An Anti-GD2 Monoclonal Antibody Enhances Apoptotic Effects of Anti-cancer Drugs against Small Cell Lung Cancer Cells via JNK (c-Jun Terminal Kinase) Activation

Small cell lung cancer (SCLC) cell lines specifically express ganglioside GD2, and anti-GD2 monoclonal antibodies (mAbs) caused suppression of cell growth and induced apoptosis of SCLC cells with single use. Here, enhancement of the cytotoxic effects of various anti-cancer drugs with an anti-GD2 mAb was demonstrated. The cytotoxicity of all six drugs examined was markedly enhanced, i.e. 2.4–7.8–fold increase of cell sensitivity in terms of IC₅₀. In particular, the combination of cisplatin (CDDP) with an anti-GD2 mAb resulted in prominent enhancement of cytotoxicity even in low-moderate GD2–expressing lines. The anti-GD2 mAb induced weak activation of c-Jun terminal kinase (JNK) in SCLC cells, and all anti-cancer drugs also induced its activation to various degrees. When CDDP and an anti-GD2 mAb were used together, significantly stronger JNK activation was observed corresponding to the cytotoxic effects, suggesting that synergistic phosphorylation of JNK with two reagents induced prominent apoptosis. The essential role of JNK in the induction of SCLC apoptosis with CDDP and anti-GD2 mAb was confirmed by experiments with a JNK inhibitor, curcumin. These results suggest that anti-GD2 mAbs would be very efficient in combination with anti-cancer drugs, both to achieve SCLC-specific cytotoxicity and to enhance its magnitude.     

Non-small and small cell lung carcinoma cell lines exhibit cell type-specific sensitivity to edelfosine-induced cell death and different cell line-specific responses to edelfosine treatment

The unique signal transduction pathways that distinguish non-small cell lung carcinoma (NSCLC) from small cell lung carcinoma (SCLC) are poorly understood. We investigated the ability of edelfosine, an inhibitor of phosphatidylinositol-specific phospholipase C (PLC) to inhibit cell viability among four NSCLC cell lines and four SCLC cell lines. The differential sensitivity of cells to edelfosine's cytostatic and cytotoxic effects has been attributed to edelfosine-induced changes in the activities of many enzymes, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinases (ERK), p38 kinase, and poly(ADP-ribose) polymerase (PARP). To investigate the role of these enzymes in edelfosine-induced cytotoxicity, we correlated edelfosine-induced changes in enzyme activity and cell viability among the different NSCLC and SCLC cell lines. We found that NSCLC cells are much more susceptible to the cytotoxic effects of this drug than are SCLC cells. Three out of the four edelfosine-sensitive NSCLC cell lines (NCI-H157, NCI-H520, NCI-H522) exhibit G2/M arrest, significant apoptosis and some degree of JNK activation in response to drug treatment. In contrast, none of the SCLC cell lines exhibit edelfosine-induced G2/M arrest or significant apoptosis. A comparison of the edelfosine-induced effects among the sensitive and resistant lung cancer lines indicates that there is little correlation between edelfosine-induced cytotoxicity and altered activities of JNK, ERK, p38, or cleavage of PARP. These results demonstrate that edelfosine-induced changes in JNK, ERK, p38, or PARP are not good predictors of cell susceptibility to edelfosine-induced cytotoxicity. Thus, edelfosine-induced inactivation of PLC may disrupt signaling cascades downstream of PLC that are unique to individual cellular environments. These findings also identify edelfosine as one of the few potential chemotherapeutic agents that has a greater cytotoxic effect against NSCLC cells than SCLC cells.     

