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Triptolide (TPL), a diterpenoid triepoxide that is extracted from a traditional Chinese herb called Tripterygium Wilfordii (also known as ‘Thunder God Vine’) has recently drawn increasing interests from pharmaceutical and biomedical researchers, especially in the aspect of its potential efficacy on multiple cancer treatment. TPL has shown significant growth and proliferation inhibition activities in a broad range of cancer cell types. Moreover, it has shown the inhibition of osteoclastogenesis by breast cancer bone metastasis. However, due to its limitation in toxicity, solubility and non-specific biodistribution, it is challenging for the application of TPL in clinical study. Besides, TPL can rapidly distribute in most vital organs and no evidences shown tissue accumulation of drug. It is indispensable to overcome those barriers and optimize the properties and performance of the promising drug molecule. Lipid-based nanocarriers such as nanostructured lipid carriers (NLC) have been extensively studied for delivery of poorly-water soluble drug compounds. They also have the potential to optimize the physicochemical properties of the drug and may enhance a targeted delivery of the drug to specific therapeutic site. Alendronate (Fosamax®), an FDA approved bisphosphonate drug for osteoporosis, osteogenesis imperfecta and several other bone diseases, has been used as a bone targeting decoration agent. Breast cancer cell line MDA-MB-231 and other type of cancer cell lines have been used to study the in vitro cytotoxicity of TPL and the carriers while MC3T3-E1 cell line was used for toxicity assessment. Rats have also been used to study the in vivo performance of the drug. After modifying and optimizing the formulation of the particle, the formulation had the ability to remain structurally and functionally stable when being in the bio-simulated media at 37 °C and in water at room temperature with high encapsulation efficiency. In vitro study illustrated that both TPL free drug (stock solution 10mg/mL dissolved in DMSO) and TPL nanoparticle without alendronate (TPL-NP) had similar cytotoxicity on MDA-MB-231 and some other type of cancer cell lines. The ALE decoration on the particle (ALE-NP-TPL) has enhanced the anti-cancer effect especially with breast cancer cell line. The in vivo study shows that after 24 hours of the dose injection at local bone site, the formulation and TPL can remained at the location without random distribution to other organs. TPL-NP has not only successfully optimized the physicochemical properties of the drug, but also shows great enhancement of therapeutic effect both in vitro and in vivo study. / Pharmaceutical Sciences

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24840号 / 医博第5008号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田, 恭之, 教授 松田, 道行, 教授 柳田, 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Planar bone scintigraphy is highly sensitive but it may not be sensitive enough to detect subtle lesions in complex bony structures such as the spine. The accurate anatomic localisation of lesions in regions such as this is also limited using planar images. Single Photon Emission Computed Tomography (SPECT) results in a higher lesion contrast resulting in an improved sensitivity for the detection of subtle lesions. SPECT also enables improved lesion localisation, often valuable in distinguishing benign from malignant disease in the spine. A number of previous studies have demonstrated that the addition of SPECT of the spine significantly enhances the value of bone scintigraphy for the detection of bone metastases compared to planar imaging alone. These studies were however not done in the African context where patients typically present with more advanced disease. In a retrospective study of 576 patients with known primary tumors sent to our institution for bone scintigraphy for the diagnosis of bone metastases, we evaluated 119 patients in whom both planar imaging and SPECT were obtained. The studies were graded for the probability of metastatic disease, and the number of spinal lesions was determined with and without SPECT. The influence of adding SPECT on the interpretation of the study was determined in terms of the reported probability of metastatic disease, the exclusion and confirmation of metastatic disease, the decisiveness of interpretation, and the number of spinal lesions. The addition of SPEeT resulted in a statistically significant change in the interpretation of studies, although the actual numbers of patients affected were relatively small. SPEeT resulted in a more decisive interpretation of bone scintigraphy. There was a significant increase in the number of spinal lesions detected after the addition of SPEeT. It was concluded that although the use of SPEeT is ideal, acceptable results could be achieved using planar imaging alone in this patient population. This is particularly relevant in the African context, where SPEeT is often unavailable or scarce and in great demand. / AFRIKAANSE OPSOMMING: Planare beenflikkergrafie is hoogs sensitief, maar moontlik nie sensitief genoeg om subtiele letsels in ingewikkelde beenstrukture soos die werwelkolom aan te toon nie. Akkurate anatomiese lokalisasie van letsels in die genoemde strukture is beperk wanneer slegs planare beelde gebruik word. Enkelfoton-uitstraling Rekenaartomografie (EFERT) lewer 'n hoër letsel kontras, wat 'n verbeterde sensitiwiteit vir die opsporing van subtiele letsels tot gevolg het. EFERT lei ook tot verbeterde letsel lokalisasie, wat dikwels van waarde is om onderskeid tussen benigne en maligne siekte in die werwelkolom te tref. Reeds met 'n aantal vorige studies is aangetoon dat die toevoeging van EFERT van die werwelkolom die waarde van beenflikkergrafie in die opsporing van beenmetastases beduidend verhoog bo dié van planare beelding alleenlik. Hierdie studies is egter nie in omstandighede eie aan Afrika gedoen nie, waar pasiënte kenmerkend met gevorderde siekte voordoen. In In terugskouende studie van 576 pasiënte met bekende primêre tumore, wat na ons instelling verwys is vir beenflikkergrafie om beenmetastases op te spoor, het ons 119 pasiënte, wat beide planare beelding en EFERT ondergaan het, ge-evalueer. Die studies is gegradeer volgens die waarskynlikheid vir metastatiese siekte, en die hoeveelheid werwelkolom letsels, met en sonder EFERT, is bepaal. Die invloed van EFERT op die vertolking van die studie is bepaal in terme van die waarskynlikheid van metastatiese siekte, die bevestiging en uitskakeling daarvan, die beslistheid van vertolking, en die hoeveelheid werwelkolom letsels. Die toevoeging van EFERT het tot 'n statisties beduidende verandering in die vertolking van studies gelei, alhoewel die werklike getal pasiënte wat hierdeur geraak is, relatief min was. EFERT het 'n meer besliste vertolking van beenflikkergrafie tot gevolg gehad. Daar was 'n beduidende toename in die hoeveelheid werwelkolom letsels wat opgespoor is na die toevoeging van EFERT. Daar is tot die slotsom gekom dat, alhoewel die gebruik van EFERT wenslik is, aanvaarbare resultate met slegs die gebruik van planare beelding in hierdie pasiënt bevolkingsgroep verkry kan word. Dit is veral van belang in Afrikaomstandighede, waar EFERT dikwels onbeskikbaar of skaars is, en ook in groot aanvraag is.

