single DOX or p53 delivery, the co-delivery of DOX and gene p53 using PDP micelles displayed higher cytotoxicity, induced higher apoptosis rate of tumor cells and blocked more effectively the migration of cancer cells in vitro. In tumor-bearing mice, co-delivery of DOX and p53 also exhibited enhanced antitumor efficacy as compared with single delivery of DOX or p53 alone. In summary, these results demonstrated that it is highly promising to use cationic PDP micelles for effectively co-delivering functional gene and chemotherapeutic agent, and thus improving antitumor efficacy and systemic toxicity.

A42: Effect of mesenchymal stem cells on the microenvironment and functional morphology of sarcoma M-1

The mechanisms of mesenchymal stem cells (MSC) action on malignant neoplasms attract much attention nowadays. Studies relating to the MSC effect on tumors are contradictory and indicate that MSC has both stimulating and suppressive actions on experimental neoplasms. We have shown previously that a single MSC administration to tumor-bearing animals has an oncomodulating effect on the growth of tumors. Short-term stimulation of the growth of tumor nodes initially after the MSC injection was usually followed by subsequent deceleration of their growth. Here, we have studied the effects of allogeneic MSC on sarcoma M-1 in rats. On day 11 after the sarcoma M-1 implantation, 1.5 million MSC grown from the bone marrow cell population of Wistar rats was administered into the tail vein of outbred rats from the experimental group. On days 17 and 30 of the tumors growth, 10 rats from each group were used to study the sarcoma M-1. To analyze the MSC distribution and localization in the tumor parenchyma, on days 11 and 13, five rats in the study were intravenously infused 2 million MSC, labeled in vitro by bromodeoxyuridine (BrdU). The techniques for studying of the tumor reaction on the systemic MSC transplantation included immunostaining for PCNA, BrdU and PECAM-1, in conjunction with computerized analysis of the microscopic images. One day after transplantation, the BrdU-labeled MSC localized in the perivascular areas of angiogenesis on the periphery of tumor nodes with the diameter of more than 6 mm, containing foci of spontaneous necrosis. Sometimes, labeled cells could be seen near the vessels situated deep in the tumor parenchyma. On day 3, cells with the low-intense BrdU immunostaining were found only rarely and mostly perivascularly. These cells were visualized in the region of pericytes’ localization, and the low BrdU-immunostaining intensity of their nuclei indicated the label dilution effect. Six days after the MSC administration, some local areas of the connective tissue enlargements on the periphery of tumor nodes were observed with a distinct vascular ingrowth into the tumor parenchyma. In the same areas, the marginal region of tumor nodes contained a considerable number of neutrophils and lymphocytes. The PCNA staining revealed foci of increased proliferative activity of tumor cells in these areas, as well as intensive proliferation of fibroblasts and vascular endothelium, which indicates the increase of angiogenesis and stroma formation. After the MSC administration the content of parenchyma with PCNA-positive nuclei significantly increased, whereas the volume fraction of the necrosis regions decreased by more than 1.5 times. On day 30 of the sarcoma M-1 growth, tumors in the experimental group rats were surrounded by layers of connective tissue. The peritumoral area was infiltrated by numerous lymphocytes and macrophages. In the terminal period of the sarcoma growth, the most quantitative parameters for tumors in the experimental group did not significantly differ from the data obtained in the control group. Only the rate of tumor cell apoptosis in animals with transplanted MSC was statistically higher than that in control rats. Reviewing the literature regarding the influence of MSC on the malignant growth revealed that this problem still remains quite unclear and disputable. One of the contradictions is their ability to have opposite effects on the repopulation activity of tumor cells. The complex interactions between MSC, tumor microenvironment and neoplastic cells seem to be crucial for the outcome of the oncological process development. Further detailed studies of the mechanisms of the cellular therapy using MSC on carcinogenesis are necessary to generate new insights into this area.

