Intraperitoneal injection of adenovirus-mediated NK4 gene suppresses peritoneal dissemination of pancreatic cancer cell line AsPC-1 in nude mice

NK4, composed of the N-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts not only as a competitive antagonist for HGF but also as a potent angiogenesis inhibitor. This study was designed to assess a therapeutic potential of adenovirus-mediated NK4 gene transfer for disseminated pancreatic cancer cells in the peritoneal lavage of nude mice. We constructed a recombinant adenovirus NK4 (Ad-NK4), which encodes a secretable form of human NK4. In vitro migration of AsPC-1 (human pancreatic cancer cell line) was stimulated by HGF, and it was completely inhibited by Ad-NK4 transfection. Weekly intraperitoneal injections of Ad-NK4 could suppress the development of tumor nodules in a nude mouse peritoneal dissemination model. NK4 expression was detected in the disseminated nodules, liver, pancreas, spleen, and mesenterium. Immunohistochemical study of the disseminated tumors showed a remarkable decrease in microvessel density and an increase in number of apoptotic tumor cells in the Ad-NK4-treated mice. Survival of the Ad-NK4-treated mice was significantly improved. This study indicates that the intraperitoneal transduction of adenovirus-mediated NK4 gene may be a useful therapeutic modality to prevent the development of peritoneal dissemination of pancreatic cancer.     

Intraperitoneal administration of an adenovirus vector carrying REIC/Dkk-3 suppresses peritoneal dissemination of scirrhous gastric carcinoma

Expression levels of the novel tumor suppressor gene REIC/Dkk-3 are reduced in many human cancers. We have previously showed that an adenovirus vector carrying REIC/Dkk-3 (Ad-REIC) induced apoptosis of cancer cells selectively and exerted bystander antitumor effects via ER stress. We examined possible effects of Ad-REIC in a peritoneal dissemination model of scirrhous gastric carcinoma (SGC). Among various types of gastric cancer, SGC continues to be associated with the worst prognosis due to a high incidence of metastases in the peritoneal cavity. We found that a single intraperitoneal injection of Ad-REIC suppressed tumor dissemination and disease progression. Immunomodulation by Ad-REIC led to recruitment of natural killer cells inside tumor nodules. We conclude that Ad-REIC gene therapy may be a potential tool in combinatorial approaches to achieve curative effects in SGC.     

Local interferon-alpha gene therapy elicits systemic immunity in a syngeneic pancreatic cancer model in hamster

The interferon (IFN) protein is a cytokine with pleiotropic biological functions that include induction of apoptosis, inhibition of angiogenesis and immunomodulation. We previously examined the two antitumor mechanisms, taking advantage of the fact that IFN-alpha did not show cross-species activity in its in vivo effect. In a nude mouse subcutaneous xenograft model using human pancreatic cancer cells, the expression of human IFN-alpha effectively induced cell death of human pancreatic cancer cells, whereas mouse IFN-alpha augmented antitumor immunity by stimulation of natural killer cells. Here, we extended our investigation to a syngeneic pancreatic cancer model, so that the integrated antitumor activity of local IFN-alpha gene therapy, including the antiproliferative, proapoptotic, antiangiogeneic and immunomodulatory effects, can be evaluated rigorously. When a recombinant hamster IFN-alpha adenovirus was injected into syngeneic subcutaneous tumors of hamster pancreatic cancer (PGHAM-1) cells in Syrian hamster, tumor growth was significantly suppressed due to cell death and T cell- and natural killer cell-mediated antitumor immunity. Moreover, in this case, tumor regression was observed not only for the injected subcutaneous tumors but also for the untreated tumors both in the peritoneal cavity and at distant sites. No significant systemic toxicity was observed in the treated hamsters. Moreover, the subcutaneous rechallenge of PGHAM-1 cells was rejected in three of four cured hamsters from the initial tumor challenge. This study further demonstrated that local IFN-alpha gene therapy is a promising therapeutic strategy for pancreatic cancer, due to its multiple mechanisms of antitumor activity and its lack of significant toxicity.     

