Unleash Multifunctional Role of miRNA Biogenesis Gene Variants (XPO5*rs34324334 and RAN*rs14035) with Susceptibility to Hepatocellular Carcinoma.

Numerous reports have explored the roles of different genetic variants in miRNA biogenesis mechanisms and the progression of various types of carcinomas. The goal of this study is to explore the association between XPO5*rs34324334 and RAN*rs14035 gene variants and susceptibility to hepatocellular carcinoma (HCC). In a cohort of 234 participants (107 HCC patients and 127 unrelated cancer-free controls) from the same geographic region, we characterized allelic discrimination using PCR-RFLP and performed subgroup analysis and multivariate regression. We found that the frequency of the XPO5*rs34324334 (A) variant was correlated with elevated risk of HCC under allelic (OR = 10.09, p-value < 0.001), recessive (OR = 24.1, p-value < 0.001), and dominant (OR = 10.1, p-value < 0.001) models. A/A genotype was associated with hepatitis C cirrhosis (p-value = 0.012), ascites (p-value = 0.003), and higher levels of alpha-fetoproteins (p-value = 0.011). Carriers of the RAN*rs14035 (T) variant were more likely to develop HCC under allelic (OR = 1.76, p-value = 0.003) and recessive (OR = 3.27, p-value < 0.001) models. Our results suggest that XPO5*rs34324334 and RAN*rs14035 variants are independent risk factors for developing HCC.

Diarylheptanoids/sorafenib as a potential anticancer combination against hepatocellular carcinoma: the p53/MMP9 axis of action.

Hepatocellular carcinoma (HCC) is a serious and potentially fatal form of cancer associated with liver damage. New anticancer drugs are increasingly needed due to the increasing number of cancer cases every year. In this study, diarylheptanoids (DAH) from Alpinia officinarum were examined for their antitumor activity against DAB-induced HCC in mice, as well as their ability to reduce liver damage. Assays for cytotoxicity were conducted using MTT. The DAB-induced HCC Swiss albino male mice were given DAH and sorafenib (SOR) either as single treatments or in combination, and the effects on tumour development and progression were monitored. Malondialdehyde (MDA) and total superoxide dismutase (T-SOD) were evaluated along with biomarkers of liver enzymes (AST, ALT, and GGT). The apoptosis-related gene (CASP8), the apoptosis-related gene (p53), the anti-inflammatory genes (IL-6), the migration-related gene matrix metalloprotease-9 (MMP9), and the angiogenesis-related gene vascular endothelial growth factor (VEGF) were assessed using qRT-PCR in the hepatic tissue. As a final step, DAH and SOR were docked with CASP8 and MMP9 via molecular docking to propose potential mechanisms of action. Our results revealed that the combination of DAH and SOR has a potent inhibitory effect on the growth and viability of the HepG2 cell line. The outcomes demonstrated that DAH and SOR-treated HCC-bearing mice displayed a reduction in the tumour burden and liver damage as demonstrated by (1) parameters of repaired liver function; (2) low levels of hepatic MDA; (3) elevated levels of hepatic T-SOD; (4) p53, IL-6, CASP8, MMP9, and VEGF downregulation; and (5) enhanced hepatic structure. The best results were revealed in mice that were co-treated with DAH (given orally) and SOR (given intraperitoneally). The docking study also proposed that both DAH and SOR could inhibit CASP8 and MMP9's oncogenic activities and had a high affinity for these enzymes. In conclusion, according to study findings, DAH enhances SOR antiproliferative and cytotoxic effects and identifies their molecular targets. Furthermore, the results revealed that DAH was able to boost the anticancer effects of the drug SOR and reduce liver damage caused by HCC in mice. This suggests that DAH could be a potential therapeutic agent against liver cancer.

Vasopressin induces apoptosis but does not enhance the antiproliferative effect of dynamin 2 or PI3K/Akt inhibition in luminal A breast cancer cells.

