白杨素增强顺铂抗肿瘤作用的研究

目的：探讨白杨素增强顺铂抗肿瘤作用的最佳条件并筛选敏感细胞。方法：设立阴性对照组、空白调零组及40 μmol/L白杨素、5.0 μg/mL顺铂、40 μmol/L白杨素+5.0 μg/mL顺铂3个处理组,选取人结直肠癌细胞（HCT-116）、人鼻咽癌细胞（CNE1）、人肝癌细胞（HepG2）、人胃癌细胞（BGC-823）分别作用24 h。采用四甲基噻唑蓝（MTT）法测定肿瘤细胞的增殖抑制率,采用Hoechst 33342荧光染色法测定诱导肿瘤细胞的凋亡率,通过抑制率和凋亡率筛选出敏感细胞。采用L₉（3³）正交设计方法,设立3个白杨素水平（10、20、40 μmol/L）,3个顺铂水平（1.3、2.5、5.0 μg/mL）,以及3种作用时间（12、24、36 h）处理敏感细胞,确定白杨素增强顺铂抗肿瘤作用的最佳作用条件。结果：与白杨素或顺铂单独处理组相比,MTT试验结果表明,白杨素联合顺铂作用组对上述4种肿瘤细胞增殖的抑制率均明显增加,差异均具有统计学意义（P均 < 0.01）;Hoechst 33342染色实验发现,白杨素联合顺铂作用组诱导各肿瘤细胞的凋亡率亦均明显增加（P均 < 0.01）。白杨素联合顺铂作用组对人胃癌细胞BGC-823的增殖抑制率为（78.0±2.0）%、诱导细胞的凋亡率为（72.3±6.5）%。在4种细胞中,人胃癌细胞株BGC-823对白杨素联合顺铂抗肿瘤作用最敏感。正交实验的体外模型结果表明,最佳作用条件为白杨素40 μmol/L联合顺铂5.0 μg/mL,作用时间24 h。结论：白杨素能增强顺铂抑制人肿瘤细胞增殖和诱导人肿瘤细胞凋亡的作用。人胃癌细胞BGC-823是白杨素和顺铂联合作用的敏感细胞株。     

树突状细胞对免疫活性细胞抑癌效应的非限制性增强作用

目的研究抗原负载的树突状细胞(dendritic cell,DC)对免疫活性细胞抑癌的增强作用。方法在建立PC体外扩增的基础上,将抗原负载的DC作用于单个核细胞及高聚金葡素活化的单个核细胞,观察其对人肺癌GLC-82和A549细胞的体外和裸鼠体内抑癌效应。结果抗原负载的DC能显著增强单个核细胞及高聚金葡素活化的单个核细胞对GLC-82和A549细胞的抑制作用。当效靶化为10∶1时,抗原负载的DC-单个核细胞对GIC-82及A549细胞抑制率分别为100%和94.2%;而对照组分别为16%和8%。该作用不受主要组织相容性复合物基因(major histocompatibility com-plex,MHC)限制。结论抗原负载的DC对免疫活性细胞的抑癌作用有MHC非限制性增强效果。     

Enhancement by vaso-active intestinal peptide of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in rats.

The effects of vaso-active intestinal peptide (VIP) on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated in Wistar rats given VIP every other day for 27 weeks after oral administration of MNNG for 25 weeks. In week 52, administration of VIP caused a significant increase in the incidence of gastric cancers, but did not influence their histological appearance. VIP significantly increased the labeling indices of the antral mucosa. Our findings indicate that VIP enhances gastric carcinogenesis, and that this effect may be related to its effect in increasing cell proliferation of the antral epithelial cells.     

