Induction of resistance-proteins and oncoproteins in murine and human tumor-cell lines after irradiation

The aim of this investigation was to analyze whether or not ionizing radiation can induce the expression of resistance- and oncoproteins in murine sarcoma 180 cells and several human carcinoma cell lines. For assessment of the proteins the streptavidin-biotin peroxidase-complex method was performed using specific antibodies. Expression of glutathione S-transferase-pi and thymidylate-synthase was increased after a single dose of irradiation which was accompanied by a lower sensitivity against cisplatin and 5-fluorouracil. No differences in protein expression could be detected for P-glycoprotein 170 and topoisomerase II. Differences in the expression of the oncoproteins c-Fos and c-ErbB1 were also found after irradiation. In contrast, a decreased expression of topoisomerase II was found after fractionated irradiation. This was confirmed by mRNA analysis.     

天然花色素类诱导肿瘤细胞凋亡机制研究进展

天然花色素类可以诱导多种肿瘤细胞凋亡。其作用机制是通过JNK信号传导通路、活化CASPases、线粒体途径、产生活性氧及阻止细胞增殖周期等多种途径实现的。现将天然花色素类诱导肿瘤细胞凋亡的主要作用机制作一综述。     

天然花色素类诱导肿瘤细胞凋亡机制研究进展

天然花色素类可以诱导多种肿瘤细胞凋亡。其作用机制是通过JNK信号传导通路、活化CASPases、线粒体途径、产生活性氧及阻止细胞增殖周期等多种途径实现的。现将天然花色素类诱导肿瘤细胞凋亡的主要作用机制作一综述。     

Free-radicals dependent and independent pathways of cddp-mediated cytotoxicity and apoptosis in ovarian tumor-cell lines

Cis-platinum (CDDP) is a chemotherapeutic drug widely used alone or in combination with other drugs in the treatment of cancer. However, many tumors become resistant to CDDP and the mechanisms of resistance are complex. The present study investigated the role of free radicals in CDDP-mediated cytotoxicity and apoptosis. Four human ovarian cancer cell lines were chosen for the study: two lines, 222 and A2780, are sensitive to CDDP and two lines, AD10 and C30 are resistant to CDDP. The importance of free radical formation was tested by the use of an inhibitor, 2,6 di-tert-butyl-4-methoxy phenol (BHA), that inhibits the production of free radicals and detoxifies free radicals. Cytotoxicity was assessed by the MTT assay and apoptosis assessed by flow cytometric analysis of DNA hypoploidy. Three of the 4 cell lines, 222, AD10, and C30, were completely inhibited by BHA in CDDP-mediated cytotoxicity. The CDDP-sensitive A2780 cell line, however, was not inhibited by BHA, even at high non-toxic concentrations of BHA. The DNA analysis for apoptosis paralleled the findings obtained in the cytotoxicity assay. In order to rule out that the A2780 cell line was not reactive to BHA, VP-16 mediated cytotoxicity was examined. All four cell lines were sensitive to VP-16 and all four were inhibited by BHA. In contrast, there was no detectable inhibition by BHA of actinomycin-D-mediated cytotoxicity in all four lines tested. Overall, the findings demonstrate that either free-radical dependent (CDDP, VP-16) or free radical-independent (CDDP, actinomycin-D) pathways of cytotoxicity and apoptosis are utilized by different drugs. Further, a single agent like CDDP can mediate killing by both a free radical-dependent and by a free-radical independent pathway. Therefore, new agents that are developed to reverse resistance by a particular drug must take into consideration alternative cytotoxic pathways mediated by the same drug.     

Induction of tumor-cell lysis by bi-specific antibody recognizing ganglioside GD2 and T-cell antigen CD3

Human tumor cells expressing ganglioside GD2 were lysed by various effector populations targeted with an anti-CD3-anti-GD2 bi-specific antibody (BAb CD3 x GD2). This antibodyheteroconjugate was prepared by chemically cross-linking the OKT-3 monoclonal antibody (MAb) reactive with CD3 antigen on T lymphocytes with the ganglioside MAb ME 361, which binds preferentially to the tumor-associated ganglioside GD2. The specificity of target-cell lysis by the cytotoxic T cells (CTL) was mediated by the specificity of the targeting antibody: GD2-negative cells were not lysed in the presence of the CD3 x GD2 BAb. A dose-dependent response was observed in a range of 10 to 10,000 ng/ml. In contrast, 2 other BAbs recognizing the tumor-associated antigens EGF-R and TKB-2 had greater potency to mediate tumor-cell lysis than the GD2 x CD3 BAb. Peripheral-blood cells (PBL) stimulated with OKT-3 MAb or with irradiated tumor cells in a mixed lymphocyte culture (MLTC) could be induced to lyse GD2-positive tumor cells in the presence of CD3 x GD2 BAb. The tumor-cell lysis could be mediated by autologous or allogeneic effector cells. NK cells had no influence on the BAb-induced cytotoxicity.     

