Invasiveness of endometrial cancer cell lines is potentiated by estradiol and blocked by a traditional medicine Guizhi Fuling at clinically relevant doses

The Traditional Chinese medicine, Guizhi Fuling (here called Fuling), has been confirmed in meta-analysis studies to reduce recurrence of endometriosis and improve pregnancy outcomes; however, the possible use of Fuling as a fertility-preserving treatment in endometrial cancer has not previously been tested. Results here are the first to demonstrate dose-dependent inhibition of cell motility by Fuling in two endometrial cancer cell lines, classified as Grade I which is responsive to progesterone treatment, and Grade III (MFE-280) which is resistant. The major outcome of this study was the novel demonstration that Fuling (30-80 µg/ml) significantly inhibits invasiveness in both high and low grades of EC cells, achieving 70-80% block of trans-barrier migration without cytotoxicity. This effective dose range is estimated to be comparable to that used in human clinical trials and traditional practice. Results here further show that clinically relevant doses of Fuling override the motility-promoting effects of estradiol in endometrial cancer cell lines. Medroxyprogesterone acetate has to date been the standard therapy to treat metastatic or inoperable endometrial cancers; however, success rates are low with high rates of recurrence, due in part to acquired resistance to medroxyprogesterone acetate therapy. The discovery here that Fuling appears to control the spread of treatment-resistant advanced cancers is an exciting prospect

Pharmacologically impeding glioblastoma tumour motility through simultaneous inhibition of aquaporin-1 and ion channels

Glioblastoma multiforme (GBM) is an invasive tumour derived from neuroglial progenitor stem cells. Despite increasingly intricate treatment strategies, rapid infiltration of glioma cells into healthy brain tissue remains a major clinical challenge. I hypothesise that therapies which target cellular motility pathways could effectively slow tumour dispersal and widen the time window for administration of frontline treatments aimed at direct eradication of primary tumours. The array of signal transduction pathways that control cellular motility include aquaporins and ion channels. These protein classes could therefore be prime candidates as pharmacological targets to restrain cell motility in glioblastoma. Identifying optimal combinations of inhibitory agents to use against selected channel targets, and developing drug delivery systems that have minimal side effects in the complex environment of the brain, could control glioblastoma motility without disrupting finely tuned activities of neuroglial networks. This thesis explores a selection of ion channel and aquaporin channel blockers as putative inhibitors of in vitro glioblastoma invasion. Results here define novel agents that potently impair GBM cell motility. Natural compounds (xanthurenic acid and caelestine C) and semi-synthetic amides (SN00756563, SN00756564 and SN00756565) decreased invasion in U87-MG and U251-MG glioblastoma cell lines, revealing previously unknown anti-invasive activities of these agents. When combined with the aquaporin-1 inhibitor AqB013, xanthurenic acid and caelestine C produced a synergistic block of invasion in both U87-MG and U251-MG. Ion channel blockers nifedipine, amiloride, apamin, 4-aminopyridine, and AMPA/kainate receptor inhibitor cyanquixaline significantly decreased invasion in U87-MG and U251-MG, effects that were additively enhanced upon co-treatment with AqB013. Interestingly, when solid tumour spheroids of U87-MG and U251-MG were treated with the same agents, no significant additive or synergistic effects were observed. Some combinations of inhibitors blocked invasion in U87-MG more effectively than in U251- MG and vice versa. This differential effect could reflect the array of protein factors that affect the motility of glioblastoma tumour cells, including unique expression patterns of aquaporins and ion channels. This work presents the novel finding that these pharmacological inhibitors, natural compounds, or semi-synthetics, administered at low doses either individually or in combinations, produced no significant cytotoxicity in cultured glioblastoma cells or astrocytes. This could indicate the capacity to minimise off-target effects associated with these agents. Implementation of site-specific drug delivery systems could further reduce off-target effects. pHsensitive gating mechanisms in many channel proteins is attributable to pore-lining histidine residues. Site-directed mutagenesis experiments described in this thesis identified sites within AQP1 where introduction or removal of histidine residues potentiated or abolished cGMPinduced ionic currents in the presence of Ni2+. Histidine could serve as a promising pH-sensing agent to be incorporated into the design of drug delivery systems that are activated by the hallmark acidity of the tumour microenvironment. Impeding glioblastoma tumour dispersal by targeting enriched signalling proteins with functions in key cellular motility pathways could constitute a powerful adjunct therapy when applied in parallel with existing procedures. Additionally, coupling these novel inhibitors of glioblastoma invasion to carrier molecules containing pH-sensitive histidine residues could maximise controlled release of treatment agents to glioblastoma tumour cells and limit off-target effects.Thesis (M.Phil.) -- University of Adelaide, Adelaide Medical School, 202

