奥曲肽对胃癌化疗药物抗肿瘤特性的影响

目的 探讨奥曲肽(OCT)对三种常见胃癌化疗药物(5-Fu、MMC、CDDP)体外抗肿瘤特性的影响。方法 用MTT法分别测定5-Fu、MMC、CDDP单一使用以及按不同比例联合OCT使用时对胃癌细胞生长的抑制率, CalcuSyn(Biosoft)软件计算出量-效曲线和半数-效应曲线的相关参数:斜率(m)、截距(y-int)IC_(50)、联合指数(CI)和剂量减少指数(DRI),并以此判断奥曲肽对这三种化疗药物的体外抗肿瘤特性的影响。结果 奥曲肽在体外单独使用时能抑制肿瘤细胞的生长;而联合用药时,典曲肽与5-Fu及CDDP间呈协同作用,与MMC联合使用呈拮抗作用。5-Fu与OCT联合使用时协同作用250:1优于25:1及2.5:1;CDDP与OCT联合使用时25:1理想于0.25:1及2.5:1;而MMC与OCT间的拮抗作用0.125:1明显于1.25:1及12.5:1。结论 奥曲肽具有一定的抗肿瘤特性;可增强5-Fu、CDDP的化疗效果,但对MMC无增效作用;正确的联合用药才能充分发挥联合化疗的优势。     

胃癌细胞株化疗药物相互作用的实验研究

目的　利用胃癌细胞株探讨化疗药物的相互作用。方法　利用MTT法测定四种化疗药的单药和联合用药对胃癌细胞株的抑制率 ,将测得的数据输入电脑 ,用CalcuSyn(Biosoft)软件处理 ,推算出有关数据 :斜率 (m )、截距 ( y int)、IC50 、联合指数 (CI)和剂量减少指数 (DRI)。结果　资料显示 ,胃癌细胞株化疗敏感性顺序 :ADM >CDDP >MMC >5Fu ;联合用药的敏感性顺序 :MMC +CDDP >5Fu +MMC >ADM +MMC >5Fu +ADM >5Fu +CDDP >ADM +CDDP。联合用药协同作用的大小依次为 :5Fu +MMC >5Fu +CDDP >5Fu +ADM >MMC +CDDP >ADM +CDDP。根据两种药物联合用药DRI分析 ,以 5Fu +MMC为最佳 ;三种药物的联合用药的DRI也明显优于两种药物的联合用药。在三种药物联合使用中 ,5Fu、MMC和ADM中 2 0 0∶1∶1的配比要比 10 0∶1∶1更理想。结论　三种药物联合用药优于两种药物联合用药 ;在两种药物联合用药中 ,5Fu +MMC最佳。     

奥沙利铂与常用化疗药物对胃癌细胞株相互作用的研究

目的　利用胃癌细胞株探讨奥沙利铂与常用化疗药物的相互作用。方法　测定奥沙利铂与 4种常用化疗药的单药和联合用药对胃癌细胞株的抑制率 ,将测得的数据输入电脑 ,用CalcuSyn(Biosoft)软件处理 ,推算出有关数据 :斜率(m )、截距 ( y int)、IC50 、联合指数 (CI)和剂量减少指数 (DRI)。结果　胃癌细胞株单药化疗敏感性顺序 :OXA >MMC >5 Fu >FUDR >CDDP ;联合用药的敏感性顺序 :CDDP +OXA >FUDR +OXA >5 Fu +OXA >MMC +OXA。联合用药协同作用的大小依次为 :FUDR +OXA >5 Fu +OXA >MMC +OXA。根据这 4组两种药物联合用药的CI和DRI综合分析 ,以FUDR +OXA和 5 Fu +OXA组较佳 ;此外 ,三种药物联用的CI和DRI也十分理想。结论　OXA与常用化疗药物联合 ,对胃癌细胞株有良好的抑制率     

