Urolithin B inhibits proliferation and migration and promotes apoptosis and necrosis by inducing G2/M arrest and targeting MMP-2/-9 expression in osteosarcoma cells.

Osteosarcoma (OS) is the most prevalent primary bone cancer, with a high morbidity and mortality rate. Over the past decades, therapeutic approaches have not considerably improved patients' survival rates, and further research is required to find efficient treatments for OS. Data from several studies have shown that urolithin B (UB), the intestinal metabolite of polyphenolic ellagitannins, is emerging as a new class of anticancer compounds, yet its effect on OS cancer cells remains elusive. Herein, we investigated UB's antimetastatic, antiproliferative, and apoptotic effects on the MG-63 OS cell line. Cell viability assay, annexin V/propidium iodide staining, cell cycle arrest analysis, determination of the gene expression of MMP-2, MMP-9, Bax, Bcl-2, and p53 messenger RNA (mRNA), evaluation of reactive oxygen species (ROS) generation and migration, and MMP-2 and MMP-9 protein expression assessments were performed. UB caused late apoptosis, necrosis, G2/M arrest, and ROS generation in MG-63 cells. It increased the mRNA expression of the p53 tumor suppressor and Bax proapoptotic genes. UB also inhibited the migration and metastatic behavior of MG-63 OS cells by downregulating mRNA and MMP-2 and MMP-9 protein expression. In general, although further in vivo investigations are warranted, the current results showed that UB might be utilized as a potential novel natural compound for OS therapy due to its nontoxic, antiproliferative, and antimetastatic nature.

Everolimus attenuates glutamate-induced PC12 cells death.

BACKGROUND: Glutamate-induced neuronal cell death plays a key role in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Some recent studies reported the potential immunomodulatory and neuroprotective properties of inhibitors of serine-threonine kinase, mTOR (mammalian target of rapamycin). However, no study was conducted about the neuroprotective potential of everolimus (EVR), a selective and potent mTOR inhibitor. Therefore, this study was planned to investigate whether EVR has protective effects against glutamate-induced toxicity in PC12 cells, which are used as model for neurons injury, and to elucidate the underlying mechanism. METHODS: PC12 cells were concurrently treated with glutamate (8 mM) and EVR (0-40 nM) for 24 h. Then, the cells viability, apoptosis rate, and apoptosis-related proteins (caspase-3, bax and bcl-2) were measured using MTT, annexin V/PI and immunoblotting assays. RESULTS: Analyzing the protective effect of different concentrations of EVR (0-40 nM) against glutamate-induced cytotoxicity revealed a significant increase in cell viability in co-treatment regimen (p < 0.01). Also, EVR (40 nM) significantly (p < 0.01) inhibited glutamate-induced apoptosis through depressing the elevation of bax/bcl-2 ratio and expression of cleaved caspase-3, concentration depend. CONCLUSION: The results demonstrated, for the first time, that EVR could protect against glutamate-mediated PC12 cell death via inhibiting apoptosis.

The Effects of Urolithin B and Auraptene on Quinolinic Acid-induced Toxicity in the SH-SY5Y Neuroblastoma Cell Line.

The pathological accumulation of quinolinic acid (QA) is often associated with neuritis and neuronal cell death in several neurodegenerative diseases, through the overproduction of free radicals. Urolithin B and auraptene have been reported to exert potent antioxidant effects - however, little is known about the protective effects of these compounds against QA-induced neurotoxicity. Therefore, this study aimed to explore the in vitro protective effects of urolithin B and auraptene against QA-induced neurotoxicity in the SH-SY5Y neuroblastoma cell line. The MTT assay was used to evaluate cell viability, and flow cytometry was carried out to evaluate effects on the cell cycle and apoptosis. The intracellular levels of reactive oxygen species (ROS) were also determined. Our findings showed that auraptene at non-toxic concentrations had no protective effect on QA-induced toxicity. However, urolithin B at concentrations of 0.6 µM and 2.5 µM enhanced the viability of cells treated with QA. Moreover, while the percentage of apoptotic cells (i.e. in the sub-G1 phase) was shown to significantly increase after QA treatment, pre-treatment with urolithin B reduced the number of these apoptotic cells. Furthermore, urolithin B, as an antioxidant, also significantly reduced QA-induced ROS production. Our findings suggest that urolithin B may possess potent antioxidant and neuroprotective effects against QA-induced neurotoxicity that merit further investigation.

