普鲁卡因对人肝癌HepG2细胞增殖影响的实验研究

目的：探讨普鲁卡因（procaine）对体外人肝癌HepG2细胞增殖的影响。方法：应用不同浓度（0-10）mmol/L普鲁卡因处理HepG2细胞,倒置显微镜观察细胞形态学改变、四甲基偶氮唑盐比色法（MTT）检测细胞存活率、流式细胞术（FCM）观察细胞周期变化情况。结果：倒置显微镜观察普鲁卡因处理后的HepG2细胞呈现缩小、空泡、脱壁等形态学特征。MTT结果分析表明普鲁卡因能显著抑制HepG2细胞增殖,其抑制率呈药物浓度及时间依赖性。FCM结果显示普鲁卡因可以使HepG2细胞的G0/G1延长,S期缩短。结论：盐酸普鲁卡因能够使HepG2细胞的生长阻滞在G0/G1期,从而显著抑制HepG2细胞的增殖,并且具有剂量、时间依赖效应。     

Expression of activins C and E induces apoptosis in human and rat hepatoma cells

Activins C and E (homodimers of the betaC and betaE subunits), which are almost exclusively expressed in the liver, are members of the transforming growth factor beta (TGFbeta) superfamily of growth factors. We examined their expression in three different hepatoma cell lines and found that, compared with normal liver or primary hepatocytes, human hepatoblastoma (HepG2), human hepatocellular carcinoma (Hep3B) and rat hepatoma (H4IIEC3) cells have either completely lost or drastically reduced the expression of activins C and E. In order to elucidate the biological function of these proteins we transiently transfected HepG2, Hep3B and H4IIEC3 cell lines with rat activin betaC or betaE cDNA to study the consequences of restoring activin expression in hepatoma cells. Transfection with activin betaA, a known inhibitor of hepatic DNA synthesis and inducer of apoptosis, served as a positive control. We found that transfection of the three cell lines with activin betaC or betaE, as well as with activin betaA, reduced the increase in cell number by up to 40% compared with cells transfected with a control plasmid. Co-culture with a CHO cell clone secreting activin C also inhibited HepG2 cell multiplication. Furthermore, the three hepatoma cell lines studied showed an enhanced rate of apoptosis and elevated levels of active caspases in response to activin transfection. These results indicate that activins C and E share the potential to induce apoptosis in liver derived cell lines with activin A and TGFbeta1.     

The effect of fatty acid-CoA ligase 4 on the growth of hepatic cancer cells

In this study, we detected the expression of FACL4 mRNA in 40 patients with hepatic carcinoma and its adjacent normal tissues by semi-quantitative RT-PCR. The changes of proliferation and apoptosis of hepatic cancer cell line HepG2 with FACL4 protein expression were examined by MTT and flow cytometry respectively after FACL4 selective inhibitor triacsin C treatment. The activity related to apoptosis of proteinases, caspase-3, caspase-8 and caspase-9, were detected by colorimetry. The expression related to apoptosis of protein, wt-p53, Bax and Bcl-2, in HepG2 cells were evaluated by S-P immunocytochemical dyeing. The results were: (1) FACL4 mRNA was expressed in 95.0% of hepatic cancer tissue, while the positive expression of FACL4 mRNA was 82.5% in cancer adjacent normal liver tissues. Moreover, there was a statistically significant increased in quantity of FACL4 mRNA in cancer tissues compared with adjacent normal liver tissues. (2) The concentration of triacsin C (0.5-2 mg/L) could inhibit the proliferation and induce the apoptosis of HepG2 cells significantly in a dose- and time-effect. (3) During the apoptosis of HepG2 cells induced by triacsin C, flow cytometry coupled with Rhodamine 123 dyeing showed that mitochondrial transmembrane potential of HepG2 declined significantly, and the activity of caspase-9 and caspase-3 increased more remarkably than caspase-8. Besides, the increased apoptosis was accompanied by increased Bax, and decreased wtp53 and Bcl-2 protein levels. The present study suggested that FACL4 might play a role in the growth of hepatic cancer cells. FACL4 selective inhibitor triacsin C leads to a marked growth inhibition of human liver tumor cells, based on the inhibition of proliferation and induction of apoptosis. The apoptotic process was mediated by intrinsic mitochondrial apoptotic pathway due to activation of caspase-9 and caspase-3. The increased apoptosis was accompanied by upregulation of Bax, and decreased wt-p53 and Bcl-2 protein level.     