The effect of systemic hyperthermia on melphalan pharmacokinetics in mice

The effect of 45 min systemic heating at 41 degrees C on plasma and RIF-1 tumour pharmacokinetics of intraperitoneally administered melphalan (MEL) was studied in C3H mice. This heat dose causes greater potentiation of MEL in tumour than in marrow cells, resulting in a therapeutic gain for the combined therapy (Honess & Bleehen, 1985). MEL (7.5 mg kg-1) was administered at the start of heating and concentrations assayed from 20-90 min by high-performance liquid chromatography (HPLC). With or without heat peak concentrations were achieved by 20 min and were 3 to 4 micrograms ml-1 in plasma and 1-3 micrograms g-1 in tumour. Higher MEL concentrations in both plasma and tumour were found in heated animals at times after 20 min from injection, but the effect was greater in plasma (2.5-4 fold) than in tumour (1.5-2 fold) where differences were not always significant. At 40 min after a dose of 7.5 mg kg-1, plasma and tumour concentrations in heated animals were equivalent to those after 12.5 mg kg-1 and 8.5 mg kg-1, respectively, without heating. Tumour/plasma ratios were usually lower in heated than in unheated animals where they often exceeded 100%. The apparent plasma elimination half-life (t1/2) was 17.5-25 min in unheated and 24-44 min in heated animals. The area under the curve (AUC) was increased by a factor of 1.2-1.5 in heated animals, at least partly due to a decrease in volume of distribution. The heat induced increase in MEL exposure may be involved in the enhanced response to the drug, but does not appear to explain the therapeutic gain compaired to MEL alone.     

Enhancement of the DNA cross-linking activity of melphalan by misonidazole in vivo.

The technique of alkaline elution has been adapted for the study of drug-induced DNA cross-link formation in vivo. Pretreatment with misonidazole (MISO) enhances the number of cross-links formed in a fibrosarcoma and in the spleen and gut of mice for periods up to 48 h following a single injection of melphalan (MEL). The tumour was sensitized by a greater factor (2.05) than either of the normal tissues (enhancement factor 1.4-1.5). This enhancement did not appear to be related to inhibition of the repair of actual cross-links. Rather, the effect was explicable in terms of one of two alternative models. Firstly, MISO pretreatment could result in a greater amount of binding of MEL to DNA at early times after injection. This may be the result of altered pharmacokinetics of MEL, or of enhanced intracellular uptake of MEL due to MISO pretreatment. Secondly, MISO may exert its affect by inhibition of the repair of cross-links or monoadducts at early times post-injection, which would not be observed in this study. The possible involvement of glutathione depletion in chemosensitization by MISO was investigated by comparison with the effect of diethyl maleate (DEM), a known thiol-depleting reagent. Glutathione depletion, while perhaps being important, could not account for all of the effects observed.     

Enhancement of experimental tumors in mice by treatment with concanavalin A.

Weanling BALB/c mice given injections of 300 micrograms concanavalin A (Con A) prior to and at frequent intervals after challenge with Moloney murine sarcoma virus (M-MuSV), 3-methylcholanthrene (MCA), or TEPC-15 plasmacytoma cells showed an enhancement of tumor induction or development. With the M-MuSV and MCA systems, this enhancement was evidenced by larger tumors and, in the MCA system, by more devastating tumors. Regression of the M-MuSV-induced tumors was more prolonged in Con A-treated mice. With the TEPC-15 system, enhancement was evidenced by a more rapid mortality rate in treated animals.     

Lack of experimental vesicant activity for the anticancer agents cisplatin,melphalan, and mitoxantrone

Summary Cisplatin and L-PAM are DNA-crosslinking anticancer agents which have not been systematically studied for vesicant potential. Mitoxantrone is a new active anthracene-based, DNA intercalator which is undergoing widespread clinical testing for antitumor efficacy in man. These three agents were tested for vesicant activity in dehaired BALB/c mice given ID injections equivalent to human clinical doses. Neither cisplatin (up to 150 mg/m²) nor L-PAM (up to 71 mg/m²) produced any skin necrosis in the mice. The L-PAM solvent (acid/alcohol in propylene glycol) was ulcerogenic if injected undiluted. Mitoxantrone (up to 14 mg/m²) was not ulcerogenic in the mice, although the skin site retained a blue drug discoloration for several weeks. It is concluded that in clinically relevant doses, cisplatin, L-PAM, and mitoxantrone are not vesicants.Supported in part by Public Health Service grants CA-23074, CA-31078 and CA-17094 from the National Cancer Institute, Department of Health and Human Services, Bethesda, MD     

Enhancement of the clinical activity of melphalan by the hypoxic cell sensitizer misonidazole

One hundred patients with non-small cell lung cancer were entered by members of the Northern California Oncology Group into a randomized Phase II trial of i.v. melphalan versus i.v. melphalan with concomitant oral misonidazole. The patients had not received prior chemotherapy. Eighty-five patients were evaluable for assessment of response and 89 were evaluable for toxicity analysis. The melphalan/misonidazole group had a superior response rate (two complete and four partial responses among 42 patients or 14%) compared to the melphalan group in which there were no responses among 43 patients (p = 0.024, two-sided Fisher exact test). Since hematological toxicity was equivalent in the two groups, there was an improvement in therapeutic index. Data from 12 patients undergoing pharmacological studies demonstrated that the plasma concentration of melphalan was 25% higher in the misonidazole group, a difference that is not statistically significant. Although the mechanism of interaction has not been fully established, this randomized trial demonstrates that a chemosensitizer can enhance the clinical antitumor activity of an alkylating agent and suggests that chemosensitizers in combination with alkylating agents should be investigated in further clinical trials.     

