P53 Gene Expression Research Articles

Protective Effects of Peroxiredoxin 6 in Pro-Inflammatory Response Model Using Raw 264.7 Macrophages.

The aim of the work was to study effects of peroxiredoxin 6 (PRDX6), a recombinant antioxidant protein, on the level of pro-inflammatory responses of RAW 264.7 macrophages to endotoxin exposure. Addition of LPS to the RAW 264.7 cell culture medium expectedly increased production of TNF-α, and addition of PRDX6 led to a significant (15-20%) decrease in its production. The level of production of another pro-inflammatory cytokine, IL-1β, which was significantly activated by endotoxin, was completely normalized under the PRDX6 action. Moreover, addition of PRDX6 reduced production of reactive oxygen species (ROS) induced by endotoxin and also prevented overexpression of the iNos gene in the RAW 264.7 cells. The results showed that PRDX6 had a suppressive effect on the expression of Nrf-2 gene and production of the transcription factor NRF-2 during the first 6 h of cell cultivation. Addition of endotoxin caused activation of the NF-κB and SAPK/JNK signaling cascades, while in the presence of PRDX6, activity of these signaling cascades decreases. It is known that the pro-inflammatory response of cells caused by exposure to bacterial LPS leads to activation of apoptosis and elimination of the damaged cells. Our studies confirm this, since exposure to LPS led to activation of the expression of P53 gene, a marker of apoptosis. Peroxiredoxin 6 added within the first hours of the development of acute pro-inflammatory response suppressed the P53 gene expression, indicating protective effect of PRDX6 that reduced apoptosis in the RAW 264.7 macrophages.

In Vitro and In Vivo Synergistic Antitumor Activity of Albumin-Coated Oleic Acid-Loaded Liposomes toward Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers, closely associated with cirrhosis and fibrosis. This study aimed to assess the antitumor activity of oleic acid-liposomes (uncoated liposomes) upon coating with albumin against HCC. The in vitro studies revealed the high safety of the prepared uncoated and albumin-coated liposomes to normal HFB-4 cells (EC100 of 35.57 ± 0.17 and 79.133 ± 2.92 µM, respectively) with significant anticancer activity against HepG-2 cells with IC50 of 56.29 ± 0.91 and 26.74 ± 0.64 µM, respectively. The albumin-coated liposomes revealed superior apoptosis induction potential (80.7%) with significant upregulation of p53 gene expression (7.0-fold), compared to OA. The in vivo study revealed that the administration of uncoated or albumin-coated liposomes (100 mg/kg) for six weeks markedly retarded the DENA-induced HCC in Wistar albino rates through regulating the liver enzymes, total bilirubin level, pro-inflammatory cytokines, and oxidative stress. Accordingly, the current study supports the in vitro and in vivo chemo-preventive feature of albumin-coated liposomes against HCC through modulation of apoptosis, improvement of the immune response, reduction of inflammation, and restoration of impaired oxidative stress, which is the first reported to the best of our knowledge.

The investigation of the effects of calcitriol on human ovarian carcinoma cells

Objective: Ovarian cancer is the fifth leading cause of cancer death in women, the leading cause of death from gynecologic malignancies, and the second most commonly diagnosed gynecologic malignancy, however the underlying pathophysiology is not clearly understood. Calcitriol, the active form of vitamin D serves its activity through binding to vitamin D receptor (VDR). Calcitriol regulates multiple signaling pathways such as proliferation, apoptosis, differentiation, inflammation, invasion, angiogenesis and metastasis. It has been found to have a broad effect on several cancer types such as colon, breast and prostate cancer. Therefore, the aim of the study was to investigate the effects of calcitriol on the human ovarian cancer cells.
 Material and methods: The human MDAH-2774 ovarian carcinoma cells were exposed to different dose ranges of calcitriol for 24 and 48 hours. Cultured cells were evaluated in terms of MTT assay and quantitative- Real time-PCR.
 Results: As evidenced by MTT assay, calcitriol treatment resulted in the reduction of cell viability in human MDAH-2774 cells. The gene expressions of VDR and p53 were increased with the calcitriol treatment compared to control. Additionally the gene expression of proapoptotic marker Bax was increased and the anti-apoptotic marker Bcl-2 decreased with the presence of the calcitriol.
 Conclusion: In conclusion calcitriol treatment decreased cell proliferation and induced apoptosis in ovarian cancer cells, therefore we can suggest that alone calcitriol or in combination with chemotherapy drugs may be useful for treatment of ovarian cancer.

