Induction of TNF-alpha,uPA, IL-8 and MCP-1 by doxorubicin in human lung carcinoma cells

Purpose We have previously demonstrated doxorubicin-induced urokinase (uPA) and interleukin-8 (IL-8) expression in human H69 small-cell lung carcinoma (SCLC) cells by a microarray technique using Human Cancer Chip version 2, in which 425 human "cancer-related" genes are spotted on the plates. The microarray analysis also revealed a significant induction of tumor necrosis factor-alpha (TNF-), and doxorubicin-induced macrophage chemoattractant protein-1 (MCP-1) expression was demonstrated by an RNase protection assay. We extended the study by testing the effects of doxorubicin on the induction of TNF-, uPA, IL-8 and MCP-1 in other types of lung carcinoma cells.Methods We investigated the effects of doxorubicin on the expression of TNF-, uPA, IL-8 and MCP-1 in 12 human lung carcinoma cell lines, including five SCLC, three adenocarcinoma and four squamous cell carcinoma cells. The surface expression of their receptors was also investigated.Results TNF- was significantly induced in three cell lines, H69, SBC-7 (SCLC) and PC-9 (adenocarcinoma), uPA in five cell lines, H69, SBC-7, EBC-1 (squamous cell), EBC-2 (squamous cell), and Sq-1 (squamous cell), IL-8 in three cell lines, H69, PC-9 and EBC-1, and MCP-1 in five cell lines, H69, SBC-3 (SCLC), SBC-7, PC-9 and Sq-1. In H69 cells, TNF- antigen levels were increased approximately fivefold in the conditioned medium of doxorubicin-treated cells, in parallel with an increase in mRNA levels. As with uPA and IL-8, the maximum induction was observed at the "sublethal" concentrations of 2 and 4 M at which cell growth was slightly inhibited 24 h after treatment. Furthermore, the cells did not express receptors including types I and II TNF- receptors, uPA receptor (uPAR), C-x-C-chemokine receptor-1 (CXCR-1), or C-C-chemokine receptor-2, corresponding to TNF-, uPA, IL-8 and MCP-1, respectively, that were induced by doxorubicin in the cells, although SBC-7 cells expressed uPAR, and EBC-1 cells expressed CXCR-1.Conclusions TNF-, uPA, IL-8 and MCP-1 induced and secreted from tumor cells upon doxorubicin stimulation may activate surrounding cells expressing the receptors such as neutrophils and monocytes/macrophages in a paracrine fashion. TNF- is a major proinflammatory cytokine, and IL-8 and MCP-1 are major chemoattractants for neutrophils and monocytes/macrophages, respectively. Furthermore, uPA activates matrix metalloproteinase 9 which can truncate and activate IL-8. Thus, the simultaneous induction of TNF-, uPA, IL-8 and MCP-1 may enhance the interaction between tumor and inflammatory/immune cells, and augment cytotoxicity.Abbreviations BSA Bovine serum albumin - C-C Cysteine-cysteine - C-x-C Cysteine-x-cysteine - CCR-2 C-C chemokine receptor-2 - CXCR-1 C-x-C chemokine receptor-1 - ELISA Enzyme-linked immunosorbent assay - IL-1 Interleukin-1 - IL-6 Interleukin-6 - IL-8 Interleukin-8 - MCP-1 Macrophage chemoattractant protein-1 - MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide - PBS Phosphate-buffered saline - ROS Reactive oxygen species - SCLC Small-cell lung cancer - TNF- Tumor necrosis factor-alpha - TNFRI Type I tumor necrosis factor-alpha receptor - TNFRII Type II tumor necrosis factor-alpha receptor - uPA Urokinase-type plasminogen activator - uPAR Urokinase-type plasminogen activator receptor     

The role of NF-κB in hepatocellular carcinoma cell

Objective To evaluate the role of nuclear factor-kappaB (NF-κB) and IκBα in hepatocellular cacinoma (HCC) SMMC7721 cells, the consequence of NF-κB inhibition in SMMC7721 cells transfected with mutated IκBα (mIκBα) plasmid and the effect of stable inhibition of NF-κB activity in combination with Doxorubicin.Methods Western blot was used to determine the expression of NF-κB and IκBα in SMMC7721 cells and normal liver cells. Nuclear protein was used to evaluate the binding of the 32P-labeled tandem κB sequence using electrophoretic mobility shift assay and the expression of NF-κB using Western blot between SMMC7721 cells transfected with mIκBα plasmid (SMMC7721-MT) and control cells. Furthermore, cell viability was plotted between SMMC7721-MT and control cells. The binding of κB sequence and cell viability between SMMC7721-MT and control cells at different concentrations of Doxorubicin were also investigated.Results Western blot analysis for nuclear extract showed more P50 (NF-κB1) and P65 (RelA) expression in SMMC7721 cells compared with normal liver cells. The expression of cytosolic IκBα protein in SMMC7721 cells was less than that in normal cells. SMMC7721-MT cells inhibited NF-κB nuclear translocation at 0, 24, 48 and 96 hours. Furthermore, NF-κB cannot be detected in the nuclear protein of SMMC7721-MT cells by Western blot. By calculating cell viability, the proliferation of SMMC7721-MT cells was shown to be suppressed more significantly than that of control cells. NF-κB in untransfected cells was activated by Doxorubicin in a dose-dependent manner, but that in SMMC7721-MT cells was not induced at low concentrations of Doxorubicin. Compared with untransfected cells, the viability of SMMC7721-MT cells was significantly suppressed at the same concentration of Doxorubicin (P<0.01）.Conclusions The present study demonstrates that upregulation of NF-κB and downregulation of inhibitory kappaB (IκBα) in SMMC7721 cells are related with the growth of hepatocellular cacinoma cells. Stable expression of mIκBα in SMMC7721-MT cells can inhibit NF-κB nuclear translocation and suppress cell growth. Furthermore, stable inhibition of NF-κB activity in combination with Doxorubicin can significantly inhibit cell proliferation in SMMC7721-MT cells. Thus, modulation of NF-κB may represent an improvement in the efficacy of HCC therapies and be worthy of further research and investigation.     

