The expression of tuftelin 1 as a new theranostic marker in early diagnosis and as a therapeutic target in hepatocellular carcinoma.

Currently, many challenges are associated with hepatocellular carcinoma (HCC) as the failure of early diagnosis, and the lack of effective therapy. This study aimed to investigate the possible role of tuftelin 1 (TUFT 1) in the early diagnosis of HCC and evaluate the potential contribution of the TUFT 1/Ca+2 /phosphinositol 3 kinase (PI3K) pathway in dantrolene sodium (Dan) therapeutic outcomes. The study was performed on two sets of rats, the staging (30 rats) and treatment sets (80 rats). HCC was induced by a single dose of diethylnitrosamine (DENA). The hepatic content of TUFT 1 protein was assayed via western blot and immunohistochemistry (IHC), while PI3K, vascular endothelial growth factor (VEGF), Cyclin D1, and matrix-metalloproteinase-9 (MMP-9) contents were assessed using enzyme-linked immunosorbent assay. Hepatic and serum calcium were measured colorimetrically. Furthermore, the nuclear proliferation marker, (Ki-67), (Kiel [Ki] where the antibody was produced in the University Department of Pathology and the original clone number is 67)-expression was assessed by IHC. TUFT 1/Ca+2 /PI3K signaling pathway was progressively activated in the 3 studied stages of HCC with subsequent upregulation of angiogenesis, cell cycle, and metastasis. More interestingly, Dan led to TUFT 1/Ca+2 /PI3K pathway disruption by diminution of the hepatic contents of TUFT 1, calcium, PI3K, VEGF, Cyclin D1, and MMP-9 in a dose-dependent pattern. TUFT 1 can serve as a theranostic biomarker in HCC. Moreover, Dan exerted an antineoplastic effect against HCC via the interruption of TUFT 1/Ca+2 /PI3K pathway.

Daidzein and Chicory Extract Arrest the Cell Cycle via Inhibition of Cyclin D/CDK4 and Cyclin A/CDK2 Gene Expression in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers, associated with a high rate of mortality. A disturbance between cell proliferation and cell death is one of the cancer hallmarks including HCC. Cell proliferation is mainly controlled by the cell cycle. The arrest of the cell cycle is one of the important targets of anticancer agents. OBJECTIVES: The present study tries to clarify the exact role of some natural products such as daidzein (DAZ) and alcoholic chicory leaf extract (CE), as possible regulators of cell cycle and apoptosis. METHODS: HCC in rats was induced using diethylnitrosamine (DENA). Ninety rats were allocated and divided equally into nine groups, treated with CE, DAZ, a combination of both, and sorafenib with non-treated control groups. RESULTS: Treatment with CE, DAZ, and their combination significantly downregulated hepatic tissue expression of cyclin D1/CDK4 axis as well as cyclin A/CDK2 axis. The suggested therapeutic protocol inhibited the proliferation and dampened Bcl-2 expression. Furthermore, the efficiency of combining CE and DAZ demonstrated a potency comparable to sorafenib in terms of cyclin D/CDK4 axis expression, as well as; this combination protocol was more potent in revealing a potentiated inhibitory effect on cyclin A and Ki-67 expression. CONCLUSION: Treatment with DAZ or CE alone, or in combination, could possess an inhibitory effect on hepatocarcinogenesis via cell cycle arrest, inhibition of proliferation through suppression of Ki-67 expression, and apoptosis induction, mediated by downregulation of Bcl-2.

Targeting the HIF-1α/Cav-1 Pathway with a Chicory Extract/Daidzein Combination Plays a Potential Role in Retarding Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most rapidly growing solid cancers, that is characterized by hypoxia. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that regulates tumor proliferation and metastasis. It induces caveolin-1 (Cav-1) expression, a glycoprotein found on the membrane surface, then Cav-1 triggers angiogenesis and metastasis in HCC. OBJECTIVE: We hypothesize that targeting HIF-1α and consequently, Cav-1 using the antioxidant natural compound such as chicoric acid and a Cav-1 inhibitor daidzein (DAZ) could be a useful approach in the management of HCC. This study was conducted to investigate the possible therapeutic efficacy of standardized chicory leaf extract (SCLE) and DAZ via modulation of HIF-1α and Cav-1 in HCC rats. METHODS: Diethyl nitrosamine (DENA) was used for HCC induction. After the induction period, four groups (10 rats for each) were treated with SCLE, DAZ, a combination of both, as well as sorafenib, all compared to the non-treated control. We assessed hepatic HIF-1α protein expression, Cav-1 gene expression, serum level of AFP, hepatic tissue content of VEGF, MMP-9, oxidative stress markers MDA and SOD. RESULTS: DAZ, SCLE, and their combination, significantly down-regulated the expression of HIF-1α, Cav-1, and consequently dampened MMP-9, VEGF, hepatic content. It has been observed that the combination treatment showed a synergistic effect compared to either treatment alone. Importantly, the combination treatment exhibited a significantly more potent effect than sorafenib. CONCLUSION: This study showed the potential role of the HIF-1α/Cav-1 pathway in HCC progression, moreover, SCLE and DAZ showed a potent efficacy in retarding HCC via modulation of this pathway.