Acovenoside A as a novel therapeutic approach to boost taxol and carboplatin apoptotic and antiproliferative activities in NSCLC: Interplay of miR-630/miR-181a and apoptosis genes

The aim of the present study is to explore the potential anticancer effect of the cardenolide; acovenoside A against non-small cell lung cancer (NSCLC), understand its molecular mechanism in inducing apoptosis and show the effect of its combination with carboplatin and taxol. MTT assay showed that the combination of acovenoside A with taxol and carboplatin caused 78.9% cytotoxicity reflecting the synergistic effect. The triple combination showed the best growth inhibition efficiency where the number of cells at the G2/M phase was decreased and boosted up apoptotic and necrotic activity. The combination also showed the most remarkable increase in gene expression of Bax and p53 and the least level of Bcl2. The gene expression of miRNA181a and miRNA630 was significantly upregulated in cell lines treated with the combination. The present study has proven that the underlying mechanism of acovenoside A is partially attributed to the upregulation of miR-630 and miR-181a gene expressions which in turn targets the intrinsic apoptosis genes as p53, Bax and Bcl2 as well as caspase 3. The present study is the first to address the valuable effect of using acovenoside A together with carboplatin and taxol in the treatment of NSCLC via exerting apoptotic, antiproliferative, and cytotoxic effects..

Regulation of tight junction proteins and cell death by peroxisome proliferator-activated receptor γ agonist in brainstem of hypertensive rats.

The decrease in tight junction proteins and their adapter proteins in the hypertensive brain is remarkable. Here, we aimed to investigate tight junction proteins and peroxisome proliferator-activated receptor (PPARγ) activation as well as inflammation factors and cell death proteins in the brainstem of hypertension models, namely spontaneously hypertensive rats (SHR) and borderline hypertensive rats (BHR). At first, SHR and BHR groups were treated with PPARγ agonist, pioglitazone. Then, occludin, claudin-1, claudin-2, claudin-12, ZO-1, and NF-κB p65 gene expression levels; pIKKβ, NF-κB p65, TNF, IL-1β, caspase-3, caspase-9 levels, and PARP-1 cleavage were evaluated. Significantly lower pIKKβ, NF-κB p65, TNF, and IL-1β levels were measured in pioglitazone-treated SHR. Results from this study confirm higher occludin (1.35-fold), claudin-2 (7.45-fold), claudin-12 (1.12-fold), and NF-κB p65 subunit (4.76-fold) expressions in the BHR group when compared to the SHR group. Pioglitazone was found effective in terms of regulating gene expression in SHR. Pioglitazone significantly increased occludin (8.17-fold), claudin-2 (2.41-fold), and claudin-12 (1.85-fold) mRNA levels, which were accompanied by decreased cleaved caspase-3, caspase-9 levels, PARP-1 activation, and proinflammatory factor levels in SHR (p ˂ 0.05). Our work has led us to conclude that alterations in tight junction proteins, particularly occludin, and cell death parameters in the brainstem following PPARγ activation may contribute to neuroprotection in essential hypertension.

Evaluation of the effect of iron oxide nanoparticles functionalized by glucose and conjugated with coumarin (Fe3O4@Glu-Coumarin NPs) on the expression of CASP8, CASP9, p53, mTOR1, and MAPK1 genes in liver cancer cell line

