Effects of linalool on postoperative peritoneal adhesions in rats.

The current study examines the effects of linalool in preventing postoperative abdominal adhesions. Twenty male Wistar rats were randomly divided into four groups. (1) Sham: in this group, the abdomen was approached, and without any manipulations, it was sutured. (2) Control: rats in this group underwent a surgical procedure to induce adhesions. This involved making three incisions on the right abdominal side and removing a 1×1-cm piece of the peritoneum on the left abdominal side. (3) Treatment groups: these groups underwent the same surgical procedure as the control group to induce adhesions. Animals in these groups received linalool orally with doses of 50 and 100 mg/kg, respectively, for a period of 14 days. Moreover, rats in the sham and control groups received normal saline via gavage for 14 days. The evaluation of TNF-α, TGF-ß, VEGF, and caspase 3 was performed using western blot and IHC methods. Furthermore, oxidative stress biomarkers such as MDA, TAC, GSH, and NO were assessed in the peritoneal adhesion tissue. The findings revealed that linalool significantly reduced peritoneal adhesions by reducing TNF-α, TGF-ß, VEGF, and caspase 3 levels. Moreover, MDA concentration was significantly decreased, while NO, TAC, and GSH levels were notably increased. Overall, linalool was effective in preventing adhesion formation and reduced inflammation, angiogenesis, apoptosis, and oxidative stress. Therefore, linalool as a potent antioxidant is suggested for reducing postoperative adhesions in rats.

Lectins and lectibodies: potential promising antiviral agents.

In nature, lectins are widely dispersed proteins that selectively recognize and bind to carbohydrates and glycoconjugates via reversible bonds at specific binding sites. Many viral diseases have been treated with lectins due to their wide range of structures, specificity for carbohydrates, and ability to bind carbohydrates. Through hemagglutination assays, these proteins can be detected interacting with various carbohydrates on the surface of cells and viral envelopes. This review discusses the most robust lectins and their rationally engineered versions, such as lectibodies, as antiviral proteins. Fusion of lectin and antibody's crystallizable fragment (Fc) of immunoglobulin G (IgG) produces a molecule called a "lectibody" that can act as a carbohydrate-targeting antibody. Lectibodies can not only bind to the surface glycoproteins via their lectins and neutralize and clear viruses or infected cells by viruses but also perform Fc-mediated antibody effector functions. These functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP). In addition to entering host cells, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein S1 binds to angiotensin-converting enzyme 2 (ACE2) and downregulates it and type I interferons in a way that may lead to lung disease. The SARS-CoV-2 spike protein S1 and human immunodeficiency virus (HIV) envelope are heavily glycosylated, which could make them a major target for developing vaccines, diagnostic tests, and therapeutic drugs. Lectibodies can lead to neutralization and clearance of viruses and cells infected by viruses by binding to glycans located on the envelope surface (e.g., the heavily glycosylated SARS-CoV-2 spike protein).

Cytotoxic effect of sea anemone pore-forming toxin on K562 chronic myeloid leukemia cells.

Chronic myelogenous leukemia (CML) is one of prevalent cancer worldwide. In spite of various designed drugs, chemoresistance remains the main obstacle in cancer cure. Therefore, developing novel strategy for treatment of CML is an urgent need. Fragaceatoxin C (FraC) is novel protein toxin from a sea anemone called actinia fragacea with great impacts against cells by pore formation and disturbing cell membrane integrity. The aim of this study was evaluation of FraC toxin toxicity against K562. The bacteria cells harboring expression||||||| vector of FraC were induced by IPTG and purified by Ni2+-NTA sepharose affinity chromatography. Then, purified toxin activity was evaluated using RBC hemolytic test. Eventually, evaluation of FraC cytotoxicity and apoptosis were performed using MTT and flow cytometery assays, respectively. Our results revealed that FraC toxin decreased K562 cells viability in a dose- and time-dependent manner with a whole destroy of cancer cells at 35.00 µg mL-1 after 72 hr. Furthermore, flow cytometery analysis indicated that FraC toxin enhanced necrosis along with apoptosis in K562 cells in a dose dependent manner. We speculated that FraC toxin could be considered as a novel candidate for cancer cell researches and treatments provided that it should be turned into a specific agent by engineering and directing to cancer cell membrane.