Distribution and immunochemical characterization of a keratin-like antigen in epithelial tumors using mouse and human monoclonal antibodies

Using conventional murine hybridoma technology, we have produced a monoclonal antibody (MAb), 89E5, which recognizes two keratin-like polypeptides (Mr 53,000 and 45,000), which are preferentially expressed by epithelial tumors. In addition to detection of tumor cells by immunohistochemistry, MAb 89E5 was able to localize to tumor xenografts in nude mice after iodination of its F(ab')2 fragments. To develop potentially less immunogenic antibodies to antigens defined by MAb 89E5, studies were performed to produce a human counterpart to the mouse MAb. The mouse 89E5 MAb was used to purify the 89E5 polypeptides from tumor cell lines. The partially purified 89E5 antigen was then used to sensitize human splenic lymphocytes in vitro. Immortalization of the sensitized cells by cell fusion resulted in a human IgM MAb, PA1, which showed the same reactivity pattern on a panel of cell lines as did the mouse MAb 89E5. Immunofluorescent studies showed that both 89E5 and PA1 had staining patterns on epithelial cells indicative of antibodies to cytokeratin. Furthermore, PA1 immunoprecipitated two polypeptides (Mr 53,000 and 45,000) which comigrated with the 89E5 polypeptides. Competitive binding assays showed that the PA1 MAb and 89E5 MAb recognized closely associated epitopes. As with the 89E5 MAb, PA1 was reactive with tumor tissues in immunohistochemical studies. These studies indicate that the PA1 MAb is a human counterpart of the mouse 89E5 MAb. Direct comparison of human MAb and mouse MAb against the same antigen could yield valuable information on the efficacy of using human MAb in vivo.     

Intravenous liposomal delivery of the short hairpin RNAs against Plk1 controls the growth of established human hepatocellular carcinoma

Polo-like kinase 1 (Plk1) is a key cell cycle regulator that is frequently overexpressed in human hepatocellular carcinomas. Blockade of the Plk1 pathway has been reported to be capable of inducing anti-tumor effect. Here, plasmids containing U6 promoter-driven shRNAs against human Plk1 were constructed and transfected in human hepatocellular carcinoma cell line HepG2. ShRNA targeting Plk1 almost completely reduced Plk1 expression in HepG2 hepatocellular carcinoma cells, as confirmed by RT-PCR and Western blot. As a consequence, HepG2 cells exhibited reduced proliferation and enhanced apoptosis in vitro. Most importantly, Treatment with Plk shRNA-DOTAP:Chol complex significantly suppressed the growth of HepG2 xenografts, accompanied with phenotypic changes in tumor cells, including proliferation inhibition and apoptosis induction. Our study suggested that shRNA-mediated silencing of Plk1 might be a novel therapeutic approach against human hepatocellular carcinoma by inhibiting tumor cells proliferation and inducing apoptosis.     

Characterization of a monoclonal antibody that antagonizes the function of human endostatin

Endostatin, a 20-kDa proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and tumor growth. The anti-angiogenic effects of endostatin include inhibition of endothelial cell migration and proliferation, and inhibition of the activity of MMP2. Structure-function analysis of endostatin that implies this contravention function buried in separate fragments of endostatin introduces new issues into the understanding of the structure-function relationship of endostatin. We developed and characterized a novel murine MAb, 4E7, to human endostatin, which antagonizes the function of endostatin. As we show here, MAb 4E7 blocks the anti-migration/adhesion effects of endostatin in vitro and the anti-angiogenesis effect of endostatin in vivo, but the inhibition effect of endostatin on endothelial cell proliferation is not affected by MAb4E7. These results suggest that the anti-migration and anti-proliferation functions of endostatin may have distinct structural foundations.     