Breast cancer cells abnormally express vasopressin (AVP) and its receptors. The effect of AVP is largely orchestrated through its downstream signaling and by receptor-mediated endocytosis (RME), in which Dynamin 2 (Dyn2) plays an integral role in vesicle closure. In this work, luminal A breast cancer cells were treated with AVP, and then Dynasore (DYN) was employed to inhibit Dyn2 to explore the combined effect of AVP and Dyn2 inhibition on the survival of breast cancer cells. The results revealed that DYN alone demonstrated a concentration-dependent cytotoxic effect in AVP untreated cells. Apoptosis developed in 29.7 and 30.3% of cells treated with AVP or AVP+DYN, respectively, compared to 32.5% in cells treated with Wortmannin (Wort, a selective PI3K pathway inhibitor). More apoptosis was observed when cells were treated with DYN+Wort in presence or absence of exogenous AVP. Besides, 2 or 4- fold increases in the expression of Bax and Caspase-3, were observed in cells exposed to AVP in absence or presence of DYN, respectively. This was associated with higher levels of the autophagy marker (LC3II protein). Meanwhile, the activation of Akt protein, sequentially decreased in the same pattern. Cell's invasion decreased when they were exposed to AVP alone or combined with DYN or/and Wort. Conclusively, although many reports suggested the proliferative effect of AVP, the results predict the antiproliferative and antimetastatic effects of 100 nM AVP in luminal A breast cancer cells. However, the hormone did not enhance the cytotoxic effect of Dyn 2 or PI3K pathway inhibition. Summary of the Dynamin 2 independent AVP antiproliferative effects. Breast cancer cells expresses AVP as a Prohormone (A). At high dose of AVP, the hormone is liganded with AVP receptor (B) to initiate RME, where the endosomed complex (C) is degraded through the endosome-lysosome system, as a part of signal management. These events consume soluble Dyn2 in neck closure and vesicle fission (D). This makes the cells more substitutable to the direct apoptotic effect of DYN (E). Alternatively, at lower AVP doses the liganded AVP may initiate cAMP-mediated downstream signaling (F) and cellular proliferation. In parallel, Wort inhibits PIP2-PIP3 conversion (G) and the subsequent inhibition of PI3K/Akt/mTOR pathway leading to cell death.

Functional Assembly of Caenorhabditis elegans Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity.

Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis-Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.

Induction of Apoptosis by Nano-Synthesized Complexes of H2L and its Cu(II) Complex in Human Hepatocellular Carcinoma Cells.

BACKGROUND: Chemotherapy is currently the most utilized treatment for cancer. Therapeutic potential of metal complexes in cancer therapy has attracted a lot of interest. The mechanisms of action of most organometallic complexes are poorly understood. OBJECTIVE: This study was designed to explore the mechanisms governing the anti-proliferative effect of the free ligand N1,N6-bis((2-hydroxynaphthalin-1-yl)methinyl)) adipohydrazone (H2L) and its complexes of Mn(II), Co(II), Ni(II) and Cu(II). METHODS: Cells were exposed to H2L or its metal complexes where cell viability determined by MTT assay. Cell cycle was analysed by flow cytometry. In addition, qRT-PCR was used to monitor the expression of Bax and Bcl-2. Moreover, molecular docking was carried out to find the potentiality of Cu(II) complex as an inhibitor of Adenosine Deaminase (ADA). ADA, Superoxide Dismutase (SOD) and reduced Glutathione (GSH) levels were measured in the most affected cancer cell line. RESULTS: The obtained results demonstrated that H2L and its Cu(II) complex exhibited a strong cytotoxic activity compared to other complexes against HepG2 cells (IC50=4.14±0.036µM/ml and 3.2±0.02µM/ml), respectively. Both H2L and its Cu(II) complex induced G2/M phase cell cycle arrest in HepG2 cells. Additionally, they induced apoptosis in HepG2 cells via upregulation of Bax and downregulation of Bcl-2. Interestingly, the activity of ADA was decreased by 2.8 fold in HepG2 cells treated with Cu(II) complex compared to untreated cells. An increase of SOD activity and GSH level in HepG2 cells compared to control was observed. CONCLUSION: The results concluded that Cu(II) complex of H2L induced apoptosis in HepG2 cells. Further studies are needed to confirm its anti-cancer effect in vivo.

Antimicrobial, Antioxidant, and Anti-Tumor Activities of Sargassum linearifolium and Cystoseira crinita from Egyptian Mediterranean Coast.

