Cisplatin-resistant Ovarian Carcinoma Cell Research Articles

120 Synthesis and cytoxicity studies of platinum nucleobase adducts

A new Au(III) coordination compound with the ligand 2-(phenylazo)pyridine has been synthesized and fully characterized by means of elemental analysis, IR, UV–visible, conductivity measurements, NMR, electrospray ionization (ESI-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES). The chemical stability of the cation in this compound, [Au(azpy)Cl2]+ (abbreviated: Au-azpy), was analyzed by means of several physicochemical methods. While stable in the solid state, stability studies performed with the gold compound in solution showed an unexpected and unprecedented reactivity. A cationic organic derivative of 2-(phenylazo)pyridine, (abbreviated: pyrium), was produced from the solution and has been isolated as its chloride salt and characterized by crystal structure determination, elemental analysis, NMR, ESI-MS and conductivity studies in solution. This cyclization reaction is reported for the first time in the case of gold coordination compounds.The Au adduct and the pyrium cation were investigated as potential cytotoxic and anticancer agents, and both show moderate to high cytotoxic properties in cisplatin-sensitive and cisplatin-resistant ovarian carcinoma cell lines, A2780; and cisplatin-sensitive and cisplatin-resistant murine lymphocytic leukemia cell lines, L1210. Significant anticancer activity against the cisplatin resistant cell lines was found for the pyrium salt, ruling out the occurrence of cross resistance phenomena.

Cisplatin May Induce Frataxin Expression

Cisplatin is a widely used drug in cancer chemotherapy and resistance to cisplatin is a major limitation for its successful application. Intracellular inactivation of cisplatin and detoxification of reactive oxygen species (ROS) by glutathione (a crucial cellular antioxidant) is a mechanism for cisplatin resistance. During cDNA microarray analyses of differential gene expression between a cisplatin-resistant A2780CP70 human ovarian carcinoma cell line and its parental A2780 cell line, we discovered that frataxin gene expression was frequently overexpressed in the cisplatin-resistant variant. Decreased expression of frataxin protein is associated with Friedreich's ataxia (FRDA) which is a neurodegenerative disease involving ROS-mediated cellular damage. Recent evidence suggests that frataxin might detoxify ROS via activation of glutathione peroxidase and elevation of thiols. To exploit potential involvement of frataxin gene in the development of resistance to cisplatin, we compared the levels of frataxin gene and protein in the cisplatin-resistant A2780CP70 ovarian carcinoma cell line and its parental A2780 cell line. We found that frataxin mRNA and protein expressions were elevated in the cisplatin-resistant cells. Our results suggest a potential role for cisplatin as an inducer of frataxin expression and implies that this gene may be a potential target for modulating the response to cisplatin. This is the first report showing an association between frataxin expression and cisplatin resistance.

Assessment of gene expression profile by cDNA microarray analysis.

With the advent of cDNA microarray technology, variations in cellular transcriptional events can now be monitored simultaneously, and expression profile at each functional state can be established and compared to one another. Here we illustrate gene expression profile analysis of a cisplatin-resistant ovarian carcinoma cell line (A2780CP)and its parental A2780 cell line, and compare them to assess changes in gene expression related to resistance to cisplatin. Equal quantities(1 μg)of RNA from the cell lines are reverse-transcribed, labeled with P radioisotpe, and hybridized to ovary specific GeneFilters carrying more than 5,000 genes(GF223; Research Genetics, Huntsville, AL). Filter images are recorded by a phosphor-imager and analyzed comparatively using Pathways 3.0 software(Research Genetics). The upregulated and downregulated genes in cisplatin-resistant cells are identified and changes in selected genes are validated by a semiquantitative RT-PCR. This approach allows us to identify groups of genes, rather than just single genes, involved in a process, and can indicate which molecular and cellular events might be critical in such complex biological processes.

Cisplatin-induced response of c-jun N-terminal kinase 1 and extracellular signal-regulated protein kinases 1 and 2 in a series of cisplatin-resistant ovarian carcinoma cell lines

The cellular response to cisplatin involves activation of multiple signal transduction pathways, including the mitogen-activated protein (MAP) kinase pathways. In this study, we compared the cisplatin-induced activation of two MAP kinases, c-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), in the cisplatin-sensitive ovarian carcinoma cell line A2780 and its derivative cisplatin-resistant cell lines CP70 and C200. Dose-dependent and time-dependent activation of JNK1 and ERK1/2 occurred in each of the three cell lines in response to cisplatin treatment. The requirement of higher concentrations of cisplatin for induction of maximum activation of JNK1 and ERK1/2 was correlated with increased levels of cisplatin resistance. In addition, inhibition of cisplatin-induced ERK activation, using the MAP/ERK kinase 1 synthetic inhibitor PD98059, resulted in enhanced sensitivity to cisplatin in all three cell lines. These results suggest that cisplatin-induced ERK1/2 activity is not responsible for the acquired cisplatin resistance in CP70 and C200 cells but rather provides a general cytoprotective effect in both cisplatin-sensitive and cisplatin-resistant cell lines. In conclusion, different patterns of cisplatin-induced JNK1 and ERK1/2 activation are observed in cell lines with different levels of cisplatin sensitivity, and inhibition of cisplatin-induced ERK1/2 activation enhances sensitivity to cisplatin in both cisplatin-sensitive and cisplatin-resistant cell lines.

