Amy J. Hanson

α-Tocopheryl Succinate, the Most Effective Form of Vitamin E for Adjuvant Cancer Treatment: A Review

In 1982, it was established that alpha-tocopheryl succinate (α-TS) was the most effective form of vitamin E in comparison to α-tocopherol, α-tocopheryl acetate and α-tocopheryl nicotinate in inducing differentiation, inhibition of proliferation and apoptosis in cancer cells, depending upon its concentration. During the last two decades, several studies have confirmed this observation in rodent and human cancer cells in culture and in vivo (animal model). The most exciting aspect of this α-TS effect is that it does not affect the proliferation of most normal cells. In spite of several studies published on the anti-cancer properties of α-TS, the value of this form of vitamin E has not drawn significant attention from researchers and clinicians. Therefore, a critical review on the potential role of α-TS in the management of cancer is needed. In addition, such a review can also provide in-depth analysis of existing literature on this subject. α-TS treatment causes extensive alterations in gene expression; however, only some can be attributed to differentiation, inhibition of proliferation and apoptosis. α-TS also enhances the growth-inhibitory effect of ionizing radiation, hyperthermia, some chemotherapeutic agents and biological response modifiers on tumor cells, while protecting normal cells against some of their adverse effects. Thus, α-TS alone or in combination with dietary micronutrients can be useful as an adjunct to standard cancer therapy by increasing tumor response and possibly decreasing some of the toxicities to normal cells.

Defects in cAMP-pathway may initiate carcinogenesis in dividing nerve cells: a review.

The mechanisms of carcinogenesis in nervous tissues are not well understood. It is now established that adenosine 3,′,5′-cyclic monophosphate (cAMP)-pathway plays a crucial role in initiating differentiation in transformed and embryonic cells of neuronal and glial origin. Therefore, we propose that defects in the cAMP-pathway may initiate the first phase of carcinogenesis (immortalization). Subsequent genetic abnormalities in oncogenes, anti-oncogenes or other cellular genes individually or in combination may lead to transformation (cancer phenotype). This hypothesis is derived from the fact that an elevation of the cAMP level in murine NB cells induces terminal differentiation in many of these cells in spite of the fact that they are highly aneuploid. Additional changes in cAMP-regulated genes responsible for initiating differentiation may make these cells resistant to cAMP or may make the cAMP-effect on differentiation reversible. Indeed, cAMP-resistant cells exist in NB cell populations, and the cAMP-effect on differentiation is reversible in glioma cells. Identification of genes that initiate, promote and maintain terminal differentiation and those which prevent differentiation following elevation of cAMP in NB cells may increase our understanding of the mechanisms of carcinogenesis. This review illustrates the following: (a) historical background leading to the discovery of cAMP as an inducer of differentiation in nerve cells; (b) identification of potential sites in cAMP-pathway that may play a crucial role in initiating the first phase of carcinogenesis (immortalization) and potential gene targets in immortalized cells whose alterations may cause neoplastic transformation of nerve cells. It is interesting to note that the cAMP pathway remains responsive to an elevated cAMP level in inducing differentiation in NB cells in spite of chromosomal anomalies and genetic changes associated with the maintenance of a cancer phenotype.

Prostaglandin-induced neurodegeneration is associated with increased levels of oxidative markers and reduced by a mixture of antioxidants

Prostaglandin E2 (PGE2), one product of inflammatory reactions, and PGA1, which is formed during PGE2 extraction, induce degeneration in adenosine 3′,5′‐cyclic monophosphate (cAMP)‐induced differentiated neuroblastoma (NB) cells in culture. The mechanisms of action of PGE2 on neurodegeneration are not well understood. To investigate this, we have utilized PGA1, which mimics the effect of PGE2 and is very stable in solution. We have assayed selected markers of oxidative stress such as heme oxygenase‐1 (HO‐1), catalase, glutathione peroxidase (GPx1), mitochondrial superoxide dismutase (Mn‐SOD‐2) and cytosolic superoxide dismutase (Cu/Zn‐SOD‐1). The results showed that the treatment of differentiated NB cells with PGA1 for a period of 48 hr increased the expression of HO‐1 and catalase, decreased the expression of GPx1 and Mn‐SOD‐2, and did not change the expression of Cu/Zn‐SOD‐1 as measured by gene array and confirmed by real‐time PCR. The protein levels of HO‐1 and GPx1 increased; however, the protein level of Mn‐SOD‐2 decreased and the levels of catalase and Cu/Zn‐SOD‐1 did not change as determined by Western blot. The increases in the levels of HO‐1 and GPx1 reflected an adaptive response to increased oxidative stress, whereas decrease in the level of Mn‐SOD‐2 may make cells more sensitive to oxidative damage. These data suggest that one of the mechanisms of action of PGA1 on neurodegeneration may involve increased oxidative stress. This was supported further by the fact that a mixture of antioxidants (α‐tocopherol, vitamin C, selenomethionine, and reduced glutathione), but not the individual antioxidants, reduced the level of PGA1‐induced degeneration in differentiated NB cells. The addition of a single antioxidant at two or four times the concentration used in the mixture was toxic.

