Low-dose paclitaxel improves the therapeutic efficacy of recombinant adenovirus encoding CCL21 chemokine against murine cancer.

Secondary lymphoid tissue chemokine (SLC/CCL21), one of the CC chemokines, exerts potent antitumor immunity by co-localizing T cells and dendritic cells at the tumor site and is currently tested against human solid tumors. Here, we investigated whether the combination of recombinant adenovirus encoding murine CCL21 (Ad-mCCL21) with low-dose paclitaxel would improve therapeutic efficacy against murine cancer. Immunocompetent mice bearing B16-F10 melanoma or 4T1 breast carcinoma were treated with either Ad-mCCL21, paclitaxel, or both agents together. Our results showed that Ad-mCCL21 + low-dose paclitaxel more effectively reduced the growth of tumors as compared with either treatment alone and significantly prolonged survival time of the tumor-bearing animals. These antitumor effects of the combined therapy were linked to altered cytokine network at the tumor site, enhanced apoptosis of tumor cells, and decreased formation of new vessels in tumors. Importantly, the combined therapy elicited a strong therapeutic antitumor immunity, which could be partly abrogated by the depletion of CD4(+) or CD8(+) T lymphocytes. Collectively, these preclinical evaluations may provide a combined strategy for antitumor immunity and should be considered for testing in clinical trials.

[Lumen morphogenesis and molecular mechanisms in tubular organs during zebrafish embryonic development].

A network tubular system is an important structure in the body and organ of metazoa. The lumen of tube is fundamental units in the structure, which serve to transport material, divide the organ into different functional compartments and separate the organ from the environment. The defects of lumen formation will lead to abnormalities of the organ morphogenesis and disorder of the function. Zebrafish (Danio rerio)is an important model for development research. Meanwhile easy observation of tubular organ, the relevant mutants, and transgene linages make zebrafish to become an excellent model to study the formation of lumen in the tubular organs, including the blood vessels, neural tube, gut, exocrine pancreas, and pronephric duct, which undergo the typical morphogenesis of lumen that is involved in the organs' development. The process of lumen formation is mainly consisted of induction of extracellular signals, polarization of epithelial cell, directional transportation in the polar cells, the aggregation and transportation of fluid in the lumen, and the reconstruction of cytoskeleton in polar cells and controlled by the precise and complicated molecular networks during embryonic development. This review will summarize our current knowledge on lumen morphogenesis in four kinds of typical tubular organs during zebrafish embryonic development and the related molecular mechanisms as well as to supply helpful reference to the future studies.

Suppression of human MDA-MB-435S tumor by U6 promoter-driven short hairpin RNAs targeting focal adhesion kinase.

PURPOSE: Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase implicated in cancer cell survival, proliferation, and in various steps in the metastatic cascade. In the present study, we took advantage of a cationic liposome as gene carrier and targeted FAK function through both in vitro and in vivo approaches. METHODS: We utilized a plasmid-encoded hairpin RNA targeting the human FAK mRNA (pGensil2-shRNA/FAK), as a means to inhibit FAK expression for evaluating its anti-tumor effect in vitro and in vivo. Human MDA-MB-435S breast cancer cells were transfected with pGensil2-shRNA/FAK and examined for apoptosis by propidium iodide staining, DNA ladder, and flow cytometric analysis. For in vivo study, subcutaneous breast carcinomatosis models in nude mice were established to evaluate the therapeutic potential of pGensil2-shRNA/FAK. Assessments of proliferation (Ki-67), apoptosis (TUNEL) and angiogenesis (CD31) were done using immunohistochemical analysis. RESULTS: Transcripts expressed from plasmid both in vitro and in vivo were identified by northern blot analysis. pGensil2-shRNA/FAK effectively down-regulated the expression of FAK as demonstrated in vitro by real time RT-PCR and western blot analysis, whereas by real time RT-PCR and IHC staining of MDA-MB-435S tumors growing subcutaneously. Breast cancer cells lacking FAK expression undergo apoptosis in vitro. Systemic delivery of cationic liposome-complexed plasmids targeting FAK, resulted in the diminishment of subcutaneous tumor growth beyond the effects observed with liposomes carrying a non-specific shRNA. This diminishment in growth was associated with elevated levels of apoptosis (TUNEL staining), decreased cell proliferation (Ki-67 staining) and diminished endothelial cell density (CD31 staining). CONCLUSION: These results indicate that the systemic delivery of plasmid DNA targeting FAK function using cationic liposome as a gene carrier, represents a promising avenue for breast cancer therapy.

[Construction of recombinant adenovirus containing full-length sense and antisense cyclin B1 cDNA and their impact on proliferation and apoptosis of HeLa cells].