Brown algae earned importance by virtue of their promising secondary metabolites of reasonable biological activities. Herein, the antioxidant, antimicrobial, and anticancer effects of crude extracts obtained from two Egyptian brown seaweeds, Sargassum linearifolium and Cystoseira crinita were evaluated. Phytochemical and GC-MS analyses revealed numerous active secondary metabolites in C. crinita cold methanolic extract (CCME) and S. linearifolium hot aqueous extract (SHAE). Both SHAE and CCME exhibited comparable DPPH (124.5 vs 125.6 µg/ml) and ABTS (257.1 vs 254.8 µg/ml) scavenging activities, respectively. Moreover, both crude extracts exhibited antimicrobial activity against various pathogenic microorganisms. Interestingly, employing MTT assay revealed cytotoxic effects of both extracts against a panel of cancer cells, where CCME showed a strong cytotoxic activity against MCF-7 cells (IC50 = 18.0 ± 0.74 µg/ml), while SHAE exhibited a moderate effect (IC50 = 31.1 ± 1.04 µg/ml). Increased mRNA and protein expression of Bax and Beclin-1 as well as the decreased expression of Bcl-2 revealed the ability of both extracts to induce apoptosis and autophagy in MCF-7 cells. Collectively, these findings provide evidence for antioxidant, antimicrobial, as well as anticancer effects driven by the two brown seaweeds that may underlay their plausible application in the therapeutic uses.

Fat mass and obesity-associated (FTO) and leptin receptor (LEPR) gene polymorphisms in Egyptian obese subjects.

BACKGROUND: Several studies addressed the contribution of fat mass and obesity-associated (FTO) and leptin receptor (LEPR) polymorphisms for the susceptibility to obesity among different ethnic subjects. The main purpose of this work is to evaluate the association of these polymorph\isms with obesity among Egyptian subjects. SUBJECTS AND METHODS: This case-control study was carried out on 110 unrelated obese Egyptian subjects who were compared with 122 controls. Their genomic DNA was genotyped using the PCR technique. RESULTS: The allelic frequencies of FTO rs9939609 (A) and LEPR rs1137101 (223R) were significantly higher in obese subjects compared with non-obese controls (p < .001). Comparing different phenotype frequencies including clinical, anthropometric, and biochemical parameters in obese subjects revealed no significant difference in relation to their genotype frequencies (p> .05). CONCLUSIONS: This study designates a strong association for FTO and LEPR variants with the risk of obesity among Egyptian subjects.

The Inter-Relation between Leptin Receptor (Q223R) Gene Polymorphism and the Risk of Egyptian Patients with HCC.

BACKGROUND: The relationship of leptin (LEP) and polymorphism of leptin receptor (LEPR) were studied in patients with hepatocellular carcinoma (HCC) and compared with those with liver cirrhosis to find out the extent of the risk of LEPR on patients with HCC. METHODS: Serum LEP level and LEPR Q223R gene polymorphism were determined in 300 patients with liver disease categorized equally into five groups' healthy volunteers, patients with hepatitis C (HCV), patients with non-alcoholic steatohepatitis (NASH),  liver cirrhosis and HCC. LEPR gene was amplified by polymerase chain reaction (PCR) then digested by the MSP1 restriction enzyme. RESULTS: The isolated 212 bp of LEPR was sequenced. The serum LEP level was reduced in patients with cirrhotic and HCC. Serum LEP level had negatively correlated with both tumor grade and size in HCC patients. The data obtained from restriction fragment length polymorphism­PCR and sequencing revealed the existence of a novel synonymous Q223R single nucleotide polymorphism (SNP) in exon 223 of LEPR gene (1137101). LEPR Gln223Arg, GG and GA genotypes were found in all studied groups. LEPR Gln223Arg, AA genotype was found in NASH, HCC, and control. LEPR Gln223Arg GA genotype is associated with some patients with HCC. CONCLUSION: GA genotype of LEPR Gln223Arg may be regarded as a probable genetic risk factor for Egyptian patients with HCC.

Osthole extracted from a citrus fruit that affects apoptosis on A549 cell line by histone deacetylasese inhibition (HDACs).

This study aims to investigate the interactions between osthole extracted from Egyptian citrus fruits as HDACs inhibitor by theoretical study and practically. Besides, osthole was assed as anti-cancer activity. In this study, osthole was extracted from the Egyptian citrus fruit and was characterized. The role of osthole as in vitro inhibitor of HDACs was estimated and evaluated the antitumor activity against human lung cancer cells (A549), Caspase-9 activity was detected. The results obtained from GC-MS indicate that the grapefruit showed the highest osthole concentration compared to the other citrus fruits. Moreover, the grapefruit osthole competitively inhibits HDACs. The inhibition constant value, (Ki=3.36 mM), indicates that osthole exerts an inhibitory effect upon HDACs activity. In vitro study of osthole could inhibit the growth of A549 cells that depend on time and concentration. It also induces apoptosis and causes an increase of caspase-9 by osthole. In conclusion, grapefruit osthole could induce the apoptosis in A549 lung cancer cells by inhibiting the histone deacetylase.

