Effects of Cisplatin on Cell Cycle Kinetics, Morphological Change, and Cleavage Pattern of DNA in Two Human Ovarian Carcinoma Cell Lines

Sekiguchi, Isao; Suzuki, Mitsuaki; Tamada, T; Shinomiya, Nariyoshi; Tsuru, Sumiaki; Murata, Masaya

doi:10.1159/000227529

Cited by 18 publications

(9 citation statements)

References 10 publications

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“…Regarding the underlying mechanisms, Dimanche-Boitrel's [29] and Itamochi's groups [31] proposed that an increased number of resting (non-proliferating) cells may contribute to elevated chemoresistance at higher seeding densities. However, although our FCM analysis supports the hypothesis that dividing cells (G2-M phase) are major targets of cisplatin [32–34], we could not verify that the cell cycle plays a central role in the density-dependent variation in IC 50 values (Figure 4B and 4C). Wu's group explained this phenomenon from the perspective of density-related apoptosis and autophagy [35].…”

Section: Discussioncontrasting

confidence: 81%

The changing 50% inhibitory concentration (IC50) of cisplatin: a pilot study on the artifacts of the MTT assay and the precise measurement of density-dependent chemoresistance in ovarian cancer

Zhu

Chen

et al. 2016

Oncotarget

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Inconsistencies in the half-maximal (50%) inhibitory concentration (IC50) data for anticancer chemotherapeutic agents have yielded irreproducible experimental results and thus reciprocally contradictory theories in modern cancer research. The MTT assay is currently the most extensively used method for IC50 measurements. Here, we dissected the critical reasons behind MTT-dependent IC50 inconsistencies. We showed that IC50 errors caused by the technical deficiencies of the MTT assay are large and not adjustable (range: 300–11,000%). To overcome severe MTT artifacts, we developed an unbiased direct IC50 measurement method, the limiting dilution assay. This detection technique led us to the discovery of the inherent density-dependent chemoresistance variation of cancer cells, which is manifold and unpredictable in its forms. The subsequent intracellular signaling pathway analysis indicated that pAkt and p62 expression levels correlated with alterations in the IC50 values for cisplatin in ovarian cancer, providing an explainable mechanism for this property. An in situ pAkt-and-p62-based immunohistochemical (IHCpAkt+p62) scoring system was thereby established. Both the limiting dilution assay and the IHCpAkt+p62 scoring system accurately predicted the primary chemoresistance against cisplatin in ovarian cancer patients. Furthermore, two distinct chemoresistant recurrence patterns were uncovered using these novel detection tools, which were linked to two different forms of density-chemoresistance relationships (positively vs. negatively correlated), respectively. An interpretation was given based on the cancer evolution theory. We concluded that the density-related IC50 uncertainty is a natural property of the cancer cells and that the precise measurement of the density-dependent IC50 spectrum can benefit both basic and clinical cancer research fields.

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Section: Discussioncontrasting

confidence: 81%

The changing 50% inhibitory concentration (IC50) of cisplatin: a pilot study on the artifacts of the MTT assay and the precise measurement of density-dependent chemoresistance in ovarian cancer

Zhu

Chen

et al. 2016

Oncotarget

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“…Activation of p53 by cisplatin-induced DNA damage has been reported to have various effects on cellular sensitivity to cisplatin. In some studies, activation of p53 has been shown to provide cytoprotection against cisplatin [21], [22] In contrast, increased resistance to cisplatin with disruption of normal WT p53 function has also been demonstrated [23].…”

Section: Discussionmentioning

confidence: 99%

Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature

Barr

Gray

Hoffmann³

et al. 2013

PLoS ONE

238

180

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IntroductionInherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin.MethodsAn isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed.ResultsCharacterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines.ConclusionOur results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer.

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“…shown to arrest cell cycle in the G2 phase across a range of tumour types and it is the lack of progression across the G2/M transition which leads to apoptotic cell death 467,472,473 . The G2 arrest could be seen as the result of the formation of platinum adducts during the S phase of the cell cycle where DNA is replicated.…”

Section: ) Cisplatin Has Beenmentioning

confidence: 99%

Role of the pro-survival molecule Bfl-1 in melanoma

Hind

Carter

Harris

et al. 2015

The International Journal of Biochemistry & Cell Biology

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Melanoma, the cancer derived from the melanocytes of the skin, is becoming an ever more common cancer in the ageing population of the developed world and displays an intrinsic resistance to current therapies. This chemo resistance makes metastatic melanoma an extremely difficult cancer to treat leading to a 5 year survival rate of just 20%. As such, it is important to unearth new potential drug targets to increase the prospects for melanoma patients.Bfl-1 is a pro-survival protein of the Bcl-2 family of apoptosis regulating proteins. It has been found to be over-expressed in a small subset of chemo resistant tumours, including melanoma. Accordingly, I characterised the molecule in melanoma cells and determined its role in protecting these cells from chemotherapy-induced apoptosis. I elucidated the expression profile and half-lives of the protein and mRNA across a panel of melanoma cell-lines and healthy melanocytes. I also confirmed, using pathway specific inhibitors, that Bfl-1 was transcriptionally regulated by the NFB pathway and concluded through pulsechase experiments that Bfl-1 protein was degraded, at least in part, by the proteasome. Subcellular fractionation and indirect immunofluorescence techniques elucidated that Bfl-1 was located mostly at the mitochondria in both resting and apoptotic cells, with a diffuse level present throughout the cytoplasm. As well as characterising the protein in both melanoma cells and healthy primary melanocytes, I determined its role in the resistance of these cells to apoptosis. Over-expressed Bfl-1 was found to protect melanoma cells from apoptosis caused by a range of chemotherapeutic agents in A375 melanoma cells, which naturally expressed very low levels of Bfl-1. Further, knock down of Bfl-1 using siRNA technology in melanoma cells revealed the dependence of these cells on Bfl-1 for their resistance to certain chemotherapeutic agents currently used in melanoma treatment, including the MEK inhibitor PD901. All together, this research contributes to our understanding of Bfl-1 and highlights it as a potentially important therapeutic target in metastatic melanoma.

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Effects of Cisplatin on Cell Cycle Kinetics, Morphological Change, and Cleavage Pattern of DNA in Two Human Ovarian Carcinoma Cell Lines

Cited by 18 publications

References 10 publications

The changing 50% inhibitory concentration (IC50) of cisplatin: a pilot study on the artifacts of the MTT assay and the precise measurement of density-dependent chemoresistance in ovarian cancer

The changing 50% inhibitory concentration (IC50) of cisplatin: a pilot study on the artifacts of the MTT assay and the precise measurement of density-dependent chemoresistance in ovarian cancer

Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature

Role of the pro-survival molecule Bfl-1 in melanoma

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