Combination Treatment with Liposomal Doxorubicin and Inductive Moderate Hyperthermia for Sarcoma Saos-2 Cells.

Despite efforts in osteosarcoma (OS) research, the role of inductive moderate hyperthermia (IMH) in delivering and enhancing the antitumor effect of liposomal doxorubicin formulations (LDOX) remains unresolved. This study investigated the effect of a combination treatment with LDOX and IMH on Saos-2 human OS cells. We compared cell viability using a trypan blue assay, apoptosis and reactive oxygen species (ROS) measured by flow cytometry and pro-apoptotic Bax protein expression examined by immunocytochemistry in response to IMH (42 MHz frequency, 15 W power for 30 min), LDOX (0.4 µg/mL), and LDOX plus IMH. The lower IC50 value of LDOX at 72 h indicated increased accumulation of the drug in the OS cells. LDOX plus IMH resulted in a 61% lower cell viability compared to no treatment. Moreover, IMH potentiated the LDOX action on the Saos-2 cells by promoting ROS production at temperatures of <42 °C. There was a 12% increase in cell populations undergoing early apoptosis with a less heterogeneous distribution of Bax after combination treatment compared to those treated with LDOX (p < 0.05). Therefore, we determined that IMH could enhance LDOX delivery and its antitumor effect via altered membrane permeabilization, ROS generation, and a lower level of visualized Bax heterogeneity in the Saos-2 cells, suggesting the potential translation of these findings into in vivo studies.

Sodium Butyrate Enhances the Cytotoxic Effect of Etoposide in HDACi-Sensitive and HDACi-Resistant Transformed Cells.

To overcome the problem of antitumor agent toxicity for normal cells, a combined therapy using drugs with synergistic effects seems to be more effective. We investigated the molecular mechanisms of the sensitization of tumor cells resistant and sensitive to histone deacetylase inhibitors (HDACis) upon etoposide treatment together with the HDACi sodium butyrate (NaBut). We showed that NaBut enhances the cytotoxic effect of etoposide in both HDACi-sensitive and HDACi-resistant cells due to the accumulation of the Bax protein and the dissociation of Ku70-Bax inhibitory complexes. In HDACi-resistant cells, NaBut causes the cytoplasmic accumulation of Bax dissociated from mitochondria in complexes with Ku70 proteins. The increased phosphorylation of the pro-apoptotic Bad protein due to the NaBut-induced activation of Erk and Akt kinases is one of the possible reasons for the accumulation of Bax in the cytoplasm. Despite the inactivation of Bax in HDACi-resistant cells, its accumulation in the cytoplasm upon NaBut treatment makes it possible to enhance the apoptotic response against agents activating the intrinsic pathway of apoptosis. Thus, HDACis involved in combined therapy mediate the sensitization of tumor cells to genotoxic drugs, regardless of the cells' resistance to HDACis.

Análise da imunoexpressão de BAX e MMP-9 no líquen plano oral / Analysis of BAX and MMP-9 immunoexpression in oral lichen planus

Introdução: O líquen plano oral é uma doença crônica imunologicamente mediada relativamente comum, que acomete a mucosa oral. Clinicamente, o LPO é classificado em seis padrões bem identificados: placa, reticular, bolhoso, atrófico, papular e erosivo.Sendo os mais comuns oos tipos reticulares e erosivos. A ativação dos linfócitos TCD4+ no LPO, pode induzir os ceratinócitos ao processo de apoptose através da respostaimunológica citotóxica. A proteína Bax desempenha uma função relevante para o processo apoptótico. Deste modo, a presente pesquisa consistiu em um estudo transversal retrospectivo, descritivo, quantitativo e comparativo. Objetivo: Avaliar a expressão imuno-histoquímica das proteínas MMP9 e Bax no LPO. Método: Foram utilizados 43 casos de LPO para análise da imunoexpressão de Bax e MMP-9. Os resultados foram analisados através dos testes estatísticos apropriados e serão considerados significativos, valores onde p<0,05. Resultado: A imunoexpressão de MMP9 foi significativamente maior nos ceratinócitos e quando analisados os subtipos de líquen plano oral, não foram observados diferenças estatísticas entre os tipos reticulares e erosivos para as proteínas analisadas. Conclusões: Com essas observações, infere-se que a alteração na expressão das proteínas estudadas sugere um distúrbio nos mecanismos apoptóticos, os quais estão associados às lesões de LPO, e podemos concluir também que as imunoexpressões dessas proteínas não apresentaram diferença, quando relacionada ao tipo clínico reticular ou erosivo. Com esse resultado pode-se contribuir para um maior entendimento sobre os possíveis mecanismos celulares envolvidos na etiopatogenia dessa lesão (AU).