Dicoumarol potentiates cisplatin-induced apoptosis mediated by c-Jun N-terminal kinase in p53 wild-type urogenital cancer cell lines

3-3'-Methylene-bis [4-hydroxycoumarin] (dicoumarol), an inhibitor of NADPH:quinone oxidoreductase 1, has been reported to possess potential antineoplastic effects and the ability to abrogate p53 protein. In the present study, we investigated the cytotoxic effects of dicoumarol in combination with cisplatin (CDDP), using four bladder (RT112, 253J, J82 and UMUC3) and two prostate (LNCap and PC3) cancer cell lines. Single treatment with 100 microM dicoumarol suppressed cell proliferation but did not induce apoptosis at 24 h in all cell lines examined. On the other hand, pretreatment with dicoumarol enhanced cytotoxicity of CDDP in three cell lines with wild type of p53 (RT112, 253J and LNCap), but not in three other cell lines with mutant p53 or in RT112 stable transfectants with a dominant-negative mutant of p53. In RT112 and LNCap, CDDP induced p53 and p21 expression, while pretreatment of dicoumarol suppressed induction of p53/p21 and resulted in sequential activation of c-Jun N-terminal kinase (JNK) in a time-dependent manner. Furthermore, inhibition of JNK, using SP600125, completely suppressed activity of caspases and poly-(ADP-ribose) polymerase cleavage, leading to suppression of enhancement of CDDP-mediated apoptosis by dicoumarol. These results suggested that dicoumarol could enhance cytotoxicity of CDDP in urogenital cancer cells with wild-type p53 through the p53/p21/JNK pathways.     

Targeting GD2 ganglioside and aurora A kinase as a dual strategy leading to cell death in cultures of human neuroblastoma cells

The mechanism of the inhibitory effect of anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (mAb) on human neuroblastoma cells survival was studied in vitro. It was recently shown in IMR-32 cells that death induced by this antibody exhibited several characteristics typical of apoptosis. In this study we used cytotoxixity assays, qRT-PCR and immunoblotting to evaluate the response of several human neuroblastoma cell lines to the anti-GD2 14G2a mAb. We showed that the mAb decreases all three aurora kinases expression and phosphorylation in IMR-32 and LA-N-1 cells. Most importantly, we show, that MK-5108 specific aurora A kinase inhibitor decreases neuroblastoma cell survival, and when used in combination with the mAb, significantly potentiates cytotoxicity against IMR-32, CHP-134, and LA-N-5 neuroblastoma cells in vitro. It was shown that downregulation of aurora A kinase by the therapeutic antibody is associated with decreased levels of MYCN protein in cytoplasm, and induced expression of PHLDA1 and P53 proteins.     

Recombinant antibodies against ganglioside expressed on tumor cells

Several gangliosides such as GM2, GD2, and GD3 have been thought of as target molecules for active or passive immunotherapy of human cancers because of their dominant expression on the tumor cell surface, especially in tumors of neuroectodermal origin. We established a number of mouse or rat monoclonal antibodies (mAbs) to a series of gangliosides to investigate the nature of the molecules on the cell surface. Some of those mAbs were converted to chimeric or humanized mAbs with the aim of developing immunotherapy for human cancer. It is desirable for mAbs to remain on the cell surface for a long time so that they can exert effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). We found that mAbs to GM2, GD2, and GD3 remain on the cell surface for ₨ min after binding, while mAbs to other types of carbohydrate such as sialy Le^a are quickly internalized. A chimeric mAb to GD3, KM871, was generated by linking cDNA sequences encoding light- and heavy-chain variable regions of mouse mAb KM641 with cDNAs encoding the constant region of human immunoglobulin ų (IgG-1). KM871 bound to a variety of tumor cell lines, especially melanoma cells, including some cell lines to which R24 failed to bind. In a preclinical study, intravenous injection of KM871 markedly suppressed tumor growth and radio-labeled KM871 efficiently targeted the tumor site in a nude mouse model. This chimeric mAb is being evaluated in a phase I clinical trial in melanoma patients. The chimeric mAb KM966 and humanized mAb KM8969 to GM2 originated from a mouse IgM mAb. When human serum and human peripheral blood mononuclear cells were used as effectors in CDC and ADCC, respectively, KM966 and KM8969 killed GM2-expressing tumor cells effectively. In addition, these mAbs may induce apoptosis of a small cell lung cancer cell line cultured under conditions mimicking physiological tumor conditions.     