Upregulation of miR-129-5p affects laryngeal cancer cell proliferation, invasiveness, and migration by affecting STAT3 expression.

microRNA-129-5p may have a relationship with laryngeal squamous cell carcinoma (LSCC), but the expression and function of miRNA-129-5p are not fully understood. This study was performed to investigate the expression and function of miRNA-129-5p in LSCC and its effects on STAT3. HBE and Hep-2 cells were cultured and analyzed by western blotting and real-time PCR. miR-129-5p expression was detected by real-time PCR. Hep-2 cells were transfected with a miR-129-5p expression vector. Cell proliferation, invasion, migration, cell cycle, and apoptosis assays were used to determine the role of miRNA-129-5p in the growth, invasion, and migration of LSCC cells and to determine the importance of STAT3 in these effects. STAT3 mRNA expression in Hep-2 cells was significantly higher than that in HBE cells (P < 0.05). miR-129-5p expression detected by real-time PCR showed that it was expressed at a lower level in Hep-2 cells than in HBE cells (P < 0.05). Compared with the control cells, the transfected cells showed lower STAT3 mRNA expression. For up to 7days in culture, the transfected cells showed lower proliferation than the control cells (P < 0.05). After 24h in culture, the apoptosis rate in miR-129-5p-transfected cells was 3.48 ± 0.38%, while the rate in control cells was 0.92 ± 0.09% (P = 0.0028), but the statistical significance was lost after 72h in culture (P = 0.3180). The invasion and migration of the cells were inhibited after 24 and 72h in culture when the miR-129-5p expression in Hep-2 cells was upregulated (P = 0.0037 and 0.00383, respectively), while there was no statistically significant difference at 48h (P = 0.0712). STAT3 expression could be suppressed by the upregulation of miR-129-5p expression. Both the proliferation and migration of tumor cells were suppressed when the level of STAT3 expression was decreased. The apoptosis rate of tumor cells was also increased. Based on these data, we suggest that miR-129-5p may directly inhibit STAT3 expression and play an important role in the development of LSCC.

Endostar combined with cryoablation for subcutaneous xenografted tumor model of lung adenocarcinoma cell line A549 in BALB/c nude mice: An experimental study

Objective To investigate the inhibitory effect of Endostar combined with cryoablation on lung adenocarcinoma cell line A549 in BALB/c nude mice, and to discuss its interaction mechanisms.Methods The lung adenocarcinoma A549 model in BALB/c nude mice were established. When the largest diameter of tumor reached 1.0 cm, a total of 24 mice were randomly and equally divided into 4 groups:control group, Endostar group, cryoablation group and cryoablation plus Endostar group. The largest diameter and the vertical diameter of the tumors were measured at different points of time after treatment. At the 21st day, the mice were sacrificed and the tumors were removed and the rate of tumor cell apoptosis, the microvessel density(MVD) and the expression level of vascular endothelial growth factor(VEGF) were determined by using immunohistochemistry method. The results were statistically analyzed. Results The tumor growth velocity of the control group, Endostar group, cryoablation group and cryoablation plus Endostar group was(236.68 ± 51.23)%,(220.02 ± 30.61)%,(159.46 ± 29.33)% and(103.34 ± 25.50)%,respectively(P 0.01). The rate of apoptosis of the four groups was(21.67 ± 2.34)%,(22.17 ± 1.47)%,(38.33 ± 1.37)% and(49.17 ± 1.72)%,respectively(P 0.01). The MVD and the expression levels of VEGF of the cryoablation plus Endostar group were significantly lower than those of the other three groups(P 0.01). Statistical analysis revealed that a positive correlation existed between the express of VEGF and MVD. Conclusion Endostar can obviously enhance the therapeutic efficacy of cryoablation on lung adenocarcinoma A549 in BALB/c nude mice. The underlying mechanisms may be the Endostar-inhibited angiogenesis through down-regulating the expression of VEGF, and the cooperative effect of Endostar and cryoablation on the promotion of tumor cell apoptosis.

