Eliciting calcium transients with UV nanosecond laser stimulation in adult patient-derived glioblastoma brain cancer cellsin vitro.

Objective.Glioblastoma (GBM) is the most common and lethal type of high-grade adult brain cancer. The World Health Organization have classed GBM as an incurable disease because standard treatments have yielded little improvement with life-expectancy being 6-15 months after diagnosis. Different approaches are now crucial to discover new knowledge about GBM communication/function in order to establish alternative therapies for such an aggressive adult brain cancer. Calcium (Ca2+) is a fundamental cell molecular messenger employed in GBM being involved in a wide dynamic range of cellular processes. Understanding how the movement of Ca2+behaves and modulates activity in GBM at the single-cell level is relatively unexplored but holds the potential to yield opportunities for new therapeutic strategies and approaches for cancer treatment.Approach.In this article we establish a spatially and temporally precise method for stimulating Ca2+transients in three patient-derived GBM cell-lines (FPW1, RN1, and RKI1) such that Ca2+communication can be studied from single-cell to larger network scales. We demonstrate that this is possible by administering a single optimized ultra-violet (UV) nanosecond laser pulse to trigger GBM Ca2+transients.Main results.We determine that 1.58µJµm-2is the optimal UV nanosecond laser pulse energy density necessary to elicit a single Ca2+transient in the GBM cell-lines whilst maintaining viability, functionality, the ability to be stimulated many times in an experiment, and to trigger further Ca2+communication in a larger network of GBM cells.Significance.Using adult patient-derived mesenchymal GBM brain cancer cell-lines, the most aggressive form of GBM cancer, this work is the first of its kind as it provides a new effective modality of which to stimulate GBM cells at the single-cell level in an accurate, repeatable, and reliable manner; and is a first step toward Ca2+communication in GBM brain cancer cells and their networks being more effectively studied.

Mitotic Dysregulation at Tumor Initiation Creates a Therapeutic Vulnerability to Combination Anti-Mitotic and Pro-Apoptotic Agents for MYCN-Driven Neuroblastoma.

MYCN amplification occurs in approximately 20-30% of neuroblastoma patients and correlates with poor prognosis. The TH-MYCN transgenic mouse model mimics the development of human high-risk neuroblastoma and provides strong evidence for the oncogenic function of MYCN. In this study, we identified mitotic dysregulation as a hallmark of tumor initiation in the pre-cancerous ganglia from TH-MYCN mice that persists through tumor progression. Single-cell quantitative-PCR of coeliac ganglia from 10-day-old TH-MYCN mice revealed overexpression of mitotic genes in a subpopulation of premalignant neuroblasts at a level similar to single cells derived from established tumors. Prophylactic treatment using antimitotic agents barasertib and vincristine significantly delayed the onset of tumor formation, reduced pre-malignant neuroblast hyperplasia, and prolonged survival in TH-MYCN mice. Analysis of human neuroblastoma tumor cohorts showed a strong correlation between dysregulated mitosis and features of MYCN amplification, such as MYC(N) transcriptional activity, poor overall survival, and other clinical predictors of aggressive disease. To explore the therapeutic potential of targeting mitotic dysregulation, we showed that genetic and chemical inhibition of mitosis led to selective cell death in neuroblastoma cell lines with MYCN over-expression. Moreover, combination therapy with antimitotic compounds and BCL2 inhibitors exploited mitotic stress induced by antimitotics and was synergistically toxic to neuroblastoma cell lines. These results collectively suggest that mitotic dysregulation is a key component of tumorigenesis in early neuroblasts, which can be inhibited by the combination of antimitotic compounds and pro-apoptotic compounds in MYCN-driven neuroblastoma.

Inhibition of mitochondrial translocase SLC25A5 and histone deacetylation is an effective combination therapy in neuroblastoma.