Background: Bone metastases occur in most patients with advanced hormone-refractory prostate cancer causing pain, pathologic fractures, and spinal cord compression. Few studies specifically address surgical treatment of metastatic spinal cord compression (MSCC) in prostate cancer. Criteria for identifying patients who may benefit from surgery are poorly defined. Most of the current knowledge regarding tumor biology in prostate cancer is based on studies of primary tumors or soft tissue metastases. The mechanisms regulating growth of bone metastases are not fully established. Aims: a) to evaluate outcome after surgery for MSCC in prostate cancer and to identify prognostic factors for survival and functional recovery; b) to evaluate current practice for referral of prostate cancer patients with MSCC; c) to analyze expression of androgen receptor (AR), cell proliferation, apoptosis, and prostate-specific antigen (PSA) in bone metastases with regard to survival after surgery for complications of bone metastases. Patients and Methods: We retrospectively evaluated the hospital records of 68 consecutive patients operated for metastatic spinal cord compression. Tumor tissue from bone metastases was obtained on spinal surgery (54 patients), fracture surgery (4 patients) and biopsy (2 patients), and analyzed by immunohistochemistry. Results: Study I: Mortality and complication rate after surgery was high. Patients with hormone-naïve disease and those with hormone-refractory disease with good performance status and without visceral metastases had more favorable survival. The ability to walk after surgery was related to better survival. Study II: A new score for prognosis of survival after surgery for spinal cord compression includes: hormone status of prostate cancer, Karnofsky performance status, evidence of visceral metastasis, and preoperative serum PSA. The score is simple, tumor specific, and easy to apply in clinical practice. Study III: Our results suggest that delays in diagnosis and treatment may have negative impact on functional outcome. Pretreatment ability to walk, hormone status of prostate cancer, and time from loss of ambulation influenced neurological recovery after surgery for spinal cord compression. Study IV: High nuclear AR immunostaining in bone metastases and high preoperative serum PSA were associated with a poor outcome after metastasis surgery in patients with hormone-refractory prostate cancer. Short-term effect of castration therapy disclosed that nuclear AR immunostaining was decreased and apoptosis was increased, but cell proliferation remained largely unaffected. Conclusion:  Prostate cancer patients with metastatic spinal cord compression represent a heterogeneous group. We identified prognostic factors for survival and functional outcome, which may help clinicians in making decisions about treatment. Our results also implicate the need for development of local and regional guidelines for treatment of patients with spinal cord compression, as well as the importance of information to patients at risk.

