Selectivity of compounds isolated from the leaves of Nerium indicum Mill. on various human cancer cell lines.

The leaves of Nerium indicum Mill. have been utilized traditionally to cure cancer. By Bioassay (BST) guided isolation method, six compounds were isolated from the CHCl3 extract of the leaves. Selectivity of these compounds (in 0.6-12,500 ng/ml) was tested on various human cancer (MCF7, EVSA-T, T47D, H226, IGROV, A498, WIDR, M19, HeLa) and normal (Vero) cells in vitro. Doxorubicin and cysplatin were used as positive controls. The result indicated that NiO2D (5alpha-oleandrin) possessed the best cytotoxic effect on HeLa cells (IC50, 8.38 x10(-6) mM) and NiO2C (16, 17-dehidrodeasetil-5alpha-oleandrin) on A498 cells (IC50, 1.43 x 10(-6) mM). Those two compounds were not cytotoxic to normal cell.

Quantification of apoptotic cells with fluorescein isothiocyanate-labeled annexin V in chinese hamster ovary cell cultures treated with cisplatin.

Plasma membrane binding of annexin V was used to detect and quantitate apoptotic cells induced by cytotoxic drug treatment in epithelial cell cultures. Chinese hamster ovary (CHO) cells were incubated for 2 h with the ID90 concentration of Cisplatin (20 microM), and 24, 48, 72, and 96 h later the unfixed cells were stained with fluorescein isothiocyanate (FITC)-conjugated annexin V. The fluorescence signal was quantitated by flow cytometry (FCM). During the early phase of the apoptotic response, the annexin V-binding frequency histograms showed two separate cell populations, a dimly and a brightly fluorescent one. At t = 96 h after drug incubation, when the process of apoptosis was completed, only the brightly fluorescent population was present. A dose-effect relationship could be established between the Cisplatin concentration used in the 2 h incubation and the binding of annexin V on the cell membrane, as estimated by FITC fluorescence. The dimly and brightly fluorescent populations were sorted on the basis of annexin V binding, and assayed for 1) DNA breaks by in situ nick translation assay and DNA content by DNA-propidium iodine fluorescence in a bivariate analysis, 2) membrane integrity by dye exclusion, and 3) morphological characteristics of apoptosis. The dimly fluorescent cell population appeared to represent apoptotic cells in the early phase of the death process, as demonstrated by intact cell membranes, normal DNA content, few DNA breaks, and chromatin condensation. The brightly fluorescent cells predominantly had sub-G1 DNA content, nuclear fragmentation, leaky cell membranes, and probably represent late apoptotic cells. These results demonstrate that cytotoxic drug-induced apoptosis can be quantitated by annexin V binding and that by using this assay early and late apoptotic cells can be identified.

Expression of the multidrug resistance-associated protein (MRP) gene in primary non-small-cell lung cancer.

BACKGROUND: One of the major problems in the cure of advanced non-small-cell lung cancer (NSCLC) is its lack of response to cytotoxic drug treatment, and the mechanisms underlying this intrinsic drug resistance are unclear. PATIENTS AND METHODS: We determined the expression of a newly recognised drug resistance gene, the Multidrug Resistance-associated Protein (MRP) gene, in normal lung tissue and in tumour biopsies from 35 surgically resected NSCLCs (11 adenocarcinomas, 24 squamous cell carcinomas). MRP mRNA levels were quantitated by RNase protection assay and expression of the MRP Mr 190,000 glycoprotein was estimated by immunohistochemistry. RESULTS: Using the MRP-specific monoclonal antibody MRPr1, MRP expression was detected by immunohistochemistry in epithelial cells lining the bronchi in normal lung. In NSCLC approximately 35% of the samples showed elevated MRP mRNA levels. Based on MRP-specific immunohistochemical staining the tumours were divided into 4 groups: 12% were scored as negative (-), 14% showed weak cytoplasmic staining of the tumour cells (+/-), 40% had a clear cytoplasmic staining (+), and in 34% a strong cytoplasmic as well as membranous staining was observed (++). MRP expression, as estimated by immunohistochemistry, correlated with the MRP mRNA levels quantitated by RNase protection assay (correlation coefficient = 0.745, p = 0.0009), with MRP mRNA levels (mean +/- SD) of 3.0 +/- 1.0 U, 3.5 +/- 0.7 U, 7.5 +/- 5.9 U, and 19.3 +/- 10.7 U, in the (-), (+/-), (+), and (++) immunohistochemistry expression groups, respectively. Among the squamous cell carcinomas a correlation was observed between MRP staining and tumour cell differentiation: the strongest MRP staining was predominantly found in the well differentiated tumours. CONCLUSIONS: Hyperexpression of MRP is frequently observed in primary NSCLC, especially in the well differentiated squamous cell carcinomas. Further studies are needed to assess the role of MRP in the mechanism of clinical drug resistance in NSCLC.

