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Vitamin D receptor gene methylation in hepatocellular carcinoma
Accepted Manuscript Vitamin D receptor gene methylation in hepatocellular carcinoma
Mai Abdalla, Eman Khairy, Manal L. Louka, Randa Ali-Labib, Eman Abdel-Salam Ibrahim PII: DOI: Reference:
S0378-1119(18)30153-7 https://doi.org/10.1016/j.gene.2018.02.024 GENE 42576
To appear in:
Gene
Received date: Revised date: Accepted date:
15 November 2017 31 January 2018 8 February 2018
Please cite this article as: Mai Abdalla, Eman Khairy, Manal L. Louka, Randa Ali-Labib, Eman Abdel-Salam Ibrahim , Vitamin D receptor gene methylation in hepatocellular carcinoma. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), https://doi.org/10.1016/ j.gene.2018.02.024
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ACCEPTED MANUSCRIPT Research article:
Vitamin D Receptor Gene Methylation in Hepatocellular Carcinoma
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Mai Abdalla1, Eman Khairy 1a, Manal L. Louka 1, Randa Ali-Labib 1, Eman Abdel-Salam Ibrahim 2 1 Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Ain Shams University, Abbassia, Cairo, Egypt, P.O. box 11381. 2 Pathology Department, Faculty of Medicine, Ain Shams University a
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Corresponding author: Eman Khairy, Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Ain Shams University, Abbassia, Cairo, Egypt, P.O. box 11381.
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Email: [email protected]
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Conflict of interest statement The authors declare that they have no competing interests.
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Abstract
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Worldwide, hepatocellular carcinoma (HCC) is the major subtype of primary liver cancers. HCC is typically diagnosed late in its course. With respect to cancer, the genomic actions of vitamin D are mediated through binding to the Vitamin D Receptor (VDR), which allows it to modulate the expression of genes in a cell-and tissue-specific manner. Epigenetics is a rapidly evolving field of genetic study applicable to HCC. Changes in DNA methylation patterns are thought to be early events in hepatocarcinogenesis. Curcumin has great potential as an epigenetic agent. Accordingly, the current study has been designed to study the methylation status of VDR gene promoter for the first time in HCC aiming to find its clinical significance and potential screening role in chronic Liver Disease (CLD). Additionally, we aimed to investigate, the effect of Curcumin on HCC cell line, aiming to discover new therapeutic targets through epigenetics. This study was conducted on 45 formalin-fixed, paraffin-embedded liver tissue blocks including 15 HCC samples (group A), 15 CLD samples (group B) and 15 apparently normal tissue taken from around benign lesions (group C). Methylation Specific Restriction Digestion and qPCR were done on all samples after DNA extraction. The percentage of VDR gene promoter methylation was significantly higher in the HCC group compared to both CLD and control groups (p˂0.01). VDR promoter methylation by (MS-qPCR) was decreased and the relative expression of VDR by (qRT-PCR) was markedly increased in a dose-dependent fashion in cells grown in Curcuminadequate medium. In conclusion, this study may open a new gate for the use of VDR promoter methylation as a potential biomarker in HCC.
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Keywords: Epigenetics; Methylation; Hepatocellular carcinoma; Vitamin D Receptor; Curcumin, MS-qPCR; Biomarkers.
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ACCEPTED MANUSCRIPT 1. Introduction
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Worldwide, HCC is the major subtype of primary liver cancers (85-90%), where it represents the fifth most common cancer and the third leading cause of cancer related death [1]. In Egypt, HCC represents an important public health problem. It constitutes approximately 70% of all tumors among Egyptians, representing the second most common malignancy after bladder cancer in men and breast cancer in women. It is also considered to be the second most common cause of death in men [2]. The prognosis of HCC is dismal with 5-year survival being 1–4% [3].
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Egypt has the highest prevalence of HCV in the world (predominantly genotype 4), which has been attributed to previous public health eradication schemes for schistosomiasis [4]. In Egypt, increasing importance of HCV infection in the etiology of liver cancer had been shown. It was estimated to account for 40–50% of cases. HCC is typically diagnosed late in its course. Indeed, patients who present with cancer symptoms and/or with vascular invasion or extra-hepatic spread have only 50% survival rate at one year [5]. Currently, the major diagnostic measurements for HCC are imaging technology and serum α-fetoprotein (AFP). However, the sensitivity and specificity of AFP is not high, and early diagnosis of HCC is still challenging [6]. 50% of the patients reported accidental discovery of their hepatic focal lesions. Thus, there is an urgent need to develop novel strategies and investigate biomarkers for early diagnosis, treatment, and prognosis of HCC [7].
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During the last decade, Vitamin D has been established to act on many mechanisms regulating cell proliferation and differentiation [8], and has immunomodulatory, anti-inflammatory and anti- fibrotic properties [9]. Also, it can regulate key mediators of apoptosis [10]. Moreover, antiangiogenic effects of Vitamin D have also been described [11].
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With respect to cancer, vitamin D deficiency has been reported to have a negative prognostic role in breast cancer, colon cancer, prostate cancer and melanoma [12]. The genomic actions of vitamin D are mediated through its binding to VDR, which allows it to modulate the expression of genes in a cell-and tissue-specific manner. The VDR directly or indirectly regulates the expression of more than 200 genes that influence cell proliferation, differentiation and apoptosis, as well as immunomodulation and angiogenesis [13]. Since VDR is not commonly mutated in cancer, most reports have focused on altered patterns of histone acetylation and DNA methylation in the VDR promoter [14]. It has been documented in colon, endometrial and breast cancers [15-16]. Increased methylation levels in the promoter region of VDR and CYP genes may result in altered gene transcription, leading to impaired vitamin D function associated with diseases [17-18].
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ACCEPTED MANUSCRIPT Curcumin (diferuloylmethane) is a natural phytochemical. Curcumin has been displayed a plethora of favorable effects, including anti-oxidant, anti-proliferative and pro-apoptotic effects [19]. Nowadays, it is under a great deal of inspection from cancer investigators because of its anti-tumorigenic properties against human malignancies. Curcumin has great potential as an epigenetic agent. Previous studies have shown that Curcumin, can reverse the hypermethylation state of crucial genes in cervical, breast and prostatic cancers [20-22].
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Importantly, Curcumin has been shown to possess anti-HCC properties in vitro and in animal studies as revealed by Chuang et al. [23] and Mann et al [24]. However, its exact mechanism of action in the control of HCC is still not well-elucidated. Accordingly, the current study has been designed to study the methylation status of vitamin D receptor gene promoter for the first time in hepatocellular carcinoma aiming to find its clinical significance and its potential screening role in chronic Liver Disease. Additionally, we aimed to investigate, the effect of Curcumin on HCC cell line, hoping to discover new therapeutic targets through epigenetics.
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2. Patients and methods. The research protocol was assessed and approved in accordance with the guidelines of institutional review board of the Faculty of Medicine ethical committee, Ain Shams University, Cairo, Egypt. The present study was conducted on 45 formalin-fixed, paraffin-embedded liver tissue blocks including 15 hepatocellular carcinoma samples (group A), 15 samples of chronic Liver Disease (group B) and 15 apparently normal control tissue taken from around benign lesions (group C). Full clinical data were obtained from the clinical sheets of the patients. HCC was diagnosed according to AASLD Practice Guidelines and BCLC Classification [25-26]. The research protocol was in accordance with the ethical standards of the Declaration of Helsinki.
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2.1. Sample collection and processing
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1 to 5 gram samples were obtained from paraffin blocks. In normal, chronic inflammatory and uniform malignant lesions as well as slides obtained from core samples, a microtome was used to collect 3 to 4 sections. In non-uniform malignant lesions, the paraffin block was melted in a water bath of 60°C for 1 hour, then a trocar needle was used to bore a core sized sample. All samples were stored in phosphate buffer solution at 4°C until extraction was performed. 2.2. DNA extraction Total DNA from paraffin-embedded liver tissue blocks was extracted using QIAmp silica membrane columns ® DNA Kits according to the manufacturer’s instructions (Qiagen catalogue no. 56404, Germany). The concentration of DNA in each sample was measured using a
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ACCEPTED MANUSCRIPT NanoDrop™1000 Spectrophotometer (Thermo Fisher Scientific, USA). A ratio of absorbance at A260/A280 of 1.8–2 was generally accepted as “pure” for DNA.
2.3. Methylation Specific Restriction Digestion and qPCR
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The EpiTect Methyl II DNA restriction kit (Qiagen, Germany) together with the Epitect Methyl VDR qPCR primers (EPHS103069-1A; Qiagen) were used to analyze the methylation status of CpG islands in the VDR gene according to the manufacturer's instruction. Briefly, four digests were performed in each sample to detect different methylated DNA fractions. The product of a mock digest contained all of the input genomic DNA, the product of the methylation-sensitive restriction enzyme digest contained hypermethylated DNA sequences, whereas the product of the methylation-dependent restriction enzyme digest contained unmethylated DNA sequences in addition to the product of a double-digest which measured the background and the success of both enzymatic digestions. Following digestion, the remaining DNA in each individual enzyme reaction is quantified by real-time PCR using Vitamin D Receptor primers. Cycling conditions for quantitative methylation PCR analysis were as follows: Initial PCR activation was performed for 5 min at 95°C followed by 2-step cycling (40 cycles). Denaturation for 15 s at 95°C followed by Annealing/Extension for 60 s at 60°C. Finally, the raw ΔCT values were put into the data analysis spreadsheet supplied by the kit, which automatically calculates the relative amount of methylated and unmethylated DNA fractions per each sample by comparing the amount in each digest with that of a mock (no enzymes added). A dissociation curve was generated to check the specificity of the VDR gene.
