Mebendazole and radiation in combination increase survival through anticancer mechanisms in an intracranial rodent model of malignant meningioma.

PURPOSE: Meningiomas are a frequent tumor of the central nervous system. Although mostly benign, approximately 5% present as atypical or malignant tumors. Treatments for atypical meningiomas include gross total resection and radiotherapy, but about 33% of patients have recurrent tumors, sometimes as a higher grade. Recently, the brain penetrant anthelmintic drug, mebendazole, has shown promise as an anticancer agent in rodent models of glioblastoma and medulloblastoma. METHODS: The half maximal inhibitory concentration (IC50) effect on colony formation, cell proliferation, and caspase-3/7 markers of apoptosis of mebendazole with and without radiation was measured in vitro. Mice intracranially implanted with KT21MG1 human meningioma were administered mebendazole alone or in combination with radiation. Survival benefit was evaluated, while tumors were investigated by immunohistochemical staining for apoptosis, cell proliferation, and vascular density. RESULTS: In vitro experiments on meningioma cell lines showed the IC50 for mebendazole in the range of 0.26-0.42 µM. Mebendazole alone induced cytotoxicity, however the combination had a greater reduction in colony formation and resulted in higher levels of cleaved caspase-3. The in vivo study showed both, mebendazole alone and the combination, to have a survival benefit with an increase in apoptosis, and decreases in tumor cell and vascular proliferation. CONCLUSION: These preclinical findings indicate that mebendazole alone or in combination with radiation can be considered for the treatment of malignant meningioma. The mechanism of action for this combination may include an increase in apoptosis, a reduction in proliferation and angiogenesis, or a combination of these effects.

Tunable Stability of Imidazotetrazines Leads to a Potent Compound for Glioblastoma.

Even in the era of personalized medicine and immunotherapy, temozolomide (TMZ), a small molecule DNA alkylating agent, remains the standard-of-care for glioblastoma (GBM). TMZ has an unusual mode-of-action, spontaneously converting to its active component via hydrolysis in vivo. While TMZ has been FDA approved for two decades, it provides little benefit to patients whose tumors express the resistance enzyme MGMT and gives rise to systemic toxicity through myelosuppression. TMZ was first synthesized in 1984, but certain key derivatives have been inaccessible due to the chemical sensitivity of TMZ, precluding broad exploration of the link between imidazotetrazine structure and biological activity. Here, we sought to discern the relationship between the hydrolytic stability and anticancer activity of imidazotetrazines, with the objectives of identifying optimal timing for prodrug activation and developing suitable compounds with enhanced efficacy via increased blood-brain barrier penetrance. This work necessitated the development of new synthetic methods to provide access to previously unexplored functionality (such as aliphatic, ketone, halogen, and aryl groups) at the C8 position of imidazotetrazines. Through synthesis and evaluation of a suite of compounds with a range of aqueous stabilities (from 0.5 to 40 h), we derive a predictive model for imidazotetrazine hydrolytic stability based on the Hammett constant of the C8 substituent. Promising compounds were identified that possess activity against a panel of GBM cell lines, appropriate hydrolytic and metabolic stability, and brain-to-serum ratios dramatically elevated relative to TMZ, leading to lower hematological toxicity profiles and superior activity to TMZ in a mouse model of GBM. This work points a clear path forward for the development of novel and effective anticancer imidazotetrazines.

A novel biologically active acid stable liposomal formulation of docosahexaenoic acid in human breast cancer cell lines.