Liquid-Solid Phase-Inversion PLGA Implant for the Treatment of Residual Tumor Tissue after HIFU Ablation

BackgroundHIFU has been shown to be a more suitable alternative for the treatment of primary solid tumors and metastatic diseases than other focal heat ablation techniques due to its noninvasive and extracorporeal nature. However, similar to other focal heat ablation techniques, HIFU is still in need of refinements due to tumor recurrence.MethodsIn this work, we investigated the effectiveness of an adjunct treatment regimen using doxorubicin (DOX)-loaded, injectable, in situ-forming, and phase-inverting PLGA as the second line of defense after HIFU ablation to destroy detrimental residual tumors and to prevent tumor recurrence. All of the statistical analyses were performed using the Statistical Package for the Social Sciences 18.0(SPSS, Inc., Chicago, IL, USA), and p< 0.05 was considered statistically significant. All of the results are presented as the means ± STDEV (standard deviation). For multiple comparisons, ANOVA (differences in tumor volumes, growth rates, apoptosis, proliferation indexes, and Bcl-2 and Bax protein levels) was used when the data were normally distributed with homogenous variance, and rank sum tests were used otherwise. Once significant differences were detected, Student-t tests were used for comparisons between two groups.ResultsOur results revealed that DOX diffused beyond the ablated tissue regions and entered tumor cells that were not affected by the HIFU ablation. Our results also show that HIFU in concert with DOX-loaded PLGA led to a significantly higher rate of tumor cell apoptosis and a lower rate of tumor cell proliferation in the areas beyond the HIFU-ablated tissues and consequently caused significant tumor volume shrinkage (tumor volumes:0.26±0.1,1.09±0.76, and 1.42±0.9cm3 for treatment, sham, and no treatment control, respectively).ConclusionsFrom these results, we concluded that the intralesional injection of DOX-loaded PLGA after HIFU ablation is significantly more effective than HIFU alone for the treatment of solid tumors.

Picosecond pulsed electric fields induce apoptosis in a cervical cancer xenograft.

The aim of the present study was to evaluate the efficacy of picosecond pulsed electric fields (psPEF) on a cervical cancer xenograft. Human cervical cancer xenografts were established in nude mice by transplantation of HeLa cells, and the tumors were then treated with psPEF. The histological changes were observed by hematoxylin-eosin staining and transmission electron microscopy. The rate of tumor cell apoptosis was determined using a terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay. The mitochondrial transmembrane potential of the tumor cells was detected by laser scanning confocal microscopy, and the activity of caspase-3, -8, -9 and -12 was determined. The inhibitory rate seven days post-psPEF treatment was also calculated. The results showed that exposure to psPEF led to an increased rate of apoptosis, collapse of mitochondrial transmembrane potential, and activation of caspases. The inhibitory rate was 9.11% at day 7. The results of the present study indicate that psPEF may induce apoptosis in a cervical cancer xenograft through the endoplasmic reticulum stress and caspase-dependent signaling pathways.

Depletion of telomerase RNA inhibits growth of gastrointestinal tumors transplanted in mice