顺铂与热疗联合对胃癌细胞系体外增殖抑制作用的研究

目的研究顺铂与热疗联合对2株胃癌细胞系SGC-7 901和BGC-823细胞体外增殖的影响。方法使用MTT法测定细胞增殖,确定顺铂的工作浓度,并以该浓度进行化疗或与热疗联合,计算24 h和48 h时的细胞生存率,根据Veleriote法判断热化疗联合24 h或48 h的作用效果;24 h时流式细胞仪检测BGC-823细胞的凋亡情况。结果以24 h时IC40~IC50的药物浓度作为试验的工作浓度,确定CDDP对SGC-7 901和BGC-823细胞的工作浓度分别为5μg/mL和1μg/mL。单纯的43℃热疗60 min在24 h时对2个细胞系均有抑制作用(P<0.05),在48 h时没有显著抑制作用。CDDP 5μg/mL化疗或与热疗联合在24 h和48 h时对SGC-7 901细胞均有显著的抑制作用(P<0.01),CDDP 5μg/mL与热疗联合作用24 h时呈明显的协同作用,在48 h时呈次加作用。CDDP 1μg/mL化疗或与热疗联合在24 h和48 h时均对BGC-823细胞都有显著的抑制作用(P<0.01),CDDP 1μg/mL与热疗联合作用24 h和48 h均呈明显的协同作用;24 h时流式细胞仪检测BGC-823细胞的凋亡情况发现,CDDP化疗组和热化疗组细胞凋亡率均较对照组显著升高(P<0.01)。结论CDDP 5μg/mL与43℃热疗60 min联合在24 h时对SGC-7901细胞的增殖抑制具有明显的协同作用,在48 h时呈次加作用;CDDP 1μg/mL与43℃热疗60 min联合对BGC-823细胞的增殖抑制在24 h和48 h时均呈明显的协同作用,这可能与细胞凋亡的增多有关。     

以Ref-1为靶点的基因治疗在大肠癌5-FU化疗增敏中的作用及机制研究

目的探讨以Ref-1为靶点的基因治疗在大肠癌5-氟尿嘧啶(5-FU)化疗增敏中的作用及其可能机制。方法构建Ad5/F35-Ref-1小干扰RNA(siRNA)重组腺病毒(siRef-1)敲低Ref-1蛋白表达,MTT法检测不同剂量5-FU作用LOVO细胞存活率的改变;TUNEL法检测LOVO细胞凋亡;Western blot检测Ref-1蛋白表达;EMSA测定核转录因子κB(NF-κB)的DNA结合活性。结果感染siRef-1腺病毒较感染对照腺病毒的LOVO细胞对5-FU的敏感性明显增强,半数抑制浓度(IC50)分别为1.69、7.04μmol/L;且siRef-1显著增加5-FU诱导的细胞凋亡。5-FU呈剂量依赖性诱导LOVO细胞Ref-1蛋白表达,同时伴有NF-κB活性增强。siRef-1抑制5-FU诱导的LOVO细胞Ref-1蛋白表达以及NF-κB激活。结论以Ref-1为靶点的治疗显著增强大肠癌细胞5-FU化疗敏感性,其机制可能主要是通过抑制大肠癌细胞NF-κB活性和碱基切除修复功能。     

维拉帕米介导食管癌细胞多药耐药及细胞凋亡的实验研究

目的探讨维拉帕米提高食管癌化疗药物敏感性及促进细胞凋亡的影响。方法采用CCK-8法检测紫杉醇、顺铂、5-氟尿嘧啶对于4种食管癌细胞系(EC109、EC9706、KYSE-450、TE-1)的IC50值(IC501);用维拉帕米(4.91μg/m L)联合上述化疗药物处理每种细胞,CCK8法检测维拉帕米联合上述化疗药物针对上述4种食管癌细胞株(EC109,EC9706,KYSE-450和TE-1)IC50值(IC502);以前期实验得出的VER逆转化疗耐药能力差异最为显著的一对食管癌细胞为研究对象,Annexin V-PI双染法检测食管癌细胞凋亡。结果紫杉醇在KYSE-450和EC9706的IC50值明显大于EC109和TE-1,顺铂在TE-1细胞中IC50值明显小于其他3种细胞,5-氟尿嘧啶在EC109中IC50值明显高于在其他3种细胞。加用维拉帕米后,4种食管癌细胞系(EC109,EC9706,KYSE-450和TE-1)针对紫杉醇、顺铂、5-氟尿嘧啶的IC50值均不同程度下降,提示维拉帕米均不同程度提高了上述3种化疗药物的敏感性。其中,顺铂组未加入维拉帕米前,EC109,EC9706,KYSE-450与TE-1相比,均存在不同程度耐药性,加入维拉帕米后,针对EC9706 IC50值变化倍数较大(15.2倍),提示逆转敏感;针对EC109IC50值变化倍数较小(1.6倍),提示逆转耐受。结论维拉帕米逆转EC9706的阿霉素效率指数最高(15.2倍),与EC109细胞相比有显著性差异(1.6倍)。提示EC9706(DDP)对维拉帕米的逆转敏感,而EC109(DDP)对维拉帕米的逆转耐受。维拉帕米有可能通过促进细胞调亡途径提高其逆转化疗耐受的能力,从而增强化疗药物敏感性。     

