Low intensity ultrasound-induced apoptosis in human gastric carcinoma cells.

AIM: To investigate the low intensity ultrasound (US)-induced apoptosis in human gastric carcinoma cells and its potential mechanism and to suggest a new therapeutic approach to gastric carcinoma. METHODS: Human SGC-7901 gastric carcinoma cells were cultured in vitro and irradiated by low intensity US for 10 min at different intensities with different incubation times after irradiation. Morphologic changes were examined under microscope with trypan blue staining and then the percentage of early apoptotic cells was detected by flow cytometry (FCM) with double staining of fluorescein isothiocyanate (FITC)-Annexin V/propidium iodide (PI). Two-dimensional electrophoresis (2DE) was used to get the protein profile and some proteins differently expressed after US irradiation were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Functional analysis was performed to investigate the mechanism of US-induced cell apoptosis. RESULTS: The percentage of apoptotic cells increased about 10% after US irradiation (12.0 W/cm(2), 12 h culture). The percentage of early apoptosis and secondary necrosis in the US-irradiated cells increased with the increased US intensity. Moreover, apoptotic cells increased with the increased culture time after US irradiation and reached its maximum at about 12 h. Several new proteins appeared after US irradiation and were up or down regulated more than 2 times. Some heat shock proteins (HSPs) were found to be associated with the signal process simulating the apoptosis of cells. CONCLUSION: Low intensity US could induce apoptosis in human gastric carcinoma cells. US-induced apoptosis is related to US intensity/culture time. US-induced apoptosis may be caspases-dependent and endoplasmic reticulum (ER) stress-triggered apoptosis may also contribute to it. Proteomic experimental system is useful in finding the protein alteration in carcinoma cells after US irradiation, helping to develop a new cancer therapy.

Protein profile of human hepatocarcinoma cell line SMMC-7721: identification and functional analysis.

AIM: To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy. METHODS: Total proteins from human hepatocarcinoma cell line SMMC-7721 were separated by two-dimensional electrophoresis (2DE). The silver-stained gel was analyzed by 2DE software Image Master 2D Elite. Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. RESULTS: We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin, endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein I, peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed. CONCLUSION: Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

Monitoring imaging of lesions induced by high intensity focused ultrasound based on differential ultrasonic attenuation and integrated backscatter estimation.

We investigated the feasibility of two monitoring imaging methods to visualize and evaluate the high intensity focused ultrasound (HIFU) induced lesions in vitro during and after their formation, which were based on differential ultrasonic parameter estimation. Firstly, ultrasonic attenuation slope of tissue sample was estimated based on the spectral analysis of ultrasound RF backscattered signals. The differential attenuation slope maps were acquired, which were interpreted as the differences between the pretreatment image and those obtained in different stages during HIFU therapy. Secondly, ultrasonic integrated backscatter (IBS), defined as the frequency average of the backscatter transfer function over the useful bandwidth, was proposed quantitatively to evaluate the extent of lesions with the same RF signals as the first method. Differential IBS maps were also acquired to visualize temporal evolution of lesion formation. It was found in pig liver in vitro that more precise definition of the treated area was obtained from the differential IBS images than from differential attenuation slope images. Dramatic increase in both attenuation and IBS value was observed during the therapy, which may be related to dramatic enhancement of cavitation due to boiling and accompanying tissue damage. Two methods to obtain one differential image were compared and the cumulative differential image was found to be able to eliminate noises and artifacts to some extent, which was the cumulation of a series of differential images acquired from the differences between the temporally adjacent RF data frames. Moreover, we presented a bidirectional color code for identification of the artifacts due to tissue movements caused by HIFU radiation force. We conclude that cumulative differential IBS images have the potential to monitor the formation of HIFU-induced lesions.

[Preparation of gelatin nanogel modified with fluoride anion and its ultrasonic triggered release characteristics].

AIM: To investigate the preparation, shape and ultrasound triggered release characteristics of gelatin nanogel modified with fluoride anion. METHODS: Adriamycin gelatin nanogel modified with fluoride anion (ADM-FMNG) was prepared by co-precipitation with fluoride anion. The content and encapsulation rate of adriamycin were measured by HPLC method. The size and shape of ADM-FMNG were determined by electron microscope. The size and distribution of ADM-FDNG before and after sonication were measured by laser size analysis device. RESULTS: The average diameter of ADM-FMNG was (46 +/- 12) nm. Adriamycin encapsulated rate and loading were 87.2% and 0.091 g x L(-1), respectively. 48.5% of adriamycin was released within 50 h while in vitro at 37 degrees C. Under the action of ultrasound that has the frequency of 20 kHz, 0.4 W x cm(-2) of power density and 7-8 min duration, 51. 5% of adriamycin in ADM-FMNG was released that was significantly higher than control group, the size of ADM-FMNG was changed from (46 +/- 12) nm to (1,212 +/- 35) nm and restored after ultrasound stopped for 3-4 min. CONCLUSION: ADM-FDNG system has the sensitive ultrasound triggered release characteristics.