Antitumor activity of a novel glyco‐nitric oxide conjugate in ovarian carcinoma

BACKGROUND Several studies have shown that nitric oxide (NO)-releasing agents can kill tumor cells. Unfortunately, currently available NO delivery molecules do not target tumor cells preferentially. To exploit the overexpression of glucose transport proteins and the high level of glucose transport characteristics of tumor cells, glucose was conjugated to S-nitroso-N-acetyl-penicillamine (2-gluSNAP) and evaluated for cytotoxicity in human ovarian carcinoma cells. METHODS The cytotoxicity of 2-gluSNAP and SNAP was assessed by clonogenic cell survival assays performed in A2780S (cisplatin sensitive) and A2780cP (cisplatin-resistant) ovarian carcinoma cells in vitro. Immunoblotting and immunohistochemistry were used to assess the expression of Glut-1 hexose transport protein in the cell lines as well as in paraffin blocks from 28 surgical specimens of epithelial ovarian carcinoma. Apoptosis was assessed by an end-labeling assay. RESULTS The ovarian carcinoma cell lines consistently were more sensitive to 2-gluSNAP than SNAP alone. The median effective doses (MEDs) for 2-gluSNAP and SNAP in the A2780s cell line were 0.0042 μM and 20.4 μM, respectively. Therefore, 2-GluSNAP was nearly 5000-fold more potent than the NO-donating moiety (SNAP) alone. In the A2780cP cells, the MED for 2-gluSNAP (0.38 μM) was 250-fold lower than that for SNAP alone (100 μM). Immunoblotting and immunohistochemistry studies showed overexpression of Glut-1 in the cell lines and in 23 of 28 epithelial ovarian carcinoma specimens. CONCLUSIONS The novel glyco-NO conjugate 2-gluSNAP exhibits a much greater cytotoxicity than the parent NO donor without the hexose moiety. These agents have the potential to target tumor cells preferentially, that overexpress Glut-1. This transporter is expressed highly in epithelial ovarian carcinoma. Cancer 2000;88:381–8. © 2000 American Cancer Society.

Antitumor activity of a novel glyco-nitric oxide conjugate in ovarian carcinoma

Several studies have shown that nitric oxide (NO)-releasing agents can kill tumor cells. Unfortunately, currently available NO delivery molecules do not target tumor cells preferentially. To exploit the overexpression of glucose transport proteins and the high level of glucose transport characteristics of tumor cells, glucose was conjugated to S-nitroso-N-acetyl-penicillamine (2-gluSNAP) and evaluated for cytotoxicity in human ovarian carcinoma cells. The cytotoxicity of 2-gluSNAP and SNAP was assessed by clonogenic cell survival assays performed in A2780S (cisplatin sensitive) and A2780cP (cisplatin-resistant) ovarian carcinoma cells in vitro. Immunoblotting and immunohistochemistry were used to assess the expression of Glut-1 hexose transport protein in the cell lines as well as in paraffin blocks from 28 surgical specimens of epithelial ovarian carcinoma. Apoptosis was assessed by an end-labeling assay. The ovarian carcinoma cell lines consistently were more sensitive to 2-gluSNAP than SNAP alone. The median effective doses (MEDs) for 2-gluSNAP and SNAP in the A2780s cell line were 0.0042 microM and 20.4 microM, respectively. Therefore, 2-GluSNAP was nearly 5000-fold more potent than the NO-donating moiety (SNAP) alone. In the A2780cP cells, the MED for 2-gluSNAP (0.38 microM) was 250-fold lower than that for SNAP alone (100 microM). Immunoblotting and immunohistochemistry studies showed overexpression of Glut-1 in the cell lines and in 23 of 28 epithelial ovarian carcinoma specimens. The novel glyco-NO conjugate 2-gluSNAP exhibits a much greater cytotoxicity than the parent NO donor without the hexose moiety. These agents have the potential to target tumor cells preferentially, that overexpress Glut-1. This transporter is expressed highly in epithelial ovarian carcinoma.

