Gerald C. Wallace

Drug resistance in glioblastoma: a mini review.

Glioblastoma multiforme (GBM) is recognized as the most common and lethal form of central nervous system cancer. Currently used surgical techniques, chemotherapeutic agents, and radiotherapy strategies have done very little in extending the life expectancies of patients diagnosed with GBM. The difficulty in treating this malignant disease lies both in its inherent complexity and numerous mechanisms of drug resistance. In this review, we summarize several of the primary mechanisms of drug resistance. We reviewed available published literature in the English language regarding drug resistance in glioblastoma. The reasons for drug resistance in glioblastoma include drug efflux, hypoxic areas of tumor cells, cancer stem cells, DNA damage repair, and miRNAs. Many potential therapies target these mechanisms, including a series of investigated alternative and plant-derived agents. Future research and clinical trials in glioblastoma patients should pursue combination of therapies to help combat drug resistance. The emerging new data on the potential of plant-derived therapeutics should also be closely considered and further investigated.

Spinal cord injury: a review of current therapy, future treatments, and basic science frontiers.

The incidence of acute and chronic spinal cord injury (SCI) in the United States is more than 10,000 per year, resulting in 720 cases per million persons enduring permanent disability each year. The economic impact of SCI is estimated to be more than 4 billion dollars annually. Preclinical studies, case reports, and small clinical trials suggest that early treatment may improve neurological recovery. To date, no proven therapeutic modality exists that has demonstrated a positive effect on neurological outcome. Emerging data from recent preclinical and clinical studies offer hope for this devastating condition. This review gives an overview of current basic research and clinical studies for the treatment of SCI.

Understanding the basic mechanisms underlying seizures in mesial temporal lobe epilepsy and possible therapeutic targets: A review

Despite years of research, epilepsy remains a poorly understood disorder. In the past several years, work has been conducted on a variety of projects with the goal of better understanding the pathogenesis and progression of mesial temporal lobe epilepsy (MTLE), in particular, and how to exploit those properties to generate innovative therapies for treatment of refractory epilepsies. This review seeks to give an overview of common morphological and biochemical changes associated with epilepsy and proposed treatments to address those changes. Furthering the understanding of ictogenesis and epileptogenesis remains an important goal for scientists seeking to find more effective treatments for MTLE.

Calpeptin attenuated inflammation, cell death, and axonal damage in animal model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model for studying multiple sclerosis (MS). Calpain has been implicated in many inflammatory and neurodegenerative events that lead to disability in EAE and MS. Thus, treating EAE animals with calpain inhibitors may block these events and ameliorate disability. To test this hypothesis, acute EAE Lewis rats were treated dose dependently with the calpain inhibitor calpeptin (50–250 μg/kg). Calpain activity, gliosis, loss of myelin, and axonal damage were attenuated by calpeptin therapy, leading to improved clinical scores. Neuronal and oligodendrocyte death were also decreased, with down‐regulation of proapoptotic proteins, suggesting that decreases in cell death were due to decreases in the expression or activity of proapoptotic proteins. These results indicate that calpain inhibition may offer a novel therapeutic avenue for treating EAE and MS.

Calpain inhibitor attenuated optic nerve damage in acute optic neuritis in rats

Optic neuritis (ON), which is an acute inflammatory autoimmune demyelinating disease of the central nervous system (CNS), often occurs in multiple sclerosis (MS). ON is an early diagnostic sign in most MS patients caused by damage to the optic nerve leading to visual dysfunction. Various features of both MS and ON can be studied following induction of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in Lewis rats. Inflammation and cell death in the optic nerve, with subsequent damage to the retinal ganglion cells in the retina, are thought to correlate with visual dysfunction. Thus, characterizing the pathophysiological changes that lead to visual dysfunction in EAE animals may help develop novel targets for therapeutic intervention. We treated EAE animals with and without the calpain inhibitor calpeptin (CP). Our studies demonstrated that the Ca2+‐activated neutral protease calpain was upregulated in the optic nerve following induction of EAE at the onset of clinical signs (OCS) of the disease, and these changes were attenuated following treatment with CP. These reductions correlated with decreases in inflammation (cytokines, iNOS, COX‐2, and NF‐κB), and microgliosis (i.e. activated microglia). We observed that calpain inhibition reduced astrogliosis (reactive astroglia) and expression of aquaporin 4 (AQP4). The balance of Th1/Th2 cytokine production and also expression of the Th1‐related CCR5 and CXCR3 chemokine receptors influence many pathological processes and play both causative and protective roles in neuron damage. Our data indicated that CP suppressed cytokine imbalances. Also, Bax:Bcl‐2 ratio, production of tBid, PARP‐1, expression and activities of calpain and caspases, and internucleosomal DNA fragmentation were attenuated after treatment with CP. Our results demonstrated that CP decreased demyelination [loss of myelin basic protein (MBP)] and axonal damage [increase in dephosphorylated neurofilament protein (de‐NFP)], and also promoted intracellular neuroprotective pathways in optic nerve in EAE rats. Thus, these data suggest that calpain is involved in inflammatory as well as in neurodegenerative aspects of the disease and may be a promising target for treating ON in EAE and MS.

