Molecular and cellular mechanisms of aCaMKII

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Fever-range hyperthermia vs. hypothermia effect on cancer cell viability, proliferation and HSP90 expression.

PURPOSE:The current study examines the effect of fever-range hyperthermia and mild hypothermia on human cancer cells focusing on cell viability, proliferation and HSP90 expression. MATERIALS AND METHODS:A549 and H1299 lung carcinoma, MCF7 breast adenocarcinoma, U87MG and T98G glioblastoma, DU145 and PC3 prostate carcinoma and MRC5 normal fetal lung fibroblasts cell lines were studied. After 3-day exposure to 34°C, 37°C and 40°C, cell viability was determined. Cell proliferation (ki67 index), apoptosis (Caspase 9) and HSP90 expression was studied by confocal microscopy. RESULTS:Viability/proliferation experiments demonstrated that MRC5 fibroblasts were extremely sensitive to hyperthermia, while they were the most resistant to hypothermia. T98G and A549 were thermo-tolerant, the remaining being thermo-sensitive to a varying degree. Nonetheless, as a universal effect, hypothermia reduced viability/proliferation in all cell lines. Hyperthermia sharply induced Caspase 9 in the U87MG most thermo-sensitive cell line. In T98G and A549 thermo-tolerant cell lines, the levels of Caspase 9 declined. Moreover, hyperthermia strongly induced the HSP90 levels in T98G, whilst a sharp decrease was recorded in the thermo-sensitive PC3 and U87MG cell lines. Hyperthermia sensitized thermo-sensitive cancer cell lines to cisplatin and temozolomide, whilst its sensitizing effect was diminished in thermo-tolerant cell lines. CONCLUSIONS:The existence of thermo-tolerant and thermo-sensitive cancer cell lines was confirmed, which further encourages research to classify human tumor thermic predilection for patient stratification in clinical trials. Of interest, mild hypothermia had a universal suppressing effect on cancer cell proliferation, further supporting the radio-sensitization hypothesis through reduction of oxygen and metabolic demands

Cell proliferation studies with AlamrBlue after 3days in various incubation temperatures.

<p>% change of relative fluorescent units (RFUs) as recorded with the AlamarBlue assay, after 3 days of exposure of cells to hypothermia (34°C) or hyperthermia (40°C) compared to normothermia (37°C).</p

Confocal immunofluorescent microscopy and western blot images of HSP90.

<p>4a: Representative confocal microcopy images showing cytoplasmic HSP90 expression changing intensity after exposure to hypothermia (34°C) or hyperthermia (40°C) compared to normothermia (37°C) (magnification x60). 4b,c: Densitometry performed on confocal microcopy images of HSP90 immunostaining. 4d: Western blot images of HSP90 expression in the thermo-tolerant T98G and the thermo-sensitive U87MG cell lines.</p

Confocal immunofluorescent microscopy images and automated quantification of ki67 proliferation marker in various cell lines.

<p>2a: changes of Ki67 proliferation index class after 3-day exposure of cells to hypothermia (34°C) or hyperthermia (40°C) compared to normothermia (37°C). 2b: Representative confocal microcopy images showing nuclear Ki67 immunostaining changing intensity after exposure to hyperthermia (40°C).</p

Confocal immunofluorescent microscopy and western blot images of Caspase9.

<p>3a: Representative confocal microcopy images showing cytoplasmic Caspase 9 expression changing intensity after exposure to hypothermia (34°C) or hyperthermia (40°C) compared to normothermia (37°C) (magnification x60). 3b,c: Densitometry performed on confocal microcopy images of Caspase 9 immunostaining after exposure to hypothermia (34°C) or hyperthermia (40°C) compared to normothermia (37°C).</p

Confocal microscopy after γΗ2ΑX immunostaining, showing red foci in the nuclei of control (6a1,6a3) and irradiated cells (6a2,6a4), with (6a3,6a4) and without (6a1,6a2) pre-incubation with SMER28 (Fig. 6a).

<p>Computerized calculation of γH2AX foci in the nuclei of cells. Statistical significance refers to comparison with control cells (Fig. 6b).</p

HUVEC confocal immunofluorescent microscopy images LC3A(green)/LC3B(red) (Fig. 1a), LC3A(green)/LAMP2a(red) (Fig. 1b), LC3B(red)/CathepsinD(green) (Fig. 1c) and p62(red) (Fig. 1d).

<p>Co-localization is presented as yellow spots. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g001" target="_blank">Figure 1a</a>: autophagosomes stained for LC3A (perinuclear localization) and LC3B (cytoplasmic localization). Irradiation-induced accumulation of both LC3A and LC3B autophagosomes at 4 and 8 post-irradiation days (1a2,1a3). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g001" target="_blank">Figures 1b</a>1 and 1c1 show an evident degree of co-localization of LC3A with LAMP2a and of LC3B with CathepsinD. At 4 and 8 post-irradiation days there was a lack of co-localization, which suggests autophagy flux suppression (1b2, 1b3, 1c2, 1c3). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g001" target="_blank">Figure 1d</a> shows poor cytoplasmic and intense nuclear presence of p62 in control cells (1d1); intense accumulation of p62 in the cytoplasm was evident 4 and 8 days after irradiation (1d2,3). Graphic presentation of fluorescent intensity of the above immunostaining is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g001" target="_blank">Figure 1e</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g001" target="_blank">Figure 1f</a> shows the western blot analysis in the pellet fraction of HUVEC.</p

Confocal images of HUVEC cultured cells after exposure to 2 Gy of IR, with and without 2 day pre-incubation with SMER28 (Fig. 4a).

<p>LC3A(green)/LC3B(red) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g003" target="_blank">Fig. 3a1,2</a>), LC3A(green)/LAMP2a(red) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g003" target="_blank">Fig. 3a3,4</a>), LC3B(red)/CathepsinD(green) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g003" target="_blank">Fig. 3a5,6</a>) and p62(red) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g003" target="_blank">Fig. 3a7,8</a>). Co-localization is shown as yellow structures. Western blot images from supernatant and pellet protein fractions is shown in Fig. 4b.</p

Confocal microscopy for LC3A/LC3B, specific suppression of LC3A (Fig. 5a2) and of LC3B (Fig. 5a3) after incubation with relevant LC3 siRNAs (Fig. 5a).

<p>Radiation dose and response curves confirm a shift to the right when a suppression of the LC3A/LC3B expression is achieved before irradiation, while the curve shifts to the right when cells are pre-incubated with non-toxic doses of the mTO- independent autophagy inducer SMER28 (Fig. 5b). Viability curves of HUVEC cells after exposure from 1–3 days at various SMER28 concentrations confirm nontoxic effect at 25 µM (Fig. 5c). Radiation dose and recovery curves comparing the cell population at the nadir (8^th day) vs. exponential growth onset (12^th day) show enhanced recovery potential when cells are pre-incubated with SMER28 (Fig. 5d).</p