Matrix metalloproteinase contribution in management of cancer proliferation, metastasis and drug targeting.

Matrix metalloproteinases (MMPs) or matrixins, are members of a zinc-dependent endopeptidase family. They cause remodeling of the extracellular matrix (ECM) leading to numerous diseases. MMPs subfamilies possess: collagenases, gelatinases, stromelysins and membrane-type MMPs (MT-MMP). They consist of several domains; pro-peptide, catalytic, linker peptide and the hemopexin (Hpx) domains. MMPs are involved in initiation, proliferation and metastasis of cancer through the breakdown of ECM physical barriers. Overexpression of MMPs is associated with poor prognosis of cancer. This review will discuss both types of MMPs and current inhibitors, which target them in different aspects, including, biosynthesis, activation, secretion and catalytic activity. Several synthetic and natural inhibitors of MMPs (MMPIs) that can bind the catalytic domain of MMPs have been designed including; peptidomimetic, non-peptidomimetic, tetracycline derivatives, off-target MMPI, natural products, microRNAs and monoclonal antibodies.

Dantrolene Potentiates the Antineoplastic Effect of Sorafenib in Hepatocellular Carcinoma via Targeting Ca+2/PI3K Signaling Pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is the 6th prevalent cancer and the 4th leading cause of cancer-related deaths all over the world. A major challenge for sorafenib, the standard chemotherapeutic agent in HCC treatment, is the chemo-resistance. OBJECTIVE: This study was conducted to evaluate the role of dantrolene as a possible antineoplastic agent in HCC, and in chemo-sensitization of sorafenib via targeting Ca+2/PI3K pathway. METHODS: HCC was induced in rats using a single dose of diethylnitrosamine (DENA) (200 mg/kg, ip), followed by phenobarbital sodium (0.05%) in drinking water for 18 weeks. At the end of the 18th week, rats were allocated into 4 groups (10 rats/each), one group was left without treatment (DENA group) and the other three groups were treated with either sorafenib, dantrolene, or their combination for further 4 weeks. One day after the last injection, serum and liver tissues were collected. Liver tissue p53, VEGF, MMP-9, Cyclin D1, PI3K, and, serum AFP were assessed using immunoassay. Hepatic and serum Ca+2 were also computed. Furthermore, Ki-67 was assessed immunohistochemically. RESULTS: Dantrolene exhibited synergistic effect when used in combination with sorafenib compared to either drug alone (p <0.05) through decreasing p53, VEGF, MMP-9, Cyclin D1, and Ki-67. In addition, dantrolene was evidenced to have an inhibitory effect on Ca+2/PI3K pathway that mediates its antineoplastic action when used alone or in combination with sorafenib. CONCLUSION: Dantrolene exerted antineoplastic effect as well as augmented sorafenib antineoplastic activity via the intervention of Ca+2/PI3K pathway, manifested by ameliorating angiogenesis, apoptosis, proliferation and metastasis.

Serum histamine and acetylcholine variations as new noninvasive biochemical markers in staging of experimental hepatocellular carcinoma.

