5-Aminolevulinic acid-based photodynamic therapy suppressed survival factors and activated proteases for apoptosis in human glioblastoma U87MG cells

Abstract

Glioblastoma is the most common astrocytic brain tumor in humans. Current therapies for this malignancy are mostly ineffective. Photodynamic therapy (PDT), an exciting treatment strategy based on activation of a photosensitizer, has not yet been extensively explored for treating glioblastoma. We used 5-aminolevulinic acid (5-ALA) as a photosensitizer for PDT to induce apoptosis in human malignant glioblastoma U87MG cells and to understand the underlying molecular mechanisms. Trypan blue dye exclusion test showed a decrease in cell viability after exposure to increasing doses of 5-ALA for 4h followed by PDT with a broad spectrum blue light (400-550 nm) at a dose of 18J/cm(2) for 1h and then incubation at 37 degrees C for 4h. Following 0.5 and 1mM 5-ALA-based PDT (5-ALA-PDT), Wright staining and ApopTag assay showed occurrence of apoptosis morphologically and biochemically, respectively. After 5-ALA-PDT, down regulation of nuclear factor kappa B (NFkappaB) and baculovirus inhibitor-of-apoptosis repeat containing-3 (BIRC-3) protein indicated inhibition of survival signals. Besides, 5-ALA-PDT caused increase in Bax:Bcl-2 ratio and mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF). Activation of calpain, caspase-9, and caspase-3 occurred in course of apoptosis. Calpain and caspase-3 activities cleaved alpha-spectrin at specific sites generating 145kD spectrin breakdown product (SBDP) and 120kD SBDP, respectively. The results suggested that 5-ALA-PDT induced apoptosis in U87MG cells by suppression of survival signals and activation of proteolytic pathways. Thus, 5-ALA-PDT can be an effective strategy for inducing apoptosis in glioblastoma.

Fig. 1

(A) Effect of 5-ALA-PDT on morphology of U87MG cells. (B) Trypan blue dye exclusion test for residual cell viability after 5-ALA-PDT. Bar graph shows residual cell viability. Significant difference from control was indicated by **p<0.001.

Fig. 2

(A) Wright staining for determination of morphological features of apoptosis in U87MG cells following 5-ALA-PDT. Bar graph shows percent apoptosis based on Wright staining. (B) ApopTag assay for determination of morphological as well as biochemical features of apoptosis following 5-ALA-PDT. Bar graph shows percent apoptosis based on ApopTag assay. Significant difference from control was indicated by **p<0.001.

Fig. 3

(A) Western blotting showed decreases in expression of 65 kD NFκB and 68 kD BIRC-3 in U87MG cells following 5-ALA-PDT. (B) Western blotting for examining changes in Bax and Bcl-2 expression and determining the Bax:Bcl-2 ratios. Significant difference from control was indicated by **p<0.001.

Fig. 4

(A) Western blotting to examine mitochondrial release of cytochrome c and AIF in U87MG cells following 5-ALA-PDT. (B) Western blotting showed formation of 76 kD active calpain, 35 kD active caspase-9, and 20 kD active caspase-3, and activities of calpain and caspase-3 in the generation of specific SBDPs.