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USP1 deubiquitinase: cellular functions, regulatory mechanisms and emerging potential as target in cancer therapy.

García-Santisteban I, Peters GJ, Giovannetti E, Rodríguez JA - Mol. Cancer (2013)

Bottom Line: Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy.We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data.Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, Leioa, Spain.

ABSTRACT
Reversible protein ubiquitination is emerging as a key process for maintaining cell homeostasis, and the enzymes that participate in this process, in particular E3 ubiquitin ligases and deubiquitinases (DUBs), are increasingly being regarded as candidates for drug discovery. Human DUBs are a group of approximately 100 proteins, whose cellular functions and regulatory mechanisms remain, with some exceptions, poorly characterized. One of the best-characterized human DUBs is ubiquitin-specific protease 1 (USP1), which plays an important role in the cellular response to DNA damage. USP1 levels, localization and activity are modulated through several mechanisms, including protein-protein interactions, autocleavage/degradation and phosphorylation, ensuring that USP1 function is carried out in a properly regulated spatio-temporal manner. Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy. This view has gained recent support with the finding that USP1 inhibition may contribute to revert cisplatin resistance in an in vitro model of non-small cell lung cancer (NSCLC). Here, we describe the current knowledge on the cellular functions and regulatory mechanisms of USP1. We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data. Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells.

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Growth inhibition curves and median-drug effect plots of two NSCLC cell lines treated with cisplatin, pimozide or their simultaneous combination. Exponentially growing SW1573 (A) and H1299 (B) NSCLC cells were seeded in 96-well plates. Twenty four hours after seeding, cells were treated with cisplatin, pimozide or a combination of both drugs. After 72 hours of drug exposure, the sulforhodamine B (SRB) chemosensitivity assay [97] was performed. Briefly, cells were fixed with ice-cold trichloroacetic acid, washed with deionized water, left to dry and stained with SRB dye. After washing with 1% acetic acid, cells were left to dry, and protein-bound dye was dissolved in 10 mM Tris base solution. OD at 492 nm was determined. Growth inhibition curves (upper panels) were generated, where each point represents mean ± SEM of at least 3 replicates. IC50 values, the drug concentration that inhibits the cell growth by 50% (summarized in Table 3), were calculated by fitting the data to a sigmoid dose–response curve using GraphPad Prism. The treatment with the two drugs in combination was carried out using a constant cisplatin:pimozide ratio (as indicated in Table 3), which was established for each cell line as the ratio of the IC50 values for each single drug. The drug concentrations on the X-axis of the growth inhibition curves for the combination treatment refer to cisplatin. In order to evaluate the interaction between cisplatin and pimozide we used the median-drug effect analysis method originally described by Chou and Talalay [95]. Using CalcuSyn, the effect of the combination was compared to the effect of each drug to determine the combination index (C.I.) at a range of Fa (fraction affected by the dose) values. The resulting C.I. values were plotted against the Fa to produce the median-drug effect plot for each cell line (lower panels).
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Figure 4: Growth inhibition curves and median-drug effect plots of two NSCLC cell lines treated with cisplatin, pimozide or their simultaneous combination. Exponentially growing SW1573 (A) and H1299 (B) NSCLC cells were seeded in 96-well plates. Twenty four hours after seeding, cells were treated with cisplatin, pimozide or a combination of both drugs. After 72 hours of drug exposure, the sulforhodamine B (SRB) chemosensitivity assay [97] was performed. Briefly, cells were fixed with ice-cold trichloroacetic acid, washed with deionized water, left to dry and stained with SRB dye. After washing with 1% acetic acid, cells were left to dry, and protein-bound dye was dissolved in 10 mM Tris base solution. OD at 492 nm was determined. Growth inhibition curves (upper panels) were generated, where each point represents mean ± SEM of at least 3 replicates. IC50 values, the drug concentration that inhibits the cell growth by 50% (summarized in Table 3), were calculated by fitting the data to a sigmoid dose–response curve using GraphPad Prism. The treatment with the two drugs in combination was carried out using a constant cisplatin:pimozide ratio (as indicated in Table 3), which was established for each cell line as the ratio of the IC50 values for each single drug. The drug concentrations on the X-axis of the growth inhibition curves for the combination treatment refer to cisplatin. In order to evaluate the interaction between cisplatin and pimozide we used the median-drug effect analysis method originally described by Chou and Talalay [95]. Using CalcuSyn, the effect of the combination was compared to the effect of each drug to determine the combination index (C.I.) at a range of Fa (fraction affected by the dose) values. The resulting C.I. values were plotted against the Fa to produce the median-drug effect plot for each cell line (lower panels).