Monoclonal antibody targeting MUC1 and increasing sensitivity to docetaxel as a novel strategy in treating human epithelial ovarian cancer

The purpose of this study was to investigate the in vitro effect of anti-MUC1 monoclonal antibody (MAb) C595 alone and in combination with docetaxel, on the growth and survival of different epithelial ovarian cancer (EOC) cell lines. MUC1 expression was assessed on EOC cell lines (OVCAR-3, IGROV-1, A2780, CAOV-3, TOV-21G, TOV-112D, SKOV-3 and OV-90) using immunofluorescence labeling and flow cytometry. The effect of MAb C595 alone or in combination with docetaxel on the cell lines was studied by proliferation, colony and TUNEL assays. Our results indicate that all primary and metastatic EOC cell lines tested were positive to MAb C595 (MUC1); MAb C595 inhibited EOC cell proliferation in a MUC1- and dose-dependent manner; low-dose MAb C595 (1/2 of IC₅₀) combined with docetaxel greatly improved efficiency of cell killing in EOC cells and induced apoptosis; the additive effect of MAb C595 was further confirmed in colony forming assays; and cell death following single or combined treatments was associated with the release of cytochrome c and increased caspase-3 activity. These results suggest that MAb C595 used either alone, or combined with docetaxel, is an attractive strategy for targeting human EOC.     

Experimental therapy of hepatoma with artemisinin and its derivatives: in vitro and in vivo activity, chemosensitization, and mechanisms of action

PURPOSE: ART and its derivatives, clinically used antimalarial agents, have recently shown antitumor activities. However, the mechanisms underlying these activities remain unclear. This study was designed to determine their antitumor efficacy and underlying mechanisms of action in human hepatoma cells. EXPERIMENTAL DESIGN: The in vitro cytotoxicities of ART, DHA, artemether, and artesunate were compared in human hepatoma cells, HepG2 (p53 wild-type), Huh-7 and BEL-7404 (p53 mutant), and Hep3B (p53 null), and a normal human liver cell line, 7702. Based on their activity and specificity, ART and DHA were further investigated for their in vitro and in vivo antitumor effects and their effects on the protein expression of genes associated with cell proliferation and apoptosis. RESULTS: ART and DHA exerted the greatest cytotoxicity to hepatoma cells but significantly lower cytotoxicity to normal liver cells. The compounds inhibited cell proliferation, induced G(1)-phase arrest, decreased the levels of cyclin D1, cyclin E, cyclin-dependent kinase 2, cyclin-dependent kinase 4, and E2F1, and increased the levels of Cip1/p21 and Kip1/p27. They induced apoptosis, activated caspase-3, increased the Bax/Bcl-2 ratio and poly(ADP-ribose) polymerase, and down-regulated MDM2. In mice bearing HepG2 and Hep3B xenograft tumors, ART and DHA inhibited tumor growth and modulated tumor gene expression consistent with in vitro observations. DHA increased the efficacy of the chemotherapeutic agent gemcitabine. CONCLUSIONS: ART and DHA have significant anticancer effects against human hepatoma cells, regardless of p53 status, with minimal effects on normal cells, indicating that they are promising therapeutics for human hepatoma used alone or in combination with other therapies.     