Overexpression of α-synuclein decreased viability and enhanced sensitivity to prostaglandin E2, hydrogen peroxide, and a nitric oxide donor in differentiated neuroblastoma cells

Increased accumulation of α‐synuclein is associated with certain neurodegenerative diseases including Parkinsons disease (PD) and Alzheimers disease (AD). One mechanism of α‐synuclein‐induced toxicity involves increased oxidative stress. It was unknown whether neurons overexpressing α‐synuclein would exhibit increased sensitivity to hydrogen peroxide (H2O2) or 3‐morpholinosydnonimine (SIN‐1; a nitrous oxide donor). To study this, we developed a murine neuroblastoma (NB) cell line that overexpresses wild‐type human α‐synuclein (NBP2‐PN54) under the control of the cytomegalovirus (CMV) promoter using a retroviral vector. Human α‐synuclein mRNA and protein were readily detectable in NBP2‐PN54 cells. Results showed that differentiated NBP2‐PN54 cells exhibited decreased viability in comparison to differentiated vector (NBP2‐PN1) and parent (NBP2) control cells. These cells also exhibited increased sensitivity to PGE2, H2O2 and SIN‐1. Because of involvement of proteasome inhibition in neurodegeneration, we also investigated whether treatment of differentiated NBP2‐PN54 cells with PGE2, H2O2 or SIN‐1 inhibits proteasome activity. Results showed that H2O2 and SIN‐1 inhibited proteasome activity, but PGE2 did not. These results suggest that overexpression of α‐synuclein not only participates directly in degeneration of neurons, but it also increases the vulnerability of neurons to other potential neurotoxins.

Overexpression of amyloid precursor protein is associated with degeneration, decreased viability, and increased damage caused by neurotoxins (prostaglandins A1 and E2, hydrogen peroxide, and nitric oxide) in differentiated neuroblastoma cells.

Inflammatory reactions are considered one of the important etiologic factors in the pathogenesis of Alzheimers disease (AD). Prostaglandins such as PGE2 and PGA1 and free radicals are some of the agents released during inflammatory reactions, and they are neurotoxic. The mechanisms of their action are not well understood. Increased levels of β‐amyloid fragments (Aβ40 and Aβ42), generated through cleavage of amyloid precursor protein (APP), oxidative stress, and proteasome inhibition, are also associated with neurodegeneration in AD brains. Therefore, we investigated the effect of PGs and oxidative stress on the degeneration and viability of cyclic AMP‐induced differentiated NB cells overexpressing wild‐type APP (NBP2‐PN46) under the control of the CMV promotor in comparison with differentiated vector (NBP2‐PN1) or parent (NBP2) control cells. Results showed that differentiated NBP2‐PN46 cells exhibited enhanced spontaneous degeneration and decreased viability in comparison with differentiated control cells, without changing the level of Aβ40 and Aβ42. PGA1 or PGE2 treatment of differentiated cells caused increased degeneration and reduced viability in all three cell lines. These effects of PGs are not due to alterations in the levels of vector‐derived APP mRNA or human APP holoprotein, secreted levels of Aβ40 and Aβ42, or proteasome activity. H2O2 or SIN‐1 (an NO donor) treatment did not change vector‐derived APP mRNA levels, but H2O2 reduced the level of human APP protein more than SIN‐1. Furthermore, SIN‐1 increased the secreted level of Aβ40, but not of Aβ42, whereas H2O2 had no effect on the level of secreted Aβ fragments. Both H2O2 and SIN‐1 inhibited proteasome activity in the intact cells. The failure of neurotoxins to alter APP mRNA levels could be due to the fact that they do not affect CMV promoter activity. These results suggest that the mechanisms of action of PGs on neurodegeneration are different from those of H2O2 and SIN‐1 and that the mechanisms of neurotoxicity of H2O2 and SIN‐1 are, at least in part, different from each other.

Regulated expression of VP16CREB in neuroblastoma cells: analysis of differentiation and apoptosis.