Effects of a novel orally administered calpain inhibitor SNJ-1945 on immunomodulation and neurodegeneration in a murine model of multiple sclerosis.

Multiple sclerosis (MS) pathology is marked by the massive infiltration of myelin‐specific T cells into the CNS. Hallmarks of T helper (Th) cells during active disease are pro‐inflammatory Th1/Th17 cells that predominate over immunoregulatory Th2/Treg cells. Neurodegeneration, a major factor in progressive MS, is often overlooked when considering drug prescription. Here, we show that oral dosing with SNJ‐1945, a novel water‐soluble calpain inhibitor, reduces experimental autoimmune encephalomyelitis clinical scores in vivo and has a two pronged effect via anti‐inflammation and protection against neurodegeneration. We also show that SNJ‐1945 treatment down‐regulates Th1/Th17 inflammatory responses, and promotes regulatory T cells (Tregs) and myeloid‐derived suppressor cells in vivo, which are known to have the capacity to suppress helper as well as cytotoxic T cell functions. Through analysis of spinal cord samples, we show a reduction in calpain expression, decreased infiltration of inflammatory cells, and signs of inhibition of neurodegeneration. We also show a marked reduction in neuronal cell death in spinal cord (SC) sections. These results suggest that calpain inhibition attenuates experimental autoimmune encephalomyelitis pathology by reducing both inflammation and neurodegeneration, and could be used in clinical settings to augment the efficacy of standard immunomodulatory agents used to treat MS.

Overexpression of melatonin membrane receptors increases calcium-binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

Abstract:  Melatonin has shown particular promise as a neuroprotective agent to prevent motoneuron death in animal models of both amyotrophic lateral sclerosis (ALS) and spinal cord injuries (SCI). However, an understanding of the roles of endogenous melatonin receptors including MT1, MT2, and orphan G‐protein receptor 50 (GPR50) in neuroprotection is lacking. To address this deficiency, we utilized plasmids for transfection and overexpression of individual melatonin receptors in the ventral spinal cord 4.1 (VSC4.1) motoneuron cell line. Receptor‐mediated cytoprotection following exposure to glutamate at a toxic level (25 μm) was determined by assessing cell viability, apoptosis, and intracellular free Ca2+ levels. Our findings indicate a novel role for MT1 and MT2 for increasing expression of the calcium‐binding proteins calbindin D28K and parvalbumin. Increased levels of calbindin D28K and parvalbumin in VSC4.1 cells overexpressing MT1 and MT2 were associated with cytoprotective effects including inhibition of proapoptotic signaling, downregulation of inflammatory factors, and expression of prosurvival markers. Interestingly, the neuroprotective effects conferred by overexpression of MT1 and/or MT2 were also associated with increases in the estrogen receptor β (ERβ): estrogen receptor α (ERα) ratio and upregulation of angiogenic factors. GPR50 did not exhibit cytoprotective effects. To further confirm the involvement of the melatonin receptors, we silenced both MT1 and MT2 in VSC4.1 cells using RNA interference technology. Knockdown of MT1 and MT2 led to an increase in glutamate toxicity, which was only partially reversed by melatonin treatment. Taken together, our findings suggest that the neuroprotection against glutamate toxicity exhibited by melatonin may depend on MT1 and MT2 but not GPR50.