Tumor-Inhibitory Effects of Zerumbone Against HT-29 Human Colorectal Cancer Cells.

Colorectal cancer (CRC) is the second cause of cancer-associated death globally. Recently, herbal medicinal products and, in particular, zerumbone have been widely studied and used for cancer treatment as they induce significant anti-cancer effects. However, there is limited information about the anti-cancer effects of zerumbone in CRC. Therefore, we aimed to investigate the in vitro anti-cancer effects of the zerumbone in CRC, focusing on cell apoptosis and migration. Anti-proliferative and anti-migratory effects of zerumbone on HT-29 cells were evaluated using MTT and scratch wound healing assay, respectively. Quantitative real-time PCR (qRT-PCR) was performed to determine the mRNA expression levels of migration and apoptosis-related genes. Apoptosis and cell cycle distribution were evaluated by flow cytometry. The intracellular level of reactive oxygen species (ROS) was measured using a ROS assay kit. Additionally, matrix metalloproteinase-2/-9 (MMP-2/-9) activity was determined using gelatin zymography. Zerumbone suppressed the viability of the HT-29 cells dose-dependently while having less cytotoxicity on normal NIH/3T3 cells. Zerumbone induced apoptosis in HT-29 cells and arrested the cell cycle in the G2/M phase. These effects were associated with alteration in the expression of apoptosis-related genes (up-regulation of Bax and down-regulation of Bcl-2 genes). Zerumbone also enhanced the generation of ROS in HT-29 cells. Furthermore, zerumbone significantly inhibited the migration of HT-29 cells and decreased MMP-2/-9 mRNA expression and activity. Our findings provide a potential use for zerumbone to induce apoptosis and suppress metastasis in HT-29 cells; thus, it could be developed as a promising natural agent for future CRC therapy.

Challenges of expressing recombinant human tissue factor as a secreted protein in Pichia pastoris.

Tissue factor (TF) is the core reagent in the prothrombin time (PT) assay. In this study, expression and α-factor mediated secretion of three forms of tissue factor (full-length TF (Full-TF), extracellular plus transmembrane domain (TED-TF), and only extracellular domain (ED-TF) were investigated in Pichia pastoris. The amino acid sequence of TF was obtained from the UniProt database, back-translated and codon-optimized for expression in Pichia pastoris. The Full-TF sequence was synthesized but the ED-TF, TED-TF coding fragments were extracted from the Full-TF by PCR. All the coding sequences were cloned into pPICZαA vector in-frame with the α-factor; and electroporated into KM71H. The culture supernatants and the cell lysates were analyzed using SDS-PAGE, dot-blotting, and Western-blotting for expression of TF. The Full-TF and TED-TF expression vector pPICZαA were successfully inserted into the KM71H, but the product was not detected in the SDS-PAGE analysis of the culture supernatant. However, ED-TF expression and secretion was verified by SDS-PAGE, dot blotting, and Western blotting. It seems that the TM domain in the Full-TF and TED-TF have an important role in impairing α-factor-mediated secretion of TF. Therefore, further investigation is necessary to overcome challenges of expressing Full-TF as a heterologous protein in P. pastoris.

Effects of Rhus coriaria L. hydroalcoholic extract on the lipid and antioxidant profile in high fat diet-induced hepatic steatosis in rats.