Paclitaxel Nanoparticles Induce Apoptosis and Regulate TXR1, CYP3A4 and CYP2C8 in Breast Cancer and Hepatoma Cells.

BACKGROUND AND OBJECTIVE: Although the anticancer potentials of water-insoluble drugs are improved by nanoformulation, other intervening factors may contribute in the drug efficacy. This work was designated to explore the effect of paclitaxel-loaded Poly(Lactic-co-Glycolic Acid) (PLGA) nanoparticles on the viability of cancer cells, the expression of Taxol Resistance gene I (TXR1) and paclitaxel metabolizing genes. METHODS: Paclitaxel loaded PLGA Nanoparticles (PTX-NPs) were prepared, physically characterized and used in the treatment of breast adenocarcinoma cells (MCF-7) and hepatoma cells (HepG2). Cells viability and apoptosis were investigated. In parallel, RNA was isolated, reverse transcribed and used to monitor the expression levels of TXR1, CYP 3A4 and CYP2C8 genes. RESULTS: PTX-NPs were characterized by transmission electron microscopy to be of a nano-size sphere-like shape. FTIR analysis revealed good coupling between PTX and PLGA. The encapsulation efficiency was 99% and the drug release demonstrated a progressive releasing phase followed by slower and sustained releasing phases. Although HepG2 cells demonstrated more resistance to PTX than MCF-7 cells, both cell types were more responsive to PTX-NPS compared to PTX. The IC50 values decreased from 19.3 to 6.7 in breast cancer cells and from 42.5 to 13.1µg/ml in hepatoma cells. The apoptosis was the key mechanism in both cells, where at least 44% of cells underwent apoptosis. The expression of TXR1 decreased when either cells were treated with PTX-NPs, respectively, meanwhile the expressions of CYP3A4 and CYP2C8 were increased. CONCLUSION: Taken together, this in vitro study reports the associations between the enhanced responsiveness of MCF-7 and HepG2 cells to PLGA-loaded paclitaxel nanoparticles and the accompanying decrease in the cells resistance to the PTX and its enhanced metabolism.

Dexamethasone simulates the anticancer effect of nano-formulated paclitaxel in breast cancer cells.

Although the chemosensitizing effect of Dexamethasone (DEX) and its ability to increase the sensitivity of breast cancer cells to chemotherapy were previously reported, this study aimed to explore how far cotreatment of breast cancer cells with paclitaxel (PTX) and DEX mimics the anticancer effect of nanoformulated PTX. To establish this goal, PTX was nanoformulated with poly (lactic-co-glycolic acid) (PLGA) and the nanoparticles (PTX-NPs) were physically authenticated. Breast cancer cells (MCF-7) were treated with PTX or PTX-NPs in presence or absence of low concentration (10 nM) of DEX. Cells viability (assessed by MTT assay), apoptosis (assessed by flow cytometry) and the expression of PTX resistance gene (TRX1) and PTX metabolizing genes (CYP2C8 and CYP3A4) were investigated. The results showed that nanoformulated PTX was validated by nano-size assessment, increased the anionic surface charge and prober conjugation with the biodegradable carrier (PLGA), as indicated by the FTIR spectroscopy. Initially, the IC50 value of PTX was 19.3 µg/ml and cotreatment with DEX minimized it to 5.22 µg/ml, whereas PTX-NPs alone inhibited cell proliferation with IC50 6.67 µg/ml. Also, in presence of DEX, PTX-NPs further decreased the IC50 to 5 µg/ml. In parallel, DEX has increased the responsiveness of cells to PTX without potentiating its apoptotic effect. Moreover, the glucocorticoid (with PTX or PTX-NPs) downregulated TXR1 gene by 26% (P < 0.01) and 28.4% (P < 0.05) respectively. Similarly, the mRNA level of CYP3A4 significantly decreased in presence of DEX. The main PTX metabolizing gene CYP2C8, in contrast, was upregulated, especially in cells cotreated with PTX/DEX (P < 0.001). Conclusively, the study reports that cotreatment of breast cancer cells with submolar concentration of DEX acts as similar as the nanoformulated PTX, possibly through its modulatory effects on the expression of the main PTX metabolizing gene (CYP2C8) and downregulating Taxol resistance gene.