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2.4. Cell cultures The HepG2 cells were kindly provided by the National Cancer Institute (Cairo University). They were cultured on RPMI 1640 Biowest media, supplied by ATCC (The American Type Culture Collection), supplemented with 10% heat inactivated fetal bovine serum, 2% Penicillin, Streptomycin and 1% Amphotericin B. Potential cytotoxicity of Curcumin, purchased from Sigma-Aldrich (St. Louis, MO, USA), on HepG2 cell lines was tested using the method of Skehan et al. [27]. HepG2 cells were plated in 96-multiwell plate (104cells/well) for 24 hours (hrs) to allow attachment of cell to the plate wall. Different concentrations of Curcumin (0, 5, 00, 20 and 30 μM) were added to the cell monolayer. Triplicate wells were prepared for each individual dose. Monolayer cells were incubated with Curcumin for 24 and 48 hrs at 37°C and in an atmosphere of 5% CO2. After incubation time, cells were fixed, washed and stained with Sulfo-Rhodamine-B stain. Excess stain was washed with acetic acid and attached stain was recovered with Tris-EDTA buffer. Color intensity was measured in an ELISA reader. Cell viability was calculated by setting the cell viabilities of non-treated cells to 100%. The relation between surviving fraction and Curcumin concentration was plotted to get the survival curve of
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HepG2 cell line. Calculation of the substance’s inhibitory concentration inducing 50% cell viability (IC50) was done. To determine the effect of different Curcumin concentrations on VDR expression, 8-well plates with HepG2 cells were cultured and incubated for 48 hrs with different concentrations of Curcumin (5, 10, 15 and 20 μM). Then, VDR promoter methylation status was determined as previously discussed and VDR relative expression was measured after RNA extraction from HepG2 cells grown in either Curcumin-deficient or Curcumin-adequate medium.
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2.5. Relative expression of VDR using qRT-PCR Total RNA was extracted from cell culture lysate by using RNeasy Mini kit (Catalogue no. 74104, Qiagen, Germany), according to manufacturers’ protocol. High capacity cDNA reverse transcription Kit (Applied biosystems, USA) was used for cDNA synthesis. QuantiTect Primer Assays were used to amplify VDR and the endogenous control (β actin). The sequence of each primer was derived from NCBI reference sequence data base (https://www.ncbi.nlm.nih.gov/refseq/). VDR forward primer was 5'AGTCTGAGGCCCAAGCTGTC-3' and the reverse primer was 5’GGAGACAGGTCCAGGGTCAC-3'. Quantitative PCR was carried out using QuantiTect SYBR® Green PCR Kit (Applied biosystems, USA) according to manufacturer's protocol. Relative expression levels of the VDR were measured using 2-ΔΔCt method with β actin used as the internal control to normalize the data [28].
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2.6. Statistical analysis All analysis was done using the Statistical Package for the Social Sciences (SPSS software version 20, Chicago, Illinois) on a personal computer. One way ANOVA was used to compare between quantitative variables that were normally distributed. Mann-Whitney and KruskalWallis tests were used for statistical comparison of the non-parametric data variables between different groups. Chi-square analysis was used to find out the relation between various qualitative data. Variables were cross tabulated in all possible combinations against each other. The correlation coefficients (r) were calculated by using the Spearman correlation. Receiver operating characteristic (ROC) curve determined the best value that gave maximum sensitivity and specificity. “P” value ˂ 0.05 was considered significant and ˂ 0.01 was considered highly significant. 3. Results 3.1. Clinical characteristics of study samples Demographic and clinico-pathological variables of the different study groups are shown in, table 1 and Laboratory parameters are shown in, table 2.
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ACCEPTED MANUSCRIPT 3.2 Methylation pattern of VDR in studied groups One Way ANOVA and Post Hoc tests showed a high significant VDR methylation level in HCC group when compared with other studied groups, table 3.
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3.3. Performance Characteristics of the Investigated Biomarkers for HCC Discrimination
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To evaluate the clinical value of VDR methylation level, ROC curves were constructed. When comparing HCC samples with non- malignant samples, the cutoff value of VDR methylation was 6.77% and AUC was 0.894. VDR gene promoter methylation positivity rate was (80%) in malignant group, (13.3%) in the CLD group and (0%) in the control group with a highly significant statistical difference (p˂0.01).
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VDR Promoter Methylation showed higher specificity (93.3%) than serum AFP (80%) with equal sensitivity. Combined VDR Gene Promoter Methylation and serum AFP provided a more sensitive method to detect HCC (93.3%) than either biomarker alone (80%).
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When comparing HCC patients with CLD patients, the cutoff value of VDR methylation was 9.3% and AUC was 0.847, figure 1 (a, b). On applying this cutoff value, the investigated biomarker achieved the same observations when compared to AFP, table 4.
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By applying any of these cutoff points, VDR gene promoter methylation as well as its positivity rates in relation to different demographic and clinico-pathological variables in HCC group showed no significant difference (p>0.05) except for the grades of differentiation (p= 0.029).
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Taken together, VDR methylation could have a discriminative power to differentiate HCC from CLD group. Also, the predicted probability (specificity and accuracy) of VDR methylation was higher than that of AFP.
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3.4. Correlation between the VDR methylation level and laboratory parameters Spearman’s test revealed a highly significant correlation between VDR methylation, and different laboratory parameters as albumin, AST, ALT and AFP in the three studied groups (p˂0.01), table 5. 3.5. Effect of Curcumin on HepG2 cell lines: The relation between surviving fraction of HepG2 cell lines and various concentrations of Curcumin after incubation for 24 and 48 hrs is shown in figure (2). Curcumin showed high inhibition of cell population growth in dose and time-dependent manner with 50 % growth inhibitory concentration (IC50) value of 18.8 μM at 48 hrs. 7
ACCEPTED MANUSCRIPT Curcumin changed pattern of both VDR promoter methylation and VDR expression in HepG2 cells. VDR promoter methylation detected by (MS-qPCR) was decreased and the relative expression of VDR measured by (qRT-PCR) was markedly increased in a dose-dependent fashion in cells grown in Curcumin-adequate medium, figure 3.
4. Discussion
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High morbidity observed in HCC is majorly attributed to lack of early detection markers and poor prognosis, which limit the options for chemotherapy, adjuvant therapies, or surgical procedures. Hence, exploration of novel frontiers in HCC diagnosis and therapeutics remain high priority research areas [29]. AFP is the most widely used tumor biomarker currently available for HCC diagnosis. However, the specificity of AFP is low, as its levels are elevated in patients with benign liver disease, such as hepatitis and cirrhosis. In addition, one third of the cases of early stages HCC are missed using AFP [30]. VDR promoter gene methylation pattern was altered in various types of cancer as reported in previous studies [15, 16, and 31].
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Based on these reports which all highlighted the need for a new sensitive and specific biomarker using different techniques, the current study was planned to evaluate VDR gene promoter methylation in HCC hoping to find a more reliable discriminating marker. In this study, DNA methylation levels in VDR gene promoter were detected in paraffin-embedded liver tissue blocks of the different investigated groups. To the best of our knowledge this study is the first one to evaluate the clinical role of VDR gene methylation in liver tissue of HCC and CLD patients aiming to suggest a potential discriminating clinical marker using hepatic core samples, routinely taken from hepatitis patients as well as carcinoma patients for histopathological examination.
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As regards to data description of subjects of the HCC group, patients with HCC grade I, grade II and grade III constituted 46.7%, 40% and 13.3% respectively. These proportions were similar to Pawlik et al. [32]. Also, patients with CLD were classified according to Hepatitis Scoring (Histology Activity Index); where the majority of patients scored at the range of (5-9) and (1014) with incidence of 46.7% and 33.3% respectively. As for the fibrosis stage, CLD patients with mild fibrosis constituted 40%, moderated fibrosis was 20% and those with severe fibrosis constituted 40%. These findings conformed to Standish et al. [33] and Sanai and Kaffee [34]. The percentage of VDR gene promoter methylation was significantly higher in the HCC group compared to both CLD and control groups (p˂0.01). However, levels of VDR gene promoter methylation in the current study showed no significant difference in relation to different demographic and clinico-pathological variables in HCC group (p>0.05) except amongst the grades of differentiation. This may suggest that VDR gene promoter methylation levels may serve as a prognostic tool in HCC patients. Brunner et al. [35] and Araújo et al. [36] have also confirmed that certain genes promoter’s methylation varied in relation to HCC differentiation. 8
ACCEPTED MANUSCRIPT In spite of non-significant difference (p>0.05), methylation mean rank increased with increasing fibrosis stage. This may need further evaluation on a larger scale to validate potential clinical use for this biomarker in pathological prognosis of severity of Hepatitis and thus identifying high risk CLD patients.
