We have recently shown hypericin dye photodynamic therapy (PDT) decreases human pancreatic cancer cell growth in vitro and in vivo. Neutral red is a vital dye that has been used to treat squamous cell cancer in vitro. When stimulated by green laser light, neutral red emissions can activate hypericin. We hypothesized that combination therapy with neutral red and hypericin will cause cascade dye activation with a decrease in pancreatic cancer cell growth and secretion of vascular endothelial growth factor (VEGF).
Methods: Emission spectra of each dye were measured by fluorescence spectroscopy. Human ductal adenocarcinoma (MiaPaCa-2 and PANC-1) were incubated with hypericin and neutral red, hypericin alone, neutral red alone, or solvent as a control. Cells were treated with KTP532 green laser light for sixty seconds at 1.0 W using a cylindrical diffuser fiberoptic tip. Tumor growth suppression was measured by MTT assays. VEGF secretion was measured by ELISA.
Results: Emission spectra for neutral red overlapped the absorption spectra for hypericin. MTT assays reveal significant growth reduction after combination neutral red/hypericin PDT compared to single agents alone. VEGF was suppressed more strongly after combination therapy compared to single agent alone. (N= 12, P<0.05 by ANOVA). No hyperthermia was observed during treatment with laser PDT. Graphs below.
Conclusion: A PDT cascade using laser excitation of neutral red to stimulate hypericin leads to photo-oxidation and greatly decreases tumor cell growth by 6.5 fold and VEGF secretion 7.7 fold vs. control. Patients with pancreatic cancer may benefit from laser PDT with combination therapy using neutral red and hypericin.
Human Pancreatic Cell Growth Inhibition and VEGF Inhibition
Control Hypericin/Neutral Red Neutral RedHypericin
Growth Inhibition 100 ± 2 % 15.4 ± 3 % * 24.4 ± 2 % * 70.3 ± 4 % *
VEGF Level 100 ± 3 % 13.0 ± 1 % * 39.9 ± 2 % * 79.9 ± 3 %
* P < 0.05 vs. Control by ANOVA
P<0.05 Hypericin/Neutral Red vs. Single Agent by ANOVA