Sarwat Fatima

Clinicopathological and prognostic significance of serum and tissue Dickkopf-1 levels in human hepatocellular carcinoma

Although Dickkopf‐1 (DKK1) is known to be a negative regulator of the Wnt/β‐catenin pathway, it has been recently found to be upregulated in cancers.

Heat shock proteins in cancer: signaling pathways, tumor markers and molecular targets in liver malignancy.

Heat shock proteins (HSPs) consist of a large group of proteins with negligible expressions under physiological conditions. Their expressions are highly induced under stress conditions and they are ubiquitously expressed in various tissues and organs. HSPs possess chaperone functions, thus facilitating the correct folding of proteins or peptides. In hepatocellular carcinoma (HCC), high expressions of HSPs are demonstrated in liver cancer tissues and are correlated clinically with the severity of tumors and poor outcomes of HCC patients. This property enables them to be used as diagnostic markers for the onset of HCC. Since their expressions are highly expressed in liver cancer conditions, inhibitors or antisense oligonucleotides of HSPs are postulated to serve as potential therapeutics in treating this liver malignancy. In this review, we will first introduce the HSP family and discuss the major signaling pathways involved for the activities of HSPs. In addition, the clinical applications of HSPs in liver cancer in the aspects of diagnosis and therapy will be summarized and discussed.

Implications of the Use of Eukaryotic Translation Initiation Factor 5A (eIF5A) for Prognosis and Treatment of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a primary liver malignancy and accounts for most of the total liver cancer cases. Lack of treatment options and late diagnosis contribute to high mortality rate of HCC. In eukaryotes, translation of messenger RNA (mRNA) to protein is a key process in protein biosynthesis in which initiation of translation involves interaction of different eukaryotic translation initiation factors (eIFs), ribosome subunits and mRNAs. Eukaryotic translation initiation factor 5A (eIF5A) is one of the eIFs involved in translation initiation and eIF5A2, one of its isoforms, is upregulated in various cancers including HCC as a result of chromosomal instability, where it resides. In HCC, eIF5A2 expression is associated with adverse prognosis such as presence of tumor metastasis and venous infiltration. Based on eIF5A2 functional studies, suppressing eIF5A2 expression by short interfering RNA alleviates the tumorigenic properties of HCC cells in vitro while ectopic expression of eIF5A2 enhances the aggressiveness of HCC cells in vivo and in vitro by inducing epithelial-mesenchymal transition. In conclusion, eIF5A2 is a potential prognostic marker as well as a therapeutic target for HCC.

Berberine ameliorates chronic relapsing dextran sulfate sodium-induced colitis in C57BL/6 mice by suppressing Th17 responses

Ulcerative colitis (UC) is an increasingly common condition particularly in developed countries. The lack of satisfactory treatment has fueled the search for alternative therapeutic strategies. In recent studies, berberine, a plant alkaloid with a long history of medicinal use in Chinese medicine, has shown beneficial effects against animal models of acute UC. However, UC usually presents as a chronic condition with frequent relapse in patients. How berberine will act on chronic UC remains unclear. In the present study, we adopted dextran sulfate sodium (DSS)-induced chronic relapsing colitis model to assess the ameliorating activity of berberine. Colitis was induced by two cycles of 2.0% DSS for five days followed by 14days of drinking water plus a third cycle consisting of DSS only for five days. The colitis mice were orally administered 20mg/kg berberine from day 13 onward for 30days and monitored daily. The body weight, stool consistency, and stool bleeding were recorded for determination of the disease activity index (DAI). At the end of treatment, animals were sacrificed and samples were collected and subjected to histological, RT-qPCR, Western blot, and LC-MS analyses. Lymphocytes were isolated from spleens and mesenteric lymph nodes (MLN) and cultured for flow cytometry analysis of IL-17 secretion from CD4(+) cells and the Th17 cell differentiation. Results showed that berberine significantly ameliorated the DAI, colon shortening, colon tissue injury, and reduction of colonic expression of tight junction (TJ) protein ZO-1 and occludin of colitis mice. Notably, berberine treatment pronouncedly reduced DSS-upregulated Th17-related cytokine (IL-17 and ROR-γt) mRNAs in the colon. Furthermore, the mRNA expression of IL-6 and IL-23, and the phosphorylation of STAT3 in colon tissues from DSS-treated mice were pronouncedly inhibited by berberine. Moreover, the up-regulation of IL-17 secretion from CD4(+) cells of spleens and MLNs caused by DSS were significantly reversed by berberine treatment. Furthermore, Th17 cell differentiation from naive CD4(+) cells isolated from above DSS colitis mice were suppressed by berberine in a concentration-dependent manner. In summary, we demonstrated for the first time that berberine reduced the severity of chronic relapsing DSS-induced colitis by suppressing Th17 responses. The demonstration of activity in this mouse model supports the possibility of clinical efficacy of berberine in treating chronic UC.

