Induction of cancer cell apoptosis by alpha-tocopheryl succinate: molecular pathways and structural requirements.

The vitamin E analog alpha-tocopheryl succinate (alpha-TOS) can induce apoptosis. We show that the proapoptotic activity of alpha-TOS in hematopoietic and cancer cell lines involves inhibition of protein kinase C (PKC), since phorbol myristyl acetate prevented alpha-TOS-triggered apoptosis. More selective effectors indicated that alpha-TOS reduced PKCalpha isotype activity by increasing protein phosphatase 2A (PP2A) activity. The role of PKCalpha inhibition in alpha-TOS-induced apoptosis was confirmed using antisense oligonucleotides or PKCalpha overexpression. Gain- or loss-of-function bcl-2 mutants implied modulation of bcl-2 activity by PKC/PP2A as a mitochondrial target of alpha-TOS-induced proapoptotic signals. Structural analogs revealed that alpha-tocopheryl and succinyl moieties are both required for maximizing these effects. In mice with colon cancer xenografts, alpha-TOS suppressed tumor growth by 80%. This epitomizes cancer cell killing by a pharmacologically relevant compound without known side effects.

Minute oxidative stress is sufficient to induce apoptotic death of NIT-1 insulinoma cells.

When cultured NIT-1 cells were subjected to a low level of oxidative stress (30 microM hydrogen peroxide for 15 min at 37 degrees C) several of their lysosomes ruptured, as demonstrated by intravital staining with the lysosomotropic weak base acridine orange. Such rupture is due to intralysosomal, iron-catalyzed oxidative reactions, since it was largely prevented by previous endocytotic uptake of desferrioxamine. The resultant limited leakage of lysosomal hydrolytic enzymes into the cytosol could be important for an apoptotic-type degradation/fragmentation process within initially intact plasma membranes. In contrast, extensive lysosomal rupture leads to necrosis. The development of the damage process was followed by light- and electron microscopy; and by the TUNEL-reaction. As a result of the applied oxidative stress, which is comparable to that expected to occur within the microenvironment surrounding activated macrophages under oxidative burst (e.g. during autoimmune insulitis), about 90% of the cells eventually died due to post-apoptotic secondary necrosis. The few surviving cells phagocytosed the debris from their fragmented neighbours and began to divide about 24 h after the insult. Thus the sensitivity to oxidative stress varies, perhaps as a consequence of varying amounts of intralysosomal redox-active iron, as we have found to be the case in several other cellular systems. Since the NIT-1 cells are highly differentiated, and in many ways like beta cells, we consider our result to be of value for the understanding of beta-cell death during the development of insulin-dependent (Type I) diabetes mellitus (IDDM).

Human islets in mixed islet grafts protect mouse pancreatic beta-cells from alloxan toxicity.

We have previously shown that human beta-cells are resistant to the toxic effects of alloxan. In order to further clarify this characteristic of human islets, we investigated whether these cells might transfer their alloxan resistance to alloxan-sensitive rat or mouse islets. Islets from two species (human-mouse or rat-mouse) were mixed into one graft, which was implanted into the subcapsular kidney space of nude mice. Alloxan or saline was injected intravenously two weeks after implantation and one week thereafter the mice were killed. The number of grafted and endogenous beta-cells were evaluated by a semi-quantitative method after immunohistochemistry. Human islet production of the scavenging enzymes extracellular superoxide dismutase and plasma glutathione peroxidase were analyzed with ELISA-techniques, and mouse and human islet hydrogen peroxide breakdown activity were monitored with a horseradish peroxidase-dependent assay. Mouse beta-cells transplanted together with human islets were protected against alloxan cytotoxicity. Rat islets did not protect mouse beta-cells against alloxan, suggesting that the mixing procedure as such did not impose the protection. Production of extracellular superoxide dismutase and plasma glutathione peroxidase by human islets was very low. Moreover, H2O2 breakdown in vitro, did not differ between human and mouse islets. Alloxan-insensitive human islets protect mouse beta-cells against alloxan-induced lesions, suggesting that yet to be identified extracellular factors are involved in human islet resistance to alloxan toxicity.

