京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第18214号 / 薬博第804号 / 新制||薬||237(附属図書館) / 31072 / 京都大学大学院薬学研究科生命薬科学専攻 / (主査)教授 金子 周司, 教授 髙倉 喜信, 教授 竹島 浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Oxaliplatin as an effective chemotherapeutic agent in FOLFOX regimens is using to treat colorectal cancer. In this study we investigate cytotoxicity of Oxaliplatin as single chemotherapeutic agent toHCT116P53+/- to identify molecular mechanism of Oxaliplatin action in induction of apoptosis pathway. Oxaliplatin exposure to HCT116P53+/- colorectal cell lines with deficiency of mismatch repair characteristic resulted to decrease the number of viable cells through apoptosis. Effective Oxaliplatin concentrations (IC50) which inhibit 50% of cell viability were determined using XTT method. Standard curve and time-dependent assay performed to confirm IC50 concentration. Western blot analysis demonstrated relocalization of Bax to mitochondria and induction of intrinsic apoptosis pathway resulted Oxaliplatin exposure. Inactivation of Bax in HCT116P53+/- will result significant reduction in number of viable cells following treatment with Oxaliplatin

Pt(II) chemotherapies, including cisplatin and oxaliplatin, have been used in cancer treatment since the 1970s, however, a full understanding of the mechanism by which these drugs function is still lacking. While the interaction between Pt(II) drugs and DNA has been extensively studied and subsequently indicted in the cellular response to Pt(II) drugs, recent data indicates non-DNA targets play important roles as well. To gain insight into the non-DNA damage-based effects induced by these drugs, MDA-MB-468 cells were treated at therapeutic concentrations of cisplatin between 30 minutes and 24 hours. Not only does this data provide insight into the complex time-dependent nature of the cellular response to cisplatin, but novel responses were also observed. First, I describe how the expression of numerous snoRNAs decreases as early as 30 minutes post-treatment with either cisplatin or oxaliplatin, and differential expression analysis indicates this occurs before activation of the DNA damage response. Since snoRNAs are necessary components in ribosome processing, we sought to determine the role snoRNAs play in the cellular response to Pt(II) drugs. A subgroup of our identified snoRNAs direct modification of helix 69 on the 28S ribosome. Quantification of methylation of helix 69 and other locations suggests cisplatin induced changes in snoRNA expression leads to dysregulation of rRNA modification, likely altering ribosome activity. I also observe varied activation of different types of DNA damage and cell cycle arrest between 3 and 12 hours of cisplatin treatment while early expression changes show downregulation of mitochondrial genes. We also identify a number of lncRNAs previously associated with TNBC that are downregulated after cisplatin treatment. This study establishes a gene expression profile induced by cisplatin treatment of triple-negative breast cancer that demonstrates the complex interplay of multiple means of stress induction. Lastly, we establish a method for analyzing direct DNA binding targets of platinum(II) chemotherapeutics. This pilot study confirms high accumulation of platinum(II) compounds on guanine-rich DNA and suggests DNA binding of significant genes leads to changes in their RNA expression. / 10000-01-01

No description available.