Due to the high morbidity and mortality of liver cancer, many researchers are seeking novel anticancer formulations. Complex iron oxide containing compounds could be used for site directed drug delivery using a magnetic force. Due to the anticancer property of Coumarin, this study aimed to study the effect of iron oxide nanoparticles functionalized by glucose and conjugated with Coumarin (Fe3O4@Glu-Coumarin NPs) on the expression of cell cycle and apoptosis regulating genes in a liver cancer cell line. Fe3O4@Glu-Coumarin NPs were synthesized and their physicochemical properties were evaluated by FT-IR, XRD (X-Ray diffraction), EDS mapping, and electron microscope imaging. Anti-proliferative activity of the NPs on HepG2 cells was studied by MTT assay. The effect of Fe3O4@Glu-Coumarin NPs on the expression of CASP8, CASP9, p53, mTOR1, and MAPK1 genes was investigated by quantitative PCR. Moreover, the apoptogenic impact of the Fe3O4@Glu-Coumarin NPs was assessed using annexin V- Fluorescein isothiocyanate (FITC) - propidium iodide (PI) staining and cell cycle analysis. The synthesized NPs were spherical, with a moderate level of aggregation and in a size range of 26–46 nm. FT-IR, XRD, and EDS mapping confirmed the proper synthesis and purity of the synthesized NPs. The zeta potential value of the NP was −23 mV. According to the MTT assay, Fe3O4@Glu-Coumarin NPs showed a considerable anti-proliferative effect on liver cancer cells, and the 50 % inhibitory concentration was determined 120 μg/mL. Quantitative PCR assay showed that the NPs significantly increased the expression of CASP8, p53, and MAPK1 genes by 1.94, 4.87, and 3.87 folds, respectively, while the mTOR1 gene was reduced by −6.33 folds. The expression of the CASP9 had an insignificant reduction by 0.06 folds. Annexin V/PI staining assay showed an increase in frequency of apoptotic cells in comparison to control cells. Cell cycle analysis confirmed an increase in the number of the cells in G0/G1step among nanoparticles treated cells. Our results showed that Fe3O4@Glu-Coumarin NPs could induce cell apoptosis by interfering with the expression of the cell regulatory genes.

Efficacy of Prunus armenica, Bee venom, and their combinations on p53 and Bcl-2 gene expression in human pancreatic and lung cancer cells

Malignancies are one of the world’s most serious health problems. Pancreatic and lung cancers are two of the most lethal types of cancer, with 5-year survival rates ranging from 10 to 20%. Despite decades of investigation, the molecular processes and selectivity of Prunus armenica and Bee venom as anticancer drugs are still largely un-understood. The goal of our study is to determine the expression levels ofp53 and Bcl-2 mRNAs obtained from human pancreatic (PANC-1) and lung (A549) cancer cells treated with Prunus armenica, Bee venom or their combinations by using MTT and real time PCR assays. We showed that in a concentration-dependent manner of Prunus armeniaca, bee venom, and a combination of both, the cell growth was significantly decreased. Our findings also revealed an adverse association between tumor suppressor (p53) and proto-oncogene (Bcl-2) gene expression in both cancer cells treated with diverse substances vs untreated. As a consequence, the tested compounds dramatically alter p53 and Bcl-2 gene expression, suggesting that they might be useful in the prevention or treatment of pancreatic and lung cancers.

Ethanolic Extract of Artemisia herba-alba Ameliorates Hyperinsulinemia and Hyperglycemia-Induced HepG2 Cells Through Cell Survival Promotion, Oxidative Stress Mitigation, and Insulin Signaling Restoration

Background: In recent years, diabetes and its risk factors were linked to an augmented occurrence of cardiovascular diseases (CVD), which are considered major causes of morbidity and mortality in diabetes mellitus subjects. Hyperinsulinemia (HI) and hyperglycemia (HG) are recognized as insulin resistance-inducers, which can trigger several alterations in cellular biogenesis besides inflammatory signaling activation. As a folk medicine in many countries, Artemisia herbaalba has been preclinically studied for treatment of type 2 diabetes mellitus (T2DM) and showed beneficial effects on hyperglycemia. Methods: In the current work, we explored the potential mechanisms underlying the antihyperglycemic behavior of the ethanolic extract of A. herba-alba and its bioactive compounds. Results: Obtained data demonstrated that ethanolic extract of A. herba-alba enhances HepG2 viability and proliferation, decreases considerably the apoptosis, through the regulation of pro- and anti-apoptotic pathways implying p53, p21 and Bcl-2 genes expression and Pan caspases activation under IR concentration at the lowest concentrations. A. herba-alba extract treatment additionally exhibited a potential antioxidant effect, evidenced by the significant decrease in total intracellular reactive oxygen species (ROS) levels and the improved mitochondrial transmembrane potential in HG/HI-challenged HepG2 cells (p < 0.001). Furthermore, A. herba-alba positively upregulated Irs1 and Irs2 as well as Akt/Pi3K pathway compared to insulin-resistant untreated cells, and subsequently stimulated glucose uptake in response to insulin infusion, as a consequence of its signal transduction restoration. Conclusion: Overall, the obtained data highlighted the beneficial effects of A. herba-alba ethanolic extract in ameliorating insulin signaling and liver cells metabolic balance, and shed the light for its use as a promising and safe therapeutic lead for the management of type 2 diabetes and underlying metabolic failures.