TRAIL联合化疗药物对人宫颈癌Hela细胞增殖与凋亡的影响

目的探讨肿瘤坏死因子相关诱导凋亡配体 (TRAIL)对人宫颈癌Hela细胞的作用 ,以及与化疗药物的协同作用。方法将Hela细胞接种至 96孔培养板后分别加入浓度为l.0、10 .0、10 0 .0 μl/L的TRAIL、0 .1、1.0、10 .0mg/L的阿霉素 (ADM )和丝裂霉素 (MMC) ,及不同匹配的TRAIL MMC和TRAIL ADM ,采用四甲基偶氮唑盐 (MTT)比色法分别检测肿瘤细胞的生存率 ;将Hela细胞接种至 12孔板 ,培育 2 4h后加入不同浓度的TRAIL、ADM、MMC、TRAIL ADM、TRAL MMC ,用流式细胞术检测不同处理组肿瘤细胞的凋亡率和死亡率。结果 10 0 μg/LTRAIL引起细胞的凋亡率为 2 0 .1% ,与无药物组 1.1%的凋亡率有非常显著性差异 (P <0 .0 1) ;单独采用 10mg/LMMC、ADM对细胞的抑制率为 3 6.0 %和 44 .1% ,而 10 0 μg/LTRAIL与 10mg/LMMC、ADM联合后对细胞的抑制率分别达到 5 8.4%和 73 .7% ,两者有协同作用 (P <0 .0 5 )。结论在体外实验中 ,TRAIL可通过诱导宫颈癌Hela细胞的凋亡而产生抗肿瘤作用 ;TRAIL与化疗药物ADM、MMC有协同抗肿瘤作用。     

恶性肿瘤的放-化联合治疗进展

放 -化联合治疗已成为临床常用方式 ,其优点已在一些肿瘤的治疗中得到证明 ,这些肿瘤包括小细胞肺癌、非小细胞肺癌、食管癌、宫颈癌、胰腺癌等。放 -化联合应用的优点主要是 :①具有时空协同作用 ,放疗治疗原发肿瘤及其卫星病灶 ,而化疗则针对治疗时可能存在的隐匿微小病灶 ;②抗肿瘤效果相加作用 ,临床已证明两种方式联合应用 ,如二者的毒性不同 ,那么 ,联合效果比单一的要好 ;③两种治疗方式的互为增强 (超相加 )作用 ,其机制仍有争议。1　临床应用方式和药物选择1.1　序贯疗法　一般采用先化疗后放疗方式 ,化疗1～ 2个周期 ,放疗仍为根…     

β1整合素表达下调对人结肠癌细胞增殖和化疗敏感性影响

目的 观察β1整合素表达下调对人结肠癌HT-29细胞增殖和化疗敏感性的影响.方法 采用反义寡核苷酸（ASODN）技术转染HT-29细胞,逆转录-聚合酶链反应（RT-PCR）检测β1整合素表达下调情况.MTT法检测HT-29细胞相对存活率.给予梯度浓度氟尿嘧啶（5-FU）,计算IC50.结果 实验组β1整合素条带吸光度积分（IAD）值/β-actin条带IAD值的比值及HT-29细胞的存活率与对照组相比显著降低（F=1 630.08,q=72.40,P〈0.01;t=4.37,P〈0.05）.5-FU＋ASODN组HT-29细胞对5-FU的IC50与5-FU组比较显著下降（t′=37.71,P〈0.05）.结论 β1整合素表达下调可抑制结肠癌细胞增殖,增强结肠癌细胞对化疗药物敏感性.     