Activation of STAT3/HIF-1α/Hes-1 axis promotes trastuzumab resistance in HER2-overexpressing breast cancer cells via down-regulation of PTEN.

BACKGROUND: Resistance to the HER2-targeted antibody trastuzumab remains to be a major clinical challenge in the treatment of HER2-positive breast cancer. Hyper-activation of STAT3 is proposed to be a predictive biomarker of trastuzumab resistance. However, the precise mechanism(s) remains poorly defined. Evidence is emerging that HIF-1α, a central downstream element of STAT3 pathway, serves a pivotal role in the complex signaling network with subsequent diverse cellular events. MATERIAL AND METHODS: We have established trastuzumab resistant SKBR3 cells (SKBR3-TR). The cell viability, apoptosis as well as western blot, siRNA transfection and co-immunoprecipitation assays were performed to evaluate the involvement of STAT3/HIF-1α in modulation of trastuzumab resistance. RESULTS: We found that in SKBR3-TR cells and conditioned medium-treated parental cells, constitutive phosphorylated STAT3 coincided with prominent up-regulation of HIF-1α which was accompanied with PTEN attenuation. Moreover, the inhibition of STAT3 activation by Stattic and/or genetically STAT3 knocking down decreased HIF-1α level in SKBR3-TR cells. Additionally, treatment with Stattic and/or STAT3 siRNA engendered the up-regulation of PTEN protein in STAT3-inhibited resistant cells. Restoration of PTEN was also observed following siRNA-mediated silencing of HIF-1α expression. Moreover, down-regulation of HIF-1α caused a reduction in the HES-1 content. Further study with HES-1 specific siRNA revealed the elevation of PTEN expression in HES-1 knock-down trastuzumab resistant cells. CONCLUSION: The impairment of STAT3-HIF-1α-HES-1 pathway restored trastuzumab sensitivity through up-regulation of PTEN protein. GENERAL SIGNIFICANCE: These findings highlighted the signal integrator role of HIF-1α in STAT3-mediated trastuzumab resistance induction which would be valuable in designing more efficient chemosensitization strategies.

Mycophenolic acid potentiates HER2-overexpressing SKBR3 breast cancer cell line to induce apoptosis: involvement of AKT/FOXO1 and JAK2/STAT3 pathways.

Trastuzumab has been successfully used as a first-line therapy specific for HER2-overexressing breast cancer patients. However, despite the effectiveness of trastuzumab, the occurrence of inherent and acquired resistance remains as the main challenge of the therapy. Thus, this has motivated efforts toward finding new therapeutic strategies including combining trastuzumab with other drugs to enhance its therapeutic efficacy. In that line, we investigated the capability of mycophenolic acid (MPA), an inhibitor of de novo guanine nucleotide synthesis with potential anti-cancer activity, on improving the response to trastuzumab among SKBR3 cells as well as trastuzumab resistant SKBR3-TR cells. Our data indicated that irrespective to trastuzumab sensitivity of cells, MPA effectively inhibited cell growth through inducing adipocyte-like cell differentiation as well as blocking cell cycle progression at G1 phase along with augmentation of p27kip expression level. Furthermore, combined treatment with trastuzumab and MPA was more potent in cell growth inhibition, cell cycle arrest and apoptosis induction, as evident by flow cytometric analyses and caspase-3 production, in both trastuzumab sensitive and resistant SKBR3 cells. Besides, western blot analysis showed that elevated apoptosis induction in both cell groups was associated with attenuation in phosphorylation of some key elements of HER2 signaling pathway including AKT, ERK, STAT3 and consequently augmentation in FOXO1 expression level in response to combination of trastuzumab and MPA. These data suggest that manipulation of intracellular GTP level by MPA and consequent molecular perturbation in some of the cell survival and pro-apoptotic relevant signaling pathways might provide an alternative clinical strategy for chemosensitization of resistant breast cancer cells to anti- HER2 therapy.

