Effect of hyperbaric oxygen on post-stroke depression.

BACKGROUND: In patients with post-stroke depression (PSD) in diabetes, the situation may be more complex, requiring simultaneous treatment of blood glucose, depressive symptoms, and neurological dysfunction. Hyperbaric oxygen (HBO) therapy can improve tissue oxygen content and improve the situation of ischemia and hypoxia, thus playing a role in protecting brain cells and restoring the function of brain cells. However, there are few studies on HBO therapy for patients with PSD. This study explores the clinical efficacy of such therapy for stroke complicated with depression and diabetes mellitus, and to provide reference and basis for clinical treatment and development through the application of relevant rating scales and laboratory test indicators. AIM: To evaluate the clinical effects of HBO therapy on patients with diabetes with PSD. METHODS: A total of 190 diabetic patients with PSD were randomly divided into observation and control groups (95 patients per group). The control group received escitalopram oxalate 10mg once a day for eight weeks. In addition, the ob-servation group was also given HBO therapy, once a day, five times a week, for eight weeks. The Montgomery Depression Rating Scale (MADRS), National Institutes of Health Stroke Scale (NIHSS), hypersensitive C-reactive protein, tumor necrosis factor (TNF)-α, and fasting glucose levels were compared. RESULTS: There were no significant differences in age, sex, or depression course between the groups (P > 0.05). After HBO treatment, MADRS scores in both groups decreased significantly (14.3 ± 5.2), and were significantly lower in the control group (18.1 ± 3.5). After HBO treatment, NIHSS scores in both groups decreased significantly, and scores in the observation group (12.2 ± 4.0) decreased more than in the control group (16.1 ± 3.4), the difference was statistically significant (P < 0.001). The levels of hypersensitive C-reactive protein and TNF-α in both groups were significantly decreased, and the observation group was significantly lower than the control group (P < 0.001). Fasting blood glucose levels in both groups decreased significantly, and those in the observation group decreased more (8.02 ± 1.10) than in the control group (9.26 ± 1.04), with statistical significance (t = -7.994, P < 0.001). CONCLUSION: HBO therapy can significantly improve depressive symptoms and neurological dysfunction in patients with PSD, and reduce the levels of hypersensitive C-reactive protein, TNF-α and fasting blood glucose.

Electrical peripheral nerve stimulation relieves bone cancer pain by inducing Arc protein expression in the spinal cord dorsal horn.

OBJECTIVE: The analgesic effect on chronic pain of peripheral nerve stimulation (PNS) has been proven, but its underlying mechanism remains unknown. Therefore, this study aimed to assess the analgesic effect of PNS on bone cancer pain in a rat model and to explore the underlying mechanism. MATERIALS AND METHODS: PNS on sciatic nerves with bipolar electrode was performed in both naïve and bone cancer pain model rats. Then, the protein levels of activity-regulated cytoskeleton-associated protein (Arc), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor 1 (GluA1), and phosphate N-methyl-d-aspartic acid-type glutamate receptor subunit 2B (pGluNR2B) in spinal cord were evaluated by immunohistochemistry and Western blotting. Thermal paw withdraw latency and mechanical paw withdraw threshold were used to estimate the analgesic effect of PNS on bone cancer pain. Intrathecal administration of Arc shRNA was used to inhibit Arc expression in the spinal cord. RESULTS: PNS at 60 and 120 Hz for 20 min overtly induced Arc expression in the spinal cord, increased thermal pain thresholds in naïve rats, and relieved bone cancer pain; meanwhile, 10 Hz PNS did not achieve those results. In addition, PNS at 60 and 120 Hz also reduced the expression of GluA1, but not pGluNR2B, in the spinal cord. Finally, the anti-nociceptive effect and GluA1 downregulation induced by PNS were inhibited by intrathecal administration of Arc shRNA. CONCLUSION: PNS (60 Hz, 0.3 mA) can relieve bone-cancer-induced allodynia and hyperalgesia by upregulating Arc protein expression and then by decreasing GluA1 transcription in the spinal cord dorsal horn.

Early care of acute hyperglycemia benefits the outcome of traumatic brain injury in rats.