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There was a highly significant correlation between the three groups regarding VDR methylation , and different laboratory parameters as albumin, AST, ALT and AFP (p˂0.01). This is relatable since these parameters reflect severity of disease, whether CLD or HCC and therefore may suggest a potential clinical value for the studied biomarker.
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The current findings regarding VDR gene methylation level were consistent with others [16, 31]. These studies reported implication of VDR methylation in various tumors, where high levels of VDR gene methylation resulting in hindering the role of Vitamin D in inhibiting proliferation and metastasis in malignant cells in vitro and in vivo revealing Vitamin D resistance in cancer. Notably, vitamin D receptor-knock-out mice shows increased sensitivity to carcinogen with more pre-neoplasic lesions in the mammary glands than in wild-type mice as reported by Zinser and Welsh [37].
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In vivo study of colon cancer confirmed increased frequency of VDR CpG methylation and after treatment with estradiol, they suggested that estrogen may interfere with the process of DNA methylation and prevent silencing of the VDR gene [31]. Another study confirmed hypermethylation state VDR gene in primary breast tumors 45% as compared to 15% in normal breast tissue, on treatment with 5'deoxy-Azacytidine (AZA) – a hypomethylating drug expression of the active longer transcripts of VDR was restored, with a concurrent increase in expression of VDRE-containing genes; thus suggesting that pharmacological reversal of VDR methylation may re-establish breast cancer cell susceptibility to differentiation therapy using Calcitriol [16]. Furthermore, malignant adrenal tumors and choriocarcinoma-derived trophoblast cell lines were also associated with promoter methylation of the VDR gene as reported by Pilon et al. [17] and Novakovic et al. [38] respectively. In contrast, parathyroid tumors showed decreased expression of VDR [39]. However, no differences in DNA methylation of VDR were observed between parathyroid tumors and healthy controls [40]. Concerning serum level of AFP, current study showed a significant difference between the three groups (p˂0.05). In addition, the ROC curve based on the values collected from patients’ data sheets of the current study revealed that the best cutoff for serum AFP to discriminate HCC group from groups with non-malignant lesions was 11ng/mL which was conforming to previous studies [41-42].On the other hand, this study also evaluated serum AFP using the standard routinely used diagnostic level, i.e. 200ng/ml as reported by Shaker et al. [3]. As AFP is frequently used for the diagnosis of HCC, the relationship between AFP and VDR gene promoter methylation level was investigated and the results showed that the expression of 9
ACCEPTED MANUSCRIPT VDR promoter methylation in HCC patients was positively associated with the corresponding AFP level. The performance characteristics of both VDR gene methylation percent and serum AFP for detection of HCC against non-malignant lesions were analyzed. Notably, the combined use of VDR gene promoter methylation with routinely used AFP improved the sensitivity to 93.3%. The correlation indicated that VDR gene promoter methylation might be a potential clinical biomarker of HCC.
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Moreover, since VDR gene promoter methylation was detected in this study in the CLD patients as well, it may suggest that VDR gene promoter methylation is likely to be involved in the process of inflammation. This suggests that these cases should be followed up, as chronic inflammation is reported to play an important role in the pathogenesis of HCC. Forner et al. [43] supported this hypothesis by stating that CLD is the major risk factor for the development of HCC and thus, preventing or treating liver disease can decrease the risk of HCC.
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In accordance, another ROC curve was performed comparing HCC and CLD groups. The combined use of VDR gene promoter methylation level with AFP increased the sensitivity to 86.7%. Accordingly, the correlation indicated that VDR gene promoter methylation might be a potential biomarker in assessing high risk CLD patients with potential of carcinogenesis.
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On the basis that VDR gene promoter methylation in hepatocellular carcinoma may account for a large portion of Vitamin D resistance in hepatic carcinomatous cells, it was aimed to use Hypomethylating Drugs and/or nutrients (with reported DNA Demethylase activity) hoping to pharmacologically reverse VDR gene promoter methylation; therefore re-establishing cancer cell susceptibility to treatment with Calcitriol
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Curcumin has the potential to be an effective hypomethylating agent, through its unique mechanism of action [44]. Moreover, as it is a natural compound with no side-effects or toxicity to normal cells. In cultured cell models, the dose and time-dependent study was performed in the current work using different concentrations of Curcumin which gave an IC50 value of approximately 18.8 μM with an incubation period of 48 hrs. In comparison with previous studies, IC50 of Curcumin in HepG2 was 45.7 μM after 24 h, 23.7 μM after 48 hrs while it was 8 μM after 72 hrs as recorded by Dai et al., [45]. Also, Wang et al., [46] reported that IC50 of Curcumin in HepG2 cell line was 17.5 μM after 24 hrs. The difference in results could be attributed to different cell culture conditions. In addition, the results of current study showed that changes in Curcumin supply to HepG2 cell line can alter VDR promoter methylation pattern and subsequently VDR gene expression profile. Our preliminary data showed that, in a dose dependent manner, Curcumin was able to significantly reactivate VDR gene expression in parallel with decrease in VDR methylation percent. These results support a potential role for Curcumin as an epigenetic modulator through DNA methylation. Also, DNA methylation is one of epigenetics mechanisms may alter VDR gene expression [17].
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ACCEPTED MANUSCRIPT The study limitations include the following; firstly the study was not sufficiently powered to assess differences in marker level between early and advanced HCC stages. Secondly, detection of VDR methylation marker in HCC patient blood sample is needed. Therefore, further large multicenteric studies and in vitro functional analysis are required to reach a more definitive conclusion to elucidate clinical significance of VDR methylation in HCC and aid in the design of potential DNA hypomethylating agents as an adjuvant therapy.
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In conclusion, this study may open a new gate for the use of VDR gene promoter methylation as a potential biomarker, side by side with other laboratory investigations such as AFP to improve detection of HCC. In addition, it may identify the high risk group in CLD patients, since epigenetic changes that can lead to cancer precede pathological carcinogenesis. Also, VDR gene promoter methylation can act as a potential prognostic tool amongst different HCC grades. Findings in current study support a potential role for Curcumin as an epigenetic modulator.
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5. Figure legends:
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Figure (1): ROC curve analysis for methylation percentage of VDR gene promoter (a) HCC versus non- malignant, (b) HCC versus CLD
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Figure (2): The survival curve for HepG2 cell line after incubation with various concentrations (0, 5, 10, 20 and 30 µM) of Curcumin; (a) for 24 hrs (b) for 48 hrs.
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Figure (3): VDR methylation percent estimated by MS-qPCR and relative expression of VDR measured by qRT-PCR from Curcumin-adequate and Curcumin-depleted HepG2 cells.
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6. Tables
Table (1): Demographic and clinico-pathological variables in the different study groups.
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Table (2): Laboratory parameters in the different study groups. Table (3): Percentage of VDR gene promoter methylation among the different study groups.
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Table (4): Performance characteristics of investigated biomarkers for HCC detection Table (5): Correlation between VDR gene promoter methylation and laboratory parameters in the different study groups 7. References 1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F (2015): Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer; 136:E359-E 386.