Abstract 2183: PF-02341066 (crizotinib), a dual ALK/c-Met inhibitor, for inhibiting growth of glioblastoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Glioblastoma is the most common and highly aggressive human brain cancer. Most of the glioblastoma patients do not survive more than a few months after the diagnosis. The prognosis for glioblastoma patients remains very poor even after aggressive treatments. Traditional therapeutic strategies are not yet satisfactory for controlling the growth of this deadly malignancy in humans. Heterogeneity of cells in glioblastoma is one of biggest hurdles in its management. Novel and innovative therapeutic agents need to be explored for targeting the key cell signaling pathways that promote proliferation and survival of different human glioblastoma cells. Receptor tyrosine kinases have been implicated in the progression of this malignancy. Anaplastic lymphoma kinase (ALK) and c-Met are two such proteins. ALK/c-Met are associated with the development and progression of many human cancers, including glioblastoma. Also, overexpression or mutation of c-ros proto-oncogene encodes a receptor tyrosine kinase, which also leads to oncogenic development of glioblastoma. In the present study, we examined the activity of the Pfizer drug crizotinib (PF-02341066, a dual ALK/c-Met inhibitor) for suppression of both ALK/c-Met pathways and induction of apoptotic death in different human glioblastoma cell lines (U87MG, T98G, U118MG, and U138MG) and fresh tumor biopsies surgically extracted from 6 glioblastoma patients. Our dose-response studies indicated that treatment with PF-02341066 for 24 h could induce morphological features (as detected by Wright staining) and also biochemical features (as detected by DNA fragmentation assay and Western blotting) of apoptosis. We used Western blotting for investigation into the molecular mechanisms of apoptotic death and found that induction of apoptosis was associated with decreases in expression of ALK, c-Met, c-Ros, Survivin, and Bcl-2 proteins after the treatment. Western blotting also demonstrated that treatment with PF-02341066 caused apoptosis due to an increase in Bax:Bcl-2 ratio and activation of caspase-3 in glionlastoma cells and tissues. Increase in activity of caspase-3 was confirmed by colorimetric assay. Most importantly, PF-02341066 significantly enhanced the anti-cancer effect of temozolomide (TMZ), a widely used anti-glioblastoma drug, in human glioblastoma cell lines (U87MG, T98G, U118MG, and U138MG) as well as in fresh tumor biopsies from 6 glioblastoma patients. Our studies also suggested that induction of apoptosis by PF-02341066 does not depend on the functional status of p53, PTEN, ALK, c-Met, or c-Ros in the glioblastoma cell lines or in the glioblastoma tissues. Further studies in different animal models of glioblastoma are warranted in the future to determine whether PF-02341066 may be useful as an anti-cancer agent alone or in combination with TMZ for the management of human glioblastomas in vivo. This investigation was supported in by Pfizer Oncology. Citation Format: Arabinda Das, Gerald C. Wallace, Catherine Haar, Michele L. DeCandio, W Alex Vandergrift, Sunil J. Patel, Swapan K. Ray, Naren L. Banik, Pierre Giglio. PF-02341066 (crizotinib), a dual ALK/c-Met inhibitor, for inhibiting growth of glioblastoma. [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 2183. doi:10.1158/1538-7445.AM2013-2183

Abstract 2350: DATS prevented tumor progression and induced apoptotic death in an ectopic model of glioblastoma in SCID mice

BACKGROUND: Glioblastoma, the most common and malignant brain tumor in humans, still remains incurable because of inefficacy of the currently available chemotherapeutic agents. Conventional chemotherapeutic agents have not been able to show promising results in glioblastoma patients for the last several decades. Therefore, new and non-conventional therapeutic agents also must be explored for controlling the growth of glioblastoma in vivo. Organosulfur compounds from garlic are well characterized and used as alternative medicine in many Asian countries. As a new treatment strategy in this investigation, we examined the anti-tumor properties of the most potent garlic compound diallyl trisulfide (DATS) in human glioblastoma U87MG xenografts in SCID mice. METHODS: We developed human glioblastoma U87MG xenografts in SCID mice for treatment with DATS for 7 days consecutively. The efficacy of DATS treatments in controlling tumor growth was assessed by histopathological examinations and also molecular analyses such as in situ immunofluorescent labeling and Western blotting. RESULTS: Our findings indicated that a range of DATS doses (10 μg/kg - 10 mg/kg) was effective in reducing tumor volume and weight in SCID mice ectopically xenografted with exponentially growing human glioblastoma U87MG cells. Histopathological examinations of the tumor sections showed significant amounts of inhibition of cell proliferation and induction of cell death. In addition, these findings were associated with a significant reduction in the number of mitotic bodies. Increase in TUNEL-positive staining was also detected in the tumor following treatment, suggesting that DATS induced apoptotic death in glioblastoma xenografts in SCID mice. Further, immunofluorescent staining of the tumor sections confirmed increase in expression of glial fibrillary acidic protein (GFAP), an acclaimed astrocyte-specific marker, in the tumors after the treatments. Our Western blotting demonstrated increases in the apoptosis promoting factors, reduction in the anti-apoptotic proteins (e.g., Bcl-2, BIRC proteins), and activation of the cysteine proteases such as calpain and caspase-3 to favor induction of apoptotic death in the tumors. It should also be mentioned that light microscopic examinations tissue sections revealed no damage in the livers and kidneys from the SCID mice following DATS treatments. CONCLUSIONS: Taken together, our results clearly demonstrated that DATS could be an effective therapeutic agent in preventing not only tumor progression but also induction of apoptosis in human glioblastoma in vivo. Success of our in vivo treatment of human glioblastoma xenografts with DATS strongly suggests that DATS should also be explored as a potential therapeutic agent for treatment glioblastoma in humans. 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 2350. doi:1538-7445.AM2012-2350