OBJECTIVE: Previous animal studies showed contradictory clinical observations on whether acute hyperglycemia contributes to poor outcome in traumatic brain injury (TBI). Herein, we tried to clarify this issue. METHODS: Striking with depths of 3.0-4.25mm at right occipitoparietal brain region and with depth of 3.75mm at right/left occipitoparietal or right/left frontoparietal brain region were performed, respectively. Blood glucose and insulin levels were traced every four hours from 1 to 72h after striking. HOMA2-%S and HOMA2-%ß were calculated. Modified neurological severity scores (mNSS) were used to evaluate neurological deficit within 72h. RESULTS: Striking with depths of 3.5-4.25mm induced increase in blood glucose lasting up to 24h after striking. The levels of blood glucose after striking with depths of 3.75-4.25mm were significantly different from that of striking with the depth of 3.0mm. Striking with depth of 3.75mm at right/left occipitoparietal region induced higher blood glucose in 24h than that at right/left frontoparietal region. Insulin concentration increased slowly during 72h after striking. Striking also induced decrease in insulin sensitivity and secretion lasting 72h. Evaluation of mNSS revealed that severe striking (beyond 3.75mm) worsened nerve function than slight striking (<3.0mm). Intervention of acute hyperglycemia could decrease the mNSS from 2 to 7 days after TBI. CONCLUSION: Our results suggested that only severe TBI could induce acute hyperglycemia by itself, and early care of acute hyperglycemia could benefit the outcome of TBI patients.

Adiponectin protects rat heart from left ventricular remodeling induced by chronic intermittent hypoxia via inhibition of TGF-ß/smad2/3 pathway.

OBJECTIVE: Obstructive sleep apnea syndrome (OSAS) is associated with many cardiovascular disorders. Chronic intermittent hypoxia (CIH) is the primary player in OSAS of the many associated factors. This study was in order to investigate the effects of the Adiponectin (Ad) on left ventricular remodeling induced by CIH. METHODS: Forty-five rats were randomly divided into three groups: normal control (NC) group, CIH group and CIH plus Ad supplemented (CIH + Ad) group. After 35 days' CIH exposure, masson analysis was used to detect the left ventricular fibrosis and western blot was used to measure the protein expression of collagen I, collagen III and TGF-ß/smad2/3 pathway. Gene analysis by RT-PCR was used to study the MMP2 and TIMP2. RESULTS: After CIH exposure, the fibrosis of left ventricular in CIH group was significantly remarkable than that in both NC and CIH + Ad groups (P<0.05), although statistical difference existed between NC and CIH + Ad groups (P<0.05). In addition, the protein expression of collagen I as well as collagen III and the ratio of mRNA levels of MMP2/TIMP2 were the highest in CIH group but the lowest in NC group, with CIH + Ad group in between. There was a significant difference among three groups (all P<0.05). The TGF-ß/smad2/3 pathway was activated obviously in CIH group, but less noticeably in CIH + Ad group (P<0.05) with a significant difference in the two groups. CONCLUSIONS: The present study showed that Ad could ameliorate the left ventricular remodeling induced by CIH via inhibition of the expression of TGF-ß/smad2/3 pathway.

CXCL1 contributes to ß-amyloid-induced transendothelial migration of monocytes in Alzheimer's disease.

BACKGROUND: Bone marrow-derived microglia that originates in part from hematopoietic cells, and more particularly from monocytes preferentially attach to amyloid deposition in brains of Alzheimer's disease (AD). However, the mechanism of monocytes recruited into the amyloid plaques with an accelerated process in AD is unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we reported that monocytes from AD patients express significantly higher chemokine (C-X-C motif) ligand 1 (CXCL1) compared to age-matched controls. AD patient's monocytes or CXCL1-overexpressing THP-1 cells had enhanced ability of ß-amyloid (Aß)-induced transendothelial migration and Aß-induced transendothelial migration for AD patient's monocytes or CXCL1-overexpressing THP-1 cells was almost abrogated by anti-CXCL1 antibody. Furthermore, monocytes derived from a transgenic mouse model of AD also expressed significantly higher CXCL1. CD11b⁺CD45(hi) population of cells that were recruited from the peripheral blood were markedly bolcked in APP mouse brain by anti-CXCL1 antibody. Accordingly, in response to Aß, human brain microvascular endothelial cells (HBMEC) significantly up-regulated CXC chemokine receptor 2 (CXCR2) expression, which was the only identified receptor for CXCL1. In addition, a high level expression of CXCR2 in HBMEC significantly promoted the CXCL1-overexpressing THP-1 cells transendothelial migration, which could be was abrogated by anti-CXCR2 antibody. Further examination of possible mechanisms found that CXCL1-overexpressing THP-1 cells induced transendothelial electrical resistance decrease, horseradish peroxidase flux increase, ZO-1 discontinuous and occludin re-distribution from insoluble to soluble fraction through interacting with CXCR2. ROCK inhibitor, Y27632, could block CXCL1-overexpressing THP-1 cells transendothelial migration, whereas other inhibitors had no effects. CONCLUSIONS/SIGNIFICANCE: The present data indicate that monocytes derived from AD patients overexpressing CXCL1, which is a determinant for Aß-induced transendothelial migration. CXCL1 expressed by monocytes and CXCR2 on HBMEC is involved in monocytes migrating from blood to brain in AD patients.