Mentions: In an attempt to extend these findings to a larger number of NSCLC cell lines, we tested the effect of pimozide in combination with cisplatin on a panel of seven NSCLC cell lines. A cell viability assay was used to determine the cytotoxic activity of cisplatin, pimozide or a combination of both agents. Figure 4 shows representative examples of the results of this assay in two NSCLC cell lines, and the data for the remaining cell lines analyzed are shown in Additional file 2: Figure S1. The C.I. was subsequently calculated using Calcusyn. The cell lines tested displayed a wide range of cisplatin sensitivity, as summarized in Table 3, and were classified as “ciplatin-sensitive” if their IC50 was below the mean IC50 of the panel (6.2 μM), or as “cisplatin-resistant” if their IC50 was above the mean. Pimozide showed a synergistic or additive effect with cisplatin in the three “resistant” cell lines (H1299, H1703 and H520). In contrast, the effect of the pimozide/cisplatin combination was antagonistic in three of the four “sensitive” cell lines (H322, SKLU-1 and SW1573). These findings are consistent with those of Chen et al. [94], and suggest that USP1 inhibition may contribute to revert cisplatin resistance in some preclinical models of NSCLC.


USP1 deubiquitinase: cellular functions, regulatory mechanisms and emerging potential as target in cancer therapy.

García-Santisteban I, Peters GJ, Giovannetti E, Rodríguez JA - Mol. Cancer (2013)

Growth inhibition curves and median-drug effect plots of two NSCLC cell lines treated with cisplatin, pimozide or their simultaneous combination. Exponentially growing SW1573 (A) and H1299 (B) NSCLC cells were seeded in 96-well plates. Twenty four hours after seeding, cells were treated with cisplatin, pimozide or a combination of both drugs. After 72 hours of drug exposure, the sulforhodamine B (SRB) chemosensitivity assay [97] was performed. Briefly, cells were fixed with ice-cold trichloroacetic acid, washed with deionized water, left to dry and stained with SRB dye. After washing with 1% acetic acid, cells were left to dry, and protein-bound dye was dissolved in 10 mM Tris base solution. OD at 492 nm was determined. Growth inhibition curves (upper panels) were generated, where each point represents mean ± SEM of at least 3 replicates. IC50 values, the drug concentration that inhibits the cell growth by 50% (summarized in Table 3), were calculated by fitting the data to a sigmoid dose–response curve using GraphPad Prism. The treatment with the two drugs in combination was carried out using a constant cisplatin:pimozide ratio (as indicated in Table 3), which was established for each cell line as the ratio of the IC50 values for each single drug. The drug concentrations on the X-axis of the growth inhibition curves for the combination treatment refer to cisplatin. In order to evaluate the interaction between cisplatin and pimozide we used the median-drug effect analysis method originally described by Chou and Talalay [95]. Using CalcuSyn, the effect of the combination was compared to the effect of each drug to determine the combination index (C.I.) at a range of Fa (fraction affected by the dose) values. The resulting C.I. values were plotted against the Fa to produce the median-drug effect plot for each cell line (lower panels).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750636&req=5