肺癌细胞株中GEM和CDDP药物敏感性相关基因的研究

背景与目的 筛选小细胞肺癌(small cell lung cancer,SCLC)和非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞株中与健择(gemcitabine hydrochloride,GEM)和顺铂(cisplatin,CDDP)药物敏感性相关的基因,有助于进一步阐明抗癌药物作用机制,为克服抗癌药物的耐药性、研制开发新的抗癌药物提供新线索,同时也将为临床的个体化治疗提供理论依据.方法 采用MTT比色分析法测定4株SCLC细胞株和6株NSCLC细胞株对CDDP和GEM的药物敏感性,同时应用cDNA macroarray技术检测10株肺癌细胞株中1 291个药物敏感性相关基因的表达状态,分析二者之间的相关性.结果 与GEM药物敏感性呈明显正相关(r≥0.632,P<0.05)的基因有6个;与CDDP药物敏感性关联的基因共有45个;与GEM、CDDP敏感性关联(r≥±0.4)的基因有41个;肺癌细胞系中与GEM和CDDP两类药物敏感性呈明显相关的基因足Metallothinein(信号转导分子)、Cathepsin B(组织蛋白酶B)和TIMP1(生长因子);肺癌细胞系中与GEM、CDDP药物敏感性相关联的基因主要分布于Metallothinein、CathepsinB、牛长因子TIMP1等类别.结论 SCLC和NSCLC细胞株中GEM、CDDP存在药物敏感性相关基因,其中Metallothinein、Cathepsin B和TIMP1基因与GEM药物敏感性呈正相关,与CDDP药物敏感性呈负相关.     

Induction of apoptosis and activation of the caspase cascade by anti-EGF receptor monoclonal antibodies in DiFi human colon cancer cells do not involve the c-jun N-terminal kinase activity

We previously reported that exposure of DiFi human colon cancer cells to the anti-epidermal growth factor (EGF) receptor monoclonal antibody (mAb) 225 resulted in apoptosis, but the mechanisms remain to be elucidated. In the present study, we investigated the effects of a panel of four anti-EGF receptor mAbs, each of which binds to different epitopes of the EGF receptor in DiFi cells, on the induction of apoptosis. We found that each of these mAbs induced apoptosis in DiFi cells. Exposure of DiFi cells to mAb 225 activated the initiation caspase-8, which was detectable between 8 and 16 h after exposure of the cells to the antibody. There was also an activation of the initiation caspase-9, which lagged a few hours behind the activation of caspase-8. Exposure of DiFi cells to mAb 225 also activated the execution caspase-3, which was accompanied temporally by evidence of cleavage of a well-characterized caspase-3 substrate, poly(ADP)ribosepolymerase (PARP). Pre-exposure of the cells to the caspase-3-specific inhibitor DEVD-CHO partially reduced the mAb 225-induced PARP cleavage and apoptosis, whereas pre-exposure of the cells to the caspase pan-inhibitor z-VAD-fmk completely inhibited mAb 225-induced apoptosis. Caspases-3, -8 and -9 were not activated in the cell lines in which mAb 225 only induced G1 phase arrest of the cell cycle. In contrast to the apoptosis of DiFi cells induced by ultraviolet irradiation, which strongly activated the c-jun N-terminal kinase-1 (JNK1) and the caspase cascade, mAb 225-induced apoptosis and activation of the caspase cascade in DiFi cells were not associated with activation of JNK1.     

