DNA-Bound Platinum Is the Major Determinant of Cisplatin Sensitivity in Head and Neck Squamous Carcinoma Cells

<div><p>Purpose</p><p>The combination of systemic cisplatin with local and regional radiotherapy as primary treatment of head and neck squamous cell carcinoma (HNSCC) leads to cure in approximately half of the patients. The addition of cisplatin has significant effects on outcome, but despite extensive research the mechanism underlying cisplatin response is still not well understood.</p><p>Methods</p><p>We examined 19 HNSCC cell lines with variable cisplatin sensitivity. We determined the <i>TP53</i> mutational status of each cell line and investigated the expression levels of 11 potentially relevant genes by quantitative real-time PCR. In addition, we measured cisplatin accumulation and retention, as well as the level of platinum-DNA adducts.</p><p>Results</p><p>We found that the IC₅₀ value was significantly correlated with the platinum-DNA adduct levels that accumulated during four hours of cisplatin incubation (p = 0.002). We could not find a significant correlation between cisplatin sensitivity and any of the other parameters tested, including the expression levels of established cisplatin influx and efflux transporters. Furthermore, adduct accumulation did not correlate with mRNA expression of the investigated influx pumps (<i>CTR1</i> and <i>OCT3</i>) nor with that of the examined DNA repair genes (<i>ATR</i>, <i>ATM</i>, <i>BRCA1</i>, <i>BRCA2</i> and <i>ERCC1</i>).</p><p>Conclusion</p><p>Our findings suggest that the cisplatin-DNA adduct level is the most important determinant of cisplatin sensitivity in HNSCC cells. Imaging with radio-labeled cisplatin might have major associations with outcome.</p></div

Spearman’s rho correlations of the mRNA expression levels of the nine genes examined in 19 HNSCC cell lines.

<p><b>Bold</b> Correlation is significant at the 0.01 level (2-tailed). <i>Italics</i> Correlation is significant at the 0.05 level (2-tailed).</p

Platinum-DNA adducts after four hours of cisplatin exposure and the percentage of adduct retention after three hours in drug free medium.

<p>Values are the means of duplicate experiments ± SEM. ND; not determined.</p

Cisplatin accumulation after four hours of cisplatin exposure and the percentage of cisplatin retention after three hours in drug free medium.

<p>Retention (%) represents the remaining cellular cisplatin concentration after three hours in drug free medium. Values are the means of at least duplicate experiments ± SEM. ND; not determined.</p

Relative mRNA expression levels of <i>CTR1</i>, <i>ATP7B</i> and <i>ERCC</i>.

<p>The mRNA expression level of eleven genes were determined in our HNSCC cell line panel. Genes were selected on basis of their involvement in cisplatin toxicity. CTR1 is believed to mediate cisplatin influx, ATP7B is a known cisplatin efflux pump and ERCC1 is a DNA repair protein. Results are medians of triplicate experiments and are presented relative to the expression of the beta-glucuronidase (<i>GUSB</i>) housekeeping gene. Error bars represent standard deviations.</p

The half-maximal inhibitory concentration of cisplatin.

<p>Nineteen HNSCC cell lines were continuously treated with 18 different concentrations of cisplatin during 72 h. Cell viability was determined and plotted against the cisplatin concentration. Cell lines VU-SCC-094 and UM-SCC-38 are presented as examples of a relatively sensitive and a relatively resistant cell line, respectively. Measurements were performed in at least three independent triplicate experiments. Error bars represent the standard error of the mean.</p

Inverse correlation between IC₅₀ value and accumulation of platinum-DNA adducts.

<p>The level of DNA-bound platinum was determined in 17 HNSCC cell lines that were incubated with cisplatin. A significant inverse correlation was found between the platinum-DNA adducts and the IC₅₀ value. Measurements were performed in duplicate and error bars represent standard deviations.</p

The Cytidine Analog Fluorocyclopentenylcytosine (RX-3117) Is Activated by Uridine-Cytidine Kinase 2 - Fig 6

<p><b>Correlation between the UCK2 mRNA expression of the cell lines in panels A and B (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162901#pone.0162901.g005" target="_blank">Fig 5</a>) with phosphorylation and the sensitivity to RX-3117</b>. The cell lines starting from highest UCK2 expression and sensitivity are: A2780, U937, A549, SW1573 and CCRF-CEM. <b>(A)</b> Correlation between sensitivity to RX-3117 and mRNA expression of UCK2. <b>(B)</b> RX-3117 phosphorylation in panel cells. RX-3117MP formation after 120 minutes correlated with UCK expression. <b>(C)</b> Protein expression of UCK1 and UCK2; the two left lanes are positive controls for UCK1 and UCK2 for which high levels of purified protein was used, which shows some cross-reactivity. Equal amounts of protein were put on the gels.</p

Rescue of cell cycle effects and proliferation inhibition of cell lines upon treatment with RX-3117 conditioned with Uridine or Cytidine.

<p><b>(A)</b> The cell cycle distribution was monitored 36 hours after exposure to 33.3 μM RX-3117 or 33.3 μM RX-3117 with 100 μM of uridine or cytidine in A549 and SW1573 cells. <b>(B)</b> Proliferation of A549 and SW1573 cell lines treated with 33.3 μM of RX-3117 during 24 hours or with RX-3117 plus 100 μM of uridine/cytidine.</p

Chemical structure of cytidine and RX-3117.

<p>Chemical structure of cytidine and RX-3117.</p