Sylvia A. Chung

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.

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.

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 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.

Dual-targeting of aberrant glucose metabolism in glioblastoma

BackgroundGlioblastoma (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.MethodsRepresentative 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.ResultsAs 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.ConclusionsThe 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.

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.

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.

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.

Abstract 1701: PENAO, a novel mitochondria-targeted agent, has shown potent antitumor effect on glioblastoma in vitro and in vivo.

Despite advances in multimodal therapy, the prognosis in the great majority of patients with glioblastoma (GBM) is still dismal as almost all tumors relapse and result in disease-related death. New therapeutic strategies are urgently needed. PENAO, (4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid) is a novel mitochondria-targeted agent that inactivates adenine nucleotide translocase (ANT) located on the inner-membrane of mitochondria. PENAO blocks ANT delivery of ATP to mitochondrial-bound hexokinase II. This blockage inhibits glucose metabolism and triggers the mitochondrial permeability transition pore to leak which results in proliferation arrest and apoptotic cell death. In vitro, we tested the efficacy of PENAO on a panel of glioblastoma cell lines including immortalized and primary glioblastoma neural stem (GNS) cell lines. PENAO demonstrated potent anti-proliferative activity in all cells. The half-maximal inhibitory concentration (IC50) values for PENAO were in the range of 0.3-6 μM. Blocking ANT with PENAO resulted in inhibition of glioblastoma cell migration at concentration of 1.4 μM. PENAO (0.3-5 μM) increased caspase 3 and 9 activity levels indicative of the intrinsic apoptotic pathway. Proper functioning of ANT is also of importance for maintaining mitochondrial integrity. Low micromolar concentrations of PENAO inhibited oxygen utilization, increased the cytosolic levels of superoxide and depolarized the mitochondrial trans-membrane potential, which are responsible for the activation of mitochondria-mediated apoptosis in glioblastoma cells. In vivo, PENAO was found to cross the intact blood-brain-barrier in mice and continuous administration of 1 mg/kg/day PENAO to 10 mice bearing subcutaneous glioblastoma tumors for 28 days resulted in 8 partial and 2 complete tumor remissions. No signs or symptoms of treatment toxicity were noticed. To further inhibit glucose metabolism of glioblastoma, we tested the effectiveness of PENAO in combination with 2-deoxy-D-glucose, a glucose analogue that inhibits glycolysis via competitive inhibition of hexokinase. This dual-targeting of glucose metabolism by inhibiting both mitochondrial respiration and glycolysis led to synergistic effects and improved efficacy when measuring proliferation arrest in vitro. By targeting the glioblastoma aberrant metabolism of glucose better treatments for this cancer may arise. Citation Format: Han Shen, Peter P. Luk, Sylvia A. Chung, Stephanie Decollogne, Pierre J. Dilda, Philip J. Hogg, Kerrie L. McDonald. PENAO, a novel mitochondria-targeted agent, has shown potent antitumor effect on glioblastoma in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1701. doi:10.1158/1538-7445.AM2013-1701

Abstract 1131: Blocking ATP delivery to hexokinase II in glioblastoma is a promising therapeutic strategy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Glioblastoma has a 5-year survival of less than 5% and is responsible for more years of life lost than any other malignancy, making it a challenging therapeutic problem. Healthy cells mainly rely on oxidative phosphorylation to catabolize glucose, while glioblastoma cells use aerobic glycolysis. The first step in glycolysis, conversion of glucose to glucose-6-phosphate using ATP, is catalyzed by hexokinase and glioblastoma tumors employ an isoform (HKII) that is bound to mitochondria via interaction with the outer-membrane voltage dependent anion channel. The prevailing theory is that this localization affords HKII preferential access to the mitochondrial ATP via inner-membrane adenine nucleotide translocase (ANT) to rapidly phosphorylate and trap glucose, thus initiating glycolysis across the cell cytosol. We have tested this hypothesis using a novel water-soluble organoarsenical, PENAO, to inactivate ANT (1). Treatment of glioblastoma cell lines and primary glioma initiating cells with PENAO inhibited acid (lactate) production, a measure of cancer cell glycolysis, at a half-maximal inhibitory concentration (IC50) of 1.5 µM, which supports the hypothesis. Proper functioning of ANT is also important for mitochondrial integrity and low micromolar concentrations of PENAO increased the cytosolic levels of superoxide, disrupted the mitochondrial trans-membrane potential and inhibited O2 utilization in glioblastoma cells. Blocking ANT with PENAO resulted in inhibition of glioblastoma cell migration at IC50 value of 1.4 µM, proliferation arrest at IC50 values of 0.3-4.5 µM and mitochondrial-mediated apoptotic cell death at PENAO concentrations of 0.3-5 µM. These effects were observed under both normoxic and hypoxic (1% O2) conditions. PENAO has up to 440- and 171-fold greater anti-proliferative activity than temozolomide or carboplatin, typical drugs used to treat glioblastoma, in glioblastoma cell lines and primary glioma initiating cells. PENAO was found to cross the intact blood-brain-barrier in mice and administration of 1 mg/kg/day PENAO to ten mice bearing subcutaneous glioblastoma tumors resulted in eight partial and two complete tumor responses. There were no signs or symptoms of treatment toxicity. These findings indicate that blocking ATP delivery to hexokinase II in glioblastoma by inhibiting mitochondrial ANT is a promising therapeutic strategy. The efficacy of PENAO in in vitro and in vivo models of glioblastoma supports its clinical development as a treatment option for this cancer. 1. Dilda PJ, Decollogne S, Weerakoon L, Norris MD, Haber M, Allen JD, Hogg PJ (2009) Optimization of the antitumor efficacy of a synthetic mitochondrial toxin by increasing the residence time in the cytosol. J Med Chem 52, 6209-6216. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1131. doi:1538-7445.AM2012-1131