Oxidative stress is related to increased fat deposition in the liver, known as hepatic steatosis. The present study is an evaluation of the anti-oxidative and antihyperlipidemic effects of the hydroalcoholic extract of Rhus coriaria L. (HARE) in rats on a high-fat diet (HFD). Twenty male Wistar rats were divided into four groups: control, HFD, HFD + HARE 50 mg/kg/day, and HFD + HARE 250 mg/kg/day for 12 weeks. Animals were weighed weekly and treated with the HARE extract for 12 weeks by gavage. Subsequently, the histopathological changes, oxidative markers, and lipid profile were evaluated. Statistical analysis was performed using the one-way analysis of variance (ANOVA) for multiple comparisons. First, the active ingredients of the extract were determined by HPLC. Then, the levels in the serum lipid profile (TG, cholesterol, HDL, and LDL) in rats fed with the HFD + HARE were analyzed where a significant reduction was observed. The HFD proved to increase the activity of the liver enzymes, the serum lipid levels, and the malondialdehyde (MDA) level. The ferric-reducing antioxidant activity power (FRAP), catalase (CAT), and superoxide dismutase (SOD) catalytic activity were reduced in the liver homogenate of HFD rats compared to the controls. Additionally, the aforementioned liver enzymes activities were reduced in response to HARE. Evaluation of oxidative stress determined a reduction in the MDA level while a raised FRAP was confirmed. In accordance with the present results, histopathological observations have also demonstrated that HARE ameliorated grade-1 hepatic steatosis induced by HFD. Taken together, the findings of this study introduce HARE as a future potential therapeutic agent in treating hepatic steatosis and reducing oxidative damages of an HFD in the liver.

Antitumor Effects of 5-Aminolevulinic Acid on Human Malignant Glioblastoma Cells.

5-Aminolevulinic acid (5-ALA) is a naturally occurring non-proteinogenic amino acid, which contributes to the diagnosis and therapeutic approaches of various cancers, including glioblastoma (GBM). In the present study, we aimed to investigate whether 5-ALA exerted cytotoxic effects on GBM cells. We assessed cell viability, apoptosis rate, mRNA expressions of various apoptosis-related genes, generation of reactive oxygen species (ROS), and migration ability of the human U-87 malignant GBM cell line (U87MG) treated with 5-ALA at different doses. The half-maximal inhibitory concentration of 5-ALA on U87MG cells was 500 µg/mL after 7 days; 5-ALA was not toxic for human optic cells and NIH-3T3 cells at this concentration. The application of 5-ALA led to a significant increase in apoptotic cells, enhancement of Bax and p53 expressions, reduction in Bcl-2 expression, and an increase in ROS generation. Furthermore, the application of 5-ALA increased the accumulation of U87MG cells in the SUB-G1 population, decreased the expression of cyclin D1, and reduced the migration ability of U87MG cells. Our data indicate the potential cytotoxic effects of 5-ALA on U87MG cells. Further studies are required to determine the spectrum of the antitumor activity of 5-ALA on GBM.

Acute and sub-acute toxicity evaluation of the root extract of Rheum turkestanicum Janisch.

Despite the widespread use of Rheum turkestanicum in herbal medicine, no study has yet examined its in vivo toxicity. The aim of this study is to evaluate the acute and sub-acute toxicity of hydroalcoholic extract of R. turkestanicum root. In acute toxicity experiment, female and male mice (n = 5/group/sex) were orally administrated with the extract at single doses of 300, 2000 and 3000 mg/kg and observed for 14 days. In the sub-acute study, the extract was orally administered daily at doses of 100 and 400 mg/kg to male rats (n = 8) for 4 weeks. During the acute toxicity test, there were no deaths or any signs of toxicity observed after administration of the R. turkestanicum extract at 300 mg/kg, which was the no-observed-adverse-effect level (NOAEL). The extract at a dose of 3000 mg/kg led to the death of one female and one male mouse (LD50 > 3000 mg/kg). In sub-acute toxicity experiment, the extract induced no mortality or significant changes in body weight, general behaviors, hematological parameters, serum biochemical factors (related to the kidney and liver function), and histopathology of the heart, liver, kidney, and brain up to the highest dose tested of 400 mg/kg (NOAEL). High-performance liquid chromatography-mass spectrometry revealed the presence of phenolic compounds, flavonoids, alkanes, and anthraquinones in the extract. In conclusion, short-term use of R. turkestanicum root does not appear to produce significant toxicity up to a dose of 400 mg/kg.

The role of HSP27 in the development of drug resistance of gastrointestinal malignancies: Current status and perspectives.