Induction bleomycin infusion in head and neck cancer

Twenty-one male patients with previously untreated advanced squamous cell carcinoma of the head and neck were treated with an induction regimen of bleomycin 15 mg/m2 I.V. bolus followed by a continuous 24-hour I.V. infusion at a dose of 15 mg/m2/day for 7 days. One week following induction therapy, patients were reevaluated for response and then received definitive therapy with surgery and/or radiation therapy. The chemotherapy yielded a major response rate of 33% (one CR, six PR). Toxic manifestations of this regimen were mild, consisting of fever, alopecia, rash, and mucositis. There was no pulmonary toxicity detected. The response rate obtained with bleomycin infusion is inferior to the combination of cis-platinum with a bleomycin infusion as induction therapy in previously untreated patients with squamous cell carcinoma of the head and neck.     

MicroRNA-138 suppresses invasion and promotes apoptosis in head and neck squamous cell carcinoma cell lines

Metastasis is a critical event in the progression of head and neck squamous cell carcinoma (HNSCC). To identify microRNAs associated with HNSCC metastasis, six paired HNSCC cell lines with different metastatic potential were examined. Using microarrays, a panel of differentially expressed microRNAs was identified, including reduction of miR-138 in highly metastatic cells. Ectopic transfection of miR-138 suppressed cell invasion and led to cell cycle arrest and apoptosis. Knockdown of miR-138 enhanced cell invasion and suppressed apoptosis. Thus, our results suggested miR-138 acts as a tumor suppresser and may serve as a therapeutic target for HNSCC patients at risk of metastasis.     

Induction chemotherapy in patients with head and neck cancer

Neoadjuvant chemotherapies for patients with advanced head and neck squamous cell carcinoma have been widely studied for twenty years. Despite a high level of activity on the primary tumor, no study has demonstrated a survival benefit suggesting the use of neoadjvant chemotherapy. One can consider that the only benefit of such strategy is for larynx preservation in patients with operable hypopharnx or larynx cancer. Nevertheless, recently the well established preservation strategy based on induction chemotherapy following according to the activity by radiotherapy has been knocked over by a strategy developed by Forastiere et al. using primary concomitant chemoradiotherapy. However, the lack of benefit reported by neoadjuvant chemotherapy has been thwarted by the recent results provided by the EORTC study which assessed the survival benefit of neoadjuvant chemotherapy by docetaxel-cisplatin-fluorouracile. Interestingly, since 2002 the clearly established strategies for patients with advanced head and neck cancer have been challenged and new options are emerging. This paper reviews the standard strategy of the past and the future proposal emerging from recent studies.     

Induction of apoptosis in hepatocellular carcinoma cell lines by emodin.

Previous experiments have shown that emodin is highly active in suppressing the proliferation of several tumor cell lines. However, it is not clear that emodin can induce growth inhibition of hepatoma cells. We have found that emodin induces apoptotic responses in the human hepatocellular carcinoma cell lines (HCC) Mahlavu, PLC/PRF/5 and HepG2. The addition of emodin to these three cell lines led to inhibition of growth in a time- and dose-dependent manner. Emodin generated reactive oxygen species (ROS) in these cells which brought about a reduction of the intracellular mitochondrial transmembrane potential (DeltaPsim), followed by the activation of caspase-9 and caspase-3, leading to DNA fragmentation and apoptosis. Our findings demonstrate that ROS and the resulting oxidative stress play a pivotal role in apoptosis. Preincubation of hepatoma cell lines with the hydrogen peroxide-scavenging enzyme, catalase (CAT) and cyclosporin A (CsA), partially inhibited apoptosis. These results demonstrate that enhancement of generation of ROS, DeltaPsim disruption and caspase activation may be involved in the apoptotic pathway induced by emodin.     

Oxaliplatin activity in head and neck cancer cell lines

Oxaliplatin (cis-[(1R,2R)-1,2-cyclohexanediamine-N,N] [oxalato(2-)-O,O] platinum; Eloxatin) is a third-generation platinum compound with a 1,2-diaminocyclohexane (DACH) carrier ligand, which has a wide spectrum of anticancer activity in vitro systems and has displayed preclinical and clinical activity in a wide variety of tumors. To investigate its in vitro activity against head and neck cancer, we exposed two head and neck cancer cell lines to the compound, created a variant resistant to cisplatin to study cross-resistance to the compound and analyzed the potential radiosensitizing effect of the drug. We report here that oxaliplatin was cytotoxic at similar doses to cisplatin in these cells. There was no cross-resistance to cisplatin, as demonstrated by different IC₅₀ values in these cell lines and the sensitivity to oxaliplatin of the cisplatin-resistant cell line. There was an effective radiosensitizer effect of the compound in either cell line. Additional in vitro and in vivo experimentation is warranted in order to support the use of oxaliplatin as a radiosensitizer in head and neck cancer patients.Magali Espinosa and Moises Martinez contributed equally to this paper.     