Background: Oral lichen planus is a relatively common immune-mediated chronic disease that affects the oral mucosa. Clinically, OLP is classified into six well-identified patterns: plaque, reticular, bullous, atrophic, papular, and erosive. The most common being the reticular and erosive types. The activation of TCD4+ lymphocytes in the OLP can induce keratinocytes to the process of apoptosis through the cytotoxic immune response. Thus, the present research consisted of a retrospective, descriptive, quantitative and comparative crosssectional study. Objective: to evaluate the immunohistochemical expression of MMP-9 and Bax proteins in OLP. Methods: We used 20 cases of Inflammatory Fibrous Hyperplasia as control. The results were analyzed through the appropriate statistical tests and will be considered significant, values where p<0.05. Results: The immunoexpression of MMP-9 was significantly higher in keratinocytes and when the subtypes of oral lichen planus were analyzed, no statistical differences were observed between the reticular and erosive types for the proteins analyzed. Conclusions: With these observations, it is inferred that the alteration in the expression of the studied proteins suggests a disturbance in the proliferative and apoptotic mechanisms, which are associated with a pathological behavior of the oral mucosa, and consequently with a repercussion on the lesions of OLP, and we can also conclude that the immunoexpression of these proteins had no difference, when related to the reticular or erosive clinical type. This research aims to contribute to a greater understanding of the possible cellular mechanisms involved in the etiopathogenesis of this lesion, thus enabling the understanding of the clinical aspects of the pathology (AU).

Gamma irradiation induces a pro-apoptotic state in longer stored platelets, without progressing to an overt apoptosis by day 7 of storage.

BACKGROUND: Although gamma-irradiation to platelet products is a standard method to prevent the risk of TA-GVHD in vulnerable recipients, it induces some proteomic and redox changes, of which irradiation-induced ROS increments may potentiate platelet mitochondrial dysfunction. However, whether these changes cause platelet apoptosis, or affect their viability during storage, is the main subject of this study. METHODS: PLT-rich plasma PC was split into two bags, one kept as control while other was subjected to gamma-irradiation. Within 7-days storage, cytosolic and mitochondrial levels of cytochrome c and pro-apoptotic molecules of Bak and Bax were evaluated by western-blotting. Intraplatelet active caspase (using FAM-DEVD-FMK) and PS-exposure were detected by flowcytometry. Caspase activity in platelet lysate was also confirmed by immunofluorescence detection of Caspase-3/7 Substrate N-Ac-DEVD-N'-MC-R110 while platelet viability was evaluated with MTT assays. RESULTS: Cytosolic cytochrome c gradually increased while its mitochondrial content steadily declined during 7 days of storage. In a contrary trend, reverse patterns were observed for Bak and Bax expressions. Gamma-irradiated platelets showed higher release of mitochondrial cytochrome c that reflected by higher cytosolic cytochrome c levels on day 7 of storage. Concurrently mitochondrial pro-apoptotic Bak and Bax proteins increased on day 7 in irradiated products. However, gamma-irradiation didn't significantly increase caspase activity or PS-exposure, nor did it decrease platelet viability. CONCLUSION: Here, consistent with studies on "gamma-irradiation-induced oxidative stress", we showed that gamma-ray also increases platelet pro-apoptotic signals during storage, although not strongly enough to affect platelet viability by overt apoptosis induction. Conclusively, whether supplementing ROS scavengers or antioxidants to irradiated platelets can improve their quality during storage may be of interest for future research.

Alpha-Pinene Exerts Antiseizure Effects by Preventing Oxidative Stress and Apoptosis in the Hippocampus in a Rat Model of Temporal Lobe Epilepsy Induced by Kainate.

Oxidative stress and apoptosis following seizures play pivotal roles in the consequences of repeated seizures. Beneficial effects of alpha-pinene (APN) have been reported in some experimental models of neurodegenerative diseases. However, its neuroprotective efficacy in a rat model of temporal lobe epilepsy (TLE) induced by kainic acid (KA) has remained unexplored. We aimed to explore the possible antiseizure effects of APN pretreatment and underlying molecular mechanisms in a rat model of TLE induced by KA. TLE was induced in male Wistar rats by intracerebroventricular injection of KA. APN at a dose of 50 mg/kg/day was intraperitoneally injected for 2 weeks before induction of TLE. One day after the induction of TLE, behavioral expressions of seizure were recorded and scored using Racine's scale. Furthermore, the hippocampal levels of oxidative stress markers, B-cell lymphoma 2 (Bcl2), BCL2-associated X protein (BAX), and c-Jun N-terminal kinase (JNK) protein levels were also assessed. Histopathological assessment in the hippocampus was performed with Nissl staining 5 days following induction of TLE. The results revealed that APN pretreatment alleviated epileptic seizures, diminished oxidative stress indicators, blocked the mitochondrial apoptotic pathway via decreasing BAX and raising BCL2 protein levels in the hippocampus at least partly through inhibiting JNK activity, and decreased neuronal death in the CA3 and hilus regions. These findings reveal that APN pretreatment mitigates KA-induced seizures by blocking oxidative stress and neuronal damage factors. It can be concluded that APN has a potent potential to be considered an antiseizure medication, but it needs further investigation.

Para­hydroxybenzaldehyde against transient focal cerebral ischemia in rats via mitochondrial preservation.