FGFR4 and β-Klotho in Metastatic Prostate Cancer by Derek LaMar Shenefelt Fibroblast growth factors and fibroblast growth factor receptors have been associated with the aggressiveness and progression of Prostate Cancer (PCa). Also, β-Klotho is a known co-receptor with FGFR4 for FGF19 in the liver however, the role of this co-receptor pair remains unclear in the setting of PCa. I demonstrated that FGFR4 and KLB mRNA and protein are highly expressed in PCa cells when compared to bone marrow stromal cells, a common site of metastasis. I also provide support for the association of FGFR4 and KLB in PCa, suggesting a functional co-receptor pair capable of altering cellular signaling. FGFR4-KLb may also provide some level of protection to PCa cells from chemotherapeutics. This analysis of FGFR4 and KLB expression and signaling in PCa has provided novel insights into phenotypic alterations during PCa progression while also providing new avenues of study to further explore the role and importance of this exciting co-receptor complex.

Nous avons montré un rôle d’un canal K+, le canal SK3, dans la migration des cellules cancéreuses de sein MDA-MB-435s et le développement de métastases ostéolytiques du cancer du sein. Lors de l’ostéolyse, la [Ca²+]ext augmente dans le microenvironnement osseux. Nous avons voulu déterminer si cette élévation de [Ca²+]ext, pouvait moduler l’expression et l’activité du canal SK3. Nous avons montré que l’augmentation de la [Ca²+]ext: i) favorise l’expression du canal SK3. Cet effet fait intervenir le récepteur au calcium (CaSR), qui en diminuant la [AMPc]int réduit l’activité de la PKA et lève ainsi son inhibition de la transcription du gène KCNN3 (codant pour SK3) ; ii) favorise la migration cellulaire dépendante du canal SK3, mécanisme impliquant également le CaSR ; iii) active le canal SK3 qui, par ailleurs, voit son activité réduite par l’élévation d’AMPc intracellulaire. De plus, l’augmentation d’AMPc délocalise un canal calcique partenaire de SK3, le canal Orai1, et diminue l’entrée constitutive de Ca²+ et la migration dépendantes du canal SK3. En conclusion, nos résultats montrent que l’expression et l’activité de SK3 sont régulées par l’AMPc et le Ca2+ extracellulaire. Ceci permet d’envisager une nouvelle stratégie thérapeutique ciblant l’AMPc pour le traitement des métastases osseuses du cancer du sein. / We showed that a K+ channel, SK3 channel, is a mediator of MDA-MB-435s breast cancer cells migration and of osteolytic bone metastasis development of breast cancer. Since [Ca²+]out rises during osteolysis, in bone microenvironment, we study if this [Ca²+]out elevation could modulate SK3 expression and activity. We show that [Ca²+]out elevation: i) increases SK3 expression threw CaSR activation which, in turn, decreases [cAMP]int and PKA activation, leading to loss of its inhibitory effect on KCNN3 transcription; ii) increases SK3-dependent migration threw CaSR activation; iii) increases SK3 channel activity that is in addition, decreased by [cAMP]int elevation. Furthermore, cAMP elevation moves the Ca2+ channel Orai1 (SK3 partner) outside of lipid rafts and reduces the SK3 dependent-constitutive Ca²+ entry and cell migration. Our results show that both SK3 expression and activity are regulated by cAMP and extracellular Ca²+. These results underscore an innovative opportunity to use therapeutic approaches targeting cAMP for the treatment of breast cancer bone metastasis.