5-Hydroxytryptamine promotes hepatocellular carcinoma proliferation by influencing β-catenin

5‐Hydroxytryptamine (5‐HT), a neurotransmitter and vasoactive factor, has been reported to promote proliferation of serum‐deprived hepatocellular carcinoma (HCC) cells but the detailed intracellular mechanism is unknown. As Wnt/β‐catenin signalling is highly dysregulated in a majority of HCC, this study explored the regulation of Wnt/β‐catenin signalling by 5‐HT. The expression of various 5‐HT receptors was studied by quantitative real‐time polymerase chain reaction (qPCR) in HCC cell lines as well as in 33 pairs of HCC tumours and corresponding adjacent non‐tumour tissues. Receptors 5‐HT1D (21/33, 63.6%), 5‐HT2B (12/33, 36.4%) and 5‐HT7 (15/33, 45.4%) were overexpressed whereas receptors 5‐HT2A (17/33, 51.5%) and 5‐HT5 (30/33, 90.1%) were reduced in HCC tumour tissues. In vitro data suggests 5‐HT increased total β‐catenin, active β‐catenin and decreased phosphorylated β‐catenin protein levels in serum deprived HuH‐7 and HepG2 cells compared to control cells under serum free medium without 5‐HT. Activation of Wnt/β‐catenin signalling was evidenced by increased expression of β‐catenin downstream target genes, Axin2, cyclin D1, dickoppf‐1 (DKK1) and glutamine synthetase (GS) by qPCR in serum‐deprived HCC cell lines treated with 5‐HT. Additionally, biochemical analysis revealed 5‐HT disrupted Axin1/β‐catenin interaction, a critical step in β‐catenin phosphorylation. Increased Wnt/β‐catenin activity was attenuated by antagonist of receptor 5‐HT7 (SB‐258719) in HCC cell lines and patient‐derived primary tumour tissues in the presence of 5‐HT. SB‐258719 also reduced tumour growth in vivo. This study provides evidence of Wnt/β‐catenin signalling activation by 5‐HT and may represent a potential therapeutic target for hepatocarcinogenesis.

Azoxystrobin Induces Apoptosis of Human Esophageal Squamous Cell Carcinoma KYSE-150 Cells through Triggering of the Mitochondrial Pathway