The Role of the 3' Untranslated Region in the Post-Transcriptional Regulation of KLF6 Gene Expression in Hepatocellular Carcinoma.

KLF6 is ubiquitously expressed in human tissues and regulates many pathways such as differentiation, development, cellular proliferation, growth-related signal transduction, and apoptosis. We previously demonstrated that KLF6 expression is altered during liver carcinogenesis. More importantly, KLF6 invalidation results in cell cycle progression inhibition and apoptosis of liver cancer cells. On the other hand, enforced expression of KLF6 variant 2 (SV2) induces cancer cell death by apoptosis. Thus, we and others demonstrated that KLF6 and its splicing variants play a critical role in liver cancer. However, little is known on the mechanisms governing KLF6 expression in HCC. In the present work, we asked whether the 3' untranslated region (3'UTR) of the KLF6 mRNA may be responsible for regulation of KLF6 expression in HCC. We found that KLF6 mRNA stability was altered in liver-derived cell lines as compared to cervical cancer-derived cell lines and human embryonic fibroblasts. Interestingly, KLF6 mRNA was highly unstable in liver cancer-derived cell lines as compared to normal hepatocytes. We next cloned the KLF6 mRNA 3'UTR into luciferase-expressing vectors and found that gene expression and activity were strongly impaired in all liver-derived cell lines tested. In addition, we found that most the KLF6 3'UTR destabilisation activity resides between nt 1,835 and nt 2,615 of the KLF6 gene. Taken together, we provide the first steps towards better understanding of the regulation of KLF6 expression in HCC. Further work is needed to identify the factors that bind to KLF6 3'UTR to regulate its expression in liver cancer-derived cell lines.

The SV2 variant of KLF6 is down-regulated in hepatocellular carcinoma and displays anti-proliferative and pro-apoptotic functions.

BACKGROUND & AIMS: KLF6 protein is a transcription factor that plays important functions in hepatocellular carcinoma (HCC), which is one of the leading causes of death by cancer worldwide. Previous studies showed the existence of three splice variants of KLF6, termed SV1, SV2, and SV3. An increased SV1/KLF6 mRNA ratio in HCC was already described. In this study, we aimed to investigate the expression of the SV2 variant in HCC samples and its role in hepatic cells. METHODS: We measured the expression of the SV2 variant in HCC and adjacent tissue samples by q-RT-PCR. We established IHH and HepG2 stable cell lines over-expressing the SV2 variant and measured cell growth and apoptotic rate. RESULTS: We observed a reduced expression of the SV2 variant in HCC samples versus surrounding tissues and normal liver. Interestingly, our findings demonstrate that the over-expression of the SV2 variant in IHH and HepG2 cells leads to a significant reduction of proliferation associated with cell death by apoptosis. We further demonstrate that the SV2 expression leads to an induction of the cell-cycle-controlling p21(CIP/WAF1) and the pro-apoptotic Bax genes, mediated by the p53 protein. We show further that the SV2 expression in IHH and HepG2 cells induces their sensitivity to the anti-cancer drug, gemcitabine. CONCLUSION: We reveal a reduced expression of the SV2 variant of KLF6 in HCC samples and describe anti-proliferative and pro-apoptotic functions for this variant in hepatic cells.

Inhibition of mammalian aspartate transcarbamylase by quinazolinone derivatives.

Quinazolinone derivatives have been studied as both in vitro and in vivo inhibitors of aspartate transcarbamylase (ATCase). In vitro treatment of mammalian ATCase with four compounds revealed that they inhibited enzyme activity and that 2-phenyl-1,3-4(H)benzothiazin-4-thione was the most potent one. This compound acts as a noncompetitive inhibitor towards both aspartate and carbamoyl phosphate. The values of the inhibition constant (K(i)) indicate that this compound exerts a potent inhibitory effect upon ATCase activity. Moreover, in vivo treatment with different doses of these derivatives showed also an inhibitory effect upon ATCase, the relative activity being decreased by 40%-58% with a 1 mg dose. These data support the inhibition of ATCase by quinazolinone derivatives as a new type of inhibitor for the enzyme.