P-395 Protective effect of metformin on the ovarian reserve of prepubertal mice under cyclophosphamide chemotherapy

Abstract Study question Can metformin reduce the side effects of cyclophosphamide on the ovaries of prepubertal mice treated with cyclophosphamide? Summary answer Yes, metformin, as an inhibitor of mTOR and, by affecting the HIPPO pathway, can exert its protective effect in the chemotherapy mice model. What is known already PI3K/AKT and HIPPO pathways are influential pathways in the process of activation of primordial follicles and proliferation of cell growth, which are affected by various agents and drugs such as cyclophosphamide. One of the side effects of cyclophosphamide is the reduction of the ovarian reserve through abnormal changes in the PI3K/AKT and HIPPO pathways. Metformin is a drug that is used to treat type 2 diabetes and at the same time regulates PI3K/AKT and HIPPO pathways and prohibits the proliferation of cancer cells. Study design, size, duration Controlled experimental study. After metformin and cyclophosphamide dose adjustment, 12 female NMRI 14-day-old mice were randomly divided into four groups: the control group (CONT), metformin group (MET), cyclophosphamide group (CYC) and metformin with cyclophosphamide group (MET-CYC). Participants/materials, setting, methods Mice were treated by intraperitoneal injection of a dose of 150mg/kg of metformin for 11 consecutive days, and/or a dose of 65mg/kg of cyclophosphamide every three days (The third, sixth and ninth days). The mice were sacrificed 24 hours after the last metformin injection or 3 days after the last cyclophosphamide injection. The ovaries were collected for stereological evaluation, and expression of Pten, Mtor, Yap-1, p53, Bcl-2, and Bax genes by qRT-PCR method. Main results and the role of chance The stereological evaluation showed that the ovarian volume and primordial follicle number in the CYC group were decreased compared to the other groups (P < 0.05), and also in the MET-CYC group reduced compared to the CONT and MET groups, but increased significantly compared to the CYC group (P < 0.05). A significantly increased number of growing follicles was observed in the CYC group compared to the other groups (P < 0.05). The molecular analysis showed that the Pten expression in the CYC group was lower than CONT and MET groups (P < 0.05). Mtor expression in the CYC group was significantly increased compared to other groups (P < 0.05). The expression of Yap-1 was significantly decreased in the MET group compared to other groups (P < 0.05), and was lower in the MET-CYC than in the CYC group (P < 0.05). The of P53 expression in the MET-CYC group was higher than CONT and MET groups (P < 0.05), and was increased in the CYC compared to the CONT group (P < 0.05). Bcl-2 expression was higher in CONT and MET groups than in MET-CYC and CYC groups (P < 0.05). The expression of Bax in the CYC group was significantly increased compared to CONT and MET groups (P < 0.05). Limitations, reasons for caution Due to ethical issues in less use of mice, dose of metformin was obtained according to the articles used in adult mice and also by using the human to animal dose conversion formula. Wider implications of the findings It can be concluded that metformin during cyclophosphamide treatment can maintains ovarian volume, reserve, and activity in prepubertal mice by regulating PI3K/AKT and HIPPO pathways. Considering the relatively acceptable cost and long-term safety of metformin, it is a promising factor to be used to preserve fertility during chemotherapy. Trial registration number not applicable

Sub-chronic exposure of Oreochromis niloticus to environmentally relevant concentrations of smaller microplastics: Accumulation and toxico-physiological responses

This study assesses the accumulation and toxic effects of environmentally relevant concentrations (0.01, 0.1 and 1 mg/L) of polystyrene MPs (1 µm) in Oreochromis niloticus (Nile tilapia) for 14 days. The results showed that 1 µm PS-MPs accumulated in the intestine, gills, liver, spleen, muscle, gonad and brain. RBC, Hb and HCT showed a significant decline, while WBC and PLT showed a significant increase after the exposure. Glucose, total protein, A/G ratio, SGOT, SGPT and ALP showed significant increments in 0.1 and 1 mg/L of PS-MPs treated groups. The increase in cortisol level and upregulation of HSP70 gene expression in response to MPs exposure indicate MPs-mediated stress in tilapia. MPs-induced oxidative stress is evident from reduced SOD activity, increased MDA levels and upregulated P53 gene expression. The immune response was enhanced by inducing respiratory burst activity, MPO activity and serum TNF-α and IgM levels. MPs exposure also led to down-regulation of CYP1A gene and decreased AChE activity, GNRH and vitellogenin levels, indicating the toxicity of MPs on the cellular detoxification mechanism, nervous and reproductive systems. The present study highlights the tissue accumulation of PS-MP and its effects on hematological, biochemical, immunological and physiological responses in tilapia with low environmentally relevant concentrations.