Inhibition of nuclear factor-kappaB suppresses peritoneal dissemination of gastric cancer by blocking cancer cell adhesion

Currently, patients with peritoneal dissemination of gastric cancer must accept a poor prognosis because there is no standard effective therapy. To inhibit peritoneal dissemination it is important to inhibit interactions between extracellular matrices (ECM) and cell surface integrins, which are important for cancer cell adhesion. Although nuclear factor-kappa B (NF-κB) is involved in various processes in cancer progression, its involvement in the expression of integrins has not been elucidated. We used a novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), to study whether NF-κB blocks cancer cell adhesion via integrins in a gastric cancer dissemination model in mice and found that DHMEQ is a potent suppressor of cancer cell dissemination. Dehydroxymethylepoxyquinomicin suppressed the NF-κB activity of human gastric cancer cells NUGC-4 and 44As3Luc and blocked the adhesion of cancer cells to ECM when compared with the control. Dehydroxymethylepoxyquinomicin also inhibited expression of integrin (α2, α3, β1) in in vitro studies. In the in vivo model, we injected 44As3Luc cells pretreated with DHMEQ into the peritoneal cavity of mice and performed peritoneal lavage after the injection of cancer cells. Viable cancer cells in the peritoneal cavities were evaluated sequentially by in vivo imaging. In mice injected with DHMEQ-pretreated cells and lavaged, live cancer cells in the peritoneum were significantly reduced compared with the control, and these mice survived longer. These results indicate that DHMEQ could inhibit cancer cell adhesion to the peritoneum possibly by suppressing integrin expression. Nuclear factor-kappa B inhibition may be a new therapeutic option for suppressing postoperative cancer dissemination.     

Specific inhibition of K-ras expression and tumorigenicity of lung cancer cells by antisense RNA

A human lung cancer cell line (H460a) with a homozygous spontaneous K-ras mutation was transfected with a recombinant plasmid that synthesizes a 2-kilobase genomic segment of the K-ras protooncogene in antisense orientation. Translation of the mutated K-ras mRNA in H460a cells was specifically inhibited, whereas expression of H-ras and N-ras was unchanged. A 3-fold growth inhibition occurred in H460a cells when expression of the mutated ras p21 protein was down-regulated by antisense RNA. However, cells remained viable despite the absence of K-ras expression. The growth of H460a tumors in nu/nu mice was substantially reduced by expressed K-ras antisense RNA.     

Peritumoral injection of adenovirus vector expressing NK4 combined with gemcitabine treatment suppresses growth and metastasis of human pancreatic cancer cells implanted orthotopically in nude mice and prolongs survival

NK4 or adenovirus vector expressing NK4 (Ad-NK4) can act bifunctionally as a hepatocyte growth factor antagonist and angiogenesis inhibitor and has potential value in cancer therapy. The aim of this study was to evaluate the therapeutic efficacy of Ad-NK4 in combination with gemcitabine (GEM) against pancreatic cancer. In vitro study showed a strong antiproliferative effect of GEM and a potent anti-invasive effect of Ad-NK4 against pancreatic cancer cells. In in vivo experiments, SUIT-2 human pancreatic cancer cells were implanted into the pancreas of nude mice. Mice were treated with Ad-NK4 by injection into the peritumoral region of the pancreas on day 5 after implantation followed by weekly i.p. injections of GEM. On day 28 after implantation, pancreatic tumor volume was significantly smaller than that in mice treated with Ad-LacZ, Ad-NK4 alone, or GEM alone. Furthermore, combination therapy completely suppressed peritoneal dissemination and liver metastases, leading to significantly increased survival. Histologic and immunohistochemical assays of primary tumors indicated that combination therapy prohibited both cell proliferation and angiogenesis, resulting in high levels of apoptosis. These results suggest that peritumoral injection of Ad-NK4 plus GEM is a potent combination therapy for pancreatic cancer.     

Intraperitoneal administration of cisplatin (CDDP) for advanced or recurrent gastric cancer with peritoneal dissemination]

This study was undertaken in order to evaluate the effect of intraperitoneal administration of CDDP on fourteen patients with peritoneal dissemination of advanced or recurrent gastric cancer. The procedure was used together specific hydration transfusion and diuretics. Two patients showed a complete response and 5 patients a partial response. Following this therapy, 2 patients have survived for more than 6 months. Alimentary symptoms (nausea, vomiting) were found but renal toxicity and serious myelosuppression were not recognized in all patients.     