Induction of apoptosis signal-regulating Kinase 1 by E2F-1 may not be essential for E2F-1-mediated apoptosis in melanoma cells.

OBJECTIVES: In the present study, we investigate the role of apoptosis signal-regulating kinase 1 (ASK1) mitogen-activated protein (MAP) kinase signal pathways in E2F-1-mediated apoptosis. METHODS: A gene expression profile in response to E2F-1 overexpression was performed by cDNA microarray analysis and confirmed by real-time reverse-transcription polymerase chain reaction. Kinase activities were assayed by Western blot analysis or kinase assay. Apoptosis was assessed by morphologic inspection and flow-cytometric analysis. Cytotoxicity was monitored by MTT assay. RESULTS: E2F-1 upregulated the expression of ASK1 8-fold compared to the Ad-LacZ-infected control in SK-MEL-2 melanoma cells, which was confirmed by reverse-transcription polymerase chain reaction. Sequence analysis showed that there are 2 putative E2F-1 DNA binding sites in the ASK1 promoter region. Truncated E2F-1 protein, which lacks the transactivation domain, failed to upregulate ASK1, suggesting that ASK1 was regulated at the transcriptional level by E2F-1. E2F-1 overexpression resulted in the transient activation of c-Jun N-terminal kinase (JNK); however, dominant negative mutant ASK1 had no effect on E2F-1 cytotoxicity and JNK activation. p38 was not activated by E2F-1, and inhibition of p38 had no effect on E2F-1-mediated cell death. The ASK1 kinase assay showed that ASK1 activity was not upregulated in response to E2F1 overexpression. The inhibition of ASK1 upstream kinase-AKT can enhance E2F-1-mediated cell death. Moreover, an adenovirus expressing truncated E2F-1 keeps the ability of inducing apoptosis in melanoma cells. CONCLUSIONS: ASK1 expression is upregulated by E2F-1 at the transcription level, but the upregulation of ASK1 expression by E2F-1 was not coordinated with an increased ASK1 activity. The ASK1-JNK/p38 pathway does not appear to play a crucial role in E2F-1-induced apoptosis.

Alteration of gene expression in melanoma cells following combined treatment with E2F-1 and doxorubicin.

BACKGROUND: The combination of E2F-1 gene therapy and chemotherapy produces a synergistic effect on melanoma cell apoptosis. However, the molecular mechanisms have not been fully elucidated. The purpose of this study was to identify novel genes or pathways that may play key roles in apoptosis when E2F-1 gene therapy is combined with doxorubicin chemotherapy. MATERIALS AND METHODS: SK-MEL-2 melanoma cells were infected with Ad-E2F-1 alone, Ad-E2F-1 plus doxorubicin, or Ad-LacZ plus doxorubicin. After 16 hours of treatment, the total RNA was extracted from these cells and subjected to microarray analysis. Quantitative real-time PCR was performed to confirm the microarray data. RESULTS: Our results showed that the combination treatment of Ad-E2F-1 and doxorubicin affected the expression of cytokines, transcription factors, as well as genes involved in signal transduction, cell cycle regulation and apoptosis. CONCLUSION: Our findings have identified, for the first time, novel molecular targets and pathways that led to apoptosis in melanoma cells when Ad-E2F-1 was combined with doxorubicin. The molecular information provided here will enhance further mechanistic studies.

Adenovirus-mediated gene transfer of FKHRL1 triple mutant efficiently induces apoptosis in melanoma cells.

