Epipharyngeal Abrasive Therapy Down-regulates the Expression of Cav1.2: A Key Molecule in Influenza Virus Entry.

BACKGROUND/AIM: Influenza A virus (IAV) infection causes an inflammatory response to the respiratory mucosa. The viral glycoprotein hemagglutinin (HA) binds to the sialylated voltage-dependent Ca2+ channel (Cav1.2) in ciliated epithelium. The binding of HA and sialylated Cav1.2 is considered essential to IAV infection, entry, and IAV-induced Ca2+ oscillation. The epipharynx comprises the ciliated epithelium, which is the initial target for viruses that cause upper respiratory tract infections. Previously, we showed that epipharyngeal abrasive therapy (EAT), a treatment for chronic epipharyngitis in Japan, which scratches the epipharyngeal mucosa with a cotton swab containing zinc chloride, induces squamous metaplasia. In this study, we evaluated whether squamous metaplasia by EAT affects the expression patterns of Cav1.2. PATIENTS AND METHODS: The study subjects were seven patients who had not been treated with EAT and 11 patients who had. For the immunohistochemical assessment of the epipharyngeal mucosa, the staining intensity of Cav1.2 was described using the immunohistochemical score (IHC score). RESULTS: The IHC scores for Cav1.2 in the EAT-treated group was 4.19-fold lower than those in the non-treated group (p=0.0034). CONCLUSION: EAT down-regulates the expression of Cav1.2, a key cell surface molecule in influenza virus entry via squamous metaplasia. Thus, EAT may be a simple method for preventing influenza infection.

Epipharyngeal Abrasive Therapy (EAT) Reduces the mRNA Expression of Major Proinflammatory Cytokine IL-6 in Chronic Epipharyngitis.

The epipharynx, located behind the nasal cavity, is responsible for upper respiratory tract immunity; however, it is also the site of frequent acute and chronic inflammation. Previous reports have suggested that chronic epipharyngitis is involved not only in local symptoms such as cough and postnasal drip, but also in systemic inflammatory diseases such as IgA nephropathy and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and Long COVID. Epipharyngeal Abrasive Therapy (EAT), which is an effective treatment for chronic epipharyngitis in Japan, is reported to be effective for these intractable diseases. The sedation of chronic epipharyngitis by EAT induces suppression of the inflammatory cytokines and improves systemic symptoms, which is considered to be one of the mechanisms, but there is no report that has proved this hypothesis. The purpose of this study was to clarify the anti-inflammatory effect of EAT histologically. The study subjects were 8 patients who were not treated with EAT and 11 patients who were treated with EAT for chronic epipharyngitis for 1 month or more. For immunohistochemical assessment, the expression pattern of IL-6 mRNA, which plays a central role in the human cytokine network, was analyzed using in situ hybridization. The expression of IL-6 in the EAT-treated group was significantly lower than those in the EAT nontreated group (p = 0.0015). In addition, EAT suppressed the expression of tumor necrosis factor alpha (TNFα), a crucial proinflammatory cytokine. As a result, continuous EAT suppressed submucosal cell aggregation and reduced inflammatory cytokines. Thus, EAT may contribute to the improvement of systemic inflammatory diseases through the suppression of IL-6 expression.

Epipharyngeal Abrasive Therapy (EAT) Has Potential as a Novel Method for Long COVID Treatment.

COVID-19 often causes sequelae after initial recovery, referred to collectively as long COVID. Long COVID is considered to be caused by the persistence of chronic inflammation after acute COVID-19 infection. We found that all long COVID patients had residual inflammation in the epipharynx, an important site of coronavirus replication, and some long COVID symptoms are similar to those associated with chronic epipharyngitis. Epipharyngeal abrasive therapy (EAT) is a treatment for chronic epipharyngitis in Japan that involves applying zinc chloride as an anti-inflammatory agent to the epipharyngeal mucosa. In this study, we evaluated the efficacy of EAT for the treatment of long COVID. The subjects in this study were 58 patients with long COVID who were treated with EAT in the outpatient department once a week for one month (mean age = 38.4 ± 12.9 years). The intensities of fatigue, headache, and attention disorder, which are reported as frequent symptoms of long COVID, were assessed before and after EAT using the visual analog scale (VAS). EAT reduced inflammation in the epipharynx and significantly improved the intensity of fatigue, headache, and attention disorder, which may be related to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). These results suggest that EAT has potential as a novel method for long COVID treatment.