Gastrodia elata (GE) Blume has been widely used for thousands of years to treat various central and peripheral nervous disorders. P-hydroxybenzaldehyde (PHBA) is a chemical component of GE. However, its role and mechanism in transient focal cerebral ischemia remain unclear. The present study aimed to investigate the protective effect of PHBA on middle cerebral artery occlusion (MCAO) rats. A total of 56 Sprague-Dawley male rats were randomly divided into control, model, PHBA-high dose (PHBA-H) and PHBA-low dose (PHBA-L) groups. The MCAO injury model was replicated in all rats except for the control group. In the control group, only the right common carotid artery was isolated without embolization. After treatment with PHBA, the protective effects (neurological deficit score, cerebral index, weight and cerebral infarct) were analyzed. Western blotting was performed to estimate the protein levels of Bcl-2, Bax and Caspase-3. Apoptotic cells were detected using hematoxylin-eosin staining and TUNEL immunofluorescence assay. Mitochondrial oxidative stress indicators, including reactive oxygen species (ROS), malondialdehyde (MDA) and total superoxide dismutase (T-SOD), while dysfunction indicators, including mitochondrial permeability transition pore (MPTP), ATP and cytochrome C oxidase, were measured using commercial kits. The ultrastructure of mitochondria was observed under an electron microscope. Once the model was successful established, the rats in the MCAO group suffered neurological damage (P<0.001), increased cerebral index (P<0.001), decreased body weight (P<0.001) and had severe cerebral infarction (P<0.001). Moreover, the number of apoptotic cells and the levels of ROS (P<0.001) and MDA (P<0.05) in mitochondria and the protein levels of Bax (P<0.001) and cleaved caspase-3 (P<0.001) were increased. The activities of T-SOD (P<0.001) and cytochrome C oxidase (P<0.001) in the mitochondria, ATP content (P<0.05) and Bcl-2 protein level (P<0.001) decreased, MPTP was stimulated to open and mitochondrial structures were damaged (P<0.001). PHBA treatment resulted in a decrease of the neurological deficit score (PHBA-H 24 h, P<0.001; PHBA-H 6 h and PHBA-L 24 h, P<0.01; PHBA-L 6 h, P<0.05), apoptotic cell number (P<0.001), mitochondrial ROS (P<0.001) and MPTP opening (P<0.001), Bax (P<0.01, P<0.001) and cleaved caspase-3 protein expression (P<0.001) in rats. And the expression of Bcl-2 protein (P<0.001) was increased. In addition, the cerebral index (P<0.05), weight loss (P<0.05), infarction rate (P<0.01) and MDA content (P<0.001) were decrease in the PHBA-H group. The level of ATP (P<0.05) and cytochrome C oxidase (P<0.05) and T-SOD activity (P<0.05) of PHBA-H group rats increased, but no significant difference was observed in the PHBA-L group. Overall, PHBA had a protective effect on transient focal cerebral ischemia in normal rats, regulated the expression of Bcl-2, Bax and cleaved caspase-3 proteins and improved the oxidative stress and dysfunction of mitochondria.

Anti-tumor effects of PEGylated-nanoliposomes containing ginger extract in colorectal cancer-bearing mice.

Objectives: This study aimed to develop a nanoliposomal formulation containing ginger ethanolic extract with a higher therapeutic effect for cancer treatment. Materials and Methods: The present study aimed to prepare PEGylated nanoliposomal ginger through the thin film hydration method plus extrusion. Physicochemical characteristics were evaluated, and the toxicity of the prepared liposomes was assessed using the MTT assay. In addition, tumor size was monitored in colorectal cancer-bearing mice. Also, the anticancer effects of liposomal ginger were evaluated by gene expression assay of Bax and Bcl-2 and cytokines including TNF-α, TGF-ß, and IFN-γ by Real-time PCR. Also, cytotoxic T lymphocytes (CTLs) and regulatory T lymphocytes (Treg cells) were counted in spleen and tumor tissue by flow cytometry assay. Results: The nanoliposomes' particle size and polydispersity index (PDI) were 94.95 nm and 0.246 nm, respectively. High encapsulation capacity (80 %) confirmed the technique's efficiency, and the release rate of the extract was 85% at pH 6.5. In addition, this study showed that liposomal ginger at 100 mg/kg/day enhanced the expression of Bax (P<0.05) and IFN-γ (P<0.01) compared with ginger extract in the mouse model. Also, the number of tumor-infiltrating lymphocytes (TILs) and CTLs cell count in tumor tissue showed a significant increase in the LipGin group compared with the Gin group (P<0.05). Conclusion: Results indicated that the liposomal ginger enhanced the antitumor activity; therefore, the prepared liposomal ginger can be used in future clinical trials.

Cytotoxic terpenoids from Tripterygium hypoglaucum against human pancreatic cancer cells SW1990 by increasing the expression of Bax protein.

ETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium hypoglaucum (Kunmingshanhaitang in Chinese) is a plant of the genus Tripterygium which have been used as anti-tumor folk medicines in Yi and Bai ethnic groups in Yunnan province, China for hundreds of years. Terpenoids from T. hypoglaucum presented therapeutic effects on multiple tumors. But there were few studies about pancreatic cancer treatment of these terpenoids. Pancreatic cancer is an aggressive malignancy and lacked of specific drugs. Currently, anti-tumor drugs have poor therapeutic effect and prognosis for pancreatic cancer. AIM OF THE STUDY: This study aimed to elucidate the terpenoids from T. hypoglaucum and illuminate their anti-pancreatic cancer bioactivities. MATERIAL AND METHODS: Terpenoids were obtained through sequential chromatographic methods including silica gel, MCI gel, Sephadex LH-20, and preparative HPLC. Their structures were determined by HRESIMS, 1D and 2D NMR spectroscopic analysis. The absolute configurations of some new diterpenoids were assigned through comparison of experimental and calculated circular dichroism spectra. The cytotoxicity of isolates was measured using the MTT method on human pancreatic cancer cells SW1990. The effects on expressions of AKT, Erk1/2, p-AKT, p-Erk1/2, and Bax proteins in human pancreatic cancer cells SW1990 of these compounds were determined by western blotting assays. RESULTS: Eleven new (compounds 1∼11) and fourteen known terpenoids (compounds 12∼25) were isolated from the underground parts of T. hypoglaucum. These compounds were belonged to abietane diterpenoids, isoprimara diterpenoids, ent-kaurane diterpenoids, oleanane triterpenoids, and friedelane triterpenoids. Compounds 5, 7, 8, 9, 16, 18, 22, 24, and 25 possessed significant cytotoxicity against SW1990 cells with IC50 values of 19.28 ± 4.39, 9.91 ± 2.23, 27.32 ± 5.89, 56.43 ± 6.92, 0.16 ± 0.05, 0.58 ± 0.15, 0.81 ± 0.04, 0.48 ± 0.11, and 10.01 ± 1.39 µM respectively. After compounds 16, 22, and 24 been treated with the pancreatic cancer cells in medium and high doses, the protein expressions of AKT, p-AKT, Erk, and p-Erk were not remarkably reduced and the expressions of Bax protein were significantly increased. CONCLUSION: This study indicated that terpenoids from T. hypoglaucum could inhibit human pancreatic cancer cells SW1990. Especially, compounds 16, 22, and 24 possessed significant cytotoxicity against SW1990 cells with low IC50 values and could increase the expressions of Bax protein. These compounds shared a wide variety of structural characteristics which provided us more candidate molecules for the development of anti-pancreatic cancer drugs and further prompted us to investigate their anti-pancreatic mechanisms.

Lysophosphatidic Acid Alters The Expression of Apoptosis Related Genes and miR-22 in Cultured and Autotransplanted Ovaries.

OBJECTIVE: The aim of this study was to evaluate the effect of lysophosphatidic acid (LPA) on the follicular development, incidence of cell death, and expressions of apoptosis related genes and miR-22 in transplanted ovaries. MATERIALS AND METHODS: In this experimental study, three-week-old mice ovaries were cultured for 24 hours in the presence and absence of LPA, and we assessed cell survival and normal follicular rates in some of the cultured ovaries. The remaining cultured ovaries were autotransplanted in the presence and absence of LPA as four experimental groups (LPA-/LPA-, LPA-/LPA+, LPA+/LPA-, LPA+/LPA+). The follicular development, immunohistochemistry for BAX, and expressions of genes related to apoptosis and miR-22 by real time reverse transcription polymerase chain reaction (RTPCR) were studied at the first oestrous cycles in the recovered ovaries. Sera 17-ß-oestradiol (E2) and progesterone (P4) levels were also assessed. RESULTS: Both cell survival and normal follicular rates were significantly higher in cultured ovaries in the presence of LPA after 24 hours (P<0.05). There was an increase in follicular development in comparison with the intact control group in the four transplanted groups (P<0.05). The LPA+/LPA- group had significantly higher follicular development, a decline in BAX positive cells, and a decrease in pro-apoptotic gene expressions in parallel with enhanced expression of anti-apoptotic and miR-22 genes and higher levels of hormones compared with the non-treated and intact control groups (P<0.05). CONCLUSION: LPA, as a survival factor, improves follicular development in transplanted ovaries by providing a balance between the anti- and pro-apoptotic genes in association with an increase in miR-22 expression.

[Effect of an antioxidant on vascular wall cell apoptosis markers after reconstructive operations]. / Vliianie antioksidanta na markery apoptoza kletok sosudistoi stenki posle rekonstruktivnykh operatsii.