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17. Pilon C, Rebellato A, Urbanet R, Guzzardo V, Cappellesso R, Sasano H, Fassina A, Fallo F (2015): Methylation Status of Vitamin D Receptor Gene Promoter in Benign and Malignant Adrenal Tumors. Int. J Endocr. (1):1-7. 18. Louka ML, Fawzy AM, Naiem AM, Elseknedy MF, Abdelhalim AE, Abdelghany MA (2017). Vitamin D and K signaling pathways in hepatocellular carcinoma. Gene. 629:108-116 19. Aggarwal BB, Sung B (2009). Pharmacological basis for the role of curcumin in chronic diseases: an age-old spice with modern targets. Trends Pharmacol Sci.30:85-94. 20. Jha AK, Nikbakht M, Parashar G (2010). Reversal of hypermethylation and reactivation of the RARβ2 gene by natural compounds in cervical cancer cell lines. Folia Biol ; 56: 195–200 21. Shu L, Khor TO, Lee JH, Boyanapalli SS, Huang Y, Wu TY, Saw CL, Cheung KL, Kong AN (2011). Epigenetic CpG demethylation of the promoter and reactivation of the expression of Neurog1 by curcumin in prostate LNCaP cells. AAPS J. 13:606–614 22. Kumar U, Sharma U, Rathi G (2017). Reversal of hypermethylation and reactivation of glutathione S-transferase pi 1 gene by curcumin in breast cancer cell line. Tumour Biol. 39(2):1010428317692258 23. Chuang SE, Cheng AL, Lin JK, Kuo ML (2000). Inhibition by curcumin of diethylnitrosamine-induced hepatic hyperplasia, inflammation, cellular gene products and cell-cycle-related proteins in rats. Food Chem Toxicol ;38:991-995 24. Mann CD, Neal CP, Garcea G, Manson MM, Dennison AR, Berry DP (2009). Phytochemicals as potential chemopreventive and chemotherapeutic agents in hepatocarcinogenesis. Eur J Cancer Prev; 18: 13-25. 25. Forner A, Reig ME, de Lope CR, Bruix J (2010): Current strategy for staging and treatment: the BCLC update and future prospects. Semin Liver Dis.; 30(1): 61-74. 26. Bruix J and Sherman M (2011): Management of hepatocellular carcinoma: an update. Hepatology. 53(3): 1020-1022 27. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR (1990): New colorimetriccytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst; 82(13): 1107–1112. 28. Livak KJ and Schmittgen TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods; 25(4): 402– 408. 29. Sidhu K, Kapoor NR, Pandey V (2015): The “macro” world of microRNAs in hepatocellular carcinoma. Front. Oncol. 5(68): 1-8. 30. Marrero JA, Feng Z, Wang Y, Nguyen MH, Befeler AS, Roberts LR, Reddy KR, Harnois D, Llovet JM, Normolle D, Dalhgren J, Chia D, Lok AS, Wagner PD, Srivastava S, Schwartz M. (2009): Alpha-fetoprotein, des-gamma carboxyprothrombin, and lectinbound alpha-fetoprotein in early hepatocellular carcinoma. Gastroenterol.37 (1):110-108. 31. Smirnoff P, Liel Y, Gnainsky J, Shany S, Schwartz B (1999): The protective effect of estrogen against chemi¬cally induced murine colon carcinogenesis is associated with decreased CpG island methylation and increased mRNA and protein expression of the colonic vitamin D receptor. Oncol Res. 11:255-264. 32. Pawlik TM, Delman KA, Vauthey JN, Nagorney DM, Ng IO, Ikai I, Yamaoka Y, Belghiti J, Lauwers GY, Poon RT, Abdalla EK (2005): Tumor size predicts vascular
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invasion and histologic grade: Implications for selection of surgical treatment for hepatocellular carcinoma. Liver Transplantation; 11(9), 1086–1092. 33. Standish RA, Cholongitas E, Dhillon A, Burroughs AK, Dhillon AP (2006): An appraisal of the histopathological assessment of liver fibrosis. Gut. 55(4): 569–578. 34. Sanai FM and Keeffe EB (2010): Liver Biopsy for Histological Assessment. Saudi J Gastroenterol. 16(2): 124–132. 35. Brunner AL, Johnson DS, Kim SW (2009): Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver. Genome Res.; 19(6): 1044–1056. 36. Araújo OC, Rosa AS, Fernandes A, Niel C, Villela-Nogueira CA, Pannain V, Araujo NM (2016): RASSF1A and DOK1 Promoter Methylation Levels in Hepatocellular Carcinoma, Cirrhotic and Non-Cirrhotic Liver, and Correlation with Liver Cancer in Brazilian Patients. PLoS One; 11(4): e0153796. 37. Zinser GM and Welsh JE (2004): Effect of vitamin D (3) receptor ablation on murine mammary gland development and tumorigenesis. J Steroid Biochem Mol Biol.; 89–90, 433– 436. 38. Novakovic B, Sibson M, Ng HK, Manuelpillai U, Rakyan V, Down T, Beck S, Fournier T, Evain-Brion D, Dimitriadis E, Craig JM, Morley R, Saffery R (2009): Placentaspecific methylation of the vitaminD 24-hydroxylase gene: implications for feedback autoregulation of active vitaminD levels at the fetomaternal interface. J. Biol.Chem.; 284:14838–14848. 39. Carling T, Rastad J, Szabó E,Westin G, kerstr m G (2000): Reduced parathyroid vitamin D receptor messenger ribonucleic acid levels in primary and secondary hyperparathyroidism. J. Clin.Endocrinol.Metab.85: 2000–2003. 40. Sulaiman L, Juhlin CC, Nilsson IL, Fotouhi O, Larsson C, Hashemi J (2013): Global and gene-specific promoter methylation analysis in primary hyperparathyroidism. Epigenetics; 8: 646–655. 41. Jiang P, Chan CW, Chan KC (2015): Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients. PNAC; 112(11): E1317–E1325. 42. Tarek M, Louka ML, Khairy E, Ali-Labib R, Zakaria Zaky D, Montasser IF (2017). Role of microRNA-7 and selenoprotein P in hepatocellular carcinoma. Tumour Biol. 39(5):1010428317698372. 43. Forner A, Llovet JM, Bruix J (2012): Hepatocellular carcinoma. Lancet. 379(9822):12451255. 44. Fu S and Kurzrock R (2010). Development of curcumin as an epigenetic agent. Cancer 116:4670–4676 45. Dai XZ, Yin HT, Sun LF, Hu X, Zhou C, Zhou Y, Zhang W, Huang XE, Li XC (2013). Potential therapeutic efficacy of curcumin in liver cancer .Asian Pac J Cancer Prev. 14(6):3855-3859. 46. Wang M, Ruan Y, Chen Q, Li S, Wang Q, Cai J (2011). Curcumin induced HepG2 cell apoptosis-associated mitochondrial membrane potential and intracellular free Ca(2+) concentration. Eur J Pharmacol. 650(1):41-47.
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List of Abbreviation American association for the study of liver diseases
AFP
Serum α-fetoprotein
ALT
Alanine transaminase
AST
Aspartate transaminase
AUC
Area under the curve
CLD
Chronic liver disease
HBV
Hepatitis B virus
HCC
Hepatocellular carcinoma
HCV
Hepatitis C virus
VDR
Vitamin D receptor
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AASLD
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ACCEPTED MANUSCRIPT Table (1): Demographic and clinico-pathological variables in the different study groups (a). Demographic and clinico-pathological variables
04 (33.3%)
14 (93.3%)
10 (66.7%)
Female
7
0 (6.7%)
0 (6.7%)
5 (33.3%)
04 (33.3%)
00(73.3%)
0 (0%)
0 (6.7%)
4 (26.7%)
05 (000%)
3 (60%)
4 2
12 (66.7%)
7
7 (46.7%)
6
Undifferentiated
2
1-4
2
5-9
3-4
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15 (100%)
-
-
6 (40.0%)
-
-
2 (13.3%)
-
-
-
2 (13.3%)
-
7
-
7 (46.7%)
-
5
-
5 (33.3%)
-
1
-
1 (6.7%)
-
6
-
6 (40%)
-
3
-
3 (20%)
-
6
-
6 (40%)
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15 (100%)
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5-6
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1-2
a
0 (0%)
< 200
15-18
0.001**
0 (0%)
3 (33.3%)
10-14
0 (0%)
05 (100%)
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Chi-square test, p>0.05= non-significant, **p˂0.01= highly significant, *p˂0.05= significant
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0.06
0.001**
6 (40%)
≥ 200
Well Differentiated Moderately Differentiated
Fibrosis Stage
0 (0%)
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< 40
p
05 (100%)
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≥ 40 AST(U/L)
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2 5 2 0 2 4 2 0
< 56
Hepatitis Scoring
Control n (%)
3 8
ALT(U/L)
HCC Grade
CLD n (%)
Male
≥ 56
Alpha feto protein (ng/mL)
HCC n (%)
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Sex
Groups n
0.04*
-
-
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HCC (n=05)
CLD (n=05)
p
Control (n=05)
Mean Rank
Median
Mean Rank
Median
Mean Rank
Albumin (mg/dL)
3.2
07.5
3.2
19.4
4.2
32
0.004**
AST(U/L)
67
33.3
57
24.9
34
10.67
0.001**
ALT(U/L)
78
31.13
73
29.27
37
8.6
0.001**
AFP (ng/mL)
26
35
8
22.3
11.7
0.001**
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Kruskal-Wallis test, **p˂0.01= highly significant
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n Groups HCC CLD Control
p (a)(b)
0.001**
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a One-Way ANOVA test, bKruskal-Wallis test, **p˂0.01= highly significant Highly Significant difference from the control, ii highly significantly difference from the CLD **p˂0.01 using ANOVA followed by post hoc test
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05 05 05
VDR Gene Methylation% Mean ± SD(a) Mean Median Rank(b) i, ii 07±00.6 08.56 34.83 3.34± 2.8 3.42 22.3 0.07±0.42 0.11 00.87
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Accuracy
≥ 6.77
80%
93.3%
85.7%
90.3%
88.9%
AFP (ng/mL)
≥11
80%
80%
66.7%
88.9%
80%
93.3%
73.3%
63.6%
95.6%
80%
Cutoff
Sensitivity
Specificity
PPV
NPV
Accuracy
VDR methylation %
≥ 9.3
73.3%
93.3%
91.7%
AFP(ng/mL)
≥16.5
73.3%
73.3%
73.3%
73.3%
73.3%
86.7%
66.7%
83.3%
76.7%
HCC and CLD group
Combined VDR meth% and AFP
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PPV: Positive Predictive Value, NPV: Negative Predictive Value
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77.8%
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Combined VDR meth% and AFP
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VDR methylation %
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Table (4): Performance characteristics of investigated biomarkers for HCC detection HCC group and nonCutoff Sensitivity Specificity PPV NPV malignant group
72.2%
83.3%
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Albumin AST ALT AFP
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r= correlation coefficient, **p˂0.01= highly significant
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VDR methylation% r p -0.520 0.001** 0.663 0.001** 0.609 0.001** 0.63 0.001**
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Table (5): Correlation between VDR gene promoter methylation and laboratory parameters in the different study groups
ACCEPTED MANUSCRIPT Highlights:
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The percentage of VDR gene promoter methylation was significantly higher in the HCC group compared to both CLD and control groups. The combined use of VDR gene promoter methylation with routinely used AFP improved the sensitivity. VDR gene promoter methylation can act as a potential prognostic tool amongst different HCC grades. The correlation indicated that VDR gene promoter methylation might be a potential biomarker in assessing high risk CLD patients with potential of carcinogenesis. Curcumin changed pattern of both VDR promoter methylation and VDR expression in HepG2 cells.