Recent studies indicate that mitochondrial pathways of apoptosis are potential chemotherapeutic target for the treatment of esophageal cancer. Azoxystrobin (AZOX), a methoxyacrylate derived from the naturally occurring strobilurins, is a known fungicide acting as a ubiquinol oxidation (Qo) inhibitor of mitochondrial respiratory complex III. In this study, the effects of AZOX on human esophageal squamous cell carcinoma KYSE-150 cells were examined and the underlying mechanisms were investigated. AZOX exhibited inhibitory effects on the proliferation of KYSE-150 cells with inhibitory concentration 50% (IC50) of 2.42 μg/ml by 48 h treatment. Flow cytometry assessment revealed that the inhibitory effect of AZOX on KYSE-150 cell proliferation occurred with cell cycle arrest at S phase and increased cell apoptosis in time-dependent and dose-dependent manners. Cleaved poly ADP ribose polymerase (PARP), caspase-3 and caspase-9 were increased significantly by AZOX. It is worth noted that the Bcl-2/Bax ratios were decreased because of the down-regulated Bcl-2 and up-regulated Bax expression level. Meanwhile, the cytochrome c release was increased by AZOX in KYSE-150 cells. AZOX-induced cytochrome c expression and caspase-3 activation was significantly blocked by Bax Channel Blocker. Intragastric administration of AZOX effectively decreased the tumor size generated by subcutaneous inoculation of KYSE-150 cells in nude mice. Consistently, decreased Bcl-2 expression, increased cytochrome c and PARP level, and activated caspase-3 and caspase-9 were observed in the tumor samples. These results indicate that AZOX can effectively induce esophageal cancer cell apoptosis through the mitochondrial pathways of apoptosis, suggesting AZOX or its derivatives may be developed as potential chemotherapeutic agents for the treatment of esophageal cancer.

Dysregulated expression of dickkopfs for potential detection of hepatocellular carcinoma

The prognosis for hepatocellular carcinoma (HCC) remains dismal due to the lack of diagnostic markers for early detection. This review will discuss the clinical potential of the dickkopf (DKK) family members as diagnostic and/or prognostic markers for HCC. In comparison to serum α-fetoprotein (AFP) level, which remains the gold standard for HCC diagnosis, high serum DKK1 levels have higher diagnostic value for HCC, especially for AFP-negative HCC, and can distinguish HCC from non-malignant chronic liver diseases. Additionally, the combination of serum DKK1 and AFP levels enhances diagnostic accuracy for HCC compared to serum DKK1 or AFP levels alone. Although DKK1 offers potential for its use in HCC diagnosis this review will discuss the challenges facing DKK1 and also shed some light on recent developments on the remaining DKK family members: DKK2, DKK3 and DKK4.

Survival benefit of a monoclonal antibody against cadherin-17 in an orthotopic liver tumor xenograft model

Aims: Monoclonal antibodies against tumor-related molecules are therapeutic agents for cancers. Promising results support the use of monoclonal antibodies in several cancers such as lung and breast cancer. However, using monoclonal antibodies as therapeutic agents to treat hepatocellular carcinoma (HCC), a major type of liver cancer, is still at the beginning. We have previously generated a monoclonal antibody against cadherin-17 (CDH17), named Lic5. This antibody has proven anti-tumor and cisplatin-sensitizing effect in HCC using a subcutaneous tumor xenograft mouse model. Original Research Article Chan et al.; JALSI, 4(3): 1-10, 2016; Article no.JALSI.22188 2 Study Design and Methodology: To further consolidate our previous finding, we tested the effect of Lic5 alone or in combination with cisplatin or epirubicin on tumor growth inhibition and animal survival using a more advanced orthotopic tumor xenograft model. Results: Single Lic5, cisplatin and epirubicin treatment inhibited growth of cultured HCC cells using cell proliferation assay, while more significant reductions were observed when cisplatin or epirubicin was used together with Lic5. Similar trends of growth inhibition were observed when the same experimental grouping was applied to treat orthotopic tumor-bearing nude mice. Treatment of Lic5 enhanced survivals of orthotopic tumor-bearing nude mice when compared to the control group. Among all experimental groups, combined Lic5 and epirubicin group yielded the best survival. For next phase antibody humanization, we also identified the complementarity determining regions (CDRs) on variable regions on the light and heavy chain of Lic5. Conclusion: Together, we have validated the preclinical use of Lic5 in an orthotopic HCC xenograft model. Our successful identification of CDRs constitutes the first step in synthesizing

