Protective effects of baicalin against phenylarsine oxide-induced cytotoxicity in human skin keratinocytes.

Phenylarsine oxide (PAO) is a known environmental pollutant and skin keratinocytes are most seriously affected. Baicalin (BCN) was reported to have antioxidant and anti-inflammatory effects, but its protective effect against PAO toxicity is unknown. This study aimed at exploring whether baicalin can reverse the toxicity of human epidermal keratinocytes that are subjected to PAO exposure and underlying mechanisms. In silico analysis from a publicly accessible HaCaT cell transcriptome dataset exposed to chronic Arsenic showed significant differential expression of several genes, including the genes related to DNA replication. Later, we performed in vitro experiments, in which HaCaT cells were exposed to PAO (500 nM) in the existence of BCN (10-50 µM). Treatment of PAO alone induces the JNK, p38 and caspase-3 activation, which were engaged in the apoptosis induction, while the activity of AKT was significantly inhibited, which was engaged in the suppression of apoptosis. PAO suppressed SIRT3 expression and induced intracellular reactive oxygen species (ROS), causing a marked reduce in cell viability and apoptosis. However, BCN treatment restored the PAO-induced suppression of SIRT3 and AKT expression, reduced intracellular ROS generation, and markedly suppressed both caspase-3 activation and apoptosis induction. However, the protective effect of BCN was significantly attenuated after pretreatment with nicotinamide, an inhibitor of SIRT3. These findings indicate that BCN protects against cell death induced by PAO via inhibiting excessive intracellular ROS generation via restoring SIRT3 activity and reactivating downstream AKT pathway. In this study, we firstly shown that BCN is an efficient drug to prevent PAO-induced skin cytotoxicity, and these findings need to be confirmed by in vivo and clinical investigations.

Vincamine Ameliorates Epithelial-Mesenchymal Transition in Bleomycin-Induced Pulmonary Fibrosis in Rats; Targeting TGF-ß/MAPK/Snai1 Pathway.

Idiopathic pulmonary fibrosis is a progressive, irreversible lung disease that leads to respiratory failure and death. Vincamine is an indole alkaloid obtained from the leaves of Vinca minor and acts as a vasodilator. The present study aims to investigate the protective activity of vincamine against EMT in bleomycin (BLM)-induced pulmonary fibrosis via assessing the apoptotic and TGF-ß1/p38 MAPK/ERK1/2 signaling pathways. In bronchoalveolar lavage fluid, protein content, total cell count, and LDH activity were evaluated. N-cadherin, fibronectin, collagen, SOD, GPX, and MDA levels were determined in lung tissue using ELISA. Bax, p53, bcl2, TWIST, Snai1, and Slug mRNA levels were examined using qRT-PCR. Western blotting was used to assess the expression of TGF-ß1, p38 MAPK, ERK1/2, and cleaved caspase 3 proteins. H & E and Masson's trichrome staining were used to analyze histopathology. In BLM-induced pulmonary fibrosis, vincamine reduced LDH activity, total protein content, and total and differential cell count. SOD and GPX were also increased following vincamine treatment, while MDA levels were decreased. Additionally, vincamine suppressed the expression of p53, Bax, TWIST, Snail, and Slug genes as well as the expression of factors such as TGF-ß1, p/t p38 MAPK, p/t ERK1/2, and cleaved caspase 3 proteins, and, at the same time, vincamine increased bcl2 gene expression. Moreover, vincamine restored fibronectin, N-Catherine, and collagen protein elevation due to BLM-induced lung fibrosis. In addition, the histopathological examination of lung tissues revealed that vincamine attenuated the fibrotic and inflammatory conditions. In conclusion, vincamine suppressed bleomycin-induced EMT by attenuating TGF-ß1/p38 MAPK/ERK1/2/TWIST/Snai1/Slug/fibronectin/N-cadherin pathway. Moreover, it exerted anti-apoptotic activity in bleomycin-induced pulmonary fibrosis.

A New EGFR Inhibitor from Ficus benghalensis Exerted Potential Anti-Inflammatory Activity via Akt/PI3K Pathway Inhibition.