大黄素在体外诱导人肝癌细胞HepG2发生凋亡的初步研究

背景与目的:大黄素(3-甲基-1,6,8-三羟蒽醌)是大黄等多种中药的有效成分之一。研究表明大黄素对于乳腺癌细胞、肺癌细胞的生长具有抑制作用,但目前大黄素抗肿瘤的作用机理尚不明确。本研究拟讨论大黄素在体外对肝癌细胞HepG2的作用及其机理。方法:应用MTT、软琼脂克隆形成、DNAladder凝胶电泳及流式细胞术等方法,研究中药单体大黄素对肝癌细胞HepG2生长增殖的影响以及作用机理。结果:大黄素在较低浓度可抑制肿瘤细胞的生长,MTT实验测得的半数抑制浓度(IC50)为(36±2.6)μg/ml。在软琼脂实验中,大黄素可以浓度依赖的方式抑制细胞克隆的形成。经DNAladder及流式细胞仪检测发现,大黄素能诱导HepG2细胞凋亡,与阴性对照相比,随着药物浓度从10μg/ml增加到20μg/ml,AnnexinV染色细胞显著增多,由27.3%增至59.6%;当药物浓度增至40μg/ml时,培养液中几乎无活细胞,由碘化丙啶和AnnexinV双重标记的凋亡后期以继发性坏死细胞为主。结论:中药单体大黄素在体外能抑制肝癌细胞HepG2的生长增殖,并能诱导该细胞凋亡。     

Silibinin efficacy against human hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is one of the most common recurrent malignancies, for which, currently, there is no effective therapy. Considering the antihepatotoxic activity of silibinin, a widely used drug and supplement for various liver disorders, together with its strong preventive and anticancer efficacy against various epithelial cancers, we investigated the efficacy of silibin against human HCC cells. EXPERIMENTAL DESIGN: Silibinin effects were examined on growth, cytotoxicity, apoptosis, and cell cycle progression in two different HCC cell lines, HepG2 (hepatitis B virus negative; p53 intact) and Hep3B (hepatitis B virus positive; p53 mutated). At molecular level, cell cycle effects of silibinin were assessed by immunoblotting and in-bead kinase assays. RESULTS: Silibinin strongly inhibited growth of both HepG2 and Hep3B cells with a relatively stronger cytotoxicity in Hep3B cells, which was associated with apoptosis induction. Silibinin also caused G1 arrest in HepG2 and both G1 and G2-M arrests in Hep3B cells. Mechanistic studies revealed that silibinin induces Kip1/p27 but decreases cyclin D1, cyclin D3, cyclin E, cyclin-dependent kinase (CDK)-2, and CDK4 levels in both cell lines. In Hep3B cells, silibinin also reduced the protein levels of G2-M regulators. Furthermore, silibinin strongly inhibited CDK2, CDK4, and CDC2 kinase activity in these HCC cells. CONCLUSION: Together, these results for the first time identify the biological efficacy of silibinin against HCC cells, suggesting the importance of conducting further investigations in preclinical HCC models, especially on in vivo efficacy, to support the clinical usefulness of silibinin against hepatocellular carcinoma in addition to its known clinical efficacy as an antihepatotoxic agent.     

Androgen receptor and E2F-1 targeted thymoquinone therapy for hormone-refractory prostate cancer

Relapse of prostate cancer after androgen ablation therapy is hormone-refractory, with continued tumor growth being dependent on the androgen receptor (AR). E2F-1, a regulator of cell proliferation and viability, reportedly plays a role in the development of hormone-refractory prostate cancer. Thymoquinone is a component of Nigella sativa, an herb used for thousands of years for culinary and medicinal purposes in Asian and Middle Eastern countries and has been reported to have an antineoplastic effect both in vitro and in vivo. We observed that thymoquinone inhibited DNA synthesis, proliferation, and viability of cancerous (LNCaP, C4-B, DU145, and PC-3) but not noncancerous (BPH-1) prostate epithelial cells by down-regulating AR and E2F-1. In LNCaP cells, this was associated with a dramatic increase in p21(Cip1), p27(Kip1), and Bax. Thymoquinone blunted progression of synchronized LNCaP cells from G1 to S phase, with a concomitant decrease in AR and E2F-1 as well as the E2F-1-regulated proteins necessary for cell cycle progression. In a xenograft prostate tumor model, thymoquinone inhibited growth of C4-2B-derived tumors in nude mice. This in vivo suppression of tumor growth, as with C4-2B cell growth in culture, was associated with a dramatic decrease in AR, E2F-1, and cyclin A as determined by Western blot of tissue extracts. Tissue immunohistochemical staining confirmed a marked reduction in E2F-1 and showed induction of apoptosis on terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay. These findings show that thymoquinone suppresses the expression of AR and E2F-1 necessary for proliferation and viability of androgen-sensitive as well as androgen-independent prostate cancer cells both in vitro and in vivo and, moreover, produced no noticeable side effects in mice. We conclude that thymoquinone, a naturally occurring herbal product, may prove to be effective in treating hormone-sensitive as well as hormone-refractory prostate cancer. Furthermore, because of its selective effect on cancer cells, we believe that thymoquinone can also be used safely to help prevent the development of prostate cancer.     

