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Characterization of opsonins with potential for engagements in removal of dying tumour cells following photodynamic therapy

University of British Columbia

It has become evident that mice bearing tumours treated with photodynamic therapy (PDT) exhibit three hallmarks of acute phase response: neutrophilia, hypothalamic-pituitary-adrenal (HPA) axis activation and the release of acute phase proteins. The latter and a subset of the innate immune system are the subjects of this research for they have been shown to play critical roles in the clearance of dead and dying cells. Among these respective proteins are participants in a rapid and non-inflammatory dead cell removal process, which is believed to be important for the outcome of tumour PDT. Therefore, innate immune proteins including early complement components (C1q, mannose-binding lectin (MBL) and ficolins), acute phase proteins including pentraxins (serum amyloid P component (SAP) & pentraxin-3 (Ptx3)), and heat shock protein-70 (Hsp70) were the focus of our investigation. These proteins are implicated to be soluble sensors of molecular patterns expressed on dying cells. Identification of the critical candidates from this selection, and modulation of their actions could optimize the PDT-induced development of tumour specific immunity. Hypothesis: Early complement components (C1q, MBL and ficolins) and pentraxins (SAP & Ptx3) are known to be involved in a rapid and non-immunogenic dead cell disposal method. Characterization of their activity and of Hsp70 when faced with the burden of clearing PDT killed solid tumour cancer cells would be beneficial in development of new therapeutic approaches. The first objective of this project was to determine the genes most involved in the removal of apoptotic cells. In vivo gene expression studies were performed using Lewis Lung Carcinomas (LLC) growing in C57B1/6J mice. By performing real-time PCR on different tissue samples collected from naïve, untreated and PDT-treated mice, the expression of seven genes were evaluated: early complement components C1q, MBL-A, ficolins A & B; pentraxins SAP and Ptx3; and Hsp70. Among these candidates, Hsp70, SAP and ficolin B showed the most pronounced gene up-regulation in vivo in response to PDT at both the local treated site (tumour) and at a systemic site (liver). These three proteins were therefore further investigated in this project. In order to pinpoint the sources responsible for the elevated expressions of Hsp70, SAP and ficolin B, in vitro gene expression studies were performed using mouse peritoneal macrophages (IC-21), mouse hepatomas (Hepa 1-6) and LLC tumour cell line. The investigated genes were found to become highly up-regulated in PDT-treated LLC cells. Moreover, untreated macrophages and hepatoma cells up-regulated their SAP and Hsp70 genes respectively, when co-incubated with PDT-treated LLC cells. Our second objective was to examine the mechanisms responsible for systemic up-regulation of Hsp70, SAP and ficolin B. Since tumour PDT activates the hypothalamic-pituitary-adrenal (HPA) axis in the host, we attempted to discover any links between the HPA activation and up-regulation of these genes in the liver. Experiments were done to test the effects of the glucocorticoid (dexamethasone), its receptor antagonist (mifepristone) and its synthesis inhibitor (metyrapone) on naïve mice and mice with PDT-treated tumours, demonstrated that the up-regulation of the investigated three genes is at least partially mediated by the activation of the HPA axis and the release of glucocorticoids. The final objective of this study was to determine whether Hsp70 would act as an acute phase protein, produced and released systemically by the liver in response to PDT induced trauma. Lower levels of Hsp70 were found in livers of mice with PDT treated tumours compared to mice with untreated tumours. This demonstrates that Hsp70 is being released from the liver in response to tumour-localized PDT-induced trauma. In addition, LLC cells treated in vitro with PDT, but not untreated LLC cells, were found to bind extracellulary added Hsp70 protein. These findings suggest that released Hsp70 is capable of binding to PDT-damaged and dying tumour cells, and indicate that this protein can have a critical role in the removal of dying cells. The results of the experiments have demonstrated that among the investigated proteins Hsp70, SAP and ficolin B are highly transcribed at the local (tumour) and at a systemic (liver) site in response to tumour PDT treatment. Based on this evidence and previously published data, the three proteins studied appear to be the main candidates responsible for the effective removal of dead cancer cells. Their activity in clearing large loads of killed tumour cells could influence the development of the adaptive immune response towards cancer cells, destroying the primary tumour as well as any metastases or re-occurrences of the same type of cancer. Further studies aimed at elucidating the activity of these proteins in dead cell removal should identify therapeutic targets with potential for improved curative outcome.

Text

2011-02-22

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http://hdl.handle.net/2429/31604

Pathology

University of British Columbia

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