A Cold-Inducible RNA Binding Protein Gene from Acanthopagrus schlegelii: Molecular Characterization, Expression, and Association with Apoptosis to Low-Temperature Stress

In this study, the complete cDNA sequence (1552 bp) of the cold-inducible RNA binding protein gene (cirbp gene) was successfully cloned from the liver in Acanthopagrus schlegelii (initial weight: 15.0 ± 2.3 g). Results showed that Ascirbp (cirbp gene from A. schlegelii) gene has 24 phosphorylation sites, no signal peptide, and no transmembrane helix structure. AsCIRBP, with a molecular weight of 18.84 ku and an isoelectric point of 9.04 was a stable protein that encodes 182 amino acids. Subcellular localization analysis of this protein showed that it was located in the nucleus. Sequence alignment results showed that the AsCIRBP amino acid sequences of various fishes including black porgy were highly conserved, especially the RNA recognition motif (RRM). Those results of real-time quantitative PCR (qRT-PCR) demonstrated that Ascirbp gene was specifically expressed in the liver tissue of black porgy and its expression was significantly increased under cold stress or cold acclimation. The RNA interference experiment results showed that Ascirbp-dsRNA could suppress the expression of Ascirbp gene in the liver of black porgy through intraperitoneal injection. After silencing the expression of Ascirbp gene, RNAi groups were more severely damaged in the structure of the liver tissue and more prone to apoptosis under cold stress than control groups. The results of the study on the linkage between Ascirbp gene expression and mitochondrial apoptosis pathways showed that changes in the expression of the Ascirbp gene had a significant effect on the expression of key genes of apoptosis. The most striking result from silencing the expression of the Ascirbp gene was that expressions of the bcl-2 and apaf-1 gene in the liver of black porgy decreased significantly, which can block the normal apoptotic process. After the disruption of the normal apoptotic process, the expressions of p53, bax, cyto-c, caspase-9, caspase-3, diablo, and caspase-1 gene were significantly affected. These results suggest that Ascirbp gene can inhibit apoptosis and protect tissue structure in the liver tissue of black porgy at low temperatures.

Effects of dietary guava leaf aqueous extract supplementation on growth, antioxidant capacity, and non-specific immunity in mud crab Scylla paramamosain

Mud crab (Scylla paramamosain) fed five different diets with varying concentrations of guava leaf aqueous extract (0 mg·kg–1, 80 mg·kg–1, 160 mg·kg–1, 320 mg·kg–1, and 640 mg·kg–1) for 30 days. Mud crabs in the 320 mg·kg–1 guava-leaf extract groups outperformed the control group in terms of survival rates (SR), weight gain rates (WGR), and specific growth rates (SGR). When compared to the control group, mud crabs in the 320 mg·kg–1 guava-leaf extract groups had significantly higher levels of lipase (LPS), pepsin, lysozyme (LZM), superoxide dismutase (SOD), acid phosphatase (ACP), and glutathione (GSH) (P < 0.05). The amylase (AMS) activity was significantly decreased in all experimental groups (P < 0.05). Malondialdehyde (MDA) content in the hepatopancreas of mud crabs in the 160 mg·kg–1, 320 mg·kg–1, and 640 mg·kg–1 guava-leaf extract groups were significantly reduced compared to the control group (P < 0.05). Additionally, real-time PCR results illustrated that the expression levels of GPx3, CAT, and JNK were all considerably increased in the 80 mg·kg–1 guava-leaf extract groups compared to the control group (P < 0.05). In the 160 mg·kg–1, 320 mg·kg–1, and 320 mg·kg–1 guava-leaf extract groups, the expression levels of SOD genes were considerably greater than the control (P < 0.05), which was consistent with the level of SOD activity. GST and P53 gene expression levels were significantly up-regulated in the 80 mg·kg–1, 160 mg·kg–1, 320 mg·kg–1, and 640 mg·kg–1 guava-leaf extract groups compared to the control group (P < 0.05). Overall, the addition of 160 mg·kg–1-320 mg·kg–1 guava-leaf extract to the feed of Scylla paramamosain promoted growth, enhanced the activities of digestive and antioxidant enzymes, and strengthened immunity.