The mitochondrion is a gatekeeper of apoptotic processes, and mediates drug resistance to several chemotherapy agents used to treat cancer. Neuroblastoma is a common solid cancer in young children with poor clinical outcomes following conventional chemotherapy. We sought druggable mitochondrial protein targets in neuroblastoma cells. Among mitochondria-associated gene targets, we found that high expression of the mitochondrial adenine nucleotide translocase 2 (SLC25A5/ANT2), was a strong predictor of poor neuroblastoma patient prognosis and contributed to a more malignant phenotype in pre-clinical models. Inhibiting this transporter with PENAO reduced cell viability in a panel of neuroblastoma cell lines in a TP53-status-dependant manner. We identified the histone deacetylase inhibitor, suberanilohydroxamic acid (SAHA), as the most effective drug in clinical use against mutant TP53 neuroblastoma cells. SAHA and PENAO synergistically reduced cell viability, and induced apoptosis, in neuroblastoma cells independent of TP53-status. The SAHA and PENAO drug combination significantly delayed tumour progression in pre-clinical neuroblastoma mouse models, suggesting that these clinically advanced inhibitors may be effective in treating the disease.

Constitutive CHK1 Expression Drives a pSTAT3-CIP2A Circuit that Promotes Glioblastoma Cell Survival and Growth.

High-constitutive activity of the DNA damage response protein checkpoint kinase 1 (CHK1) has been shown in glioblastoma (GBM) cell lines and in tissue sections. However, whether constitutive activation and overexpression of CHK1 in GBM plays a functional role in tumorigenesis or has prognostic significance is not known. We interrogated multiple glioma patient cohorts for expression levels of CHK1 and the oncogene cancerous inhibitor of protein phosphatase 2A (CIP2A), a known target of high-CHK1 activity, and examined the relationship between these two proteins in GBM. Expression levels of CHK1 and CIP2A were independent predictors for reduced overall survival across multiple glioma patient cohorts. Using siRNA and pharmacologic inhibitors we evaluated the impact of their depletion using both in vitro and in vivo models and sought a mechanistic explanation for high CIP2A in the presence of high-CHK1 levels in GBM and show that; (i) CHK1 and pSTAT3 positively regulate CIP2A gene expression; (ii) pSTAT3 and CIP2A form a recursively wired transcriptional circuit; and (iii) perturbing CIP2A expression induces GBM cell senescence and retards tumor growth in vitro and in vivo. Taken together, we have identified an oncogenic transcriptional circuit in GBM that can be destabilized by targeting CIP2A. IMPLICATIONS: High expression of CIP2A in gliomas is maintained by a CHK1-dependent pSTAT3-CIP2A recursive loop; interrupting CIP2A induces cell senescence and slows GBM growth adding impetus to the development of CIP2A as an anticancer drug target.

A case study of a long-term glioblastoma survivor with unmethylated MGMT and hypermutated genotype.

Effective treatments that extend survival of malignant brain tumor glioblastoma (GBM) have not changed in more than a decade; however, there exists a minority patient group (<5%) whose survival is longer than 3 yr. We herein present a case report of a long-term surviving 51-yr-old female diagnosed with a MGMT unmethylated GBM. The patient was progression-free for 23 mo. Fresh primary and recurrent tumor samples were collected and processed for patient-derived model development. Whole-genome sequencing (WGS) was performed concurrently with additional standard of care diagnostics. WGS revealed a hypermutated genotype in the germline tissue and in both the primary and recurrent tumor samples. Specific to the matched tumors, an average of 30 cancer driver genes were mutated. Noteworthy was the identification of a nonsynonymous mutation in the POLE gene. As a possible instigator of the hypermutational genotype observed in the tumors, we identified nonsynonymous germline mutations within the mismatch repair genes, MLH1 and PMS2 Mutations within these genes are often indicative of the pan-cancer phenotype known as Lynch syndrome; however, their pathogenicity remains unreported. We performed a drug screen of 165 compounds, which identified one compound, YM155, an experimental survivin inhibitor, that showed effectivity to the patient-derived cell lines of both tumors. Treatment selection based on a patient's genome to individualize treatment for GBM patients could potentially be useful in the clinic. This is a promising avenue for further translational research, with larger databases and integrated platforms to increase the efficiency of analyzing and interpreting the individual genomic data of GBM.