Constitutive expression of the c-H-ras oncogene inhibits doxorubicin-induced apoptosis and promotes cell survival in a rhabdomyosarcoma cell line.

Drugs used in anti-cancer chemotherapy are thought to exert their cytotoxic action by induction of apoptosis. Genes have been identified which can mediate or modulate this drug-induced apoptosis, among which are c-myc, p53 and bcl-2. Since expression of oncogenic ras genes is a frequent observation in human cancer, we investigated the effects of the c-H-ras oncogene on anti-cancer drug-induced apoptosis. Apoptosis induced by a 2 h doxorubicin exposure was measured by in situ nick translation and flow cytometry in a rat cell line (R2T24) stably transfected with the c-H-ras oncogene and in a control cell line (R2NEO) transfected only with the antibiotic resistance gene neo. Both cell lines (R2T24 and R2NEO) had nearly identical growth characteristics, including cell doubling time, distribution over the cell cycle phases and plating efficiency in soft agar. Doxorubicin exposure of the R2NEO cells led to massive induction of apoptosis. In contrast, R2T24 cells, expressing the c-H-ras oncogene, showed significantly less apoptosis after doxorubicin incubation. Doxorubicin induced approximately 3- to 5-fold less cytotoxicity in the R2T24 cells than in the R2NEO cells, as determined by clonogenic assay in soft agar. No difference was observed in intracellular doxorubicin accumulation between the two cell lines, indicating that the classical, P-glycoprotein-mediated multidrug resistance phenotype is not involved in the observed differences in drug sensitivity. In conclusion, our data show that constitutive expression of the c-H-ras oncogene suppresses doxorubicin-induced apoptosis and promotes cell survival, suggesting that human tumours with ras oncogene expression might be less susceptible to doxorubicin treatment.

Expression of the multidrug resistance-associated protein (MRP) in acute and chronic leukemias.

We determined the expression of the multidrug resistance-associated protein (MRP), a new putative transmembrane drug transporter, in peripheral blood cells from healthy volunteers as well as from 60 patients with acute or chronic leukemia, using an RNase protection assay. MRP appeared to be ubiquitously expressed at low levels in all nonmalignant hemopoietic cell types, reflecting its basal constitutive expression. In acute myelocytic leukemia (AML) (n = 16), one of nine untreated patients and two of seven patients with prior chemotherapy showed significant hyperexpression of MRP. In chronic lymphocytic leukemia (CLL) (n = 21), either treated (n = 8) or untreated (n = 13), a high percentage (15 of 21: 71% had relatively high expression levels of the MRP gene. In contrast, low MRP expression levels were detected in acute lymphocytic leukemia (n = 14), and in chronic myelocytic leukemia (n = 9). DNA analysis by Southern blotting did not reveal amplification of the MRP gene in the leukemia samples, including those with elevated MRP mRNA levels. We conclude that relatively high expression of MRP is occasionally observed in AML and at high frequency in CLL, irrespective of treatment, probably due to transcriptional activation and/or increased mRNA stability.