不同抗癌药物配伍作动脉灌注和栓塞治疗肝癌的疗效比较观察

抗癌药物联用增加药物杀伤癌细胞的协同作用,减少和延缓耐药性的优越性已为人所知晓。但抗癌药物的组配法可不相同。本文用阿霉素(ADM)加丝裂霉素(MMC)与 ADM 加MMC 和顺氯胺铂(CDDP)两种组配法,对34例肝癌作一次肝动脉灌注化疗和栓塞治疗的疗效比较观察。现报道如下。     

Low-dose all-trans retinoic acid enhances cytotoxicity of cisplatin and 5-fluorouracil on CD44+ cancer stem cells

Cis-diamminedichloridoplatinum(II)(CDDP)-based combination chemotherapy is frequently used in gastrointestinal cancer. The synergistic mechanism of all-trans retinoic acid (ATRA), cisplatin (CDDP) and 5-fluorouracil (5-FU) in combination remains unclear. Despite their potent antitumor properties, resistance to CDDP and 5-FU develops frequently in tumors. To clarify this mechanism, we determined the sensitivity to each drug and their combination in two gastrointestinal cancer stem cells (CSCs) subpopulation.Here, we report the identification and separation of CD44⁺ cells from human gastric carcinoma (AGS) and human esophageal squamous cell carcinoma (KYSE-30) cancer cell lines by magnetic activated cell sorting (MACS). We allowed the CD44^± cells to grow 6 days at a subtoxic concentration of ATRA and then treated with different concentration of CDDP and 5-FU for 24 h. The cytotoxicity was examined by cell proliferation MTT assay. Additionally, AO/EB staining was used for detection of apoptotic cells. In order to determine whether the growth inhibition was also associated with changes in cell cycle distribution, cell cycle analysis was performed using flow cytometry.Low concentration of ATRA (1 μM, 6days) followed by 5-FU and CDDP was found to be more effective than either drugs alone, thus resulting in synergistic cytotoxicity in Kyse-30 and AGSCD44^± cells. Furthermore, there was an indication that the combination of ATRA with 5FU and CDDP caused an increase in cell cycle arrest in G2/M and G0/G1.We conclude that low concentration of ATRA enhances the cytotoxicity of CDDP and 5FU by facilitating apoptosis and cell cycle arrest in gastrointestinal CSCs and provide a rational basis for the design of novel, well-tolerated CDDP- and 5FU-based chemotherapy in human gastrointestinal carcinoma.     

Efficacy of oncolytic herpesvirus NV1020 can be enhanced by combination with chemotherapeutics in colon carcinoma cells

NV1020, an oncolytic herpes simplex virus type 1, can destroy colon cancer cells by selectively replicating within these cells, while sparing normal cells. NV1020 is currently under investigation in a clinical phase I/II trial as an agent for the treatment of colon cancer liver metastases, in combination with conventional chemotherapeutic agents such as 5-fluorouracil (5-FU), SN38 (the active metabolite of irinotecan), and oxaliplatin. To study the synergy of NV1020 and chemotherapy, cytotoxicity and viral replication were evaluated in vitro by treating various human and murine colon carcinoma cell lines, using a colorimetric viability assay, a clonogenic assay, and a plaque-forming assay. In vivo experiments, using a subcutaneous syngeneic CT-26 tumor model in BALB/c mice, were performed to determine the efficacy of combination therapy. In vitro studies showed that the efficacy of NV1020 on human colon carcinoma cell lines HT-29, WiDr, and HCT-116 was additively or synergistically enhanced in combination with 5-FU, SN38, or oxaliplatin. The sequence of application was not important and effects were still apparent after a 21-day incubation period. Three intra-tumoral treatments with NV1020 (1 x 10(7) plaque-forming units), followed by three subcutaneous treatments with 5-FU (50 mg/kg), resulted in substantially higher inhibition of tumor growth and prolongation of survival compared with monotherapies (NV1020/5-FU vs. NV1020, p = 0.027). On WiDr cells, reduced replication of NV1020, in combination with 5-FU, indicated that additive and synergistic effects of combination therapy must be independent from viral replication. These results suggest that NV1020, in combination with chemotherapy, is a promising therapy for treating patients with metastatic colorectal cancer of the liver. We hypothesize that infection of cells with NV1020 sensitizes the infected cells for the cytotoxic effect of the chemotherapeutics.     