VEGF receptor antisense therapy inhibits angiogenesis and peritoneal dissemination of human gastric cancer in nude mice

The efficacy of a phosphorothioate antisense oligonucleotide (ASO) for KDR/Flk-1 (KDR/Flk-1-ASO), an endothelial cell-specific vascular endothelial growth factor (VEGF) receptor, was investigated on the peritoneal dissemination and angiogenesis of a human gastric cancer cell line in nude mice. Green fluorescent protein (GFP)-transduced NUGC-4 (NUGC-4-GFP) human gastric cancer cells were implanted into the peritoneal cavity of nude mice. KDR/Flk-1-ASO, -SO, or phosphate-buffered saline was administrated from days 7 to 14, 200 microg/mouse, once a day. The mice were sacrificed on day 28. Disseminated peritoneal tumor nodules expressing GFP were visualized by fluorescence microscopy. KDR/Flk-1-ASO significantly decreased the extent of peritoneal dissemination of the tumors. The number of cells undergoing apoptosis was significantly increased in the KDR/Flk-1-ASO-treated tumors. Microvessel density was significantly reduced in the KDR/Flk-1-ASO-treated tumor nodules. The KDR/Flk-1 antisense strategy, therefore, decreases tumor dissemination apparently by inhibiting angiogenesis.     

Intraperitoneal radioimmunotherapy in treating peritoneal carcinomatosis of colon cancer in mice compared with systemic radioimmunotherapy

Peritoneal spread is one of major causes of mortality in colorectal cancer patients. In the current investigation, the efficacy of radioimmunotherapy (RIT) with i.p. administration of an anti-colorectal cancer IgG1, 131I-A7, was compared to that with i.v. administration in BALB/c female mice bearing peritoneal nodules of LS180 human colon cancer cells, at the same toxicity level. Distribution of either i.p. or i.v. administered 131I-A7 and i.p. administered irrelevant 131I-HPMS-1 was assessed. Based on the results of toxicity determination at increments of 2 MBq and estimated dosimetry, an i.p. dose of 11 MBq and an i.v. dose of 9 MBq were chosen for treatment. Mice were monitored for long-term survival: untreated mice (n = 11), mice undergoing i.p. RIT with 131I-A7 (n = 11), mice undergoing i.v. RIT with 131I-A7 (n = 11) and mice undergoing non-specific i.p. RIT with 131I-HPMS-1 (n = 5). Intraperitoneal injection of 131I-A7 produced faster and greater tumor accumulation than i.v. injection: 34.2 +/- 16.5% of the injected dose per g (% ID/g) and 11.1 +/- 3.6% ID/g at 2 h, respectively (P < 0.0001). Consequently, cumulative radioactivity in tumors was 1.73-fold higher with i.p. injection. 131I-HPMS-1 did not show specific accumulation. Non-specific RIT with 131I-HPMS-1 (mean survival, 26.0 +/- 2.5 days) did not affect the survival as compared to no treatment (26.7 +/- 1.9 days). Intravenous RIT with 131I-A7 prolonged the survival of mice to 32.8 +/- 1.8 days (P < 0.01). Intraperitoneal RIT with 131I-A7 improved the survival more significantly and attained cure in 2 of 11 mice (P < 0.05 vs. i.v. RIT). In conclusion, i.p. RIT is more beneficial in treating peritoneal carcinomatosis of colon cancer than i.v. RIT in a murine model.     

A pilot study of intraperitoneal gemcitabine for reduction of peritoneal dissemination in advanced pancreatic cancer

We schemed intraperitoneal gemcitabine (ipGEM) for reduction of peritoneal dissemination to three patients with unresectable and one patient with recurrent pancreatic cancer, followed by intraperitoneal catheter and subcutaneous port placement. Two locally advanced cases were performed for intra-operative radiotherapy, and one of 2 locally advanced cases was performed for gastrojejunostomy. And another locally advanced case had ileostomy. The recurrent case was resected for metastatic tumor of abdominal wall. In one of locally advanced cases, we couldn't perform ipGEM for progression of disease. In two remaining locally advanced cases, we could keep on doing ipGEM, and the patients did not experience with abdominal discomfort or hematological toxicity.     