肿节风注射液抗肿瘤及与阿霉素联合用药的实验研究

目的了解肿节风注射液抗肿瘤及与阿霉素联合用药的效果。方法采用四氮唑盐法(MTT法)测定药物的体外抑制作用,用IC50评测直接抗肿瘤效果,用金氏公式进行联合用药分析。结果肿节风注射液能抑制人肝癌细胞Bel7402、人结肠癌细胞HCT-8的增殖,其IC50分别为33.13,52.39mg/mL。3.125,6.25,12.5mg/mL的肿节风注射液与阿霉素联合对HCT-8细胞体外抑制呈相加作用;25,50mg/mL的肿节风注射液与阿霉素联合对HCT-8细胞体外抑制呈协同作用。结论肿节风注射液对Bel7402、HCT-8细胞有一定的抑制肿瘤生长的作用,它与阿霉素联合应用对HCT-8细胞可产生相加或增强的协同抑制效果,尤其是高浓度的肿节风注射液与阿霉素的协同作用更为显著。     

Pegylated liposomal doxorubicin: metamorphosis of an old drug into a new form of chemotherapy

Pegylated liposomal doxorubicin (Doxil, Caelyx) is a formulation of doxorubicin in poly(ethylene glycol)-coated (stealth) liposomes with a prolonged circulation time and unique toxicity profile. We review the preclinical and clinical pharmacology as well as recent clinical data obtained in specific cancer types. Doxil liposomes retain the drug payload during circulation and accumulate preferentially in tissues with increased microvascular permeability, as often is the case of tumors. Doxil toxicity profile is drastically different from that of doxorubicin, and is characterized by dominant and dose-limiting mucocutaneous toxicities, mild myelosupression, minimal alopecia, and no apparent cardiac toxicity. Although the single maximum tolerated dose (MTD) of Doxil is actually lower than that of conventionally administered doxorubicin, the cumulative MTD dose of Doxil may be substantially greater than that of free doxorubicin. Doxil is probably one of the most active agents in AIDS-related Kaposi's sarcoma and has a definite role in management of recurrent ovarian cancer. The potential of Doxil in the treatment of other cancer types and the opportunities it offers in combination with other drugs and therapeutic modalities are under active investigation.     

Effects of amifostine on perfused isolated rat heart and on acute doxorubicin-induced cardiotoxicity

Purpose: To determine the effects of amifostine on an isolated perfused rat-heart model and its protective activity with regard to cardiotoxic doxorubicin perfusion. Methods: Langendorff constant-pressure isolated rat-heart preparations were used to analyze the effects of the drugs during a 40-min period of perfusion after a 20-min stabilization interval. The first study was conducted with amifostine alone (controls and 10⁻⁶, 10⁻⁵, and 10⁻⁴ M amifostine; n=6 in each group). The second study was conducted with amifostine and doxorubicin (controls, 2.5 × 10⁻⁵ M doxorubicin, 2.5 × 10⁻⁵ M doxorubicin and 10⁻⁵ M amifostine, and 2.5 × 10⁻⁵ M doxorubicin and 10⁻⁴ M amifostine; n=4 in each group). Results: Amifostine had no significant effect on hemodynamic parameters at 10⁻⁶, 10⁻⁵, and 10⁻⁴ M concentrations. However, amifostine increased the coronary flow expressed as a percentage ± SEM of the baseline flow as follows: 82 ± 4% for controls, 95 ± 6% for 10⁻⁶ M amifostine, (P=0.13), 111 ± 4% for 10⁻⁵ M amifostine (P < 0.01), and 104 ± 3% for 10⁻⁶ M amifostine (P < 0.01). When we commenced an amifostine perfusion 20 min in advance of and then during a 40-min perfusion with doxorubicin, at a cardiotoxic concentration of 2.5 × 10⁻⁵ M the left ventricular pressures (LVDP, expressed as percentages ± SEM of the baseline LVDP before doxorubicin) were 55 ± 3% for the doxorubicin controls, 68 ± 2% for doxorubicin with 10⁻⁵ M amifostine (P=0.05), and 80 ± 3% for doxorubicin with 10⁻⁴ M amifostine (P < 0.01). Whether this protective effect might be related to the known free-radical-scavenging activity of amifostine remains to be determined. Conclusion: On a Langendorff-type model of rat heart, 10⁻⁵ and 10⁻⁴ M amifostine alone induced a coronary dilation and, when associated with a cardiotoxic concentration of 2.5 × 10⁻⁵ M doxorubicin, 10⁻⁵ and 10⁻⁴ M amifostine displayed a cardioprotective effect. Received: 9 March 1998 / Accepted: 6 July 1998     

