Kedar N. Prasad

DIFFERENTIATION OF NEUROBLASTOMA CELLS IN CULTURE

1. An elevation of the intracellular level of cyclic AMP in neuroblastoma cells by prostaglandin E1 by an inhibitor of cyclic AMP phosphodiesterase, or by analogues of cyclic AMP irreversibly induces many differentiated functions which are characteristic of mature neurones. These include formation of long neurites, increase in size of soma and nucleus associated with a rise in total RNA and protein contents, increase in activities of specific neural enzymes, loss of malignancy, increase in sensitivity of adenylate cyclase to catecholamines and blockade of cells in G1‐stage of the cell cycle.

Effect of sodium butyrate on mammalian cells in culture: A review

SummarySodium butyrate produces reversible changes in morphology, growth rate, and enzyme activities of several mammalian cell types in culture. Some of these changes are similar to those produced by agents which increase the intracellular level of adenosine 3′,5′-cyclic monophosphate (cAMP) or by analogs of cAMP. Sodium butyrate increases the intracellular level of cAMP by about two fold in neuroblastoma cells; therefore, some of the effects of sodium butyrate on these cells may in part be mediated by cAMP. Sodium butyrate appears to have properties of a good chemotherapeutic agent for neuroblastoma tumors because the treatment of neuroblastoma cells in culture causes cell death and “differentiation”; however, it is either innocuous or produces reversible morphological and biochemical alterations in other cell types.

Butyric acid: A small fatty acid with diverse biological functions

Abstract Butyric acid, a 4-carbon fatty acid, affects morphology, growth rate and gene expression in mammalian cells in culture. Sodium butyrate (0.5 to 3 mM) produces reversible growth inhibition in several mammalian tumor cells in culture, but it causes cell death only in human neuroblastomas and human glioma cells in culture. Sodium butyrate in combination with currently used tumor therapeutic agents produced a synergistic, an additive or no effect on growth of mouse neuroblastoma cells and rat glioma cells in culture. At least in NB cells, the cell death and growth inhibition may be related to the reduction in anaerobic glycolysis. Sodium butyrate increases the expression of one or more differentiated functions in mouse NB cells, mouse erythroleukemic cells, human epidermoid carcinoma, human colon carcinoma cells and Chinese hamster ovary cells. The induction of differentiation by butyrate may in part be related to an increase in the cellular cyclic AMP level. Sodium butyrate increases the activities of several enzymes, whereas, it decreases the activities of some. The increase of some enzymes appears to be correlated to hyperacetylation of histones. In vitro studies suggest that sodium butyrate may be useful in the management of neoplasms by causing selective cell death, and/or cell differentiation and by increasing the cell killing effect in conjuction with currently used tumor therapeutic agents. Sodium butyrate can also be used as a tool to study the regulation of gene expression in mammalian cells.

Overexpression of human α-synuclein causes dopamine neuron death in rat primary culture and immortalized mesencephalon-derived cells

Parkinsons disease (PD) is a neurodegenerative disorder characterized by the appearance of intracytoplasmic inclusions called Lewy bodies (LB) in dopamine neurons in the substantia nigra and the progressive loss of these neurons. Recently, mutations in the alpha-synuclein gene have been identified in early-onset familial PD, and alpha-synuclein has been shown to be a major component of LB in all patients. Yet, the pathophysiological function of alpha-synuclein remains unknown. In this report, we have investigated the toxic effects of adenovirus-mediated alpha-synuclein overexpression on dopamine neurons in rat primary mesencephalic cultures and in a rat dopaminergic cell line - the large T-antigen immortalized, mesencephalon-derived 1RB3AN27 (N27). Adenovirus-transduced cultures showed high-level expression of alpha-synuclein within the cells. Overexpression of human mutant alpha-synuclein (Ala(53)Thr) selectively induced apoptotic programmed cell death of primary dopamine neurons as well as N27 cells. The mutant protein also potentiated the neurotoxicity of 6-hydroxydopamine (6-OHDA). By contrast, overexpression of wild-type human alpha-synuclein was not directly neurotoxic but did increase cell death after 6-OHDA. Overexpression of wild-type rat alpha-synuclein had no effect on dopamine cell survival or 6-OHDA neurotoxicity. These results indicate that overexpression of human mutant alpha-synuclein directly leads to dopamine neuron death, and overexpression of either human mutant or human wild-type alpha-synuclein renders dopamine neurons more vulnerable to neurotoxic insults.

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.

High Doses of Multiple Antioxidant Vitamins: Essential Ingredients in Improving the Efficacy of Standard Cancer Therapy

Numerous articles and several reviews have been published on the role of antioxidants, and diet and lifestyle modifications in cancer prevention. However, the potential role of these factors in the management of human cancer have been largely ignored. Extensive in vitro studies and limited in vivo studies have revealed that individual antioxidants such as vitamin A (retinoids), vitamin E (primarily alpha-tocopheryl succinate), vitamin C (primarily sodium ascorbate) and carotenoids (primarily polar carotenoids) induce cell differentiation and growth inhibition to various degrees in rodent and human cancer cells by complex mechanisms. The proposed mechanisms for these effects include inhibition of protein kinase C activity, prostaglandin E1-stimulated adenylate cyclase activity, expression of c-myc, H-ras, and a transcription factor (E2F), and induction of transforming growth factor-beta and p21 genes. Furthermore, antioxidant vitamins individually or in combination enhance the growth-inhibitory effects of x-irradiation, chemotherapeutic agents, hyperthermia, and biological response modifiers on tumor cells, primarily in vitro. These vitamins, individually, also reduce the toxicity of several standard tumor therapeutic agents on normal cells. Low fat and high fiber diets can further enhance the efficacy of standard cancer therapeutic agents; the proposed mechanisms for these effects include the production of increased levels of butyric acid and binding of potential mutagens in the gastrointestinal tract by high fiber and reduced levels of growth promoting agents such as prostaglandins, certain fatty acids and estrogen by low fat. We propose, therefore, a working hypothesis that multiple antioxidant vitamin supplements together with diet and lifestyle modifications may improve the efficacy of standard and experimental cancer therapies.