To explore effects of telomerase RNA-targeting phosphorothioate antisense oligodeoxynucleotides (PS-ASODN) on growth of human gastrointestinal stromal tumors transplanted in mice. A SCID mouse model for transplantation of human gastrointestinal stromal tumors (GISTs) was established using tumor cells from a patient who was diagnosed with GIST and consequently had been treated with imatinib. GIST cells cultured for 10 passages were used for inoculation into mice. Transfection of PS-ASODN was carried out with Lipotap Liposomal Transfection Reagent. GISTs that subsequently developed in SCID mice were subjected to intra-tumoral injection once daily from day 7 to day 28 post-inoculation, and mice were divided into the following four groups according to treatment: PS-ASODN group (5.00 μmoL/L of oligonucleotide, each mouse received 0.2 mL once daily); imatinib group (0.1 mg/g body weight); liposome negative control group (0.01 mL/g); and saline group (0.01 mL/g). On day 28, the mice were sacrificed, and tumor attributes including weight and longest and shortest diameters were measured. Tumor growth was compared between treatment groups, and telomerase activity was measured by enzyme-linked immunosorbent assay. Apoptosis was examined by flow cytometry. Real-time polymerase chain reaction was used to detect expression of the mRNA encoding the apoptosis inhibition B-cell leukemia/lymphoma 2 (bcl-2) gene. In the PS-ASODN group, tumor growth was inhibited by 59.437%, which was markedly higher than in the imatinib group (11.071%) and liposome negative control group (2.759%) [tumor inhibition = (mean tumor weight of control group--mean tumor weight of treatment group)/(mean tumor weight of control group) × 100%]. Telomerase activity was significantly lower (P < 0.01) in the PS-ASODN group (0.689 ± 0.158) compared with the imatinib group (1.838 ± 0.241), liposome negative control group (2.013 ± 0.273), and saline group (2.004 ± 0.163). Flow cytometry revealed that the apoptosis rate of tumor cells treated with PS-ASODN was 20.751% ± 0.789%, which was higher (P < 0.01) than that of the imatinib group (1.163% ± 0.469%), liposome negative control group (1.212% ± 0.310%), and saline group (1.172% ± 0.403%). Expression of bcl-2 mRNA in the transplanted GISTs was markedly downregulated (P < 0.01) in the PS-ASODN group (7.245 ± 0.739) compared with the imatinib group (14.153 ± 1.618) and liposome negative control group (16.396 ± 1.351). PS-ASODN can repress GIST growth, mediated perhaps by inhibition of telomerase activity and downregulation of bcl-2 expression.

Luciferase bioluminescence imaging monitoring gene therapeutic effect of apoptosis-inducing ligand for lung cancer A549 cells nude mice transplantation tumor in vivo

Objective To detect the expression and effect of human tumor necrosis factor related apoptosis-inducing ligand (hTRAIL) in vivo,by using a novel double expressing adenoviral vector encoding hTRAIL and firefly luciferase ( luc ) gene ( ad -luc-hTRAIL),in which luc was used as reporter gene.Methods Lung cancer A549 cell xenografts in 16 nude mice models were established in subcutaneous inoculation way,the adenovirus vectors ( ad-luc-hTRAIL,ad-hTRAIL,ad-luc) and phosphate buffer saline (PBS) (n =4) as control were injected into tumor respectively.The size of the tumor was measured at different time points (4,7,10,14,21,28 d)after injection.The activity of luciferase in surface of the tumor was detected in vivo by using high-sensitivity cooled-charged coupled device(CCD) camera.The expression of hTRAIL was demonstrated by immunohistochemistry staining after sacrificing the animals at different time points,and immunohistochemical scores (IHS) were measured. The apoptosis rate of tumor cells was detected by using TUNEL and calculated. Analysis of variance,the paired t test and linear correlation analysis was used for the statistics. Results The growing speed of tumour xenografts was more slowly in ad-luc-hTRAIL and ad-hTRAIL groups than PBS group (t =2.71,2.72,P ＜ 0.05 ).The tumor volumes of ad-luc-hTRAIL,ad-hTRAIL,ad-luc and PBS groups 28 days after injection were (208.4 ± 42.3 ),( 181.5 ±23.9),( 403.1 ± 54.0 ) and ( 427.0 ± 59.3 ) mm3, respectively. There was no significant difference between ad-luc group and PBS group(t =2.07,P ＞ 0.05).The expression of luciferase in ad-luc-hTRAILgroup reached its peak at 7th day ( 1.37 ± 1.04),and then decreased quickly.The IHS and apoptosis rate in ad-luc-hTRAIL and ad-hTRAIL groups reached their peaks at 7th day,the peak values of IHS were 6.25 ±2.06 and 6.5 ± 2.89,the peak values of apoptosis rate were (60.75 ± 8.06 ) ％ and ( 61.50 ± 8.47 ) ％,respectively.The amount of luciferase expression ( absolute number of photons detected by CCD camera)was linear positive correlated with IHS and apoptosis rate ( rphotons/IHs =0.942,rph /rate =0.842,rIHs/rate =0.887,P ＜ 0.05 ).Conclusion The target gene hTRAIL can be transfected into lung cancer A549 cell xenografts nude mice models efficiently with a high level expression,and the therapeutic effect of hTRAIL can be monitored by detecting the expression of luc. Key words: Luminescent proteins; Tumor necrosis factors; Fluorescein; Genes