Inflammation is a critical defensive mechanism mainly arising due to the production of prostaglandins via cyclooxygenase enzymes. This study aimed to examine the anti-inflammatory activity of fatty acid glucoside (FAG), which is isolated from Ficus benghalensis against lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The cytotoxic activity of the FAG on RAW 264.7 macrophages was evaluated with an MTT assay. The levels of PGE2 and NO and the activity of iNOS, COX-1, and COX-2 enzymes in LPS-stimulated RAW 264.7 cells were evaluated. The gene expression of IL-6, TNF-α, and PGE2 was investigated by qRT-PCR. The expression of epidermal growth factor receptor (EGFR), Akt, and PI3K proteins was examined using Western blotting analysis. Furthermore, molecular docking of the new FAG against EGFR was investigated. A non-cytotoxic concentration of FAG increased NO release and iNOS activity, inhibited COX-1 and COX-2 activities, and reduced PGE2 levels in LPS-stimulated RAW 264.7 cells. It diminished the expression of TNF-α, IL-6, PGE2, EGFR, Akt, and PI3K. Furthermore, the molecular docking study proposed the potential direct binding of FAG with EGFR with a high affinity. This study showed that FAG is a natural EGFR inhibitor, NO-releasing, and COX-inhibiting anti-inflammatory agent via EGFR/Akt/PI3K pathway inhibition.

Inhibition of NF-kB/IL-6/JAK2/STAT3 Pathway and Epithelial-Mesenchymal Transition in Breast Cancer Cells by Azilsartan.

Metastatic breast cancer is an incurable form of breast cancer that exhibits high levels of epithelial-mesenchymal transition (EMT) markers. Angiotensin II has been linked to various signaling pathways involved in tumor cell growth and metastasis. The aim of this study is to investigate, for the first time, the anti-proliferative activity of azilsartan, an angiotensin II receptor blocker, against breast cancer cell lines MCF-7 and MDA-MB-231 at the molecular level. Cell viability, cell cycle, apoptosis, colony formation, and cell migration assays were performed. RT-PCR and western blotting analysis were used to explain the molecular mechanism. Azilsartan significantly decreased the cancer cells survival, induced apoptosis and cell cycle arrest, and inhibited colony formation and cell migration abilities. Furthermore, azilsartan reduced the mRNA levels of NF-kB, TWIST, SNAIL, SLUG and bcl2, and increased the mRNA level of bax. Additionally, azilsartan inhibited the expression of IL-6, JAK2, STAT3, MMP9 and bcl2 proteins, and increased the expression of bax, c-PARP and cleaved caspase 3 protein. Interestingly, it reduced the in vivo metastatic capacity of MDA-MBA-231 breast cancer cells. In conclusion, the present study revealed, for the first time, the anti-proliferative, apoptotic, anti-migration and EMT inhibition activities of azilsartan against breast cancer cells through modulating NF-kB/IL-6/JAK2/STAT3/MMP9, TWIST/SNAIL/SLUG and apoptosis signaling pathways.

Dual Topoisomerase I/II Inhibition-Induced Apoptosis and Necro-Apoptosis in Cancer Cells by a Novel Ciprofloxacin Derivative via RIPK1/RIPK3/MLKL Activation.