Combating trastuzumab resistance by targeting thioredoxin-1/PTEN interaction.

Trastuzumab is by far the drug of choice for treatment of human epidermal growth factor receptor 2 (Her2) overexpressing breast cancer patients. However, frequently, the therapy remains ineffective due to the induced drug resistance. In spite of various reported mechanisms, we hypothesize that the acquired resistance to trastuzumab might be attributed to the failure of the drug to activate phosphatase and tensin homolog (PTEN) mainly due to the high level of reduced thioredoxin-1 protein among the resistant cells. In the present study, the effect(s) of PX-12, a Trx-1 inhibitor, was examined on proliferation of breast cancer cells which are unresponsive to trastuzumab. Treatment of the cells with PX-12 (5 µM) and trastuzumab (10 µg/ml) reduced cells viabilities, p-Akt, and Bcl2 levels while increasing the levels of reactive oxygen species (ROS) and p-JNK with consequent higher levels of G1 arrest and apoptosis among the resistant cells compared to parental trastuzumab sensitive cells. The most significant observation was that PX-12/trastuzumab co-treatment enhanced the cell membrane localization of PTEN which is believed to be the active biological form of the signal. Our data confirmed that Trx-1 inhibition is required for chemosensitization of resistant breast cancer cells to anti-Her2 therapy, and this approach might offer an alternative clinical strategy for preventing acquired resistance.

Anti-apoptotic and anti-inflammatory effects of Silybum marianum in treatment of experimental steatohepatitis.

In this study, we were aimed to evaluate the probable effect of the crud extract of Silybum marianum, with high polyphenolic content, on experimental nonalcoholic steatohepatitis (NASH). To induce NASH, a methionine and choline deficient (MCD) diet was given to N-Mary rats for 8 weeks. After NASH development, MCD-fed rats were divided into two groups: MCD groups received MCD diet and MCD+S group was fed MCD diet plus crude extract of S. marianum orally for 3 weeks. Control group was fed a normal diet for 11 weeks. Finally, all rats were sacrificed. Plasma alanine amino transferase (ALT) and aspartate amino transferase (AST) levels were evaluated. In addition, the following hepatic factors were also evaluated: liver histology, malondialdehyde (MDA) and reduced glutathione (GSH) contents, gene expressions of TNF-α and TGF-ß and immunoblot evaluations of caspase-3, ERK/p-ERK, JNK/pJNK and p38/pp38. Histopathological evaluations of the liver samples revealed that treatment with the S. marianum extract has abated the severity of NASH among the MCD-fed rats. Also, a significant reduction was observed in the sera ALT and AST activities. In addition, the extract caused dramatic reduction in the elevated hepatic TNF-α and TGF-ß mRNA and MDA levels along with an increase in the GSH content. Moreover, the plant extract treatments significantly lowered activation of procaspase-3 to active caspase-3 and also lowered the phosphorylated form of JNK among the same group of rats. These results suggest that the S. marianum crude extract beneficial effects on NASH are mainly due to its antioxidant and anti-inflammatory activities.

Teucrium polium reversed the MCD diet-induced liver injury in rats.

