Figure 3 - uploaded by Marina De Rosa
Content may be subject to copyright.
Effect of LiCl and high energy photon irradiation on p53, Bax, Survivin and β-catenin protein expression in T88 cells. (A) Representative western blot images revealing the protein expression levels of p53, Bax and Survivin in treated and untreated T88 cells. GAPDH was used as the loading control. The graph shows the densitometric analysis of (B) p53, (C) Bax, (D) Survivin and (E) β-catenin compared with GAPDH. Bar graphs represent mean ± SEM (3 independent experiments). * P<0.05 and *** P<0.0001 vs. untreated cells, using Kruskal-Wallis test. LiCl, lithium chloride.

Effect of LiCl and high energy photon irradiation on p53, Bax, Survivin and β-catenin protein expression in T88 cells. (A) Representative western blot images revealing the protein expression levels of p53, Bax and Survivin in treated and untreated T88 cells. GAPDH was used as the loading control. The graph shows the densitometric analysis of (B) p53, (C) Bax, (D) Survivin and (E) β-catenin compared with GAPDH. Bar graphs represent mean ± SEM (3 independent experiments). * P<0.05 and *** P<0.0001 vs. untreated cells, using Kruskal-Wallis test. LiCl, lithium chloride.

Source publication
Article
Full-text available
Colorectal cancer (CRC) is the third most prevalent type of cancer worldwide. It is also the second most common cause of cancer‑associated mortality; it accounted for about 9.2% of all cancer deaths in 2018, most of which were due to resistance to therapy. The main treatment for CRC is surgery, generally associated with chemotherapy, radiation ther...

Contexts in source publication

Context 1
... apoptosis and death escape, such as p53, Bax, and Survivin. To this aim, we performed Western blot assay on total protein extracts from untreated cells, cells treated with LiCl or high energy photons, and cells irradiated with high-energy photons after pretreatment with LiCl. Samples were collected 48 h after completion of treatments. As shown in Fig. 3A-C, p53 and Bax protein expression was upregulated in both LiCl and LiCl plus high-energy photon treated cells, and the highest increase was observed after combined treatment. On the contrary, Survivin expression was greatly reduced after each treatment ( Fig. 3A and D). As LiCl induces GSK-3β inhibition, we investigated β-catenin expression ...
Context 2
... with LiCl. Samples were collected 48 h after completion of treatments. As shown in Fig. 3A-C, p53 and Bax protein expression was upregulated in both LiCl and LiCl plus high-energy photon treated cells, and the highest increase was observed after combined treatment. On the contrary, Survivin expression was greatly reduced after each treatment ( Fig. 3A and D). As LiCl induces GSK-3β inhibition, we investigated β-catenin expression level in treated and untreated cells. As shown in Fig. 3A and in Fig. 3E, β-catenin expression was stabilized in both LiCl-treated cells and in cells treated with LiCl plus high-energy irradiation. Interestingly, in LiCl-treated cells, western blot immunostaining ...
Context 3
... in both LiCl and LiCl plus high-energy photon treated cells, and the highest increase was observed after combined treatment. On the contrary, Survivin expression was greatly reduced after each treatment ( Fig. 3A and D). As LiCl induces GSK-3β inhibition, we investigated β-catenin expression level in treated and untreated cells. As shown in Fig. 3A and in Fig. 3E, β-catenin expression was stabilized in both LiCl-treated cells and in cells treated with LiCl plus high-energy irradiation. Interestingly, in LiCl-treated cells, western blot immunostaining showed upregulation of an isoform of β-catenin a little higher in molecular weight than that observed in cells irradiated without LiCl ...
Context 4
... apoptosis and death escape, such as p53, Bax, and Survivin. To this aim, we performed Western blot assay on total protein extracts from untreated cells, cells treated with LiCl or high energy photons, and cells irradiated with high-energy photons after pretreatment with LiCl. Samples were collected 48 h after completion of treatments. As shown in Fig. 3A-C, p53 and Bax protein expression was upregulated in both LiCl and LiCl plus high-energy photon treated cells, and the highest increase was observed after combined treatment. On the contrary, Survivin expression was greatly reduced after each treatment ( Fig. 3A and D). As LiCl induces GSK-3β inhibition, we investigated β-catenin expression ...
Context 5
... with LiCl. Samples were collected 48 h after completion of treatments. As shown in Fig. 3A-C, p53 and Bax protein expression was upregulated in both LiCl and LiCl plus high-energy photon treated cells, and the highest increase was observed after combined treatment. On the contrary, Survivin expression was greatly reduced after each treatment ( Fig. 3A and D). As LiCl induces GSK-3β inhibition, we investigated β-catenin expression level in treated and untreated cells. As shown in Fig. 3A and in Fig. 3E, β-catenin expression was stabilized in both LiCl-treated cells and in cells treated with LiCl plus high-energy irradiation. Interestingly, in LiCl-treated cells, western blot immunostaining ...
Context 6
... in both LiCl and LiCl plus high-energy photon treated cells, and the highest increase was observed after combined treatment. On the contrary, Survivin expression was greatly reduced after each treatment ( Fig. 3A and D). As LiCl induces GSK-3β inhibition, we investigated β-catenin expression level in treated and untreated cells. As shown in Fig. 3A and in Fig. 3E, β-catenin expression was stabilized in both LiCl-treated cells and in cells treated with LiCl plus high-energy irradiation. Interestingly, in LiCl-treated cells, western blot immunostaining showed upregulation of an isoform of β-catenin a little higher in molecular weight than that observed in cells irradiated without LiCl ...