Highly malignant neuroblastoma tumors generally have defects in differentiation and apoptotic pathways. For a better understanding of these events, we use a murine neuroblastoma cell line (NBP2) that terminally differentiates into mature neurons in response to elevated levels of cAMP. Because one of the main downstream effectors of the cAMP signaling pathway is cAMP‐response element binding (CREB), we reasoned that it might affect the expression of genes associated with differentiation and apoptotic events in NBP2 cells. To investigate this, we established tetracycline‐regulated expression (TetOff) of VP16CREB, which constitutively transactivates promoters containing the CRE sequence motif. Using this system, we found that inducible expression of VP16CREB in NBP2 cells results in 1) morphological differentiation that is characterized by the formation of neurites and growth cones, 2) reversible cell differentiation unlike cAMP‐induced terminal differentiation, 3) cell cycle arrest at G1, 4) no apoptosis in the presence of partial inhibition of proteasome unlike an increase in cAMP levels, and 5) changes in the expression of many genes, including down‐regulation of N‐myc, cyclin B1, Dickkopf‐1, and Mad‐2 and up‐regulation of tyrosine hydroxylase, c‐fos, N10, and ICER genes. Although VP16CREB expression and activation of the cAMP pathway impart many similar effects in NBP2 cells, they also bear some distinct genetic and morphological differences. Our data suggest that increased levels of cAMP function through not only CREB but also other signaling pathways that account for the additional cAMP‐induced effects, including irreversible differentiation and onset of apoptosis during partial inhibition of proteasome in NBP2 cells.

Altered expression of genes regulating cell growth, proliferation, and apoptosis during adenosine 3',5'-cyclic monophosphate-induced differentiation of neuroblastoma cells in culture.

SummaryAn elevation of the intracellular levels of adenosine 3′,5′-cyclic monophosphate (cAMP) induces terminal differentiation in neuroblastoma (NB) cells in culture; however, genetic alterations during differentiation have not been fully identified. To investigate this, we used Mouse Genome U74A microarray containing ∼6000 functionally characterized genes to measure changes in gene expression in murine NB cells 30 min and 4,24, and 72 h after treatment with cAMP-stimulating agents. Based on the time of increase in differentiated functions and their status (reversible versus irreversible) after treatment with cAMP-stimulating agents, the induction of differentiation in NB cells was divided into three distinct phases: initiation (about 4 h after treatment when no increase in differentiated functions is detectable), promotion (about 24 h after treatment when an increase in differentiated functions occurs, but they are reversible upon the removal of cAMP), and maintenance (about 72 h after treatment when differentiated functions are maximally expressed, but they are irreversible upon the removal of cAMP). Results showed that alterations in expression of genes regulating cell growth, proliferation, apoptosis, and necrosis occurred during cAMP-induced differentiation of NB cells. Genes that were upregulated during the initiation, promotion, or maintenance phase were called initiators, promoters, or maintainers of differentiation. Genes that were downregulated during the initiation, promotion, or maintenance phase were called suppresors of initiation, promotion, or maintenance phase. Genes regulating growth may act as initiators, promoters, maintainers, or suppressors of these phases. Genes regulating cell proliferation may primarily act as suppressors of promotion. Genes regulating cell cycle may behave as suppressors of initiation or promotion, whereas those regulating apoptosis and necrosis may act as initiators or suppressors of initiation or promotion. The fact that genetic signals for differentiation occurred 30 min after treatment with cAMP, whereas cell-cycle genes were downregulated at a later time, suggests that decision for NB cells to differentiate is made earlier and not at the cell-cycle stage, as commonly believed.

Selenomethionine Prevents Degeneration Induced by Overexpression of Wild-Type Human α-Synuclein during Differentiation of Neuroblastoma Cells

Objective: High levels of wild-type α-synuclein are found in autopsied brain samples of idiopathic Parkinson’s disease (PD), some familial PD, some Alzheimer’s disease (AD) and Down’s syndrome with dementia. Therefore, we have investigated whether overexpression of wild-type α-synuclein causes degeneration during adenosine, 3′,5′-cyclic monophosphate (cAMP)-induced differentiation of murine neuroblastoma (NB) cells in culture. We have also studied whether selenomethionine can modify the effect of overexpression of α-synuclein during differentiation of NB cells. Methods: To study these issues, we established a murine neuroblastoma (NB) clone (NBP2-PN54-C20) that expressed high levels of wild-type human α-synuclein as determined by real time PCR and Western blot. We have utilized RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, and prostaglandin A1 (PGA1), a stimulator of adenylate cyclase, or RO20-1724 and dibutyryl cAMP to induce terminal differentiation in over 95% of the cell population by elevating the intracellular levels of cAMP in NB cells. The viability of cells was determined by MTT assay and LDH leakage assay, and the degeneration was documented by photomicrographs. Results: The results showed that overexpression of human wild-type α-synuclein decreased viability and increased degenerative changes in comparison to those observed in vector control cells, when differentiation was induced by treatment with RO20-1724 and PGA1, but not with RO20-1724 and dibutyryl cAMP. When selenomethionine was added to NB cells overexpressing α-synuclein immediately after the addition of RO20-1724 and PGA1, the viability and degenerative changes were markedly reduced, suggesting the involvement of increased oxidative stress in the mechanism of action of α-synuclein. This protective effect was not observed after treatment with sodium selenite or methionine. Conclusions: Data suggested that Overexpression of wild-type human α-synuclein-decreased viability and increased the levels of degenerative changes during differentiation of NB cells were reduced by selenomethionine treatment. This suggest that one of mechanisms of action α-synuclein may involve increased oxidative stress.