Combination of palbociclib and radiotherapy for glioblastoma.

The cyclin-dependent kinase inhibitor, palbociclib has shown compelling efficacy in breast cancer patients. Several pre-clinical studies of glioblastoma (GBM) have also shown palbociclib to be efficacious. In this study, we investigated palbociclib in combination with radiation therapy (RT) for treating GBM. We tested palbociclib (with and without RT) on four patient-derived cell lines (PDCLs; RB1 retained; CDKN2A loss). We investigated the impact of therapy on the cell cycle and apoptosis using flow cytometry, in vitro. Balb/c nude mice were intracranially injected with the PDCL, GBM-L1 and treated orally with palbociclib (with and without RT). Overall survival was measured. Palbociclib treatment resulted in a significant increase in the percentage of cells in the G1 cell cycle phase. Apoptotic cell death, measured by Annexin V was induced. Palbociclib combined with RT acted synergistically with the significant impediment of colony formation. The oral treatment of mice with palbociclib did not show any significant survival advantage when compared to control mice, however when combined with RT, a survival advantage of 8 days was observed. Our results support the use of palbociclib as an adjuvant treatment to RT and warrant translation to the clinic.

Localization of bone morphogenetic protein 13 in human intervertebral disc and its molecular and functional effects in vitro in 3D culture.

Our laboratory has demonstrated that bone morphogenetic protein 13 prevented the effects of annular injury in an ovine model, maintaining intervertebral disc height, cell numbers and increasing extracellular matrix production compared to degenerated controls. The present study sought to examine the molecular effects of bone morphogenetic protein 13 on human degenerated disc cells and localize its expression in both human degenerate and scoliotic disc tissue. Effect of bone morphogenetic protein 13 on human derived nucleus pulposus, annulus fibrosus and endplate cells cultured in alginate beads was evaluated by changes in proteoglycan and collagen content. Migratory potential of disc cells towards bone morphogenetic protein 13 was also examined. Bone morphogenetic protein 13 induced significant proteoglycan accumulation in nucleus (18%), annulus (21%) and endplate (23%) cells cultured in alginate beads (p<0.05) compared to controls. Further bone morphogenetic protein 13 increased collagen I and II protein expression in nucleus and endplate cells. Nucleus cells displayed a significant chemotactic response towards bone morphogenetic protein 13. The endogenous expression of bone morphogenetic protein 13 in degenerate disc tissue was not different to scoliotic disc. Bone morphogenetic protein 13 has the potential to enhance extracellular matrix accumulation and induce cell migration in certain disc cells.

Alterations in the mitochondrial responses to PENAO as a mechanism of resistance in ovarian cancer cells.

OBJECTIVE: The purpose of this study was to test PENAO, a promising new organoarsenical that is in phase 1 testing in patients with solid tumours, on a range of ovarian cancer cell lines with different histotypes, and to understand the molecular basis of drug resistance exhibited by the endometrioid ovarian cancer cell line, SKOV-3. METHODS: Proliferation arrest and cell death induced by PENAO in serous (OVCAR-3), endometrioid (SKOV-3, TOV112D), clear cell (TOV21G) and mucinous (EFO27) ovarian cancer cells in culture, and anti-tumour efficacy in a murine model of SKOV-3 and OVCAR-3 tumours, were measured. Cells were analysed for cell cycle arrest, cell death mechanisms, reactive oxygen species production, mitochondrial depolarisation, oxygen consumption and acid production. RESULTS: PENAO demonstrated promising anti-proliferative activity on the most common (serous, endometrioid) as well as on rare (clear cell, mucinous) subtypes of ovarian cancer cell lines. No cross-resistance with platinum-based drugs was evident. Endometrioid SKOV-3 cells were, however, shown to be resistant to PENAO in vitro and in a xenograft mouse model. This resistance was due to an ability to cope with PENAO-induced oxidative stress, notably through heme oxygenase-1 induction, and a shift in metabolism towards glycolysis. The adaptive glycolytic shift in SKOV-3 was targeted using a mTORC1 inhibitor in combination with PENAO. This strategy was successful with the two drugs acting synergistically to inhibit cell proliferation and to induce cell death via apoptosis and autophagy. CONCLUSION: Mitochondria/mTOR dual-targeting therapy may constitute a new approach for the treatment of recurrent/resistant forms of epithelial ovarian cancer.

