Topoisomerase I‐Mediated Antiproliferative Activity of 10‐Substituted and 12‐Substituted Homocamptothecins

Homocamptothecin (hCPT) is an E‐ring modified camptothecin (CPT) analogue, which showed pronounced inhibitory activity of topoisomerase I. In search of novel hCPT‐type anticancer agents, two series of hCPT derivatives were synthesized and evaluated in vitro against three human tumor cell lines. The results indicated that the 10‐substituted hCPT derivatives had a considerably higher cytotoxic activity than the 12‐substituted ones. Among the 10‐substituted compounds, 8a, 8b, 9b , and 9i showed an equivalent or even more potent activity than the positive control drug topotecan against the lung cancer cell line A‐549. Moreover, the hCPT analogues 8a and 8b exhibited a higher topoisomerase I inhibitory activity than CPT at a concentration of 100 μM .     

Topotecan Treatment and Its Toxic Effects on Hematologic Parameters and Trace Elements

The aim of this study was to investigate the effects of topotecan, a topoisomerase I-inhibiting anticancer agent, on hematologic parameters and serum levels of trace elements. The study was conducted on three groups consisting of 16 and 18 rabbits in the study groups and 15 rabbits in the control group. Rabbits in group I (n = 16) received high-dose topotecan intravenously (i.v.; 0.5 mg/kg once daily), while rabbits in group II (n = 18) received low-dose topotecan i.v. (0.25 mg/kg once daily) for 3 days. The 15 rabbits comprising the control group did not receive topotecan. Serum samples were collected from each rabbit on the first day, before the treatment, and on the 15th day of treatment. Erithrocytes, hemoglobin, white blood cell count, thrombocyte count, and trace elements such as selenium, copper, lead, zinc, and cobalt were analyzed. Hemoglobin levels and erythrocyte counts were lower in both study groups than in the control group. However, thrombocyte and leukocyte counts were similar in all three groups (p > 0.005). Serum trace element levels (copper, lead, zinc, and cobalt) did not differ significantly between groups. However, serum selenium levels were significantly lower in both study groups than the control group (p < 0.001). The results revealed that topotecan treatment causes a decrease in erythrocyte counts and hemoglobin levels due to bone marrow suppression, and these effects must be taken into account during treatment. In addition, selenium supplementation might be helpful in cancer patients receiving topotecan to increase the effect of the chemotherapeutic agent.     

Separation methods for camptothecin and related compounds

This paper reviews working procedures for the analytical determination of camptothecin and analogues. We give an overview of aspects such as the chemistry, structure–activity relationships, stability and mechanism of action of these antitumor compounds. The main body of the review describes separation techniques. Sample treatment and factors influencing high-performance liquid chromatography development are delineated. Published high-performance liquid chromatographic methods are summarized to demonstrate the variability and versatility of separation techniques and a critical evaluation of separation efficiency, detection sensitivity and specificity of these methods is reported.     

Differential gene expression in anticancer drug- and TRAIL-mediated apoptosis in renal cell carcinomas

Renal cell carcinomas (RCC) exhibit marked differences in susceptibility towards anticancer drug- and TRAIL-induced apoptosis. However, the underlying mechanisms determining apoptosis-sensitivity or -resistance are not well understood. The purpose of this study was to compare gene expression patterns induced by DNA-damage- and death receptor-induced apoptosis and to detect differentially expressed genes responsible for differences in apoptosis-susceptibility. Therefore, we performed a comparative cDNA-array analysis in an apoptosis-resistant and an apoptosis-sensitive RCC cell line. In the sensitive cell line an upregulation of multiple E2F1- and p53-inducible proapaptotic and cell-cycle regulating target genes by Topotecan as well as TRAIL was observed. Interestingly, several antiapoptotic NFκB-dependent target genes were also induced. In the resistant cell line, however, only a small number of E2F1-, p53- and NFκB-dependent target genes were differentially regulated. Conclusively, anticancer drug- as well as TRAIL-sensitivity go along with an upregulation of multiple proapoptotic genes. In contrast, the mechanisms of apoptosis-resistance are—at least in part—located upstream of gene induction and seem not to depend upon upregulation of de-novo-synthesized antiapoptotic genes. Conclusively, the proapoptotic stimuli are confronted with a cellular context which allows apoptosis to be conducted—in the sensitive cell line—or not—in the resistant cell line. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Sebastian Heikaus and Ercan Casliskan contributed equally to this work. This work was supported by the ’Deutsche Forschungsgemeinschaft (DFG).     