Fluoroquinolones (FQs) are synthetic broad-spectrum antimicrobial agents that have been recently repurposed to anticancer candidates. Designing new derivatives of FQs with different moieties to target DNA topoisomerases could improve their anticancer efficacy. The present study aimed to synthesize a novel ciprofloxacin derivative, examine its anticancer activity against HepG2 and A549 cancer cells, and investigate the possible molecular mechanism underlying this activity by examining its ability to inhibit the topo I/II activity and to induce the apoptotic and necro-apoptotic pathways. Molecular docking, cell viability, cell migration, colony formation, cell cycle, Annexin V, lactate dehydrogenase (LDH) release, ELISA, and western blotting assays were utilized. Molecular docking results showed that this novel ciprofloxacin derivative exerted dual topo I and topo II binding and inhibition. It significantly inhibited the proliferation of A549 and HepG2 cancer cells and decreased their cell migration and colony formation abilities. In addition, it significantly increased the % of apoptotic cells, caused cell cycle arrest at G2/M phase, and elevated the LDH release levels in both cancer cells. Furthermore, it increased the expression of cleaved caspase 3, RIPK1, RIPK3, and MLKL proteins. This novel ciprofloxacin derivative exerted substantial dual inhibition of topo I/II enzyme activities, showed antiproliferative activity, suppressed the cell migration and colony formation abilities for A549 and HepG2 cancer cells and activated the apoptotic pathway. In addition, it initiated another backup deadly pathway, necro-apoptosis, through the activation of the RIPK1/RIPK3/MLKL pathway.

Carpachromene Ameliorates Insulin Resistance in HepG2 Cells via Modulating IR/IRS1/PI3k/Akt/GSK3/FoxO1 Pathway.

Insulin resistance contributes to several disorders including type 2 diabetes and cardiovascular diseases. Carpachromene is a natural active compound that inhibits α-glucosidase enzyme. The aim of the present study is to investigate the potential activity of carpachromene on glucose consumption, metabolism and insulin signalling in a HepG2 cells insulin resistant model. A HepG2 insulin resistant cell model (HepG2/IRM) was established. Cell viability assay of HepG2/IRM cells was performed after carpachromene/metformin treatment. Glucose concentration and glycogen content were determined. Western blot analysis of insulin receptor, IRS1, IRS2, PI3k, Akt, GSK3, FoxO1 proteins after carpachromene treatment was performed. Phosphoenolpyruvate carboxykinase (PEPCK) and hexokinase (HK) enzymes activity was also estimated. Viability of HepG2/IRM cells was over 90% after carpachromene treatment at concentrations 6.3, 10, and 20 µg/mL. Treatment of HepG2/IRM cells with carpachromene decreased glucose concentration in a concentration- and time-dependant manner. In addition, carpachromene increased glycogen content of HepG2/IRM cells. Moreover, carpachromene treatment of HepG2/IRM cells significantly increased the expression of phosphorylated/total ratios of IR, IRS1, PI3K, Akt, GSK3, and FoxO1 proteins. Furthermore, PEPCK enzyme activity was significantly decreased, and HK enzyme activity was significantly increased after carpachromene treatment. The present study examined, for the first time, the potential antidiabetic activity of carpachromene on a biochemical and molecular basis. It increased the expression ratio of insulin receptor and IRS1 which further phosphorylated/activated PI3K/Akt pathway and phosphorylated/inhibited GSK3 and FoxO1 proteins. Our findings revealed that carpachromene showed central molecular regulation of glucose metabolism and insulin signalling via IR/IRS1/ PI3K/Akt/GSK3/FoxO1 pathway.

A star-shaped molecularly imprinted polymer derived from polyhedral oligomeric silsesquioxanes with improved site accessibility and capacity for enantiomeric separation via capillary electrochromatography.

A star-shaped molecularly imprinted coating was prepared starting from octavinyl-modified polyhedral oligomeric silsesquioxanes (Ov-POSS). It possesses a relatively open structure and has good site accessibility and a larger capacity even at lower cross-linking. The imprinted coating was prepared from S-amlodipine (S-AML) as the template and analyte, Ov-POSS as the cross-linker, and methacrylic acid as the functional monomer. The preparation and chromatographic parameters were optimized, including ratio of template to functional monomer, apparent cross-linking degree, pH value, ACN content and salt concentration in the mobile phase. The best resolution in enantiomer separation by means of capillary electrochromatography reaches a value of 33. A good recognition ability (α = 2.60) was obtained and the column efficiency for S-AML was 54,000 plates m-1. The use of Ov-POSS as a cross-linker significantly improves the column capacity and thus the detection sensitivity. The results show that Ov-POSS is an effective cross-linker for the preparation of imprinted polymers with good accessibility and large capacity. Graphical abstract Schematic presentation of the preparation of star-shaped imprinted polymer using octavinyl-modified polyhedral oligomeric silsesquioxanes (Ov-POSS) and by using methacrylic acid (MAA) as functional monomer. The best enantiometric resolution (33) for amlodipine (AML) can be achieved in capillary chromatography (CEC).