Dual-targeting of aberrant glucose metabolism in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common and malignant primary brain tumor. In contrast to some other tumor types, aberrant glucose metabolism is an important component of GBM growth and chemoresistance. Recent studies of human orthotopic GBM in mice and in situ demonstrated GBM cells rely on both glycolysis and mitochondrial oxidation for glucose catabolism. These observations suggest that the homeostasis of energy metabolism of GBM cells might be further disturbed by dual-inhibition of glucose metabolism. The present study aimed to evaluate the efficacy and the mechanisms of dual-targeting therapy in GBM cells. METHODS: Representative GBM cells (immortalized GBM cell lines and patient-derived GBM cells) and non-cancerous cells were treated with 4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid (PENAO), an in-house designed novel arsenic-based mitochondrial toxin, in combination with dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor. The efficacy of this combinatorial therapy was evaluated by MTS assay, clonogenic surviving assay and apoptotic assays. The underlying mechanisms of this dual-targeting treatment were unraveled by using mitochondrial membrane potential measurements, cytosol/mitochondrial ROS detection, western blotting, extracellular flux assay and mass spectrometry. RESULTS: As monotherapies, both PENAO and DCA induced proliferation arrest in a panel of GBM cell lines and primary isolates. PENAO inhibited oxygen consumption, induced oxidative stress and depolarized mitochondrial membrane potential, which in turn activated mitochondria-mediated apoptosis. By combining DCA with PENAO, the two drugs worked synergistically to inhibit cell proliferation (but had no significant effect on non-cancerous cells), impair the clonogenicity, and induce mitochondria-mediated apoptosis. An oxidative stress of mitochondrial origin takes a prominent place in the mechanism by which the combination of PENAO and DCA induces cell death. Additionally, PENAO-induced oxidative damage was enhanced by DCA through glycolytic inhibition which in turn diminished acid production induced by PENAO. Moreover, DCA treatment also led to an alteration in the multidrug resistance (MDR) phenotype of GBM cells, thereby leading to an increased cytosolic accumulation of PENAO. CONCLUSIONS: The findings of this study shed a new light with respect to the dual-targeting of glucose metabolism in GBM cells and the innovative combination of PENAO and DCA shows promise in expanding GBM therapies.

The effect of running exercise on intervertebral disc extracellular matrix production in a rat model.

STUDY DESIGN: Using a running rat model, the effects of physical exercise on cellular function and intervertebral disc (IVD) extracellular matrix were studied. OBJECTIVE: To investigate whether 3-weeks treadmill running exercise can stimulate matrix production and cellular proliferation of the IVD. SUMMARY OF BACKGROUND DATA: Appropriate physical exercise plays an important role in the treatment of patients with low back pain-associated IVD disorder. However, it is unknown how regular exercise affects the disc at the cellular level. METHODS: Twelve Sprague-Dawley rats underwent a daily treadmill exercise regime for a total of 3 weeks. Twelve nonexercised rats served as controls. The spinal lumbar IVD were collected and paraffin embedded for histologic analysis. Cell counts were determined on hematoxylin-eosin- and Masson-Trichrome-stained paraffin sections. Protein expression of collagen-I, collagen-II, aggrecan, Sox-9, and Sox-6 was evaluated with immunohistochemical staining. mRNA expression of Sox-9 and collagen-2 were studied by in situ hybridization. Proteoglycans were visualized with Alcian blue. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. RESULTS: The cell numbers in the anulus fibrosus (AF) increased by 25% (P < 0.05) after 3 weeks of exercise. Collagen-2 and Sox-9 mRNA were strongly expressed in the nucleus pulposus (NP) samples of the running group, but weakly expressed in the controls. An increase in collagen-II, aggrecan, and Sox-9 protein expression in NP and AF regions of the disc was detected in the exercised rats compared with controls. Quantification of Alcian blue staining demonstrated increased proteoglycan in both NP (8-fold) and AF (7-fold) in the exercised group compared with controls (P < 0.05). In addition, no significant differences were observed between the experimental groups in cellular apoptosis, collagen-I, or Sox-6 expression. CONCLUSION: In this study, increased extracellular matrix production and cell proliferation with no induction of disc cell apoptosis was observed in the lumbar IVD after a 3-week running regimen in rats, suggesting that regular exercise may have an augmentative effect on cells and matrix production.