Heat-shock protein 27 (HSP27) is a chaperone molecule that plays a critical role in the refolding and activity of several proteins responsible for cancer cell drug toxicity. Upregulation of HSP27 is associated with decreased drug sensitivity as well as poorer survival in gastrointestinal (GI) malignancies. It is, therefore, possible that HSP27 may be of value in the assessment of prognostic and therapeutic efficacy in the treatment of GI cancers. Pharmacological and biological inhibitors of HSP27 enhance tumor cell chemosensitivity. This review summarizes the potential role of HSP27 in chemotherapy drug resistance and the therapeutic potential of HSP27 inhibitors as a novel strategy in the treatment of GI cancers.

5'-Adenosine monophosphate-activated protein kinase: A potential target for disease prevention by curcumin.

Curcumin (diferuloylmethane), a yellowish agent extracted from turmeric, is a bioactive compound known for its anti-inflammatory, antiproliferative, antidiabetic, and anticancer activities. Multiple lines of evidence have indicated that curcumin regulates several regulatory proteins in the cellular signal transduction pathway. AMP-activated protein kinase (AMPK) is one of the central regulators of cellular metabolism and energy homeostasis, which is activated in response to increasing cellular adenosine monophosphate/adenosine triphosphate ratio. AMPK plays a critical role in regulating growth and reprogramming metabolism and is linked to several cellular processes including apoptosis and inflammation. Recently, it has been demonstrated that AMPK is a new molecular target affected by curcumin and its derivatives. In this review, we discuss recent findings on the targeting of AMPK signaling by curcumin and the resulting impact on the pathogenesis of proinflammatory diseases. We also highlight the therapeutic value of targeting AMPK by curcumin in the prevention and treatment of proinflammatory diseases, including cancers, atherosclerosis, and diabetes.

Current status and future prospective of Curcumin as a potential therapeutic agent in the treatment of colorectal cancer.

Colorectal cancer (CRC) is the third most common cause of cancer-related deaths worldwide. Hence there is a need to identify new therapeutic agents that improve the current repertoire of chemotherapeutic drugs. The antitumor activity of curcumin has been reported for several tumors, including CRC. A recent phase I trial showed that curcumin is safe and tolerable adjunct to FOLFOX (5-fluorouracil, folinic acid and oxaliplatin) chemotherapy in patient-derived colorectal liver metastases at doses up to 2 g daily. Another trial revealed the effect of combining curcumin with FOLFOX in patients with inoperable colorectal cancer. The aim of current review was to summarize the current knowledge about possible molecular mechanisms of curcumin in CRC with particular emphasis on preclinical and early clinical studies of colorectal cancer.

Auraptene inhibits migration, invasion and metastatic behavior of human malignant glioblastoma cells: An in vitro and in silico study.

Objective: The present work examined the anti-metastatic effects of auraptene and their underlying mechanisms of action in U87 Glioblastoma multiforme (GBM) cells. Materials and Methods: To test the hypothesis, cell culture, Matrigel invasion assay, scratch wound healing assay, gelatin zymography assay, qRT-PCR, and western blot experiments were conducted. Results: At sublethal concentrations of 12.5 and 25 µg/ml, auraptene exhibited a significant reduction in cell invasion and migration of U87 cells, as assessed using scratch wound healing and Transwell tests, respectively. The qRT-PCR and zymography experiments demonstrated a significant decrease in both mRNA expression and activities of MMP-2 and MMP-9 following auraptene treatment. Western blot analysis also showed that MMP-2 protein level and phosphorylation of metastasis-related proteins (p-JNK and p-mTOR) decreased in auraptene-treated cells. Molecular docking studies consistently demonstrated that auraptene exhibits a significant affinity towards MMP-2/-9, the ATP binding site of mTOR and JNK1/2/3. Conclusion: Auraptene inhibited the migration and invasion of GBM cells. This inhibitory effect was induced by modulating specific mechanisms, including suppressing MMPs, JNK, and mTOR activities.

Notice of retraction: Auraptene-induced cytotoxicity mechanisms in human malignant glioblastoma (U87) cells: role of reactive oxygen species (ROS).

[This retracts the article DOI: 10.17179/excli2019-1136.].

Alpha-Lipoic Acid, Auraptene, and Particularly Their Combination Prevent the Metastasis of U87 Human Glioblastoma Cells.