Similar publications

Article
Full-text available
Cell pluripotency, spatial restriction, and development are spatially and temporally controlled by epigenetic regulatory mechanisms that occur without any permanent loss or alteration of genetic material, but rather through modifications “on top of it.” These changes modulate the accessibility to transcription factors, either allowing or repressing...
Article
Full-text available
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with limited therapeutic options. The current model suggests that chronic or repetitive "micro-injuries" of the alveolar epithelium lead to activation and proliferation of fibroblasts and excessive extracellular matrix (ECM) deposition. Disruption of alveolar type II (ATII)...

Citations

... To set up the optimal conditions and discriminate apoptotic cells from live cells, we used RKO and HEK 293T untreated cells as internal controls in these experiments. When these cells were induced to undergo apoptosis by a 24 h treatment with 60 mM LiCl, a GSK3β inhibitor known to induce apoptosis in several human cell lines [35][36][37], they exhibited a high percentage of subdiploid cells ( Figure 3A). In contrast, when Dox-treated RKO and HEK 293T cells were analyzed for their DNA content, they presented only a negligible percentage of the subdiploid peak ( Figure 3A), indicating that they are not subjected to apoptosis. ...
... To set up the optimal conditions and discriminate apoptotic cells from live cells, we used RKO and HEK 293T untreated cells as internal controls in these experiments. When these cells were induced to undergo apoptosis by a 24 h treatment with 60mM LiCl, a GSK3β inhibitor known to induce apoptosis in several human cell lines [35][36][37], they exhibited a high percentage of subdiploid cells ( Figure 3A). In contrast, when Dox-treated RKO and HEK 293T cells were analyzed for their DNA content, they presented only a negligible percentage of the subdiploid peak ( Figure 3A), indicating that they are not subjected to apoptosis. ...
Article
Full-text available
Dyskerin is an evolutionarily conserved nucleolar protein implicated in a wide range of fundamental biological roles, including telomere maintenance and ribosome biogenesis. Germline mutations of DKC1, the human gene encoding dyskerin, cause the hereditary disorders known as X-linked dyskeratosis congenita (X-DC). Moreover, dyskerin is upregulated in several cancers. Due to the pleiotropic functions of dyskerin, the X-DC clinical features overlap with those of both telomeropathies and ribosomopathies. In this paper, we evaluate the telomerase-independent effects of dyskerin depletion on cellular physiology by using inducible DCK1 knockdown. This system allows the downregulation of DKC1 expression within a short timeframe. We report that, in these cellular systems, dyskerin depletion induces the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum, which in turn induces the activation of the PERK branch of the unfolded protein response. We also demonstrate that the PERK-eIF2a-ATF4-CHOP signaling pathway, activated by dyskerin downregulation, triggers a functional autophagic flux through the inhibition of the PI3K/AKT/mTOR pathway. By revealing a novel unpredicted connection between the loss of dyskerin, autophagy and UPR, our results establish a firm link between the lowering of dyskerin levels and the activation of the ER stress response, that plays a key role in the pathogenesis of several diseases.
... In light of this previous evidence, in this study, we have analyzed the effects of LPE using primary human colon T88 and T93 cancer cells, which have been previously characterized [30][31][32]. These primary colon cancer cells were isolated and established from the tumor tissues of two patients affected by sporadic colon adenocarcinoma. ...
