Cytoprotective role of nitric oxide in HepG2 cell apoptosis induced by hypocrellin B photodynamic treatment
Graphical Abstract
Introduction
Hypocrellin B (HB), a natural perylenequinone pigment from a traditional Chinese medicinal fungus Shiraia bambusicola, is a promising second-generation photosensitizer for photodynamic therapy (PDT) due to its high singlet oxygen yield, low dark toxicity and rapid metabolic rate in vivo [1]. Originally, hypocrellins (primarily HA and HB) were used clinically to treat numerous cutaneous diseases such as psoriasis, vitiligo, keloid, tinea capitis, lichen amyloidosis and white lesions of the vulva [2]. Recently, growing evidence showed that light-activated HB could induce cytotoxicity and apoptosis of many kinds of tumor cells [3], [4]. To develop HB as a novel photosensitizer for clinical utilization, many researchers made efforts to improve its solubility via chemical modification and novel drug-delivery systems [5], [6]. Although the mechanism of action of HB is still under investigation, the generation of reactive oxygen species (ROS) upon irradiation is considered an important factor to initiate apoptotic cell death [3], [4].
Over the last 10 years, it has been suggested that PDT-mediated apoptosis in carcinoma cells may also be modulated by nitric oxide (NO) in addition to oxidative stress [7]. Intracellular biosynthesis of NO is catalyzed by enzymes of the nitric oxide synthase (NOS) family, which were found to be mediated by photosensitive chemicals in PDT process [8], [9]. The role of NO in PDT is complex and dualistic. It has been reported that higher concentrations of NO (> 500 nM) could promote the anti-tumor effects and induce cell apoptosis, while lower concentration NO (< 100 nM) acted as a cytoprotective antioxidant to foster cell survival under the oxidative challenge by PDT [10], [11]. Nevertheless, it is now recognized that NO has a major influence on the outcome of PDT [7]. Recently emerging evidence has shown that HB can directly destroy cancer cells through the activation of apoptosis [3], [4]. However, the role of the generated NO has not yet been reported in apoptosis induced by HB in PDT. In the present study, HB/light treatment of human hepatocellular carcinoma (HepG2) cells caused a significant upregulation of inducible NOS (iNOS) with the generation of NO. The antiapoptotic role of NO was revealed by the strong death-promoting effect of NOS inhibitor Nω-monomethyl-l-arginine (l-NMMA) and NO scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). We herewith demonstrated that the elevated endogenous NO can act cytoprotectively on cell apoptosis. This is the first reported evidence for NO-mediated cytoprotection in HB-PDT.
Section snippets
General Materials
HB (purity, > 98%) was purchased from the Institute of Chemistry, Chinese Academy of Sciences (Beijing, China). The compound was dissolved in dimethyl sulfoxide (DMSO) at 5.0 mM as stock solution and diluted according to experimental requirements. Low glucose DMEM, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT), acridine orange (AO), propidium iodide (PI) and 3-amino, 4-aminomethyl-2′, 7′-difluorescein, diacetate (DAF-FM DA) were purchased from Sigma (St. Louis, MO, USA),
HB/Light Inhibits HepG2 Cells Growth
We initiated our study by examining the phototoxicity of HB using MTT assay on HepG2 cells incubated with HB (0–5 μM) for 4 h and then exposed to LED light at 463 nm for 0–3 h (Fig. 1). As shown in Fig. 1A, HB/light treatment caused HepG2 cells shrinkage and partial detachment over a 24-h period. Fluorescence microscopy with AO/EB staining confirmed many non-viable cells with nuclei stained orange in the HB/light-treated group. Although HB alone had little effected on the cell morphology, even at
Discussion
HB is one of the main photosensitive pigments of hypocrellin, a kind of peryloquinone from fungus Shiraia bambusicola[2]. The photochemical study of HB using electron paramagnetic resonance (EPR) spin-trapping and spin-counteraction techniques provided evidence for the generation of active oxygen (1O2,O2⋅− , ⋅ OH) in HB photosensitization process [16]. It has been universally established that reactive oxygen species (ROS) generated photosensitizers is vital for PDT effects [17]. Flow cytometry
Acknowledgments
The authors are grateful to the Graduate Program of Higher Education in Jiangsu Province (No. CXLX13-841), Suzhou Scholar Program (No. 14317363) and the National Natural Science Foundation of China (No. 81473183) for financial support of this work.
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