Effect of rapamycin alone and in combination with antiangiogenesis therapy in an orthotopic model of human pancreatic cancer.

PURPOSE: The overall 5-year survival of patients with pancreatic cancer remains <5%. Novel therapeutic strategies are needed. We examined the effect of rapamycin, alone and in combination with antiangiogenesis therapy, on pancreatic cancer in vivo. EXPERIMENTAL DESIGN: Human pancreatic cancer AsPC-1 cells were orthotopically injected into severe combined immunodeficient/beige mice to evaluate primary tumor growth and liver metastasis after treatment with rapamycin alone or in combination with anti-vascular endothelial growth factor antibody 2C3. Tumor cell proliferation was determined by bromodeoxyuridine incorporation. To detect tumor cell apoptosis, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was used. Tumor angiogenesis was investigated by using a monoclonal anti-CD31 antibody. All statistical tests were two-sided. RESULTS: Rapamycin, alone and in combination with 2C3, strongly inhibited primary and metastatic tumor growth in an orthotopic pancreatic cancer animal model. Furthermore, the combination therapy significantly improved the effect on liver metastasis compared with single treatment with either rapamycin (P = 0.0128) or 2C3 (P = 0.0099). Rapamycin alone inhibited pancreatic tumor cell proliferation, induced apoptosis, and decreased tumor angiogenesis. Nevertheless, the combination therapy showed a significant, stronger inhibition of tumor cell proliferation (P = 0.0002 versus rapamycin alone and P < 0.0001 versus 2C3 alone). The induction of apoptosis was significantly higher than in the rapamycin-treated group (P = 0.0039). Additionally, the combination therapy further improved suppression of tumor cell angiogenesis compared with rapamycin treatment (P = 0.029) CONCLUSIONS: Our studies propose new therapeutic strategies to inhibit both primary and metastatic tumor growth in pancreatic cancer. Considering the fact that liver metastasis is a crucial problem in advanced stages of pancreatic cancer, the combination therapy of rapamycin plus anti-vascular endothelial growth factor antibody 2C3 is a significant advantage compared with single treatment with rapamycin.     