Induction chemotherapy for head and neck epidermoid carcinomas

The standard treatment for head and neck inoperable squamous cell carcinoma is an association of radiotherapy and platinum. However, only one patient out of three remains alive five years after diagnosis. The interest in induction chemotherapy was renewed by the introduction of taxanes combined with cisplatinum and 5-fluoro-uracile. The triple association taxane-cisplatinum-5-fluoro-uracile yielded improved survival when compared to cisplatinum-5-fluoro-uracile. Wider use of taxane-cisplatinum-5-fluoro-uracile is limited by its toxicity and the lack of randomized comparison with a concomitant chemoradiotherapy scheme including optimal doses of platinum. Until the results of new phase III trials are published, the choice between induction chemotherapy followed by concomitant chemoradiotherapy or concomitant chemoradiotherapy alone has to be made on an individualized basis, taking into account the patient's medical condition, the ability of the medical team to deal with intensive treatment regimens, and the clinical/pathological characteristics of the tumour.     

多糖诱导肿瘤细胞凋亡机制的研究进展

多糖是普遍存在于动物、植物和微生物中的大分子物质,在细胞识别、信号传导、调节肌体免疫及细胞的迁移、增殖、分化和代谢等方面均有十分重要的作用。近年来众多研究结果表明,多糖可抗肿瘤活性,抑制多种肿瘤细胞生长。究其机制,多糖除可诱导免疫因子生成,增强机体免疫功能外,还能直接杀死肿瘤细胞。通过阐述动物、植物和微生物多糖可以诱导多种肿瘤细胞凋亡,说明多糖具有广谱抑制肿瘤细胞生长的作用;初步探讨了多糖诱导肿瘤细胞凋亡的机制,例如激活死亡受体途径和线粒体途径,调控凋亡基因,调节端粒酶和拓扑酶等。     

Induction of apoptosis by morphine in human tumor cell lines in vitro

Most previous studies of the induction of tumor cell apoptosis by morphine have been conducted with concentrations very much higher than those used clinically. An investigation of the ability of morphine to induce apoptosis at its clinical concentration (10(-8) M) was therefore undertaken. Cytotoxicity was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, induction of early apoptosis and necrosis by fluorescence-activated cell sorter (FACS) analysis with Annexin V and propidium iodide (PI), activation of caspase -2, -3, -8 and -9 by cleavage of specific substrates, DNA fragmentation by agarose gel electrophoresis, radical intensity and O2- scavenging activity by ESR spectroscopy. Millimolar concentrations of morphine showed higher cytotoxicity against human tumor cell lines (HL-60, A549, MCF7) than against normal human cells (HGF, HPC, HPLF). The clinical concentration of morphine produced early apoptotic markers in HL-60 and A549 cells whereas it induced higher numbers of necrotic cells in MCF7 cells, both in a naloxone-sensitive manner. The clinical concentration of morphine failed to activate any caspase species and induced only trace amounts of internucleosomal DNA fragmentation, in contrast to cytotoxic concentrations of morphine. Morphine, with a C-3 hydroxyl group, showed higher cytotoxicity and O2- scavenging activity than codeine, in which the hydroxyl group at C-3 was replaced with a methoxy group, suggesting the involvement of a radical-mediated reaction. The present report may offer new strategies for treatment and prevention of cancer using a clinical concentration of morphine not only as an anti-nociceptive, but also as an apoptosis or necrosis inducer.     

E1A-mediated suppression of EGFR expression and induction of apoptosis in head and neck squamous carcinoma cell lines