Oncofetal Molecules as Biomarkers and Drug Targets for Hepatic Cancer

Hepatocellular carcinoma (HCC) is a major type of liver cancer prevalent in Asia and Africa, with a global increase in numbers in western countries. Despite decades of efforts in improving management of this malignancy, prognosis of patients still remains suboptimal. Frontline surgical treatments and traditional diagnostic methods suffer from own limitations. To alleviate this clinical dismal, research for alternated and supplemental methods are imperative. Different studies have discovered a panel of molecules related to tumorigenesis. Among them, a class of oncofetal molecules, characterized by their abundance in fetal livers and HCC but not in adult healthy livers, seems to serve as biomarkers and therapeutic targets for HCC. Tumorigenesis and embryogenesis share common characteristics and undergo similar processes in terms of proliferation, division, plasticity, motility, and convergence of mechanistic pathways. This chapter reviews several oncofetal molecules of livers including alpha-fetoprotein (AFP), glypican-3 (GPC3), insulin-like growth factor II mRNA binding protein 3 (IMP3), survivin, Golgi protein 73 (GP73), cadherin-17 (CDH17), and granulin-epithelin precursor (GEP) for their diagnostic and prognostic values. In addition, how these molecules can be used for developing therapies for HCC is also discussed. Most of the mentioned oncofetal molecules are found associating with poor disease conditions, while some of them have been studied for their potential capability in treating tumors in preclinical animal models. In summary, oncofetal molecules belong to an emerging class of candidates with potential application in improving current methods of diagnosis, prognosis, and treatment of HCC.

5-HT promotes hepatocellular carcinoma by influencing β-catenin

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA 5-hydroxytryptamine (5-HT), a neurotransmitter and vasoactive factor, has been reported to promote proliferation of serum-deprived human hepatocellular carcinoma cells (HCC) but the detailed intracellular mechanism is still unknown. As Wnt/β-catenin signaling is highly dysregulated in a majority of HCC, this study explored the regulation of Wnt/β-catenin signaling by 5-HT. 5-HT promoted proliferation of serum-deprived HuH-7 and HepG2 cells and also increased total β-catenin levels, and active β-catenin protein levels compared to just control cells under serum free medium without 5-HT. Furthermore, 5-HT increased β-catenin levels in the presence of cycloheximide, a protein synthesis inhibitor, suggesting increased β-catenin levels due to inhibition of β-catenin degradation. Quantitative real-time polymerase chain reaction (qPCR) showed increased β-catenin downstream target genes, Axin1, cyclin D1, dickoppf-1 (DKK1) and glutamine synthetase (GS), in serum-deprived HCC cell lines treated with 5-HT. We next studied the expression of various 5-HT were receptors (5-HT1D, 5-HT2A, 5-HT2B, 5-HT5 and 5-HT7) by qPCR in 33 pairs of HCC tumors and corresponding adjacent non-tumor tissues. Receptors 5-HT1D (21/33, 64%), 5-HT2B (12/33, 36%) and 5-HT7 (15/33, 45%) were overexpressed in HCC tumour tissues whereas receptor 5-HT5 was reduced (30/33, 91%) in HCC tumour tissues. Receptor 5-HT2A did not show any significant statistical difference between HCC tumour and corresponding non-tumour tissues. We further investigated whether antagonists of the 5-HT receptors attenuated the activity of 5-HT both in vitro and in vivo and we narrowed our study to 5-HT7 antagonist as the expression of 5-HT7 was found to be significantly associated to liver histology and venous infiltration. Antagonist of receptor 5-HT7, SB258719, attenuated growth of serum-deprived HCC cell lines and primary tumor tissues in the presence of 5-HT. Furthermore, SB258719 reduced tumor growth in a xenograft mouse model accompanied by reduced β-catenin and increased GSK-3β levels as observed by immunohistochemical analysis. Conclusion: This study provides evidence of Wnt/β-catenin signaling activation in 5-HT induced proliferation of serum-deprived HCC cells. The proliferation is inhibited by 5-HT7 antagonist, SB258719, which may represent a potential therapeutic target for hepatocarcinogenesis. Citation Format: Sarwat Fatima, Shi Xiaoki, Lin Zesie, Chen Guo, John W. Ho, Nikki P. Lee, Xiang Bian Zhao. 5-HT promotes hepatocellular carcinoma by influencing β-catenin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 58. doi:10.1158/1538-7445.AM2015-58