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Figure 1
Figure 2
Figure 3
Mai Abdalla, Eman Khairy, Manal L. Louka, Randa Ali-Labib, Eman Abdel-Salam Ibrahim PII: DOI: Reference:
S0378-1119(18)30153-7 https://doi.org/10.1016/j.gene.2018.02.024 GENE 42576
To appear in:
Gene
Received date: Revised date: Accepted date:
15 November 2017 31 January 2018 8 February 2018
Please cite this article as: Mai Abdalla, Eman Khairy, Manal L. Louka, Randa Ali-Labib, Eman Abdel-Salam Ibrahim , Vitamin D receptor gene methylation in hepatocellular carcinoma. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), https://doi.org/10.1016/ j.gene.2018.02.024
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ACCEPTED MANUSCRIPT Research article:
Vitamin D Receptor Gene Methylation in Hepatocellular Carcinoma
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Mai Abdalla1, Eman Khairy 1a, Manal L. Louka 1, Randa Ali-Labib 1, Eman Abdel-Salam Ibrahim 2 1 Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Ain Shams University, Abbassia, Cairo, Egypt, P.O. box 11381. 2 Pathology Department, Faculty of Medicine, Ain Shams University a
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Corresponding author: Eman Khairy, Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Ain Shams University, Abbassia, Cairo, Egypt, P.O. box 11381.
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Email: [email protected]
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Conflict of interest statement The authors declare that they have no competing interests.
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Abstract
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Worldwide, hepatocellular carcinoma (HCC) is the major subtype of primary liver cancers. HCC is typically diagnosed late in its course. With respect to cancer, the genomic actions of vitamin D are mediated through binding to the Vitamin D Receptor (VDR), which allows it to modulate the expression of genes in a cell-and tissue-specific manner. Epigenetics is a rapidly evolving field of genetic study applicable to HCC. Changes in DNA methylation patterns are thought to be early events in hepatocarcinogenesis. Curcumin has great potential as an epigenetic agent. Accordingly, the current study has been designed to study the methylation status of VDR gene promoter for the first time in HCC aiming to find its clinical significance and potential screening role in chronic Liver Disease (CLD). Additionally, we aimed to investigate, the effect of Curcumin on HCC cell line, aiming to discover new therapeutic targets through epigenetics. This study was conducted on 45 formalin-fixed, paraffin-embedded liver tissue blocks including 15 HCC samples (group A), 15 CLD samples (group B) and 15 apparently normal tissue taken from around benign lesions (group C). Methylation Specific Restriction Digestion and qPCR were done on all samples after DNA extraction. The percentage of VDR gene promoter methylation was significantly higher in the HCC group compared to both CLD and control groups (p˂0.01). VDR promoter methylation by (MS-qPCR) was decreased and the relative expression of VDR by (qRT-PCR) was markedly increased in a dose-dependent fashion in cells grown in Curcuminadequate medium. In conclusion, this study may open a new gate for the use of VDR promoter methylation as a potential biomarker in HCC.
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Keywords: Epigenetics; Methylation; Hepatocellular carcinoma; Vitamin D Receptor; Curcumin, MS-qPCR; Biomarkers.
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Worldwide, HCC is the major subtype of primary liver cancers (85-90%), where it represents the fifth most common cancer and the third leading cause of cancer related death [1]. In Egypt, HCC represents an important public health problem. It constitutes approximately 70% of all tumors among Egyptians, representing the second most common malignancy after bladder cancer in men and breast cancer in women. It is also considered to be the second most common cause of death in men [2]. The prognosis of HCC is dismal with 5-year survival being 1–4% [3].
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Egypt has the highest prevalence of HCV in the world (predominantly genotype 4), which has been attributed to previous public health eradication schemes for schistosomiasis [4]. In Egypt, increasing importance of HCV infection in the etiology of liver cancer had been shown. It was estimated to account for 40–50% of cases. HCC is typically diagnosed late in its course. Indeed, patients who present with cancer symptoms and/or with vascular invasion or extra-hepatic spread have only 50% survival rate at one year [5]. Currently, the major diagnostic measurements for HCC are imaging technology and serum α-fetoprotein (AFP). However, the sensitivity and specificity of AFP is not high, and early diagnosis of HCC is still challenging [6]. 50% of the patients reported accidental discovery of their hepatic focal lesions. Thus, there is an urgent need to develop novel strategies and investigate biomarkers for early diagnosis, treatment, and prognosis of HCC [7].
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During the last decade, Vitamin D has been established to act on many mechanisms regulating cell proliferation and differentiation [8], and has immunomodulatory, anti-inflammatory and anti- fibrotic properties [9]. Also, it can regulate key mediators of apoptosis [10]. Moreover, antiangiogenic effects of Vitamin D have also been described [11].
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With respect to cancer, vitamin D deficiency has been reported to have a negative prognostic role in breast cancer, colon cancer, prostate cancer and melanoma [12]. The genomic actions of vitamin D are mediated through its binding to VDR, which allows it to modulate the expression of genes in a cell-and tissue-specific manner. The VDR directly or indirectly regulates the expression of more than 200 genes that influence cell proliferation, differentiation and apoptosis, as well as immunomodulation and angiogenesis [13]. Since VDR is not commonly mutated in cancer, most reports have focused on altered patterns of histone acetylation and DNA methylation in the VDR promoter [14]. It has been documented in colon, endometrial and breast cancers [15-16]. Increased methylation levels in the promoter region of VDR and CYP genes may result in altered gene transcription, leading to impaired vitamin D function associated with diseases [17-18].
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ACCEPTED MANUSCRIPT Curcumin (diferuloylmethane) is a natural phytochemical. Curcumin has been displayed a plethora of favorable effects, including anti-oxidant, anti-proliferative and pro-apoptotic effects [19]. Nowadays, it is under a great deal of inspection from cancer investigators because of its anti-tumorigenic properties against human malignancies. Curcumin has great potential as an epigenetic agent. Previous studies have shown that Curcumin, can reverse the hypermethylation state of crucial genes in cervical, breast and prostatic cancers [20-22].
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Importantly, Curcumin has been shown to possess anti-HCC properties in vitro and in animal studies as revealed by Chuang et al. [23] and Mann et al [24]. However, its exact mechanism of action in the control of HCC is still not well-elucidated. Accordingly, the current study has been designed to study the methylation status of vitamin D receptor gene promoter for the first time in hepatocellular carcinoma aiming to find its clinical significance and its potential screening role in chronic Liver Disease. Additionally, we aimed to investigate, the effect of Curcumin on HCC cell line, hoping to discover new therapeutic targets through epigenetics.
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2. Patients and methods. The research protocol was assessed and approved in accordance with the guidelines of institutional review board of the Faculty of Medicine ethical committee, Ain Shams University, Cairo, Egypt. The present study was conducted on 45 formalin-fixed, paraffin-embedded liver tissue blocks including 15 hepatocellular carcinoma samples (group A), 15 samples of chronic Liver Disease (group B) and 15 apparently normal control tissue taken from around benign lesions (group C). Full clinical data were obtained from the clinical sheets of the patients. HCC was diagnosed according to AASLD Practice Guidelines and BCLC Classification [25-26]. The research protocol was in accordance with the ethical standards of the Declaration of Helsinki.
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2.1. Sample collection and processing
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1 to 5 gram samples were obtained from paraffin blocks. In normal, chronic inflammatory and uniform malignant lesions as well as slides obtained from core samples, a microtome was used to collect 3 to 4 sections. In non-uniform malignant lesions, the paraffin block was melted in a water bath of 60°C for 1 hour, then a trocar needle was used to bore a core sized sample. All samples were stored in phosphate buffer solution at 4°C until extraction was performed. 2.2. DNA extraction Total DNA from paraffin-embedded liver tissue blocks was extracted using QIAmp silica membrane columns ® DNA Kits according to the manufacturer’s instructions (Qiagen catalogue no. 56404, Germany). The concentration of DNA in each sample was measured using a
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ACCEPTED MANUSCRIPT NanoDrop™1000 Spectrophotometer (Thermo Fisher Scientific, USA). A ratio of absorbance at A260/A280 of 1.8–2 was generally accepted as “pure” for DNA.