NRF2/HO-1 axis, BIRC5, and TP53 expression in ESCC and its correlation with clinical pathological characteristics and prognosis.

Many types of cancer exhibit high nuclear factor erythroid 2-related factor 2 (NRF2), which is effective in resisting drugs and radiation. However, the role of NRF2 gene expression in predicting the prognosis of esophageal squamous cell carcinoma (ESCC) remains unclear. The association between NRF2, heme oxygenase-1 (HO-1), baculovirus IAP repeat 5 (BIRC5), P53 gene expression levels and their relationship to immune-infiltrating cells were assessed using the Cancer Genome Atlas dataset, the Human Protein Atlas and the TISDB database. The expression of NRF2, HO-1, BIRC5, and TP53 in 118 ESCC patients was detected by immunohistochemistry, and the relationship between their expression level and clinicopathological parameters and prognosis was analyzed. In ESCC, NRF2 overexpression was significantly associated with Han ethnicity, lymph node metastasis, and distant metastasis. HO-1 overexpression was significantly associated with differentiation, advanced clinical staging, lymph node metastasis, nerve invasion, and distant metastasis. BIRC5 overexpression was significantly associated with Han ethnicity and lymph node metastasis. TP53 overexpression was significantly associated with Han ethnicity and T staging. The NRF2/HO-1 axis expression was positively correlated with BIRC5 and TP53. Kaplan-Meier and multivariate Cox regression analysis showed that NRF2, BIRC5, and TP53 genes co-expression was an independent prognostic risk factor. TISIDB dataset analysis showed that immune-infiltrating cells were significantly negatively correlated with NRF2 and BIRC5. NRF2, BIRC5, and TP53 axis gene expressions are predictors of poor prognosis for ESCC. The overexpression of the NRF2/HO-1/BIRC5 axis may not be related to immune-infiltrating cells.

Vincamine Ameliorates Epithelial-Mesenchymal Transition in Bleomycin-Induced Pulmonary Fibrosis in Rats; Targeting TGF-β/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-β1/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-β1, 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-β1, 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-β1/p38 MAPK/ERK1/2/TWIST/Snai1/Slug/fibronectin/N-cadherin pathway. Moreover, it exerted anti-apoptotic activity in bleomycin-induced pulmonary fibrosis.

Influence of Rho/ROCK inhibitor Y-27632 on proliferation of equine mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) isolated form bone marrow and adipose tissue are the most common cells used for cell therapy of orthopedic diseases. MSC derived from different tissues show differences in terms of their proliferation, differentiation potential and viability in prolonged cell culture. This suggests that there may be subtle differences in intracellular signaling pathways that modulate these cellular characteristics. The Rho/ROCK signaling pathway is essential for many cellular functions. Targeting of this pathway by the ROCK inhibitor Y-27632 has been shown to be beneficial for cell viability and proliferation of different cell types. The aim of this study was to investigate the effects of Rho/ROCK inhibition on equine MSC proliferation using bone marrow-derived MSC (BMSC) and adipose-derived MSC (ASC). Primary ASC and BMSC were stimulated with or without 10 ng/mL TGF-β3 or 10 μM Y-27632, as well as both in combination. Etoposide at 10 μM was used as a positive control for inhibition of cell proliferation. After 48 h of stimulation, cell morphology, proliferation activity and gene expression of cell senescence markers p53 and p21 were assessed. ASC showed a trend for higher basal proliferation than BMSC, which was sustained following stimulation with TGF-β3. This included a higher proliferation with TGF-β3 stimulation compared to Y-27632 stimulation (p < 0.01), but not significantly different to the no treatment control when used in combination. Expression of p21 and p53 was not altered by stimulation with TGF-β3 and/or Y-27632 in either cell type. In summary, the Rho/ROCK inhibitor Y-27632 had no effect on proliferation activity and did not induce cell senescence in equine ASC and BMSC.

Epigallocatechin gallate, folic acid, vitamin B12, and hyaluronic acid significantly increase apoptosis and p53 expression in HeLa cells.