DNA polymerase η (pol η), a crucial component of the cellular translesion synthesis pathway, allows cells to bypass and thereby temporarily tolerate DNA damage. Inherited deficiency of pol η, as reported in the variant form of xeroderma pigmentosum, predisposes to UV light-induced skin cancers. To date, pol η is the only DNA polymerase shown to exhibit a causal link to the formation of cancers in humans. However, the role of pol η in the cellular response to forms of DNA damage other than UV-induced lesions is largely unknown. In the first part of this thesis, it is shown that cells deficient in pol η are resistant to ionising radiation. Deficiency in the polymerase was associated with accumulation of cells in S phase of the cell cycle. Cells deficient in pol η demonstrated increased homologous recombination-directed repair of DNA double-strand breaks created by ionising radiation, and depletion of the homologous recombination protein X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells compared to pol η-complemented cells. These findings suggest that homologous recombination mediates S phase-dependent radioresistance associated with pol η-deficiency. In the second part of this thesis, it is shown that pol η-deficient cells have increased sensitivity to the chemotherapeutic compound, oxaliplatin, compared to pol η-deficient expressing cells, but not to the drug 5-fluorouracil that is usually administered in combination with oxaliplatin in the clinical setting. Despite the importance of pol η for cellular survival following exposure to oxaliplatin, the drug did not upregulate the enzyme after either short-term or long-term exposure. Inhibition of pol η activity by siRNA-mediated knockdown of the protein sensitised cells to oxaliplatin treatment, and partially reversed acquired resistance in oxaliplatin-resistant tumour cell lines. These data suggest that pol η is an interesting target whose function can potentially be interfered with to optimise oxaliplatin-based chemotherapy. In the third part of this thesis, clinical samples obtained from oesophageal cancer patients before and after treatment with oxaliplatin-containing chemotherapy were analysed for POLH mRNA levels encoding pol η protein. Malignant tissue specimens obtained before treatment demonstrated a significantly higher level of POLH mRNA than matched normal oesophageal tissue samples. Contrary to the preclinical data, high POLH mRNA expression before therapy was shown to correlate with increased overall and disease-free survival of the patient cohort in the clinical trial. Additionally, patients with high POLH mRNA-expressing cancers had better therapeutic responses (measured by PET-CT) to oxaliplatin-based treatment than those with low levels. These data suggest that POLH mRNA expression should be tested as a biomarker to predict survival and therapeutic responses in oesophageal cancer patients treated with oxaliplatin-containing chemotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic cancer resistant to current treatments. A better understanding of the genetic basis of PDAC progression is urgently needed to improve treatment options. The ARF tumor suppressor is inactivated in ~45% of PDAC. My thesis lab identified a new, uncharacterized ARF binding protein, Partner of ARF isoform 1A (Parf-1A). This thesis explores the hypothesis that Parf-1A plays an important role in PDAC and ARF tumor suppressor signaling Initial studies sought to develop a novel mouse xenograft model of PDAC metastasis that would expedite testing of putative PDAC genes. Human PDAC cell lines stably expressing luciferase were generated and introduced by intracardiac injection into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored non-invasively by bioluminescence imaging and found to recapitulate PDAC tumor formation and metastatic distribution. The model was validated by the ability of ARF to suppress PDAC cancer cell migration in vitro and reduce tumor cell colonization in vivo; establishing a new bioluminescent mouse model for rapidly assessing the significance of suspected PDAC genes. Using human PDAC cell lines and tumor specimens, we investigated the role and significance of Parf-1A to PDAC. RNAi analyses demonstrated Parf-1A is required for PDAC cell survival, proliferation and resistance to the PDAC therapeutic, oxaliplatin. PDAC cells are ARF-null; therefore these tumor promoting activities of Parf-1A were independent of ARF. Notably, immunohistochemical analyses of Parf-1A in human PDAC tumors showed Parf-1A expression is a prognostic marker of poor survival in PDAC patients. These data suggest Parf-1A is a novel biomarker of PDAC and potential target for anticancer therapy. Other studies tested how Parf-1A influenced ARF signaling. Parf-1A depletion and overexpression showed it inhibits ARF anti-proliferative activity by mobilizing ARF from the nucleus (where it is functional) into the cytoplasm. These data show Parf-1A is a new inhibitor of ARF. Considered with findings that Parf-1A can act independent of ARF to promote PDAC tumorigenesis, such results suggest Parf-1A is a novel oncoprotein that acts through multiple pathways to facilitate tumorigenesis. Thus, Parf-1A may have broad relevance to many types of human cancers.

Doctor of Philosophy (PhD) / The H69CIS200 cisplatin-resistant and H69OX400 oxaliplatin-resistant cell lines developed as part of this study, are novel models of low-level platinum resistance. These resistant cell lines do not have common mechanisms of platinum resistance such as increased expression of glutathione or decreased platinum accumulation. Rather, these cell lines have alterations in their cell cycle allowing them to proliferate rapidly post drug treatment in a process known as ‘regrowth resistance’. This alteration in cell cycle control has come at the expense of DNA repair capacity. The resistant cell lines show a decrease in nucleotide excision repair and homologous recombination repair, the reverse of what is normally associated with platinum resistance. The alterations in these DNA repair pathways help signal the G1/S checkpoint to allow the cell cycle to progress despite the presence of DNA damage. The decrease in DNA repair capacity has also contributed to the development of chromosomal alterations in the resistant cell lines. Similarities in chromosomal change between the two platinum resistant cell lines have been attributed to inherent vulnerabilities in the parental H69 cells rather than part of the mechanism of resistance. The H69CIS200 and H69OX400 resistant cells are cross-resistant to both cisplatin and oxaliplatin. This demonstrates that oxaliplatin does not have increased activity in low-level cisplatin-resistant cancer. Oxaliplatin resistance also developed more rapidly than cisplatin resistance suggesting that oxaliplatin may be less effective than cisplatin in the treatment of SCLC. The resistant cell lines have also become hypersensitive to taxol but show no alterations in the expression, polymerisation or morphology of tubulin. Rather, the PI3K/Akt/mTOR pathway is involved in both platinum resistance and taxol sensitivity as both are reversed with rapamycin treatment. mTOR is also phosphorylated in the resistant cell lines indicating that platinum resistance is associated with an increase in activity of this pathway. The mechanism of regrowth resistance in the platinum-resistant H69CIS200 and H69OX400 cells is a combination of activation of PI3K/Akt/mTOR signalling and alterations in control of the G1/S cell cycle checkpoint. However, more work remains to determine which factors in these pathways are governing this novel mechanism of platinum resistance.