Cytotoxic effects of 15d-PGJ2 against osteosarcoma through ROS-mediated AKT and cell cycle inhibition

Polo-like kinase 1 (PLK1), a critical cell cycle regulator, has been identified as a potential target in osteosarcoma (OS). 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2), a prostaglandin derivative, has shown its anti-tumor activity by inducing apoptosis through reactive oxygen species (ROS)-mediated inactivation of v-akt, a murine thymoma viral oncogene homolog, (AKT) in cancer cells. In the study analyzing its effects on arthritis, 15d-PGJ2 mediated shear-induced chondrocyte apoptosis via protein kinase A (PKA)-dependent regulation of PLK1. In this study, the cytotoxic effect and mechanism underlying 15d-PGJ2 effects against OS were explored using OS cell lines. 15d-PGJ2 induced significant G2/M arrest, and exerted time- and dose-dependent cytotoxic effects against all OS cell lines. Western blot analysis showed that both AKT and PKA-PLK1 were down-regulated in OS cell lines after treatment with 15d-PGJ2. In addition, transfection of constitutively active AKT or PLK1 partially rescued cells from 15d-PGJ2-induced apoptosis, suggesting crucial roles for both pathways in the anti-cancer effects of 15d-PGJ2. Moreover, ROS generation was found treatment with 15d-PGJ2, and its cytotoxic effect could be reversed with N-acetyl-l-cysteine. Furthermore, inhibition of JNK partially rescued 15d-PGJ2 cytotoxicity. Thus, ROS-mediated JNK activation may contribute to apoptosis through down-regulation of the p-Akt and PKA-PLK1 pathways. 15d-PGJ2 is a potential therapeutic agent for OS, exerting cytotoxicity mediated through both AKT and PKA-PLK1 inhibition, and these results form the basis for further analysis of its role in animal studies and clinical applications.     

Chemoimmunosensitization of the T24 human bladder cancer line to Fas-mediated cytotoxicity and apoptosis by cisplatin and 5-fluorouracil.

Previous studies have demonstrated that cisplatin (CDDP) sensitizes bladder cancer cells to Fas-mediated cytotoxicity and that CDDP also enhances the cytotoxic effect of 5-fluorouracil (5-FU). These agents mediate apoptosis and may share common intracellular pathways leading to cell killing. We reasoned that combination treatment with CDDP, 5-FU and anti-Fas monoclonal antibody (mAb) might overcome the drug-resistance. We investigated whether CDDP, 5-FU and anti-Fas mAb synergize in cytotoxicity and apoptosis against the T24 bladder cancer line. Cytotoxicity was monitored by a one day microculture tetrazolium dye assay. Treatment of T24 cells with anti-Fas mAb in combination with CDDP or 5-FU resulted in a synergistic cytotoxic effect. In addition, combination treatment of T24 cells with CDDP, 5-FU and anti-Fas mAb further enhanced the synergistic cytotoxicity achieved by two agents. The synergy achieved in cytotoxicity with CDDP, 5-FU and anti-Fas mAb was also achieved in apoptosis. The current study demonstrates that combination treatment of bladder cancer cells with CDDP, 5-FU and anti-Fas mAb overcomes their resistance. In addition, the sensitization required low concentrations of CDDP and 5-FU, and thus supporting the potential in vivo application of combination of CDDP, 5-FU and immunotherapy in the treatment of drug-resistant bladder cancer.     

The GD2-specific 14G2a monoclonal antibody induces apoptosis and enhances cytotoxicity of chemotherapeutic drugs in IMR-32 human neuroblastoma cells