Cytotoxicity of HSVtk and hrTNF-alpha fusion genes with IRES in treatment of gastric cancer.

The efficacy of the suicide gene therapy by using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system for the treatment of cancer is limited because of the insufficient gene transfer and the low killing activity. To enhance the anti-tumor activity, we probed into whether recombinant retroviral expression vector PLXSN expressing both HSVtk and TNF-alpha genes could potentiate the destruction of SGC7901. The pL(tk-TNF-alpha)SN harboring HSVtk and TNF-alpha genes in sequence was constructed with a bicistronic unit including the internal ribosomal entry site, the recombinant retroviruses were transferred into SGC7901 cells by lipofectamine, and pEGFP and Western blot analysis were used to detect the expression of fusion genes in transfected SGC7901 cells, and then apoptosis of the transfected cells were detected by using the TdT-mediated dUTP nick end labeling, flow cytometric analysis and transmission electron microscopy. In vitro study, the transfected gastric cancer cells were maintained in the GCV-contained medium, to assay the cell killing effect and bystander effect. In vivo experiments, retroviral serum plasmids were transfected into tumor-bearing nude mice, to observe the changes of tumor volumes and survival of the mice. In vitro there was no significant difference of cell survival rate between the three groups. However, in vivo results showed that tk/GCV, tk-TNF-alpha/GCV and TNF-alpha could inhibit the tumor growth, and the obvious anti-tumor effect was shown in tk-TNF-alpha/GCV group, and TNF-alpha obviously enhanced the anti-tumor effect in vivo. The pathologic examination showed necrosis of the cancer in the treated groups.

The alteration of protein profile of Walker 256 carinosarcoma cells during the apoptotic process induced by ultrasound.

The objective of this study was to investigate the alteration of the protein profile in cells after sonication and to identify the key proteins involved in the process of cell apoptosis. Walker 256 carinosarcoma cells were exposed to focused ultrasound (US) at the intensity of 2.0, 7.0, 10.2, 14.2 and 17.0 W/cm2 (I(spta)) for 10 min in vitro and the morphologic and functional changes of the cells were detected by hematoxylin & eosin staining and flow cytometry, with double staining of fluorescein isothiocyanate (FITC)-labeled Annexin V/propidium iodide (PI). The protein compositions in the cells after sonication were detected by 2-D SDS polyacrylamide gel electrophoresis. Our results showed that apoptosis of Walker 256 carinosarcoma cells could be induced by US. The percentage of early apoptosis and secondary necrosis increased with increasing intensity of US irradiation. Comparing with the protein patterns of cells before sonication, it was found that around 420 new protein spots were present in the gel after sonication. Among them, Hsp60 and Bcl-2 like protein 13 were found to be involved in the process of cell apoptosis and US-induced apoptosis of the cells was probably performed through the pathway of promoting the activation of caspase-3.

Pulmonary functional MRI: an animal model study of oxygen-enhanced ventilation combined with Gd-DTPA-enhanced perfusion.

BACKGROUND: The assessment of regional pulmonary ventilation and perfusion is essential for the evaluation of a variety of lung disorders. Pulmonary ventilation MRI using inhaled oxygen as a contrast medium can be obtained with a clinical MR scanner, without additional equipment, and has been demonstrated to be a feasible means of assessing ventilation in animal models and some clinical patients. However, few studies have reported on MR ventilation-perfusion imaging. In this study, we evaluated the usefulness of oxygen-enhanced ventilation in combination with first-pass Gd-DTPA-enhanced perfusion MRI in a canine model of pulmonary embolism and airway obstruction. METHODS: Peripheral pulmonary embolisms were produced in eight dogs by intravenous injection of gelfoam strips at the pulmonary segmental arterial level, and airway obstructions were created in five of the dogs by inserting a self-designed balloon catheter into a secondary bronchus. Oxygen-enhanced MR ventilation images were produced by subtracting images from before and after inhalation of pure oxygen. Pulmonary perfusion MR images were acquired with a dynamic three-dimensional fast gradient-echo sequence. MR ventilation and perfusion images were read and contrasted with results from general examinations of pathological anatomy, ventilation-perfusion scintigraphy, and pulmonary angiography. RESULTS: Regions identified as having airway obstructions matched using both MR ventilation and perfusion imaging, but regions of pulmonary embolisms were mismatched. The area of airway obstruction defects was smaller using MR ventilation imagery than that using ventilation scintigraphy. Abnormal perfusion regions due to pulmonary embolisms were divided into defective regions and reduced regions based on the time course of signal intensity changes. In the diagnosis of pulmonary embolisms with the technique of ventilation and perfusion MRI, sensitivity and specificity were 75.0% and 98.1%, respectively, and the diagnostic results of this MRI technique were in agreement with the results of ventilation-perfusion scintigraphy and pulmonary angiography (K: 0.899, 0.743). CONCLUSIONS: Oxygen-enhanced ventilation in combination with pulmonary perfusion MRI can be used to diagnose abnormalities of airways and blood vessels in the lungs, and can provide regional functional information with high spatial and temporal resolution. This method possesses great potential value for clinical applications.

