Effects of Silencing Heme Biosynthesis Enzymes on 5‐Aminolevulinic Acid‐mediated Protoporphyrin IX Fluorescence and Photodynamic Therapy

Abstract

Aminolevulinic acid (ALA)-mediated protoporphyrin IX (PpIX) production is being explored for tumor fluorescence imaging and photodynamic therapy (PDT). As a prodrug, ALA is converted in heme biosynthesis pathway to PpIX with fluorescent and photosensitizing properties. To better understand the role of heme biosynthesis enzymes in ALA-mediated PpIX fluorescence and PDT efficacy, we used lentiviral shRNA to silence the expression of porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD) and ferrochelatase (FECH) in SkBr3 human breast cancer cells. PBGS and PBGD are the first two cytosolic enzymes involved in PpIX biosynthesis, and FECH is the enzyme responsible for converting PpIX to heme. PpIX fluorescence was examined by flow cytometry and confocal fluorescence microscopy. Cytotoxicity was assessed after ALA-mediated PDT. Silencing PBGS or PBGD significantly reduced ALA-stimulated PpIX fluorescence, whereas silencing FECH elevated basal and ALA-stimulated PpIX fluorescence. However, compared with vector control cells, the ratio of ALA-stimulated fluorescence to basal fluorescence without ALA was significantly reduced in all knockdown cell lines. PBGS or PBGD knockdown cells exhibited significant resistance to ALA-PDT, while increased sensitivity to ALA-PDT was found in FECH knockdown cells. These results demonstrate the importance of PBGS, PBGD and FECH in ALA-mediated PpIX fluorescence and PDT efficacy.

Fluorouracil Enhances Photodynamic Therapy of Squamous Cell Carcinoma via a p53-Independent Mechanism that Increases Protoporphyrin IX levels and Tumor Cell Death

“…ALAD and PBGD were also decreased, but those enzymes are expected to be less rate-limiting than CPO and FC which lie immediately upstream and downstream relative to PpIX in the heme pathway. Our results broadly agree with other studies performed by various groups using a gene-silencing approach, which amply demonstrated that downregulation of ALAD or PBGD reduced the PpIX levels, but silencing of FC was the only approach that elevated PpIX levels in target cells (31–33). …”

ABCG2 transporter inhibitor restores the sensitivity of triple negative breast cancer cells to aminolevulinic acid-mediated photodynamic therapy

“…Cell suspensions were centrifuged and cell pellets were re-suspended in PBS for PpIX or Pha measurement. PpIX or Pha fluorescence was measured with a FACSCalibur flow cytometer (BD Biosciences) in the FL3 channel (488 nm excitation, 650 nm long-pass emission), which captured the second emission band of PpIX 30 . About 20,000 cells were measured and recorded for each experiment.…”

Chemical approaches for the enhancement of 5-aminolevulinic acid-based photodynamic therapy and photodiagnosis

“…Moreover, it has been observed that differences in the accumulation of PpIX in healthy cells and malignant cells are due to differences in the activity of enzymes in the haem biosynthetic pathway such as porphobilinogen deaminase and ferrochelatase. 51 Molecules such as 2-allyl-2-isopropylacetamide are known to stimulate ALA synthase and ALA dehydratase activities, 52 while molecules that inhibit protoporphyrinogen oxidase 53 or upregulate coproporphyrinogen oxidase 54 have been identified which show positive results in ALA-PDT. While this highlights the potential for targeting various steps in the heme cycle in order to enhance intracellular PpIX production most attention has been paid to targeting the insertion of ferrous iron (Fe 2+ ) into the PpIX core, which is catalysed by ferrochelatase in the last step of heme biosynthesis.…”