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. The majority of children suffers from high risk neuroblastoma and has disseminated disease at the time of diagnosis. Despite recent advances in chemotherapy, the prognoses for children with high risk NB remain poor. Therefore, new treatment modalities are urgently needed. GD2 ganglioside is an antigen that is highly expressed on NB cells with only limited distribution on healthy tissues. Consequently, it appears to be an ideal target for both active and passive immunotherapy. The immunological effector mechanisms mediated by anti-GD2 monoclonal antibodies (mAbs) have been already well characterized. However, a growing number of reports suggest that GD2-specific antibodies may exhibit anti-proliferative effects without the immune system involvement. Here, we have shown that anti-GD2 14G2a mAb is capable of decreasing survival of IMR-32 human neuroblastoma cells in a dose-dependent manner. Death induced by this antibody exhibited several characteristics typical for apoptosis such as increased number of Annexin V- and propidium iodide-positive cells, cleavage of caspase 3 and prominent rise in caspase activity. The use of a pan caspase inhibitor Z-VAD-fmk suggested that the killing potential of this mAb is partially caspase-dependent. 14G2a mAb was rapidly endocytosed upon antigen binding. Employment of chloroquine, an inhibitor of lysosomal degradation, did not rescue IMR-32 cells from antibody-induced cell death suggesting lack of ceramide involvement in the observed effect. Most importantly, our studies showed that at particular drug concentrations 14G2a mAb exerts a synergistic effect with doxorubicin and topotecan, as well as an additive effect with carboplatin in killing IMR-32 cells in vitro. Our results provide guidance regarding how to best combine GD2-specific 14G2a antibody with existing cancer therapeutic agents to improve available treatment modalities for neuroblastoma.     

Immunoliposomal fenretinide: a novel antitumoral drug for human neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In advanced disease stages, prognosis is poor and treatments have limited efficacy, thus novel strategies are warranted. The synthetic retinoid fenretinide (HPR) induces apoptosis in NB and melanoma cell lines. We reported an in vitro potentiation of HPR effects on melanoma cells when the drug is incorporated into GD2-targeted immunoliposomes (anti-GD2-SIL-HPR). Here, we investigated the antitumor activity of anti-GD2-SIL-HPR against NB cells, both in vitro and in vivo. Anti-GD2-immunoliposomes (anti-GD2-SIL) showed specific, competitive binding to, and uptake by, various NB cell lines. Moreover, anti-GD2-SIL-HPR presented increased selectivity and efficacy in inhibiting NB cell proliferation through the induction of apoptosis, compared to free drug and SL-HPR. In an in vivo NB metastatic model, we demonstrated that anti-GD2-SIL-HPR completely inhibited the development of macroscopic and microscopic metastases in comparison to controls. However, similar, but significantly less potent antitumor effect was observed also in mice treated with anti-GD2 immunoliposomes without HPR (anti-GD2-SIL-blank) or anti-GD2 mAb alone (P=0.0297 and P=0.0294, respectively, vs. anti-GD2-SIL-HPR). Moreover, our results clearly demonstrated that, although anti-GD2 mAb had a strong antitumor effect in this in vivo NB model, 100% curability was obtained only following treatment with anti-GD2-SIL-HPR (P<0.0001). Anti-GD2 liposomal HPR should receive clinical evaluation as adjuvant therapy of neuroblastoma.     

The idiotype (Id) cascade in mice elicited the production of anti-R24 Id and anti-anti-Id monoclonal antibodies with antitumor and protective activity against human melanoma

Gangliosides have been considered as potential targets for immunotherapy because they are overexpressed on the surface of melanoma cells. However, immunization with purified gangliosides results in a very poor immune response, usually mediated by IgM antibodies. To overcome this limitation, we immunized mice with R24, a monoclonal antibody (mAb) that recognizes the most tumor-restricted ganglioside (GD3); our goal was to obtain anti-idiotype (Id) antibodies bearing the internal image of GD3. Animals produced anti-Id and anti-anti-Id antibodies. Both anti-Id and anti-anti-Id antibodies were able to inhibit mAb R24 binding to GD3. In addition, the anti-anti-Id antibodies were shown to recognize GD3 directly. Anti-Id and anti-anti-Id mAb were then selected from two fusion experiments for evaluation. The most interesting finding emerged from the characterization of the anti-anti-Id mAb 5.G8. It was shown to recognize two different GD3-expressing human melanoma cell lines in vitro and to mediate tumor cell cytotoxicity by complement activation and antibody-dependent cellular cytotoxicity. The biological activity of the anti-anti-Id mAb was also tested in a mouse tumor model, in which it was shown to be a powerful growth inhibitor of melanoma cells. Thus, activity of the anti-anti-Id mAb 5.G8 matched that of the prototypic anti-GD3 mAb R24 both in vitro and in vivo. Altogether, our results indicate that the idiotype approach might produce high affinity, specific and very efficient antitumor immune responses.     