Beta-cells, oxidative stress, lysosomal stability, and apoptotic/necrotic cell death.

Reactive oxygen intermediates (ROI) may be involved in the destruction of pancreatic beta-cells during the development of insulin-dependent diabetes mellitus (IDDM). To investigate the possible role of lysosomes in this process, normal mouse beta-cells were cultured as monolayers at D-glucose concentrations of 1.6 (pronounced crinophagy), 11 or 28 mM (minimal crinophagy), subjected to a low level of oxidative stress and returned to standard culture conditions. Some cultures were exposed to desferrioxamine (Des) before the oxidative stress. As a result of such stress, many of the cells' lysosomes ruptured with consequent apoptosis or necrosis. Cells kept at 1.6 mM glucose were rich in secretory granules, showed crinophagy/autophagy, were very sensitive to oxidative stress, and had the least stable lysosomes. Cells kept at 28 mM glucose did not show crinophagy, contained fewer secretory granules, were less sensitive to oxidative stress, and had more stable lysosomes. Des-treated cells behaved almost as cells not exposed to oxidative stress at all. The findings suggest that iron may occur together with zinc within the secretory granules and that it sensitizes crinophagic lysosomes to oxidative stress. The stress that was applied in this study may be comparable to what occurs within the vicinity of activated macrophages during autoimmune insulitis.

A short exposure to a high-glucose milieu stabilizes the acidic vacuolar apparatus of insulinoma cells in culture to ensuing oxidative stress.

It was recently suggested that extracellular hydrogen peroxide, after diffusing into and throughout adjacent cells--which may be the case if they have only a weak capacity to degrade hydrogen peroxide--labilizes their lysosomal compartment due to its content of low-molecular-weight iron in redox-active form. The iron would be present as a consequence of normal autophagocytotic degradation of various iron-containing metalloproteins. Beta- and insulinoma cells are especially vulnerable to oxidative stress, since they possess only low capacity to degrade hydrogen peroxide, and, perhaps, since they normally have a certain degree of autophagocytotic degradation of secretory granules with some iron content--crinophagy. The toxicity to beta cells of oxidative stress, such as an exposure to alloxan, that results in extracellular formation of hydrogen peroxide, is considerably reduced if animals are initially given an intravenous bolus dose of glucose, temporarily bringing up the blood level to about 20 mM. In this study it was demonstrated that already as short an exposure as 30 min to 20 mM D-glucose reduces the sensitivity of HIT and NIT insulinoma cells in culture to a subsequent exposure to hydrogen peroxide. In parallel, exposure to such a high-glucose medium also reduces their desferrioxamine-available amount of iron and, moreover, stabilizes their lysosomal membranes against oxidative stress--thus preventing diffusion to the cytosol of damaging lysosomal contents following iron-catalyzed, Fenton-type, intralysosomal reactions. We suggest that both general autophagocytotic turnover and, in particular, crinophagy of secretory granules are decreased by an increased glucose concentration of the surrounding milieu, with attendant reduced amounts of intralysosomal low-molecular-weight iron and, thus, diminished sensitivity to oxidative stress.

Fas receptor is expressed in human lung squamous cell carcinomas, whereas bcl-2 and apoptosis are not pronounced: a preliminary report.