Mitochondrial translocation of p53 mediates release of cytochrome c and hippocampal CA1 neuronal death after transient global cerebral ischemia in rats.

Although p53 is a key modulator of cellular stress responses, the mechanism of p53-mediated apoptosis is ambiguous. p53 can mediate apoptosis in response to death stimuli by transcriptional activation of proapoptotic genes and transcriptional-independent mechanisms. Recent studies have shown that the p53 protein can directly induce permeabilization of the outer mitochondrial membrane by forming a inhibitory complex with a protective Bcl-2 family protein, resulting in cytochrome c release. However, how the mitochondrial p53 pathway mediates neuronal apoptosis after cerebral ischemia remains unclear. We examined the interaction between the mitochondrial p53 pathway and vulnerable hippocampal CA1 neurons in rats using a transient global cerebral ischemia (tGCI) model. Western blot analysis and immunofluorescent staining revealed mitochondrial p53 translocation after tGCI in the hippocampal CA1 neurons. Coimmunoprecipitation revealed that translocated p53 bound to Bcl-X(L) in the mitochondrial fraction. To examine the effect of a specific p53 inhibitor on the mitochondrial p53 pathway and apoptotic cell death after tGCI, we intravenously administered pifithrin-alpha (PFT). Mitochondrial p53 translocation and interaction between p53 and Bcl-X(L) were prevented by treatment with PFT. Moreover, cytochrome c release from mitochondria and subsequent apoptotic CA1 neuronal death were decreased with PFT treatment. These results suggest that the mitochondrial p53 pathway is one of the novel mechanisms mediating delayed death of vulnerable hippocampal CA1 neurons after tGCI.

Overexpression of SOD1 in transgenic rats attenuates nuclear translocation of endonuclease G and apoptosis after spinal cord injury.

Spinal motor neurons are selectively vulnerable after spinal cord injury (SCI). Recent studies suggest they undergo apoptosis after caspase activation through a mitochondria-dependent apoptosis pathway, and that oxidative stress after SCI is likely to play a role. However, other signaling pathways of apoptosis that involve mitochondria have not been thoroughly studied after SCI. Apoptosis-inducing factor (AIF) and endonuclease G (EndoG) are mitochondrial apoptogenic proteins that are capable of inducing neuronal apoptosis when translocated from mitochondria to nuclei through a caspase-independent pathway. In this study, we examined translocation of these proteins and apoptotic cell death of motor neurons. The role of oxidative stress was also studied using transgenic (Tg) rats that overexpress the intrinsic antioxidant copper/zinc-superoxide dismutase (SOD1). Western blots and an activity assay demonstrated that a greater amount of SOD1 and higher activity of SOD presented in mitochondria of Tg rats compared with wild-type (Wt) rats. Immunohistochemistry and Western blots showed translocation of EndoG and AIF from mitochondria to nuclei in motor neurons 1 day after SCI in both groups of rats. However, there was significantly less translocation of EndoG in the Tg rats compared with the Wt rats. Less apoptotic cell death was detected in the Tg rats than in the Wt rats 3 days after SCI. These results suggest that translocation of EndoG and AIF from mitochondria to nuclei may initiate a caspase-independent pathway of apoptosis. An increased level of SOD1 in mitochondria conceivably reduces oxidative stress, thereby attenuating EndoG translocation, and resulting in reduction of caspase-independent apoptosis.

Interferon-beta fails to protect in a model of transient focal stroke.

BACKGROUND AND PURPOSE: Compelling evidence supporting the role of inflammation in the development of cerebral infarction has focused attention on the potential of antiinflammatory treatment strategies for stroke. Interferon (IFN)-beta, an immunomodulatory agent approved for treatment of multiple sclerosis, is being evaluated in a phase I clinical trial in acute ischemic stroke. In the present study, we evaluated the effects of wild-type rat IFN-beta and its pegylated counterpart (PEG-IFN-beta) in a model of focal ischemia and reperfusion. METHODS: After 60 minutes of middle cerebral artery occlusion, rats (n=12/group) were treated with IV tail injections of 8 or 16 mug of IFN-beta in 300 muL of PBS once daily for 3 or 7 days or with IV or SC injections of PEG-IFN-beta for 1 day. The animals were assessed daily for weight and for neurological findings. Additional animals underwent complete hematology and chemistry profiles, as well as complete multiorgan necropsy studies. All of the brain tissue was evaluated for assessment of infarct areas, neutrophil infiltration, and presence of hemorrhagic transformations. RESULTS: IFN-beta and PEG-IFN-beta failed to protect against experimental ischemic brain injury as assessed by histopathology and neurological outcome. Furthermore, IFN-beta treatment was associated with significant weight loss and alterations in hematology and chemistry profiles. CONCLUSIONS: Our results suggest that additional preclinical studies are warranted.