Delivering antisense telomerase RNA by a hybrid adenovirus/ adeno-associated virus significantly suppresses the malignant phenotype and enhances cell apoptosis of human breast cancer cells

Activated telomerase is frequently detected in cancer cells and is able to maintain and stabilize the integrity of telomeres; it also contributes to unlimited divisions in cancer cells. Recently, a new generation of selective anticancer strategies is under development targeting the blockage of telomerase activity either at the protein level or telomerase RNA. Here, we report suppression of the malignant phenotype by the expression of the full-length antisense human telomerase RNA (hTR) delivered by a novel hybrid vector recombining adenovirus and adeno-associated virus (vAd-AAV). The hybrid vector vAd-AAV retained the unique traits from two parental viruses, such as high efficiency of gene transfer in mammalian cells and the ability to integrate into the genomic DNA of host cells. The stable expression of antisense hTR in MCF-7 cells significantly suppressed telomerase activity and progressively shortened telomere length for 30 population doublings (PD30). Expression of antisense hTR leads to a telomere-based growth arrest and the induction of spontaneous apoptosis. Antisense hTR decreased soft agar colony formation and reduced the cell proliferation, leading to exit from the cell cycle at G1 at PD15. The expression of antisense hTR also sensitized MCF-7 cells to apoptosis induced by sodium butyrate or serum starvation. Our study demonstrates that delivering antisense hTR by the hybrid Ad/AAV vector is an effective antineoplastic gene therapeutic strategy, which significantly suppresses the malignant phenotype and enhances apoptosis of human breast cancer cells.     

Interleukin-10 gene transfer to peritoneal mesothelial cells suppresses peritoneal dissemination of gastric cancer cells due to a persistently high concentration in the peritoneal cavity

Interleukin (IL)-10 has potent biological properties including an inhibitory action on the proliferation and metastasis of various cancer cells. However, it is difficult to maintain a high concentration of this cytokine as it has a short half life. In this study, we evaluated whether peritoneal mesothelial cells (PMCs) could be suitable for maintaining a high concentration of IL-10 using adenoviral gene transfer. We also evaluated the therapeutic effects of an intraperitoneal injection with adenoviral vector containing mouse IL-10 gene (Ad-mIL-10) using a mouse peritoneal dissemination model of MKN45 gastric cancer cells. We demonstrated that in vitro transfection efficiency of a recombinant adenovirus containing the bacterial beta-galactosidase gene (Ad-LacZ) was approximately 10-fold higher for primarily isolated PMCs than MKN45. The entire peritoneum was transfected until 3 weeks after an intraperitoneal Ad-LacZ injection. Ad-mIL-10 treatment increased intraperitoneal IL-10 levels until 3 weeks after treatment, and then significantly inhibited peritoneal cancer growth by inhibiting angiogenesis. This treatment also improved cachexia and prolonged mice survival. We thus concluded that IL-10 gene transfer in PMCs could be a new strategy for the prevention of peritoneal dissemination of gastric cancer due to the resulting persistently high IL-10 concentration in the peritoneal cavity.     

Intraperitoneal administration of paclitaxel solubilized with poly(2-methacryloxyethyl phosphorylcholine-co n-butyl methacrylate) for peritoneal dissemination of gastric cancer