We report a pilot study on the Fas receptor (APO-1, CD95) in vivo in 15 human squamous cell (non-small) carcinomas and ten normal bronchial specimens. The principal aim was to investigate whether the so-called death receptor, Fas, is present in these tumours. Ligation of Fas promptly induces apoptosis, particularly in T Jurkat cells in vitro, and expression of Fas on human cancer would thus theoretically be of great interest. The immunoreactivity for the anti-apoptotic protein Bcl-2 was also investigated, and the degree of apoptosis was evaluated by TdT dUTP nick end labelling (TUNEL) and conventional morphological criteria. Fas was present in all initial tumours but absent in control tissue, that is in the potential precursor cells of bronchial epithelium (P = 0.001). Fas was not detectable after radiotherapy (P = 0.03). We propose that radiotherapy induces an early selection of tumour cells rather than a down-regulation of Fas. Both Bcl-2 and apoptosis (TUNEL) were generally expressed at a modest level. In agreement with other studies, we did not find any significant correlation between Bcl-2 and prognosis, or between Bcl-2 and TUNEL. Hence, in this preliminary report, we have demonstrated Fas receptor in human squamous cell carcinomas in vivo. This is a novel finding, and the apparent absence of Fas after radiotherapy may have important therapeutic implications.

Starvation-induced autophagocytosis paradoxically decreases the susceptibility to oxidative stress of the extremely oxidative stress-sensitive NIT insulinoma cells.

Glucose and amino acid starvation of cells in culture generally enhances their sensitivity to oxidative stress. This is explained by compensatory autophagocytosis, which results in increased amounts of lysosomal low-molecular-weight, redox-active iron, due to the degradation of metallo-proteins, with a potential increase in iron-catalyzed, intralysosomal oxidative reactions. Such reactions diminish the stability of lysosomal membranes, with resultant leakage of hydrolytic enzymes into the cytosol and ensuing cellular degeneration, often of apoptotic type. However, starvation of NIT insulinoma cells, which are normally remarkably sensitive to oxidative stress, actually attenuated the sensitivity to such stress. We found that starved NIT cells rapidly synthesized ferritin. Moreover, ferritin was found to be autophagocytosed, and the lysosomes were stabilized, as assayed by the acridine orange relocation test. We hypothesize that compensatory autophagocytosis during starvation increases the cytosolic pool of redox-active iron, as a reflection of enhanced transportation of low-molecular-weight iron from autophagic lysosomes to the cytosol, resulting in ferritin induction. The newly formed ferritin would, in turn, become autophagocytosed and bind redox-active lysosomal iron in a non-redox-active form. We also suggest that the proposed mechanism may be a way for oxidative stress-sensitive cells to compensate partly for their failing capacity to degrade hydrogen peroxide before it leaks into the acidic vacuolar apparatus and induces intralysosomal oxidative stress. The insulin-producing beta cell may belong to this type of cells.

Starvation-induced autophagocytosis enhances the susceptibility of insulinoma cells to oxidative stress.

We have previously shown insulinoma (HIT-T15 and RINm5F) cells in culture to be very sensitive, in comparison with a reference cell line (J-774), to the oxidative stress that is created when alloxan reacts extracellularly with reducing agents, forming superoxide and hydrogen peroxide. The toxic effects are prevented by catalase added to the medium, suggesting that alloxan does not need to be taken up in order to affect cells. Rather, alloxan seems to exert its action through extracellular formation of hydrogen peroxide that influences the stability of the cells' lysosomes following diffusion into them. To further analyse the mechanisms in operation, we studied the influence of induced autophagocytosis on the sensitivity to ensuing oxidative stress. Starvation for 60-120 min in PBS at 37°C markedly enhanced autophagocytosis and, in parallel, increased the cytotoxic effect and lysosomal vulnerability of ensuing exposure to hydrogen peroxide, while not significantly changing the antioxidative status or the energy balance. Autophagocytosis increased the size of the intralysosomal pool of reactive, low-molecular-weight, iron, probably by degradation of metallo-proteins, as shown by autometallography and HPLC demonstration of desferrioxamine-reactive intracellular iron. Moreover, exposure to the iron-chelator desferrioxamine before treatment with hydrogen peroxide prevented lysosomal destabilization and cellular death of both starved and control cells, further proving the importance of intralysosomal iron for the response to oxidative stress. We hypothesize that ß-cells which, like insulinoma cells, have a weak antioxidative defence system under conditions of enhanced general autophagocytosis, or crinophagy, might become vulnerable to even low, or moderate, oxidative stress.