Angiogenesis is a major prerequisite for hepatocellular carcinoma (HCC) development and progression. The present study aims to assess the potential role of two endogenous regulators of angiogenesis histamine (His) and acetylcholine (Ach), as possible biochemical markers for staging of HCC. Five groups of rats were used in this study: a control healthy group (I), another 4 intoxicated groups used for the induction of HCC with a high dose of diethyl nitrosamine (DENA, 200 mg/kg, single I.P. dose), (II, III, IV, and V). Groups II, III, IV, and V were killed following 8, 16, 24, and 32 weeks after DENA injection, respectively. Serum level of His and Ach was estimated using high-performance liquid chromatography technique coupled with diode array detector (HPLC-DAD), and alpha-fetoprotein (AFP) was measured using ELISA technique along with liver histological examination for all groups. Progression of HCC was estimated by histopathological examination. The results exhibited prominent increase in serum His and Ach levels during the early stages of HCC in group II, III in comparison with the control, and then His serum level declined to the normal level during the last stage of HCC development (group V).However, Ach elevation continued. AFP serum level showed marked increase, till 32 weeks after hepatocarcinogenesis. The decreased histamine level, combined to elevated AFP, indicates an early stage, while continued elevation of Ach with decreased His levels indicates a later stage of HCC. The combination of these two neurotransmitters to AFP may contribute to a noninvasive biochemical staging for HCC.

Expression of thioredoxin and glutaredoxin in experimental hepatocellular carcinoma-Relevance for prognostic and diagnostic evaluation.

Hepatocellular carcinoma (HCC), represents more than 85% of liver cancers. The diagnosis of HCC may be delayed due to the absence of early, sensitive and specific biomarkers. This study was conducted to investigate whether the expression of thioredoxin (Trx) and glutaredoxin (Grx) is helpful for HCC diagnosis in an experimental model. Twenty male albino rats were equally divided into two groups (HCC and control). Hepatocarcinogenesis was performed by single intraperitoneal (i.p) injection of 200 mg/kg of diethylnitrosamine (DENA). Two weeks later, 0.05% of phenobarbital (PB) was supplied in the drinking water for other 14 weeks. HCC was diagnosed by measuring serum alpha-fetoprotein (AFP) level and histopathological examination. Our results found that hepatic indices alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin were elevated but decreased total protein level. Lipid peroxidation was elevated through increasing hepatic content of MDA with decreased antioxidant parameters like hepatic SOD, CAT activities and GSH. The current study also found that Trx and Grx tissue genes were overexpressed in HCC group significantly, compared to control group. This study substantiated that increased expression of these enzymes may be predictive of outcomes in HCC.

Herbal management of hepatocellular carcinoma through cutting the pathways of the common risk factors.

Hepatocellular carcinoma (HCC) is considered the most frequent tumor that associated with high mortality rate. Several risk factors contribute to the pathogenesis of HCC, such as chronic persistent infection with hepatitis C virus or hepatitis B virus, chronic untreated inflammation of liver with different etiology, oxidative stress and fatty liver disease. Several treatment protocols are used in the treatment of HCC but they also associated with diverse side effects. Many natural products are helpful in the co-treatment and prevention of HCC. Several mechanisms are involved in the action of these herbal products and their bioactive compounds in the prevention and co-treatment of HCC. They can inhibit the liver cancer development and progression in several ways as protecting against liver carcinogens, enhancing effects of chemotherapeutic drugs, inhibiting tumor cell growth and metastasis, and suppression of oxidative stress and chronic inflammation. In this review, we will discuss the utility of diverse natural products in the prevention and co-treatment of HCC, through its capturing of the common risk factors known to lead to HCC and shed the light on their possible mechanisms of action. Our theory assumes that shutting down the risk factor to cancer development pathways is a critical strategy in cancer prevention and management. We recommend the use of these plants side by side to recent chemical medications and after stopping these chemicals, as a maintenance therapy to avoid HCC progression and decrease its global incidence.

Chemosensitizing effect of Alpinia officinarum rhizome extract in cisplatin-treated rats with hepatocellular carcinoma.