Therapeutic effect of ovarian intra-arterial infusion of GE7-delivery system-mediated HSVl-tk/ganciclovir gene therapy in a rat model of malignant ovarian tumor

To observe the gene expression of herpes simplex virus type 1 thymidine kinase (HSVl-tk) in rat malignant ovarian tumor tissues and the therapeutic effect of ganciclovior (GCV) after intra-arterial infusion of HSVl-tk gene therapy mediated by GE7-delivery system. A GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 4-element complex was constructed. Eighteen rats with DMBA-induced ovarian tumor were divided into 3 groups as Atk, ANS and Vtk groups. The 4-element complex GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 was injected via the ovarian artery into the rats of Atk group, saline buffer was injected in the ANS groups, and the 4-element complex was injected via the tail vein into the rats of Vtk group. All rats received intraperitoneal injection of GCV in a dose of 50 mg/kg daily for 10 days. The rats were sacrificed 3 days after the final dose of GCV, and the tumor weight was measured and tumor growth inhibition rate was calculated. Flow cytometry was used to assess the cell cycle and apoptosis. The tumor weight in the rats of Atk group was (4.77 ± 2.31) g, significantly lower than that of ANS group [(14.66 ± 6.26) g, P < 0.01] and Vtk group [(17.53 ± 7.19) g, P < 0.01]. The tumor growth inhibition rate of the Atk group was 67.5%, while that of Vtk group was -19.6%. The flow cytometry showed that S-phase tumor cells in the Atk group were (54.32 ± 9.65)%, significantly higher than that in the ANS (27.43 ± 9.22)% and (30.16 ± 11.57)% in the Vtk group (both P < 0.01). The tumor cell apoptosis rate in the Atk group was (39.15 ± 12.16)%, significantly higher than that in the ANS group [(11.86 ± 5.28)%, P < 0.01] and Vtk group [(14.32 ± 6.43)%, P < 0.01]. HSV1-tk/GCV gene therapy system mediated by GE7 non-viral delivery system via ovarian arterial infusion effectively causes cell cycle arrest at S phase and enhances cell apoptosis, therefore, exerts an inhibitory effect on tumor growth.

Effects of celecoxib combined with radiotherapy on apoptosis of CNE-2Z cell lines

To investigate the effects of celecoxib combined with radiotherapy on apoptosis of CNE-2Z cell lines and the potential mechanisms. Four groups were used, a control, celecoxib (25 micromol/L celecoxib), irradiation (8 Gy X ray) and celecoxib plus irradiation. The radiosensitising effect was detected by clone formation experiment. Flow cytometry was used to detect the apoptosis rate of cells. The expressions of Bcl-2 and Bax were assessed by immunocytochemistry. Western blot was used to examine the expression of Caspase-3. Celecoxib enhanced the radiosensitivity of CNE-2Z cells. In experimental group, the mean surviving fraction and the mean lethal dose of CNE-2Z cells were 0.50 and 2.36 respectively. Compared with the irradiated group, there was significant differences between the two groups (P < 0.01). Celecoxib combined with radiotherapy up-regulation the expression of Bax. The score of the expression of Bax in the control group and the experimental group were 1.221 +/- 0.116 and 2.758 +/- 0.256 respectively. Celecoxib combined with radiotherapy could inhibit the expression of the protein of Bcl-2. The score of the expression of Bcl-2 in the control group and the experimental group were 2.559 +/- 0.144 and 1.253 +/- 0.114 respectively, with significant differences (P < 0.01). Celecoxib combined with radiotherapy could increase the apoptosis rate of tumor cells with significant differences (F = 7.63, P < 0.01). Western blot showed that the expression of Caspase-3 was strengthened. Celecoxib combined with radiotherapy could induce apoptosis and enhance the radiosensitivity of human nasopharyngeal carcinoma CNE-2Z cell lines.