Cancer targeting Gene-Viro-Therapy of liver carcinoma by dual-regulated oncolytic adenovirus armed with TRAIL gene

Liver cancer is a common and aggressive malignancy, but available treatment approaches remain suboptimal. Cancer targeting Gene-Viro-Therapy (CTGVT) has shown excellent anti-tumor effects in a preclinical study. CTGVT takes advantage of both gene therapy and virotherapy by incorporating an anti-tumor gene into an oncolytic virus vector. Potent anti-tumor activity is achieved by virus replication and exogenous expression of the anti-tumor gene. A dual-regulated oncolytic adenoviral vector designated Ad·AFP·E1A·E1B (Δ55) (Ad·AFP·D55 for short thereafter) was constructed by replacing the native viral E1A promoter with the simian virus 40 enhancer/α-fetoprotein (AFP) composite promoter (AFPep) based on an E1B-55K-deleted construct, ZD55. Ad·AFP·D55 showed specific replication and cytotoxicity in AFP-positive hepatoma cells. It also showed enhanced safety in normal cells when compared with the mono-regulated vector ZD55. To improve the anti-hepatoma activities of the virus, the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene was introduced into Ad·AFP·D55. Ad·AFP·D55-TRAIL exhibited remarkable anti-tumor activities in vitro and in vivo. Treatment with Ad·AFP·D55-TRAIL can induce both autophagy owing to the Ad·AFP·D55 vector and caspase-dependent apoptosis owing to the TRAIL protein. Therefore, Ad·AFP·D55-TRAIL could be a potential anti-hepatoma agent with anti-tumor activities due to AFP-specific replication and TRAIL-induced apoptosis.     

Interleukin-28B acts synergistically with cisplatin to suppress the growth of head and neck squamous cell carcinoma

Interleukin-28B (IL-28B), also referred to as interferon-λ3, belongs to the type III interferon family. Earlier studies showed that IL-28B suppresses proliferation of some tumor cells in vitro. IL-28B gene transfection ex vivo also resulted in growth retardation of tumor cells in mice, through either direct antiproliferative action or induction of antitumor immunity. However, it has not been reported whether in vivo therapeutic administration of recombinant IL-28B can inhibit the growth of a pre-established tumor. Here, we found that repetitive subcutaneous administration of recombinant mouse IL-28B significantly induced tumor-specific cytotoxic T lymphocytes and augmented natural killer cytolytic activity, leading to moderate suppression of the growth of a murine head and neck squamous cell carcinoma (HNSCC) cell line that was completely resistant to the direct antiproliferative effect of IL-28B. Moreover, co-administration of recombinant mouse IL-28B and cisplatin (CDDP) more significantly inhibited in vivo growth of the tumor that had been established in syngenic mice and induced tumor-specific cytotoxic T lymphocytes. The CDDP treatment induced the expression of major histocompatibility complex class I and Fas molecules on the surface of HNSCC cells both in vitro and in vivo; this may be the mechanism underlying the synergistic tumor suppression activity of IL-28B and CDDP. Unlike type I interferon, IL-28B did not suppress growth of bone marrow cells in culture. Therefore, IL-28B may be useful as a tool for a novel multidisciplinary therapy against cancer, significantly potentiating innate and adaptive antitumor immune responses, especially when co-administrated with CDDP, which is currently the first choice chemotherapeutic agent against various tumors including HNSCCs.     

Transcriptional control of viral gene therapy by cisplatin

Ionizing radiation (IR) and radical oxygen intermediates (ROIs) activate the early growth response-1 (Egr1) promoter through specific cis-acting sequences termed CArG elements. Ad.Egr.TNF.11D, a replication-deficient adenoviral vector containing CArG elements cloned upstream of the cDNA for human recombinant TNF-alpha was used to treat human esophageal adenocarcinoma and rat colon adenocarcinoma cells in culture and as xenografts in athymic nude mice. Cisplatin, a commonly used chemotherapeutic agent, causes tumor cell death by producing DNA damage and generating ROIs. The present studies demonstrate induction of TNF-alpha production in tumor cells and xenografts treated with the combination of Ad.Egr.TNF.11D and cisplatin. The results show that the Egr1 promoter is induced by cisplatin and that this induction is mediated in part through the CArG elements. These studies also demonstrate an enhanced antitumor response without an increase in toxicity following treatment with Ad.Egr.TNF.11D and cisplatin, compared with either agent alone. Chemo-inducible cancer gene therapy thus provides a means to control transgene expression while enhancing the effectiveness of commonly used chemotherapeutic agents.     

