Comparison of cisplatin and carboplatin cytotoxicity in human ovarian cancer cell lines using the MTT assay

doi:10.1016/0090-8258(90)90416-I

Gynecologic Oncology

Volume 39, Issue 2, November 1990, Pages 119-122

https://doi.org/10.1016/0090-8258(90)90416-I Get rights and content

Abstract

In this study, we compared the cytotoxicity of cisplatin and carboplatin against a panel of human ovarian cancer cell lines using the MTT assay, a rapid colorimetric test that can be used to evaluate the number of residual viable tumor cells following chemotherapy. The established human ovarian cancer cell line OVCAR-3 and the recently isolated and characterized A721, A90, A286, A1, and A121A cell lines were evaluated for chemosensitivity. Each cell line was treated separately with cisplatin and carboplatin at concentrations ranging from 500 to 0.16 μg/ml. Various chemotherapeutic exposure periods (1, 4, 24, and 48 hr) were tested to determine maximal efficacy. All cell lines were more susceptible to cisplatin than carboplatin at all drug concentrations and all exposure periods tested (P = 0.005). The overall median 50% inhibitory concentration (ID₅₀) for cisplatin was 107 μg/ml compared with 490 μg/ml for carboplatin (P = 0.005). For both cisplatin and carboplatin a 24-hr exposure was significantly more cytotoxic than a 1-hr exposure (P = 0.003 and P = 0.006, respectively). These in vitro results suggest that cisplatin is significantly more cytotoxic than carboplatin against human ovarian cancer cell lines and that cisplatin should not be replaced by carboplatin in the treatment of advanced epithelial ovarian cancer until randomized trials using maximum dosing of the cisplatin-containing regimen are performed.

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Specific point mutations in key redox enzymes are associated with chemoresistance in epithelial ovarian cancer
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Citation Excerpt :
The point mutation induced cells were then utilized for assessment of IC50 for cisplatin by regression analysis. Viability was determined using the TACS MTT Cell Proliferation Assay (Trevigen) per the manufacturer's protocol, and as described [41]. A standard curve was constructed using each respective cell line.
Oxidative stress plays an important role in the pathophysiology of ovarian cancer. Resistance to chemotherapy presents a significant challenge for ovarian cancer treatment. Specific single nucleotide polymorphisms (SNPs) in key redox enzymes have been associated with ovarian cancer survival and progression. The objective of this study was to determine whether chemotherapy induces point mutations in key redox enzymes that lead to the acquisition of chemoresistance in epithelial ovarian cancer (EOC). Human EOC cell lines and their chemoresistant counterpart were utilized for this study. Specific SNPs in key redox enzymes were analyzed by TaqMan SNP Genotyping. Activities and levels of key redox enzymes were determined by real-time RT-PCR, ELISA and a greiss assay. Point mutations in key redox enzymes were introduced into sensitive EOC cells via the CRISPR/Cas9 system. Cell viability and IC₅₀ for cisplatin were determined by the MTT Cell Proliferation Assay. Data was analyzed with SPSS using Student's two-tailed t-tests and One-way ANOVA followed by Dunnett's or Tukey's post hoc tests, p<0.05. Here, we demonstrate that chemoresistant EOC cells are characterized by a further enhancement in oxidative stress as compared to sensitive counterparts. Additionally, chemoresistant EOC cells manifested specific point mutations, which are associated with altered enzymatic activity, in key redox enzymes that are not detected in sensitive counterparts. Supplementation of an antioxidant was able to successfully sensitize EOC cells to chemotherapeutics. Causality was established by the induction of these point mutations in sensitive EOC cells, which resulted in a significant increase in the level of chemoresistance. These findings indicate that chemotherapy induces specific point mutations in key redox enzymes that contribute to the acquisition of chemoresistance in EOC cells, highlighting a potential novel mechanism. Identification of targets for chemoresistance with either biomarker and/or screening potential will have a significant impact for the treatment of this disease.
URI Is an Oncogene Amplified in Ovarian Cancer Cells and Is Required for Their Survival
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Citation Excerpt :
For cell viability MTT assay, cells were seeded at high density in a 96-well format (NUNC) and were treated for 24 hr with the indicated concentrations of cisplatin (SIGMA). MTT viability assay was performed as described elsewhere (Fanning et al., 1990). Details regarding cell culture and immunoblotting can be found in the Supplemental Experimental Procedures.
Abrogation of negative feedback control represents a fundamental requirement for aberrantly activated signaling pathways to promote malignant transformation and resistance to therapy. Here we identify URI, which encodes a mitochondrial inhibitor of PP1γ and PP1γ-mediated feedback inhibition of S6K1-BAD survival signaling, as an oncogene amplified and overexpressed in ovarian cancer cell lines and human ovarian carcinomas. URI is an “addicting” oncogene selectively required for the survival of ovarian cancer cells with increased URI copy number. By constitutively detaining PP1γ in inactive complexes, URI sustains S6K1 survival signaling under growth factor–limiting conditions and mediates resistance of cells to cisplatin. Thus, oncogenic activation of URI defines an important mechanism for activating mitochondrial S6K1-BAD signaling and promoting cell survival through disabling PP1γ-dependent negative feedback inhibition.
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Background: To determine the effect of granulocyte colony-stimulating factor (G-CSF) on tumor growth and cisplatin cytotoxicity in human epithelial ovarian cancer. Methods: Six human epithelial ovarian cancer cell lines were treated with media (control) for 24 hr, G-CSF for 24 hr, cisplatin for 24 hr, simultaneous cisplatin and G-CSF for 24 hr, media (control) for 48 hr, cisplatin for 24 hr and sequential media for 24 hr, cisplatin for 24 hr, and sequential G-CSF for 24 hr. Following incubation, the percentage cell lysis was determined by particle concentration fluorescein immunoassay chemosensitivity assay. Results: G-CSF resulted in tumor cell growth (7–19%) in three cell lines. Simultaneous G-CSF decreased cisplatin cytotoxicity in five cell lines (7–45%). Sequential G-CSF increased cisplatin cytotoxicity in all six cell lines (5–108%). Conclusions: G-CSF may result in ovarian cancer proliferation. Simultaneous G-CSF may decrease cisplatin cytotoxicity, while sequential G-CSF appears to increase cisplatin cytotoxicity.
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Regular articleComparison of cisplatin and carboplatin cytotoxicity in human ovarian cancer cell lines using the MTT assay

Abstract

Gynecol. Oncol.

Cancer Treat. Rev.

Eur. J. Cancer Clin. Oncol.

Ca

Regular article
Comparison of cisplatin and carboplatin cytotoxicity in human ovarian cancer cell lines using the MTT assay