阻断肝再生增强因子的表达对人肝癌细胞株HepG2增殖的抑制作用

背景与目的:前期研究中已发现人肝再生增强因子(humanaugmenterofliverregeneration,hALR)在肝癌细胞中呈高表达,在正常肝细胞中几乎不表达,并在体外能刺激肝癌细胞株增殖,而对正常肝细胞无影响。本研究通过运用hALR的siRNA和hALR单克隆抗体阻断人肝癌细胞株HepG2表达,探讨hALR对细胞增殖的影响。方法:设计、构建siRNA表达质粒pSIALR-A及其阴性对照质粒pSIALR-B。将构建的pSIALR-A和pSIALR-B分别转染HepG2细胞。荧光显微镜观察绿色荧光蛋白的表达,以计算转染效率。转染48h后,免疫细胞化学及RT-PCR法检测HepG2细胞中hALR的表达。用3H-TdR掺入法检测hALR的siRNA和特异性单克隆抗体对HepG2细胞增殖的影响。结果:HepG2细胞中有hALR的表达。成功构建了针对hALR基因编码区的siRNA表达质粒pSIALR-A。转染入细胞后发现,pSIALR-A能明显抑制hALR的表达(mRNA的表达量减少83%),而随机序列的siRNA却无此作用。hALR的siRNA在体外能显著抑制人肝癌细胞株HepG2增殖,分别转染质粒pSIALR-A和pSIALR-B后HepG2细胞的cpm值为67687±6548和104807±5713(P<0.05)。抗hALR单克隆抗体特异性地阻断hALR的作用后,可部分抑制肿瘤细胞自主性生长(P<0.05)。结论:HepG2细胞高表达hALR;针对hALR的siRNA能显著、特异性地抑制hALR表达,进而抑制HepG2细胞的生长。抗hALR单克隆抗体也能部分抑制HepG2细胞自主性生长。     

Naringenin (Citrus Flavonone) Induces Growth Inhibition, Cell Cycle Arrest and Apoptosis in Human Hepatocellular Carcinoma Cells

Search for new substances with antiproliferative activity and apoptosis inducing potential towards HepG2 cells is important since HCC is notoriously resistant to conventional chemotherapy. Dietary phytochemicals with significant anti-proliferative and apoptosis inducing potential are considered as agents promising for cancer therapy. Naringenin, a common dietary flavonoid abundantly present in fruits and vegetables, is believed to possess strong cytotoxic activity in numerous types of cancer cells. However, the detailed molecular mechanisms of its antiproliferative effects and apoptosis induction are still unclear. In this study, we investigated antiproliferative and apoptosis-inducing effect of naringenin in human hepatocellular carcinoma HepG2 cells. Naringenin was shown to inhibit the proliferation of HepG2 cells resulted partly from an accumulation of cells in the G0_/G1 and G2/M phase of the cell cycle. Naringenin induced a rapid accumulation of p53, which might account for the naringenin-induced G0_/G1 and G2/M phase arrests in Hep G2 cells. In addition, naringenin have been shown to induce apoptosis as evidenced by nuclei damage and increased proportion of apoptotic cells detected by flow cytometry analysis. Naringenin triggered the mitochondrial-mediated apoptosis pathway as shown by an increased ratio of Bax/Bcl-2, subsequent release of cytochrome C, and sequential activation of caspase-3. Our results showed that naringenin had inhibitory effect on the growth of HepG2 cell line through inhibition of cell proliferation and apoptosis induction. The elucidation of the drug targets of naringenin on inhibition of tumor cells growth should enable further development of naringenin for liver cancer therapy.     

Targeted constitutive activation of signal transducer and activator of transcription 3 in human hepatocellular carcinoma cells by cucurbitacin B

Purpose  To determine the effect of cucurbitacin B on human hepatocellular carcinoma cell growth and apoptosis, and to explore the potential mechanisms. Methods  In vitro viability of human hepatocellular carcinoma cell line (HepG2) was investigated using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Morphologic changes of cells were evaluated through light microscopy. Cell cycle distribution was evaluated with flow cytometry following PI staining. Apoptosis was evaluated respectively with flow cytometry and fluorescent microscopy following Annexin V-FITC/PI and Hoechst 33258 staining. Western blot assays were performed to determine the expression of pSTAT3 and Bcl-2. Finally, in vivo effect of cucurbitacin B on the growth of HepG2 cells was determined in nude mice. Results  The MTT assay showed that cucurbitacin B inhibited HepG2 cell viability in a dose and time-dependent manner. Cucurbitacin B treatment resulted in accumulation of cells at the S phase of cell cycle as well as apoptosis. Marked morphological changes, including condensation of chromatin, nuclear fragmentation and apoptotic bodies were clearly shown on Hoechst 33258 staining. Western blot showed that cucurbitacin B inhibited STAT3 phosphorylation and down-regulated the expression of Bcl-2. Growth of HepG2 tumor in nude mice was also inhibited by cucurbitacin B. Conclusion  Our results suggest that cucurbitacin B may have a therapeutic value in suppressing the growth of human hepatocellular carcinoma. The mechanism may be attributable to the suppression of STAT3 phosphorylation.     

