Bipin Kumar

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.

Scientific Rationale for Using High-Dose Multiple Micronutrients as an Adjunct to Standard and Experimental Cancer Therapies

We have hypothesized that high-dose multiple micronutrients, including antioxidants, as an adjunct to standard (radiation therapy and chemotherapy) or experimental therapy (hyperthermia and immunotherapy), may improve the efficacy of cancer therapy by increasing tumor response and decreasing toxicity. Several in vitro studies and some in vivo investigations support this hypothesis. A second hypothesis is that antioxidants may interfere with the efficacy of radiation therapy and chemotherapy. This hypothesis is based on the concept that antioxidants will destroy free radicals that are generated during therapy, thereby protecting cancer cells against death. None of the published data on the effect of antioxidants in combination with radiation or chemotherapeutic agents on tumor cells supports the second hypothesis. Scientific rationale in support of a micronutrient protocol to be used as an adjunct to standard or experimental cancer therapy is presented.

D-Alpha-Tocopheryl Succinate (Vitamin E) Enhances Radiation-Induced Chromosomal Damage Levels in Human Cancer Cells, but Reduces it in Normal Cells

Objective: The purpose of this study was to measure and compare the effect of d-α-tocopheryl succinate (α-TS) in modifying radiation-induced chromosomal damage in human normal cells and cancer cells in culture. Methods: Three human normal fibroblast cell lines (GM2149, AG1522 and HF19) and three human cancer cell lines, cervical cancer (HeLa) and ovarian carcinoma cells (OVG1 and SKOV3) were treated with α-TS (37.6 μM) 20 hours before 100 cGy γ-irradiation. After 30 minutes of irradiation, colcemid was added and cells were fixed. One hundred randomly selected metaphase cells were scored for the presence of chromatid gaps and breaks. To study the cellular accumulation of α-TS, cells were incubated in the presence of α-TS (18.8 and 37.6 μM) for 24 hours, and α-TS was extracted with hexane using α-tocopheryl acetate as an internal standard. The levels of α-TS were determined by HPLC. Results: Results showed that α-TS induced chromosomal damage in both human cervical cancer cells and ovarian cancer cells, but not in human normal fibroblasts in culture. In addition, α-TS enhanced the level of radiation-induced chromosomal damage in cancer cells, but it protected normal cells against such damage. Both cancer cells and normal cells accumulated similar levels of α-TS, suggesting that increased sensitivity of cancer cells to α-TS is acquired during transformation. Conclusion: The use of α-TS during radiation therapy may improve the efficacy of radiation therapy by enhancing tumor response and decreasing some of the toxicities on normal cells.

Multiple antioxidants in the prevention and treatment of neurodegenerative disease: analysis of biologic rationale.

Parkinsons disease and Alzheimers disease are major progressive neurologic disorders, the risk of which increases with advancing age (65 years and over). In familial cases, however, early onset of disease (35-65 years) is observed. In spite of extensive basic and chemical research on Parkinsons disease and Alzheimers disease, no preventive or long-term effective treatment strategies are available. The analysis of existing literature suggests that oxidative stress is a major intermediary risk factor for the action of diverse groups of neurotoxins that are involved in these neurodegenerative diseases. In this review, it is proposed that the epigenetic components (mitochondria, other organelles, membranes, protein modification) rather than nuclear genes of neurons are the primary targets for the action of neurotoxins, including free radicals. In addition, a scientific rationale for using multiple antioxidants in clinical trials for the prevention of Parkinsons disease and Alzheimers disease among high-risk populations, and as an adjunct to standard therapy in the treatment of these diseases is presented.

Effects of altered cyclophilin A expression on growth and differentiation of human and mouse neuronal cells.

Abstract1. Cyclophilin A (CyP-A), a soluble cytoplasmic immunophilin, is known for its involvement in T cell differentiation and proliferation. Although CyP-A has a pivotal role in the immune response, it is most highly concentratedin brain, where its functions are largely unknown.2. We reported previously that a murine neuroblastoma (NB-P2) cellline can partially differentiate into neurons when treated with cyclosporin A (CyS-A), implicating a role for CyP-A in neuronal differentiation (Hovland et al. [1999]. Neurochem. Int. 3:229–235).3. The role of CyP-A in regulating neuronal growth and differentiation is not well defined. To investigate this, we first tested the utility of retroviral-mediated gene transfer and expression in human embryonic brain (HEB) and NB-P2 cells. Second, we examined the effects of retroviral-mediated overexpression or antisense-mediated reduction of CyP-A in HEB and NB-P2 cells.4. Our data show that retroviral vectors are efficient for stable gene transfer and expression in both cell lines. Moreover, neither overexpression nor reduction of CyP-A expression in NB-P2 cells altered the growth rate or induced differentiation. More importantly, the up- or down-regulation of CyP-A expressiondid not affect the magnitude of cAMP-induced NB-P2 differentiation. However, overexpression of CyP-A increased the growth rate of HEB cells.5. In summary, the utility of retroviral vectors for stable gene expression in human embryonic brain and murine neuroblastoma cells was shown. Furthermore,a novel role for CyP-A in augmenting the proliferation of human embryonic braincells was demonstrated in vitro.

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.

Differentiation Genes: Are They Primary Targets for Human Carcinogenesis?

In spite of extensive research in molecular carcinogenesis, genes that can be considered primary targets in human carcinogenesis remain to be identified. Mutated oncogenes or cellular growth regulatory genes, when incorporated into normal human epithelial cells, failed to Immortalize or transform these cells. Therefore, they may be secondary events In human carcinogenesis. Based on some experimental studies we have proposed that downregulation of a differentiation gene may be the Primary event in human carcinogenesis. Such a gene could be referred to as a tumor-initiating gene. Downregulation of a differentiation gene can be accomplished by a mutation in the differentiation gene, by activation of differentiation suppressor genes, and by inactivation of tumor suppressor genes. Downregulation of a differentiation gene can lead to immortalization of normal cells. Mutations in cellular proto-oncogenes, growth regulatory genes, and tumor suppressor genes in immortalized cells can lead to transformation. Such genes could be called tumor-promoting genes. This hypothesis can be documented by experiments published on differentiation of neuroblastoma (NB) cells in culture. The fact that terminal differentiation can be induced In NB cells by adenosine 3′,5′-cyclic monophosphate (cAMP) suggests that the differentiation gene in these cells Is not mutated, and thus can be activated by an appropriate agent. The fact that cAMP-resistant cells exist In NB cell populations suggests that a differentiation gene is mutated in these cancer cells, or that differentiation regulatory genes have become unresponsive to cAMP. In addition to cAMP, several other differentiating agents have been identified. Our proposed hypothesis of carcinogenesis can also be applied to other human tumors such as melanoma, pheochromocytoma, medulloblastoma, glioma, sarcoma, and colon cancer.