2.3. Methylation Specific Restriction Digestion and qPCR
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The EpiTect Methyl II DNA restriction kit (Qiagen, Germany) together with the Epitect Methyl VDR qPCR primers (EPHS103069-1A; Qiagen) were used to analyze the methylation status of CpG islands in the VDR gene according to the manufacturer's instruction. Briefly, four digests were performed in each sample to detect different methylated DNA fractions. The product of a mock digest contained all of the input genomic DNA, the product of the methylation-sensitive restriction enzyme digest contained hypermethylated DNA sequences, whereas the product of the methylation-dependent restriction enzyme digest contained unmethylated DNA sequences in addition to the product of a double-digest which measured the background and the success of both enzymatic digestions. Following digestion, the remaining DNA in each individual enzyme reaction is quantified by real-time PCR using Vitamin D Receptor primers. Cycling conditions for quantitative methylation PCR analysis were as follows: Initial PCR activation was performed for 5 min at 95°C followed by 2-step cycling (40 cycles). Denaturation for 15 s at 95°C followed by Annealing/Extension for 60 s at 60°C. Finally, the raw ΔCT values were put into the data analysis spreadsheet supplied by the kit, which automatically calculates the relative amount of methylated and unmethylated DNA fractions per each sample by comparing the amount in each digest with that of a mock (no enzymes added). A dissociation curve was generated to check the specificity of the VDR gene.
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2.4. Cell cultures The HepG2 cells were kindly provided by the National Cancer Institute (Cairo University). They were cultured on RPMI 1640 Biowest media, supplied by ATCC (The American Type Culture Collection), supplemented with 10% heat inactivated fetal bovine serum, 2% Penicillin, Streptomycin and 1% Amphotericin B. Potential cytotoxicity of Curcumin, purchased from Sigma-Aldrich (St. Louis, MO, USA), on HepG2 cell lines was tested using the method of Skehan et al. [27]. HepG2 cells were plated in 96-multiwell plate (104cells/well) for 24 hours (hrs) to allow attachment of cell to the plate wall. Different concentrations of Curcumin (0, 5, 00, 20 and 30 μM) were added to the cell monolayer. Triplicate wells were prepared for each individual dose. Monolayer cells were incubated with Curcumin for 24 and 48 hrs at 37°C and in an atmosphere of 5% CO2. After incubation time, cells were fixed, washed and stained with Sulfo-Rhodamine-B stain. Excess stain was washed with acetic acid and attached stain was recovered with Tris-EDTA buffer. Color intensity was measured in an ELISA reader. Cell viability was calculated by setting the cell viabilities of non-treated cells to 100%. The relation between surviving fraction and Curcumin concentration was plotted to get the survival curve of
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HepG2 cell line. Calculation of the substance’s inhibitory concentration inducing 50% cell viability (IC50) was done. To determine the effect of different Curcumin concentrations on VDR expression, 8-well plates with HepG2 cells were cultured and incubated for 48 hrs with different concentrations of Curcumin (5, 10, 15 and 20 μM). Then, VDR promoter methylation status was determined as previously discussed and VDR relative expression was measured after RNA extraction from HepG2 cells grown in either Curcumin-deficient or Curcumin-adequate medium.
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2.5. Relative expression of VDR using qRT-PCR Total RNA was extracted from cell culture lysate by using RNeasy Mini kit (Catalogue no. 74104, Qiagen, Germany), according to manufacturers’ protocol. High capacity cDNA reverse transcription Kit (Applied biosystems, USA) was used for cDNA synthesis. QuantiTect Primer Assays were used to amplify VDR and the endogenous control (β actin). The sequence of each primer was derived from NCBI reference sequence data base (https://www.ncbi.nlm.nih.gov/refseq/). VDR forward primer was 5'AGTCTGAGGCCCAAGCTGTC-3' and the reverse primer was 5’GGAGACAGGTCCAGGGTCAC-3'. Quantitative PCR was carried out using QuantiTect SYBR® Green PCR Kit (Applied biosystems, USA) according to manufacturer's protocol. Relative expression levels of the VDR were measured using 2-ΔΔCt method with β actin used as the internal control to normalize the data [28].
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2.6. Statistical analysis All analysis was done using the Statistical Package for the Social Sciences (SPSS software version 20, Chicago, Illinois) on a personal computer. One way ANOVA was used to compare between quantitative variables that were normally distributed. Mann-Whitney and KruskalWallis tests were used for statistical comparison of the non-parametric data variables between different groups. Chi-square analysis was used to find out the relation between various qualitative data. Variables were cross tabulated in all possible combinations against each other. The correlation coefficients (r) were calculated by using the Spearman correlation. Receiver operating characteristic (ROC) curve determined the best value that gave maximum sensitivity and specificity. “P” value ˂ 0.05 was considered significant and ˂ 0.01 was considered highly significant. 3. Results 3.1. Clinical characteristics of study samples Demographic and clinico-pathological variables of the different study groups are shown in, table 1 and Laboratory parameters are shown in, table 2.
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3.3. Performance Characteristics of the Investigated Biomarkers for HCC Discrimination
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To evaluate the clinical value of VDR methylation level, ROC curves were constructed. When comparing HCC samples with non- malignant samples, the cutoff value of VDR methylation was 6.77% and AUC was 0.894. VDR gene promoter methylation positivity rate was (80%) in malignant group, (13.3%) in the CLD group and (0%) in the control group with a highly significant statistical difference (p˂0.01).
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VDR Promoter Methylation showed higher specificity (93.3%) than serum AFP (80%) with equal sensitivity. Combined VDR Gene Promoter Methylation and serum AFP provided a more sensitive method to detect HCC (93.3%) than either biomarker alone (80%).
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When comparing HCC patients with CLD patients, the cutoff value of VDR methylation was 9.3% and AUC was 0.847, figure 1 (a, b). On applying this cutoff value, the investigated biomarker achieved the same observations when compared to AFP, table 4.
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By applying any of these cutoff points, VDR gene promoter methylation as well as its positivity rates in relation to different demographic and clinico-pathological variables in HCC group showed no significant difference (p>0.05) except for the grades of differentiation (p= 0.029).
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Taken together, VDR methylation could have a discriminative power to differentiate HCC from CLD group. Also, the predicted probability (specificity and accuracy) of VDR methylation was higher than that of AFP.
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3.4. Correlation between the VDR methylation level and laboratory parameters Spearman’s test revealed a highly significant correlation between VDR methylation, and different laboratory parameters as albumin, AST, ALT and AFP in the three studied groups (p˂0.01), table 5. 3.5. Effect of Curcumin on HepG2 cell lines: The relation between surviving fraction of HepG2 cell lines and various concentrations of Curcumin after incubation for 24 and 48 hrs is shown in figure (2). Curcumin showed high inhibition of cell population growth in dose and time-dependent manner with 50 % growth inhibitory concentration (IC50) value of 18.8 μM at 48 hrs. 7
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4. Discussion
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High morbidity observed in HCC is majorly attributed to lack of early detection markers and poor prognosis, which limit the options for chemotherapy, adjuvant therapies, or surgical procedures. Hence, exploration of novel frontiers in HCC diagnosis and therapeutics remain high priority research areas [29]. AFP is the most widely used tumor biomarker currently available for HCC diagnosis. However, the specificity of AFP is low, as its levels are elevated in patients with benign liver disease, such as hepatitis and cirrhosis. In addition, one third of the cases of early stages HCC are missed using AFP [30]. VDR promoter gene methylation pattern was altered in various types of cancer as reported in previous studies [15, 16, and 31].
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Based on these reports which all highlighted the need for a new sensitive and specific biomarker using different techniques, the current study was planned to evaluate VDR gene promoter methylation in HCC hoping to find a more reliable discriminating marker. In this study, DNA methylation levels in VDR gene promoter were detected in paraffin-embedded liver tissue blocks of the different investigated groups. To the best of our knowledge this study is the first one to evaluate the clinical role of VDR gene methylation in liver tissue of HCC and CLD patients aiming to suggest a potential discriminating clinical marker using hepatic core samples, routinely taken from hepatitis patients as well as carcinoma patients for histopathological examination.
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As regards to data description of subjects of the HCC group, patients with HCC grade I, grade II and grade III constituted 46.7%, 40% and 13.3% respectively. These proportions were similar to Pawlik et al. [32]. Also, patients with CLD were classified according to Hepatitis Scoring (Histology Activity Index); where the majority of patients scored at the range of (5-9) and (1014) with incidence of 46.7% and 33.3% respectively. As for the fibrosis stage, CLD patients with mild fibrosis constituted 40%, moderated fibrosis was 20% and those with severe fibrosis constituted 40%. These findings conformed to Standish et al. [33] and Sanai and Kaffee [34]. The percentage of VDR gene promoter methylation was significantly higher in the HCC group compared to both CLD and control groups (p˂0.01). However, levels of VDR gene promoter methylation in the current study showed no significant difference in relation to different demographic and clinico-pathological variables in HCC group (p>0.05) except amongst the grades of differentiation. This may suggest that VDR gene promoter methylation levels may serve as a prognostic tool in HCC patients. Brunner et al. [35] and Araújo et al. [36] have also confirmed that certain genes promoter’s methylation varied in relation to HCC differentiation. 8
ACCEPTED MANUSCRIPT In spite of non-significant difference (p>0.05), methylation mean rank increased with increasing fibrosis stage. This may need further evaluation on a larger scale to validate potential clinical use for this biomarker in pathological prognosis of severity of Hepatitis and thus identifying high risk CLD patients.