Activation of the Akt/GSK3beta signaling pathway mediates survival of vulnerable hippocampal neurons after transient global cerebral ischemia in rats.

Recent studies have revealed that the phosphatidylinositol 3-kinase (PI3-K) pathway is involved in apoptotic cell death after experimental cerebral ischemia. The serine-threonine kinase, Akt, functions in the PI3-K pathway and prevents apoptosis by phosphorylation at Ser473 after a variety of cell death stimuli. After phosphorylation, activated Akt inactivates other apoptogenic factors, including glycogen synthase kinase-3beta (GSK3beta), thereby inhibiting cell death. However, the role of Akt/GSK3beta signaling in the delayed death of hippocampal neurons in the CA1 subregion after transient global cerebral ischemia (tGCI) has not been clarified. Transient global cerebral ischemia for 5 mins was induced by bilateral common carotid artery occlusion combined with hypotension. Western blot analysis showed a significant increase in phospho-Akt (Ser473) and phospho-GSK3beta (Ser9) in the hippocampal CA1 subregion after tGCI. Immunohistochemistry showed that expression of phospho-Akt (Ser473) and phospho-GSK3beta (Ser9) was markedly increased in the vulnerable CA1 subregion, but not in the ischemic-tolerant CA3 subregion. Double staining with phospho-GSK3beta (Ser9) and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed different cellular distributions in the CA1 subregion 3 days after tGCI. Phosphorylation of Akt and GSK3beta was prevented by LY294002, a PI3-K inhibitor, which facilitated subsequent DNA fragmentation 3 days after tGCI. Moreover, transgenic rats that overexpress copper/zinc-superoxide dismutase, which is known to be neuroprotective against delayed hippocampal CA1 injury after tGCI, had enhanced and persistent phosphorylation of both Akt and GSK3beta after tGCI. These findings suggest that activation of the Akt/GSK3beta signaling pathway may mediate survival of vulnerable hippocampal CA1 neurons after tGCI.

Modulation of proline-rich akt substrate survival signaling pathways by oxidative stress in mouse brains after transient focal cerebral ischemia.

BACKGROUND AND PURPOSE: A proline-rich Akt substrate (PRAS) contributes to the regulation of apoptosis after a variety of cell death stimuli, as well as in an in vivo transient focal cerebral ischemia (tFCI) model. We reported previously that overexpression of copper/zinc-superoxide dismutase (SOD1) reduces apoptotic cell death after tFCI. Our present study was designed to clarify the relationship between the PRAS signaling pathway and oxidative stress in the regulation of apoptosis after tFCI. METHODS: We used a tFCI model with SOD1 transgenic mice and wild-type littermates to examine the expression of phosphorylated PRAS (pPRAS) by Western blotting and immunohistochemistry and the interaction of pPRAS with phosphorylated Akt (pPRAS/pAkt) or the 14-3-3 protein (pPRAS/14-3-3) by coimmunoprecipitation. Direct oxidation of the carbonyl groups, an indication of oxidative injury to total and individual proteins caused by tFCI, was examined using a 2,4-dinitrophenylhydrazone reaction assay. RESULTS: Expression of pPRAS, pPRAS/pAkt, and pPRAS/14-3-3 decreased 2 hours after tFCI. Oxidized hydroethidine did not colocalize with expression of pPRAS. Individual oxidized carbonyls in pPRAS remarkably increased 2 hours after tFCI but were significantly reduced by SOD1 2 hours after tFCI. Expression of pPRAS, pPRAS/pAkt, and pPRAS/14-3-3 was promoted by SOD1 during the same time course. CONCLUSIONS: These results suggest that overexpression of SOD1 may affect the PRAS pathway after tFCI by reducing the direct oxidative reaction to pPRAS after reperfusion injury.