The human papilloma virus (HPV) is the etiological agent of cervical cancer in more than 95% of cases worldwide. Although most HPV infections clear up on their own and most pre-cancerous lesions spontaneously resolve, in some cases, they can persist, leading to lesions which may progress towards invasive cervical cancer. We evaluated the effects of the association of epigallocatechin gallate (EGCG) + folic acid (FA) + vitamin B12 (B12) + hyaluronic acid (HA) on HPV-positive cervical cancer cells (HeLa). The association of EGCG + FA + B12 + HA induced a significant increase of apoptosis and p53 gene expression with a concomitant decrease of E6/E7 gene expression, a marker of HPV infection. This study provides for the first-time evidence on the potential additive activity of EGCG + FA + B12 + HA in counteracting HPV infection, by increasing apoptosis and p53 expression in HPV-infected cervical HeLa cells.

Commercial next generation sequencing in a real-world cohort of patients with metastatic breast cancer.

e13082 Background: Clinicians increasingly order next generation sequencing (NGS) of tissue or liquid (ctDNA) biopsies from patients with metastatic breast cancer. Although some genetic information is clinically actionable, some, particularly non-ERBB2 gene amplifications, is not. We reviewed NGS data from a real-world cohort of metastatic breast cancer patients to evaluate the prevalence of somatic abnormalities using commercial NGS technology. Methods: We evaluated Foundation One NGS data from 44 tissue and 17 liquid biopsies from patients seen in a medical oncology practice between 2016 and 2022. We used KM Plotter to measure overall survival in early-stage patients by relative gene expression. Results: 76% of patients had ductal and 24% had lobular carcinomas. 59% of patients had luminal (ER-positive) and 32% of patients had basal breast cancer (ER-negative). Only 9% of patients had HER2-positive disease. 48% of the tumors had a p53 abnormality, either a point (59%) or frameshift mutation (36%). 50% tumors had a somatic mutation in a tumor suppressor gene such as PTEN, RB or APC. One patient had a germline BRCA1 mutation and 5 patients had germline BRCA2 mutations. 39% of patients carried a somatic mutation in PIK3CA, predominantly H1047R (45%) in the kinase domain or E545X (20%) in the helicase domain. KM plotter showed worse OS in early-stage patients with low p53 gene expression or high PIK3CA expression. 10/11 patients with lobular carcinoma had somatic mutations in CDH1, 70% of which were frameshift mutations. 8/27 patients with luminal cancer had an ESR1 mutation, predominantly Y537 or D538. 9 patients, predominantly luminal, had amplifications of the 11q13 (CCND1). 7 patients had amplifications of 8q24 (MYC). 20q13 (AURKA) amplifications were only seen in luminal cancers. 12p13 (CCND2) amplifications were only seen in basal cancers. 27% of lobular cancers (vs. 6-17% of ductal cancers) demonstrated amplification of the 11q13, 8q24 and/or 8p11 amplicons. 20q13 and 12p13 amplifications were only seen in ductal cancers. Liquid biopsies were more likely to detect point mutations than tissue biopsies. p53 mutations were seen in 65% of liquid vs. 48% of tissue biopsies. 47% of liquid biopsies showed two p53 mutations. PIK3CA mutations were seen in 71% of liquid vs. 39% of tissue biopsies. 18% of liquid biopsies showed two PIK3CA mutations. 93% of basal cancers had at least one p53 mutation. 44% of luminal cancers had a PIK3CA mutation. ESR1, GATA3 and AKT1 mutations were only seen in luminal cancers. While ESR1 mutations were seen in ductal and lobular cancers, GATA3 and AKT1 mutations were only seen in ductal cancers. Conclusions: Commercial NGS testing of a real-world cohort yields actionable mutations that can impact clinical management and outcome of metastatic breast cancer patients. NGS testing demonstrates the range of gene amplifications and corresponds closely with what has been reported in the published literature.

The anti-tumoral role of Hesperidin and Aprepitant on prostate cancer cells through redox modifications.

Prostate cancer is the second prevalent cancer in men. While the anti-cancer effect of Hesperidin and (Aprepitant) AP on prostate cancer cells is well documented, their combined effect and their mechanism of action are not fully investigated. Therefore, this study aimed to investigate the anti-cancer effects of Hesperidin and AP alone and in combination on prostate cancer cells. PC3 and LNCaP cell lines were treated with Hesperidin and AP alone and in combination. The Resazurin test was used for assessing cell viability. The ROS (reactive oxygen Species) level, P53, P21, Bcl-2, and Survivin gene expression were assessed. Also, a trypan blue assay was done. Hesperidin and AP reduced cell viability and increased apoptosis in PC3 and LNCaP cells. The ROS level reduced after treating the PC3 and LNCaP cells with AP with or without Hesperidin. P53 and P21 gene expression increased after treatment with Hesperidin with or without AP compared to the untreated group in the PC3 cell line. Bcl-2 and Survivin gene expression decreased with AP with or without Hesperidin in the PC3 and LNCaP cells. The current study showed the synergic anti-cancer effect of Hesperidin and AP in both PC3 and LNCaP cell lines.