Checkpoint kinase 2 is dispensable for regulation of the p53 response but is required for G2/M arrest and cell survival in cells with p53 defects under heat stress.

Hyperthermia induced by heat stress (HS) is known to inhibit proliferation and induce cell death in cancer. We previously demonstrated that checkpoint kinase 1 (Chk1) contributes to G2/M arrest and cell survival under HS; however, the role of Chk2, a functional analog of Chk1, in regulation of the cell cycle and cell death under HS is still unknown. Here, we addressed the role of Chk2 using Molt-4 cells with p53-targeted shRNA (Molt-4/shp53) and parental control cells (Molt-4/V). Chk2 inhibition suppressed C-terminal acetylation of p53 and delayed the induction of p53-target genes in Molt-4/V cells under HS; however, Chk2 inhibition failed to inhibit apoptosis induced by HS, indicating that Chk2 was dispensable for p53-dependent apoptosis under HS. In contrast, Chk2 inhibition abrogated G2/M arrest and promoted cell death induced by HS in HeLa cells and Molt-4/shp53 cells. Thus, we demonstrated for the first time that Chk2 was required for cell cycle arrest and cell survival, particularly in cells with p53 defects under HS. These findings indicated that Chk2 may be a selective target for p53-mutated or -deficient cancer treated with hyperthermia.

Enhancement of hyperthermia-induced apoptosis by 5Z-7-oxozeaenol, a TAK1 inhibitor, in Molt-4 cells.

PURPOSE: Transforming growth factor-ß-activated kinase1 (TAK1) plays an anti-apoptotic role in response to multiple stresses. TAK1 inhibitor, 5Z-7-oxozeaenol (OZ) has been studied for its apoptotic effects. However, the combined effect of OZ with physical stresses remains to be elusive. Therefore, in this study we focussed to determine the combined effects of OZ with hyperthermia (HT) using Molt-4 cell line. MATERIALS AND METHODS: Molt-4 cells were pre-treated with OZ for 1 h followed by heat exposure (44 °C, 10 min) and harvested 24 h after incubation at 37 °C, apoptosis was measured by Annexin V-FITC/PI double staining assay using flow cytometry and cell growth was observed by cell counting assay. Further mechanism involved in the combination was investigated by measuring mitochondrial membrane potential (MMP), intracellular ROS generation, expression of apoptosis related protein, intracellular calcium ion level and Fas activity. RESULTS: Combination of OZ with HT significantly enhances MMP loss and superoxide generation. Furthermore, OZ pre-treatment promotes caspase-8 cleavage, Fas externalisation, caspase 3 activity and intracellular calcium ion levels. OZ pre-treatment decreased the expression of HT-induced Bcl-2 and increased the expression of pro-apoptotic Bax, while markedly suppressed the phosphorylation of JNK and p38. In addition, increased expression of CHOP following combined treatment indicates that ER stress may also involve in the enhancement of HT-induced apoptosis. CONCLUSION: Our data showed for the first time that OZ sensitizes Molt-4 cells to HT-induced apoptosis via extrinsic and intrinsic apoptotic pathways. Furthermore, ROS and ER stress may also play role in the enhancement of HT-induced apoptosis by OZ.

Serum C-reactive protein acted as a prognostic biomarker for overall survival in metastatic prostate cancer patients.

