Tazemetostat decreases ß-catenin and CD13 protein expression in HEPG-2 and Hepatitis B virus-transfected HEPG-2 with decreased cell viability.

Hepatocellular carcinoma (HCC) is one of the global health concerns. Hepatitis B virus (HBV) is one of the major causes of HCC. Poor clinical outcome of HCC patients is attributed to a small population of cancer cells known as cancer stem cells (CSCs). In this work, we studied the effect of inhibiting the enhancer of zeste homologue 2 (EZH2), a histone methyltransferase known to be overexpressed in CSCs, using tazemetostat (Taz). The effect of Taz was assessed in the HCC cell line (HEPG2) and Hepatitis B virus-transfected HEPG2 (HBV/HEPG2) cells. MTT assay showed a significant decrease in HEPG2 cells viability after 48 h treatment with either 0.5, 1, 4 or 6 µM Taz. HEPG2 and HBV/HEPG2 cells were incubated with either 0.5 or 1 µM Taz for 48 h, and then, the cells and supernatants were collected for protein expression analysis of EZH2, CD13, epithelial cell adhesion molecule (EpCAM) and ß-catenin using enzyme-linked immunosorbent assay (ELISA). Taz showed a significant dose-dependent inhibition of EZH2, CD13 and ß-catenin in HEPG2 and HBV/HEPG2 cells. Also, EpCAM protein levels were significantly decreased in HBV/HEPG2 but not in HEPG2 cell line alone. Our results indicate that Taz inhibition of EZH2 leads to downregulation of ß-catenin signaling and eventually decreased expression of CD13 and EpCAM, which are characteristic for CSCs. The present study suggests that Taz could be a promising treatment for HCC including HBV-induced HCC that might be used in combination with radio/chemotherapy to target CSCs and prevent tumor relapse.

Rapamycin Improves Adipose-Derived Mesenchymal Stem Cells (ADMSCs) Renoprotective Effect against Cisplatin-Induced Acute Nephrotoxicity in Rats by Inhibiting the mTOR/AKT Signaling Pathway.

OBJECTIVE: Because the poor survival of transplanted cells in a hostile microenvironment limits stem cell therapy, in the current study, we investigated the effect of rapamycin (Rapa)-preactivated autophagy on the survival and homing of transplanted adipose mesenchymal stem cells (ADMSCs) in a rat model of cisplatin (Cis)-induced nephrotoxicity, as well as the possible role of the mTOR/AKT signaling pathway. MATERIALS AND METHODS: In vitro, ADMSCs isolated from rats were treated with 50 nmol/L rapamycin for 2 h, after which the cytoprotective and autophagy-inducing effects of Rapa were investigated. The cis-induced acute nephrotoxicity rat model was constructed in vivo. ADMSCs and Rapa-ADMSCs were administered into the tail vein before Cis therapy. At 3, 7, and 10 days after Cis injection, all animals were euthanized. The renal functions and morphology as well as autophagy response were assessed. RESULTS: The pretreatment of cultured ADMSCs with Rapa caused a significant increase in autophagic activities and lysosome production of the cells, with a significant increase in the secretion of SDF-1, IL-10 and autophagy promoter LC3 and Beclin from these cells, while mTOR/AKT pathways were inhibited. In addition, the transplantation of Rapa-pretreated ADMSCs restored the kidney functions and morphology dramatically. Renal expression of SDF-1 and HIF1 was upregulated, while expression of IL-6, NF-kB and TGF-ß1 was downregulated. CONCLUSIONS: We concluded that the preactivation of autophagy with Rapa improves the survival and differentiation of the transplanted ADMSCs by inhibiting the mTOR/AKT signaling pathway, which in turn could significantly attenuate the Cis-induced acute renal injury.

Chemopreventive and hepatoprotective effects of genistein via inhibition of oxidative stress and the versican/PDGF/PKC signaling pathway in experimentally induced hepatocellular carcinoma in rats by thioacetamide.

OBJECTIVE: Genistein is a recognized isoflavone present in soybeans with antioxidant, anti-inflammatory, antiangiogenic and antitumor activities. This study aimed to test ability of genistein in modulating versican/platelet derived growth factor (PDGF) axis in HCC. METHODS: HCC was experimentally induced in male Sprague-Dawley rats then treated with 25 or 75 mg/kg genistein. Antioxidant activities of genistein was assessed by measuring the gene expression of Nrf2 and the hepatic levels of malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione. Expression of versican, PDGF, protein kinase C (PKC) and ERK-1 protein was assessed by Western blotting and immunostaining. RESULTS: HCC induced an elevation in oxidative stress, PDGF, versican, PKC and ERK protein expression levels. Genistein significantly reduced an HCC-induced increase in oxidative stress. Moreover, genistein dose-dependently reduced HCC-induced elevation of PDGF, versican, PKC and ERK protein expression levels. Moreover, genistein helped retain a normal hepatocyte structure and reduced fibrous tissue deposition, especially in high dose. CONCLUSIONS: Genistein exerted antitumor and antioxidant effects and therefore suppress HCC development via inhibition of the PDGF/versican bidirectional axis, suppressing both ERK1 and PKC as downstream regulators. Therefore, genistein is a potential novel therapeutic candidate for improving the outcome of patients with HCC.