重组人血管内皮抑制素对裸鼠骨肉瘤的抑瘤作用

目的 研究Rh-Endostatin对人骨肉瘤细胞裸鼠移植瘤的抑瘤作用.方法 建立人骨肉瘤细胞OS-732皮下移植瘤裸鼠动物模型,分为4组,分别给予(a)生理盐水;(b、c、d) Rh-Endostatin低、中和高剂量,分别为2.5、5.0和10mg/kg;为腹腔注射给药,1次/天,4周后裸鼠全部处死,称量肿瘤重量计算抑瘤率,用药疗效.对肿瘤标本进行微血管密度计数和细胞凋亡指数检测.结果 Rh-Endostatin单药2.5、5和10mg/kg治疗抑瘤率分别为25.3%、34.1%和35.7%;微血管密度:所有治疗组与对照组比较,差异均具有统计学意义(P＜0.01).凋亡指数:治疗组与对照组比较,差异均具有统计学意义(P＜0.01).结论 Rh-Endostatin单药对骨肉瘤具有明显的抑瘤作用,抗血管生成治疗骨肉瘤具有潜在的临床应用价值,值得进一步临床试验评估其疗效.     

经靶动脉灌注碳酸氢钠提高部分抗肿瘤药物疗效

目的：经靶动脉向恶性肿瘤组织灌注碳酸氢钠，以调节恶性肿瘤细胞组织的pH值，观察其对白细胞介素-2或IFN-α2b和阿霉素介入治疗的增效作用。方法：分层随机抽取37例中晚期原发性肝癌，用Seldinger技术穿刺经股动脉进入肿瘤动脉血管，碳酸氢钠 rIL-2／IFN-α2b 阿霉素，药物按一定比例稀释后灌注。介入治疗2个疗程后判断疗效。结果：2个疗程后总有效率(CR PR)为76％(28／37)。ⅢA、ⅣA、ⅣB期中位缓解期分别为7、13和6个月，ⅢA、ⅣA、ⅣB期中位生存期分别为10、14和8个月，ⅢA、ⅣA、ⅣB期生存6个月分别为7、5和3例，ⅢA、ⅣA、ⅣB期生存12个月分别为3、3和2例，ⅢA、ⅣA、ⅣB期生存24个月分别为1、3和0例，ⅢA、ⅣA、ⅣB期3～4年生存期分别为1、1和1例。白细胞下降Ⅲ级3例恢复较快。消化道反应Ⅳ级1例、脱发Ⅲ级5例、发热Ⅲ级13例。结论：向恶性肿瘤组织灌注碳酸氢钠可提高白细胞介素-2、IFN-α2b和ADM的疗效。     

Enhancement of an anti-tumor effect of interferon by dipyridamole in established human malignant melanoma cell lines.

Enhancement of the anti-proliferative effect of human interferon (HuIFN) preparations (alpha, beta and gamma) by dipyridamole was detected in a human malignant melanoma cell line, MM-ICB, which we originally established. Cell growth was inhibited by HuIFN alone, but a marked increase in inhibition was noted in vitro and in vivo when dipyrydamole was added. Cellular DNA synthesis, as determined by 3H-deoxythymidine incorporation into the acid-insoluble cellular fraction, was more inhibited by combined treatment than by any of the agents used alone. Two other melanoma cell lines that we established, MM-2CB and MM-3CB, also exhibited sensitivity to combined treatment both in vitro and in vivo. Furthermore, the HMV-I and SEKI melanoma cell lines were susceptible to the combination. Even non-cytotoxic concentrations of dipyridamole could enhance the effect of HuIFN on MM-ICB, MM-2CB, and SEKI cells.     