Les métastases osseuses sont des complications de nombreux cancers tels que le cancer du poumon, du sein et de la prostate. Chez la femme, 70% des patientes présentant un cancer du sein avancé développent des métastases principalement ostéolytiques. Ce type de métastases est associé à une destruction importante de l'os causant chez les patientes des douleurs osseuses et des fractures pathologiques. Les traitements actuellement dispensés tels que les bisphosphonates et le denosumab ne sont pas curatifs mais seulement palliatifs, il est donc indispensable d'identifier de nouveaux facteurs impliqués dans la progression métastatique afin d'améliorer le pronostic et d'imaginer de nouvelles approches thérapeutiques. Dans ce contexte, une analyse transcriptomique comparative a montré que les gènes codant les récepteurs de guidage axonal ROBO1 et ROBO4 étaient surexprimés dans une sous-population de cellules de cancer du sein MDA-MB-231 qui métastasent spécifiquement à l'os (nommée B02). De manière intéressante, une expression élevée de ROBO4 dans les tumeurs primaires de patientes atteintes d'un cancer du sein corrèle avec un mauvais pronostic et un risque accru de rechute à l'os. Chez la souris, une réduction de 50% de la charge tumorale a été observée lorsque les cellules B02 déplétées en ROBO4 ont été inoculées dans la glande mammaire de souris ; et le ciblage des cellules parentales B02 avec un anticorps anti-ROBO4 avant l'injection intra-tibiale diminue l'incidence de la formation de colonies de cellules tumorales. In vitro, la co-culture des cellules B02 avec des cellules ostéoblastiques murines MC3T3-E1 a entraîné une augmentation de la production de SLIT2, le ligand naturel des récepteurs ROBO, par les cellules MC3T3-E1. De plus, le traitement avec un anticorps anti-ROBO4 réduit considérablement l'adhésion des cellules B02 sur les cellules MC3T3-E1. Par ailleurs, la surexpression de ROBO4 dans d'autres modèles cellulaires de cancer du sein a conduit à mettre en évidence la présence d'une glycoforme de ROBO4. L'étude des deux isoformes de ROBO4 (glycosylée et non glycosylée) a révélé le caractère agressif de la forme non glycosylée comparée à la forme glycosylée / Bone metastasis is a complication of many cancers such as lung, breast and prostate. In women, 70% of patients with advanced breast cancer predominantly develop osteolytic metastases. This type of metastasis is associated with significant bone destruction in patients causing bone pain and pathological fractures. The current treatments, such as bisphosphonates and denosumab, are not curative but only palliative, so it is essential to identify new factors involved in the metastatic progression to improve prognosis and to devise new therapeutic approaches. In this context, a comparative transcriptomic analysis demonstrated that axon guidance ROBO1 and ROBO4 receptors were overexpressed in a subpopulation of MDA-MB-231 breast cancer cells that only metastasize to bone (referred to as B02). Interestingly, high ROBO4 expression in primary tumors from patients with breast cancer correlated with poor prognosis and increased risk of relapse to bone. In mice, a 50% reduction in tumor burden was observed when ROBO4-depleted B02 cells were inoculated in the mammary fat pad of mice; and targeting of parental B02 cells with an anti-ROBO4 antibody before intra-tibia inoculation decreased the incidence of tumor cell colony formation. In vitro, the co-culture of B02 cells with murine MC3T3-E1 osteoblastic cells increased SLIT2 production, the primary ligand of ROBO receptors, by MC3T3 cells. Further, the treatment with an anti-ROBO4 antibody dramatically reduced B02 cell adhesion to MC3T3-E1 cells. Moreover, over-expression of ROBO4 in other models of breast cancer led to the discovery of a glycoform of ROBO4. The study of the two ROBO4 isoforms (glycosylated and non-glycosylated) revealed the aggressive nature of the non-glycosylated form compared to the glycosylated form. These results provide strong evidence that the axon guidance receptor ROBO4 is involved in bone metastasis formation and that the use of an antibody directed against the non-glycosylated form of this receptor could lead to the development of innovative therapies to prevent metastasis formation in the bone marrow

<p>In this study, we first examined the roles of metabolic signaling, specifically global AMPK modulators and mitochondria-specific AMPK inhibitor (Mito-AIP), as well as mechanical force in beta catenin signaling through interaction between osteocytes and precursor osteoblasts as well as osteocytes and breast cancer cells. We also evaluated the role of metabolic signaling in Rho GTPases including RhoA, Rac1 and Cdc42. We observed that AMPK activator (A769662) and Mito-AMPK stimulated beta catenin translocation to the nucleus, indicating the activation of Wnt signaling, while Mito-AIP did not significantly affect beta catenin activation in osteoblasts. We also observed that osteocyte conditioned medium (CM) treated with Mito-AIP substantially increased beta catenin signaling in osteoblasts, while decreasing beta catenin signaling in breast cancer cells. CM of osteocytes treated with fluid flow increased beta catenin signaling in breast cancer cells. A769662 and Mito-AIP also decreased the activities of RhoA, Rac1, and Cdc42 in cancer cells which are known to regulate cancer cell migration.</p><p>Additionally, we evaluated the roles of intracellular and extracellular moesin (MSN) protein in well-established oncogenic signaling proteins, such as FAK, Src, and RhoA as well beta catenin signaling. Constitutively active MSN (MSN+) significantly increased FAK and Src activities in cancer cells, but decreased the activity of RhoA. Surprisingly, CM of mesenchymal stem cells treated with MSN+ decreased the activities of FAK, Src, and RhoA, suggesting the inhibitory role of extracellular MSN in tumor-promoting signaling. Our results suggest the distinct role of AMPK signaling, specifically at mitochondria of osteocytes, in the activities of beta-catenin signaling in osteoblasts and breast cancer cells and the distinct role of intracellular and extracellular MSN in these two types of cell.</p>

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