PURPOSE: The risk of breast cancer can be influenced by certain dietary components, such as the amount and type of dietary fatty acids ingested. Docosahexaenoic acid (DHA), a component of fish oil, is known to suppress rat mammary carcinogenesis, reduce cell growth and induce apoptosis in human breast cancer cell lines. The purpose of this study was to develop a novel nanoliposomal formulation that would encapsulate a concentrated amount of DHA and utilize lipids that could protect DHA from pH fluctuations and oxidation. METHODS: We developed an acid stable liposome formulation of DHA by utilizing ether and phytanyl lipids similar in structure to those found in Archaea, known to endure high acidity and temperature; we compared its biological activities with free DHA in human breast cancer cells. RESULTS: The mean size of the liposomal DHA was 137 ± 12 nm with a slightly negative charge; the encapsulation efficiency of DHA in the liposomes as determined by LC-MS/MS ranged from 60 to 80%; our formulation is resistant to oxidation and stable over a range of pH (1.0-7.4) at 37 °C for a duration of two hours. In MCF-7 cells, liposomal DHA (IC50 38.8 µM) significantly reduced cell viability more effectively than free DHA (IC50 72.5 µM, p = 0.0017). In MDA-MB-231 cells, liposomal DHA was also marginally more effective. Liposomal DHA was more effective than free DHA in inducing apoptosis in both cell lines. It altered proteins involved in cell growth, cell cycle, and apoptosis more effectively than free DHA in both cell lines; it up-regulated p21 and cleaved PARP, while P-AKT and P-S6 were down-regulated. CONCLUSIONS: We developed a novel biologically active acid stable liposomal DHA as a potentially useful formulation for breast cancer prevention.

Combination of Antiestrogens and Omega-3 Fatty Acids for Breast Cancer Prevention.

The molecular and biological heterogeneity of human breast cancer emphasizes the importance of a multitargeted approach for effective chemoprevention. Targeting the estrogen receptor pathway alone with the antiestrogens, Tamoxifen and Raloxifene reduces the incidence of estrogen receptor positive tumors but is ineffective against the development of hormone independent cancers. Our preclinical data indicate that the administration of omega-3 fatty acids potentiates the antitumor effects of Tamoxifen by inhibiting multiple proliferative and antiapoptotic pathways, several of which interact with estrogen receptor signaling. The complementarity in the mechanism of antitumor action of Tamoxifen and omega-3 fatty acids is well supported by our signaling, genomic, and proteomic studies. Furthermore, administration of omega-3 fatty acids allows the use of lower and, hence, likely less toxic doses of Tamoxifen. If these findings are supported in the clinical setting, the combination of omega-3 fatty acids and anteistrogens may emerge as a promising, effective, and safe chemopreventive strategy to be tested in a large multi-institutional trial using breast cancer incidence as the primary endpoint.

Proteomic changes induced by effective chemopreventive ratios of n-3:n-6 fatty acids and tamoxifen against MNU-induced mammary cancer in the rat.

We used a proteomic approach to gain insights into the mechanisms of protection at the protein level by a high n-3:n-6 ratio in the absence and presence of Tamoxifen. Four groups were treated with 1-methyl-1-nitrosourea (MNU) and fed the following diets with varied n-3:n-6 ratios; group 1 = 1:1; groups 2 and 3 = 10:1 and 25:1, respectively; group 4: (25:1) plus Tamoxifen (1 mg/kg diet). The plasma from six rats/group was pooled and analyzed with the isobaric tags for relative and absolute quantitation method; 148 proteins were identified with 95% confidence by ProteinPilot 4.0. In plasma of rats fed 10:1, 25:1 n-3:n-6, and 25:1 plus Tamoxifen, the number of proteins that met our criteria (P ≤ 0.05, error factor ≤ 2) were 10, 14, and 19 proteins, respectively. Selected proteins were further validated by Western blotting. Compared to 1:1, both 10:1 and 25:1 diets upregulated vitamin D binding protein, gelsolin, and 14-3-3 sigma, reported to have tumor suppressive effects, whereas alpha-1B-glycoprotein, which has been reported to be elevated in the serum of breast cancer patients was decreased. Compared to 25:1, the 25:1 plus Tamoxifen diet downregulated apolipoprotein E, haptoglobin, and inter-α-inhibitor H4 heavy chain. Ingenuity pathway analysis determined that the trends of specific proteins were related to lipid metabolism in the 25:1 n-3:n-6 group, whereas the 25:1 n-3:n-6 plus Tamoxifen group included proteins involved in cancer and inflammation. Our results show that several proteins were altered in a manner consistent with chemoprevention. Such proteins may serve as biomarkers to monitor efficacy of n-3 and Tamoxifen in future clinical chemoprevention trials.