Morphological and biochemical study in X-ray- and dibutyryl cyclic AMP-induced differentiated neuroblastoma cells☆

Abstract X-irradiation or dibutyryl adenosine 3′:5′-cyclic monophosphate (DBcAMP) induced axon formation in mouse neuroblastoma cells in vitro. The differentiated cells showed morphological maturation, as shown by an increase in the cellular and nuclear size. Neuroblastoma cells in the presence of DBcAMP grew at a slightly reduced rate for 2 days; however, at day 3 they reached plateau phase. The removal of DBcAMP and readdition of fresh growth medium at day 4 did not cause renewal of cell division, indicating that morphological cell differentiation for the most part was irreversible. Na-butyrate, 3′5′ cyclic AMP, 5′ AMP, ATP, ADP, 3′5′ cyclic guanosine monophosphate caused inhibition of cell growth but did not produce morphological differentiation. Acetylcholinesterase activity in both the X-ray- and DBcAMP-induced differentiated cells increased by a factor of 10. Na-butyrate and 3′5′ cyclic AMP which inhibited cell division but did not cause morphological differentiation also increased AChE activity to a similar extent.

Inhibitors of cyclic-nucleotide phosphodiesterase induce morphological differentiation of mouse neuroblastoma cell culture☆

Abstract Inhibitors of phosphodiesterase, papaverine, 4-(3-butoxy-4-methoxybenzyl)-2 imidazolidinone (RO 20-1724) and 4-(3,4-dimethoxybenzyl)-2-imidazolidinone (RO-7-2956) induce morphological differentiation of mouse neuroblastoma cells in culture as shown by the formation of axon-like processes. These differentiated cells showed morphological maturation as revealed by an increase in the size of soma and nucleus. On removal of papaverine and re-addition of fresh growth medium 1 day after treatment, the morphological differentiation was reversible; however, when the drug was removed 3 days after treatment, the morphological differentiation for the most part was irreversible. Although a maximal differentiation of cells was seen 24 h after papaverine treatment, a maximal inhibition of cell division was observed 2 days after treatment. This observation further supports the hypothesis that the inhibition of cell division may be secondary to the induction of differentiation.

Modification of the effect of tamoxifen, cis‐platin, DTIC, and interferon‐α2b on human melanoma cells in culture by a mixture of vitamins

The effect of a mixture of vitamins in modifying the efficacy of commonly used drugs in the treatment of human melanoma has not been studied. Vitamin C and d-alpha-tocopheryl succinate (alpha-TS) alone reduced the growth of human melanoma (SK-30) cells in culture, whereas beta-carotene (BC), 13-cis-retinoic acid (RA), or sodium selenite alone was ineffective. RA caused morphological changes, as evidenced by flattening of cells and formation of short cytoplasmic processes. A mixture of four vitamins (vitamin C, BC, alpha-TS, and RA) was more effective in reducing growth of human melanoma cells than a mixture of three vitamins. The growth-inhibitory effect of cis-platin, decarbazine, tamoxifen, and recombinant interferon-alpha 2b was enhanced by vitamin C alone, a mixture of three vitamins (BC, alpha-TS, and RA), and a mixture of four vitamins (vitamin C, BC, alpha-TS, and RA) that contained 50 micrograms/ml of vitamin C. These data show that a mixture of three or four vitamins can enhance the growth-inhibitory effect of currently used chemotherapeutic agents on human melanoma cells.

ALUMINUM-INDUCED OXIDATIVE EVENTS IN CELL LINES: GLIOMA ARE MORE RESPONSIVE THAN NEUROBLASTOMA

Aluminum, a trivalent cation unable to undergo redox reactions, has been linked to many diseases such as dialysis dementia and microcytic anemia without iron deficiency. It has also been implicated in Alzheimers disease although this is controversial. Because cell death due to oxidative injury is suspected to be a contributory factor in many neurological diseases and aluminum neurotoxicity, glioma (C-6) and neuroblastoma (NBP2) cells were utilized to assess early changes in oxidative parameters consequent to a 48-h exposure to aluminum sulfate. A 500-microM concentration of this salt produced a significant increase in reactive oxygen species (ROS) production and a significant decrease in glutathione (GSH) content in glioma cells. However, the same concentration of the aluminum salt did not lead to any significant changes in the neuroblastoma cells. Mitochondrial respiratory activity in glioma cells was also found to be significantly higher in the aluminum treated cells. As judged by morin-metal complex formation, aluminum can enter glioma cells much more readily than neuroblastoma cells. Thus, it is possible that the cerebral target following an acute exposure to aluminum may be glial rather than neuronal.