Cytotoxicity of HSVtk and hrTNF-alpha fusion genes with IRES in treatment of gastric cancer.

The efficacy of the suicide gene therapy by using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system for the treatment of cancer is limited because of the insufficient gene transfer and the low killing activity. To enhance the antitumor activity, we probed into whether recombinant ritroviral expression vector PLXSN expressing both HSVtk and TNF-alpha genes could potentiate the destruction of SGC7901. The PL(tk-TNF-alpha)SN harboring HSVtk and TNF-alpha genes in sequence was constructed with a bicistronic unit including the internal ribosomal entry site, the recombinant retroviruses were transferred into SGC7901 cells by lipofectamine, and pEGFP and Western blot analysis were used to detect the expression of fusion genes in transfected SGC7901 cells, and then apoptosis of the transfected cells were detected by using the TdT-mediated dUTP nick end labeling, flow cytometric analysis and transmission electron microscopy. In vitro study, the transfected gastric cancer cells were maintained in the GCV-contained medium, to assay the cell killing effect and bystander effect. In vivo experiments, retroviral serum plasmids were transfected into tumor-bearing nude mice, to observe the changes of tumor volumes and survival of the mice. In vitro there was no significant difference of cell survival rate between the three groups. However, in vivo results showed that tk/GCV, tk-TNF-alpha/GCV and TNF-alpha could inhibit the tumor growth, and the obvious anti-tumor effect was shown in tk-TNF-alpha/GCV group, and TNF-alpha obviously enhanced the anti-tumor effect in vivo. The pathologic examination showed necrosis of the cancer in the treated groups.

Effects of ultrasound and additives on the function and structure of trypsin.

Encapsulating proteins in polymeric microspheres is a useful mode of drug delivery, but the proteins are subjected to damage in the process of ultrasound emulsion microencapsulation. The objective of this study was to investigate the effects of ultrasound power and duration on the function and structure of trypsin, and the reason of protein denaturation when it was irradiated by 20 kHz ultrasound. The relatively stable enzyme, trypsin, was dissolved in aqueous solution in the presence and absence of additives to study the stability of trypsin during the ultrasound irradiation. The damage of the molecular structure of trypsin was detected via combined high performance liquid chromatogram and electrospray ionization mass spectrometry (HPLC-ESI-MS). The results showed that the activity of trypsin decreased with increasing ultrasound power from 100 to 500 W or extending the irradiation time from 1 to 20 min. This effect could be enhanced via aerating the solution for a duration 10 min at 300 W. Fragments of trypsin were detected in the treatment (300 W, 10 min) by HPLC-ESI-MS. The additives, Tween 80 and mannitol, could protect trypsin against the inactivation caused by ultrasound. The reason of inactivation was partly from the alteration of the molecular conformation and partly from the modification or damage of trypsin's molecular structure.

Construction and identification of recombinant vectors carrying herpes simplex virus thymidine kinase and cytokine genes expressed in gastric carcinoma cell line SGC7901.

AIM: To construct and identify the recombinant vectors carrying herpes simplex virus thymidine kinase (HSV-TK) and tumor necrosis factor alpha (TNF-alpha) or interleukin-2 (IL-2) genes expressed in gastric carcinoma cell line SGC7901. METHODS: The fragments of HSV-TK, internal ribosome entry sites (IRES) and TNF-alpha or IL-2 genes were inserted in a TK-IRES-TNF-alpha or TK-IRES-IL-2 order into pEGFP-N(3) and pLXSN to generate the therapeutic vectors pEGFP-TT, pEGFP-TI, pL(TT)SN and pL(TI)SN respectively, which were structurally confirmed by the digestion analysis of restriction endonuclease. The former two plasmids were used for the transient expression of recombinant proteins in the target cells while pL(TT)SN and pL(TI)SN were transfected into SGC7901 cells by lipofectamine for the stable expression of objective genes through G418 selection. The protein products expressed transiently and stably in SGC7901 cells by the constructed vectors were confirmed by fluorescent microscopy and Western blot respectively. RESULTS: The inserted fragments in all constructed plasmids were structurally confirmed to be consistent with that of the published data. In the transient expression, both pEGFP-TT and pEGFP-TI were shown expressed in nearly 50% of the transfected SGC7901 cells. Similarly, the G418 selected vectors PL(TT)SN and PL(TI)SN were confirmed to be successful in the stable expression of the objective proteins in the target cells. CONCLUSION: The constructed recombinant vectors in the present study that can express the suicide gene TK in combination with cytokines genes may serve as the potential tools to perform more effective investigations in future for the gene therapy of gastric carcinoma.