The PTEN/Akt signal pathway plays an important role in tumorigenesis. Mutations or deletions of PTEN have been observed in up to 60% of melanoma cell lines, resulting in PI3K/Akt activation. The Forkhead family of transcription factors induce apoptosis in their unphosphorylated forms and were recently reported to be a substrate of Akt kinase. In the present study, an adenovirus expressing a triple mutant (TM) of FKHRL1, which cannot be phosphorylated by Akt, was assessed for its ability to induce apoptosis in melanoma cells. Marked overexpression of FKHRL1/TM was evident in the SK-MEL-2 cell line 24 hours after infection with Ad-FKHRL1/TM by Western blot analysis. The expression of FKHRL1/ TM was moderately delayed in SK-MEL-28 cells. Overexpression of FKHRL1/TM can efficiently inhibit melanoma cell growth and result in rapid loss of cell viability. Cell cycle analysis showed overexpression of FKHRL1/TM in both melanoma cell lines resulted in development of a Sub-G1 population, indicating apoptosis by Ad-FKHRL1/TM infection. Apoptosis was confirmed by morphologic inspection, poly-ADP-ribosepolymerase (PARP) cleavage assay, and annexin V-PE analysis. After Ad-FKHRL1/TM infection, the expression of Bax and Bak did not differ markedly, whereas Mcl-1 and Bcl-x(L) levels decreased markedly. Involvement of caspase 3 and 6 in FKHRL1/TM-mediated apoptosis was demonstrated by cleavage of caspase 3/CPP32 and PARP as well as fragmentation of the caspase 6 substrate lamin B in SK-MEL-2 cells as early as 24 hours after Ad-FKHRL1/ TM infection, but those events were delayed 72 hours in SK-MEL-28. In addition, we found that p27(kip1) was cleaved in SK-MEL-2 cells at 24 hours after treatment with Ad-FKHRL1/TM. This cleavage was observed in SK-MEL-28 cells until 72 hours after infection with Ad-FKHRL1/TM. Our data suggest that adenovirus expressing a FKHRL1 triple mutant could be a useful vector for gene therapy of cancers resistant to chemotherapy and radiotherapy induced by hyperactivity of PI3K/Akt.

SW-620 cells treated with topoisomerase I inhibitor SN-38: gene expression profiling.

BACKGROUND: The goal of this study was to evaluate changes in gene expression in SW-620 cells in response to SN-38 in order to further elucidate the mechanisms by which SN-38 causes apoptosis and cell cycle arrest. METHODS: We used a quantitative gene expression microarray assay to identify the genes regulated by SN-38 treatment in colon cancer cells and confirmed our results with RT-PCR. By gene expression profiling, we first screened a proprietary list of about 22,000 genes. RESULTS: Treatment with SN-38 cells resulted in two-fold or greater alteration in the level of expression of 192 genes compared to control treatment. Most of the affected genes were not known to be responsive to SN-38 prior to this study. SN-38 treatment of these cells was found to affect the expression of various genes involved in DNA replication, transcription, signal transduction, growth factors, cell cycle regulation, and apoptosis, as well as other genes with unknown function. Changes in expression of 14 genes were confirmed by quantitative real-time polymerase chain reaction (RT-PCR). CONCLUSION: This study leads to an increased understanding of the biochemical pathways involved in SN-38-induced apoptosis and possibly to the identification of new therapeutic targets.

Increased mdm-2 expression in a p53-independent manner blocks UV-induced cell cycle arrest and apoptosis in human osteosarcoma cells.

DNA damage results in an increase in P53 levels, which is required to initiate a P53-mediated cell cycle arrest and/or apoptosis. P53 and MDM-2 form a feedback control loop: while P53 can transactivate the MDM-2 gene, high levels of MDM-2 inhibit P53 transactivation as well as promote rapid degradation of P53. In the present study, we investigated the interaction between endogenous MDM-2 and P53 following UV-induced DNA damage in an MDM-2 overexpression cell line. A human osteosarcoma cell line (OsACL, which contains wild-type P53 and overexpresses MDM-2 protein) was used in this study. Here we show that following UV treatment, P53 levels increased in the OsACL cells despite the presence of high-level endogenous MDM-2; however, CAT assays using a P53 reporter system revealed that this P53 was transcriptionally inactive. Although P53 transactivation was inhibited, MDM-2 levels rose markedly following UV irradiation. Northern blot analysis revealed that the increase in MDM-2 protein levels was a result of increased levels of MDM-2 mRNA, possibly due to increased transcription. Cell cycle analysis revealed that OsACL cells were markedly resistant to UV-induced apoptosis. Transfection of OsACL cells with an anti-sense MDM-2 plasmid dowregulated MDM-2 expression and increased UV-induced apoptosis. In conclusion, MDM-2 overexpression can block UV-induced cell cycle arrest and apoptosis by inhibiting P53 transcriptional activity. Furthermore, increased expression of MDM-2 in OsACL cells following UV irradiation appears to be related to P53-independent mechanisms.