This study was conducted to estimate the preventing and sensitizing efficiency of Alpinia officinarum rhizome extract (AORE) in an experimental model of hepatocellular carcinoma (HCC) +/- cisplatin. HCC was induced by a single intraperitoneal (i.p) dose of diethylnitrosamine (DENA, 200mg/kg). After 14 days, phenobarbitone (PB, 0.05%) was added to drinking water for 14 weeks to promote hepatocarcinogenesis. Cisplatin (CP) was given in a dose of 1.5 mg/kg (i.p), twice a week, alone or with AORE (400 mg/kg daily, orally) for 21 days. AORE was tried as a protective before the induction of HCC for three weeks as well. Results revealed that DENA/PB elevated hepatic indices as ALT and AST and total bilirubin with declining serum total protein. It increased oxidative stress, as hepatic malondialdehyde (MDA) with depressed hepatic reduced glutathione (GSH) contents, superoxide dismutase (SOD) and catalase activities. This was accompanied by an increase in hepatic expression of antioxidant genes (thioredoxin and glutaredoxin). Hepatocarcinogenesis was detected by histopathological changes in liver sections and the elevation of serum alpha-fetoprotein (AFP) level. Treatment with CP partially restored altered hepatic functions and oxidative stress markers. It also showed a partial decrease in the expression of antioxidant genes, improving histopathological changes in the liver and AFP level in serum. The treatment with AORE alone or AORE+CP enhanced hepatic function and oxidative stress markers. It also caused a decrease in the expression of antioxidant genes and improved histopathological changes in liver and serum AFP level. This effect is more potent than the treatment with CP alone. Our study suggested that AORE can be used as a promising natural chemoprevention or a chemosensitizing agent against hepatocarcinogenesis.

Isolates from Alpinia officinarum Hance attenuate LPS-induced inflammation in HepG2: Evidence from in silico and in vitro studies.

In an attempt to connect the legacy of centuries of invaluable knowledge from traditional medicine and the current understanding to the molecular mechanism of diseases, we took the advantage of the emergence of in silico screening as a promising tool for identification of potential leads from libraries of natural products. Traditional Chinese Medicine database was subjected to structure based virtual screening for identification of anti-inflammatory compounds using the 3D crystal structure of p38 alpha mitogen activated protein kinase. The molecular docking studies revealed the potential activity of several classes of compounds known to be the constituents of the rhizomes of Alpinia officinarum Hance (Lesser galangal). Five compounds, galangin, kaempferide, isorhamnetin, and two diarylheptanoids, were isolated from the rhizomes of the plant using vacuum liquid chromatography and flash chromatography techniques. The anti-inflammatory activity of these compounds was investigated on HepG2 cells stimulated by lipopolysaccharide. The latter induced the gene expression of proinflammatory cytokines; interleukin-1ß, interleukin-6, tumor necrosis factor alpha. Addition of the 5 isolated compounds downregulated this increased gene expression in a dose dependent manner. Thus, these results indicate that the isolated compounds from A. officinarum could be used as a beneficial source for preventing and treating inflammatory diseases.

Cytokines as important playmakers of experimental hepatocarcinogenesis confounded by diabetes.

PURPOSE: To explore the possible intermediary pathways through which diabetes mellitus (DM) adversely worsens hepatocellular carcinoma (HCC), focusing on cell life controllers as some transcription factors and inflammatory mediators. MATERIAL AND METHODS: Forty male albino rats were divided into four groups, control, cancer [given single intra-peritoneal (IP) dose of diethyl nitrosamine, NDEA, 125 mg/kg body weight], diabetic (given single dose of streptozotocin, STZ, 65 mg/kg) and cancer diabetic. HCC was initiated with NDEA, 3 weeks later, DM was induced with STZ. At 14th week, animals were sacrificed. Serum ALT, AST, GGT activities, AFP, IL-6, TNF-α levels and liver tissue Bax and Bcl2 proteins were measured. Liver sections were stained for histological examination. Both histological and AFP variations were chosen to prove cancer development. RESULTS: NDEA group showed significant increase in liver weight, serum ALT, AST, GGT, AFP, TNF-α, IL-6 and liver Bcl2 protein with decrease in total body weight, liver Bax protein and Bax/Bcl2 ratio. These effects were more pronounced in DENA plus STZ group. IL-6, TNF-α and Bcl2 were positively correlated while Bax and Bax/Bcl2 ratio were negatively correlated to AFP levels reflecting potential diagnostic value. CONCLUSION: Co-induction of DM in the course of hepatocarcinogenesis can dramatically influence disease progression through inflammation and retarded apoptosis. The suggested apoptotic and inflammatory markers seem to be beneficial diagnostic tools for HCC and improve the diagnostic performance of AFP.

Novel biochemical pathways for 5-Fluorouracil in managing experimental hepatocellular carcinoma in rats.