Novel doxorubicin / folate-targeted trans-ferulic acid-loaded PLGA nanoparticles combination: In-vivo superiority over standard chemotherapeutic regimen for breast cancer treatment.

AIM: Doxorubicin/Cyclophosphamide (AC) is one of the standard adjuvant anthracycline-containing regimens that is still in use for breast cancer treatment. Cancer cell resistance and AC-induced side effects make treatment suboptimal and worsen patients' quality of life. This study aimed to improve trans-ferulic acid's (TFA) efficiency via loading into folate-receptor-targeted-poly lactic-co-glycolic acid nanoparticles (FA-PLGA-TFA NPs). Also, investigating both the antitumor efficacy of Doxorubicin (Dox)/FA-PLGA-TFA NPs combination against dimethylbenz[a]anthracene (DMBA)-induced breast cancer and its safety profile. METHODS: FA-PLGA-TFA NPs were optimally fabricated and characterized. Levels of Notch1, Hes1, Wnt-3a, ß-catenin, MMP-9, cyclin D1, Permeability-Glycoprotein (P-gp), ERα, PR, and HER2 were assessed as a measure of the antitumor efficacy of different treatment protocols. Histopathological examination of heart and bone, levels of ALT, AST, ALP, CK-MB, and WBCs count were evaluated to ensure the combination's safety profile. KEY FINDINGS: Dox/FA-PLGA-TFA NPs not only inhibited Notch signaling but also suppressed Notch synergy with Wnt, estrogen, progesterone, and HER2 pathways. Interestingly, Dox/FA-PLGA-TFA NPs decreased P-gp level and preserved heart, bone, and liver health as well as WBCs count. SIGNIFICANCE: Dox/FA-PLGA-TFA NPs reduced the side-effects of each single drug, and at the same time exerted excellent antitumor activity that surpass the AC regimen in evading cancer cell resistance and having a superior safety profile.

Pevonedistat attenuates cisplatin-induced nephrotoxicity in mice by downregulating the release of inflammatory mediators.

Pevonedistat (MLN4924) is a specific NEDD8-activating enzyme inhibitor that inactivates cullin-RING ligases involved in ubiquitylation and turnover of different signaling molecules. In the current study, we evaluated the effect of pevonedistat on cisplatin (CIS)-induced nephrotoxicity in mice. Serum creatinine and urea levels were analyzed in different groups. Histopathological examination of renal tissue was done using hematoxylin and eosin staining. In addition, renal IL-6 and TNF-α expressions were analyzed using the enzyme-linked immunosorbent assay technique, and IL-1ß and NF-κB expressions were analyzed by immunohistochemical staining of renal tissue. Caspase-3, A20, ß-catenin, and Nrf2 gene expressions in renal tissue were analyzed using the reverse-transcription polymerase chain reaction technique. Western blot analysis was adopted to assess cleaved caspase-3 and ß-catenin expressions in renal tissue. Pevonedistat coadministration with CIS improved kidney functions and attenuated CIS-induced nephrotoxicity as indicated by the significant decrease in serum creatinine and urea levels. In addition, pevonedistat coadministration with CIS showed a significant decrease in caspase-3 and a significant increase in A20, ß-catenin, and Nrf2 gene expressions. Also, pevonedistat decreased caspase-3 cleavage to p19 in mice treated with CIS. Moreover, pevonedistat decreased CIS-induced upregulation of IL-6, TNF-α, IL-1ß, and NF-κB protein expressions in renal tissue. Thus, pevonedistat alleviated CIS-induced nephrotoxicity that might be attributed to suppression of the inflammation induced in renal tissue.

Effect of zinc oxide nanoparticles and ferulic acid on renal ischemia/reperfusion injury: possible underlying mechanisms.