The aim of this study was to determine whether baseline serum C-reactive protein (CRP) levels were associated with overall survival in patients with metastatic prostate cancer in the Chinese population. A total of 135 patients with metastatic prostate cancer were retrospectively reviewed. Both Kaplan-Meier product-limit method and multivariable analysis by Cox regression model were used to assess the prognostic role of serum CRP levels on overall survival of patients with metastatic prostate cancer. There were 51 patients (37.8%) with higher values of baseline serum CRP levels (≥10 mg/L). Kaplan-Meier product-limit method and log-rank test showed that patients with high serum CRP level (≥10 mg/L) had significantly worse overall survival than those patients with normal serum CRP level (<10 mg/L) (P < 0.001). The multivariable analysis by Cox regression model further showed that high serum CRP level (≥10 mg/L) was a significantly independent predictor of overall survival (hazard ratio (HR) = 2.39; 95% confidence interval (95% CI) 1.56-2.39, P < 0.001). In addition, high Gleason score (≥8) also was an independent predictor of overall survival (HR = 1.80; 95% CI 1.16-2.79, P = 0.008). In conclusion, serum CRP level is useful to predict the prognosis of metastatic prostate cancer patients, and high serum CRP level is a significantly independent predictor of worse overall survival.

Isofraxidin, a potent reactive oxygen species (ROS) scavenger, protects human leukemia cells from radiation-induced apoptosis via ROS/mitochondria pathway in p53-independent manner.

Ionizing radiation (IR) leads to oxidizing events such as excessive reactive oxygen species (ROS) in the exposed cells, resulting in further oxidative damage to lipids, proteins and DNA. To screen the potential radio-protective drug, the intracellular ROS was measured in irradiated U937 cells pretreated with 80 candidate traditional herbal medicine, respectively. Isofraxidin (IF) was one possible radio-protector in these 80 drugs. This study investigated the radio-protective role of IF, a Coumarin compound, in human leukemia cell lines, for the first time. Results indicate that IF protects against IR-induced apoptosis in U937 cells in the time- and concentration- dependent manner. IF decreases IR-induced intracellular ROS generation, especially hydroxyl radicals formation, inhibits IR-induced mitochondrial membrane potential loss and reduces IR-induced high intracellular Ca(2+) levels regardless of ER stress. IF down-regulates the expression of caspase-3, phospho-JNK, phospho-p38 and activates Bax in mitochondria. IF inhibits cytochrome c release from mitochondria to cytosol. IF also moderates IR-induced Fas externalization and caspase-8 activation. IF also exhibits significant protection against IR-induced cell death in other leukemia cell lines such as Molt-4 cells and HL60 cells regardless of p53. Taken together, the data demonstrate that IF protects leukemia cells from radiation-induced apoptosis via ROS/mitochondria pathway in a p53-independent manner.

Arbutin, an intracellular hydroxyl radical scavenger, protects radiation-induced apoptosis in human lymphoma U937 cells.

Ionizing radiation (IR) can generate reactive oxygen species (ROS). Excessive ROS have the potential to damage cellular macromolecules including DNA, proteins, and lipids and eventually lead to cell death. In this study, we evaluated the potential of arbutin, a drug chosen from a series of traditional herbal medicine by measuring intracellular hydroxyl radical scavenging ability in X-irradiated U937 cells. Arbutin (hydroquinone-ß-D-glucopyranoside), a naturally occurring glucoside of hydroquinone, has been traditionally used to treat pigmentary disorders. However, there are no reports describing the effect of arbutin on IR-induced apoptosis. We confirmed that arbutin can protect cells from apoptosis induced by X-irradiation. The combination of arbutin and X-irradiation could reduce intracellular hydroxyl radical production and prevent mitochondrial membrane potential loss. It also could down-regulate the expression of phospho-JNK, phospho-p38 in whole cell lysate and activate Bax in mitochondria. Arbutin also inhibits cytochrome C release from mitochondria to cytosol. To verify the role of JNK in X-irradiation-induced apoptosis, the cells were pretreated with a JNK inhibitor, and found that JNK inhibitor could reduce apoptosis induced by X-irradiation. Taken together, our data indicate that arbutin plays an anti-apoptotic role via decreasing intracellular hydroxyl radical production, inhibition of Bax-mitochondria pathway and activation of the JNK/p38 MAPK pathway.

Hernandezine promotes cancer cell apoptosis and disrupts the lysosomal acidic environment and cathepsin D maturation.