... The T93 cell line exhibited a chromosomal instability (CIN) phenotype and the T88 cell line exhibited a microsatellite instability (MSI) phenotype. It has been previously demonstrated that these cells both underwent EMT from epithelial adenocarcinoma cells; indeed, they simultaneously expressed epithelial and mesenchymal markers, such as cytokeratin and Ecadherin, and Vimentin and N-cadherin, respectively, together with high levels expression of EMT-associated transcription factors, stemness markers, and the Cyclooxygenase-2 (Cox-2) enzyme [30][31][32]. ...
... Furthermore, the data reported here show that LPE is able to inhibit the rIL-6-dependent cell migration and invasiveness in human primary T88 and T93 colon cancer cells [30][31][32] via the up-regulation of MMP-2 expression, and that the observed effects correlate with the STAT3 phosphorylation levels. The protective effects showed by plant polyphenols against different types of cancer [36] include their anti-inflammatory and antioxidant properties [26][27][28][29]. ...
Article
Full-text available
Among matrix metalloproteinases (MMPs), MMP-9/2 are key enzymes involved in the proteolysis of extracellular matrices in the inflammatory process and in cancer. Since MMP-9/2 expression levels, activity, and secretion is up-regulated during inflammation in response to pro-inflammatory cytokines, such as interleukin-6 (IL-6), many efforts have been devoted to identifying factors that could inhibit the IL-6-induced MMP-9/2 expression. Up to now, several reports indicated that polyphenols from fruits and vegetables are among the major components of health promotion for their antioxidant properties and also for their anti-inflammatory and anti-cancer agents. Among plant derived polyphenols, lemon (Citrus limon) peel extract (LPE) shows anti-cancer properties in various cancer types. In our previous work, we demonstrated that LPE can reduce IL-6-induced migration/invasiveness and MMP-9/2 up-regulation in some gastric cancer cell lines. This study aims to exploit the anti-cancer properties of LPE using an in vitro system model of inflammation, consisting of IL-6-exposed human primary colon cancer cells. We first analyzed the effect of LPE on IL-6-induced cell migration and invasiveness by wound healing and Boyden chamber assay, respectively. The MMP-2 mRNA expression levels and gelatinolytic activity in the cell culture media were determined by q-PCR analysis and gelatin zymography, respectively, and finally, the effects of LPE on IL-6-induced JAK2/STAT3 signaling pathways have been investigated by Western blotting analysis. Our results show that LPE is able to inhibit the IL-6-dependent cell migration and invasiveness associated with the up-regulation of MMP-2 expression levels and that these effects are correlated to the STAT3 phosphorylation in human primary T88 and T93 colon cancer cells.
... Furthermore, the rats treated with lithium chorlide and CCl 4 showed a significant increase in the apoptotic cell count compared to those treated with CCl 4 only. These results are in accordance with Zhang et al. [63], Yao et al. [64] and Cammarota et al. [65] who reported that lithium chloride up-regulated the expression of proapoptotic proteins such as apoptosis regulator BAX, p53, and Survivin and down-regulated the expression of proteins involved in survival. In addition, these rats showed significant up-regulation of α-SMA expression compared to the rats treated with CCl 4 only. ...
Article