M-VCA方案治疗晚期鼻咽癌35例疗效分析

目的 探讨Ｍ－ＶＣＡ方案治疗晚期鼻咽癌的疗效及毒副反应,方法：对３５例晚期鼻咽癌患者（初治诱导化疗者１１例,复发或转移者２４例）采用ＭＴＸ＋ＶＣＲ＋ＤＤＰ＋ＡＤＭ联合化疗方案治疗。结果 评定其化疗２疗程后的客观疗效,总有效率为７５．５１％,无ＣＲ病例,其中初治肿瘤有效率达１００％,复发者为６６．６７％,远处转移总有效率为７０．０％,肺转移疗效最好（８８．８９％）,主要毒副反应是脱发,轻到中度的骨髓     

Effect of Phosphatidic Acid on Human Breast Cancer Cells Exposed to Doxorubicin

We previously demonstrated that phosphatidic acid (PA) induces chemotactic migration of highly metastatic breast cancer cells MDA-MB-231. The widely used anticancer drug doxorubicin was reported to induce apoptosis of cancer cells. Growth factors such as epidermal growth factor (EGF) and bioactive lipids such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (SPP) have been shown to enhance viability and to protect cancer cells against apoptosis. In this study, we investigated the effect of PA on MDA-MB-231 cells exposed to the anticancer drug doxorubicin. Cell migration toward PA was partially inhibited by doxorubicin treatment, and PA moderately diminished cell cycle arrest of cells exposed to doxorubicin. Although PA itself was not able to induce apoptosis of MDA-MB-231 cells, apoptosis of cells exposed to doxorubicin was markedly enhanced by PA treatment. Thus, PA is able to increase the apoptotic potential of doxorubicin, and may regulate the effects of doxorubicin used for chemotherapy.     

Interleukin-1 signaling mediates acute doxorubicin-induced cardiotoxicity

Doxorubicin (DOX) is a potent anticancer drug, which is widely used in the treatments of a variety of solid and hematopoietic tumours, but its use is limited by its cardiotoxicity and dose-dependent congestive heart failure. After we found the high expression of interleukin-1 receptor I (IL-1RI) gene in mice cardiac tissues with DOX-induced cardiotoxicity, we assumed interleukin-1 (IL-1) signaling might mediate acute DOX-induced cardiotoxicity. In this report, Balb/c mice were intraperitoneally injected with different dosage of DOX followed by different days of study. We found both IL-1β and interleukin-1 receptor antagonist (IL-1Ra) concentrations in serum were highly induced after DOX treatment, associated with increased IL-1RI expression in cardiac tissues. Furthermore, IL-1 signaling was found to express higher with the increased dosage of DOX treatment. Histology score of cardiac tissues showed obvious cardiac damage after DOX treatment, while assessment with the Spearman rank correlation coefficient showed that histology score was closely correlated with IL-1β and IL-1Ra concentrations in serum and IL-1RI expression in cardiac tissue. Our results reveal a potential role of IL-1 signaling in acute DOX-induced cardiotoxic injury and lead to an assumption that this signal system might be a potential candidate agent that inhibits cardiomyocyte-toxicity in DOX-exposed patients.     

铁碳复合磁性载体体内特征的研究

目的 通过吸附阿霉素的纳米铁碳复合磁性载体经猪肝动脉插管介入,观察靶区肝组织、非靶区肝组织及血清中阿霉素的药代动力学变化.方法 将香猪20头随机分为5组,分别经肝左动脉内注入阿霉素水溶液、外加磁场的高中低剂量载药铁碳复合磁性载体及不加用磁场的高剂量载药铁碳复合磁性载体,在不同时间点分别取靶区肝组织、非靶区肝组织及静脉血,质谱法测定阿霉素浓度.结果 外加磁场载药铁碳复合磁性载体组靶区肝组织内阿霉索浓度明显高于非靶区肝组织内阿霉素浓度,最高达101.4倍;也高于阿霉素水溶液组及未加用磁场组,最高达23.8倍及28.3倍.在不同剂量磁靶向治疗组中靶区肝组织内阿霉素浓度呈剂量依赖性增高,持续时间延长.结论 载药铁碳复合磁性载体在外加磁场引导下易聚集于靶区,在局部释放药物,对周围组织器官影响较小.