Two areas of particular importance in prostate cancer progression are primary tumour development and metastasis. These processes involve a number of physiological events, the mediators of which are still being discovered and characterised. Serine proteases have been shown to play a major role in cancer invasion and metastasis. The recently discovered phenomenon of their activation of a receptor family known as the protease activated receptors (PARs) has extended their physiological role to that of signaling molecule. Several serine proteases are expressed by malignant prostate cancer cells, including members of the kallikreinrelated peptidase (KLK) serine protease family, and increasingly these are being shown to be associated with prostate cancer progression. KLK4 is highly expressed in the prostate and expression levels increase during prostate cancer progression. Critically, recent studies have implicated KLK4 in processes associated with cancer. For example, the ectopic over-expression of KLK4 in prostate cancer cell lines results in an increased ability of these cells to form colonies, proliferate and migrate. In addition, it has been demonstrated that KLK4 is a potential mediator of cellular interactions between prostate cancer cells and osteoblasts (bone forming cells). The ability of KLK4 to influence cellular behaviour is believed to be through the selective cleavage of specific substrates. Identification of relevant in vivo substrates of KLK4 is critical to understanding the pathophysiological roles of this enzyme. Significantly, recent reports have demonstrated that several members of the KLK family are able to activate PARs. The PARs are relatively new members of the seven transmembrane domain containing G protein coupled receptor (GPCR) family. PARs are activated through proteolytic cleavage of their N-terminus by serine proteases, the resulting nascent N-terminal binds intramolecularly to initiate receptor activation. PARs are involved in a number of patho-physiological processes, including vascular repair and inflammation, and a growing body of evidence suggests roles in cancer. While expression of PAR family members has been documented in several types of cancers, including prostate, the role of these GPCRs in prostate cancer development and progression is yet to be examined. Interestingly, several studies have suggested potential roles in cellular invasion through the induction of cytoskeletal reorganisation and expression of basement membrane-degrading enzymes. Accordingly, this program of research focussed on the activation of the PARs by the prostate cancer associated enzyme KLK4, cellular processing of activated PARs and the expression pattern of receptor and agonist in prostate cancer. For these studies KLK4 was purified from the conditioned media of stably transfected Sf9 insect cells expressing a construct containing the complete human KLK4 coding sequence in frame with a V5 epitope and poly-histidine encoding sequences. The first aspect of this study was the further characterisation of this recombinant zymogen form of KLK4. The recombinant KLK4 zymogen was demonstrated to be activatable by the metalloendopeptidase thermolysin and amino terminal sequencing indicated that thermolysin activated KLK4 had the predicted N-terminus of mature active KLK4 (31IINED). Critically, removal of the pro-region successfully generated a catalytically active enzyme, with comparable activity to a previously published recombinant KLK4 produced from S2 insect cells. The second aspect of this study was the activation of the PARs by KLK4 and the initiation of signal transduction. This study demonstrated that KLK4 can activate PAR-1 and PAR-2 to mobilise intracellular Ca2+, but failed to activate PAR-4. Further, KLK4 activated PAR-1 and PAR-2 over distinct concentration ranges, with KLK4 activation and mobilisation of Ca2+ demonstrating higher efficacy through PAR-2. Thus, the remainder of this study focussed on PAR-2. KLK4 was demonstrated to directly cleave a synthetic peptide that mimicked the PAR-2 Nterminal activation sequence. Further, KLK4 mediated Ca2+ mobilisation through PAR-2 was accompanied by the initiation of the extra-cellular regulated kinase (ERK) cascade. The specificity of intracellular signaling mediated through PAR-2 by KLK4 activation was demonstrated by siRNA mediated protein depletion, with a reduction in PAR-2 protein levels correlating to a reduction in KLK4 mediated Ca2+mobilisation and ERK phosphorylation. The third aspect of this study examined cellular processing of KLK4 activated PAR- 2 in a prostate cancer cell line. PAR-2 was demonstrated to be expressed by five prostate derived cell lines including the prostate cancer cell line PC-3. It was also demonstrated by flow cytometry and confocal microscopy analyses that activation of PC-3 cell surface PAR-2 by KLK4 leads to internalisation of this receptor in a time dependent manner. Critically, in vivo relevance of the interaction between KLK4 and PAR-2 was established by the observation of the co-expression of receptor and agonist in primary prostate cancer and prostate cancer bone lesion samples by immunohistochemical analysis. Based on the results of this study a number of exciting future studies have been proposed, including, delineating differences in KLK4 cellular signaling via PAR-1 and PAR-2 and the role of PAR-1 and PAR-2 activation by KLK4 in prostate cancer cells and bone cells in prostate cancer progression.