AIM: This study was aimed at determining Bcl-2 and Bax proteins expression before and after reconstructive-repairing operations in patients with atherosclerosis obliterans of lower extremities and at assessing the effect of an antioxidant (vitamin E at a dose of 100 mg once daily for 1 month after surgery) on the dynamics of changes of Bcl-2 and Bax proteins in the postoperative period. PATIENTS AND METHODS: The study included a total of 60 patients with stage III-IV lower limb atherosclerosis obliterans. All patients underwent reconstructive-repairing operations on the arteries of the aortofemoral segment. After surgery the patients were divided into two groups. Group A included 30 patients who during 1 month received additionally to basic therapy vitamin E at a daily dose of 100 mg. Group B was composed of 30 patients receiving basic therapy alone according to the National guidelines of managing patients with peripheral artery disease. All patients before, on POD 1, and 1 month after surgery were subjected to venous blood test aimed at determining Bcl-2 and Bax apoptosis proteins expression by means of enzyme-linked immunosorbent assay. RESULTS: In patients of groups A and B, the baseline level of Bcl-2 protein (4.75 and 4.2 ng/ml, respectively) was comparable with that in apparently healthy volunteers (5.3 ng/ml). The baseline levels of Bax protein in patients of the operated groups (26.9 and 26.0 ng/ml, respectively) were increased compared with the values in healthy volunteers (16.5 ng/ml). On POD 1 there was increased expression of Bax protein in Group A and B patients to 39.4 and 30.2 ng/ml, respectively. One month after surgery, Group B patients demonstrated a decrease in the Bcl-2 values below the baseline level - 1.1 ng/ml (p=0.003), with the Bax level continuing to increase - 36 ng/ml (p=0.004). In turn, Group A patients after 1 month were found to have increased levels of the Bcl-2 protein - 5.75 ng/ml, with the Bax level returning to the baseline values - 27.4 ng/ml. CONCLUSION: In stage III and IV lower limb obliterating atherosclerosis, the level of the Bax proapoptoric protein was higher than that in healthy volunteers. On POD 1, there occurred increased expression of the pro-apoptotic protein Bax and activation of apoptosis markers. On the background of using vitamin E at a dose of 100 mg once daily for 1 month, there was a decrease in level of the Bax propapoptotic protein (p=0.003) and an increase in level of the anti-apoptotic Bcl-2 protein level (p=0.0007).

Follicular development and the expression of BAX and vascular endothelial growth factor in transplanted ovaries in uni- and bilateral ovariectomized mice: An experimental study.

BACKGROUND: Several conflicting results have been reported on the survival and function of transplanted ovaries. OBJECTIVE: Evaluation of the follicular development and the expression of vascular endothelial growth factor (VEGF) and Bcl-2-associated X protein (BAX) in ovaries transplanted into uni- and bilaterally ovariectomized mice. MATERIALS AND METHODS: In this experimental study, 40 female NMRI mice (21-days-old, 12-15 gr) were ovariectomized uni- and bilaterally (n = 20/ group), while the 8-wk-old mice were considered as intact control group (n = 6). 5 weeks after transplantation at the proestrus stage, the morphology of recovered transplanted ovaries and the proportion of follicles were studied at different developmental stages. The apoptosis cell death by pro-apoptotic protein BAX and the expression of VEGF were evaluated using immunohistochemistry. RESULTS: In the bilaterally ovariectomized mice, among the 455 counted normal follicles, a lower rate of primordial and primary follicles and a higher rate of preantral and antral follicles were observed (p = 0.002). However, the percentages of preantral and antral follicles, and the corpus luteum were significantly lower in the intact control group (among the 508 counted normal follicles in this group) compared to other transplanted groups (p = 0.002). The number of BAX-positive cells in all groups was not significantly different. The VEGF expression was prominent in vessels of the corpus luteum, and also in the theca layer of large follicles of studied groups. CONCLUSION: Early discharge of ovarian reserve was prominent in the bilaterally ovariectomized group but the incidence of apoptotic cells and VEGF expression as angiogenic factor did not differ in both ovariectomized mice. Thus, unilaterally ovariectomy has less side effects on the ovarian reserve compared to bilateral ovariectomy.

Total flavonoids of hawthorn leaves promote motor function recovery via inhibition of apoptosis after spinal cord injury.

Flavonoids have been reported to have therapeutic potential for spinal cord injury. Hawthorn leaves have abundant content and species of total flavonoids, and studies of the effects of the total flavonoids of hawthorn leaves on spinal cord injury have not been published in or outside China. Therefore, Sprague-Dawley rats were used to establish a spinal cord injury model by Allen's method. Rats were intraperitoneally injected with 0.2 mL of different concentrations of total flavonoids of hawthorn leaves (5, 10, and 20 mg/kg) after spinal cord injury. Injections were administered once every 6 hours, three times a day, for 14 days. After treatment with various concentrations of total flavonoids of hawthorn leaves, the Basso, Beattie, and Bresnahan scores and histological staining indicated decreases in the lesion cavity and number of apoptotic cells of the injured spinal cord tissue; the morphological arrangement of the myelin sheath and nerve cells tended to be regular; and the Nissl bodies in neurons increased. The Basso, Beattie, and Bresnahan scores of treated spinal cord injury rats were increased. Western blot assays showed that the expression levels of pro-apoptotic Bax and cleaved caspase-3 were decreased, but the expression level of the anti-apoptotic Bcl-2 protein was increased. The improvement of the above physiological indicators showed a dose-dependent relationship with the concentration of total flavonoids of hawthorn leaves. The above findings confirm that total flavonoids of hawthorn leaves can reduce apoptosis and exert neuroprotective effects to promote the recovery of the motor function of rats with spinal cord injury. This study was approved by the Ethics Committee of the Guangxi Medical University of China (approval No. 201810042) in October 2018.