Gene therapy with secretory leukoprotease inhibitor promoter-controlled replication-competent adenovirus for non-small cell lung cancer

Secretory leukoprotease inhibitor (SLPI) is highly expressed in almost all non-small cell lung cancers (NSCLCs), but not in the majority of other tumor types. In an attempt to create a specific gene therapy for NSCLC, we constructed AdSLPI.E1AdB, an adenovirus vector with a double expression cassette consisting of E1A driven by the SLPI promoter gene followed by E1B-19K under the control of the cytomegalovirus (CMV) promoter that can selectively replicate only in NSCLC cells. Infection with AdSLPI.E1AdB yielded E1A protein expression and adenovirus replication resulting in a >100-fold increase of the virus titers only in SLPI-producing NSCLC cells (A549, H358, and HS24 cells). In contrast, neither E1A protein nor replication was detected in non-SLPI-producing HepG2 cells. Treatment with AdSLPI.E1AdB significantly inhibited the proliferation of NSCLC cells in vitro in a dose-dependent manner, whereas the cell growth of HepG2 or normal human bronchial epithelial cells was not affected by AdSLPI.E1AdB infection. Direct injection of AdSLPI.E1AdB into A549 and H358 tumors in nude mice resulted in a marked reduction in tumor growth compared with controls (A549, 57%, P < 0.02; H358, 67%, P < 0.03). Histological examination revealed the replication of AdSLPI.E1AdB and strong induction of necrosis and apoptosis. In addition, we evaluated the combination of AdSLPI.E1AdB and AdCMV.NK4 encoding NK4 protein, which has strong antiangiogenic activity. E1A expressed by AdSLPI.E1AdB trans-acts on the replication of AdCMV.NK4 and thus increases the expression of NK4. Injection of these two vectors into H358 tumors resulted in a more striking reduction of tumor growth compared with single injection of each vector. These results suggest that AdSLPI.E1AdB could provide a selective therapeutic modality for NSCLC and that the combination of AdSLPI.E1AdB and AdCMV.NK4 may be a more effective gene therapy for NSCLC.     

Murine monoclonal anti-idiotypic antibody as a surrogate antigen for human Her-2/neu

The anti-idiotypic (Id) monoclonal antibody (MAb) 520C9-6b (IgG1k), raised in syngenic mice against the murine anti-Her2/neu MAb 520C9 (Ab1), functionally mimics a human Her2/neu epitope and serves as a surrogate for the protein antigen. Immunization of allogeneic C57BL/6 mice and rabbits with 520C9-6b (Ab2) induced anti-Her2/neu-specific antibodies that react with antigen-positive SKBr3 cells by ELISA and FACS analysis. The immune sera inhibited binding between Ab1 and Ab2 and vice versa (binding of Ab2 to Ab1), indicating that it was a true anti-anti-Id (Ab3) antibody. The Ab3 sera or purified Ab3 specifically lysed Her2/neu-positive SKBr3 cells, but no significant lysis was observed in antigen-negative LS174T cells in an antibody-dependent cellular cytotoxicity assay. An Id-specific cellular immune response was also demonstrated in an in vitro lymphocyte proliferation assay. Furthermore, a panel of tumor tissues and tumor cells was screened for the presence of the Her2/neu epitope by its reactivity with Ab1 and Ab3 using immunohistochemistry and FACS analysis. Identical results were obtained using either Ab1 or Ab3 (Ab1'). The data indicated that anti-Id 520C9-6b can induce Her2/neu-specific antibody in experimental animals and may serve as a potential network antigen for the treatment of patients bearing Her2/neu-positive tumors.