Das Mammakarzinom ist weltweit eine der häufigsten Malignomerkrankung der Frau. Im fortgeschrittenen Stadium der Erkrankung entwickeln bis zu 75 % der Patientinnen ossäre Metastasen. Basierend auf der Erkenntnis, dass GnRH-Analoga in vitro die Migration und Invasion von Mammakarzinomzellen während der Kokultur mit humanen Osteoblasten bzw. der Osteoblasten-ähnlichen Zellreihe MG-63 hemmen, sollten in dieser Arbeit die dem zugrundeliegenden molekularen Mechanismen näher betrachtet werden. Es ist bekannt, dass GnRH-Analoga in vielen verschiedenen GnRH-Rezeptor exprimierenden Tumorzelllinen die Wachstumsfaktor- und Wachstumsfaktorrezeptor-Expression beeinflussen und somit z.B. antiproliferative oder Apoptose-induzierende Effekte vermitteln. In dieser Arbeit wurde die humane Mammakarzinomzelllinie MCF-7 mit der osteoblasten-ähnlichen Zellreihe MG-63 kokultiviert. Zu bestimmten Zeitpunkten erfolgte die Behandlung der Mammakarzinomzellen mit drei verschiedenen GnRH-Analoga. Dem GnRH-I-Agonisten Triptorelin, dem GnRH-I-Antagonisten Cetrorelix und dem GnRH-II-Agonisten [D-Lysin]-GnRH-II. Neben einer nicht-kokultivierten Kontrolle jeder Zelllinie wurde eine nicht behandelte Kokulturkontrolle mitgeführt. Nach 48 h (bzw. nach 72 h od. 96 h) wurde der Versuch beendet und die mRNA-Expression von EGF, EGFR, TGF-beta und TGFBRI und -II in MCF-7 und MG-63 mittels PCR bestimmt. Zudem wurde die mRNA-Expression von EGF, EGFR und TGF-beta auch nach längerer Kokultur- und Behandlungszeit sowie unter Stressbedingungen beobachtet. Es hat sich gezeigt, dass es weder in MG-63 noch in MCF-7 signifikante Expressionsunterschiede von EGF, EGFR und TGF-beta im Vergleich zur Kokulturkontrolle gab. Dies hat sich auch weder im Zeitverlauf noch unter Stressbedingungen geändert. In MG-63 konnte man ebenfalls keine Expressionsunterschiede der TGFB-Rezeptoren beobachten. Dagegen konnte erstmals gezeigt werden, dass alle drei GnRH-Analoga die mRNA-Expression des TGFBR-I und -II während der Kokultur mit der osteoblasten-ähnlichen Zellreihe MG-63 signifikant hemmen. Während man in anderen Gewebetypen bereits eine verminderte Expression von TGFBR-I und -II nach Behandlung mit GnRH-Analoga nachweisen konnte, ist dies erstmals in einer Mammakarzinomzelllinie gelungen. Desweiteren kann man aus den vorliegenden Ergebnissen den Schluss ziehen, dass die verminderte Expression der TGFB-Rezeptoren eine Rolle in der bereits nachgewiesene GnRH-Analoga-vermittelten Migrations- und Invasionshemmung spielt.

