AT101

AT101-Loaded Cubosomes as an Alternative for Improved Glioblastoma Therapy.[Pubmed:33116479]

Int J Nanomedicine. 2020 Oct 5;15:7415-7431.

Introduction: AT101, the R-(-)-enantiomer of the cottonseed-derived polyphenol gossypol, is a promising drug in glioblastoma multiforme (GBM) therapy due to its ability to trigger autophagic cell death but also to facilitate apoptosis in tumor cells. It does have some limitations such as poor solubility in water-based media and consequent low bioavailability, which affect its response rate during treatment. To overcome this drawback and to improve the anti-cancer potential of AT101, the use of cubosome-based formulation for AT101 drug delivery has been proposed. This is the first report on the use of cubosomes as AT101 drug carriers in GBM cells. Materials and Methods: Cubosomes loaded with AT101 were prepared from glyceryl monooleate (GMO) and the surfactant Pluronic F-127 using the top-down approach. The drug was introduced into the lipid prior to dispersion. Prepared formulations were then subjected to complex physicochemical and biological characterization. Results: Formulations of AT101-loaded cubosomes were highly stable colloids with a high drug entrapment efficiency (97.7%) and a continuous, sustained drug release approaching 35% over 72 h. Using selective and sensitive NMR diffusometry, the drug was shown to be efficiently bound to the lipid-based cubosomes. In vitro imaging studies showed the high efficiency of cubosomal nanoparticles uptake into GBM cells, as well as their marked ability to penetrate into tumor spheroids. Treatment of GBM cells with the AT101-loaded cubosomes, but not with the free drug, induced cytoskeletal rearrangement and shortening of actin fibers. The prepared nanoparticles revealed stronger in vitro cytotoxic effects against GBM cells (A172 and LN229 cell lines), than against normal brain cells (SVGA and HMC3 cell lines). Conclusion: The results indicate that GMO-AT101 cubosome formulations are a promising basic tool for alternative approaches to GBM treatment.

AT101 [(-)-Gossypol] Selectively Inhibits MCL1 and Sensitizes Carcinoma to BH3 Mimetics by Inducing and Stabilizing NOXA.[Pubmed:32824203]

Cancers (Basel). 2020 Aug 15;12(8). pii: cancers12082298.

Anti-apoptotic BCL2 proteins are important targets for cancer therapy as cancers depend on their activity for survival. Direct inhibitors of MCL1 have entered clinical trials, although their efficacy may be limited by toxicity. An alternative approach may be to induce the pro-apoptotic protein NOXA which selectively inhibits MCL1 in cells. Many compounds originally proposed as inhibitors of the BCL2 family were subsequently found to induce the pro-apoptotic protein NOXA through the unfolded protein response. In the present study, we compared various putative BH3 mimetics across a panel of carcinoma cell lines and measured expression of NOXA protein and mRNA, as well as the kinetics of NOXA induction. We found that AT101 [(-)-gossypol] induces high levels of NOXA in carcinoma cell lines yet cells survive. When combined with an appropriate BCL2 or BCL-XL inhibitor, NOXA-dependent sensitization occurs. NOXA protein continues to accumulate for many hours after AT101 is removed, providing a window for administering these combinations. As MCL1 promotes drug resistance and overall survival, we propose that NOXA induction is an alternative therapeutic strategy to target MCL1 and either kill cancer cells that are dependent on MCL1 or sensitize cancer cells to other BCL2 inhibitors.

Combined treatment of AT101 and demethoxycurcumin yields an enhanced anti-proliferative effect in human primary glioblastoma cells.[Pubmed:31844979]

J Cancer Res Clin Oncol. 2020 Jan;146(1):117-126.

PURPOSE: Glioblastoma multiforme (GBM) is a poorly curable disease due to its profound chemoresistance. Despite recent advances in surgery, radiotherapy and chemotherapy, the efficient treatment of GBMs is still a clinical challenge. Beside others, AT101, the R-(-) enantiomer of gossypol, and demethoxycurcumin (DMC), a curcumin-related demethoxy compound derived from Curcuma longa, were considered as possible alternative drugs for GBM therapy. METHODS: Using different human primary GBM cell cultures in a long-term stimulation in vitro model, the cytotoxic and anti-proliferative effects of single and combined treatment with 5 microM AT101 and 5 microM or 10 microM DMC were investigated. Furthermore, western blots on pAkt and pp44/42 as well as JC-1 staining and real-time RT-PCR were performed to understand the influence of the treatment at the molecular and gene level. RESULTS: Due to enhanced anti-proliferative effects, we showed that combined therapy with both drugs was superior to a single treatment with AT101 or DMC. Here, by determination of the combination index, a synergism of the combined drugs was detectable. Phosphorylation and thereby activation of the kinases p44/42 and Akt, which are involved in proliferation and survival processes, were inhibited, the mitochondrial membrane potential of the GBM cells was altered, and genes involved in dormancy-associated processes were regulated by the combined treatment strategy. CONCLUSION: Combined treatment with different drugs might be an option to efficiently overcome chemoresistance of GBM cells in a long-term treatment strategy.