Late immune and haemopoietic functions in plasmacytoma-bearing mice cured by melphalan

Alkylating agents can cause latent and permanent damage to the bone marrow. We compared the long term effects of melphalan on a number of immune and haemopoietic functions of plasmacytoma bearing BALB/c mice with that of normal mice treated with a similar dose of melphalan. The drug administered orally at a dose of 250 micrograms and 400 micrograms on day 14 and 24 following i.m. inoculation of MOPC-315 plasmacytoma cells resulted in cure of the mice. Their spleen cells showed a permanent impairment of MLR activity, T-cell number and IL-2 production as well as a mild suppression of NK activity for one year after cessation of melphalan therapy. The number of B cells was elevated. In contrast, plasmacytoma-free mice treated with melphalan retained long term normal immune functions, although shortly after melphalan therapy a temporary suppression was noted. On the other hand, melphalan was responsible for bone marrow myeloid stem cell damage since the number of myeloid progenitor cell (CFU-GM) colonies was reduced in both melphalan-treated groups compared to untreated normal controls. Plasmacytoma bearing mice had a shorter survival. These results demonstrate that some late sequelae of alkylating agents are not due to the drug alone; shorter survival and T-cell deficiency are related to the previous presence of the tumour.     

Application of cytopathology in a sensitivity test for anti-tumor agents. I. An experimental study

Although the human tumor clonogenic assay (HTCA) is extremely reliable in determining clinical correlations, it is a complicated process requiring considerable time in order to obtain results. Thus, an experimental study on cytopathologic observation (cytologic assay) and comparative evaluation between it and HTCA were performed in order to establish a more rapid and accurate drug sensitivity test. Materials included Colon 26, a cell line established in our department, malignant effusion and surgical specimens. In carrying out HTCA according to the Hamburger-Salmon method, the cell suspension samples following exposure to anti-tumor agents (MMC, L-PAM, ADM, CDDP) were cultivated in test tubes for 3-8 hours and stained by the Papanicolaou and Giemsa methods. According to Tokita's criteria, when cellular changes showed as nuclear pyknosis and nuclear destruction were found to have increased significantly in comparison with a control group, the cells were judged to be sensitive. Very similar and parallel results were obtained between HTCA and cytologic assay in this study, with a significant correlation. Cytologic assay was proved to be an easy, rapid and accurate method for testing drug sensitivity and its clinical application can be expected in the future.     

Cyclophosphamide and melphalan as immunopotentiating agents in cancer therapy

The murine plasmacytoma, MOPC-315, has been used as a tumor model to investigate the immunopotentiating effect of a low dose of cyclophosphamide (CY) or melphalan (l-PAM). Each drug was shown to shift the balance in mice bearing a late-stage tumor from a state of immunosuppression to that of potent T-cell-dependent antitumor immunity against tumor-associated antigens. The resultant immunity eradicated the extensive tumor burden not already eradicated by the direct tumoricidal activity of the drug and brought about the cure of the mice. The immunity responsible for tumor eradication, as well as the immunity responsible for the resistance of the cured mice to further tumor challenge, was mediated by the Lyt 2 subset of T-cells which contains cytotoxic T-cells. The principle of using a low dose of drug to selectively decrease suppressor cell activity so as to allow the development of antitumor immunity with the aid of autologous tumor vaccine or interleukin-2 has been exploited successfully by clinicians in therapeutic protocols for human melanoma.     

Cyclophosphamide and melphalan as immunopotentiating agents in cancer therapy

The murine plasmacytoma, MOPC-315, has been used as a tumor model to investigate the immunopotentiating effect of a low dose of cyclophosphamide (CY) or melphalan (L-PAM). Each drug was shown to shift the balance in mice bearing a late-stage tumor from a state of immunosuppression to that of potent T-cell-dependent antitumor immunity against tumor-associated antigens. The resultant immunity eradicated the extensive tumor burden not already eradicated by the direct tumoricidal activity of the drug and brought about the cure of the mice. The immunity responsible for tumor eradication, as well as the immunity responsible for the resistance of the cured mice to further tumor challenge, was mediated by the Lyt 2 subset of T-cells which contains cytotoxic T-cells. The principle of using a low dose of drug to selectively decrease suppressor cell activity so as to allow the development of antitumor immunity with the aid of autologous tumor vaccine or interleukin-2 has been exploited successfully by clinicians in therapeutic protocols for human melanoma.     