E2F-1 overexpression sensitizes colorectal cancer cells to camptothecin.

Topoisomerase I inhibitors have been shown to have clinical activity against human colorectal cancer. Previous studies showed that the cytotoxicity of camptothecin, a topoisomerase I inhibitor, occurs mainly in the S -phase of the cell cycle and is protectable by aphidicolin, an inhibitor of replicative DNA polymerase in some camptothecin-sensitive colorectal cells. Transcription factor E2F-1 regulates the G1/S transition, and recent studies have shown that E2F-1 potentiated the cytotoxicity of some cell-cycle-related drugs. Therefore, the present study was designed to investigate the effect of adenovirus-mediated E2F-1 gene transfer on chemosensitivity of colorectal cancer to camptothecin, in vitro and in vivo. Two human colorectal cancer cells, SW620 (mutant p53) and RKO (wild-type p53), were treated with camptothecin, alone or in combination with adenoviral vectors expressing beta-galactosidase (Ad-LacZ), or E2F-1 (Ad-E2F-1). E2F-1 overexpression was confirmed by Western blot analysis. Ad-E2F-1 gene transfer at low doses (less than the LD(20) dose) markedly increased the sensitivity of human colorectal cancer cells to camptothecin in vitro, which is because of induction of apoptosis. Aphidicolin did not have any protective effect on the Ad-E2F-1/camptothecin-mediated cytotoxicity. The level of topoisomerase I expression was not affected by combination treatment as well, suggesting that DNA replication and topoisomerase I activity may not account for the molecular mechanism of cell killing in response to Ad-E2F-1/camptothecin treatment. Fas and Fas ligand expression were not altered by treatment with camptothecin and/or Ad-E2F-1. Moreover, combination of camptothecin and Ad-E2F-1 has an additive antitumor effect in an in vivo nude mouse xenograft model. When combined with camptothecin, E2F-1 adenovirus therapy resulted in a 95.7% decrease in tumor size compared to control groups (P<.05). These results suggest a chemosensitization strategy that may have clinical utility in human colorectal cancer.

E2F-1 gene therapy induces apoptosis and increases chemosensitivity in human pancreatic carcinoma cells.

Pancreatic cancer is often resistant to conventional chemotherapy. In this study, we examined the role of adenovirus-mediated overexpression of E2F-1 in inducing apoptosis and increasing the sensitivity of pancreatic cancer cells to chemotherapeutic agents. MIA PaCa-2 pancreatic head exocrine adenocarcinoma cells (mutant p53) were treated by mock infection or adenoviruses expressing beta-galactosidase or E2F-1 (Ad-E2F-1) alone or in combination with sublethal concentrations of each chemotherapeutic drug. Cell growth and viability were assessed at selected time points. Apoptosis was evaluated by flow cytometry, characteristic changes in cell morphology and poly (ADP-ribose) polymerase (PARP) cleavage. Western blot analysis was used to examine the expression of E2F-1 and Bcl-2 family member proteins and PARP cleavage. Western blot analysis revealed marked overexpression of E2F-1 at a multiplicity of infection (MOI) of 20 and 70. By 3 days after infection, Ad-E2F-1 treatment at an MOI of 70 resulted in approximately a 20-fold reduction in cell growth and 60% reduction in cell viability as compared to mock-infected cells. Cell cycle analysis, PARP cleavage and changes in cell morphology supported apoptosis as the mechanism of cell death in response to E2F-1. In order to test the efficacy of treatment with a combination of gene therapy and chemotherapy, we utilized concentrations of Ad-E2F-1 which reduced viability to 50% in combination with each chemotherapeutic agent. Cotreatment of the cells with E2F-1 virus and roscovitine (ROS) or etoposide resulted in an additive effect on cell growth inhibition and induction of apoptosis. Interestingly, 5-fluorouracil did not cooperate with Ad-E2F-1 in the mediation of tumor death or inhibition of cell growth. Immunoblotting for Bcl-2 family members revealed no significant changes in the expression levels of Bcl-2, Bcl X(L), Bax or Bak following gene or 'chemogene' therapy with E2F-1. However, a Bax cleavage product was noted which was substantially increased by cotreatment with ROS or etoposide. E2F-1 overexpression initiates apoptosis and suppresses growth in pancreatic MIA PaCa-2 cells in vitro. E2F-1-mediated apoptosis was not associated with significant changes in the expression of Bcl-2 family member proteins in these pancreatic cancer cells. ROS and etoposide, when combined with E2F-1 overexpression, induce apoptosis in an additive manner. This chemogene combination may provide a potentially useful therapeutic strategy for advanced pancreatic cancer.