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<p>Prostate cancer is the second leading cause of cancer-related death among American men. Prostate tumor cells exhibit significant tropism for the bone and once metastasis occurs, survival rates fall significantly. Current treatment options are not curative and focus on symptom management. Immunotherapies are rapidly emerging as a possible therapeutic option for a variety of cancers including prostate cancer, however, variable patient response remains a concern. Chemotherapies, like cabozantinib, can have immune-priming effects which sensitize tumors to immunotherapies. Additionally, lower doses of chemotherapy can be used in this context which can reduce patient side effects. It was hypothesized that a combination of chemotherapy (cabozantinib) and immunotherapy (Interleukin-27 (IL-27)) could treat bone-metastatic prostate cancer and also exert pro-osteogenic effects. IL-27 is a multi-functional cytokine, which promotes immune cell recruitment to tumors, while also promoting bone repair. To test this hypothesis, <i>in vivo</i> experiments were performed where syngeneic C57BL/6J mice were implanted intratibially with TRAMP-C2ras-Luc cells able to form tumors in bone. Immunotherapy was administered in the form of intramuscular gene therapy, delivering plasmid DNA encoding a reporter gene (Lucia), or a therapeutic gene (IL-27). Ultrasound was used to aid gene delivery. Various gene delivery methods were tested and optimized through <i>in vivo</i> studies, with microbubbles in combination with ultrasound (sonoporation) emerging as the best method. Following immunotherapy, the animals received either cabozantinib or a vehicle control by oral gavage. Bioluminescence imaging was used to monitor tumor size over time. Combinatorial therapy inhibited tumor growth and improved survival. Further, RNA sequencing and cytokine arrays were used to investigate the mechanisms involved. Microcomputed tomography and differentiation assays indicated that the combination therapy improved bone health by improving osteoblast differentiation and inhibiting osteoclast differentiation. Our conclusion is that a chemo-immunotherapy approach such as the one examined in this work has potential to emerge as a novel therapeutic strategy for treating bone-metastatic prostate cancer. This approach should enable a significant reduction in chemotherapy-associated toxicity, improving sensitivity to immunotherapy, and simultaneously improving bone quality.</p>

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At present, there is no cure for bone metastasis. The current goals in patient care are to palliate pain, prevent pathological bone fracture and increase the strength and function of bone, so as to extend the life expectancy and maintain a good quality of life. Bisphosphonate treatment is the currently standard therapy of bone metastasis and is commonly used by physicians; it alleviates the tumour-induced hypercalcemia in 90% of patients and reduces the metastatic bone pain in 50% of patients. Moreover, it also prevents the pathological fracture of the affected bones. However, while effective, bisphosphonate injections are very costly, though its oral formulation is less expensive it is also less efficacious, and causes gastrointestinal discomfort. Furthermore, prolonged use of bisphosphonate treatment may lead to certain adverse effects, including hypocalcemia. These factors will prohibit the longterm use of such medication as it can negatively affect the treatment outcome. / Based on enormous medical potentials illustrated by the aforementioned findings, BBYNG deserves wider clinical application, large-scale clinical study on its preventive effect against bone metastasis and detailed investigation of its mode(s) of action in the body. / Based on the above-described understanding of Chinese medicine and bone metastasis, supplementing the kidney and strengthening bone could be the basic principle for the treatment of bone metastasis using Chinese medicine. In view of this theory, and in addition to the clinical observation and a thorough search of the available literature, we selected relevant kidney-tonifying Chinese herbs, namely (Fructus Ligustri Lucidi), (Rhizoma Drynariae), (Herba Epimedii), (Psoralea Corylifolia) and wide-spectrum anticancer herbs (Herba Hedyotidis Diffusae) for the preparation of a combined formula--BBYNG. / Chinese medicine has long been used to treat cancers. Its advantages reside in its holistic properties, which bring palliative, corrective and convalescing functions against damage caused by radiotherapy, chemotherapy and surgery. These features position Chinese medicines as the adjuvant to orthodox cancer treatment. During the late stage of tumour development, when standard therapy is no longer effective, Chinese medicine plays a critical role as an integrated therapy. Searching for a safe, inexpensive and effective Chinese medicine preparation suitable for prolonged use as adjunct therapy in late cancer cases is of paramount importance. / Clinical results. Both Chinese medicine and Western medicine treated patients showed no significant change in their blood parameters or liver and kidney examinations before and after drug administration; Male subjects on BBYNG, their bone mass density remained stable after 6 months treatment and the subjects on OSTAC showed slightly decreased In females, subjects on BBYNG remained stable, but subjects on OSTAC slightly increased. / Clinical study. The study was designed as a randomized, parallel-group comparison between BBYNG formula and Bisphosphonate. The patients who meet the inclusion/exclusion criteria were randomly assigned to receive either BBYNG granules, which was prepared by a GMP manufacturer, or Clodronic acid. The