Differentiation of rodent bone marrow mesenchymal stem cells into intervertebral disc-like cells following coculture with rat disc tissue.

This study aimed to evaluate whether rat mesenchymal stem cells (rMSCs) could be differentiated in vitro into disc-like cells by coculturing with intervertebral disc tissue. rMSCs were cultured with rodent intervertebral disc for up to 30 days in transwell plates. The differentiation of rMSCs was evaluated by immunostaining, Western blot, real-time RT-PCR, Northern blot, and electron microscopy. The potentials of multilineage differentiation and proteoglycan and collagen synthesis were also investigated. rMSCs underwent morphological changes to form three-dimensional micromasses and expressed collagen-2, aggrecan, and sox-9 at RNA and protein levels after 14 days of coculture. These changes were not detected in the samples of rMSCs cultured alone. Cocultured rMSCs also showed other characteristic features of disc-like cells, including the extracellular matrix formation, and proteoglycan and collagen synthesis. In addition, cellular contact between cocultured rMSCs and disc tissue was observed by electron microscopy. Committed rMSCs still retained their differentiation ability into mesoderm lineages of adipocytes or osteocytes when the local environment was altered. This study supports that MSCs are a promising source for cell therapy and tissue engineering in disc regeneration, and highlights that rMSCs can be induced into nucleus pulposus-like cells in vitro under the direct influence of intact disc tissue.

The fate of transplanted xenogeneic bone marrow-derived stem cells in rat intervertebral discs.

Intervertebral disc degeneration is a major cause and a risk factor for chronic low back pain. The potential of using stem cells to treat disc degeneration has been raised. The aims of our study were to assess whether xenogeneic bone-marrow derived stem cells could survive in a rat disc degeneration model and to determine which cell types, if any, survived and differentiated into disc-like cells. Human bone-marrow derived CD34(+) (hematopoietic progenitor cells) and CD34(-) (nonhematopoietic progenitor cells, including mesenchymal stem cells) cells were isolated, fluorescent-labeled, and injected into rat coccygeal discs. The rats were sacrificed at day 1, 10, 21, and 42. Treated discs were examined by histological and immunostaining techniques and compared to control discs. The survival of transplanted cells was further confirmed with a human nuclear specific marker. Fluorescent labeled CD34(-) cells were detected until day 42 in the nucleus pulposus of the injected discs. After 3 weeks these cells had differentiated into cells expressing chondrocytic phenotype (Collagen II and Sox-9). In contrast, the fluorescent labeled CD34(+) cells could not be detected after day 21. No fluorescence-positive cells were detected in the noninjected control discs. Further, no inflammatory cells infiltrated the nucleus pulposus, even though these animals had not received immunosuppressive treatment. Our data provide evidence that transplanted human BM CD34(-) cells survived and differentiated within the relative immune privileged nucleus pulposus of intervertebral disc degeneration.

Bone morphogenetic protein-7 protects human intervertebral disc cells in vitro from apoptosis.