Five fluorouracil (5-FU) is extensively used in the treatment of hepatocellular carcinoma (HCC). It is well documented that 5-FU and its metabolites inhibit DNA synthesis through inhibition of thymidylate synthetase. Little is known about additional pathways for 5-FU in managing HCC. The present experiment was mainly designed to study possible biochemical pathways that can be added to 5-FU's mechanisms of action. Four groups of rats constituted a control group (given saline only), a trichloroacetic acid group (TCA, 0.5 g/kg/day for 5 days, orally), a 5-FU-positive group (75 mg/kg body weight, intraperitoneally, once weekly for 3 weeks) and a TCA-treated with 5-FU group (24 h from last TCA dose). We executed both biochemical-serum alpha-fetoprotein (AFP), liver tissue contents of total glycosaminoglycan (TGAGs), collagen (represented as hydroxyproline), total sialic acid (TSA), free glucosamine (FGA) and proteolytic enzyme activity (as pepsin and free cathepsin-D-and histological examinations of the liver tissue. The results revealed histological changes such as central vein congestion and irregularly shaped, substantially enlarged, vesiculated and binucleated hepatocytes. The nuclei were mostly polymorphic and hyperchromatic, and several vacuolation was noticed in the cytoplasm encircling the nucleus with masses of acidophilic material. 5-FU greatly corrected these changes, except that some necrotic and cytotoxic effects of 5-FU were still shown. AFP was significantly elevated in TCA-intoxicated, but reversed in 5-FU-treated, groups. Increased proteolytic activity by TCA was reversed by 5-FU, which also restored TGAG contents to normal; but both TCA and 5-FU depleted collagen content. TCA significantly elevated FGA but depressed TSA; this action was reversed by 5-FU treatment. In conclusion, it is possible that proteolytic activity, expressed as upregulated pepsin and free cathepsin-D activities, is increased in HCC. This is accompanied by extracellular matrix macromolecular disturbance, manifested as decreased TGAGs, collagen and TSA, with marked increase in FGA liver tissue content. The elevated FGA with depressed TSA content of liver tissue may be attributed to a cancer-hampered N-acetylation of FGA into SA. 5-FU administration markedly depressed hepatic tissue proteolysis, possibly reactivated N-acetylation of FGA into SA and elevated TGAGs without stopping tissue fibrosis as collagen was not affected. This study explores additional pathways for the mechanism of action of 5-FU, through conservation of extracellular matrix composition in situ, inhibiting invasion and metastasis in addition to its DNA-disturbing pathway.

Premalignant variations in extracellular matrix composition in chemically induced hepatocellular carcinoma in rats.

Chemical composition of extracellular matrix (ECM) plays a pivotal role in cellular and tissue development, regeneration, and differentiation. It also plays a key role in pathogenesis of hepatocellular carcinoma (HCC). This study explored premalignant changes in the liver tissue content of collagen (as hydroxyproline, HP), total glycosaminoglycans (TGAGs), free glucosamine (FGA), total sialic acid (TSA), lysosomal membrane integrity variations (calculated as total and free cathepsin D activities), and liver histology. Serum alfa-fetoprotein (AFP) level was used as an early marker for HCC in two groups of Wistar rats. One group of rats served as control and was provided normal saline orally. The other group was provided trichloroacetic acid (TCA) as 0.5 g/kg/day for five consecutive days by oral gavage. Animals were killed before tumor development. The treatment revealed dysplastic changes in addition to microsteatosis (fatty changes). Both sinusoids and the portal vein among dysplastic cells were dilated and congested. These dysplastic foci are believed to be premalignant and may be precancerous lesions. The following things were observed: a highly significant increase in serum AFP (as a key marker for HCC), a significant decrease in HP and TSA, a significant increase in FGA, nonsignificant decrease in TGAGs, significant up-regulation of free cathepsin D, nonsignificant decrease in total cathepsin D activities, and destabilization of lysosomal membrane integrity. Down-regulation of HP, TSA, and TGAGs seems to be a prerequisite for cancer development. This might be stimulated by up-regulation of free cathepsin D activity. Perhaps tissue fibrosis is not a condition for developing HCC because collagen was significantly depressed. Up-regulated FGA could be assumed to be a defense mechanism against TCA-induced proteolysis of membrane proteins because it is frequently reported to be of value in cancer chemotherapy. Studied ECM perturbations can be assumed as preliminary changes during chemical hepatocarcinogenesis at the tissue level. Prospective studies on blood levels of cathepsins, TGAGs, and individual ECM variables such as TSA, FGA, and Hp in patients at risk for HCC, performed in parallel with assessments of AFP, may provide a cost-effective way to find new links between tissue changes and circulation that would permit early prediction of disease. It may also provide a way to monitor HCC and compensate for the missed peak AFP values.