Insulin receptor substrate 1 may play divergent roles in human colorectal cancer development and progression.

BACKGROUND: Despite effective prevention and screening methods, the incidence and mortality rates associated with colorectal cancer (CRC) are still high. Insulin receptor substrate 1 (IRS-1), a signaling molecule involved in cell proliferation, survival and metabolic responses has been implicated in carcinogenic processes in various cellular and animal models. However, the role of IRS-1 in CRC biology and its value as a clinical CRC biomarker has not been well defined. AIM: To evaluate if and how IRS-1 expression and its associations with the apoptotic and proliferation tumor markers, Bax, Bcl-xL and Ki-67 are related to clinicopathological features in human CRC. METHODS: The expression of IRS-1, Bax, Bcl-xL and Ki-67 proteins was assessed in tissue samples obtained from 127 patients with primary CRC using immunohistochemical methods. The assays were performed using specific antibodies against IRS-1, Bax, Bcl-xL, Ki-67. The associations between the expression of IRS-1, Bax, Bcl-xL, Ki-67 were analyzed in relation to clinicopathological parameters, i.e., patient age, sex, primary localization of tumor, histopathological type, grading, staging and lymph node spread. Correlations between variables were examined by Spearman rank correlation test and Fisher exact test with a level of significance at P < 0.05. RESULTS: Immunohistochemical analysis of 127 CRC tissue samples revealed weak cytoplasmatic staining for IRS-1 in 66 CRC sections and strong cytoplasmatic staining in 61 cases. IRS-1 expression at any level in primary CRC was associated with tumor grade (69% in moderately differentiated tumors, G2 vs 31% in poorly differentiated tumors, G3) and with histological type (81.9% in adenocarcinoma vs 18.1% in adenocarcinoma with mucosal component cases). Strong IRS-1 positivity was observed more frequently in adenocarcinoma cases (95.1%) and in moderately differentiated tumors (85.2%). We also found statistically significant correlations between expression of IRS-1 and both Bax and Bcl-xL in all CRC cases examined. The relationships between studied proteins were related to clinicopathological parameters of CRC. No significant correlation between the expression of IRS-1 and proliferation marker Ki-67, excluding early stage tumors, where the correlation was positive and on a high level (P = 0.043, r = 0.723). CONCLUSION: This study suggests that IRS-1 is co-expressed with both pro- and antiapoptotic markers and all these proteins are more prevalent in more differentiated CRC than in poorly differentiated CRC.

Effect and mechanism of allicin combined with 5-fluorouracil on proliferation and apoptosis of the MEC-1 cell line in mucoepidermoid carcinoma / 口腔疾病防治

Objective @#To investigate the effect and mechanism of allicin combined with 5-FU on proliferation inhibition and apoptosis of the mucoepidermoid carcinoma MEC-1 cell line in mucoepidermoid carcinoma in order to provide the corresponding basis for subsequent clinical drug application.@*Methods @# MEC-1 cells in the logarithmic growth phase were randomly divided into control groups and experimental groups. The control groups were PBS groups containing 0.1% DMSO, while the experimental groups were the allicin group, 5-FU group and combined drug group (the allicin combined with the 5-FU group). The proliferation inhibition rates of allicin, 5-FU and allicin combined with 5-FU in MEC-1 cells were detected by the CCK8 method at different concentrations (0, 25, 50, and 75 mg/L) for 24 h, and the IC50 value of allicin and 5-FU after 24 hours was calculated. The apoptotic rate of MEC-1 cells treated with allicin, 5-FU and allicin combined with 5-FU at different concentrations (0, 25, 50, and 75 mg/L) for 24 hours was measured by flow cytometry. The expression of Bax and Bcl-2 protein was determined by Western blot analysis of the IC50 concentration of allicin and 5-FU alone and in combination with MEC-1 cells for 24 hours. @*Results@#The growth inhibition rate and apoptosis rate of MEC-1 cells in the combined drug group were higher than those in the allicin group and the 5-FU alone group (P < 0.01). Allicin and 5-FU alone and in combination downregulated Bcl-2 protein and upregulated Bax protein expression, and the combined drug group had the largest ratio of Bax/Bcl-2 (P < 0.05). @*Conclusion @#Allicin and 5-FU both alone and in combination can inhibit the proliferation of and induce apoptosis in MEC-1 cells, and allicin can enhance the apoptosis of 5-FU in MEC-1 cells, which may be related to the apoptosis of the mitochondrial pathway.

