Effective adoptive transfer of haploidentical tumor-specific T cells in B16-melanoma bearing mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Adoptive transfer of allogeneic tumor-specific T cells often results in severe graft-versus-host disease (GVHD). Here, we sought to maximize graft-versus-tumor and minimize GVHD by using haploidentical T cells in pre-irradiated B16-melanoma bearing mice.</p><p><b>METHODS</b>C57BL/6 mice bearing B16-melanoma tumors were irradiated with 0, 5, or 7 Gy total body irradiation (TBI), or 7 Gy TBI plus bone marrow transplantation. Tumor areas were measured every 3 days to assess the influence of irradiation treatment on tumor regression. B16-melanoma bearing mice were irradiated with 7 Gy TBI; sera and spleens were harvested at days 1, 3, 5, 7, 9, 11, and 13 after irradiation. White blood cell levels were measured and transforming growth factor β1 (TGF-b1) and interleukin 10 (IL-10) levels in serum were detected using enzyme-linked immunosorbent assay (ELISA) kits. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry were performed to test TGF-b1, IL-10 and Foxp3 mRNA levels and the proportion of CD4+CD25+Foxp3+ T-regulatory cells (Tregs) in spleens. B16-melanoma bearing C57BL/6 mice were irradiated with 7 Gy TBI followed by syngeneic (Syn1/Syn2) or haploidentical (Hap1/Hap2), dendritic cell-induced cytotoxic T lymphocytes (DC-CTLs) treatment, tumor areas and system GVHD were observed every 3 days. Mice were killed 21 days after the DC-CTLs adoptive transfer; histologic analyses of eyes, skin, liver, lungs, and intestine were then performed.</p><p><b>RESULTS</b>Irradiation with 7 Gy TBI on the B16-melanoma-bearing mice did not influence tumor regression compared to the control group; however, it down-regulated the proportion of Tregs in spleens and the TGF-b1 and IL-10 levels in sera and spleens, suggesting inhibition of autoimmunity and intervention of tumor microenvironment. Adoptive transfer of haploidentical DC-CTLs significantly inhibited B16-melanoma growth. GVHD assessment and histology analysis showed no significant difference among the groups.</p><p><b>CONCLUSION</b>Adoptive transfer of haploidentical tumor-specific T cells in irradiation-pretreated B16-melanoma bearing mice preserved antitumor capacity without causing a GVHD response.</p>

Therapeutic effects of signal transducer and activator of transcription 3 siRNA on human breast cancer in xenograft mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Signal transducer and activator of transcription 3 (STAT3) is usually constitutively activated in a variety of malignancies. It directly contributes to tumorigenesis, invasion, and metastasis. The surgical treatment of breast cancer has made no breakthroughs in terms of treatment effect, in spite of its long history. Current biotherapies bring a note of optimism to breast cancer treatment. To explore the possibility of a siRNA targeted STAT3 blocking treatment for over-activated tumor cells, we evaluated the efficacy of a STAT3 siRNA on human breast cancer cells in vitro and in vivo.</p><p><b>METHODS</b>Three MCF-7 human breast cancer cell lines were tested: control MCF-7 cells, non-specific siRNA transfected MCF-7 cells and STAT3 siRNA transfected MCF-7 cells. Expression of STAT3 in MCF-7 cells was inhibited by RNA interference (RNAi). The STAT3 mRNA and protein levels were detected by semi-quantity RT-PCR and Western blotting. Cell proliferation and apoptosis were determined by MTT method and flow cytometry. The three groups of MCF-7 cells mentioned above were transplanted subcutanuously into nude mice and their tumorgenic ability observed. The STAT3 mRNA and protein levels of the samples from tumors in different groups were determined by semi-quantity RT-PCR and Western blotting and compared.</p><p><b>RESULTS</b>In STAT3 siRNA transfected MCF-7 cells, the expressions (STAT3/β-actin) of STAT3 mRNA (0.327 ± 0.020) and protein (0.153 ± 0.006) were significantly lower than that in control MCF-7 cells (mRNA 1.093 ± 0.018, protein 1.374 ± 0.022) and non-specific siRNA transfected MCF-7 cells (mRNA 1.035 ± 0.050, protein 1.320 ± 0.033) (P < 0.05). MTT showed that cell proliferation was significantly reduced and the cell growth inhibition ratio in the STAT3-siRNA group was (44.00 ± 5.10)%, significantly higher than that in non-specific siRNA transfected MCF-7 cells ((16.10 ± 1.05)%, P < 0.05). Flow cytometry results showed that more apoptosis was observed in the STAT3-siRNA group. The rate of apoptosis was (14.79 ± 0.22)%, much higher than in control MCF-7 cells (7.06 ± 0.71) and non-specific siRNA transfected MCF-7 cells (8.45 ± 0.43) (P < 0.05). The tumor growth in the STAT3 siRNA transfected MCF-7 cells was significantly slower than in the two control groups. On the 22th day after transplantation the tumor weight ((21.40 ± 10.57) mg) and volume ((41.15 ± 12.17) mm(3)) in the STAT3 siRNA transfected group were significantly lower than in control group (weight (88.60 ± 12.16) mg, volume (118.45 ± 24.68) mm(3)) and non-specific siRNA transfected group (weight (57.20 ± 21.86) mg, volume (101.36 ± 21.90) mm(3)) (P < 0.05). Both the STAT3 mRNA and protein levels in the tumors from the STAT3 siRNA transfected group were significantly lower than in the tumors from the two control groups.</p><p><b>CONCLUSIONS</b>STAT3 siRNA can effectively silence the STAT3 gene in vitro and in vivo, increase cell apoptosis rate and significantly decrease cell proliferation, which inhibits the growth of breast cancer cell in vitro. Tumor growth of xenograft mice is significantly inhibited. The results obtained in vivo are in consistency with those in vitro. STAT3 may be a novel therapeutic target for breast cancer and RNA interference has potential clinical application.</p>

