Abstract
Current clinical failure to cure primary glioblastoma multiforme in virtually all adult patients is due to genetic aberrations, molecular heterogeneity, and clonal evolution of tumor stem and differentiated cells within the core tumor, leading to their migration, invasion and proliferation in normal surrounding and in distant cerebral tissue sites. These factors are the causes of targeted drug resistance, inadequate surgical removal, and inadequate radio-therapeutic interventions. Resolution of this clinical conundrum may be found in administration of Withaferin A alone or in combination with pleiotropic drugs which address aberrant molecules and pathways promoting tumor cell motility, migration, invasion and proliferation.
Primary glioblastoma multiforme (PGM) is a common migratory, invasive heterogeneous malignant brain tumor in adults with a median survival of 14.6 months, following surgical ablation, radiotherapy and targeted chemotherapy (1). The current clinical failure to completely cure this tumor is due to several factors: the core tumor is composed of both tumor stem cells and differentiated tumor cells with differing genotypic and phenotypic characteristics promoting cellular motility, migration and invasion. Proteolytic digestion of the surrounding normal cerebral tissues also permits invasion and proliferation of tumor at distant sites in the brain (2).
The tumor cell heterogeneity with multiple molecular pathway alterations, genetic changes and clonal evolution promoting motility, migration and invasion, is the result of deregulated tumor genomes. Detailed analysis of PGM has revealed deletion of tumor suppressor genes, receptor tyrosine kinase amplifications and mutational aberrations, characterized by augmented survival pathways, defects in the apoptosis signaling machine and most important, a propensity to migration and invasion (1).
To improve outcomes in patients with glioblastoma multiforme, a therapeutic approach addressing clonal evolution, cellular heterogeneity, a plethora of mutations, and altered molecular pathways is required. Treatment capable of inhibiting tumor growth, motility, migration and invasion ultimately may prevent the development of recurrent tumors. Single targeting drugs, such as temozolamide, does not address all these issues, indicating a need for a novel medicinal compound or a combination of pleiotropic drugs (3).
There are several drugs, which, in pre-clinical studies, have shown that they inhibit molecular components promoting motility, migration, invasion, and proliferation at invaded sites, causing tumor recurrence (Tables I and II). Among natural products, withanolide extracts have been extensively studied in pre-clinical in vitro studies and in animal models of neural tumors (4).
Withanolides
Withanolides are naturally-occurring C28 steroidal lactones built on an ergostane framework, in which C22 and C26 are oxidized to form a six-member lactone ring. Withanolides are a potential drug source accommodating pleiotropic requirements. The most active pleiotropic drug of this class is withaferin A, a major constituent of Withania somnifera. This natural product has a wide range of pharmacological properties, including cardio-protective, anti-inflammatory, immuno-modulatory, anti-lactone, and anti-angiogenic activities (5). Withaferin A is a 4-hydroxy steroid, an enone, an epoxide, an ergostanoid, and a secondary alcohol. It has α,β-unsaturated carbonyl moieties, permitting it to act as a Michael acceptor and a thiol modifier which contribute to its pleiotrophic interactions with glioblastoma-specific migration/invasive biochemical processes (6).
These interactions serve as the mechanistic basis for its anti-tumoral properties, prohibiting tumor cell motility, and migratory/invasive characteristics, wherein the tumor cells to leave the primary site and invade normal brain parenchyma. The inhibition of glioblastoma growth, migration and invasion into normal cerebral tissue translates into control of tumor dispersal (6). Withaferin A modulates several oncogenic targets simultaneously (7). It induces cell cycle arrest and apoptosis in glioma cells (8, 9). In U 87 human glioblastoma cell line, the 50% inhibition concentration (IC50) was found to be 1.4 μg/mL, equivalent to 2.97 μM (Figure 1, unpublished observation, Sabinsa corporation). Withaferin A was also found to induce apoptosis in glioblastoma cells, which was evident within 24 hours of treatment. The number of total apoptotic cells increased from 7.2% to 28.1% in treated cells (Figure 1, unpublished observation, Sabinsa corporation).
Drugs inhibiting migration/invasion of glioblastoma cells.
Drugs promoting migration/invasion of glioblastoma cells.
Withaferin A is purified from Withania somnifera and formulated in capsules containing 10 mg of active compound (10). Withaferin A is reported to have an oral bioavailability of 32.4±4.8%, resulting in approximately 1.25 μM concentration in the blood (11, 12). The Tmax of W. somnifera extract having 0.045% withaferin A was 20 min and the T1/2 was found to be 59.9±15.9 min, while purified withaferin A showed a higher half-life of 7.6±3.3 h (13, 14). Considering these PK parameters in rodents, 10 mg dose of withaferin A is likely to achieve the therapeutic dose in human in order to have a biological effect.
Inhibiting effects of Withaferin A on putative targets leading to inhibition of migration invasion and proliferation of glioblastoma cells.
Mechanistic Targets of Migration-invasion and Interacting Drugs
Administration of withaferin A, a polyploidic compound as an oral anti-migration/invasion agent for patients with glioblastoma requires several characteristics to be proved as clinically safe and effective. Assessment of the oral bioavailability of Withaferin A is important since the intestinal flora and epithelium may have a role in the absorption, transportation and possible inactivation of this drug. There is published pre-clinical evidence that ingestion of oral Withaferin A results in a high concentration in the plasma (4). The effect of withaferin A on molecules and pathways inhibiting migration, invasion, and proliferation of glioblastoma multiforme are given in Tables III and IV.
Discussion
Failure to improve the outcomes of contemporary treatment of glioblastoma multiforme is due to its migratory and invasive characteristics, enhanced by a number of genetic mutations in both tumor stem and differentiated cells compounded by clonal evolution, and a plethora of heterogeneous molecules and pathways promoting cellular motility, migration, invasion and growth in normal cerebral tissues proximal to and at distance from the core tumor.
Effect of Withaferin A on inhibition of migration, invasion and proliferation glioblastoma cells.
Due to the limitations of curative surgery, radiotherapy, and targeted chemotherapy, addressing this problem requires application of one or a combination of pleiotropic drugs prior to or concomitant with surgery and radiotherapy. It is suggested, that the drug Withaferin A, can be applied in combination with other drugs, such as sutinib, lapatinib, KU-60019, dexamethasone, cedarinib, and temozolamide to inhibit migration/invasion of glioblastoma cell lines (14, 15).
Anti-proliferative and apoptotic activity of 30% withaferin A in glioblastoma cells (U97MG). The anticancer activity of 30% withaferin A was assessed in glioblastoma cells (U97MG) in vitro by MTT assay. Glioblastoma cells were treated with 1.4 μg/mL (IC50 concentration) of 30% withaferin A for 48 hours and apoptosis was studied by flow cytometry using annexin – FITC and propidium iodide (PI) staining. Annexin positive cells were considered to be in early apoptosis while double positive cells (annexin +PI) were late apoptotic cells. UT: Untreated, WA: 30% withaferin A.
Footnotes
Authors' Contributions
LH wrote and conceived the study. MM and scientists in Sami Labs, India performed the in vitro studies of Withaferin A.
Conflicts of Interest
LH has no financial involvement with Sabinsa Inc. MM is Founder and Managing Director of Sami Labs Limited and Sabinsa Corporation, which manufactures withaferin A for potential human trials.
- Received May 3, 2019.
- Revision received June 3, 2019.
- Accepted June 19, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