Auraptene has neuroprotective and memory enhancing effects in a rat model of Alzheimer’s disease

@#Background: Auraptene is a simple coumarin that exhibits multiple protective activities in the brain. Alzheimer’s disease is a complex, multifactorial, and progressive neurodegenerative disease. Microinjection of the β-amyloid peptide (Aβ) into the hippocampus of rat has been recognized as a reliable and stable animal model of Alzheimer’s disease, which mimics the memory deficits. In the present study, the memory enhancing effects of auraptene were studied in rats that Aβ was injected into their hippocampus to create a model of Alzheimer’s disease. Methods: Different doses of auraptene (5, 10 and 25 mg/kg) were administered intraperitoneally to male Wistar rats. The spatial memory performance was tested by Morris water maze after Alzheimer`s induction. The hippocampal expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins were calculated for evaluating the neuroprotective and anti-apoptotic effects of Auraptene in the brain tissue. Results: In comparison with the control group, auraptene significantly decreased the escape latency time in the treated rats. In addition, auraptene increased the percentage of time spent and traveled pathway in the target quadrant. Molecular data showed that auraptene attenuated the Bax/Bcl-2 ratio in the hippocampus of rats. Conclusion: This study demonstrated the memory enhancing effect of Aur after Aβ injection, which could be through inhibiting the apoptotic pathways in the hippocampus of rats.

Somatostatin Type 2 Receptor Antibody Enhances Mechanical Hyperalgesia in the Dorsal Root Ganglion Neurons after Sciatic Nerve-pinch Injury: Evidence of Behavioral Studies and Bax Protein Expression.

BACKGROUND: Our previous study has indicated that somatostatin potently inhibits neuropathic pain through the activation of its type 2 receptor (SSTR2) in mouse dorsal root ganglion and spinal cord. However, the underlying mechanism of this activation has not been elucidated clearly. OBJECTIVE: The aim of this study is to perform the pharmacological studies on the basis of sciatic nerve-pinch mice model and explore the underlying mechanism involving SSTR2. METHODS: On the basis of a sciatic nerve-pinch injury model, we aimed at comparing the painful behavior and dorsal root ganglion neurons neurochemical changes after the SSTR2 antibody (anti- SSTR2;5µl,1µg/ml) administration in the mouse. RESULTS: After pinch nerve injury, we found that the mechanical hyperalgesia and severely painful behavior (autotomy) were detected after the application of SSTR2 antibody (anti-SSTR2; 5µl, 1µg/ml) on the pinch-injured nerve. The up-regulated phosphorylated ERK (p-ERK) expression and the apoptotic marker (i.e., Bax) were significantly decreased in DRGs after anti-SSTR2 treatment. CONCLUSION: The current data suggested that inhibitory changes in proteins from the apoptotic pathway in anti-SSTR2-treated groups might be taking place to overcome the protein deficits caused by SSTR2 antibody and supported the new therapeutic intervention with SSTR2 antagonist for neuronal degeneration following nerve injury.

Dl-3-n-butylphthalide inhibits phenytoin-induced neuronal apoptosis in rat hippocampus and cerebellum.

Rats were divided into six groups: sham/control , Dl-3-n-butylphthalide, P1 (low phenytoin, 100 mg/kg), P2 (high phenytoin, 200 mg/kg), NP1 (Dl-3-n-butylphthalide 80 mg/kg, phenytoin 100 mg/kg), NP2 (Dl-3-n-butylphthalide 80 mg/kg, phenytoin 200 mg/kg). Hematoxylin/eosin and Nissl staining showed that, compared to the sham/control group, the Dl-3-n-butylphthalide group had no obvious hippocampal and cerebellar neuron loss, but there was a significant neuron loss in the P1 and P2 groups (P < 0.05), which was more obvious in the P2 group (P < 0.05). The positive expression of Bax and Bcl-2 proteins in hippocampal and cerebellar neurons was not significantly different between sham and Dl-3-n-butylphthalide groups; however, compared to sham, Bax expression was significantly increased and Bcl-2 was significantly decreased in the hippocampal and cerebellar neurons of rats in both P1 and P2 groups (P < 0.05), being more obvious in the P2 group (P < 0.05). Furthermore, the administration of Dl-3-n-butylphthalide attenuated the deleterious effects of phenytoin (P < 0.05). Our results indicate that phenytoin causes apoptosis of hippocampal and cerebellar neurons in rats in a dose-dependent manner, with the effect of a higher dose being more obvious, whereas, Dl-3-n-butylphthalide inhibits the phenytoin-induced apoptosis of neurons and has a neuroprotective role.

Study on the mechanism of Sorafenib combined with Bevacizumab on the tumor inhibition of cervical cancer cell line U14 transplantation in mice / 国际外科学杂志