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There was a highly significant correlation between the three groups regarding VDR methylation , and different laboratory parameters as albumin, AST, ALT and AFP (p˂0.01). This is relatable since these parameters reflect severity of disease, whether CLD or HCC and therefore may suggest a potential clinical value for the studied biomarker.
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The current findings regarding VDR gene methylation level were consistent with others [16, 31]. These studies reported implication of VDR methylation in various tumors, where high levels of VDR gene methylation resulting in hindering the role of Vitamin D in inhibiting proliferation and metastasis in malignant cells in vitro and in vivo revealing Vitamin D resistance in cancer. Notably, vitamin D receptor-knock-out mice shows increased sensitivity to carcinogen with more pre-neoplasic lesions in the mammary glands than in wild-type mice as reported by Zinser and Welsh [37].
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In vivo study of colon cancer confirmed increased frequency of VDR CpG methylation and after treatment with estradiol, they suggested that estrogen may interfere with the process of DNA methylation and prevent silencing of the VDR gene [31]. Another study confirmed hypermethylation state VDR gene in primary breast tumors 45% as compared to 15% in normal breast tissue, on treatment with 5'deoxy-Azacytidine (AZA) – a hypomethylating drug expression of the active longer transcripts of VDR was restored, with a concurrent increase in expression of VDRE-containing genes; thus suggesting that pharmacological reversal of VDR methylation may re-establish breast cancer cell susceptibility to differentiation therapy using Calcitriol [16]. Furthermore, malignant adrenal tumors and choriocarcinoma-derived trophoblast cell lines were also associated with promoter methylation of the VDR gene as reported by Pilon et al. [17] and Novakovic et al. [38] respectively. In contrast, parathyroid tumors showed decreased expression of VDR [39]. However, no differences in DNA methylation of VDR were observed between parathyroid tumors and healthy controls [40]. Concerning serum level of AFP, current study showed a significant difference between the three groups (p˂0.05). In addition, the ROC curve based on the values collected from patients’ data sheets of the current study revealed that the best cutoff for serum AFP to discriminate HCC group from groups with non-malignant lesions was 11ng/mL which was conforming to previous studies [41-42].On the other hand, this study also evaluated serum AFP using the standard routinely used diagnostic level, i.e. 200ng/ml as reported by Shaker et al. [3]. As AFP is frequently used for the diagnosis of HCC, the relationship between AFP and VDR gene promoter methylation level was investigated and the results showed that the expression of 9
ACCEPTED MANUSCRIPT VDR promoter methylation in HCC patients was positively associated with the corresponding AFP level. The performance characteristics of both VDR gene methylation percent and serum AFP for detection of HCC against non-malignant lesions were analyzed. Notably, the combined use of VDR gene promoter methylation with routinely used AFP improved the sensitivity to 93.3%. The correlation indicated that VDR gene promoter methylation might be a potential clinical biomarker of HCC.
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Moreover, since VDR gene promoter methylation was detected in this study in the CLD patients as well, it may suggest that VDR gene promoter methylation is likely to be involved in the process of inflammation. This suggests that these cases should be followed up, as chronic inflammation is reported to play an important role in the pathogenesis of HCC. Forner et al. [43] supported this hypothesis by stating that CLD is the major risk factor for the development of HCC and thus, preventing or treating liver disease can decrease the risk of HCC.
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In accordance, another ROC curve was performed comparing HCC and CLD groups. The combined use of VDR gene promoter methylation level with AFP increased the sensitivity to 86.7%. Accordingly, the correlation indicated that VDR gene promoter methylation might be a potential biomarker in assessing high risk CLD patients with potential of carcinogenesis.
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On the basis that VDR gene promoter methylation in hepatocellular carcinoma may account for a large portion of Vitamin D resistance in hepatic carcinomatous cells, it was aimed to use Hypomethylating Drugs and/or nutrients (with reported DNA Demethylase activity) hoping to pharmacologically reverse VDR gene promoter methylation; therefore re-establishing cancer cell susceptibility to treatment with Calcitriol
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Curcumin has the potential to be an effective hypomethylating agent, through its unique mechanism of action [44]. Moreover, as it is a natural compound with no side-effects or toxicity to normal cells. In cultured cell models, the dose and time-dependent study was performed in the current work using different concentrations of Curcumin which gave an IC50 value of approximately 18.8 μM with an incubation period of 48 hrs. In comparison with previous studies, IC50 of Curcumin in HepG2 was 45.7 μM after 24 h, 23.7 μM after 48 hrs while it was 8 μM after 72 hrs as recorded by Dai et al., [45]. Also, Wang et al., [46] reported that IC50 of Curcumin in HepG2 cell line was 17.5 μM after 24 hrs. The difference in results could be attributed to different cell culture conditions. In addition, the results of current study showed that changes in Curcumin supply to HepG2 cell line can alter VDR promoter methylation pattern and subsequently VDR gene expression profile. Our preliminary data showed that, in a dose dependent manner, Curcumin was able to significantly reactivate VDR gene expression in parallel with decrease in VDR methylation percent. These results support a potential role for Curcumin as an epigenetic modulator through DNA methylation. Also, DNA methylation is one of epigenetics mechanisms may alter VDR gene expression [17].
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ACCEPTED MANUSCRIPT The study limitations include the following; firstly the study was not sufficiently powered to assess differences in marker level between early and advanced HCC stages. Secondly, detection of VDR methylation marker in HCC patient blood sample is needed. Therefore, further large multicenteric studies and in vitro functional analysis are required to reach a more definitive conclusion to elucidate clinical significance of VDR methylation in HCC and aid in the design of potential DNA hypomethylating agents as an adjuvant therapy.
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In conclusion, this study may open a new gate for the use of VDR gene promoter methylation as a potential biomarker, side by side with other laboratory investigations such as AFP to improve detection of HCC. In addition, it may identify the high risk group in CLD patients, since epigenetic changes that can lead to cancer precede pathological carcinogenesis. Also, VDR gene promoter methylation can act as a potential prognostic tool amongst different HCC grades. Findings in current study support a potential role for Curcumin as an epigenetic modulator.
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5. Figure legends:
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Figure (1): ROC curve analysis for methylation percentage of VDR gene promoter (a) HCC versus non- malignant, (b) HCC versus CLD
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Figure (2): The survival curve for HepG2 cell line after incubation with various concentrations (0, 5, 10, 20 and 30 µM) of Curcumin; (a) for 24 hrs (b) for 48 hrs.
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Figure (3): VDR methylation percent estimated by MS-qPCR and relative expression of VDR measured by qRT-PCR from Curcumin-adequate and Curcumin-depleted HepG2 cells.
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6. Tables
Table (1): Demographic and clinico-pathological variables in the different study groups.
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Table (2): Laboratory parameters in the different study groups. Table (3): Percentage of VDR gene promoter methylation among the different study groups.
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Table (4): Performance characteristics of investigated biomarkers for HCC detection Table (5): Correlation between VDR gene promoter methylation and laboratory parameters in the different study groups 7. References 1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F (2015): Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer; 136:E359-E 386.
