RUNX3Expression Is Associated With Pathogenesis And Sensitivity To Pheophorbide A Based Photodynamic Therapy In Keloid

Keloid disease (KD), a special type of scar disease, is well known as a connective tissue tumor. Morphologically, it is characterized by ridges on the surface of the skin having a smooth surface, hard texture, and irregular margins. Proliferating fibroblasts and increased production of extracellular matrix components, such as the deposition of excessive amounts of collagen, are among the histological characteristics of KD. A keloid can extend the boundaries of the original wound and cause invasive growth into adjacent normal tissues. Keloids are not degenerate, and recurrence is frequently found after surgical resection alone in patients with KD. Clinically, keloids cause varying degrees of itching and pain, and their functional, aesthetic, and psychosocial impact can seriously impair quality of life for patients with KD.Photodynamic therapy (PDT) is a widely used treatment option for tumor lesions. PDT confines damage to tumor tissue by relying on light irradiation and photosensitizers that selectively accumulate in tumor tissue, thereby preventing harm to the surrounding normal tissue. Because the effects of PDT depend directly on the characteristics of the photosensitizer, identification of reliable photosensitizers is required. Pa, a newly developed photosensitizer, has been reported by some researchers to have antitumor activity and minimal toxicity. Following Pa-based PDT (Pa-PDT), increased apoptosis and decreased proliferation ability of cancer cells have been observed. However, the efficacy and underlying mechanism of Pa-PDT have not yet been widely investigated in keloids. In this study, we determined the therapeutic effect of Pa-PDT in keloids.A recent trend in tumor therapy is the personalized treatment of patients. Molecular analysis of each patient with KD is required to develop and implement effective personal therapy. Recently, some investigators have shown that runt-related transcription factor3(RUNX3) is a molecular target for predicting sensitivity to Pa-PDT.Objects:To elucidate the role of RUNX3in keloid pathogenesis, investigate the therapeutic effect of Pa-PDT in keloids, and identify the relationship between RUNX3expression and the sensitivity of Pa-PDT in keloids.Establish a nudemice model for keloid with keloid fibroblast.Methods:1) keloid fibroblasts (KFs) were cultured from keloid tissues;2) Vimentin, a-SMA and type I collagen expression were detected by immunocytochemistry in KFs;3) MTT assay was performed in KFs for detect the toxicity of both Pa and Pa-PDT;4) Quantitative real-time PCR analysis was done for keloid pathogenesis and apoptosis-related genes:PCNA and Type I collagen expression were comparatively investigated in KFs treated with Pa alone and those treated with Pa-PDT;5) ROS generation was assayed:a fluorescent probe,2’,7’-dichlorofluorescin diacetate dye, was used to detect the intracellular level of ROS in each group of KFs;6) Apoptotic cell death in KFs was assessed after Pa-PDT and compared between KFs treated with Pa alone and those treated with Pa-PDT;7) Apoptosis-related protein expression was assayed in KFs post-Pa-PDT:Caspase-3, cleaved caspase-3, cleaved poly (adenosine diphosphate ribose), and polymerase (PARP) expressions were compared between the two groups of KFs by western blot analysis;8) RUNX3expression was detected by immunohistochemistry in keloid tissue samples and the clinicopathological siginificance was also investigated;9) RUNX3expression was detected by immunocytochemistry in KFs;10) RUNX3down-regulation was performed by siRNA transfection and the quantitative real-time PCR analysis was done for determine PCNA expression in the KFs;11) quantitative real-time PCR analysis was performed for determine RUNX3expression in KFs treated with Pa-PDT;12) PCNA, type I collagen expression, and ROS generation as well as apoptotic cell death were comparatively investigated between KFs with and without RUNX3expression;13) The effect of Pa-PDT were comparatively investigated in RUNX3down-regulated KFs and related control cells;14) Establish keloid fibroblast nudemice model:The mixed external keloid fibroblast and basal membrane matrigel was inoculated in the subcutaneous of the nudemice with cell density of1.0×107/ml,3.0×107/ml,5.0x107/ml, the total amount was0.1ml. And then, to observe the formation and growth condition and to screen an appropriate inoculated cell density, to conduct internal transfer with the established keloid tumor body, then establish a transplant model. Results:1) Established5KFs due to primary culture program and numbered as KF1, KF2, KF3, KF4and KF5;2) Vimentin, α-SMA and type Ⅰ collagen expression were detected in all5KFs;3) Minimal cytotoxic effect and cellular accumulation of Pa was found in all4KFs;4) Cell viability was significantly reduced after Pa-PDT in all four KFs;5) Type Ⅰ collagen, PCNA expressions were significantly decreased in KFs post Pa-PDT (P<0.05);6) ROS generation was significantly increased in KFs after Pa-PDT(P<0.05);7) Apoptotic cell death was significantly increased in KFs after Pa-PDT(P<0.05);8) Both cleaved caspase-3and cleaved PARP expression were significantly increased in KFs after Pa-PDT;9) Nuclear expression of RUNX3was detected in16(50%) keloid tissue samples but not in the dermis of6normal skin samples. RUNX3expression is significantly related to the increased proliferating ability of KFs and the presence of symptoms, such as pain and pruritus in KD patients(P=0.002and P=0.013, respectively);10) RUNX3expression was found in both KF1and KF2but not in KF3, KF4and KF5;11) PCNA expression was significantly reduced by RUNX3down-regulation;12) RUNX3expression was significantly reduced in KFs after Pa-PDT;13) Significant difference was found between KFs with and without RUNX3expression in the relative fold change of PCNA and type Ⅰ collagen expression, and ROS generation and apoptotic cell death(P<0.05);14) after Pa-PDT, the toxicity was decreased in RUNX3down-regulated KFs than control cells (P<0.05);15) Establish keloid fibroblast nudemice model successfully and screen a more appropriate cell inoculated density as5.0×107/ml. The tumor body was still enlarging through internal transfer. There was no significant difference between the two histopathology.Conclusion:1) In KFs, both ROS generation and apoptotic cell death are significantly increased after Pa-PDT, and cell viability is significantly decreased after Pa-PDT;2) In keloid tissues, RUNX3is expressed in the nucleus of KFs, and it is related to the increased proliferating ability of KFs and to the presence of symptoms such as pain and pruritus in KD patients;3) Cell viability is increased in RUNX3-downregulated KFs compared to control cells following Pa-PDT;4) Both ROS generation and apoptotic cell death increased in RUNX3-positive KFs compared to RUNX3-negative KFs;5) It was concluded that inoculating human keloid fibroblast in the subcutaneous of the nudemice could establish a keloid nudemice model.We conclude that Pa-PDT is a potential therapeutic modality for KD and that RUNX3, as a possible contributor to keloid pathogenesis, may improve sensitivity to Pa-PDT in KFs. RUNX3should be further investigated as a therapeutic target and predictive biomarker for the assessment of sensitivity to Pa-PDT in KD.