C-terminal deletion mutant p21(WAF1/CIP1) enhances E2F-1-mediated apoptosis in colon adenocarcinoma cells.

The present study was designed to investigate the efficacy of combination gene therapy using adenoviral vectors expressing gene products shown to possess apoptotic activity: E2F-1 (Ad-E2F-1) and a C-terminal deletion mutant of p21(WAF1/cIP1) (Ad-p21(-PCNA)), on growth inhibition and apoptosis of human colon cancer cells in vitro and in vivo. Marked E2F-1 and p21(-PCNA) overexpression in response to adenovirus infection was evident by Western blot analysis. IC(25) concentrations of each virus were used for each treatment in vitro to detect cooperative effects on cell death. Coexpression of E2F-1 and p21(-PCNA) resulted in an additive effect on cell death compared to infection with either virus alone. Cell cycle analysis, poly(ADP-ribose) polymerase (PARP) cleavage and analysis of cell morphology also revealed that coinfection with both Ad-E2F-1 and Ad-p21(-PCNA) enhanced cellular apoptosis compared to either virus alone. Interestingly, E2F-1 protein expression was markedly enhanced in the E2F-1/p21(-PCNA) adenovirus combination compared to Ad-E2F-1 infection alone. However, these same effects were not evident in cells coinfected with Ad-E2F-1 and an adenovirus expressing wild-type human p21(WAF1/CIP1) (Ad-p21(WT)). The increase in E2F-1 expression with coexpression of E2F-1 and p21(-PCNA) was not a result of increased E2F-1 protein stability, but was related to increased transcriptional activity from the CMV promoter. Cell cycle analysis revealed G1 arrest 72 hours following single-gene therapy with either the wild-type or mutant p21, whereas increased accumulation of cells in G2/M phase was demonstrated in the E2F-1-overexpressing cells. In the combined therapies, E2F-1/p21(-PCNA) treatment still resulted in G1 arrest, but E2F-1 was able to counteract the G1 arrest when coinfected with p21(WT). These results provide further evidence of the importance of the p21:PCNA-binding domain in mediating the complex cell cycle interaction between E2F-1 and p21. Simultaneous intratumoral injection of Ad-E2F-1 and Ad-p21(-PCNA) dramatically reduced tumor burden of SW620 xenografts compared to either treatment alone in our in vivo model but not in HT-29 colon cancer xenografts. When combined with Ad-p21(-PCNA), E2F-1 adenovirus therapy resulted in approximately 95% decrease in tumor volume of SW620 tumor xenografts compared with controls (P<.05). In conclusion, although simultaneous delivery of E2F-1 and p21(-PCNA) transgenes results in increased E2F-1 expression and enhanced apoptosis of both SW620 and HT-29 colon cancer cells in vitro, this combination was only effective in the treatment of SW620 metastatic colon cancer in vivo. This may represent a potentially useful combination gene therapy strategy for metastatic colon cancer.

Adenovirus-mediated E2F-1 gene transfer sensitizes melanoma cells to apoptosis induced by topoisomerase II inhibitors.