Intraperitoneal (i.p.) administration of paclitaxel (PTX) is a hopeful therapeutic strategy for peritoneal malignancy. Intravenously (i.v.) injected nanoparticle anticancer drugs are known to be retained in the blood stream for a long time and favorably extravasated from vessels into the interstitium of tumor tissue. In this study, we evaluated the effect of i.p. injection of PTX (PTX-30W), which was prepared by solubulization with water-soluble amphiphilic polymer composed of PMB-30W, a co-polymer of 2-methacryloxyethyl phosphorylcholineand n -butyl methacrylate, for peritoneal dissemination of gastric cancer. In a peritoneal metastasis model with transfer of MKN45P in nude mice, the effct of i.p. administration of PTX-30W was compared with conventional PTX dissolved in Cremophor EL (PTX-Cre). The drug accumulation in peritoneal nodules was evaluated with intratumor PTX concentration and fluorescence microscopic observation. PTX-30W reduced the number of metastatic nodules and tumor volume significantly more than did conventional PTX dissolved in Cremophor EL (PTX-Cre), and prolonged the survival time ( P  < 0.05). PTX concentration in disseminated tumors measured by HPLC was higher in the PTX-30W than in the PTX-Cre group up to 24 h after i.p. injection. Oregon green–conjugated PTX-30W, i.p. administered, preferentially accumulated in relatively hypovascular areas in the peripheral part of disseminated nodules, which was significantly greater than the accumulation of PTX-Cre. I.p. administration of PTX-30W may be a promising strategy for peritoneal dissemination, due to its superior characteristics to accumulate in peritoneal lesions. ( Cancer Sci  2009; 100: 1979–1985)     

K-ras和IGF-IR反义寡核苷酸联合转染对人胰腺癌Patu8988细胞增殖和凋亡的影响

背景与目的:已有研究表明K-ras基因点突变及胰岛素样生长因子Ⅰ受体(insulin-like growth factor receptor type1,IGF-IR)过表达在胰腺癌的发生发展过程中起着重要作用,针对K-ras mRNA和IGF-IR mRNA的反义寡核苷酸(antisense oligodeoxynucleotide,ASODN)均可抑制胰腺癌细胞的增殖。本研究旨在探讨针对K-ras基因12密码子点突变的硫代反义寡核苷酸(K-ras ASODN)联合IGF-IR硫代反义寡核苷酸(IGF-IR ASODN)对人胰腺癌Patu8988细胞体内外增殖和凋亡的影响。方法:顺序特异引物聚合酶链反应(polymerase chain reaction using special sequence primers,PCR-SSP)法和基因测序检测Patu8988细胞K-ras点突变形式,根据点突变形式设计并合成K-ras ASODN。K-ras ASODN和IGF-IR ASODN分别单独和联合作用Patu8988细胞后,通过四甲基偶氮唑蓝(MTT)法、克隆形成实验及透射电镜观察其对Patu8988细胞增殖及形态结构的影响;流式细胞术(FCM)检测K-ras和IGF-IR蛋白表达和细胞凋亡;RT-PCR检测K-ras和IGF-IR mRNA表达水平;以Patu8988细胞建立裸鼠人胰腺癌模型,观察K-ras ASODN与IGF-IR ASODN联合应用在体内的抗肿瘤效果。结果:PCR-SSP法和基因测序检测表明Patu8988细胞存在K-ras基因第12密码子点突变,其突变方式为GGT→GTT。2～32mg/L的K-ras ASODN和2～32mg/L的IGF-IR ASODN单独应用均能一定程度抑制Patu8988细胞增殖、诱导细胞凋亡,两者联合应用较单独应用抑制作用更为显著(P<0.01)。体内实验表明,K-ras ASODN与IGF-IR ASODN联合应用较单独应用能更有效地抑制BALB/c裸鼠胰腺癌的生长(P<0.01)。结论:K-ras ASODN和IGF-IF ASODN两种反义寡核苷酸对人胰腺癌Patu8988细胞增殖的抑制具有协同作用,其机制可能是联合下调K-ras、IGF-IR蛋白及K-ras、IGF-IR mRNA表达水平。     

Intraperitoneal administration of paclitaxel and oral S-1 for a patient with peritoneal dissemination and hydronephrosis due to advanced gastric cancer

We report a patient with type 3 gastric cancer with peritoneal dissemination and hydronephrosis who was successfully treated with intraperitoneal infusion of paclitaxel and oral administration of S-1. He was diagnosed with unresectable gastric cancer with severe peritoneal dissemination by staging laparoscopy. We selected combined chemotherapy with both paclitaxel and S-1. Paclitaxel at 60 mg/m² was administered intraperitoneally on days 1 and 8, and S-1 at 100 mg/body was administered orally for 14 days, followed by 7 days’ rest, as one course. After five courses, primary tumor reduction was confirmed and no cancer cells were detected on pathocytological investigation at second-look laparoscopy. The patient underwent total gastrectomy with lymph node dissection. He died from liver metastasis 29 months after the initial treatment, but he had not suffered from peritoneal metastases and had kept a good quality of life (QOL) since that treatment. This chemotherapy can be applied as one of the promising candidates for the treatment of patients with peritoneal metastasis of gastric cancer.     

