Enhanced gene therapy of lung and liver cancer by controlling autophagy and apoptosis

Abstract

Even with the development in medical technologies, lung and liver remained as difficult organs for early diagnosis for cancers. Therefore, morbidity and mortality figures still remain high. As cancer is not a disease cause by dysregulation of single gene or protein, it is hard to expect a complete recovery. Autophagy is a cellular mechanism controlling both cell survival and death. The thesis covers the therapeutic strategies to enhance the efficacy in aspects of controlling autophagy and introducing 2A peptide cleavage sequence to induce synergistic anti-tumor effects. In Part I, beclin1 which plays a major role in autophagy has been chosen as a target. Beclin1 has been cloned into lentivirus vector and delivered to lungs of K-rasLA1 lung cancer model mouse twice a week for 4 weeks to induce prolonged activation of autophagy. As beclin1 has been delivered to the target organ, which is lung, beclin1, LC3-II levels have been increased and in turn, p62 has been decreased. Number and size of the tumors in the lungs of K-rasLA1 mice decreased after beclin1 delivery, and progression of tumorigenesis towards adenocarcinoma has been delayed in histopathological analysis. Under transmission electron microscopy, autophagic vacuoles have been detected as a typical outcome of autophagy activation, and add to the result, abnormal morphology of mitochondria has been detected. When apoptosis activates, nuclear changes come first and number of mitochondria increases as a compensatory mechanism. This phenomenon has been found in the lungs of K-rasLA1 mice after delivery of beclin1. However, the amount of mitochondrial 12S RNA, which participates in generation of ATP in the mitochondria and counts as a standard of mitochondrial function, decreased after all. Mitochondria-related apoptotic proteins, Bax, Apaf-1, c-PARP and cytochrome, and TUNEL positive cells have been significantly increased. As a final consequence, not only autophagy, but also apoptosis were activated in lungs of K-rasLA1 mice. In Part II, beclin1 was delivered to lungs of K-rasLA1 lung cancer model mice together with fractionated radiation to induce synergistic anti-tumor effect. Radiotherapy is known to induce radioresistance and to activate autophagy for cell survival. However, as proved in Part I, prolonged activation of autophagy can lead to autophagic cell death. Fractionated radiation opening to thorax (2 Gy, 5 times) to minimize the side effect and inhalation of beclin1 synergistically induced cancer cell death in lung cancers. Increase in number of autophagic vacuoles and increase in protein levels of beclin1, ATG5, LC3-II were observed. Radiation together with beclin1 inhalation activated the dissociation of beclin1-bcl2 complex and suppressed phosphorylation of Akt1 at Serine473 and Threonine308. It also affected both raptor in mTORC1 and rictor in mTORC2. Prolonged activation of autophagy induced positive feedback, suppressed Akt-mTOR pathway and maximized anti-cancer effects. In Part III, 2A-peptide self-cleavage sequence was introduced to livers of H-ras12V liver cancer model mice, to activate two or more genes at the same time with equal efficiency. Using galactosylated-poly(ethylene glycol)-chitosan-graft-spermine (GPCS), which is a liver targeting non-viral vector, LETM1-2A-CTMP was made into complex and delivered to the target organ. After four weeks of repeated exposures, 2A-peptide-mediated delivery was able to induce synergic effect of LETM1 and CTMP. The hepatocytes in the tumor area remained as altered foci, not being progressed as hepatocellular adenoma (HCA) or hepatocellular adenocarcinoma (HCC). Under transmission electron microscopy, the cristae of mitochondria have been disrupted and abnormal morphology in nuclear membrane (indentation and disruption of nuclear membrane) was observed. Protein levels of mitochondria-related apoptosis, including Apaf-1, bax and cytochrome c, as well as number of TUNEL positive cells were increased, confirming the activation of apoptosis in liver cancer. Development of strategies minimizing side effects and maximizing therapeutic efficacy is important. Introduction of beclin1 to control autophagy and combination use of fractionated radiation proved to enhance the therapeutic efficacy. Also, linkage of LETM1 and CTMP using 2A peptide cleavage sequence induced synergistic outcome. Intervention of therapeutic mechanism which is known to bring side effects and introduction of techniques to control two or more genes are promising strategies to bring more effective outcome in development of gene therapeutics.