In the present study, we evaluated the ability of Teucrium polium ethyl acetate fraction, with high antioxidant activity, in the treatment of nonalcoholic steatohepatitis (NASH) in rats and its possible effect on factors involved in pathogenesis of the disease. To induce NASH, a methionine and choline deficient (MCD) diet was given to N-Mary rats for 8 weeks. After NASH development, MCD-fed rats were divided into 2 groups: NASH group that received MCD diet and NASH + T group which was fed MCD diet plus ethyl acetate fraction of T. polium orally for 3 weeks. Histopathological evaluations revealed that treatment with the extract has abated the severity of NASH among the MCD-fed rats. In addition, the fraction reduced the elevated levels of hepatic tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-ß) gene expression and also the elevated level of malondialdehyde (MDA). In addition, the extract increased the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and enhanced the level of hepatic glutathione (GSH). Moreover, the fraction treatments lowered caspase-3 level and the phosphorylated form of C-Jun N-terminal kinase (JNK) and augmented the phosphorylated level of extracellular regulated kinase1/2 (ERK1/2). These results indicate that the ethyl acetate fraction of T. poium effectively reversed NASH, mainly due to its strong antioxidant and anti-inflammatory properties.

Effects of Teucrium polium on Insulin Resistance in Nonalcoholic Steatohepatitis.

Nonalcoholic fatty liver disease, the most common chronic liver disorder, is frequently associated with the clinical features of metabolic syndrome such as insulin resistance. We aimed to determine the effect of the crude and the ethyl acetate extracts of Teucrium polium on insulin resistance in rats with nonalcoholic steatohepatitis. Rats were divided into four groups. Group A was fed a normal diet for 11 weeks. Nonalcoholic steatohepatitis was induced in the remaining groups using a methionine/choline deficient (MCD) diet for 8 weeks. After nonalcoholic steatohepatitis development, group B continued with receiving the MCD diet alone; group C rats were given the MCD diet along with crude extract of T. polium (equivalent to 1 g leaves powder/kg body weight/day); group D rats were given the ethyl acetate fraction of T. polium by intragastric administration for 3 weeks. MCD diet led to grade 1 liver steatosis. In group C and D, these factors abated to grade 0 in 80% of the rats. In the groups receiving the extract, lipoprotein profiles were significantly improved relative to those not receiving the extract. Also, a dramatic reduction was observed in sera alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase activities. In addition, in groups C and D, an increase in the activities of liver superoxide dismutase, glutathione peroxidase and glutathione reductase enzymes were also associated with a decrease in the malondialdehyde level relative to group B. Moreover, both extracts significantly decreased plasma glucose and insulin levels along with insulin resistance. In conclusion, both extracts of T. polium could reverse the adverse effects of an MCD diet.

Inhibition of JNK along with activation of ERK1/2 MAPK pathways improve steatohepatitis among the rats.

BACKGROUND & AIMS: Oxidative stress-induced hepatocyte apoptosis is implicated in the onset of non-alcoholic steatohepatitis (NASH). Regarding the pronounced roles of mitogen activated protein kinases (MAPKs) in oxidative stress-induced cellular damages and the development of NASH, we evaluated the effect of Teucrium polium ethyl acetate (EtoAc) extract on rats with NASH and on the modulation of MAPKs activities. METHODS: To induce NASH, a methionine choline deficient diet (MCD) was given to N-Mary rats. These animals were then compared to the control group receiving normal diet and/or MCD diet plus EtoAc extract (0.5g/kg/day). After 8 weeks, liver histopathology, malondialdehyde (MDA) and immunoblot analyses of caspase-3 cleavage, ERK/pERK and JNK/pJNK were examined. RESULTS: Simultaneous treatments with MCD diet and the EtoAc extract resulted in pronounced improvements in liver steatosis, ballooning degeneration and inflammation. Elevated MDA and caspase-3 levels among MCD-fed rats were also markedly decreased. In addition, the EtoAc extract treatments caused significant reduction in the phosphorylated form of JNK along with an increase in the phosphorylated level of ERK1/2. CONCLUSION: These results indicate that the anti-apoptotic effect of T. polium extract is mediated through inhibition of ROS generation. Moreover, the inhibitory effect of T. polium on the development and progression of NASH is apparently governed by the attenuation of JNK activation and augmentation of ERK1/2 activity through phosphorylation.