• Identifying the key players in osteopontin/alpha(v)beta(3)-mediated migration and invasion of metastatic prostate cancer cells

      Desai, Bhavik; Chellaiah, Meenakshi A. (2008)
      Advanced stages of prostatic carcinoma have a high incidence of metastases to the bones. Our research examines the mechanisms that can facilitate local invasion of prostate cancer (PC) cells inside the extracellular matrix (ECM) of bone. PC3 is a stable cancer cell line derived from bony metastasis of prostate cancer. Osteopontin (OPN), which is an autocrine motility factor secreted by both osteoclasts and osteoblasts, is an important component of the ECM in bone. PC3 cells degrade ECM by secretion of matrix metalloproteinases (MMP-s), primarily through secretion of active MMP-9. Experimental approaches that employed both an over expression and knockdown of endogenous OPN in PC3 cells demonstrated that OPN regulates migration of PC3 cells by increasing active MMP-9 secretion. OPN-mediated upregulation of MMP-9 expression and secretion occurs in response to activation of the hyaluronan receptor CD44. Bisphosphonate-mediated inhibition of Rho kinase, which is an upstream activator of CD44, attenuated the stimulatory effects of OPN on both migration and secretion of MMP-9. In addition to a standard form, multiple CD44 isoforms exist in several cell systems. We have identified CD44 isoforms, which exist in PC3 cells. At the cellular level, migration is a consequence of increased MMP activity in conjunction with changes in the actin cytoskeleton. PC3 cells exhibited punctate evaginations (similar to invadopodia), enriched in actin, which could degrade the underlying gelatin matrix. MMP-9 activity was essential for the invasiveness of invadopodia, but had no role in their formation. The WAVE protein family member WASP was observed to have a role in actin polymerization in PC3 cells. Active MMP-9 was demonstrated to be associated with WASP, thereby pointing out to a mechanistic possibility of WASP's association to MMP-9, with or without an intervening adaptor protein. OPN increased the incidence of invadopodia formation as well as WASP co-localization with invadopodia. The contiguous activation from OPN, CD44, and MMP-9 to WASP and dynamic actin changes provides deeper insight into OPN-mediated PC3 cell invasion and paves the way for therapeutically targeting this pathway in future.
    • Methylsulfonylmethane: Possible Role in Bone Remodeling

      Aljohani, Hanan; Chellaiah, Meenakshi A. (2020)
      Methylsulfonylmethane (MSM) is a popular dietary supplement to assist with various conditions. The anti-inflammatory and osteogenic capabilities of MSM makes it an excellent material for inducing bone formation and promoting osteointegration. MSM is a non-toxic, naturally occurring sulfur-containing compound. This thesis investigated the effect of MSM on osteogenesis in vitro and in vivo. We first used oral stem cells derived from the human exfoliated deciduous teeth (SHED) to elucidate the effect of MSM on osteogenic differentiation using MC3T3-E1 and UMR-106 cells as positive controls. MSM reproduced the results of the osteogenic medium in the osteogenic differentiation of SHED cells. Osteogenic differentiation of SHED cells was determined by an increase in the expression of differentiation markers such as osterix, RUNX2, osteopontin (OPN), and collagen type 1 (Col 1), at both mRNA and protein levels. Moreover, MSM increased the activity of the alkaline phosphatase enzyme, which is vital in the maturation of the extracellular matrix and the formation of mineralized nodules. Very interestingly, the addition of mineralized bone particles enhanced the MSM’s effect on mineralization compared with MSM alone or MSM with demineralized bone particles. Therefore, MSM can act as a cost-effective osteoinductive material for reinforcing bone regeneration. Secondly, we determined the role of transglutaminase-2 (TG2) enzyme in the calcification process via cross-linking of matrix proteins. TG2 is a multifunctional enzyme implicated in matrix stabilization and maturation. MSM treated SHED cells showed a time-dependent increase in TG2 protein expression from 7 to 21 days. Furthermore, immunoprecipitation and immunostaining analyses showed an increase in TG2 colocalization with two prominent osteogenic markers (OPN, Col 1) in a time- dependent manner. An inhibitor to TG2 reduced not only the differentiation of SHED cells but also the mineralization processes by reducing the interaction of TG2 with OPN and Col 1. Our studies demonstrated the effect of MSM on osteogenesis in vitro in TG2 mediated cross-linking of matrix proteins. Thirdly, we identified the effect of MSM on osteogenesis in vivo using aging mice model. We injected aging C57BL/6 female mice (36 weeks old) subcutaneously with MSM and PBS for 13 weeks. Micro-computed tomography (Micro-CT), histological, and immunohistochemistry analyses were done extensively in the bone sections of mandibles isolated from aging mice injected with PBS and MSM. Comparative studies were also done in the tibial and femoral bones of long bones. An increase in the mandibular bone density at the inter-radicular area was observed in mice injected with MSM. The increase was either little or not seen in the femoral or tibial bones analyzed by Micro-CT or in bone sections stained with H&E and TRAP-stains. Immunohistochemistry analyses demonstrated an increase in osteocalcin (OCN) staining in osteoblast-like cells and a decrease in CD105, which is a marker for stem cells. Additionally, we found that MSM has an osteogenic effect via not only increasing the osteogenesis potential of osteoblast- like cells but also the differentiation potential of stem cells into osteoblast-like cells. More experiments are needed to further confirm whether the increase in bone density is a result of the induction of bone formation by osteoblasts or reduction of bone resorption by osteoclasts. MSM is a sulfur-containing non-toxic natural nutrient found in small quantities in many foods. It is commonly used as a supplement to treat arthritis and other inflammatory conditions. This is the first study to show the in vivo effect of MSM on bone remodeling in an aging mice model. We trust, our results may ultimately impact the treatment of other bone loss-associated diseases, including rheumatoid arthritis and periodontitis, which share several pathologic features with osteoporosis.
    • Role of Lipopolysaccharide and RANKL in Osteoclastogenesis: Potential Inhibitory Effects of C-Phycocyanin on the Respective Molecular Pathways of Osteoclastogenesis