treatment period was 6 months (24 weeks). For both groups, various clinical parameters such as body functions, blood examinations, bone density (BMD) assessment, X-ray examinations, pain intensity and quality of life were evaluated and compared. / Conclusions. (1) As an adjuvant to patients with bone metastases, BBYNG is effective in relieving the metastatic bone pain, improving the quality of life. (2) In the animal model, BBYNG reduced the metastatic bone damage, prolonged the survival and enhanced the T lymphocyte immunity in the tumour-bearing mice. (3) In vitro study on the breast and lung cancer cell lines showed that BYYNG could induce apoptosis and prevent tumour cell invasion. It suggests that BYYNG may restrict tumour growth and development, thus reducing the occurrence of bone metastasis. / In accordance with Chinese medicine, bone metastasis can be categorized into "bone tumour" "bone erosion" "bone wilting" "bone necrosis" and "bone impediment". The main cause of bone metastasis is twofold: cancer toxicity, and in Chinese medicine theory, the kidney governs the bone marrow, if the kidney is not functioning in balance, then the bone will become weak. Cancer toxicity is the "pathogenic cause" to skeletal metastases, while kidney weakness decreases the body defence against the cancer. A vicious cycle ensues when cancer and kidney deficiency and bone weakness occurs simultaneously coincidently and worsens the conditions. / In vitro study on tumour cell lines. The anticancer effects of different concentrations of BBYNG formula and various single components against human breast cancer and lung cancer cell lines were evaluated by cell viability test (MTT assay), cell apoptosis test and invasion suppression test. / In vitro study results. BBYNG and the aqueous extracts of its component herbs at very low drug concentrations stimulated the growth of three tumour cell lines tested. When the concentrations were slightly increased, they showed an inhibitory effect on cancer cell proliferation. As the drug concentrations further increased, the extracts showed cytotoxic effects on these tumor cells. At the noncytotoxic dose, the extracts could trigger apoptosis and enhance the caspase-3 activity in all three tumour cell lines. In addition, at this "non toxic" concentration, the extracts markedly inhibited the in vitro invasive property of the 4T1 breast cancer cell lines in our Matrigel invasion model. Thus these in vitro results suggested that BBYNG possess anticancer, invasion-inhibitory and anti-metastatic activities. / In vivo animal study results. (Tumour growth was slower in the BBYNG treatment group when compared to the OSTAC and control groups, but this was not significantly difference) BBYNG significantly delayed tumour growth in tumour bearing mice, but it did not minimize the tumour size markedly. Moreover, BBNYG did minimize the mobility restriction caused by tumours, reduce the damage to bones, prolong the survival time and enhanced the T lymphocyte immunity. / In vivo animal study. A well-established animal model for breast cancer was used to evaluate and compare the pharmacological effects of BBYNG formula and Clodronic acid, as shown by different indicators such as tumour progression, animal's mobility, survival time, bone metastasis-induced fracture intensity and the immunological status of the tumour-bearing mice. / Malignant tumour is characterized by early metastasis. Among them 37 to 80 (depending on which type of cancer) patients show tendency of bone metastasis. Bone metastasis is usually accompanied by various complications, such as severe pain, pathological bone fracture, hypercalcemia, and bone marrow suppression, which can substantially affect the quality of life of the patients. Thus, the prevention and treatment of bone metastasis in cancer is an issue worth pursuing. / Malignant tumours leading to high mortality and morbidity are a serious threat to human health. It is the leading cause of death in China. In Hong Kong, there are over 20 thousand new cancer cases and more than 1100 people die due to cancers every year. / Study objectives. To elucidate the efficacy and some pharmacological aspects of BBYNG in regard to the treatment of bone metastasis through clinical observation and different laboratory experiments. This study would be of significant reference value to the disease-oriented drug formulation and application, mechanistic study and research methodology of the treatment of bone metastasis using Chinese medicines. / The clinical and laboratory experimental results are summarized as below: / The research study is composed of three parts, the clinical study, in vivo animal study and in vitro study on tumour cell lines. The research methods used are as follows: / Those on BBYNG treatment showed more a stable and satisfactory quality of life than those in the Western medicine-treated group. For the Clodronic acid treatment group, patients generally showed worsened symptoms and quality of life deteriorated. The ECOG index of the BBYNG group was statistically better than that of the Clodronic acid group. Within the 72-week clinical observational period, the mortality of Clodronic acid group is significantly higher that of the BBYNG group. The effects of BBYNG group as presented in relieving the pain-induced influence on patients' emotion, interpersonal relationship and entertainment was more pronounced than that in the Clodronic acid group. / Wu Ka. / 論文(哲學博士)--香港中文大學, 2006. / 參考文獻(p. 299-324). / Adviser: Leung Ping Chung. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1570. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in Chinese and English. / School code: 1307. / Lun wen (zhe xue bo shi)--Xianggang Zhong wen da xue, 2006. / Can kao wen xian (p. 299-324). / Wu Ka.