Role of the adenosine 3',5'-cyclic monophosphate (cAMP) in enhancing the efficacy of siRNA-mediated gene silencing in neuroblastoma cells.

Gene-silencing activity mediated by siRNA has been demonstrated in mammalian cells; however, the mechanism of its regulation is not well understood. Since downregulation of a number of genes occurs during adenosine 3′,5′-cyclic monophosphate (cAMP)-induced differentiation of neuroblastoma (NB) cells, it is possible that cAMP may play a role in regulating siRNA activity during differentiation. To study this, we utilized an NB cell line (NBP2-PN25) that expresses a short-lived green fluorescent protein (d2EGFP) under the CMV promoter. These cells were transfected with a retroviral plasmid that expresses U6 promoter-driven expression of siRNA targeted to d2EGFP and then were treated with cAMP-elevating agents (200 μg/ml RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, and 1 μg/ml prostaglandin A1, a stimulator of adenylate cyclase) for 2 or 24 h. The siRNA activity was measured by determining the level of intensity of d2EGFP protein by flow cytometry, and the level of d2EGFP mRNA by real-time PCR. The results showed that cAMP-elevating agents enhanced U6-driven siRNA activity directed towards d2EGFP in NB cells 24 h after treatment. One of the mechanisms of action of cAMP is mediated via phosphatidylinositol 3-kinase (PI3K) inhibition; therefore, we have investigated the effect of a PI3K inhibitor on siRNA activity. This study showed that inhibition of PI3K also enhanced U6-driven siRNA activity towards d2EGFP. cAMP-stimulating agents increased U6 transcript levels, perhaps suggesting that increased siRNA activity may in part be due to an increase in transcriptional activity. When NB cells were transfected with a synthetic siRNA directed to d2EGFP, both cAMP elevation and PI3K inhibition similarly enhanced siRNA activity. Sodium butyrate, which inhibits the growth of NB cells similar to the effect produced by cAMP, did not affect U6-driven siRNA activity towards d2EGFP. Protein kinase C (PKC) activation or inhibition also failed to affect siRNA activity in NB cells. This study also showed that cAMP elevation and PI3K inhibition increases U6-driven siRNA activity directed towards an endogenous gene, p53. Our data suggest a role for the cAMP pathway in affecting the efficacy of siRNA system during differentiation of NB cells.

Concomitant Differentiation and Partial Proteasome Inhibition Trigger Apoptosis in Neuroblastoma Cells

Proteasome activity is essential during cAMP-induced terminal differentiation of a murine neuroblastoma cell line (NBP2). However, the mechanisms through which proteasome affects NBP2 differentiation have not been characterized. We hypothesized that proteasome is required to implement the differentiation-mediated effects on cell cycle, and its partial inhibition during differentiation may have adverse consequences. Here we show that partial inhibition of proteasome during cAMP-induced differentiation of NBP2 cells causes apoptosis. Whereas differentiation induced growth arrest at G1 phase, partial proteasome inhibition during differentiation resulted in the accumulation of cells at G2M phase. Cell cycle data correlated with the level of cyclin-dependent kinase inhibitors p21WAF and p27Kip1, and cyclin A. While the level of p21 and p27 increased, the level of cyclin A decreased upon differentiation. In contrast, cells treated with proteasome inhibitor in the presence of cAMP-inducing agents showed increased levels of p21 and cyclin A early in the course of differentiation. However, the level of p21 and p27, but not cyclin A, decreased later during concomitant differentiation and partial proteasome inhibition when cells were undergoing apoptosis. Our data suggest that differentiation-mediated growth arrest is dependent on the temporal activity of cell cycle proteins. Partial inhibition of proteasome interferes with differentiation events partly by stabilizing cell cycle proteins and this triggers apoptosis. Thus, differentiating drugs combined with partial proteasome inhibition may impart higher therapeutic efficacy than differentiating agents alone for the treatment of neuroblastoma tumors.