Do there exist synergistic antitumor effects by coexpression of herpes simplex virus thymidine kinase with cytokine genes on human gastric cancer cell line SGC7901?

AIM: To evaluate the synergistic antitumor effects of herpes simplex virus thymidine kinase (HSV-TK) together with tumor necrosis factor alpha (TNF-alpha) or interleukin-2 (IL-2) gene expression on gastric cancer cell line SGC7901. METHODS: Recombinant vectors pL(TT)SN and pL(TI)SN, which express TK-IRES-TNF-alpha and TK-IRES-IL-2 genes separately, as well as the control plasmids pL(TK)SN and pLXSN were employed to transfect PA317 cells respectively to generate the viruses that can stably express the objective genes through G418 selection. The gastric cancer cells were then transfected by the retroviral serum from the package cells and maintained in culture to determine the cell growth and apoptosis. The cytotoxic effects of HSV-TK together with TNF-alpha or IL-2 gene expression on the transfected cancer cells were evaluated by the cell viability and bystander effects in the presence of GCV supplemented in the cultural medium. RESULTS: Expression of recombinant proteins including TNF-alpha and IL-2 by stable transfectants was confirmed by Western blotting. The percentage of cell apoptosis in the SGC/0, SGC/TK-TNF-alpha, SGC/TK-IL-2 and SGC/TK clone was 2.3%, 12.3%, 11.1% and 10.9% respectively at 24 h post-transfection. Cell growth status among all the experimental groups as judged by cell absorbance (A) at 570nm did not exhibit any significant difference (P>0.05); although it was noted to be slightly lower in the SGC/TT group. Cell survival rate in SGC/TI, SGC/TT and SGC/TK group was significantly decreased in a dose-dependent manner of GCV compared with that of the SGC/0 group (P<0.05-0.01). Among all studied cells, the SGC/TT was shown most sensitive to GCV with a half lethal dose of 0.5 mg/L(-1). In contrast, the survival rate of SGC/0 cells was not affected by the presence of GCV with the doses less than 10 mg/L(-1). The half lethal dose of GCV for SGC/0 cells was more than 100 mg/L(-1). Marked bystander effect induced by SGC/TI, SGC/TT and SGC/TK cells was confirmed by the fact that 20% of these stable transfectants could kill 50% of the co-cultured cells, in which the most prominent bystander effect was found in the circumstance of SGC/TT presence. However, no significant difference of these variables was found among SGC/TI, SGC/TT and SGC/TK cells (P>0.05). CONCLUSION: The synergistic antitumor effects produced by the co-expression of HSV-TK with TNF-alpha or IL-2 genes were not present in the transfected SGC7901 cells. The mechanism underlying these phenomena was not known.

Effects of ultrasound on the structure and function of tumor necrosis factor-alpha.

The objective of this study was to investigate the effects of ultrasound on the structure and function of human tumor necrosis factor-alpha (TNF-alpha) and to study whether TNF-alpha underwent a denaturation process and the molecular structure was damaged when it was irradiated by ultrasound. The samples of TNF-alpha were dissolved in aqueous solution and filled into polystyrene tubes. High intensity ultrasound processor (20 kHz frequency, burst mode, 0.5 duty factor, 100-500 W total electrical power, 0-20 min total treatment time) was used during the treatment. The biologic activity of TNF-alpha was determined by its toxic activity towards TNF-alpha sensitive cell line L929 in the presence of actinomycin D. The methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) were used to detect the integrity of TNF-alpha molecule after it was irradiated by ultrasound. The results showed TNF-alpha could keep its biological activity, instead of undergoing a denaturation process, when it is irradiated by ultrasound in the aqueous solution; at the same time, the aggregates of TNF-alpha formed by the recombinant DNA E. coli could be dissociated through the molecular vibration induced by ultrasound energy. The biologic activity of TNF-alpha was not reduced, but small quantities of TNF-alpha molecular structure were damaged during the process of sonication. These features of TNF-alpha molecule irradiated by ultrasound probably gave TNF-alpha the advantage in being used in the drug microencapsulation and provided a new drugs formulation for tumor therapy.