Figure 4: Growth inhibition curves and median-drug effect plots of two NSCLC cell lines treated with cisplatin, pimozide or their simultaneous combination. Exponentially growing SW1573 (A) and H1299 (B) NSCLC cells were seeded in 96-well plates. Twenty four hours after seeding, cells were treated with cisplatin, pimozide or a combination of both drugs. After 72 hours of drug exposure, the sulforhodamine B (SRB) chemosensitivity assay [97] was performed. Briefly, cells were fixed with ice-cold trichloroacetic acid, washed with deionized water, left to dry and stained with SRB dye. After washing with 1% acetic acid, cells were left to dry, and protein-bound dye was dissolved in 10 mM Tris base solution. OD at 492 nm was determined. Growth inhibition curves (upper panels) were generated, where each point represents mean ± SEM of at least 3 replicates. IC50 values, the drug concentration that inhibits the cell growth by 50% (summarized in Table 3), were calculated by fitting the data to a sigmoid dose–response curve using GraphPad Prism. The treatment with the two drugs in combination was carried out using a constant cisplatin:pimozide ratio (as indicated in Table 3), which was established for each cell line as the ratio of the IC50 values for each single drug. The drug concentrations on the X-axis of the growth inhibition curves for the combination treatment refer to cisplatin. In order to evaluate the interaction between cisplatin and pimozide we used the median-drug effect analysis method originally described by Chou and Talalay [95]. Using CalcuSyn, the effect of the combination was compared to the effect of each drug to determine the combination index (C.I.) at a range of Fa (fraction affected by the dose) values. The resulting C.I. values were plotted against the Fa to produce the median-drug effect plot for each cell line (lower panels).
Mentions: In an attempt to extend these findings to a larger number of NSCLC cell lines, we tested the effect of pimozide in combination with cisplatin on a panel of seven NSCLC cell lines. A cell viability assay was used to determine the cytotoxic activity of cisplatin, pimozide or a combination of both agents. Figure 4 shows representative examples of the results of this assay in two NSCLC cell lines, and the data for the remaining cell lines analyzed are shown in Additional file 2: Figure S1. The C.I. was subsequently calculated using Calcusyn. The cell lines tested displayed a wide range of cisplatin sensitivity, as summarized in Table 3, and were classified as “ciplatin-sensitive” if their IC50 was below the mean IC50 of the panel (6.2 μM), or as “cisplatin-resistant” if their IC50 was above the mean. Pimozide showed a synergistic or additive effect with cisplatin in the three “resistant” cell lines (H1299, H1703 and H520). In contrast, the effect of the pimozide/cisplatin combination was antagonistic in three of the four “sensitive” cell lines (H322, SKLU-1 and SW1573). These findings are consistent with those of Chen et al. [94], and suggest that USP1 inhibition may contribute to revert cisplatin resistance in some preclinical models of NSCLC.

Bottom Line: Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy.We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data.Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, Leioa, Spain.

ABSTRACT
Reversible protein ubiquitination is emerging as a key process for maintaining cell homeostasis, and the enzymes that participate in this process, in particular E3 ubiquitin ligases and deubiquitinases (DUBs), are increasingly being regarded as candidates for drug discovery. Human DUBs are a group of approximately 100 proteins, whose cellular functions and regulatory mechanisms remain, with some exceptions, poorly characterized. One of the best-characterized human DUBs is ubiquitin-specific protease 1 (USP1), which plays an important role in the cellular response to DNA damage. USP1 levels, localization and activity are modulated through several mechanisms, including protein-protein interactions, autocleavage/degradation and phosphorylation, ensuring that USP1 function is carried out in a properly regulated spatio-temporal manner. Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy. This view has gained recent support with the finding that USP1 inhibition may contribute to revert cisplatin resistance in an in vitro model of non-small cell lung cancer (NSCLC). Here, we describe the current knowledge on the cellular functions and regulatory mechanisms of USP1. We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data. Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells.

Show MeSH
Related in: MedlinePlus