Epo protects SOD2-deficient mouse astrocytes from damage by oxidative stress.

Erythropoietin (Epo) expression, which regulates erythropoiesis, has been shown in rat and mouse brain after hypoxia. A previous study from our laboratory showed that astrocytes from manganese-superoxide dismutase (SOD2) homozygous knockout (SOD2(-/-)) mice can survive under 5% O(2), but not under normal aerobic conditions. However, the mechanism involved is not clear. Our preliminary study using reverse transcriptase-polymerase chain reaction showed increased Epo mRNA expression in astrocytes cultured with 5% hypoxia compared with astrocytes under normal conditions. After administration of anti-sense Epo, protection decreased with time. Dose-dependent administration of Epo to SOD2(-/-) mouse astrocytes improved their survivability under normal conditions. Survivability of heterozygous SOD2(-/+) mutant and wild-type mouse astrocyte cultures was the same under normal conditions but, after administration of 2 mM of paraquat, a reactive oxygen species generator, survivability of the SOD2(-/+) astrocytes decreased remarkably compared with the wild-type cells. Epo administration 24 h before exposure to paraquat significantly improved the survivability of the SOD2(-/+) astrocytes. Western blot studies suggest that Jak-Stat signal transduction pathways are involved in this process. Our study demonstrates an important role for Epo in the protection of astrocytes from reactive oxygen species. We suggest that Epo can compensate in part for the antioxidant properties of mitochondrial SOD2 deficiency.

Oxidative stress and neuronal death/survival signaling in cerebral ischemia.

It has been demonstrated by numerous studies that apoptotic cell death pathways are implicated in ischemic cerebral injury in ischemia models in vivo. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and the numerous reports suggest the involvement of cell survival/death signaling pathways in the pathogenesis of apoptotic cell death in ischemic lesions. In these models, reoxygenation during reperfusion provides oxygen as a substrate for numerous enzymatic oxidation reactions and for mitochondrial oxidative phosphorylation to produce adenosine triphosphate. Oxygen radicals, the products of these biochemical and physiological reactions, are known to damage cellular lipids, proteins, and nucleic acids and to initiate cell signaling pathways after cerebral ischemia. Genetic manipulation of intrinsic antioxidants and factors in the signaling pathways has provided substantial understanding of the mechanisms involved in cell death/survival signaling pathways and the role of oxygen radicals in ischemic cerebral injury. Future studies of these pathways could provide novel therapeutic strategies in clinical stroke.

Superoxide dismutase 1 overexpression reduces MCP-1 and MIP-1 alpha expression after transient focal cerebral ischemia.

Proinflammatory cytokines and chemokines are quickly upregulated in response to ischemia/reperfusion (I/R) injury; however, the relationship between I/R-induced oxidative stress and cytokine/chemokine expression has not been elucidated. We investigated the temporal profile of cytokine and chemokine gene expression in transient focal cerebral ischemia using complementary DNA array technology. Among 96 genes studied, 10, 4, 11, and 5 genes were increased at 6, 12, 24, and 72 h of reperfusion, respectively, whereas, 4, 11, 8, and 21 genes, respectively, were decreased. To clarify the relationship between chemokines and oxidative stress, we compared the gene and protein expression of monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) in wild-type (WT) mice and copper/zinc-superoxide dismutase (SOD 1) transgenic (Tg) mice. Monocyte chemoattractant protein-1 and MIP-1 alpha mRNA were significantly upregulated at 6 to 12 h of reperfusion. In the SOD 1 Tg mice, however, MCP-1 and MIP-1 alpha mRNA expression was significantly decreased 12 h postinsult. In the WT mice, MCP-1 and MIP-1 alpha protein expression peaked 24 h after onset of reperfusion determined by immunohistochemistry. In the SOD 1 Tg mice, MCP-1 and MIP-1 alpha immunopositive cells were reduced, as were concentrations of these proteins (measured by enzyme-linked immunosorbent assay) at 24 h of reperfusion. Our results suggest that MCP-1 and MIP-1 alpha expression is influenced by I/R-induced oxidative stress after transient focal stroke.

Modulation of p53 degradation via MDM2-mediated ubiquitylation and the ubiquitin-proteasome system during reperfusion after stroke: role of oxidative stress.

