Melanogenesis.

<p>Total melanin content (μg/ml) and tyrosinase enzymatic activity (Units/mg protein) measurements in WM35 (upper panels) and M1-15 (lower panels) were done by spectrophotometry. Each bar represents mean ± standard deviation (n = 3). ir = irradiated cells; # = not significant, * = p<5.0E-02, ** = p<1.0E-02, *** = p<1.0E-03.</p

Protein expression measured by Western Blot.

<p>Protein expressions of NF-κB, pNF-kB, IκKα, IκKβ, pIκK α/β, HIF1α, tyrosinase and MITF in melanoma cells treated with GaPc-PDT + Metformin, left panel (WM35), right panel (M1-15) were measured by WB. Image analysis of WB bands was done by densitometry, results were normalised to GAPDH.—WB images (upper panels) 1 = control, 2 = irradiated control, 3 = Metformin, 4 = irradiated Metformin, 5 = GaPc, 6 = GaPc-PDT, 7 = GaPc and Metformin, 8 = GaPc-PDT and Metformin; graphical representation of quantitative WB results for WM35 and M1-15 (lower panels); ir = irradiated cells; # = not significant, * = p<5.0E-02, ** = p<1.0E-02, *** = p<1.0E-03. Each bar represents mean ± standard deviation (n = 3).</p

Oxidative stress assessment.

<p>Malondyaldehide (MDA), nitric oxide (NO) levels (nM/mg protein) measurements in WM35 (upper panels) and M1-15 (lower panels) were done by spectrophotometry. Each bar represents mean ± standard deviation (n = 3). ir = irradiated cells; # = not significant, * = p<5.0E-02, ** = p<1.0E-02, *** = p<1.0E-03.</p

Assessment of inflamatory and angiogenetic markers.

<p>Protein expressions of TNF-α, TRAIL, VEGF (pg/ml) in melanoma cultures exposed to GaPc-PDT + Metformin were determined by ELISA, upper panels (WM35), lower panels (M1-15); Each bar represents mean ± standard deviation (n = 3), ir = irradiated cells; # = not significant, * = p<5.0E-02, ** = p<1.0E-02, *** = p<1.0E-03.</p

Viability testing after GaPc-PDT and Metformin exposure.

<p>WM35 (a, b), M1-15 (c, d) melanoma cell cultures exposed to GaPc-PDT (a, c), respectively GaPc-PDT+ Metformin (b, d). OD 540 graphs were generated using GraphPad Software and show mean values ± standard deviation, n = 3 for each sample. Cell viability of both cell lines was decreased by GaPc-PDT depending on GaPc concentration and irradiation dose; Metformin increased the efficacy of GaPc-PDT especially in WM35 cells. e-images of WM35 (upper panels) and M1-15 (lower panels) cells subjected to GaPc-PDT and Metformin. Cells exposed to GaPc w/o Metformin, showed a normal morphology, compared to controls, while PDT irradiation of treated cells induced loss of cell adhesion, pleiomorphysm with spherical or bipolar shaped cells, signs of treatment induced photo-toxicity.</p

Chemical formula.

<p>Chloro-gallium (III) phthalocyanine (a) and Metformin (b).</p

Viability testing after Metformin exposure.

<p>Melanoma cell cultures exposed to different concentrations of Metformin or Metformin and irradiation (a)WM35 and (b)M1-15. OD 540 graphs were generated using GraphPad Software and show mean values ± standard deviation, n = 3 for each sample. Cell viability of both cell lines was decreased by increasing concentrations of Metformin, in a dose dependent manner; irradiation had no effect on the cell viability.</p

Cell death assessment.

<p>Comparative FACS analysis following GaPc-PDT + Metformin treatment versus controls in WM35 (a) and M1-15 (b) cells; c- confocal microscopy images of treated WM35 cells stained with annexin V-FITC/ PI (upper panels) and phalloidin-FITC and DRAQ5 (lower panels), original magnification 63x; PDT exposed cells showed annexin V positive (green) and some exhibit PI positive red fluorescence, while Metformin addition increased the number of annexin V/PI positive cells, there are also present stress related morphological changes; phalloidin staining showed cytoskeleton alterations like increased condensations of actin filaments, retraction of dendrites, spherical shaped cells and loss of cell adhesion in PDT w/o Metformin treated cells. d, e quantitative FACS results for WM35 (d) and M1-15 (e) are expressed as % of total dead cells—annexin V and PI positive cells, from the total cell number; ir = irradiated cells; # = not significant, * = p<5.0E-02, ** = p<1.0E-02, *** = p<1.0E-03. Each bar represents mean ± standard deviation (n = 3).</p