      Al Qranei, Mohammed; Chellaiah, Meenakshi A. (2020)
      Many skeletal diseases are characterized by excessive bone loss. Bone loss is mediated by osteoclasts, which are differentiated from cells of the monocyte/macrophage lineage upon stimulation of two indispensable factors, the RANKL and M-CSF. Lipopolysaccharide (LPS), a bacterial pathogenic factor, has also been shown to engage in osteoclastogenesis during inflammatory events actively. C-phycocyanin (C-PC) is a phycobiliprotein found in the blue-green algae that showed many therapeutic effects, including anti-arthritic and anti-inflammatory properties. However, the exact mechanism by which LPS regulates osteoclastogenesis and also the impact of C-PC on osteoclastogenesis needs further elucidations. We studied the osteoclast differentiation process in vitro using RAW 264.7 macrophage cell line. First, we showed that LPS induced osteoclastogenesis in RANKL-primed cells in vitro. LPS elicited osteoclastogenic mechanism by signaling through the TLR4 receptor, which is expressed in osteoclast precursors. Here we also found that TNF-α secreted by osteoclast precursors in response to TLR4 stimulation regulated the processes of osteoclastogenesis via TNF-R signaling. Second, we tested the inhibitory effect of C-PC on osteoclastogenesis. We showed here that C-PC strongly inhibited the early stage of osteoclast differentiation, thus significantly suppressing RANKL- and LPS- mediated osteoclastogenesis. Nonetheless, osteoblast differentiation and activity were not affected by C-PC. Reactive oxygen species (ROS) are generated during RANKL-mediated osteoclast differentiation. While studying the possible mechanisms of osteoclast differentiation, we found that C-PC a) attenuated RANKL-induced ROS; and b) interfered with RANKL-stimulated NF-κB signaling by preventing the degradation of cytosolic IκB-α; subsequently, these resulted in the loss of sequential activation of the osteoclastogenic downstream markers such as c-Fos and NFATc1. We propose that a unique mechanism of osteoclastogenesis is mediated by bacterial LPS that can be targeted during inflammatory-mediated bone loss. Also, C-PC could potentially be used as a therapeutic compound in osteolytic diseases caused by osteoclast activation without affecting osteoblast function.
    • The Role of Osteopontin in Prostate Cancer Progression

      Robertson, Brian W.; Chellaiah, Meenakshi A. (2009)
      Our research investigates the relationship between the local environment of bone and how it influences the proliferation and metastasis of prostate cancer. Our focus centers on the bone extracellular matrix protein Osteopontin (OPN), which has previously been demonstrated to be an autocrine motility factor secreted by both osteoclasts and osteoblasts. In order to investigate the role of OPN in prostate cancer progression, we primarily employ PC3 prostate cancer cells derived from bony metastasis, but also look at the role of OPN in DU145 prostate cancer cells, and the lowly tumorigenic prostate cancer cell line LNCaP. OPN has been shown to activate Akt, and here we thoroughly define the OPN induced Akt cell survival pathway. Investigating the downstream players of activated Akt, we show that OPN induces an Akt-mediated increase in β-Catenin. Here we reveal a novel role for OPN, in inducing increased transcription of the TCF/LEF family of genes, of which β-catenin enhances as a co-factor after nuclear import. In an effort to more fully understand the role of OPN in prostate cancer progression, we investigated its role in MAPK signaling. Over-expression of OPN in PC3 cells resulted in the induced activation of the Erk1/2 pathway. Recently the field has determined that the metastatic potential of tumors or cancer cell lines can be measured through the scope of an Epithelial to Mesenchymal Transition(EMT). An EMT is typically characterized by a loss of E-Cadherin, an increase in N-Cadherin, and an increase in Vimentin. In order to more fully characterize the role of OPN in prostate cancer cell motility and metastasis, we probed the levels of EMT associated proteins. Surprisingly we found that OPN induces the opposite of an MET, a Mesenchymal to Epithelial Transition (MET), which is characterized by a more epithelial phenotype. Our results demonstrate that OPN can play many roles in prostate cancer progression, OPN increases survival of prostate cancer cells, OPN increases the expression of proteins important for cancer progression, and OPN helps to provide a means in which cancerous cells can aggregate and colonize a site of distant metastasis.