BACKGROUND CONTEXT: Disc degeneration includes dysfunction and loss of disc cells leading to a decrease in extracellular matrix (ECM) components. Apoptosis has been identified in degenerated discs. Bone morphogenetic protein-7 (BMP-7) has been reported to stimulate ECM synthesis in the intervertebral disc (IVD), but its effect on disc cell viability is unknown. PURPOSE: To investigate whether BMP-7 can protect disc cells from programmed cell death while enhancing ECM production. STUDY DESIGN: An in vitro study to examine the effect of BMP-7 on apoptosis of IVD cells. METHODS: Human nucleus pulposus (NP) cells were cultured in monolayer, and human recombinant pure BMP-7 (rhBMP-7) was added to the medium when the cells were in the second passage. Thereafter, apoptosis was induced by either tumor necrosis factor-alpha (TNF-alpha) or hydrogen peroxide (H(2)O(2)). Cellular apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and caspase-3 activity. ECM synthesis was assessed by immunofluorescence for collagen-2 and aggrecan. To study the possibility of bone induction by rhBMP-7 in disc cells, alkaline phosphatase activity and Alizarin red-S staining were evaluated. RESULTS: Apoptosis was induced by both TNF-alpha and H(2)O(2). Addition of rhBMP-7 resulted in inhibition of the apoptotic effects caused by both inducers. Further, BMP-7 decreased caspase-3 activity. In the presence of BMP-7, ECM production was maintained by the cells despite being in an apoptotic environment. No osteoblastic induction of the disc cells was seen. CONCLUSIONS: BMP-7 was demonstrated to prevent apoptosis of human disc cells in vitro. One of the antiapoptotic effects of BMP-7 on NP cells might be a result of its inactivation of caspase-3. Collagen production was maintained by addition of rhBMP-7 in an apoptotic environment.

Nucleus pulposus cellular longevity by telomerase gene therapy.

STUDY DESIGN: Nonviral transfection of nucleus pulposus cells with a telomerase expression construct to assess the effects on cellular lifespan, function, karyotypic stability, and transformation properties. OBJECTIVES: To investigate whether telomerase gene therapy can extend the cellular lifespan while retaining functionality of nucleus pulposus cells in a safe manner. SUMMARY OF BACKGROUND DATA: Degeneration of the intervertebral disc is an age-related condition in which cells responsible for the maintenance and health of the disc deteriorate with age. Telomerase can extend the cellular lifespan and function of other musculoskeletal tissues, such as the heart, bones, and connective tissues. Therefore, extension of the cellular lifespan and matrix production of intervertebral disc cells may have the potential to delay the degeneration process. METHODS: Ovine nucleus pulposus cells were lipofectamine transfected in vitro with a human telomerase reverse transcriptase (hTERT) expression construct. Cellular lifespan and matrix transcript levels were determined by cumulative population doublings and real-time RT-PCR, respectively. G1-cell cycle checkpoint, p53 functionality, growth of transfected cells in anchorage-independent or serum starvation conditions, and karyotypic analysis were performed. RESULTS: Transfection was achieved successfully with 340% +/- 7% (mean +/- SD) relative telomerase activity in hTERT-transfected cells. hTERT transfection enabled a 50% extension in mean cellular lifespan and prolonged matrix production of collagen 1 and 2 for more than 282 days. Karyotypic instability was detected but G1-cell cycle checkpoint and p53 was functionally comparable to parental cells with no growth in serum starvation or anchorage-independent conditions. CONCLUSIONS: Telomerase can extend the cellular lifespan of nucleus pulposus cells and prolong the production of extracellular matrix. Safety is still unresolved, as karyotypic instability was detected but no loss of contact inhibition, mitogen dependency, or G1-cell cycle checkpoint control was evident.

The molecular basis of intervertebral disk degeneration.

At present, the molecular evidence involved in the degeneration of the IVD is still in its primitive stage. In general, however, intrinsic, extrinsic, and generic factors all have shown a contribution to the initiation of degeneration. These factors may stimulate cytokines and directly or indirectly generate the cellular events thus far detected and observed. Such changes discussed have been the cellularity, matrix degradation, matrix quality, and synthesis of enzymes involved in the breakdown of the matrix, including fibronectin fragments of the degraded matrix which can contribute to further degradation. The involvement of cytokines and other inflammatory mediators in the generation of vascularization and stimulation of pain receptors are still controversial but may reveal the pathway to the symptomatic conditions of IVD degeneration.