The inhibitory effects of rh-endostatin (YH-16) in combination with radiotherapy on lung adenocarcinoma A549 in mice and the underlying mechanisms

In order to investigate the inhibitory effects of Endostar (rh-endostatin, YH-16) in combination with radiotherapy on lung adenocarcinoma A549 in mice and the interaction mechanisms of combined therapy, the transplantation tumor models of A549 lung adenocarcinoma were established. When the largest diameter of tumor reached 1.0 cm, all nude mice were randomly divided into 4 groups: Endostar group, radiotherapy group, radiotherapy plus Endostar (combined treatment) group, and control group (n=6 in each group). The largest diameter and the vertical diameter of tumor were measured at different time points. At the 16th day, mice were executed, and the tumors were applied to analysis of rate of tumor cell apoptosis, and the expression levels of basic fibroblast growth factor (bFGF) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR) and those of vascular endothelial growth factor (VEGF) by immunohistochemistry. The results demonstrated that the rate of tumor inhibition in combined treatment group was higher than that in other groups. And the rate of tumor cell apoptosis in combined treatment group was also higher than that in other groups. Meanwhile, the levels of bFGF mRNA and VEGF expression in combined treatment group were lower than those in other groups. It was concluded that Endostar obviously enhanced the curative effectiveness of radiotherapy on lung adenocarcinoma A549 in mice. The underlying mechanisms may involve the down-regulation of bFGF mRNA and VEGF expression to inhibit angiogenesis by Endostar and the cooperative effect of Endostar and radiotherapy to synergistically promote tumor cell apoptosis. And Endostar inhibits angiogenesis by down-regulating the expression of bFGF mRNA and VEGF.

A retrovirus‐based system to stably silence GDF‐8 expression and enhance myogenic differentiation in human rhabdomyosarcoma cells

Myostatin, also called GDF-8, a secreted growth and differentiating factor that belongs to the transforming growth factor-beta superfamily, is a known negative regulator of myogenesis in vivo. Overexpression of GDF-8 contributes to the lack of differentiation in human rhabdomyosarcoma (RMS) cells. We investigated whether a retrovirus-based RNA interference (RNAi) system against GDF-8 expression in human RMS cells would enhance myogenic differentiation. A retrovirus-based RNAi system was developed that utilized the U6-RNA polymerase III promoter to drive efficient expression and deliver the GDF8-specific short hairpin RNAs (shRNAs) in human RMS cell A204. In this system, the retrovirus vector was integrated into the host cell genome and allowed stable expression of shRNAs. GDF-8 expression was determined by real-time polymerase chain reaction and western blotting analysis. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the cell proliferation. Myogenic differentiation markers were monitored by western blotting analysis. Cell cycle and apoptosis was determined by propidium iodide staining and analysed in a flow cytometer. In the siGDF8 A204 cell pools, the levels of both GDF-8 mRNA and protein were dramatically reduced by this RNAi system. In differentiation conditions, inhibition of myostatin synthesis led to enhanced cell cycle withdrawal, consequently stimulated myogenic differentiation and increased the rate of tumor cell apoptosis. The results demonstrate that deactivation of myostatin by using retrovirus-based RNAi thus may be useful for therapy in rhabdomyosarcomas.