Background Liver fibrosis is a serious health problem which may lead to advanced liver cirrhosis and hepatocellular carcinoma. Objective The present study aimed to investigate the role of Wnt/β-catenin signaling pathway and glutamine aminohydrolase enzyme (l-glutaminase) in the pathogenesis of liver fibrosis and the potential benefits of niclosamide in treating liver fibrosis. Methods Ninety male Albino rats were divided into 6 equal groups (n = 15) as follows: a normal control group (NC), CCl4-only treated group (Fib.) which received 1 mg/kg CCl4 two times weekly, niclosamide-treated group (Niclo.) which received 5 mg/kg of niclosamide one time daily, lithium chloride-treated group (LiCl) which received 100 mg/kg of LiCl one time daily, niclosamide-and-CCl4-treated group (Niclo. + Fib.) which received same doses of niclosamide and CCl4 given to other groups, and finally lithium chloride-and-CCl4-treated rat group (LiCl + Fib.) which received same doses of LiCl and CCl4 given to other groups. All treatments were administered orally for 8 weeks. Liver tissue was assessed for l-hydroxyproline, beta-catenin (β-catenin), l-glutaminase activity, as well as the gene expression of transforming growth factor beta-1 (TGF-β1) and Dishevelled-2 (Dvl2). Histopathological and immunohistochemical analyses of alpha smooth muscle actin α-SMA were performed. Serum alanine transaminase (ALT), aspartate transaminase (AST), and total bilirubin were measured. Results The group of niclosamide-and-CCl4-treated rats showed a significant decrease in total bilirubin, ALT and AST, β-catenin, l-hydroxyproline, l-glutaminase activity, and gene expression of TGF-β1 and Dvl2. Moreover, the liver tissue in this group of rats showed mild α-SMA reactivity compared with the rats treated with CCl4 only (fibrosis group). On the other hand, lithium chloride-and-CCl4-treated rats showed a significant increase in liver indices, TGF-β1 expression, β-catenin, l-hydroxyproline, and l-glutaminase activity with severe α-SMA reactivity and apoptosis in the liver tissue. Conclusions Niclosamide protected rats against liver fibrosis by inhibiting the Wnt/β-catenin pathway and glutaminolysis.
... Cell viability assay. cell viability was analysed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTT) assay as previously described (15). cell suspensions (500 µl) from the RKO-PBS and RKO-Nitrodi cells, containing 3x10 4 viable cells, were plated in 24 multi-well plates. ...
Article
Full-text available
Balneotherapy and spa therapy have been used in the treatment of ailments since time immemorial. Moreover, there is evidence to suggest that the beneficial effects of thermal water continue for months following the completion of treatment. The mechanisms through which thermal water exerts its healing effects remain unknown. Both balneological and hydroponic therapy at 'the oldest spa in the world', namely, the Nitrodi spring on the Island of Ischia (Southern Italy) are effective in a number of diseases and conditions. The aim of the present study was to investigate the molecular basis underlying the therapeutic effects of Nitrodi spring water in low‑grade inflammation and stress‑related conditions. For this purpose, an in vitro model was devised in which RKO colorectal adenocarcinoma cells were treated with phosphate‑buffered saline or phosphate‑buffered saline prepared with Nitrodi water for 4 h daily, 5 days a week for 6 weeks. The RKO cells were then subjected to the following assays: 3‑(4,5‑Dimethylthiazol‑2‑yl)‑2,5‑diphenyl‑2H‑tetrazolium bromide assay, Transwell migration assay, western blot analysis, the fluorimetric detection of protein S‑nitrosothiols and S‑nitrosylation western blot analysis. The results revealed that Nitrodi spring water promoted cell migration and cell viability, and downregulated protein S‑nitrosylation, probably also the nitrosylated active form of the cyclooxygenase (COX)‑2 protein. These results concur with all the previously reported therapeutic properties of Nitrodi spring water, and thus reinforce the concept that this natural resource is an important complementary therapy to traditional medicine.