Influence of the proto-oncogene c- fos on cisplatin sensitivity

Cisplatin resistance has been associated with overexpression of the c- fos gene in a human ovarian carcinoma cell line. To determine whether the correlation between c- fos overexpression and cisplatin resistance was limited to this cell line or was a more generalized phenomenon, we investigated cisplatin sensitivity in rat fibroblast cells that overexpressed the c- fos gene. The cisplatin ic 50 values for two different c- fos transfectants, CMVc- fos and L1–3c- fos, were 7.6 ± 0.8 and 5.6 ± 1.0 μM, respectively, whereas the cisplatin ic 50 value for the parental line, 208F, was 2.4 ± 0.1 μM. This represented a 3.2- and 2.3-fold resistance to cisplatin for CMVc- fos and L1–3c- fos cells, respectively. The correlation between c -fos expression and cisplatin resistance also was examined in a human ovarian carcinoma cell line, 2008, and its cisplatin-resistant variant, C13∗. Expression of c- fos was elevated slightly at both the mRNA and protein levels in the C13∗ cells compared with 2008 cells, and c-Fos protein levels were induced in C13∗ cells following cisplatin treatment. In addition, it was observed that C13∗ cells were significantly more sensitive than 2008 cells to a c- fos antisense oligonucleotide. The ic 50 values for the c- fos antisense oligonucleotide were 19.9 ± 5.0 pmol for C13∗ cells and 58.1 ± 6.0 pmol for 2008 cells ( P = 0.0012). Furthermore, combinations of c- fos antisense and cisplatin reduced the amount of cisplatin required to kill 50% of the C13∗ cells, although the interaction was not synergistic. These results suggest that expression of the c- fos gene can influence cisplatin sensitivity, and that c- fos antisense oligonucleotide based therapy may be effective at killing parental and cisplatin-resistant ovarian carcinoma cells, either alone or in combination with cisplatin.

Comparative genomic hybridization analysis of cisplatin-resistant ovarian carcinoma cells.

Comparative genomic hybridization analysis of cisplatin-resistant ovarian carcinoma cells.

Expression and intracellular localization of heat shock proteins in multidrug resistance of a cisplatin resistant human ovarian cancer cell line.

TYK-R10 is a cisplatin resistant human ovarian carcinoma cell line and showed a cross resistance to various anti-cancer drugs including adriamycin (ADR), vincristine (VCR) and etoposide, despite a lack of multidrug phenotype. Under normal conditions, various heat shock proteins (HSPs) were expressed in TYK-R10 but not in parental line (TYK-nu). Non-lethal short-term heat shock treatment induced a high tolerance for cisplatin and VCR in TYK-R10 and ADR, and VCR in TYK-nu. This treatment induced and/or enhanced the expression of various types of HSPs in various intracellular localizations in both TYK-R10 and TYK-nu, with minor differences. These findings indicate that combined expression and intracellular localization of HSPs may play an important role in drug resistance of TYK-R10.

Radiotherapy as a cisplatin-sensitizer in a resistant ovarian carcinoma cell line

Stage III ovarian carcinoma has shown resistance to adjuvant chemotherapy following surgical cytoreduction. With recurrence of ovarian carcinoma, cell lines may develop resistance to previously used chemotherapy. This contributes to the fact that survival rates for patients with ovarian carcinoma have not been dramatically improved in decades. The objective of this study is to evaluate radiotherapy as a cisplatin-sensitizer in a cisplatin-resistant ovarian carcinoma cell line. In vitro OVCAR-3 human ovarian carcinoma cells were irradiated with external beam radiation (XRT) at doses of 500, 1,500, and 4,500 centigray (cGy) in a single fractionation. Twelve hours after XRT, cells were treated with a dose of cisplatin for 2 hours (0, 1, 3, 9, and 90 micrograms/mL). Cell attachment was determined by cell counts using a hemocytometer under phase-contrast microscopy. Analysis of variance followed by the Student Newman Keuls Test were used for statistical analysis. Dose-response curves demonstrate the results of this study as follows: (1) XRT has a significant direct effect on cell attachment of OVCAR-3 cells in a dose-response relationship. (2) cisplatin has no effect on cell attachment in the absence of XRT. (3) When cells are exposed to XRT, cisplatin demonstrates a dose-response effect on cell attachment with a dose of XRT as low as 500 Gy. This in vitro study suggests that XRT sensitizes cisplatin-resistant OVCAR-3 to cisplatin. This occurred with doses of radiation low enough to suggest a potential clinical role in treating resistant ovarian carcinoma.

Cyclosporin A suppresses cisplatin-induced c-fos gene expression in ovarian carcinoma cells.

Cisplatin-resistant A2780DDP human ovarian carcinoma cells, 1 and 3 h following cisplatin treatment, display 3.2-6.1-fold enhancement of mRNA expression for oncogenes c-fos and c-H-ras and for enzymes necessary for DNA synthesis and repair. In contrast, c-myc mRNA was partially decreased. Increased transcription for the aforementioned genes accounts for the higher levels of mRNA since the enhancement is susceptible to actinomycin D pretreatment. Cyclosporin A (CSA) has been shown to reverse resistance to a variety of antineoplastic agents. An 18-h treatment with CSA yielded a 6-fold improved sensitivity to cisplatin in A2780DDP cells. When A2780DDP cells are pretreated with CSA followed by cisplatin, there is no increase in mRNA for c-fos and c-H-ras and the genes for thymidine synthesis and DNA repair. Their expression approaches the levels demonstrated in A2780S cells. In contrast, c-myc mRNA was elevated 7.4-fold in the presence of CSA in A2780DDP cells. Long term weekly exposures of A2780DDP cells to CSA resulted in the evolution of a revertant cell line, A2780DDP/CSA, with decreased c-fos and dTMP synthase mRNA. Thus, cisplatin treatment in A2780DDP cells differentially induces expression of certain nuclear oncogenes (c-fos/c-myc), as well as genes possibly involved in the repair of cisplatin-mediated DNA damage, an induction suppressed by CSA treatment.