Isolation And Identification Of Bamboo Yellow And The Mechanism Of Photodynamic Induction Of Apoptosis By Hypocrellin

Shiraia bambusicola is a kind of fungi which parasitize on certain species of bamboo,and its fruit bodies are often used as an important Chinese medicine. Hypocrellins are the main active chemicals isolated from Shiraia bambusicola. Hypocrellin, a class of perylenequinoid pigments, was first discovered by Chinese researchers by extraction from fruiting bodies of Shiraia bambusicola. Recently, with the development of pharmacological research of hypocrellins, Shiraia bambusicola shows great application prospects and attracts researchers’ attention.As present, Shiraia bambusicola and hypocrellins still come from limited natural resource which is restricted to geographical distribution, season, host plant and other factors.In order to achieve hypocrellin mass production under artificial conditions, it is necessary to isolate a strain suitable for production. By applying a variety of culture medium, such as PDA,etc., combined with tissue separation method and color reaction of perylenequinone, a perylenequinone-producing strain was isolated from Shiraia bambusicola fruit bodies, and was named as S-4201. The crude pigment extracts were separated by reversed-phase high performance liquid chromatograph. Compared with the extracted hypocrellin reference standard,it was confirmed that the main component of the pigment produced by S-4201 was hypocrellin A (HA). The PCR amplification was carried out using universal primer ITS5/ITS4. ITS rDNA was 533 bp,a total of 13 sequences alignment were compared. The number of loci was 508 bp, of which 143 (28.15%) are variable sites and 365 (71.85%) are conserved sites. The results of rDNA cluster analysis showed that strain S-4201 had a close genetic relationship with Shiraia bambusicola in GenBank. Based on its hypocrellin synthesize feature, together with colony morphology and microstructure, this isolated stain was proved to belong to the genus Shiraia.Photodynamic therapy is a form of therapy systemically or topically applying photosensitizer with visible light irradiation at certain wavelength to eliminate cancer cells or non-cancer cells. At present, there are still controversial debates about cell death pathway induced by hypocrellins. Further study at molecular level is still needed for the development and utilization of hypocrellin as an anticancer drug. HA-mediated cytotoxicity and apoptosis in human lung adenocarcinoma A549 cells were evaluated after photodynamic therapy. A temporal quantitative proteomics approach by iTRAQ (isobaric tag for relative and absolute quantitation) 2D-LC-MS/MS was introduced to help clarify molecular cytotoxic mechanisms and identify candidate targets of HA-induced apoptotic cell death. Specific caspase inhibitors were used to further elucidate molecular pathway underlying apoptosis in PDT-treated A549 cells. Finally, down-stream apoptosis-related proteins were evaluated. Apoptosis induction mediated by HA was associated with cell shrinkage, externalization of cell membrane phosphatidylserine, DNA fragmentation, and mitochondrial disruption, which were preceded by increased intracellular ROS generations. Five important differentially expressed proteins(over 1.2-fold) involved in the apoptosis pathway were identified from the proteins collected from PDT post-treated cells compared with untreated cells. Further studies showed markedly blocked apoptosis by pan-caspase and caspase-9 inhibitors, along with mitochondrial disruption, cytochrome C release, and activation of caspase-3, -9,-7,implying that mitochondria might be the primary site of HA acting in A549 cancer cells when excited by irradiation at 470 nm. Taken together, these findings suggested that HA is a possible therapeutic agent directed toward mitochondria and might be a promising photodynamic anticancer candidate for further evaluation.Because of the poor water solubility of hypocrellin,it is easy to cause aggregation after intravenous injection into blood. It also can make drug administration process complicated and affect biodistribution, therefore the efficacy of photodynamic therapy is decreased. The hydrophobicity of hypocrellin A and its photodegeradation and dark cytotoxity hamper its clinical application. Furthermore, chemical modified hypocrellin often has a high dark toxicity or low singlet oxygen yield, so it become a research focus to use appropriate pharmaceutical technology to improve the water solubility of hypocrellin. In this present study, an oil-in-water (O/W) emulsion solvent evaporation technique was employed to fabricate water-dispersible hypocrellin A-loaded PLGA (poly(lactic-co-glycolic acid))nanoparticles. The physiochemical properties and morphological characteristics of asprepared nanoparticles were evaluated,as well as the photostability,uptake and in vitro phototoxicity and dark toxicity toward A549 cells. SEM (scanning electron microscopy) and CLSM(confocal laser microscopy) images, combined with DLS (dynamic light scattering)measurements and surface charge (ζ-potential) results, showed that the as-prepared HA-loaded nanoparticles were narrowly dispersed in 20～200 nm with a surface charge of -5.8 mV. By means of DSC (differential scanning calorimetry), XRD (X-ray powder diffractometry) and FTIR (fourier transform infrared) spectroscopy,it is confirmed that HA was successfully encapsulated into PLGA . The encapsulation efficiency and drug content were 55.1% and 5.0%, respectively. UV-vis spectra and the results from photobleaching experiments indicated that encapsulation could enhance the photostability of HA. In vitro experiments demonstrated that HA-loaded PLGA nanoparticles was taken up by A549 cells and exhibited significantly reduced dark cytotoxicity while maintaining excellent anti-tumor and apoptosis-reducing property. Further studies revealed that the light-induced cytotoxicity was closely related to ROS production ability.