Cisplatin activates Akt in small cell lung cancer cells and attenuates apoptosis by survivin upregulation

The inhibitor of apoptosis protein (IAP) survivin is overexpressed in many tumors but is absent in most normal adult tissues. We report high levels of survivin expression in small cell lung cancer (SCLC), and describe the role of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in survivin upregulation. Moreover, the cytoprotective function of survivin in response to the anti-cancer agent cisplatin (CDDP) was investigated. Negative modulation of PI3K/Akt using pharmacological inhibitors or dominant negative Akt (DN-Akt) decreased Akt kinase activity and resulted in decreased survivin expression and phosphorylation on Thr34, whereas transfection of constitutively active Akt (CA-Akt) increased survivin expression and phosphorylation. Interestingly, we found that treatment of SCLC cells with CDDP further increased survivin expression in a cell cycle independent manner by activation of Akt. CA-Akt or lentiviral survivin also inhibited apoptosis induced by CDDP, whereas DN-Akt or survivin-specific RNA interference sensitized cells to CDDP. We identified survivin as an anti-apoptotic protein in SCLC cells that is regulated by Akt, and demonstrate that treatment with the DNA damaging agent CDDP activates the PI3K/Akt/survivin pathway that in part protects cells from drug-induced apoptosis.     

Effects of interleukin-6 (IL-6) on chemotherapy-induced apoptosis in human ovarian cancer cell lines

Background. Apoptosis is a cascade of events that is regulated by many factors. We studied the effects of interleukin-6 (IL-6) on chemotherapy-induced apoptosis in three human ovarian cancer cell lines (JV, GG, and NF). Methods. Cells were treated with the anticancer drugs cis-diamminedichloroplatinum (II) (CDDP) and paclitaxel (Taxol; Bristol-Myers Squibb Pharmacessticals, Noble Park, Australia) over a period of 72 h. The treatments were repeated in combination with human recombinant IL-6 or anti-IL-6 monoclonal antibody (anti-IL-6 mAb). The induction of cell death was examined by morphology and by internucleosomal DNA fragmentation. Results. Reduced cytotoxicity and fewer apoptotic cells were observed after treatment with CDDP or Taxol combined with IL-6 compared with treatment with CDDP or Taxol alone. However, treatment with CDDP or Taxol combined with anti-IL-6 mAb enhanced the cytotoxic effects of the drugs and increased the number of apoptotic cells. These findings indicated that apoptosis caused by CDDP or Taxol was influenced negatively by high doses of IL-6. Conclusion. The use of CDDP or Taxol combined with anti-IL-6 mAb may have therapeutic value for patients with ovarian cancer. Received: April 27, 1998 / Accepted: October 28, 1998     

Enhanced sensitivity to cis-diamminedichloroplatinum(II) of a human carcinoma cell line with mutated p53 gene by cyclin-dependent kinase inhibitor p21(WAF1) expression