Effect of Apremilast on LPS-induced immunomodulation and inflammation via activation of Nrf2/HO-1 pathways in rat lungs

Lipopolysaccharides (LPS), the lipid component of gram-negative bacterial cell wall, is recognized as the key factor in acute lung inflammation and is found to exhibit severe immunologic reactions. Phosphodiesterase-4 (PDE-4) inhibitor: “apremilast (AP)” is an immune suppressant and anti-inflammatory drug which introduced to treat psoriatic arthritis. The contemporary experiment designed to study the protective influences of AP against LPS induced lung injury in rodents. Twenty-four (24) male experimental Wistar rats selected, acclimatized, and administered with normal saline, LPS, or AP + LPS respectively from 1 to 4 groups. The lung tissues were evaluated for biochemical parameters (MPO), Enzyme Linked Immunosorbent Assay (ELISA), flowcytometry assay, gene expressions, proteins expression and histopathological examination. AP ameliorates the lung injuries by attenuating immunomodulation and inflammation. LPS exposure upregulated IL-6, TNF-α, and MPO while downregulating IL-4 which were restored in AP pretreated rats. The changes in immunomodulation markers by LPS were reduced by AP treatment. Furthermore, results from the qPCR analysis represented an upregulation in IL-1β, MPO, TNF-α, and p38 whereas downregulated in IL-10 and p53 gene expressions in disease control animals while AP pretreated rats exhibited significant reversal in these expressions. Western blot analysis suggested an upregulation of MCP-1, and NOS-2, whereas HO-1, and Nrf-2 expression were suppressed in LPS exposed animals, while pretreatment with AP showed down regulation in the expression MCP-1, NOS-2, and upregulation of HO-1, and Nrf-2 expression of the mentioned intracellular proteins. Histological studies further affirmed the toxic influences of LPS on the pulmonary tissues. It is concluded that, LPS exposure causes pulmonary toxicities via up regulation of oxidative stress, inflammatory cytokines and stimulation of IL-1β, MPO, TNF-α, p38, MCP-1, and NOS-2 while downregulation of IL-4, IL-10, p53, HO-1, and Nrf-2 at different expression level. Pretreatment with AP controlled the toxic influences of LPS by modulating these signaling pathways.

Multiparametric analysis of etoposide exposed mesenchymal stem cells and Fanconi anemia cells: implications in development of secondary myeloid malignancy.

Secondary acute myeloid leukemia (sAML) may develop following a prior therapy or may evolve from an antecedent hematological disorder such as Fanconi Anemia (FA). Pathophysiology of leukemic evolution is not clear. Etoposide (Eto) is a chemotherapeutic agent implicated in development of sAML. FA is an inherited bone marrow (BM) failure disease characterized by genomic instability and xenobiotic susceptibility. Here, we hypothesized that alterations in the BM niche may play a critical/driver role in development of sAML in both conditions. Expression of selected genes involved in xenobiotic metabolism, DNA double-strand break response, endoplasmic reticulum (ER) stress, heat shock response and cell cycle regulation were determined in BM mesenchymal stem cells (MSCs) of healthy controls and FA patients at steady state and upon exposure to Eto at different concentrations and in recurrent doses. Expression of CYPA1, p53, CCNB1, Dicer1, CXCL12, FLT3L and TGF-Beta genes were significantly downregulated in FA-MSCs compared with healthy controls. Eto exposure induced significant alterations in healthy BM-MSCs with increased expression of CYP1A1, GAD34, ATF4, NUPR1, CXCL12, KLF4, CCNB1 and nuclear localization of Dicer1. Interestingly, FA-MSCs did not show significant alterations in these genes upon Eto exposure. As opposed to healthy MSCs DICER1 gene expression and intracellular localization was not altered on FA BM-MSCs after Eto treatment. These results showed that Eto is a highly potent molecule and has pleiotropic effects on BM-MSCs, FA cells show altered expression profile compared to healthy controls and Eto exposure on FA cells shows differential profile than healthy controls.