Doctor of Philosophy (PhD) / The H69CIS200 cisplatin-resistant and H69OX400 oxaliplatin-resistant cell lines developed as part of this study, are novel models of low-level platinum resistance. These resistant cell lines do not have common mechanisms of platinum resistance such as increased expression of glutathione or decreased platinum accumulation. Rather, these cell lines have alterations in their cell cycle allowing them to proliferate rapidly post drug treatment in a process known as ‘regrowth resistance’. This alteration in cell cycle control has come at the expense of DNA repair capacity. The resistant cell lines show a decrease in nucleotide excision repair and homologous recombination repair, the reverse of what is normally associated with platinum resistance. The alterations in these DNA repair pathways help signal the G1/S checkpoint to allow the cell cycle to progress despite the presence of DNA damage. The decrease in DNA repair capacity has also contributed to the development of chromosomal alterations in the resistant cell lines. Similarities in chromosomal change between the two platinum resistant cell lines have been attributed to inherent vulnerabilities in the parental H69 cells rather than part of the mechanism of resistance. The H69CIS200 and H69OX400 resistant cells are cross-resistant to both cisplatin and oxaliplatin. This demonstrates that oxaliplatin does not have increased activity in low-level cisplatin-resistant cancer. Oxaliplatin resistance also developed more rapidly than cisplatin resistance suggesting that oxaliplatin may be less effective than cisplatin in the treatment of SCLC. The resistant cell lines have also become hypersensitive to taxol but show no alterations in the expression, polymerisation or morphology of tubulin. Rather, the PI3K/Akt/mTOR pathway is involved in both platinum resistance and taxol sensitivity as both are reversed with rapamycin treatment. mTOR is also phosphorylated in the resistant cell lines indicating that platinum resistance is associated with an increase in activity of this pathway. The mechanism of regrowth resistance in the platinum-resistant H69CIS200 and H69OX400 cells is a combination of activation of PI3K/Akt/mTOR signalling and alterations in control of the G1/S cell cycle checkpoint. However, more work remains to determine which factors in these pathways are governing this novel mechanism of platinum resistance.

Colorectal cancer is the third most common cancer in the United States with a 5-year late-stage survival rate of only 14%. Due to the lack of translation between animal models and clinical trials as well as the inefficacy of many chemotherapeutics in initial clinical trials, researchers are turning to in vitro drug screening models in an effort to mimic the conditions in vivo. This research project aimed to validate an in vitro tumor culture model within a microfluidic device using a clinically relevant chemotherapy drug. The first experiment consisted of a cell density and drug concentration study to determine the correct cell density and oxaliplatin concentration combinations that would result in a spectrum of quantifiable effects on the tumor cells. This experiment was then converted from a monolayer cell culture on glass into a 2D culture on top of a fibrin extracellular matrix (ECM) to ensure that the cells would respond in a similar way to the drug in the presence of an ECM as they did in the first experiment. The third experiment involved SW620 cells cultured within the fibrin hydrogel to create a 3D tumor model that better mimics the growing conditions in vivo. The goal of this experiment was again to ensure that the cells would respond in the same way to the oxaliplatin treatments as the previous experiments when adding complexity to the model. The final experiment was then to convert this 3D experiment performed in chamber slides into a 3D culture within a microfluidic device with media and oxaliplatin treatments perfused through the chamber using a syringe pump. The purpose of this experiment was to assess whether tumor cells could grow and survive within a microfluidic device with interstitial flow as well as determining if they responded as expected to the oxaliplatin treatment. The first three experiments performed within chamber slides showed that tumor count and average tumor size decreased with increasing oxaliplatin concentrations as expected, which is comparable to the in vivo tumor response to the drug. The fourth experiment demonstrated that, although cells are able to grow within the microfluidic device, this model did not accurately replicate the in vivo condition and future work needs to be aimed at improving the design of the device as well as optimizing parameters within the experiment.

no / Chemotherapy drugs usually inflict a lethal dose to tumour cells with the consequence that these cells are being killed by cell death. However, each round of chemotherapy also causes damage to normal somatic cells. The DNA cross-linking agent oxaliplatin which causes DNA double-strand breaks and vinflunine which disrupts the mitotic spindle are two of these chemotherapy drugs which were evaluated in vitro using peripheral lymphocytes from colorectal cancer patients and healthy individuals to determine any differential response. Endpoints examined included micronucleus (MN) induction using the cytokinesis-blocked micronucleus (CBMN) assay and pancentromeric fluorescence in situ hybridisation. Also, survivin expression was monitored since it regulates the mitotic spindle checkpoint and inhibits apoptosis. Oxaliplatin produced cytogenetic damage (MN in binucleated cells) via its clastogenic but also previously unknown aneugenic action, possibly through interfering with topoisomerase II, whilst vinflunine produced MN in mononucleated cells because of incomplete karyokinesis. Survivin expression was found to be significantly reduced in a concentration-dependent manner by not only oxaliplatin but surprisingly also vinflunine. This resulted in large numbers of multinucleated cells found with the CBMN assay. As survivin is upregulated in cancers, eliminating apoptosis inhibition might provide a more targeted chemotherapy approach; particularly, when considering vinflunine, which only affects cycling cells by inhibiting their mitotic spindle, and alongside possibly other pro-apoptotic compounds. Hence, these newly found properties vinflunine – the inhibition of survivin expression - might demonstrate a promising chemotherapeutic approach as vinflunine induces less DNA damage in normal somatic cells compared to other chemotherapeutic compounds. / Saudi Arabian Government