Hernandezine (Her), a bisbenzylisoquinoline alkaloid extracted from Thalictrum flavum, is recognized for its range of biological activities inherent to this herbal medicine. Despite its notable properties, the anti-cancer effects of Her have remained largely unexplored. In this study, we elucidated that Her significantly induced cytotoxicity in cancer cells through the activation of apoptosis and necroptosis mechanisms. Furthermore, Her triggered autophagosome formation by activating the AMPK and ATG5 conjugation systems, leading to LC3 lipidation. Our findings revealed that Her caused damage to the mitochondrial membrane, with the damaged mitochondria undergoing mitophagy, as evidenced by the elevated expression of mitophagy markers. Conversely, Her disrupted autophagic flux, demonstrated by the upregulation of p62 and accumulation of autolysosomes, as observed in the RFP-GFP-LC3 reporter assay. Initially, we determined that Her did not prevent the fusion of autophagosomes and lysosomes. However, it inhibited the maturation of cathepsin D and increased lysosomal pH, indicating an impairment of lysosomal function. The use of the early-stage autophagy inhibitor, 3-methyladenine (3-MA), did not suppress LC3II, suggesting that Her also induces noncanonical autophagy in autophagosome formation. The application of Bafilomycin A1, an inhibitor of noncanonical autophagy, diminished the recruitment of ATG16L1 and the accumulation of LC3II by Her, thereby augmenting Her-induced cell death. These observations imply that while autophagy initially plays a protective role, the disruption of the autophagic process by Her promotes programmed cell death. This study provides the first evidence of Her's dual role in inducing apoptosis and necroptosis while also initiating and subsequently impairing autophagy to promote apoptotic cell death. These insights contribute to a deeper understanding of the mechanisms underlying programmed cell death, offering potential avenues for enhancing cancer prevention and therapeutic strategies.

TAK1 promotes cell survival by TNFAIP3 and IL-8 dependent and NF-κB independent pathway in HeLa cells exposed to heat stress.

PURPOSE: Transforming growth factor-ß-activated kinase 1 (TAK1) plays a role in inhibiting apoptosis in response to multiple stresses. In the present study, we investigated the role of TAK1 in cell death induced by heat stress (HS). MATERIALS AND METHODS: TAK1 knockdown HeLa cells and their parental cells were exposed to HS at 44 °C for 15, 30, 45 min followed by colony formation assay. Heat shock proteins (HSPs) induction, NF-κB phosphorylation, and caspase-3 cleavage were estimated by western blotting using specific antibodies. Global gene expression analysis was performed using the GeneChip® system. The anti-apoptotic roles of the identified genes were elucidated using small interfering RNAs targeting those genes. RESULTS: Heat sensitivity estimated by colony formation assay and caspase-3 cleavage increased in TAK1 knockdown cells. This sensitisation was not due to alterations in HSP induction or NF-κB phosphorylation as the expression levels of these proteins did not differ significantly between the TAK1 knockdown and the parent cells after HS exposure. The GeneChip® analysis revealed differences in gene expression between both cell variants after HS exposure and defined the genetic network associated with cell death. TNF-α interacting protein 3 (TNFAIP3) and Interleukin 8 (IL-8) are two of the identified genes. RNA interference against these genes increased the cleavage of caspase-3 and cell death after HS exposure. CONCLUSION: Our findings reveal the role of TAK1 in thermoresistance and show that the mediation is independent of NF-κB phosphorylation but is dependent on TNFAIP3 and IL-8 induction.

Renal epithelioid angiomyolipoma: A case report.

Epithelioid angiomyolipoma (EAML) of the kidney is an uncommon neoplasm with malignant potential. It can occur sporadically or be associated with tuberous sclerosis. EAML is a monotypic variant of angiomyolipoma (AML), which is classified as neoplasm of the perivascular epithelioid cell or perivascular epithelioid cell tumor. Due to its epithelioid nature and paucity of fat components, unlike classic AML, which has abundant adipose tissue with characteristic features on CT scans, it is difficult to distinguish EAML from renal cell carcinoma and fat-poor AML on CT or MRI preoperatively, which may lead to misdiagnosis and unnecessary nephrectomy. The present report describes two cases of renal EAML, which were successfully treated by laparoscopic surgery. Preoperative diagnosis had not been achieved until surgery was performed and histological analysis was accomplished. No local recurrence or distal metastasis was observed during follow-up. Although the differential diagnosis was challenging preoperatively, a diagnosis of EAML should be considered and surgical excision was the preferred treatment strategy for the patients with localized tumors.