The tumor suppressor gene p53 plays an important role in the regulation of apoptosis through transcriptional activation of cell cycle control. Degradation of p53 hinders its role in apoptosis regulation. Recent studies have shown that MDM2-mediated ubiquitylation and the ubiquitin-proteasome system are critical regulating systems of p53 ubiquitylation. However, the mechanism regulating p53-mediated neuronal apoptosis after cerebral ischemia remains unknown. We examined the MDM2 pathway and the ubiquitin-proteasome system using a transient focal cerebral ischemia (tFCI) model and analyzed the interaction between p53 regulation and superoxide using copper/zinc superoxide dismutase (SOD1) transgenic mice after tFCI. p53 degradation and ubiquitylation were detected after tFCI. The accumulation of ubiquitylated p53 was inhibited and p53 degradation was facilitated by SOD1. Nuclear translocation and MDM2/Akt interaction were detected after tFCI and were inhibited by phosphatidylinositol 3-kinase inhibition and promoted by SOD1. Cytosolic translocation of the p53/MDM2 complex was detected after tFCI and was promoted by SOD1. Moreover, accumulation of multiubiquitin chains and direct oxidative injury to a proteasome were detected and inhibited by SOD1 after tFCI. These results suggest that SOD1 promotes the MDM2 pathway and the ubiquitin-proteasome system after tFCI and that production of reactive oxygen species after tFCI prevents p53 degradation by inhibiting both systems.

Oxidative stress affects the integrin-linked kinase signaling pathway after transient focal cerebral ischemia.

BACKGROUND AND PURPOSE: The integrin-linked kinase (ILK) signaling pathway contributes to regulation of cellular adhesion, migration, and differentiation, and to apoptotic cell death after a variety of cell death stimuli. We have reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduces apoptotic cell death by promoting the phosphatidylinositol 3-kinase (PI3-K)/Akt survival pathway after transient focal cerebral ischemia (tFCI). However, the role of the ILK pathway after tFCI and the role of oxygen free radicals in regulation of apoptosis remain unclear. METHODS: To clarify these issues, we used an in vivo tFCI model with SOD1 transgenic mice and wild-type mice. We administered the PI3-K inhibitor, LY294002, into mouse brains after tFCI and examined the role of PI3-K in the ILK pathway and expression of the ILK/Akt complex by immunohistochemistry, Western blot analysis, and coimmunoprecipitation. RESULTS: A transient increase in ILK was detected early after tFCI and was prevented by treatment with LY294002, but promoted by SOD1. Coimmunoprecipitation revealed that the direct reaction of ILK/Akt transiently increased concurrent with the increase in ILK after tFCI. Moreover, the ILK/Akt complex was prevented by LY294002, but promoted by SOD1. CONCLUSIONS: These results suggest that the ILK pathway mediated by PI3-K is affected by tFCI and by SOD1.

Modulation of the Omi/HtrA2 signaling pathway after transient focal cerebral ischemia in mouse brains that overexpress SOD1.

Omi/HtrA2 is a novel protein that contributes to the regulation of mitochondrial apoptosis after a variety of cell death stimuli in vitro and is thought to negatively control the inhibitor-of-apoptosis protein (IAP) family. However, the Omi/HtrA2 pathway remains unknown in apoptotic neuronal cell death in vivo. To examine the role of the Omi/HtrA2 pathway and its relationship to oxidative stress after reperfusion following cerebral ischemia, we used a transient focal cerebral ischemia (tFCI) model in copper/zinc-superoxide dismutase (SOD1) transgenic mice and wild-type mice. We evaluated the link between the Omi/HtrA2 pathway and the caspase cascade reaction after tFCI by administration of a pan-caspase inhibitor, Z-VAD-FMK. We observed the time-dependent expression of Omi/HtrA2 and its binding to X-chromosome-linked IAP (Omi/XIAP) by immunohistochemistry, Western blotting and coimmunoprecipitation. Translocation of Omi/HtrA2 into the cytosolic space was detected during the early period after tFCI and was not affected by Z-VAD-FMK administration, but it was prevented by SOD1 overexpression. Coimmunoprecipitation revealed that Omi/XIAP transiently increased and that it was prevented by SOD1 overexpression. These results suggest that the Omi/HtrA2 pathway may play an important role in the progress of apoptotic neuronal cell death and that overexpression of SOD1 may attenuate this apoptotic cell death by preventing the Omi/HtrA2 cell signaling pathway.