Enhancement of melphalan activity by inhibition of DNA polymerase-α and DNA polymerase-β

Our previous studies exploring melphalan resistance in the human rhabdomyosarcoma xenograft TE-671 MR revealed elevation of DNA polymerase-α and DNA polymerase-β . The present study evaluated the alteration of melphalan activity in TE-671 (melphalan-sensitive) and TE-671 MR (melphalan-resistant) subcutaneous xenografts in nude mice after DNA polymerase-α was inhibited using aphidicolin glycinate (AG) and DNA polymerase-β was inhibited using dideoxycytidine (DDC). Administration of AG or DDC did not produce toxicity or demonstrate antineoplastic activity when given alone. AG (90 mg/m²) enhanced the activity of melphalan against TE-671, with growth delays increasing by 8.4, 15.8, and 21.2 days over the regimen with melphalan only. AG (180 mg/m²) only modestly increased melphalan activity against TE-671 MR, with the growth delays increasing from 9.6 and 12.1 days using melphalan alone to 12.1 and 14.5 days using melphalan plus AG. AG (180 mg/m²) plus melphalan (the dose lethal to 10% of animals) produced greater weight loss compared with melphalan alone, whereas DDC plus melphalan produced no additional toxicity. DDC modestly enhanced the activity of melphalan plus AG against TE-671 MR. AG plus O ⁶-benzylguanine did not increase the activity of 1,3-bis(2-chloroethyl)-1-nitrosourea against TE-671 or TE-671 MR. AG (90 mg/m² and 180 mg/m²) inhibited DNA polymerase-α to 80% and 72% of control in TE-671 and 64% and 37% in TE-671 MR, and DDC inhibited DNA polymerase-β to 59% in TE-671 and 48% in TE-671 MR. These results suggest a role for AG-mediated enhancement of melphalan activity, particularly in the treatment of newly diagnosed, melphalan-sensitive tumors. Received: 19 June 1995 / Accepted: 2 November 1995     

靶动脉灌注NaHCO3提高部分抗肿瘤药物疗效的基础及临床研究

Objective: To observe the influence of pH value on the proliferation of LAK cells and on the killing effect of rIL-2,IFN-α2b, TNF-α, LAK cells and doxorubicin on malignant tumor cells, and investigate the possibility of increasing the efficacy of rIL-2 or IFN-α2b and doxorubicin by infusing sodium bicarbonate (NaHCO3) through target arteries. Methods: Separating single nucleus cells from peripheral blood of healthy men, and observing the influence of pH on the activation of single nucleus cells by rIL-2. MTT assay was used to measure the killing effect of rIL-2, IFN-α2b and TNF-α on 7404 cells and the increased effect of doxorubicin on rIL-2 and IFN-α2b, the cytotoxity of LAK cells in different pH. Forty-two patients with advanced primary liver cancer were obtained by stratified random, NaHCO3, rIL-2/IFN-α2b and doxorubicin were infused through target arteries. The efficacy was estimated after two cycles. Results: The conditions of pH 7.3 and pH 7.6 in vitro helped the proliferation of LAK cells and the killing effect of rIL-2, IFN-α2b and LAK cells on 7404 cells. In the condition of pH 6.8 there was almost no killing effect for LAK cells. In the condition of pH 7.0, 7.2, 7.4 and 7.6, the killing rate of TNF-α to 7404 cells increased by degrees, and in pH 7.4 the killing effect was the optimum. After two cycles treatments in the 42 patients with advanced primary liver cancer,the response rate (CR PR) was 88% (37/42). The median overall response and median overall survival were increased, and no complication associated with infusing sodium bicarbonate was observed. Conclusion: The killing effect of rIL-2, IFN-α2b, TNF-αand doxorubicin on malignant tumor cells was enhanced by increasing the pH value.     

Thermally-enhanced tumor regression in mice treated with melphalan

Mice were implanted subcutaneously with one of three types of tumor, each isogeneic to the respective host. These were two mammary adenocarcinomas designated DBAH and MT2, and a spindle cell sarcoma designated TEC. Tumor-bearing mice were treated with melphalan alone, locally-applied microwave hyperthermia alone (42-43 degrees C) or with a combination of these two modalities. Following treatment, tumor growth and regression, and survival of host were recorded. Only mice bearing the DBAH tumor were cured by either modality alone. The MT2 and TEC tumors responded only slightly to melphalan treatment alone. Approximately half of the TEC tumors responded to prolonged treatment by hyperthermia alone, yielding total regressions; the MT2 tumor proved to be resistant to this modality. Doses of hyperthermia which had no effect on tumor growth when applied alone were able to induce a thermal potentiation of melphalan. All 3 tumor types were cured by this combined treatment, although different doses of hyperthermia were required for each tumor type.