Melanoma has proven to be resistant to conventional chemotherapy; however,the mechanism of chemoresistance is still unclear. Recent reports show that the transcription factor, E2F-1, may play a role in mediating cytotoxicity of certain chemotherapeutic agents. We have shown in a previous study that adenovirus-mediated overexpression of E2F-1 can efficiently induce apoptosis in melanoma cells. In the present study, the effect of E2F-1 expression on drug sensitivity of melanoma cells was evaluated. Two human melanoma cell lines, SK-MEL-28 and SK-MEL-2, were treated with drugs (etoposide, Adriamycin, roscovitine, cisplatin, 5-fluorouracil, or cycloheximide), alone or in combination with adenoviral vectors expressing beta-galactosidase (Ad-LacZ) or E2F-1 (Ad-E2F-1) at a multiplicity of infection of 1 in vitro. E2F-1 expression was confirmed by Western blot analysis. Sublethal concentrations of each drug alone or infection with Ad-E2F-1 alone produced <5% apoptosis by 3 days posttreatment. Conversely, cotreatment with Ad-E2F-1 and low concentrations of etoposide or Adriamycin markedly sensitized melanoma cells to apoptotic cell death. A slight enhancement of the cytotoxicity of roscovitine was demonstrated in combination with E2F-1 overexpression, but not to cisplatin, 5-fluorouracil, or cycloheximide. Ad-LacZ infection showed no obvious effects on drug sensitivity. Overexpression of p21 can block apoptosis induced by the combination chemogene therapy of Ad-E2F-1 and topoisomerase II poisons and does not require its proliferating cell nuclear antigen-binding ability. The protein synthesis inhibitor cycloheximide also has a cytotoxicity-protective effect against topoisomerase II inhibitor/E2F-1-induced apoptosis and suggests that new protein synthesis is required for this process. Topoisomerase II inhibitors also cooperated with Ad-E2F-1 to enhance antitumor activity in an in vivo model using xenografts in nude mice. When combined with Adriamycin or etoposide, E2F-1 adenovirus therapy resulted in an 87% or 91% decrease in tumor size, respectively, compared with controls (P < 0.002). Our results show that adenovirus-mediated E2F-1 gene transfer can sensitize melanoma cells to some chemotherapeutic agents, particularly topoisomerase II poisons, in vitro and in vivo. These results suggest a new chemosensitization strategy for melanoma gene therapy.

Inhibition of cyclin A kinase activity in E2F-1 chemogene therapy of colon cancer.

Adenoviral-mediated gene transfer of the apoptotic gene E2F-1 has been shown to induce apoptosis in a variety of tumor cells and acts in an additive or cooperative fashion with several specific chemotherapeutic agents to induce tumor cell death. The apoptotic function of E2F-1 is dependent on its ability to bind DNA; cyclin A kinase activity has been shown to negatively regulate the DNA-binding capacity of E2F-1. In the present study, we sought to determine whether cyclin A kinase activity is involved in mediating the interaction between E2F-1 and chemotherapeutic agents in colon cancer cells. Therefore, human colon adenocarcinoma (SW620) cells were treated with an adenovirus expressing E2F-1 (Ad-E2F-1, multiplicity of infection 20). Immediately following infection, a panel of conventional chemotherapeutic agents with varying modes of cytotoxic action were administered at LD(25 )doses. Three days following treatment, viability and growth inhibition were determined by trypan blue exclusion assay. Apoptosis was confirmed using cellular morphology, poly (ADP-ribose) polymerase cleavage, and flow-cytometric analysis. E2F-1 overexpression and cyclin A protein expression were monitored by immunoblot, and cyclin A kinase activity was determined by kinase assay. Vincristine (VIN), camptothecin (CPT), and actinomycin D were found to have a cooperative (>38% over the additive single therapy values) effect on E2F-1-mediated apoptosis. Etoposide, cisplatin (CIS), and 5-fluorouracil (5-FU) showed the least cooperation ( 0.1) compared to Ad-E2F-1 treatment alone. Combination of Ad-LacZ/5-FU and Ad-LacZ/actinomycin D significantly inhibited cyclin A kinase activity compared to Ad-LacZ treatment alone (p < 0.005). No other Ad-LacZ/drug combinations significantly affected cyclin A kinase activity (p > 0.05). In conclusion, combinations of E2F-1 adenovirus and VIN, CPT, or actinomycin D at LD(25 )had significant cooperative effects on colon cancer apoptotic cell death in vitro. Although inhibition of cyclin A kinase activity was observed in most Ad-E2F-1/drug combination treatments compared to Ad-E2F-1 treatment alone, there was no consistent correlation between degree of inhibition of cyclin A kinase activity and the cooperative effect. Nonetheless, inhibition of cyclin A kinase activity may be an important mechanism by which the chemogene therapy effects involving E2F-1 are modulated.