Previous studies have shown early region 1A (E1A) gene to inhibit the proliferation of tumour cells with wild-type, but not mutant, p53. E1A has also been shown to downregulate c-erb-B-2/neu expression, resulting in inhibition of growth in c-erb-B-2/neu overexpressing tumour cells. In this study, we have investigated the effect of E1A expression on four head and neck squamous cell carcinoma (HNSCC) cell lines that do not overexpress c-erb-B-2/neu. Cell cycle and Western blot analysis show E1A-mediated induction of apoptosis in all cell lines examined. This induction of apoptosis was independent of the p53 status as it occurred in the cell lines with wild-type, mutated or deleted p53. However, there was no evidence of E1A-induced apoptosis in a p53(+ve) normal human fibroblast cell line, 1BR3. Analysis of apoptosis in the SCC cell lines demonstrated E1A-mediated downregulation of EGFR, which was overexpressed in each of these cell lines. Overexpression of an exogenously introduced EGFR, under the control of an E1A-insensitive heterologous promoter, blocked E1A induction of apoptosis in these cells. Therefore, E1A-mediated downregulation of EGFR expression appears to be the cause, rather than a consequence of E1A-induced apoptosis in these SCC cell lines. Previous studies have shown downregulation of EGFR expression by PML. Interestingly, E1A expression in the HNSCC cells altered the pattern of PML distribution and induced the level of PML protein, thus suggesting that E1A-mediated downregulation of EGFR may occur via direct or indirect interactions with PML. These findings demonstrate a novel pathway by which E1A can induce apoptosis and identify EGFR as a potential target for the development of therapeutic strategies against epithelial malignancies, the majority of which have abnormal EGFR expression.     

Induction of plasminogen activator inhibitor type-1 (PAI-1) by hypoxia and irradiation in human head and neck carcinoma cell lines

Background  

Squamous cell carcinoma of the head and neck (SCCHN) often contain highly radioresistant hypoxic regions, nonetheless, radiotherapy is a common treatment modality for these tumours. Reoxygenation during fractionated radiotherapy is desired to render these hypoxic tumour regions more radiosensitive. Hypoxia additionally leads to up-regulation of PAI-1, a protein involved in tumour progression and an established prognostic marker for poor outcome. However, the impact of reoxygenation and radiation on PAI-1 levels is not yet clear. Therefore, we investigated the kinetics of PAI-1 expression and secretion after hypoxia and reoxygenation, and determined the influence of ionizing radiation on PAI-1 levels in the two human SCCHN cell lines, BHY and FaDu.     

Induction of apoptosis in human head and neck cancer cell lines by an active component of OK-432 through p53-independent pathway via toll-like receptor (TLR) 4 signaling

OK-PSA, an active component of OK-432, induces anti-tumor immunity via Toll-like receptor (TLR) 4/MD-2 complex. In the current study, we evaluated the effect of the OK-PSA on human head and neck cancer cell lines. Twelve cancer cell lines including 7 squamous cell carcinoma (SCC) cell lines and 5 salivary gland cancer (SGC) cell lines were examined. The quantitative real-time PCR analysis revealed that TLR4 mRNA was expressed in all 12 cell lines, and that MD-2 mRNA was expressed in 5 cell lines. OK-PSA stimulation resulted in the activation of NF-kappaB in the 4 SCC cell lines which express both TLR4 and MD-2 genes, and in 5 SGC cell lines which express at least TLR4 gene independently of MD-2 expression. In these OK-PSA-responsive cell lines, OK-PSA activated caspase-1, caspase-3 and caspase-8, and induced apoptosis. OK-PSA-induced apoptosis were observed even in a SGC cell line in which p53 is mutated and its function is impaired. These findings strongly suggest that OK-PSA induces apoptosis by the activation of caspases through p53-independent pathway via TLR4 signaling in head and neck cancer cells.     

Incidence of serum antibody reactivity to autologous head and neck cancer cell lines and augmentation of antibody reactivity following acid dissociation and ultrafiltration

Serum antibody reactivity to squamous cell carcinoma of the head and neck (SCCHN) was evaluated in 41 autologous serum-tumor cell line combinations using the protein A hemadsorption assay. Autologous antibody reactivity (median titer of 1:4) was detected in sera from 24 of the patients tested. In 10 cases autologous antibody reactivity could be detected only in undiluted serum precluding further analysis. Analysis of higher titer sera from one patient revealed antibodies that define an antigen expressed on autologous tumor cells cultured from both the primary tumor (UM-SCC-17A) and from a metastasis (UM-SCC-17B). Absorption analysis showed that this antigen was also expressed on 6 of 10 allogeneic SCCHN cell lines but not on autologous fibroblasts or on allogeneic melanoma cell lines. Due to the low titer of autologous antibody reactivity in most sera, we sought to determine if dissociation of immune complexes through acidification and ultrafiltration of serum might enhance detectable antibody reactivity as has been done in previous studies in melanoma. Twelve serum samples from eight patients were subjected to acid dissociation and ultrafiltration (AD-U). Only six of the untreated sera had detectable antibody reactivity against the autologous SCCHN cell line whereas following AD-U all 12 sera had enhanced IgG reactivity against autologous SCCHN. Specificity analysis of one serum sample after dissociation revealed that the antibody detected an antigen common to SCCHN cell lines as well as melanoma, glioma, renal, and colon carcinoma cell lines. Circulating immune complexes may provide a reservoir of antibody with potential diagnostic and therapeutic applications.