Objective According to effect of Sorafenib combined Bevacizumab in mice U14 cervical cancer cell lines,observe the changes of cell lines of transplanted tumor U14 cell structure and adjustment effect of Bcl-2 and Bax protein expression.Methods Sixty female 615 mice of healthy inbreeding line,aged from 4 to 6 weeks,average weight is 20.4 g,range from 18 to 25 g.The animals were divided into blank control group (group A)with 10 mice,and U14 cervical cancer cell lines by vaccinating mice a tumor-burdened mice model was established,on the 6th day after subcutaneous inoculation of U14,40 tumor-burdened mice with tumor diameter ≥ 5 mm were selected.The mice were divided into 5 groups according to the completely randomized grouping method:model group (group B),Sorafenib group (group C),low-dose Bevacizumab combined with Sorafenib group (group D),the middle dose Bevacizumab combined with Sorafenib group (group E),and the high dose Bevacizumab combined with Sorafenib group (group F),8 mice in each group.The treatment regimen consisted of 1 ml of 0.9％ sodium chloride solution in group A and group B,Sorafenib 12 mg/kg in group C,and Bevacizumab 2.5 mg/kg + Sorafenib 12 mg/kg in group D,Bevacizumab 5 mg/kg + Sorafenib 12 mg/kg in group E,and Bevacizumab 10 mg/ kg + Sorafenib 12 mg/kg in group F.All mice were injected intraperitoneally and sacrificed by dislocation 24 days later.The tumor weight (g) was measured every 6 days,and the body weight of each group was observed,and calculated the tumor inhibition rate of each group of mice.The histopathological changes of the transplanted mice were observed by hematoxylin-eosin staining.The transplantation of each group of mice was observed by electron microscope.Morphological changes of tumor tissue;the expression of Bcl-2 and Bax protein in each group were observed by immunohistochemistry.The measurement data were expressed as mean standard deviation (Mean ± SD),univariate analysis of variance was used for inter-group comparison,and LSD method was used for pairwise comparison.Results At the beginning of the intraperitoneal injection of the drug,the body weight of the mice began to increase slowly and continuously,and the trend of the mice in each group was basically the same.The tumor inhibition rate of group C was 8.02％,4.92％ in group D,11.10％ in group E,and 5.42％ in group F.Group E had the highest tumor inhibition rate and the best effect.The difference in tumor weight between group A and the other groups was statistically significant (P ＜ 0.05).The results of electron microscopy showed that the cell structure changes in C,D,E and F groups were similar,and the apoptosis of tumor cells appeared after treatment.The apoptosis of group E was the best.The apoptosis-related proteins in group C,D,E and F were observed.The expression of Bax was up-regulated,and the positive number in group E was the highest.The expression of proto-oncogene Bcl-2 was down-regulated in group C,D,E and F,and the number of positive in group E was the least,and the difference was statistically significant (P ＜ 0.05).Conclusions Sorafenib combined with Bevacizumab can promote tumor cell apoptosis by up-regulating Bax and down-regulating Bcl-2 protein expression.Among them,Sorafenib combined with Bevacizumab at medium dose promotes tumor cell apoptosis better than other methods,providing a basis for clinical treatment.

rTMS ameliorated depressive-like behaviors by restoring HPA axis balance and prohibiting hippocampal neuron apoptosis in a rat model of depression.

Repetitive transcranial magnetic stimulation (rTMS) has been widely used to treat depression. The mechanistic basis for the effects of rTMS is not well understood, although previous studies have suggested that it involves the regulation of hypothalamic-pituitary-adrenocortical (HPA) axis and protection of hippocampal neurons. We investigated this in the present study using a chronic unpredictable mild stress (CUMS) paradigm in Sprague-Dawley rats. The rats were subjected to rTMS for 15 consecutive days, and body weight, sucrose consumption, and locomotor activity were evaluated. B cell lymphoma-2-associated X protein (Bax) expression was assessed by immunohistochemistry; cell morphology was examined by Nissl staining; and adrenocorticotropic hormone (ACTH) and cortisol (CORT) levels in the hippocampus were measured by enzyme-linked immunosorbent assay. CUMS decreased body weight and sucrose consumption in rats along with horizontal/vertical distance traveled in the open field test. Rats subjected to CUMS also showed increased levels of Bax as well as ACTH and CORT; the hippocampal neurons in these animals had abnormal morphology and were reduced in number. rTMS reversed these changes and improved depression-like behaviors. Thus, rTMS abrogates the loss of hippocampal neurons and restores the balance of the HPA axis in the treatment of depression.

Bax protein structure and related research progress of eutectic structure / 国际生物医学工程杂志

Apoptosis involves multiple signaling pathways. The intrinsic signaling pathway is the mitochondrial apoptotic pathway, which is caused by a series of processes. First, the pro-apoptotic factors such as Bax are activated by signaling molecules and transfer to the mitochondrial outer membrane forming protein pores, thus the mitochondrial membrane permeability is affected, and then the downstream caspase-9 is activated and the apoptosis initiation is induced by releasing cytochrome C. In order to explore the apoptosis initiation activated by small molecules, the specific structural changes of Bax in apoptosis were studied. The results showed that there is a hydrophobic pocket structure near the C-terminal S184 of the Bax protein. This structure can be combined with certain small molecular substances specifically remove phosphorylation S184, and regulate Bax protein to promote apoptosis activity. At present, the nuclear magnetic structure of Bax protein has been obtained and the crystal structure has not been revealed. The eutectic structure formed by corresponding with other proteins in the Bcl-2 family has been resolved, which can be used to study the interactions between proteins and to understand the specific structural changes in the formation of heterologous dimers during apoptosis, site changes, etc. In this paper, the Bax protein structure resolved by nuclear magnetic structure was reviewed to learn the change of the sites in the induced apoptosis so as to promote the research on apoptosis initiation.