Isofraxidin enhances hyperthermia­induced apoptosis via redox modification in acute monocytic leukemia U937 cells.

The cell­killing potential of most chemotherapeutic agents is enhanced by a temperature elevation. Isofraxidin (IF) is a coumarin compound widely found in plants, such as the Umbelliferae or Chloranthaceae families. IF induces anticancer effects in lung and colorectal cancer. To the best of our knowledge, the combined effects of hyperthermia (HT) and IF on heat­induced apoptosis have not been reported. Acute monocytic leukemia U937 cells were exposed to HT with or without IF pre­treatment. Apoptosis was measured by Annexin V­FITC/PI double staining assay using flow cytometry and cell viability was observed by cell counting kit assay, DNA fragmentation. The mechanism involved in the combination was explored by measuring changes in the mitochondrial membrane potential, (MMP), intracellular ROS generation, expression of apoptosis related protein, and intracellular calcium ion level. It was demonstrated that IF enhanced HT­induced apoptosis in U937 cells. The results demonstrated that combined treatment enhanced mitochondrial membrane potential loss and transient superoxide generation increased protein expression levels of caspase­3, caspase­8 and phosphorylated­JNK and intracellular calcium levels. Moreover, the role of caspases and JNK was confirmed using a pan caspase inhibitor (zVAD­FMK) and JNK inhibitor (SP600125) in U937 cells. Collectively, the data demonstrated that IF enhanced HT­induced apoptosis via a reactive oxygen species mediated mitochondria/caspase­dependent pathway in U937 cells.

Ras homolog gene family, member A promotes p53 degradation and vascular endothelial growth factor-dependent angiogenesis through an interaction with murine double minute 2 under hypoxic conditions.

BACKGROUND: Tumor neovascularization (TNV) is a common pathologic basis for malignant growth and metastasis. However, the mechanism of TNV pathogenesis is not fully understood. Ras homolog gene family, member A (RhoA), a Rho guanosine triphosphatase (GTPase) family member, may be involved in a hypoxia-induced vascular endothelial growth factor (VEGF) pathway that regulates TNV angiogenesis through an unclear mechanism. METHODS: The regulation of RhoA on p53, the p53 binding protein homolog murine double minute 2 (MDM2), and VEGF was analyzed in hypoxic MCF-7 cells using Western blot analysis, real-time polymerase chain reaction (PCR) analysis, coimmunoprecipitation, and immunofluorescence staining assays. Changes in proliferation, invasion, migration, stress fiber formation, and tube formation were detected in an MCF-7 human umbilical vein endothelial cell (HUVEC) coculture system. Correlations of RhoA expression with MDM2, wild-type p53 (wt-p53), and VEGF expression in breast cancer tissues and relations between RhoA and breast cancer clinical features were analyzed by immunohistochemistry. RESULTS: Activated RhoA down-regulated p53 protein, which increased VEGF expression in hypoxic MCF-7 cells; whereas p53 messenger RNA levels were not altered. In addition, the ubiquitin-mediated degradation of p53 was enhanced by active RhoA. RhoA and MDM2 colocalized in the cytoplasm of hypoxic MCF-7 cells and interacted with each other physically. Furthermore, nutlin-3, a specific MDM2 inhibitor, was capable of reducing activated RhoA-induced p53 protein stability and attenuating VEGF accumulation. In an MCF-7-HUVEC coculture system, nutlin-3 effectively inhibited HUVEC proliferation, invasion, migration, stress fiber formation, and tube formation mediated by activated RhoA under hypoxic conditions. Data from 129 clinical breast cancer specimens with wt-p53 revealed that high RhoA expression was correlated with high MDM2 expression, low wt-p53 expression, and high VEGF expression. CONCLUSIONS: The current data suggested that activated RhoA promotes VEGF expression and hypoxia-induced angiogenesis through the up-regulation of MDM2 to decrease p53 stability.

