Novel Insights into the Effect of Hyperforin and Photodynamic Therapy with Hypericin on Chosen Angiogenic Factors in Colorectal Micro-Tumors Created on Chorioallantoic Membrane

Abstract

Photodynamic therapy with hypericin (HY-PDT) and hyperforin (HP) could be treatment modalities for colorectal cancer (CRC), but evidence of their effect on angiogenic factors in CRC is missing. Convenient experimental model utilization is essential for angiogenesis research. Therefore, not only 2D cell models, but also 3D cell models and micro-tumors were used and compared. The micro-tumor extent and interconnection with the chorioallantoic membrane (CAM) was determined by histological analyses. The presence of proliferating cells and HY penetration into the tumor mass were detected by fluorescence microscopy. The metabolic activity status was assessed by an colorimetric assay for assessing cell metabolic activity (MTT assay) and HY accumulation was determined by flow cytometry. Pro-angiogenic factor expression was determined by Western blot and quantitative real-time polymerase chain reaction (RT-qPCR). We confirmed the cytotoxic effect of HY-PDT and HP and showed that their effect is influenced by structural characteristics of the experimental model. We have pioneered a method for analyzing the effect of HP and cellular targeted HY-PDT on pro-angiogenic factor expression in CRC micro-tumors. Despite the inhibitory effect of HY-PDT and HP on CRC, the increased expression of some pro-angiogenic factors was observed. We also showed that CRC experimental micro-tumors created on quail CAM could be utilized for analyses of gene and protein expression.

Potentiality, Limitations, and Consequences of Different Experimental Models to Improve Photodynamic Therapy for Cancer Treatment in Relation to Antiangiogenic Mechanism

“…So far, it has been more than one hundred years since the first note of cell culture cultivation [ 70 ]. Since then, the cell cultivating protocol quality has improved extensively, and we can observe a persisting effort in the development of new protocols and cultivation techniques [ 14 , 71 , 72 ]. Now, established malignant and non-malignant experimental cell lines are extensively utilized in a wide range of biological analyses.…”

“…Therefore, the consideration of the third dimension seems to be a very convenient approach for mimicking spatial tumor characteristics [ 14 , 86 , 87 , 88 , 89 , 90 ]. It is important to state that prevalently decreased proangiogenic growth factors, like PD-ECGF and FGF-2 protein expression after PDT with hypericin (HY-PDT) in HCT 116 cells [ 14 ], VEGF after PDT with aminolevulinic acid (ALA-PDT) in SW620 [ 61 , 62 ], and PDT with meta-tetra(hydroxyphenyl)chlorin (mTHPC-PDT) in VB6 and H376 cells [ 64 ], were observed if a 2D cell experimental model was utilized. This could be associated with the above-mentioned aspects of sufficient PS penetration in the whole targeted cell population [ 14 ].…”

“…It is important to state that prevalently decreased proangiogenic growth factors, like PD-ECGF and FGF-2 protein expression after PDT with hypericin (HY-PDT) in HCT 116 cells [ 14 ], VEGF after PDT with aminolevulinic acid (ALA-PDT) in SW620 [ 61 , 62 ], and PDT with meta-tetra(hydroxyphenyl)chlorin (mTHPC-PDT) in VB6 and H376 cells [ 64 ], were observed if a 2D cell experimental model was utilized. This could be associated with the above-mentioned aspects of sufficient PS penetration in the whole targeted cell population [ 14 ]. However, there are also opposite findings documented when prostaglandin COX-2 upregulation as another type of angiogenic factor after PDT with HY [ 59 ] photofrin (PT) [ 28 , 29 ] and piropheophorbide-a methyl ester (PPME) [ 68 ] were detected with the utilization of 2D cell models.…”

“…However, the increasing number of novel findings makes us pose novel questions. Nowadays, the effect of photosensitizers (PS) alone or in a combination with PDT has been analyzed from different aspects [ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. In this context, the effect on angiogenesis represents one of the most studied issues (see Table 1 , Table 2 and Table 3 ).…”

“…Based on these facts, we assume that the targeting potential of PDT represents a fundamental condition for the extension of applied potential for a wide range of tumors of different histological origins. Simultaneously, the development of novel experimental models convenient for angiogenic research now represents a very interesting and examined area in the field of tumor angiogenesis research [ 14 , 21 , 22 ]. Moreover, an appreciable effort has been dedicated to a PDT study as a combined therapeutic approach with multiple antiangiogenic agents utilized [ 15 , 17 , 20 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ].…”