Potent antitumor activity of oncolytic adenovirus expressing mda-7/IL-24 for colorectal cancer

It has been demonstrated that interleukin 24 (IL-24, also called melanoma differentiation associated gene 7) exerts antitumor activity. In this study, we investigated whether oncolytic adenovirus-mediated gene transfer of IL-24 could induce strong antitumor activity. A tumor-selective replicating adenovirus expressing IL-24 (ZD55-IL-24) was constructed by insertion of an IL-24 expression cassette into the ZD55 vector, which is based on deletion of the adenoviral E1B 55-kDa gene. ZD55-IL-24 could express substantially more IL-24 than Ad-IL-24 because of replication of the vector. It has been shown that ZD55-IL-24 exerted a strong cytopathic effect and significant apoptosis in tumor cells with p53 dysfunction. Moreover, no cytotoxic and apoptotic effects could be seen in normal cells infected with ZD55-IL-24. Expression of IL-24 did not interfere with viral replication induced by oncolytic adenovirus. Activation of caspase 3 and caspase 9, and induction of bax gene expression, were involved in tumor cell apoptosis induced by ZD55-IL-24. Treatment of established tumors with ZD55-IL-24 showed much stronger antitumor activity than that induced by ONYX-015 or Ad-IL- 24. These data indicated that oncolytic adenovirus expressing IL-24 could exert potential antitumor activity and offer a novel approach to cancer therapy.     

Oncolytic adenovirus co-expressing IL-12 and IL-18 improves tumor-specific immunity via differentiation of T cells expressing IL-12Rβ2 or IL-18Rα

The oncolytic adenovirus (Ad) is currently being advanced as a promising antitumor remedy as it selectively replicates in tumor cells and can transfer and amplify therapeutic genes. Interleukin (IL)-12 induces a potent antitumor effect by promoting natural killer (NK) cell and cytotoxic T cell activities. IL-18 also augments cytotoxicity of NK cells and proliferation of T cells. This effect further enhances the function of IL-12 in a synergistic manner. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral administration of oncolytic Ad co-expressing IL-12 and IL-18, RdB/IL-12/IL-18. Intratumoral administration of RdB/IL-12/IL-18 improved antitumor effects, as well as increased survival, in B16-F10 murine melanoma model. The ratio of T-helper type 1/2 cytokine as well as the levels of IL-12, IL-18, interferon-γ and granulocyte-macrophage colony-stimulating factor was markedly elevated in RdB/IL-12/IL-18-treated tumors. Mice injected with RdB/IL-12/IL-18 also showed enhanced cytotoxicity of tumor-specific immune cells. Consistent with these results, immense necrosis and infiltration of NK cells, as well as CD4+ and CD8+ T cells, were observed in RdB/IL-12/IL-18-treated tumor tissues. Importantly, tumors treated with RdB/IL-12/IL-18 showed an elevated number of T cells expressing IL-12Rβ2 or IL-18Rα. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-18 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.     

In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic adenovirus

Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.     

Effect of 5-fluorouracil, Optison and ultrasound on MCF-7 cell viability

The aim of this study was to analyze cell viability and expression of apoptotic-related signaling proteins in MCF-7 breast cancer cells induced by combinations of ultrasound, the anticancer drug 5-fluorouracil (5-FU) and the ultrasound contrast agent Optison. MCF-7 cells were treated with 5-FU and sonicated at the frequency of 3.0 MHz and intensity of 3.0 W/cm2 for 1 min in the presence of Optison. The cells were analyzed for lactate dehydrogenase (LDH) release (a measure of cytotoxicity) and cell proliferation (by MTT assays). The LDH/MTT ratio was used for assessment of cell death. Expression of the apoptotic-related proteins, Bax and p27kip1, as well as phosphorylated forms of ERK and Akt proteins was assessed by Western blot analysis. We demonstrate that, immediately after treatment, cell death was most dependent on Optison; however, 24 h after treatment, cell death was more dependent on 5-FU. Ultrasound duty cycle increased cell death associated with either Optison or 5-FU. Furthermore, we show that treatment with 5-FU and ultrasound increased the levels of the Bax and p27kip1 proteins, but the addition of Optison appears to suppress apoptotic protein expression.