Inhibition of checkpoint kinase 1 abrogates G2/M checkpoint activation and promotes apoptosis under heat stress.

Hyperthermia induced by heat stress (HS) inhibits the proliferation of cancer cells and induces their apoptosis. However, the mechanism underlying HS-induced apoptosis remains elusive. Here, we demonstrated a novel evidence that checkpoint kinase 1 (Chk1) plays crucial roles in the apoptosis and regulation of cell cycle progression in cells under HS. In human leukemia Jurkat cells, interestingly, the ataxia telangiectasia and Rad-3 related (ATR)-Chk1 pathway was preferentially activated rather than the ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2) pathway under HS. The selective inhibitors of ATR or Chk1 abrogated HS-induced apoptosis in human leukemia Jurkat cells whereas the inhibition of ATM or Chk2 caused only marginal effects. Inhibition of ATR and Chk1 also abrogated G2/M checkpoint activation by HS in Jurkat cells. The effects of small interfering RNA targeting Chk1 were similar to those of the selective inhibitor of Chk1. In addition, the efficiencies of Chk1 inhibition on G2/M checkpoint abrogation and apoptosis induction were confirmed in the adherent cancer cell lines HeLa, HSC3, and PC3, suggesting that the targeting of Chk1 can be effective in solid tumors cells. In conclusion, these findings indicate a novel molecular basis of G2/M checkpoint activation and apoptosis in cells exposed to HS.

Molecular mechanisms of apoptosis induction by 2-dodecylcyclobutanone, a radiolytic product of palmitic acid, in human lymphoma U937 cells.

The irradiation of fat-containing food forms 2-dodecylcyclobutanone (2-DCB) from palmitic acid (PA). In this study, we investigated whether 2-DCB and PA induce apoptosis in human lymphoma U937 cells. We found that cell viability decreased by 2-DCB and apoptosis was induced by 2-DCB and PA. 2-DCB and PA significantly enhanced the formation of intracellular reactive oxygen species (ROS). Apoptosis induced by 2-DCB and PA was strongly prevented by an antioxidant, N-acetyl-L: -cysteine. The treatment with 2-DCB and PA resulted in the loss of mitochondrial membrane potential, and Fas, caspase-8 and caspase-3 activation. Pretreatment with a pan-caspase inhibitor (z-VAD) significantly inhibited apoptosis induced by 2-DCB and PA. Moreover, 2-DCB and PA also induced Bax up-regulation, the reduction in Bcl-2 expression level, Bid cleavage and the release of cytochrome c from the mitochondria to the cytosol. In addition, an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) was observed after the treatment with 2-DCB and PA. Our results indicated that intracellular ROS generation, the modulation of the Fas-mitochondrion-caspase-dependent pathway and the increase in [Ca(2+)](i) involved in apoptosis are induced by 2-DCB and PA in U937 cells.

Castleman's disease in the pelvic retroperitoneum: A case report.

Castleman's disease (CD) is an uncommon lymphoproliferative disorder, which mostly occurs in the chest and neck. Lesions originating in the pelvic retroperitoneum are rare. It is important to consider CD as a differential diagnosis when a pelvic lesion is found. The present study reports a unique case of CD in the pelvic retroperitoneum, where the tumor was demonstrated to have a highly vascular nature on CT scanning. The preoperative diagnosis was uncertain and a vascular-derived tumor was considered. Laparoscopic surgery was performed and the mass was completely resected along with regional lymphadenectomy. The pathological diagnosis was the hyaline vascular type of CD. The patient was free of recurrence after 1 year of follow-up. As the underlying etiology remains elusive and the differential diagnosis is challenging preoperatively, surgical excision is the preferred treatment strategy for this type of benign lesion.