Topotecan enhances immune clearance of gliomas

Despite aggressive surgery, radiation therapy, and chemotherapy, glioblastoma multiforme (GBM) is refractory to therapy, recurs quickly, and results in a median survival time of only 14 months. The modulation of the apoptotic receptor Fas with cytotoxic agents could potentiate the response to therapy. However, Fas ligand (FasL) is not expressed in the brain and therefore this Fas-inducing cell death mechanism cannot be utilized. Vaccination of patients with gliomas has shown promising responses. In animal studies, brain tumors of vaccinated mice were infiltrated with activated T cells. Since activated immune cells express FasL, we hypothesized that combination of immunotherapy with chemotherapy can activate Fas signaling, which could be responsible for a synergistic or additive effect of the combination. When we treated the human glioma cell line U-87 and GBM tumor cells isolated from patients with TPT, Fas was up regulated. Subsequent administration of soluble Fas ligand (sFasL) to treated cells significantly increased their cell death indicating that these Fas receptors were functional. Similar effect was observed when CD3⁺ T cells were used as a source of the FasL, indicating that the up regulated Fas expression on glioma cells increases their susceptibility to cytotoxic T cell killing. This additive effect was not observed when glioma cells were pre-treated with temozolomide, which was unable to increase Fas expression in tumor. Inhibition of FasL activity with the antagonistic antibody Nok-1 mitigated these effects confirming that these responses were specifically mediated by the Fas-FasL interaction. Furthermore, the CD3⁺ T cells co-cultured with topotecan treated U-87 and autologous GBM tumor cells showed a significant increase in expression in IFN-γ, a key cytokine produced by activated T cells, and accordingly enhanced tumor cytotoxicity. Based on our data we conclude that drugs, such as topotecan, which cause up regulation of Fas on glioma cells can be potentially exploited with immunotherapy to enhance immune clearance of tumors via Fas signaling. Jun Wei and Guillermo DeAngulo are Co-lead authors.     

High-performance liquid chromatographic assay for the determination of a semisynthetic avermectin analog (eprinomectin) in bovine milk at parts per billion levels--method development and validation

A reversed-phase HPLC method for the quantitative determination of total topotecan in human whole blood and unwashed erythrocytes has been developed and validated in terms of sensitivity, specificity, precision and accuracy. Linear calibration curves were constructed in the range of 0.20 to 50.0 ng/ml. The sample pre-treatment for whole blood involved a two-step extraction with methanol and perchloric acid. Prior to extraction, erythrocytes were separated from other blood components by centrifugation in MESED instruments. Separations were achieved on an Inertsil ODS-80A analytical column (150x4.6 mm, 5 microm particle size), eluted at 50 degrees C and a flow-rate of 1.00 ml/min, with a mixture of 100 mM ammonium acetate (pH 6.0)-tetrahydrofuran (94.6:5.4, v/v). Fluorescence detection was performed using excitation and emission wavelengths of 381 and 525 nm, respectively. With the applied method, 80% of topotecan was extracted out of whole blood. The lower limit of quantitation in whole blood was established at 0.20 ng/ml with within-run and between-run precisions, respectively, ranging from 1.7 to 9.3% and 1.5-6.1%, while the accuracy ranged from 100 to 113%. The described method will be used in clinical studies to explore the role of erythrocytes in the overall kinetic behavior of topotecan.     

Exploration of the intercellular heterogeneity of topotecan uptake into human breast cancer cells through compartmental modelling

A mathematical multi-cell model for the in vitro kinetics of the anti-cancer agent topotecan (TPT) following administration into a culture medium containing a population of human breast cancer cells (MCF-7 cell line) is described. This non-linear compartmental model is an extension of an earlier single-cell type model and has been validated using experimental data obtained using two-photon laser scanning microscopy (TPLSM). A structural identifiability analysis is performed prior to parameter estimation to test whether the unknown parameters within the model are uniquely determined by the model outputs. The full model has 43 compartments, with 107 unknown parameters, and it was found that the structural identifiability result could not be established even when using the latest version of the symbolic computation software Mathematica. However, by assuming that a priori knowledge is available for certain parameters, it was possible to reduce the number of parameters to 81, and it was found that this (Stage Two) model was globally (uniquely) structurally identifiable. The identifiability analysis demonstrated how valuable symbolic computation is in this context, as the analysis is far too lengthy and difficult to be performed by hand.     

Topotecan对癌细胞系SUD4和DOHH2的拓扑异构酶(Ⅰ,Ⅱ)的毒性作用

Ｔｏｐｏｔｅｃａｎ（ＴＰＴ）是类似天然抗癌药物喜树碱的半合成新药．为了阐明ＴＰＴ对拓扑异构酶的毒性作用,将癌细胞系ＳＵＤ４及ＤＯＨＨ２培养在不同浓度ＴＰＴ的培养基中,分别在培养８ｈ、１８ｈ后取样进行测试．结果表明：ＴＰＴ不仅作用于拓扑酶Ⅰ,也作用于拓扑酶Ⅱ,尽管对酶Ⅱ的影响非常小．应用新的流体细胞测量仪（ＦＡＣＳ－Ｖａｎｔａｇｅ）及免疫分析法对细胞培养时间、ＴＰＴ的浓度与拓扑酶中毒程度的动力学关系进行了研究．上述研究显示ＴＰＴ能影响拓扑异构酶Ⅱ（新观点）,同时强调在抗癌化学治疗中应结合使用酶Ⅰ及酶Ⅱ抑制剂．