Oxidative stress is associated with XIAP and Smac/DIABLO signaling pathways in mouse brains after transient focal cerebral ischemia.

BACKGROUND AND PURPOSE: The interaction of X chromosome-linked inhibitor-of-apoptosis protein (XIAP) with second mitochondria-derived activator of caspase (Smac)/direct inhibitor-of-apoptosis protein-binding protein with low pI (DIABLO) contributes to regulation of apoptosis after a variety of cell death stimuli, and in our reported in vivo transient focal cerebral ischemia (tFCI) model. We have also reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduces apoptotic cell death after tFCI. Our present study was designed to clarify the relationship between the XIAP signaling pathway and oxidative stress in the regulation of apoptosis after tFCI. METHODS: We used a tFCI model of SOD1 transgenic mice and wild-type littermates to examine the expression of XIAP and Smac/DIABLO by Western blotting and the interaction of XIAP with Smac/DIABLO (XIAP/Smac) or caspase-9 (XIAP/caspase-9) by coimmunoprecipitation. The direct oxidation of carbonyl groups, an indication of oxidative injury to total and individual proteins caused by tFCI, was examined using a 2,4-dinitrophenylhydrazone reaction assay. RESULTS: Direct oxidative injury to cytosolic and mitochondrial proteins was reduced by SOD1 after tFCI. The individual oxidized carbonyls in XIAP, mitochondrial Smac/DIABLO, and caspase-9 were also reduced by SOD1. Expression of XIAP and XIAP/caspase-9 was promoted, whereas translocation of Smac/DIABLO and XIAP/Smac was reduced, by SOD1 after tFCI. CONCLUSIONS: These results suggest that overexpression of SOD1 may affect the XIAP pathway after tFCI by reducing the direct oxidative reaction to XIAP regulators after reperfusion injury.

Oxidative damage to the endoplasmic reticulum is implicated in ischemic neuronal cell death.

The endoplasmic reticulum (ER), which plays important roles in apoptosis, is susceptible to oxidative stress. Because reactive oxygen species (ROS) are robustly produced in the ischemic brain, ER damage by ROS may be implicated in ischemic neuronal cell death. We induced global brain ischemia on wild-type and copper/zinc superoxide dismutase (SOD1) transgenic rats and compared ER stress and neuronal damage. Phosphorylated forms of eukaryotic initiation factor 2 alpha (eIF2 alpha) and RNA-dependent protein kinase-like ER eIF2 alpha kinase (PERK), both of which play active roles in apoptosis, were increased in hippocampal CA1 neurons after ischemia but to a lesser degree in the transgenic animals. This finding, together with the finding that the transgenic animals showed decreased neuronal degeneration, indicates that oxidative ER damage is involved in ischemic neuronal cell death. To elucidate the mechanisms of ER damage by ROS, we analyzed glucose-regulated protein 78 (GRP78) binding with PERK and oxidative ER protein modification. The proteins were oxidatively modified and stagnated in the ER lumen, and GRP78 was detached from PERK by ischemia, all of which were attenuated by SOD1 overexpression. We propose that ROS attack and modify ER proteins and elicit ER stress response, which results in neuronal cell death.

Combined stimulation with interferon alpha and retinoic acid synergistically inhibits proliferation of the glioblastoma cell line GB12.

Since malignant glioma displays moderate resistance to conventional therapy, a new treatment modality is needed to improve the outcome of patients with these tumors. In this study, we examined whether combination stimulation with interferon alpha (IFN-alpha) and retinoic acid (RA) affected proliferation of the glioblastoma cell line GB 12 in vitro. Stimulation with IFN-alpha alone inhibited the GB 12 cell proliferation in a dose/time-dependent fashion, as assessed by WST-1 assay and uptake of 3H-thymidine, while RA limited it only slightly. The anti-proliferative action of IFN-alpha against glioblastoma cells was enhanced by the addition of RA. The IFN-alpha/RA combination also induced apoptosis in a substantial portion of the cells, compared with either reagent alone. Bcl-2 family proteins, regulating apoptosis, were altered by these stimuli: Bcl-2 was down-regulated, while Bax-alpha was up-regulated, especially by the combination. These findings suggest that the IFN-alpha/RA combination would synergistically affect glioblastoma cell growth, probably through apoptosis induction as well as a decreased cellular DNA synthesis.