p53-mediated induction of p21(WAF1), a cyclin-dependent protein kinase inhibitor, is known to protect cancer cells from the cytotoxic effects of anti-cancer drugs or gamma-irradiation. Since the p53 gene is frequently inactivated in cancer cells, we examined whether p21(WAF1) expression may alter the sensitivity of cancer cells with mutated p53 gene to anti-cancer drugs. Cells of a colon cancer cell line DLD-1 were transfected with p21(WAF1) expression vector controlled by a tetracycline-repressable promoter and transfectants were cloned (Dp21-1). p21(WAF1) expression induced by removal of tetracycline from culture media repressed cell proliferation and resulted in altered cell shape, suggesting induction of differentiation. Dp21-1 cells with p21(WAF1) expression were more sensitive to cis-diamminedichloroplatinum(II) (CDDP) (IC(50) value, 10 microM) than those without p21(WAF1) expression (IC(50), 22 microM). Sensitivity to doxorubicin was not different between Dp21-1 cells with and without p21(WAF1) expression. DNA ladder formation was observed in Dp21-1 cells treated with CDDP, indicating that the enhanced sensitivity to CDDP involves apoptosis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosolic protein revealed that subunit protein bands with M(r) 55 kDa and 44 kDa were markedly increased in cells with p21(WAF1) expression. By immunoblotting, these proteins were identified as c-Jun N-terminal kinase (JNK) 2 and p38 mitogen-activated protein kinase (MAPK) delta, respectively, both of which are believed to be involved in apoptosis induction by CDDP. These results suggest that p21(WAF1) may enhance the sensitivity of colon cancer cells with mutated p53 gene to CDDP, possibly through the JNK and p38 MAPK pathways.     

Differential effects of all-trans-retinoic acid, docosahexaenoic acid, and hexadecylphosphocholine on cisplatin-induced cytotoxicity and apoptosis in a cisplantin-sensitive and resistant human embryonal carcinoma cell line

Apart from modulation of tumor-cell drug sensitivity, induction of differentiation might be an alternative in the treatment of tumors resistant to cytotoxic drugs. In this report the capacity to induce differentiation and to modulate the cis-diamminedichloroplatinum(II) (CDDP) sensitivity of all-trans-retinoic acid (RA), docosahexaenoic acid (DCHA), and hexadecylphosphocholine (HePC) is examined in human germ-cell tumor cell lines. In the embryonal carcinoma cell line Tera-2 and its 3.7-fold CDDP-resistant subline Tera2-CP, we evaluated the effects of 96 h of pretreatment with RA (0.1 μM ), DCHA (23 μM ), and HePC (25 μM ) on differenctiation induction and on CDDP-induced cytotoxicity, DNA platination (4-h incubation), and apoptosis (continuous incubation). Without drug treatment, Tera2-CP showed less apoptosis than Tera-2. Pretreatment with RA decreased the cytotoxicity and apoptosis induced by CDDP without resulting in decreased DNA platination and increased differentiation in both cell lines. DCHA enhanced CDDP-induced cytotoxicity and apoptosis and did not affect the embryonal character of either cell line. HePC did not affect CDDP cytotoxicity or differentiation in either cell lines. Effects of the modulators on differentiation and on CDDP-induced cytotoxicity, DNA platination, and apoptosis did not differ between Tera-2 and Tera2-CP. RA can be applied for differentiation induction in CDDP-resistant germ-cell tumor models. However, in this model, RA reduced the apoptotic susceptibility. DCHA potentiated CDDP cytotoxicity in vitro; its in vivo modulatory capacity in germ-cell tumor cells deserves further study. Received: 1 June 1997 / Accepted: 22 October 1997     

Immunology and immunotherapy of neuroblastoma

Purpose

This review demonstrates the importance of immunobiology and immunotherapy research for understanding and treating neuroblastoma.

Principal results

The first suggestions of immune system–neuroblastoma interactions came from in vitro experiments showing that lymphocytes from patients were cytotoxic for their own tumor cells and from evaluations of tumors from patients that showed infiltrations of immune system cells. With the development of monoclonal antibody (mAb) technology, a number of mAbs were generated against neuroblastoma cells lines and were used to define tumor associated antigens. Disialoganglioside (GD2) is one such antigen that is highly expressed by virtually all neuroblastoma cells and so is a useful target for both identification and treatment of tumor cells with mAbs. Preclinical research using in vitro and transplantable tumor models of neuroblastoma has demonstrated that cytotoxic T lymphocytes (CTLs) can specifically recognize and kill tumor cells as a result of vaccination or of genetic engineering that endows them with chimeric antigen receptors. However, CTL based clinical trials have not progressed beyond pilot and phase I studies. In contrast, anti-GD2 mAbs have been extensively studied and modified in pre-clinical experiments and have progressed from phase I through phase III clinical trials. Thus, the one proven beneficial immunotherapy for patients with high-risk neuroblastoma uses a chimeric anti-GD2 mAb combined with IL-2 and GM-CSF to treat patients after they have received intensive cyto-reductive chemotherapy, irradiation, and surgery. Ongoing pre-clinical and clinical research emphasizes vaccine, adoptive cell therapy, and mAb strategies. Recently it was shown that the neuroblastoma microenvironment is immunosuppressive and tumor growth promoting, and strategies to overcome this are being developed to enhance anti-tumor immunotherapy.

