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Yayun Liang,1,2,* Sandy Goyette,1,2,* Salman M Hyder1,2 1Department of Biomedical Sciences, 2Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO, USA *These authors contributed equally to this work Abstract: Clinical trials and studies have shown that postmenopausal women undergoing combination hormone replacement therapy containing estrogen and progestin have an increased risk of breast cancer compared with women taking estrogen or placebo alone. Using animal models, we have previously shown that synthetic progestins, including medroxyprogesterone acetate (MPA), which is widely used clinically, accelerate breast cancer tumor growth and promote metastasis. Furthermore, we have found that MPA elevates CD44 protein expression and aldehyde dehydrogenase (ALDH) activity, two markers of cancer stem cells (CSCs), and increases mammosphere formation, another hallmark of stem cells, in hormone-dependent T47-D human breast cancer cells. Herein, we show that RO 48-8071 (RO), an inhibitor of cholesterol synthesis, reduced MPA-induced CD44 protein expression in two hormone-dependent human breast cancer cell lines, T47-D and BT-474. Because we have previously shown that MPA induction of CD44 is progesterone receptor (PR) dependent, we examined RO’s effects on PR protein and mRNA expressions in T47-D cells. PR mRNA levels remained unchanged after RO treatment; however, RO significantly reduced the protein expression of both PR receptor isoforms, PR-A and PR-B. Using the proteasome inhibitor MG-132, we demonstrated that RO decreases PR protein expression in T47-D cells via the proteasomal degradation pathway. Importantly, treatment of T47-D cells with RO abolished MPA-induced mammosphere formation. Based on our observations, we contend that RO may represent a novel means of preventing MPA-induced CSC expansion. RO could be used clinically to both treat and prevent hormone-dependent breast cancers, which represent the majority of human breast cancers. RO may also have clinical utility in reducing resistance to antihormone therapy. Keywords: breast cancer, cholesterol biosynthesis inhibitor, RO 48-8071, medroxyprogesterone acetate, cancer stem cells, CD44

We have studied how various drugs increasing the rate of nicotinic acetylcholine receptors (nAChRs) lateral diffusion affect the depression of ACh-induced current in land snail Helix lucorum neurons responsible for defensive behavior. The acetylcholine (ACh) iontophoretic application protocol imitated the behavioral habituation protocol for the intact animal. We found that the drugs decreasing cholesterol level in cell membranes as methyl-β-cyclodextrin 1 mM and Ro 48-8071 2 µM, and polyclonal antibodies to actin-binding proteins as spectrin 5 µg/ml and merlin 2.5 µg/ml have changed the dynamic of ACh-current depression. The nAChRs lateral diffusion coefficient was obtained by fluorescence recovery after photobleaching. A curve fitting model specially created for analysis of short-term choline sensitivity depression in snail neurons helped us evaluate separately the contribution of nAChRs lateral diffusion, their endocytosis and exocytosis to observed effects during electrophysiological experiments. Taken together, we hypothesize that nAChRs lateral diffusion plays an important role in the cellular correlate of habituation in land snail Helix lucorum neurons. [ABSTRACT FROM AUTHOR]

Clinical trials and studies have shown that postmenopausal women undergoing combination hormone replacement therapy containing estrogen and progestin have an increased risk of breast cancer compared with women taking estrogen or placebo alone. Using animal models, we have previously shown that synthetic progestins, including medroxyprogesterone acetate (MPA), which is widely used clinically, accelerate breast cancer tumor growth and promote metastasis. Furthermore, we have found that MPA elevates CD44 protein expression and aldehyde dehydrogenase (ALDH) activity, two markers of cancer stem cells (CSCs), and increases mammosphere formation, another hallmark of stem cells, in hormone-dependent T47-D human breast cancer cells. Herein, we show that RO 48-8071 (RO), an inhibitor of cholesterol synthesis, reduced MPA-induced CD44 protein expression in two hormone-dependent human breast cancer cell lines, T47-D and BT-474. Because we have previously shown that MPA induction of CD44 is progesterone receptor (PR) dependent, we examined RO's effects on PR protein and mRNA expressions in T47-D cells. PR mRNA levels remained unchanged after RO treatment; however, RO significantly reduced the protein expression of both PR receptor isoforms, PR-A and PR-B. Using the proteasome inhibitor MG-132, we demonstrated that RO decreases PR protein expression in T47-D cells via the proteasomal degradation pathway. Importantly, treatment of T47-D cells with RO abolished MPA-induced mammosphere formation. Based on our observations, we contend that RO may represent a novel means of preventing MPA-induced CSC expansion. RO could be used clinically to both treat and prevent hormone-dependent breast cancers, which represent the majority of human breast cancers. RO may also have clinical utility in reducing resistance to antihormone therapy. [ABSTRACT FROM AUTHOR]

Tobacco BY-2 cell suspensions are our preferred model for studying isoprenoid biosynthesis pathways, due to their easy genetic transformation and the efficient absorption of metabolic precursors, intermediates, and/or inhibitors. Using this model system, we have analyzed the effects of chemical and genetic blockage of cycloartenol synthase (CAS, EC 5.4.99.8), an oxidosqualene cyclase that catalyzes the first committed step in the sterol pathway of plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Short-term treatments (24 h) resulted in accumulation of oxidosqualene with no changes in the final sterol products. Interestingly, long-term treatments (6 days) induced down-regulation in gene expression not only of CAS but also of the SMT2 gene coding sterol methyltransferase 2 (EC 2.1.1.41). This explains some of the increase in 24-methyl sterols at the expense of the 24-ethyl sterols stigmasterol and sitosterol. In our alternative strategy, CAS gene expression was partially blocked by using an inducible artificial microRNA. The limited effectiveness of this approach might be explained by some dependence of the machinery for RNAi formation on an operating MVA/sterol pathway. For comparison we checked the effect of RO 48-8071 on a green cell suspension of Arabidopsis and on seedlings, containing a small spectrum of triterpenes besides phytosterols. Triterpenes remained essentially unaffected, but phytosterol accumulation was clearly diminished. [ABSTRACT FROM AUTHOR]

The morbidity and mortality of pancreatic cancer have been continuously increasing, causing seven deaths per 100,000 individuals/year. At present, effective therapies are severely lacking, thus, highlighting the importance of developing novel therapeutic approaches. The present study aimed to investigate the inhibitory roles of the 2,3-oxidosqualene cyclase inhibitor, RO 48-8071 (RO), on pancreatic ductal adenocarcinoma. RO was used to treat the pancreatic cancer cell line (PANC-1) in vitro to examine the effects of RO on cell viability, as well as to determine its potential molecular mechanism. Moreover, experiments in a xenograft model of subcutaneous tumors generated by injecting PANC-1 cells hypodermically into nude mice were performed to observe the inhibition of RO on tumor growth. It was found that RO inhibited PANC-1 cell viability when treatment was given for 24, 48 and 72 h. The in vivo study demonstrated that RO markedly inhibited subcutaneous tumor growth in nude mice. Further studies revealed that RO could induce cell cycle arrest in the G1 phase by regulating p27, cyclin B1 and cyclin E expression to inhibit PANC-1 cell viability. Moreover, RO inactivated the JNK and ERK MAPK signaling pathway by decreasing the phosphorylation levels of JNK and ERK. Collectively, the present study demonstrated that RO served anti-pancreatic cancer roles in vitro and in vivo, which may provide new ideas and facilitate the development of novel treatment options for pancreatic cancer. [ABSTRACT FROM AUTHOR]