CD147反义RNA表达质粒的构建及克隆鉴定

目的　构建CD14 7反义RNA表达质粒载体。方法　用DNA重组技术将人CD14 7基因反向克隆到真核表达质粒PCD NA3.1中 ,构建成CD14 7反义RNA表达质粒PCDNAasCD14 7。用阳离子脂质体介导转染人卵巢癌细胞系 8910 ,经G4 18筛选后获得的克隆进行鉴定。结果　CD14 7反义RNA表达质粒载体PCDNAasCD14 7经限制性酶切及部分序列分析证明基因插入正确。流式细胞仪及免疫组化SP法染色均证实 :转染细胞 8910 /PCDNAasCD14 7,与亲本细胞相比 ,CD14 7的表达明显下降。结论　成功构建了CD14 7反义RNA表达质粒载体PCDNAasCD14 7,此实验结果为进一步研究CD14 7蛋白分子的生物学功能以及为卵巢癌的基因治疗研究奠定了基础。     

Amphiregulin Antisense RNA Delivered by Adnovirus Suppresses Growth of Breast Cancer Cells In Vitro and In Vivo

OBJECTIVE To investigate the therapeutic potential of amphiregulin antisense RNA delivered by adenoviral vector in a human breast cancer model.METHODS Human amphiregulin cDNA was subcloned in the opposite orientation to the cytomegaloviral promoter and inserted into an E1/E3-deleted type 5 adenoviral vector to obtain an AdA4 construct which expresses amphiregulin antisense mRNA. Both in vitro and in vivo antiproliferative effects of the antisense RNA were studied by infecting transformed human breast epithelial NS2T2A1 cells and tumors.RESULTS Amphiregulin protein expression was inhibited dramatically in the NS2T2A1 cells after infection with AdA4. The in vitro cell growth was inhibited significantly at day 4 post-AdA4 infection compared with control empty virus AdC1 at a MOl of 200 and 400 pfu/cell to 69.3% and 49.8%, respectively (P<0.02, P<0.005). After 3 intra-tumoral injections of 109 pfu AdA4, tumor volumes were reduced to 40.6% of that of the control group at day 35 (P<0.005).CONCLUSION The transfer of amphiregulin RNA antisense by adenoviral vector is effective for amphiregulin targeting strategy, leading to an inhibition of in vitro cell proliferation and in vivo tumor growth in this breast cancer model.     

Regression of colon cancer and induction of antitumor immunity by intratumoral injection of adenovirus expressing interleukin-12

Interleukin-12 (IL-12) has been shown to possess potent immunoregulatory and antitumoral effects. We have evaluated the anti-oncogenic potential and the mechanisms of the antitumoral effect of in vivo adenovirus-mediated transfer of IL-12 gene in a murine model of colon cancer. AdCMVIL-12 was constructed to permit coordinated production of p40 and p35 subunits of IL-12 gene to obtain the maximum IL-12 bioactivity. Infection of murine colon cancer CT-26 cells in vitro with AdCMVIL-12 resulted in the production of high levels of IL-12. In vivo gene therapy of colon cancer nodules by intratumoral injection of AdCMVIL-12 induced a local increase in IL-12 and interferon-gamma levels and a complete regression of the tumor in 26 of 34 (76%) mice. Tumor disappeared between days 7 and 10 after vector administration. The antitumoral effect was mediated by CD8+ T cells and was associated with the generation of cytotoxic T lymphocytes against colon cancer cells. Animals that eliminated the tumor were protected against a second administration of neoplastic cells. Treatment with AdCMVIL-12 of one tumor nodule also caused regression of established tumors at distant sites. These data demonstrate that AdCMVIL-12 is a useful therapeutic tool for established colon cancer in mice and should be considered for application in humans.