Establishment of a glioblastoma in vitro (in)complete resection dual co-culture model suitable for drug testing.[Pubmed:31726206]

Ann Anat. 2020 Mar;228:151440.

BACKGROUND: The treatment of glioblastomas (GBM) is still a clinical challenge. Current GBM therapeutic plans focus on the development of new strategies for local drug administration in the tumor cavity to realize an efficient long-term treatment with small side-effects. Here, different amounts of residual GBM cells and healthy brain cells define the microenvironment of the tumor cavity after individual surgical GBM resection (complete or incomplete). METHODS: We evaluated available in vivo data and determined the required amounts and numerical ratios of GBM and healthy brain cells for our in vitro (in)complete resection dual co-culture model. We applied a generic two-drug treatment [Temozolomide (TMZ) in combination with AT101, followed by single AT101 treatment] strategy and analyzed the results in comparison with appropriate mono-culture systems to prove the applicability of our model. RESULTS: We established a suitable GBM dual co-culture model, mimicking the complete and incomplete resection in vitro, giving stable and reliable results on drug testing. Both dual co-culture conditions protectively influenced on cell death and growth rates of primary GBMs when treated with TMZ+AT101/AT101, although the treatment strategy per se was still efficient. Cell death of astrocytes correlated with amounts of increasing GBM cell numbers in the incomplete resection model upon drug treatment, and probably GBM-released chemokine and cytokines were involved in this interplay. CONCLUSIONS: Our results suggest that this dual co-culture model provides a biologically relevant platform for the discovery and compound screening of local GBM treatment strategies.

Anaplastic Lymphoma Kinase Confers Resistance to BRAF Kinase Inhibitors in Melanoma.[Pubmed:31229894]

iScience. 2019 Jun 28;16:453-467.

Melanoma frequently harbors oncogenic mutations in the BRAF gene, which drives melanoma growth. Therefore, BRAF kinase inhibitors (BRAFi) are developed and approved for treating BRAF-mutant melanoma. However, the efficacy of BRAFi is limited due to acquired resistance, and in over 40% of melanoma, the causes of BRAFi resistance remain unknown. Here, using a human phospho-receptor tyrosine kinase array we identified Anaplastic Lymphoma Kinase (ALK) as a driver of acquired BRAFi resistance in melanoma. We found that ALK ligand FAM150A was necessary for ALK activation and ALK via the PI3K/AKT pathway was sufficient to confer resistance to BRAFi. ALK inhibitor (ALKi) ceritinib inhibited BRAFi-resistant melanoma in cell culture and mice. Residual BRAFi and ALKi dual resistant melanoma cells from ceritinib-treated mice were sensitive to a broad-spectrum anti-apoptotic protein inhibitor, AT101. Collectively, our results provide a framework for treating BRAF-mutant melanoma that sequentially uses different targeted therapies based on post-treatment tumor evolution.

AT101 induces G1/G0 phase arrest via the betacatenin/cyclin D1 signaling pathway in human esophageal cancer cells.[Pubmed:30483745]

Oncol Rep. 2019 Feb;41(2):1415-1423.

AT101, an orally available and welltolerated natural panBcl2 family protein inhibitor, has been reported to be effective against a variety of cancers. However, the mechanisms whereby AT101 exhibits anticancer activity have not been fully elucidated. In this study, we demonstrated that AT101 reduced the cell viability of human esophageal cancer cells by inducing G1/G0 phase arrest and apoptosis. Apoptotic cell death occurred later than cell cycle arrest, as evidenced by an increase in the proportion of Annexin Vpositive cells and cleaved caspase3, 9 and PARP protein levels. AT101 markedly downregulated the protein levels of phosphoretinoblastoma (Ser 780) and cyclin D1, whereas it elevated protein levels of p53 and p21Waf1/Cip1, contributing to the inhibition of cell cycle progression. Moreover, AT101 substantially reduced betacatenin expression. XAV939, a small molecule that inhibits the Wnt/betacatenin signaling pathway by facilitating betacatenin degradation, lowered betacatenin and cyclin D1 protein expression to an extent similar to AT101. XAV939 alone resulted in G1/G0 phase arrest and further induced cell cycle arrest in combination with AT101, suggesting that the betacatenin/cyclin D1 signaling pathway mediated, at least in part, the cell cycle arrest induced by AT101. The present study may shed new light on the anticancer activity of AT101 in relation to cell cycle arrest as well as apoptosis in human esophageal cancer cells.