OBJECTIVE: To construct a recombinant adenovirus vector carrying the full-length sense and antisense cyclin B1 cDNA of human, and to investigate the influence of the recombinant adenoviruses on the cultured human cervical carcinoma cell line HeLa. METHODS: The recombinant adenovirus vectors were constructed by homologous recombination technique. The recombinant adenoviruses were packaged and amplified in 293A cells. Then the recombinant adenoviruses were used to infect HeLa cells. The influence was determined by cell counting and flow cytometry. RESULTS: The HeLa cells infected with antisense recombinant adenovirus exhibited significant cell apoptosis and growth inhibition compared to the normal HeLa cells and the cells infected with sense recombinant adenovirus. CONCLUSION: Recombinant adenoviruses vectors containing the full-length sense and antisense cDNA of human cyclin B1 is successfully constructed, which has laid a foundation for further studies on its anticancer functions.

Acute toxicity evaluation of biodegradable in situ gel-forming controlled drug delivery system based on thermosensitive PEG-PCL-PEG hydrogel.

In this work, a biodegradable poly(ethylene glycol)-poly(epsilon-caprolactone)-poly (ethylene glycol) (PEG-PCL-PEG, PECE) triblock copolymer was successfully synthesized. The aqueous solution of such PECE copolymer displayed special sol-gel-sol transition as temperature increase, which is a flowing sol at low-temperature and turns into a nonflowing gel at body temperature. The cytotoxicity of PECE copolymer was evaluated by cell viability assay using HEK 293 cells. In vivo gel formation and degradation test based on intraperitoneal and subcutaneous administration was conducted, respectively. The acute toxicity test and histopathological study were performed in BALB/c mice by intrapleural, intraperitoneal, or subcutaneous administration of PECE hydrogel (30 Wt %), respectively. The dose of intrapleural, intraperitoneal, or subcutaneous administration was up to 10 g/kg body weight (b.w.), 25 g/kg b.w., and 25 g/kg b.w., respectively, and the mice were observed continuously for 14 days. For histopathologic study, samples including heart, liver, lung, kidneys, spleen, stomach, intestine, and tissue of injection site were prepared for histochemical analysis and were stained with hematoxylin-eosin. No mortality or significant signs of acute toxicity was observed during the whole observation period and there is no significant lesion to be shown in histopathologic study of major organs. Therefore, the maximum tolerance dose of PECE hydrogel by intrapleural, intraperitoneal, or subcutaneous administration was calculated to be higher than 10 g/kg b.w., 25 g/kg b.w., and 25 g/kg b.w., respectively. The results indicated that the prepared PECE hydrogel was nontoxic after intrapleural, intraperitoneal, or subcutaneous administration, and it could be a safe candidate for in situ gel-forming controlled drug delivery system.

CXC-chemokine-ligand-10 gene therapy efficiently inhibits the growth of cervical carcinoma on the basis of its anti-angiogenic and antiviral activity.

Epidemiological studies have demonstrated that high-risk human papillomavirus (HPV) is involved in causing cervical carcinoma. The HPV oncoproteins E6 and E7 immortalize human keratinocytes is mostly resulted from inactivation of tumor suppressor proteins p53 and pRB, which also play an important role in regulating the expression of pro- and antiangiogenic factors. The present study was conducted to determine whether IFN--inducible protein 10 (IP-10)/CXC chemokine ligand 10(CXCL10), one of the potent antiangiogenic chemokines, can inhibit the growth of cervical cancer. Plasmid DNA encoding CXCL10 was encapsulated with cationic liposomes, mice were treated with DNA-liposome mixture 6 times with the 5-day interval. Our results demonstrated that CXCL10 could reduce the level of HPV oncoproteins E6 and E7 in cervical cancer cells. In vivo study showed that CXCL10 could inhibit the growth of tumor in the immunodeficiency mice. Immunohistology analysis revealed that CXCL10 downregulated the microvessel density and the expression of PCNA in tumor tissues. TUNEL staining demonstrated CXCL10 significantly increase the apoptotic rate. Our data suggest that CXCL10 can inhibit the growth of cervical carcinoma through modulating the formation of microvessel and the expression of HPV oncoproteins E6 and E7. The present findings also provide further evidence of the anti-tumor effects of CXCL10, and may be of importance for the further exploration of the potential application of this molecule in the treatment of cervical carcinoma.

Synergistic antitumor effect of CXCL10 with hyperthermia.