OBJECTIVES: The present study examined the effects of ferulic acid (FA) and Zinc oxide nanoparticles (ZnO-NPs) and a combination of both on renal ischemia/reperfusion injury (IRI) in rats and their possible underlying mechanisms. METHODS: two-hundreds male Sprague Dawley rats were randomly allocated into the 5 groups; i) sham group, ii) control (IRI) group (occlusion of the left renal pedicle for 45 min), iii) FA group as IRI group with FA (100 mg/Kg oral 24 hrs before ischemia), iv) ZnO-NPs group as IRI group with ZnO-NPs single 5 mg/Kg i.p. 2 hrs before ischemia and v) FA + ZnO-NPs group as IRI group with both FA and ZnO-NPs in the same previous doses. According to the reperfusion times, each group was further subdivided into 4 hr, 24 hr, 48 hr and 7 days reperfusion subgroups. RESULTS: administration of either FA or ZnO-NPs caused significant improvement in the elevated serum creatinine and BUN and malondialdehyde (MDA) concentrations and expression of TNF-α, Bax, caspase-3 in kidney tissues with significant rise in the creatinine clearance, the activities of catalase (CAT) and superoxide dismutase (SOD) and the expression of HO-1, HIF-1α genes and proliferation marker (ki67) in kidney tissues compared to IRI group (p < 0.05). Moreover, a combination of both agents produced more significant improvement in the studied parameters than each agent did alone (p < 0.05). CONCLUSIONS: Both FA and ZnO-NPs exerted cytoprotective effects against ischemic kidney injury and a combination of both exhibited more powerful renoprotective effect. This renoprotective effect might be due to suppression of oxidative stress, enhancement of cell proliferation (ki67), upregulation of antioxidant genes (Nrf2, HO-1 and HIF-1α) and downregulation of inflammatory cytokine (TNF-α) and apoptotic genes (caspase-3 and Bax).

Fucoidan Ameliorates Hepatocellular Carcinoma Induced in Rats: Effect on miR143 and Inflammation.

Fucoidan is sulfated polysaccharide of brown seaweed. It offers various pharmacological actions like anti-inflammatory, anti-bacterial and anti-tumor activities. Therefore, we aimed to investigate the effect of targeting microRNA-143 and inflammatory pathway by Fucoidan on experimentally induced hepatocellular carcinoma (HCC) in rats. HCC is experimentally induced in Sprague Dawley by thioacetamide. Rats were treated with 100 mg/kg and 200 mg/kg Fucoidan. Hepatic sections were stained with hematoxylin/esosin for investigation of cell integrity. Moreover, hepatic sections were immunohistochemically stained with antibodies for ki67, TNF-α, and IL-1ß. Finally, hepatic tissues were investigated for expression of miR-143, NF-κB, TNF-α, and IL-1ß. We found that treating HCC with Fucoidan significantly reduced HCC-induced elevation in oxidative stress. Moreover, Fucoidan reduced HCC-induced in expression of miR-143, NF-κB, TNF-α, and IL-1ß. Finally, Fucoidan attenuated pseudohepatic lobules, broad fibrous septa and vacuolar to ballooning degeneration associated with reduction of immunostaining of ki67, TNF-α, and IL-1ß. Fucoidan elevated the survival of HCC rats and reduced their serum AFP. In addition, Fucoidan treatment revealed reduction in the expression of miR-143 associated with antioxidant and anti-inflammatory activities in HCC rats.

Radio-sensitizing effect of a cocktail of phytochemicals on HepG2 cell proliferation, motility and survival.

Radio-resistance is a major hurdle challenging oncologist worldwide. Despite their anti-cancer characteristics, the implication of phytochemicals in clinical trials is still limited. This study is designed to evaluate the anticancer characteristics and radio-sensitizing effect of a cocktail of seven phytochemicals on HepG2 cells. Characterization of phytochemicals combination phenolic and flavonoids content as well as their scavenging activity were tested. The effective concentration of BSG that will be used as a radio-sensitizing dose was calculated using AlamarBlue assay. Treatment of HepG2 cells with BSG and/or ionizing radiations led to significant downregulation at cell proliferation as indicated by the decrease of colony formation ratio, proliferation marker (Ki67) expression as well as G2/M cell cycle arrest. The combined treatment stimulated P53-dependent apoptosis which was indicated by the significant increase of early apoptosis marker (Annexin V) expression, DNA fragmentation, expression of P53 & Bax and downregulation of Bcl2 expression. Combined treatment significantly attenuated HepG2 cell motility which was validated using wound healing migration assay and the significant reduction at CD95 expression. This study demonstrates the anti-cancer effect of BSG and its fundamental role in provoking cell responsiveness to IR leading to a significant inhibition at HepG2 cell proliferation, survival and migration.

The interplay of the inhibitory effect of nifuroxazide on NF-κB/STAT3 signaling attenuates acetic acid-induced ulcerative colitis in rats.

