In vitro Study on the Long-term Fate of HepG2 Cells after Photodynamic Therapy

未完整的癌症治療往往引致癌細胞再生。胱天蛋白酶3一直被認為是無法逆轉的細胞凋亡檢測點，可是近年研究發現細胞縱使激活了胱天蛋白酶3還可以逆轉凋亡，引起推測這個逆轉過程是引致癌症復發。光動力療法是近來醫學上認可的一種治療癌症方法。此療法以光去激活在癌細胞內的光増感劑來殺死癌細胞。雖然光動力療法有比傳統癌症治療有更高的針對性和療效，但是還未有研究去證明光動力療法可以避免癌症復發。因此，是次研究肝癌細胞HepG2在以脫鎂葉綠酸鹽A作増感劑的光動力療法後的細胞表現。在光動力療法後、細胞的形態、生存率和細胞增殖分別以活細胞影像、MTT法和羧基熒光素二醋酸琥珀酰亞胺酯標籤去檢測。細胞在Z動力療法48小時後能由凋亡形態回復，並於72小時增殖。凋亡特徵，包括粒線體膜去極化、胱天蛋白酶3激活、磷脂酰絲氨酸曝露、多聚ADP核糖聚合酶裂解和DNA斷裂，以流式細胞法、西方墨點法和彗星試驗去測試。上述的凋亡特徵皆在逆轉晚期凋亡時消失。凋亡細胞以細胞表面曝露磷脂酰絲氨酸作標籤來細胞分選。這些分選出來的凋亡細胞能夠逆轉凋亡和培養，其細胞形態則有改變。分選的細胞中的信使核糖核酸表達指出癌細胞抗藥性，癌幹細胞演化和侵入性可能與逆轉凋亡有關。綜合以上結果所得，光動力療法研究方向應由對癌細胞直接毒性改向由抑制血管新生和免疫調節著手。Incomplete cancer treatment often results in a rapid repopulation of tumour cells after radiotherapy and chemotherapy. While caspase-3 activation is generally regarded as the “point of no return”, results of recent studies led to a hypothesis that tumour cells could revive from late stage apoptosis, leading to cancer recurrence. Photodynamic therapy (PDT) is one latest anti-cancer strategy available in clinic. It involves light activation of non-toxic photosensitizer at the tumour site. Although PDT is renowned for having a higher potency and specificity than the traditional anti-cancer treatment, whether PDT can overcome the challenge of cancer recurrence remains to be determined. Herein, we investigate the fate of the hepatocellular carcinoma HepG2 cells after PDT using Pheophorbide a (Pa), an apoptosis-inducing photosensitizer. The cellular morphology, viability and proliferation of post-Pa-PDT HepG2 cells were examined by live-cell imaging, MTT cell viability assay and CFSE cell trace analysis. Morphological recovery of tumour cells from apoptosis was observed 48 h post PaPDT, followed by tumour cell repopulation at 72 h. Apoptotic hallmarks, including mitochondria depolarization, caspase-3 activation, phosphatidylserine (PS) exposure, poly (ADP-ribose) polymerase cleavage and DNA breakage, were analysed by flow cytometry, Western blot analysis and comet assay. Disappearance of these hallmarks suggested that tumour cells could rescue themselves upon initiation of apoptosis even to an advanced stage. The apoptotic cells were sorted out using PS as marker. While the PS-sorted cells could be cultured and proliferate, aberrant morphology was observed in these revived cells. These findings showed that Pa-PDT treated cells could revitalize from advanced stage of apoptosis. The mRNA expression of the sorted apoptotic cells changed and suggested that reversibility of apoptosis in tumour cells may be associated with drug resistance, prevalence of cancer stem cells and increase in the invasiveness. Thus, instead of focusing on the direct cytotoxicity in PDT, the anti-angiogenic and immunomodulatory effects should also be emphasised in anticancer treatment.Yau, Wing Ki.Thesis M.Phil. Chinese University of Hong Kong 2016.Includes bibliographical references (leaves ).Abstracts also in Chinese.Title from PDF title page (viewed on …).Detailed summary in vernacular field only