Conclusions

Our understanding of the immunobiology of neuroblastoma has increased immensely over the past 40 years, and clinical translation has shown that mAb based immunotherapy can contribute to improving treatment for high-risk patients. Continued immunobiology and pre-clinical therapeutic research will be translated into even more effective immunotherapeutic strategies that will be integrated with new cytotoxic drug and irradiation therapies to improve survival and quality of life for patients with high-risk neuroblastoma.     

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.     

Etoposide-loaded immunoliposomes as active targeting agents for GD2-positive malignancies

Systemic chemotherapeutics remain the standard of care for most malignancies even though they frequently suffer from narrow therapeutic index, poor serum solubility, and off-target effects. In this study, we have encapsulated etoposide, a topoisomerase inhibitor effective against a wide range of cancers, in surface-modified liposomes decorated with anti-GD2 antibodies. We characterized the properties of the liposomes using a variety of methods including dynamic light scattering, electron microscopy, and Fourier transformed infrared spectroscopy. We examined whether these immunoliposomes were able to target cell lines expressing varying levels of surface GD2 and affect cellular proliferation. Anti-GD2 liposomes were generally targeted in a manner that correlated with GD2 expression and inhibited proliferation in cell lines to which they were efficiently targeted. The mechanism by which the immunoliposomes entered targeted cells appeared to be via clathrin-dependent uptake as demonstrated using flow cytometry and confocal microscopy. These studies suggest that anti-GD2-targeted, etoposide-loaded liposomes represent a potential strategy for more effective delivery of anti-cancer drugs that could be used for GD2 positive tumors.     

The evaluation of gastric cancer sensitivity to 5-FU/CDDP in terms of induction of apoptosis: time- and p53 expression-dependency of anti-cancer drugs

Clinical in vivo and in vitro studies have revealed pronounced gastric cancer activity using the combination of 5-fluorouracil (5-FU) and cisplatin (CDDP). In addition, the combination of 5-fluorouracil plus cisplatin (FP treatment) possesses synergistic cytotoxicity against gastric cancer. Sensitivity of two gastric cancer cell lines to anti-cancer drugs, 5-fluorouracil (5-FU) and/or cisplatinum (CDDP), was evaluated by use of either flow cytometric analysis (FACS) or morphological observation in terms of induction of apoptosis. In morphological observation, a new experimental technique was used in which cancer cells were distributed in thin collagen gel as one or two cell layers, and cultured with anti-cancer drugs. Thereafter, cells were stained with fluorescent Hoechst 33258 (Ho) and photographed, then stained with hematoxylin and eosin (H&E) and photographed again. Cell death patterns were determined by combining observations of Ho- and H&E-stained cells. While combined administration of 5-FU and CDDP did not induce apoptosis of MKN-28 (mutant-type p53), apoptotic cells were markedly observed in the case of MKN45 (wild-type p53). In addition, consecutive administration of CDDP for 3 h and 5-FU for 21 h effectively induced apoptosis of MKN45. These results indicated that the type of p53 expression in cancer cells could be a promising factor in predicting response to FP therapy and the administration of CDDP prior to 5-FU may be more effective in inducing apoptosis of gastric cancer cells with wild-type p53 expression. These data may provide evidence to support the idea that p53 expression is related to multidrug resistance (MDR) in FP therapy of gastric cancer cell lines.