Novel Ion Channel Targets and Drug Delivery Tools for Controlling Glioblastoma Cell Invasiveness

Comprising more than half of all brain tumors, glioblastoma multiforme (GBM) is a leading cause of brain cancer-related deaths worldwide. A major clinical challenge is presented by the capacity of glioma cells to rapidly infiltrate healthy brain parenchyma, allowing the cancer to escape control by localized surgical resections and radiotherapies, and promoting recurrence in other brain regions. We propose that therapies which target cellular motility pathways could be used to slow tumor dispersal, providing a longer time window for administration of frontline treatments needed to directly eradicate the primary tumors. An array of signal transduction pathways are known to be involved in controlling cellular motility. Aquaporins (AQPs) and voltage-gated ion channels are prime candidates as pharmacological targets to restrain cell migration in glioblastoma. Published work has demonstrated AQPs 1, 4 and 9, as well as voltage-gated potassium, sodium and calcium channels, chloride channels, and acid-sensing ion channels are expressed in GBM and can influence processes of cell volume change, extracellular matrix degradation, cytoskeletal reorganization, lamellipodial and filopodial extension, and turnover of cell-cell adhesions and focal assembly sites. The current gap in knowledge is the identification of optimal combinations of targets, inhibitory agents, and drug delivery systems that will allow effective intervention with minimal side effects in the complex environment of the brain, without disrupting finely tuned activities of neuro-glial networks. Based on published literature, we propose that co-treatments using AQP inhibitors in addition to other therapies could increase effectiveness, overcoming some limitations inherent in current strategies that are focused on single mechanisms. An emerging interest in nanobodies as drug delivery systems could be instrumental for achieving the selective delivery of combinations of agents aimed at multiple key targets, which could enhance success in vivo

Pharmacological Inhibition of Membrane Signaling Mechanisms Reduces the Invasiveness of U87-MG and U251-MG Glioblastoma Cells In Vitro

Impairing the motility of glioblastoma multiforme (GBM) cells is a compelling goal for new approaches to manage this highly invasive and rapidly lethal human brain cancer. Work here characterized an array of pharmacological inhibitors of membrane ion and water channels, alone and in combination, as tools for restraining glioblastoma spread in human GBM cell lines U87-MG and U251-MG. Aquaporins, AMPA glutamate receptors, and ion channel classes (shown to be upregulated in human GBM at the transcript level and linked to mechanisms of motility in other cell types) were selected as pharmacological targets for analyses. Effective compounds reduced the transwell invasiveness of U87-MG and U251-MG glioblastoma cells by 20–80% as compared with controls, without cytotoxicity. The compounds and doses used were: AqB013 (14 μM); nifedipine (25 µM); amiloride (10 µM); apamin (10 µM); 4-aminopyridine (250 µM); and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; 30 µM). Invasiveness was quantified in vitro across transwell filter chambers layered with extracellular matrix. Co-application of each of the ion channel agents with the water channel inhibitor AqB013 augmented the inhibition of invasion (20 to 50% greater than either agent alone). The motility impairment achieved by co-application of pharmacological agents differed between the GBM proneural-like subtype U87-MG and classical-like subtype U251-MG, showing patterns consistent with relative levels of target channel expression (Human Protein Atlas database). In addition, two compounds, xanthurenic acid and caelestine C (from the Davis Open Access Natural Product-based Library, Griffith University QLD), were discovered to block invasion at micromolar doses in both GBM lines (IC50 values from 0.03 to 1 µM), without cytotoxicity, as measured by full mitochondrial activity under conditions matching those in transwell assays and by normal growth in spheroid assays. Mechanisms of action of these agents based on published work are likely to involve modulation of glutamatergic receptor signaling. Treating glioblastoma by the concurrent inhibition of multiple channel targets could be a powerful approach for slowing invasive cell spread without cytotoxic side effects, potentially enhancing the effectiveness of clinical interventions focused on eradicating primary tumors