DNA Damage and Cell Cycle Arrest Induced by Protoporphyrin IX in Sarcoma 180 Cells

Abstract

Background: Porphyrin derivatives have been widely used in photodynamic therapy as effective sensitizers. Protoporphyrin IX (PpIX), a well-known hematoporphyrin derivative component, shows great potential to enhance light induced tumor cell damage. However, PpIX alone could also exert anti-tumor effects. The mechanisms underlying those direct effects are incompletely understood. This study thus investigated the putative mechanisms underlying the anti-tumor effects of PpIX on sarcoma 180 (S180) cells. Methods: S180 cells were treated with different concentrations of PpIX. Following the treatment, cell viability was evaluated by the 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) assay; Disruption of mitochondrial membrane potential was measured by flow cytometry; The trans-location of apoptosis inducer factor (AIF) from mitochondria to nucleus was visualized by confocal laser scanning microscopy; DNA damage was detected by single cell gel electrophoresis; Cell cycle distribution was analyzed by DNA content with flow cytometry; Cell cycle associated proteins were detected by western blotting. Results: PpIX (≥ 1 µg/ml) significantly inhibited proliferation and reduced viability of S180 cells in a dose-dependent manner. PpIX rapidly and significantly triggered mitochondrial membrane depolarization, AIF (apoptosis inducer factor) translocation from mitochondria to nucleus and DNA damage, effects partially relieved by the specific inhibitor of MPTP (mitochondrial permeability transition pore). Furthermore, S phase arrest and upregulation of the related proteins of P53 and P21 were observed following 12 and 24 h PpIX exposure. Conclusion: PpIX could inhibit tumor cell proliferation by induction of DNA damage and cell cycle arrest in the S phase.

Protoporphyrin IX Induces a Necrotic Cell Death in Human THP-1 Macrophages through Activation of Reactive Oxygen Species/c-Jun N-Terminal Protein Kinase Pathway and Opening of Mitochondrial Permeability Transition Pore

“…Previous studies have demonstrated that both apoptosis and necrosis could be induced by PpIX [9,10,11]. In our study, macrophages died with features of early loss of membrane integrity, intracellular ATP depletion and LDH release to the supernatant indicating necrosis is the major form of cell death instead of apoptosis.…”

“…Consistent with these, studies in vitro have indicated that PpIX could induce cell death. For instance, in sarcoma 180 cells, PpIX caused cell death with characteristics of apoptosis [9]. PpIX can interact with p53 protein and induce human colon cancer cell apoptosis in both p53-dependent and -independent manners [10].…”

p53‐Dependent and p53‐Independent Responses of Cells Challenged by Photosensitization

“…Interestingly, we also made a new observation concerning the effects induced by DMMB/light treatment: There is a clear cell cycle arrest at S phase in the cell lines, in accordance with the results of other studies using different cells and photosensitizers (26,46,47). Our data also indicate that accumulation of cells in S phase was not affected by the expression level of p53 protein, as had been previously reported for HCT116 cells treated with hypericin (26).…”

Reactivation of TAp73 tumor suppressor by protoporphyrin IX, a metabolite of aminolevulinic acid, induces apoptosis in TP53-deficient cancer cells

“…Our previous findings showed that protoporphyrin IX binds to p53 and activates p53dependent and independent cell death in colon cancer cells 12 . Consistently, PpIX was shown to induce cancer cell death in sarcoma cells 25 . In line with our data (Figure 1d Recently, detailed analysis of the oligomeric state of TAp73 alpha by Melino and Dötsch labs revealed a high structure homology between p53 and p73 tetramers 26 .…”