Antioxidant pathways and human leukaemia chemotherapeutic sensitivity

Cancer is uncontrolled cellular proliferation devoid of normal biological regulatory mechanisms. Therapeutic treatment of cancers relies on overcoming such proliferation, to allow cytotoxic destruction of cancer cells. Many of the recent therapeutic breakthroughs have been in the targeting of various blood cancers: eg imatinib has effectively eradicated chronic myeloid leukaemia (CML) morbidity. However, acute myeloid leukaemia (AML) remains essentially incurable for the vast majority of patients. The current gold-standard treatment for AML has remained relatively unchanged for decades, consisting of the antimetabolite cytarabine (ara-C) and the anthracycline cytotoxic antibiotic daunorubicin (DNR). The cytoprotective gene haem oxygenase-1 (HO-1) has been found regulated in various forms of cancers (including AML) and implicated in chemotherapeutic drug-resistance. HO-1 protects AML cells from induced apoptosis via its transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2). We show a role for HO-1 in regulating apoptosis in AML cells in response to ara-C and DNR. HO-1 expression was increased in response to both cytotoxic agents. Upon micro RNA (miRNA) silencing of HO-1 expression, both ara-C and DNR stimulated greater apoptotic responses in these silenced AML cells. A concurrent induction in reactive oxygen species (ROS) generation was observed. Studies with ara-C-resistant AML cell lines (THP1araC(1)) showed there to be significantly suppressed levels of Nrf2 and HO-1. A miRNA screen in these resistant cells revealed reduced basal miR-196a expression. One of miR-196a’s targets is the Nrf2-inhibitory protein Bach1 (BTB and CNC homology 1), which was found to be elevated in these cells. Exogenous replacement of miR-196a with the introduction of a miR-mimic, suppressed the Bach1 overexpression, elevated HO-1 expression, and reintroduced sensitivity towards ara-C. These findings suggest HO-1 inhibition in conjunction with chemotherapy would improve the number of cases who reach complete remission (CR)

Évaluation des analogues de la désoxycytidine dans la thérapie génique du cancer

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Mechanism-based rational design of cisplatin analogues

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2005.Vita.Includes bibliographical references.The success of cisplatin as an anticancer drug is attributed to the ability of the platinum compound to damage DNA and subsequently induce apoptosis. Details of the cellular processing of cisplatin-damaged DNA can provide invaluable insight into the rational design of cisplatin analogues or combination therapies. Chapter I provides a survey of recent developments in the understanding of the mechanism of cisplatin action and summarizes relevant platinum-based anticancer compounds. Chapter 2 describes a series of estrogen-tethered platinum(IV) complexes (BEPn, n=l -5) that were synthesized, evaluated for their ability to upregulate HMGB1 and screened for cytotoxicity against human breast cancer cell lines. All BEPn complexes induced the overexpression of HMGB I in ER(+) MCF-7 cells. BEP3 was nearly twice as cytotoxic in ER(+) MC'F-7 cells than in ER(-) HCC-1937 cells. This result suggests the possibility of using compounds in this class specifically to target ER(+) malignancies, such as breast and ovarian cancers. In addition, the series of BEPn compounds provide an example of a useful strategy in the development of platinum-containing anticancer agents, namely, using mechanistic insights to aid in the rational design of new complexes.(cont.) The strategy of exploiting estrogen-induced HMGBI upregulation to sensitize ER(+) cells to platinum was further pursued in work described in chapters 3 and 4. Chapter 3 reports the synthesis and characterization of a series of platinum(IV)-estrogen conjugates derived from carboplatin. Although these BECPn complexes were moderately cytotoxic in ER(+) MCF-7 human breast cancer cells, no differential cytotoxicity was observed as compared to ER(-) HCC- 1937 cells. However, these compounds represent the first example of a biomolecule-tethered platinum(IV) complex that reduces to yield carboplatin in cells. The platinum estrogen conjugate described in chapter 4 was designed not only to induce upregulation of HMGB I but also to enter ER(+) cells selectively. Unlike the BEP and BECP compounds, BEEP was designed to maintain affinity for the estrogen receptor and by tethering platinum to estradiol through the 17c-position of the steroid ring. Compounds with affinity for the estrogen receptor, which is overexpressed in breast and ovarian cancers, are selectively taken up into ER(+) cells. Unexpectedly, BEEP had very low affinity for the estrogen receptor and was therefore equally cytotoxic in ER(+) and ER(-) human breast cancer cell lines.(cont.) A common feature of many cancers is overexpression of the folate receptor, which is responsible for the uptake of folic acid. Therefore targeting the folic receptor is an attractive method for achieving selective uptake in cancer cells. Chapter 5 describes the synthesis and biological activity of a folic acid-tethered platinum(lV) compound, which demonstrates the validity of this premise. The nuclear protein HMGBI has recently been discovered to function as an extracellular signaling agent. Because of oxygen deprivation, the core of a solid tumor dies by necrosis and passively releases HMGB I into the extracellular environment. This characteristic of solid tumors leads to the hypothesis that extracellular HMGB I is taken up by surrounding viable tumor tissue and mediates cisplatin sensitivity. The final chapter investigates the capability of exogenously administered HMGB to modulate the cytotoxicity of cisplatin and trans-DDP in human cancer cells. The Appendix sections provide detailed experimental protocols for several useful laboratory methods. In Appendix A, a procedure for isolation of nuclei from cisplatin-treated cells is presented.(cont.) The nuclei were subsequently used by our collaborators to examine the post- translational modifications of histones induced by cisplatin exposure. A protocol for isolation of protein extracts from formalin-fixed paraffin-embedded tissue is described in Appendix B. In addition, the extracts were probed by western blot analysis to examine the expression levels of HMGB1 in clinical testicular seminoma samples. Appendix C provides a solid-phase synthetic methodology for the preparation of peptide-conjugated platinum(IV) compounds.by Katie R. Barnes.Ph.D