Background: The primary malignant brain tumor glioblastoma multiforme (GBM) is most commonly detected in individuals over 60 years old. The standard therapeutic approach for GBM is radiotherapy combined with temozolomide. Recently, herbal products, such as alpha-lipoic acid (ALA) and auraptene (AUR), have shown promising anticancer effects on various cancer cells and animal models. However, it is not well understood how ALA, AUR, and their combination in GBM work to combat cancer. Thus, the purpose of this study was to investigate the antimetastatic effects of the ALA-AUR combination on U87 human glioblastoma cells. Methods: The inhibitory effects of ALA, AUR, and the ALA/AUR combination on the migration and metastasis of U87 cells were evaluated using a wound healing test and gelatin zymography. The expression levels of matrix metalloproteinase MMP-2 and MMP-9 were assessed at the transcriptional and translational levels using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. Results: Our findings revealed that combination therapy reduced cell migration and metastasis, which was indicated by the reduction in MMP-2/-9 expression both at mRNA and protein levels, as well as their enzymatic activity in U87 cells. Conclusion: This study demonstrated that the combination of ALA and AUR effectively inhibited the migration and metastasis of U87 cells. Thus, given their safety and favorable specifications, the combination of these drugs can be a promising candidate for GBM treatment as primary or adjuvant therapy.

Advances in the treatment of human T-cell lymphotropic virus type-I associated myelopathy.

INTRODUCTION: Nearly 2-3% of those 10 to 20 million individuals infected with the Human T-cell lymphotropic virus type-1 (HTLV-1); are predisposed to developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is a neuro-inflammatory disease; differentiated from multiple sclerosis based on the presence of typical neurologic symptoms, confirmation of HTLV-1 infection, and other molecular biomarkers. AREAS COVERED: A brief review of the epidemiology, host immune responses, and molecular pathogenesis of HAM/TSP is followed by detailed discussions about the host-related risk factors for developing HAM/TSP and success/failure stories of the attempted management strategies. EXPERT OPINION: Currently, there is no effective treatment for HAM/TSP. Anti-retroviral therapy, peculiar cytokines (IFN-α), some anti-oxidants, and allograft bone marrow transplantation have been used for treating these patients with limited success. Under current conditions, asymptomatic carriers should be examined periodically by a neurologist for early signs of spinal cord injury. Then it is crucial to determine the progress rate to adapt the best management plan for each patient. Corticosteroid therapy is most beneficial in those with acute myelitis. However, slow-progressing patients are best managed using a combination of symptomatic and physical therapy. Additionally, preventive measures should be taken to decrease further spread of HTLV-1 infection.

Rigosertib is more potent than wortmannin and rapamycin against adult T-cell leukemia-lymphoma.

Human T lymphotropic virus type 1 (HTLV-1) infection can cause adult T-cell lymphoblastic leukemia (ATLL), an incurable, chemotherapy-resistant malignancy. In a quest for new therapeutic targets, our study sought to determine the levels of AKT, mTOR, and PI3K in ATLL MT-2 cells, HTLV-1 infected NIH/3T3 cells (Inf-3T3), and HTLV-1 infected patients (Carrier, HAM/TSP, and ATLL). Furthermore, the effects of rigosertib, wortmannin, and rapamycin on the PI3K/Akt/mTOR pathway to inhibit the proliferation of ATLL cells were examined. The results showed that mRNA expression of Akt/PI3K/mTOR was down-regulated in carrier, HAM/TSP, and ATLL patients, as well as MT-2, and Inf-3T3 cells, compared to the healthy individuals and untreated MT-2 and Inf-3T3 as controls. However, western blotting revealed an increase in the phosphorylated and activated forms of AKT and mTOR. Treating the cells with rapamycin, wortmannin, and rigosertib decreased the phosphorylated forms of Akt and mTOR and restored their mRNA expression levels. Using these inhibitors also significantly boosted the expression of the pro-apoptotic genes, Bax/Bcl-2 ratio as well as the expression of the tumor suppressor gene p53 in the MT-2 and Inf-3T3cells. Rigosertib was more potent than wortmannin and rapamycin in inducing sub-G1 and G2-M cell cycle arrest, as well as late apoptosis in the Inf-3T3 and MT-2 cells. It also synergized the cytotoxic effects of vincristine. These findings demonstrate that HTLV-1 downregulation of the mRNA level may occur as a negative feedback response to increased PI3K-Akt-mTOR phosphorylation by HTLV-1. Therefore, using rigosertib alone or in combination with common chemotherapy drugs may be beneficial in ATLL patients.