Ulcerative colitis (UC) is a disease of increased worldwide prevalence. UC progression is associated with serious complications that leave the patient with considerable health burdens. Nifuroxazide is an oral nitrofuran antibiotic used as antidiarrheal medication. The current study places an emphasis on investigating the potential therapeutic effectiveness of nifuroxazide (10 mg/kg) and (20 mg/kg) against acetic acid (AA)-induced UC. Intra-rectal AA induced a significant colonic injury and impairment of colonic biochemical and functional incidences. Nifuroxazide in a dose-dependent manner significantly corrected UC associated injury. Macroscopic scoring of UC, serum lactate dehydrogenase (LDH) activity, C-reactive protein (CRP) titer, colon malondialdehyde (MDA) and total nitric oxide (NOx) contents significantly declined. Meanwhile, serum total antioxidant capacity (TAC) and colon catalase, superoxide dismutase (SOD) and glutathione transferase (GST) activities and reduced glutathione (GSH) concentration significantly increased in a dose-dependent way. Ultimately, histopathological, immunohistochemical and ultramicroscopic analysis of colon specimen revealed significant improvement. To pinpoint the mechanistic pathway underlying the curative effect of nifuroxazide, colon expression of NF-κB, caspase-3 was evaluated along with STAT-3 activation. Nifuroxazide induced a dose-dependent significant suppression of NF-κB and caspase-3 signaling together with STAT3 signaling. In conclusion; nifuroxazide can be proposed as a therapeutic candidate to attenuate UC and its associated symptoms. The potential underlying mechanism involves suppression of NF-κB/STAT-3/caspase- signaling.

Selective cytotoxic activity and protective effects of sodium ascorbate against hepatocellular carcinoma through its effect on oxidative stress and apoptosis in vivo and in vitro.

Objectives: Hepatocellular carcinoma (HCC) is characterized by elevated in oxidative stress and inflammatory cytokines, which enhance destructive effects of the tumor. Therefore, we conducted this study to investigate the protective effects of sodium ascorbate against thioacetamide-induced HCC in rats through studying its effect on the apoptotic pathway in rats. In addition, in vitro activity of sodium ascorbate was investigated on HepG2 and compared with cisplatin.Methods: HCC was experimentally induced by injecting rats with 200 mg/kg thioacetamide intraperitoneally twice weekly for 16 weeks. Part of HCC rats was concomitantly treated with 100 mg/kg sodium ascorbate intraperitoneally during the 16-week period. Hepatic tissues were used for the determination of NFκB, Nrf2, TNF-α, caspase-3, caspase-8 and caspase-9.Results: Sodium ascorbate significantly attenuated HCC-induced reduction in the expression of NrF2 associated with a reduction in concentrations of hydrogen peroxide and superoxide anion. In addition, sodium ascorbate blocked HCC-induced increase in the expression of NFκB and TNF-α. Sodium ascorbate slightly increased the activity of caspase-3, -8 and -9 in vitro but inhibited their activities in vivo.Conclusion: In spite of the antioxidant and anti-inflammatory activity of sodium ascorbate, it produced selective cytotoxic activity via direct activation of the apoptotic pathway in cancer cells without affecting the apoptotic pathway in normal hepatic cells.

Nanoformulated natural therapeutics for management of streptozotocin-induced diabetes: potential use of curcumin nanoformulation.

AIM: The goal of this study was to improve curcumin (CUR) aqueous solubility and bioavailability via nanoformulation, and then study its activity and mechanism of action as an antidiabetic agent. METHODS: CUR-loaded pluronic nanomicelles (CURnp) were prepared and characterized. Biochemical assessments were performed as well as histological, confocal and RTPCR studies on pancreatic target tissues. RESULTS: CURnp with a diameter of 333 ± 6 nm and ζ potential of -26.1 mv were obtained. Antidiabetic action of CURnp was attributed to significant upregulation of Pdx-1 and NKx6.1 gene expression and achievement of optimum redox balance, which led to alleviation of streptozotocin-induced ß-cell damage via a significant upregulation in insulin gene expression proved by RTPCR studies and by the presence of 40% insulin positive cells through confocal microscope studies on pancreatic tissue.

A newly developed silymarin nanoformulation as a potential antidiabetic agent in experimental diabetes.

AIM: This study aimed to develop a new stable nanoformulation of silymarin (SM) with optimum enhanced oral bioavailability and to evaluate its effect as well as mechanism of action as a superior antidiabetic agent over native SM using streptozotocin-induced diabetic rats. MATERIALS AND METHODS: SM-loaded pluronic nanomicelles (SMnp) were prepared and fully characterized. Biochemical parameters were performed as well as histological, confocal and reverse-transcription polymerase chain reaction studies on pancreatic target tissues. RESULTS & CONCLUSION: SMnp were found to improve significantly the antihyperglycemic, antioxidant and antihyperlipidemic properties as compared with native SM. In addition, SMnp was found to be a more efficient agent over SM in the management of diabetes and its associated complications due to its superior bioavailability in vivo, and the controlled release profile of SM. [Formula: see text].