Inhibition of K-RAS~(Asn12) Expression by Vector-based RNA Interference in Human Pancreatic Cancer Cell Line / 中国生物工程杂志

To silence the expression of K-RASAsn12 in human pancreatic cancer cell line by vector-based RNAi(RNA interference) technique,two single-strand DNA sequences encoding mutant-specific shRNA (short haipin RNA) for K-RASAsn12 were synthesized and then inserted into pSilenCircle. The recombinant plasmid was called pSC-K-RASAsn12. According to the same method, pSC-GFP encoding shRNA for GFP was gained. Both recombinant plasmids were transfected into human pacreatic cancer cell line AsPC-1 and BxPC-3. The expression level of K-RASAsn12 was detected by semi-quantitative RT-PCR and Western blot. The result indicated that the recombinant plasmid edcoding mutant-specific shRNA for K-RASAsn12 can inhibit significantly the expression of K-RASAsn12 without affection of wild-type K-RAS(K-RASWT)in Human Pancreatic Cancer Cell Line.

Experimental study of individualized cancer immunotherapy based on dendritic cells against gastric cancer / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the antitumor effects of cytotoxic T lymphocytes (CTLs) induced by autologous dendritic cells that were inspired by autologous tumor lysates (ATLs-mDCs).</p><p><b>METHODS</b>Primary gastric cancer cells prepared by short-term culture were used as targets. ATLs-mDCs were subjected to activate autologous T cells to generate CTLs. The immunological functions of DCs were evaluated by flow cytometry and by mixed leukocyte response (MLR) assay. The antitumor outcome of tumor antigen specific CTLs was tested by cytotoxicity assay. Concentrations of IL-12 in cultured DCs and INF-gamma in CTLs were measured by ELISA.</p><p><b>RESULTS</b>The expressions of MHC-II, CD80, CD83 and CD86 were significantly up-regulated in ATLs-mDCs, moreover, the ATLs-mDCs obtained the capability of stimulating the proliferation of autologous T cells with high efficiency. The secretion of IL-12 in ATLs-mDCs was significantly higher than that in pure mature DCs (t = 15.47, P < 0.01) and in immature DCs (t = 28.44, P < 0.01). The secretion of INF-gamma in CTLs activated by ATLs-mDCs was significantly higher than that in CTLs by pure mature DCs (t = 4.84, P < 0.05) and in CTLs by immature DCs (t = 13.74, P < 0. 01). The antigen specific cytotoxicity of CTLs induced by ATLs-mDCs was significantly higher against autologous tumor cells [(84 +/- 11)%] than that against two allogeneic tumor cell lines [(19 +/- 7)% and (19 +/- 11)%; t = 54.18 and 56.46, P < 0.01, respectively].</p><p><b>CONCLUSIONS</b>ATLs-mDCs might mediate the antigen specific CTLs against autologous gastric cancer cells ex vivo with high efficiency.</p>