Micro-Environmental Mechanical Stress Controls Tumor Spheroid Size and Morphology by Suppressing Proliferation and Inducing Apoptosis in Cancer Cells

Compressive mechanical stress produced during growth in a confining matrix limits the size of tumor spheroids, but little is known about the dynamics of stress accumulation, how the stress affects cancer cell phenotype, or the molecular pathways involved.We co-embedded single cancer cells with fluorescent micro-beads in agarose gels and, using confocal microscopy, recorded the 3D distribution of micro-beads surrounding growing spheroids. The change in micro-bead density was then converted to strain in the gel, from which we estimated the spatial distribution of compressive stress around the spheroids. We found a strong correlation between the peri-spheroid solid stress distribution and spheroid shape, a result of the suppression of cell proliferation and induction of apoptotic cell death in regions of high mechanical stress. By compressing spheroids consisting of cancer cells overexpressing anti-apoptotic genes, we demonstrate that mechanical stress-induced apoptosis occurs via the mitochondrial pathway.Our results provide detailed, quantitative insight into the role of micro-environmental mechanical stress in tumor spheroid growth dynamics, and suggest how tumors grow in confined locations where the level of solid stress becomes high. An important implication is that apoptosis via the mitochondrial pathway, induced by compressive stress, may be involved in tumor dormancy, in which tumor growth is held in check by a balance of apoptosis and proliferation

In vitro modulation of inflammatory cytokine and IgG levels by extracts of Perna canaliculus

BACKGROUND: Inflammation is a predominant characteristic of autoimmune diseases which is characterized by the increased expression of pro-inflammatory cytokines. Soon to be published work from our laboratory has shown that ingestion of Perna canaliculus prevents the development of autoimmune diseases such as Systemic Lupus Erythematosus and rheumatoid arthritis in laboratory animals. The current paper attempts to illustrate how Perna can alleviate inflammation by modulating inflammatory cytokines, cyclooxygenase enzymes and Immunoglobulin-G (IgG) levels. METHODS: In the present study, hydrochloric acid [HCl] and Tween-20 were used to develop extracts of Perna. These extracts were assayed for protein content. Increasing concentrations of these extracts were then tested in cell culture for modulation of inflammatory cytokine, cyclooxygenase enzymes and IgG levels. Parallel tests were run using an available glycogen extract of Perna as a comparison to our in-house laboratory preparations. RESULTS: Tween-20 Perna extracts were found to be more stable and less toxic in cell culture than HCl digest of Perna. They also assayed higher in protein content that HCl extracts. Although both extracts inhibited IgG production in V2E9 hybridomas, Tween-20 extracts were more consistent in IgG suppression than HCl extracts. Overall Tween-20 extracts effectively decreased levels of TNF-α, IL-1, IL-2 and IL-6 as observed using cytokine bioassays. Twenty micrograms of Tween-20 Perna extracts induced such significant decreases in inflammatory cytokine production that when tested on sensitive cell lines, they very nearly abolished the decrease in viability induced by these cytokines. Tween-20 extracts effectively inhibited both COX-1 and COX-2 cyclooxygenase activity. As a comparison, the glycogen extract also demonstrated a similar though weaker effect on COX-1 and COX-2 enzymes. The active components of both extracts (Tween-20 and glycogen) were observed to possess molecular weights above 100 kDa. Although the anti-cytokine activity of the Tween-20 extract was destroyed by Proteinase-K treatment, the anti-COX-1 and anti-COX-2 activity of both the extracts were not sensitive to protease treatment. CONCLUSION: We have successfully demonstrated modulation in the levels of inflammatory cytokines, cyclooxygenase enzymes and immunoglobulins by our in-house laboratory preparations of Perna canaliculus, whereby suggesting an immunomodulatory role of Perna canaliculus in regulating inflammation

Advancing our understanding of functional genome organisation through studies in the fission yeast

Significant progress has been made in understanding the functional organisation of the cell nucleus. Still many questions remain to be answered about the relationship between the spatial organisation of the nucleus and the regulation of the genome function. There are many conflicting data in the field making it very difficult to merge published results on mammalian cells into one model on subnuclear chromatin organisation. The fission yeast, Schizosaccharomyces pombe, over the last decades has emerged as a valuable model organism in understanding basic biological mechanisms, especially the cell cycle and chromosome biology. In this review we describe and compare the nuclear organisation in mammalian and fission yeast cells. We believe that fission yeast is a good tool to resolve at least some of the contradictions and unanswered questions concerning functional nuclear architecture, since S. pombe has chromosomes structurally similar to that of human. S. pombe also has the advantage over higher eukaryotes in that the genome can easily be manipulated via homologous recombination making it possible to integrate the tools needed for visualisation of chromosomes using live-cell microscopy. Classical genetic experiments can be used to elucidate what factors are involved in a certain mechanism. The knowledge we have gained during the last few years indicates similarities between the genome organisation in fission yeast and mammalian cells. We therefore propose the use of fission yeast for further advancement of our understanding of functional nuclear organisation

Lysophosphatidic Acid Acyltransferase β (LPAATβ) Promotes the Tumor Growth of Human Osteosarcoma

Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2) in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated.Endogenous expression of LPAATβ in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATβ and silencing LPAATβ expression is employed to determine the effect of LPAATβ on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATβ is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATβ promotes osteosarcoma cell proliferation and migration, while silencing LPAATβ expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATβ effectively promotes tumor growth, while knockdown of LPAATβ expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma.Our results strongly suggest that LPAATβ expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATβ may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This is especially attractive given the availability of selective pharmacological inhibitors