Co-incubation of cisplatin with isopropanol leads to enhanced apoptosis without an increase in intercellular cisplatin concentration.

<p>(A) Expression levels of cleaved PARP, an apoptotic marker, as measured by western blot for cells incubated with 50mM isopropanol plus 10μM cisplatin, with 100μM menthol plus 10μM cisplatin or with 10μM cisplatin alone along with cisplatin free controls (B) Levels of intracellular cisplatin were measured using optical emission spectrometry for the three treatments, 50mM isopropanol plus 10μM cisplatin, with 100μM menthol plus 10μM cisplatin or with 10μM cisplatin alone. The differences between the three treatments are not statistically significant (n = 8 trials). Also shown are predicted levels of cisplatin obtained by assuming that intracellular cisplatin that is directly proportional to the external concentration determines the extent of cell death (as described in Methods). The solid line denotes the predicted mean theoretical value and corresponding dashed lines denote error bounds. (C) Levels of intracellular cisplatin for UMSCC1 and the more resistant cell line Me-180pt treated with 10μM cisplatin. The solid line as described previously denotes predicted levels given the assumptions stated in 4B and dashed lines indicate error bounds.</p

Changes in transition temperature in GPMVs correlate with the cell lines resistance to cisplatin.

<p>(A) GPMVs were isolated from four cell-lines as described in the Methods section. The transition temperature shifts are reported by comparing the transition temperatures of GPMVs probed in the presence of 10 μM cisplatin to untreated GPMVs. (B) Data points in panel A were plotted against a previously reported measure of surviving fraction to cisplatin obtained using clonogenic assays for the same four cell lines [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140925#pone.0140925.ref008" target="_blank">8</a>]. Surviving fraction was measured using a clonogenic survival assay. Surviving fractions were measured 72 hours after treatment with 10uM cisplatin. The straight line is drawn to visually distinguish sensitive and resistant celllines. Transition temperature shifts upon incubation with 10 μM cisplatin or exposure to 10 Gy irradiation for GPMVs isolated from ME-180 pt cells (C) and RBL cells (D). In all cases, points represent the average of at least 3 independent measurements and error bounds represent the standard error of the mean. Significance between transition temperature shift measurements were evaluated using t-tests.</p

Modulating transition temperature affects cisplatin mediated cellular response in UMSCC1 cells.

<p>(A) Transition temperature shifts measured for GPMVs isolated from UMSCC1 in the presence of 50mM isopropanol or 100 μM menthol or each of these treatments in combination with 10μM cisplatin. (B) Efficacy of 50mM isopropanol and 100μM menthol action on UMSCC1 cells calculated as the number of cells present after 24h of treatment divided by the number of cells present in an untreated control. (C) Efficacy of cisplatin action as a function of the transition temperature shift effected by the treatment in isolated GPMVs shown in A. Efficacy of cisplatin action on UMSCC1 cells as above was computed as above in (B) by dividing the number of cells present after 24h of treatment compared to the number of cells present in an untreated control. (D) Plots show relative cell counts as a function of cisplatin concentration either in the presence or absence of 50mM isopropanol or 100μM menthol. Each point represents the average and SEM of at least 4 independent measurements, and lines are fit to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140925#pone.0140925.e001" target="_blank">Eq 1</a>. (E) Average IC₅₀ values as determined by fitting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140925#pone.0140925.e001" target="_blank">Eq 1</a> to individual dose response curves. Values represent an average and SEM over at least 4 independent measurements.</p