PURPOSE: IFN-inducible protein 10 (IP-10)/CXCL10 (CXC chemokine ligand 10) has been described as an antiangiogenic chemokine and displays a potent antitumor activity in vivo. In the present study, we try to investigate whether the combination therapy of hyperthermia, a physical antiangiogenic modality, with CXCL10 would completely eradicate the established solid tumors. METHODS: Immunocompetent BALB/c mice bearing Meth A fibrosarcoma were established. Mice were treated with either CXCL10 at 25 microg/kg once a day for 20 days, hyperthermia was given twice (at 42 degrees C for 1 h, on day 6 and 12 after the initiation of CXCL10), or together. Tumor volume and survival time were observed. The microvessel density was determined by CD31 immunofluorescence. Histologic analysis and assessment of apoptotic cells were also conducted in tumor tissues. RESULTS: The results showed that CXCL10 and hyperthermia inhibited the growth of Meth A fibrosarcoma and interestingly, the combination therapy enhanced the antiangiogenic effects and completely eradicated the established solid tumors. Histological examination revealed that CXCL10 + hyperthermia led to increased induction of apoptosis, tumor necrosis, and elevated lymphocyte infiltration compared with the controls. Moreover, the tumor eradicated animals developed a protective T-cell-dependent antitumor memory response against Meth A tumor cells rechallenge. CONCLUSIONS: Our finding is that the combination therapy can achieve a synergistic antitumor efficacy, supporting the idea that the combination of two antiangiogenic agents may lead to improved clinical outcome. These findings could open new perspectives in clinical antitumor therapy.

[Construction of recombinant adenovirus vector carrying secondary lymphoid organ chemokine].

OBJECTIVE: To construct a recombinant adenovirus vector carring the SLC gene for further studies on gene therapy for carcinoma. METHODS: The SLC gene was amplified from the pORF5 vector with polymerase chain reaction (PCR) technique. The amplified gene was cloned into the pENTR11 vector. With the pENTR11-SLC plasmid and the backbone plasmid pAd/CMV/V5-DEST, the homologous recombination reaction took place in vitro. The reaction mixture was transferred into TOP10 E. coli strains (without F' episome). The recombination adenovirus plasmid was then generated. The recombinant adenoviruses were packaged and amplified in 293A cells. RESULTS: The SLC gene was successfully cloned into the pAd/CMV/V5-DEST plasmid and the recombinant adenoviruses carrying SLC gene were detected by PCR, with a viral titer of 2. 6 X 10(8) pfu/mL. CONCLUSION: The constructed recombinant adenovirus can introduce SLC gene into tumor tissues, which provides a foundation for the study of antitumor efficacy of SLC.

[Cloning, expression, distribution and subcellular localization of AK078438 gene].

OBJECTIVE: To investigate the molecular cloning, tissue distribution, tumor distribution and the subcellular localization of AK078438 gene. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) technique was applied to amplify the open reading frame of AK078438 gene. Prokaryotic expressing vector and affinity purification were used to get the fusion protein. At mRNA expressing levels semi-quantitative RT-PCR was employed for the investigation of its distribution in normal rat tissues, tumor cell and mesenchymal stem cell (MSC) lines. The eukaryotic expression green fluorescence protein (GFP) fusion protein vector was transfected into cells and identified subcellular localization. RESULTS: The full open reading frame of AK078438 gene with 1065 bp, 355aa were identified. mRNA of the gene distributed in brain, uterus, colon, bone marrow, testis and kidney, but not in stomach, liver, the lung, spleen and heart. Murine colon adenocarcinoma C26, melanoma B16, Lewis lung carcinoma LL/2 and MSC had the gene and mouse myeloma cell line NS-1 and Hepa mouse hepatoma cell line had no the gene. The protein was localized in cytoplasm. CONCLUSION: Protein expression, expressing profile and subcellular localization of AK078438 gene set the basis for further exploration of its functions.

[Isolation and purification of BMScs of GFP transgenic mouse using the method of adhering to cuture plastic in different time].

OBJECTIVE: To adopt the method of adhering to culture plastic in different time for cultivating and purifying BMSCs of green fluorescent protein (GFP) transgenic mice. METHODS: Bone marrow cells isolated from GFP transgenic mice are directly planted in culture flask and an exchange of the total volume medium is made at different time. Then the cells adhering to culture plastic are differently counted according to the cell types and are examined by immunohistochemistry using the antibodies of CD44, CD45 and CD54 in three days. Moreover, the cells after the exchange of the total volume medium in 4 hours, 8 hours and 24 hours are selected and successively subcultured down to the fifth passage. Then the result of amplification is calculated and the cells are examined by immunohistochemistry using the antibodies of CD44, CD45 and CD54. RESULTS: With the extending of the time for the first exchange of medium, the density of cells adhering to culture plastic increased accordingly, but the BMSCs proportion decreased. The cells after first exchange of medium in 4 hours had high BMSCs proportion but low BMSCs density, and the cells in 24 hours had high BMSCs density and low BMSCs proportion. However, the cells in 8-10 hours had high BMSCs density and also high BMSCs proportion. The subcultured BMSCs could stably express GFP. CONCLUSION: The method of adhering to culture plastic in different time for cultivating and purifying BMSCs of GFP transgenic mice is effective. It is suitable to make the first exchange of total volume medium in 8-10 hours. The subcultured cell has the capacity for amplification and will probably be a seed cell for the research of tissue engineering and gene therapy.