Analysis of Cytotoxic Effects of Zerumbone in Malignant Glioblastoma Cells.

Glioblastoma multiforme (GBM) is an aggressive tumor in the central nervous system with a poor prognosis. Currently, the main interventions include surgery, chemotherapy, and radiotherapy. Recently, several natural products have been reported as potentially effective and safer treatment options. Here, we studied the effects of zerumbone, a sesquiterpene compound derived from Zingiber zerumbet Smith rhizomes, on human GBM U-87 MG cells in vitro. To meet this purpose, we used a cytotoxicity assay, as well as a quantitative polymerase chain reaction of apoptosis-related genes and western blot analysis of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a transcription factor that controls the production of cytokines and molecules involved in cell survival.

Neuroprotective effects of the fractions of Ocimum basilicum in seizures induced by pentylenetetrazole in mice.

Objective: Neuroprotective and antioxidant effects of Ocimum basilicum (O. basilicum) against pentylenetetrazole (PTZ)-induced seizures were investigated. Materials and Methods: Mice were divided as follows: (Group 1) Control, (Group 2) PTZ, (Groups 3-5) 50,100 and 200 mg/kg hydro-ethanolic (HE) extract, and (Groups 6-8) 200 mg/kg ethyl-acetate (EAF), N-hexane (NHF) and water (WF) fractions. Minimal clonic seizures (MCS) and generalized tonic-clonic seizures (GTCS) latencies were measured. Biochemical and histological studies were done. Results: MCS and GTCS latency in HE groups were longer than the PTZ group (p<0.05 to p<0.001). EAF and NHF prolonged the onset of MCS and GTCS (p<0.001). PTZ increased malondialdehyde (MDA) and dark neuron (DN) production while decreased thiol, catalase (CAT) and superoxide dismutase (SOD) (p<0.05 to p<0.001). Pre-treatment by HE and all fractions of the plant attenuated MDA and DN while increased thiol, CAT and SOD (p<0.01 to p<0.001). Conclusion: EAF and NHF had anticonvulsant properties. The extract and fractions protected the brain from PTZ-induced oxidative damages and showed neuroprotective effects.

Impact of Curcumin on Hepatic Low-Density Lipoprotein Uptake.

Elevated levels of plasma low-density lipoprotein cholesterol (LDL-C) are causally related to atherosclerotic cardiovascular disease. Enhancing the removal of LDL particles from the plasma, mainly by the liver, is the most efficient strategy for reducing LDL-C and the ensuing atherosclerosis. In this context, polyphenolic compounds like curcumin have generated interest owing to their lipid-modifying capacity. The promising effect of curcumin has been studied in attenuating atherosclerosis (in experimental models), and correcting dyslipidemia (in clinical studies). The underlying mechanisms of the effects of curcumin are relatively unknown, and the impact of curcumin on hepatic LDL uptake warrants further investigations. Here, we present a protocol to assess the effects of curcumin on LDL uptake in hepatocytes.

Anticancer Mechanisms of Berberine: A Good Choice for Glioblastoma Multiforme Therapy.

The most typical malignant brain tumor, glioblastoma multiforme (GBM), seems to have a grim outcome, despite the intensive multi-modality interventions. Literature suggests that biologically active phytomolecules may exert anticancer properties by regulating several signaling pathways. Berberine, an isoquinoline alkaloid, has various pharmacological applications to combat severe diseases like cancer. Mechanistically, it inhibits cell proliferation and invasion, suppresses tumor angiogenesis, and induces cell apoptosis. The antitumoral effect of berberine in GBM is increasingly recognized. This review sheds new light on the regulatory signaling mechanisms of berberine in various cancers, proposing its potential role as a therapeutic agent for GBM.