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17. Pilon C, Rebellato A, Urbanet R, Guzzardo V, Cappellesso R, Sasano H, Fassina A, Fallo F (2015): Methylation Status of Vitamin D Receptor Gene Promoter in Benign and Malignant Adrenal Tumors. Int. J Endocr. (1):1-7. 18. Louka ML, Fawzy AM, Naiem AM, Elseknedy MF, Abdelhalim AE, Abdelghany MA (2017). Vitamin D and K signaling pathways in hepatocellular carcinoma. Gene. 629:108-116 19. Aggarwal BB, Sung B (2009). Pharmacological basis for the role of curcumin in chronic diseases: an age-old spice with modern targets. Trends Pharmacol Sci.30:85-94. 20. Jha AK, Nikbakht M, Parashar G (2010). Reversal of hypermethylation and reactivation of the RARβ2 gene by natural compounds in cervical cancer cell lines. Folia Biol ; 56: 195–200 21. Shu L, Khor TO, Lee JH, Boyanapalli SS, Huang Y, Wu TY, Saw CL, Cheung KL, Kong AN (2011). Epigenetic CpG demethylation of the promoter and reactivation of the expression of Neurog1 by curcumin in prostate LNCaP cells. AAPS J. 13:606–614 22. Kumar U, Sharma U, Rathi G (2017). Reversal of hypermethylation and reactivation of glutathione S-transferase pi 1 gene by curcumin in breast cancer cell line. Tumour Biol. 39(2):1010428317692258 23. Chuang SE, Cheng AL, Lin JK, Kuo ML (2000). Inhibition by curcumin of diethylnitrosamine-induced hepatic hyperplasia, inflammation, cellular gene products and cell-cycle-related proteins in rats. Food Chem Toxicol ;38:991-995 24. Mann CD, Neal CP, Garcea G, Manson MM, Dennison AR, Berry DP (2009). Phytochemicals as potential chemopreventive and chemotherapeutic agents in hepatocarcinogenesis. Eur J Cancer Prev; 18: 13-25. 25. Forner A, Reig ME, de Lope CR, Bruix J (2010): Current strategy for staging and treatment: the BCLC update and future prospects. Semin Liver Dis.; 30(1): 61-74. 26. Bruix J and Sherman M (2011): Management of hepatocellular carcinoma: an update. Hepatology. 53(3): 1020-1022 27. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR (1990): New colorimetriccytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst; 82(13): 1107–1112. 28. Livak KJ and Schmittgen TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods; 25(4): 402– 408. 29. Sidhu K, Kapoor NR, Pandey V (2015): The “macro” world of microRNAs in hepatocellular carcinoma. Front. Oncol. 5(68): 1-8. 30. Marrero JA, Feng Z, Wang Y, Nguyen MH, Befeler AS, Roberts LR, Reddy KR, Harnois D, Llovet JM, Normolle D, Dalhgren J, Chia D, Lok AS, Wagner PD, Srivastava S, Schwartz M. (2009): Alpha-fetoprotein, des-gamma carboxyprothrombin, and lectinbound alpha-fetoprotein in early hepatocellular carcinoma. Gastroenterol.37 (1):110-108. 31. Smirnoff P, Liel Y, Gnainsky J, Shany S, Schwartz B (1999): The protective effect of estrogen against chemi¬cally induced murine colon carcinogenesis is associated with decreased CpG island methylation and increased mRNA and protein expression of the colonic vitamin D receptor. Oncol Res. 11:255-264. 32. Pawlik TM, Delman KA, Vauthey JN, Nagorney DM, Ng IO, Ikai I, Yamaoka Y, Belghiti J, Lauwers GY, Poon RT, Abdalla EK (2005): Tumor size predicts vascular
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invasion and histologic grade: Implications for selection of surgical treatment for hepatocellular carcinoma. Liver Transplantation; 11(9), 1086–1092. 33. Standish RA, Cholongitas E, Dhillon A, Burroughs AK, Dhillon AP (2006): An appraisal of the histopathological assessment of liver fibrosis. Gut. 55(4): 569–578. 34. Sanai FM and Keeffe EB (2010): Liver Biopsy for Histological Assessment. Saudi J Gastroenterol. 16(2): 124–132. 35. Brunner AL, Johnson DS, Kim SW (2009): Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver. Genome Res.; 19(6): 1044–1056. 36. Araújo OC, Rosa AS, Fernandes A, Niel C, Villela-Nogueira CA, Pannain V, Araujo NM (2016): RASSF1A and DOK1 Promoter Methylation Levels in Hepatocellular Carcinoma, Cirrhotic and Non-Cirrhotic Liver, and Correlation with Liver Cancer in Brazilian Patients. PLoS One; 11(4): e0153796. 37. Zinser GM and Welsh JE (2004): Effect of vitamin D (3) receptor ablation on murine mammary gland development and tumorigenesis. J Steroid Biochem Mol Biol.; 89–90, 433– 436. 38. Novakovic B, Sibson M, Ng HK, Manuelpillai U, Rakyan V, Down T, Beck S, Fournier T, Evain-Brion D, Dimitriadis E, Craig JM, Morley R, Saffery R (2009): Placentaspecific methylation of the vitaminD 24-hydroxylase gene: implications for feedback autoregulation of active vitaminD levels at the fetomaternal interface. J. Biol.Chem.; 284:14838–14848. 39. Carling T, Rastad J, Szabó E,Westin G, kerstr m G (2000): Reduced parathyroid vitamin D receptor messenger ribonucleic acid levels in primary and secondary hyperparathyroidism. J. Clin.Endocrinol.Metab.85: 2000–2003. 40. Sulaiman L, Juhlin CC, Nilsson IL, Fotouhi O, Larsson C, Hashemi J (2013): Global and gene-specific promoter methylation analysis in primary hyperparathyroidism. Epigenetics; 8: 646–655. 41. Jiang P, Chan CW, Chan KC (2015): Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients. PNAC; 112(11): E1317–E1325. 42. Tarek M, Louka ML, Khairy E, Ali-Labib R, Zakaria Zaky D, Montasser IF (2017). Role of microRNA-7 and selenoprotein P in hepatocellular carcinoma. Tumour Biol. 39(5):1010428317698372. 43. Forner A, Llovet JM, Bruix J (2012): Hepatocellular carcinoma. Lancet. 379(9822):12451255. 44. Fu S and Kurzrock R (2010). Development of curcumin as an epigenetic agent. Cancer 116:4670–4676 45. Dai XZ, Yin HT, Sun LF, Hu X, Zhou C, Zhou Y, Zhang W, Huang XE, Li XC (2013). Potential therapeutic efficacy of curcumin in liver cancer .Asian Pac J Cancer Prev. 14(6):3855-3859. 46. Wang M, Ruan Y, Chen Q, Li S, Wang Q, Cai J (2011). Curcumin induced HepG2 cell apoptosis-associated mitochondrial membrane potential and intracellular free Ca(2+) concentration. Eur J Pharmacol. 650(1):41-47.
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List of Abbreviation American association for the study of liver diseases
AFP
Serum α-fetoprotein
ALT
Alanine transaminase
AST
Aspartate transaminase
AUC
Area under the curve
CLD
Chronic liver disease
HBV
Hepatitis B virus
HCC
Hepatocellular carcinoma
HCV
Hepatitis C virus
VDR
Vitamin D receptor
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AASLD
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ACCEPTED MANUSCRIPT Table (1): Demographic and clinico-pathological variables in the different study groups (a). Demographic and clinico-pathological variables
04 (33.3%)
14 (93.3%)
10 (66.7%)
Female
7
0 (6.7%)
0 (6.7%)
5 (33.3%)
04 (33.3%)
00(73.3%)
0 (0%)
0 (6.7%)
4 (26.7%)
05 (000%)
3 (60%)
4 2
12 (66.7%)
7
7 (46.7%)
6
Undifferentiated
2
1-4
2
5-9
3-4
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15 (100%)
-
-
6 (40.0%)
-
-
2 (13.3%)
-
-
-
2 (13.3%)
-
7
-
7 (46.7%)
-
5
-
5 (33.3%)
-
1
-
1 (6.7%)
-
6
-
6 (40%)
-
3
-
3 (20%)
-
6
-
6 (40%)
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15 (100%)
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1-2
a
0 (0%)
< 200
15-18
0.001**
0 (0%)
3 (33.3%)
10-14
0 (0%)
05 (100%)
3
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0.06
0.001**
6 (40%)
≥ 200
Well Differentiated Moderately Differentiated
Fibrosis Stage
0 (0%)
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< 40
p
05 (100%)
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2 5 2 0 2 4 2 0
< 56
Hepatitis Scoring
Control n (%)
3 8
ALT(U/L)
HCC Grade
CLD n (%)
Male
≥ 56
Alpha feto protein (ng/mL)
HCC n (%)
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Sex
Groups n
0.04*
-
-
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HCC (n=05)
CLD (n=05)
p
Control (n=05)
Mean Rank
Median
Mean Rank
Median
Mean Rank
Albumin (mg/dL)
3.2
07.5
3.2
19.4
4.2
32
0.004**
AST(U/L)
67
33.3
57
24.9
34
10.67
0.001**
ALT(U/L)
78
31.13
73
29.27
37
8.6
0.001**
AFP (ng/mL)
26
35
8
22.3
11.7
0.001**
2
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Kruskal-Wallis test, **p˂0.01= highly significant
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n Groups HCC CLD Control
p (a)(b)
0.001**
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a One-Way ANOVA test, bKruskal-Wallis test, **p˂0.01= highly significant Highly Significant difference from the control, ii highly significantly difference from the CLD **p˂0.01 using ANOVA followed by post hoc test
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05 05 05
VDR Gene Methylation% Mean ± SD(a) Mean Median Rank(b) i, ii 07±00.6 08.56 34.83 3.34± 2.8 3.42 22.3 0.07±0.42 0.11 00.87
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Accuracy
≥ 6.77
80%
93.3%
85.7%
90.3%
88.9%
AFP (ng/mL)
≥11
80%
80%
66.7%
88.9%
80%
93.3%
73.3%
63.6%
95.6%
80%
Cutoff
Sensitivity
Specificity
PPV
NPV
Accuracy
VDR methylation %
≥ 9.3
73.3%
93.3%
91.7%
AFP(ng/mL)
≥16.5
73.3%
73.3%
73.3%
73.3%
73.3%
86.7%
66.7%
83.3%
76.7%
HCC and CLD group
Combined VDR meth% and AFP
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PPV: Positive Predictive Value, NPV: Negative Predictive Value
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77.8%
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Combined VDR meth% and AFP
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VDR methylation %
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Table (4): Performance characteristics of investigated biomarkers for HCC detection HCC group and nonCutoff Sensitivity Specificity PPV NPV malignant group
72.2%
83.3%
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Albumin AST ALT AFP
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r= correlation coefficient, **p˂0.01= highly significant
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VDR methylation% r p -0.520 0.001** 0.663 0.001** 0.609 0.001** 0.63 0.001**
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Table (5): Correlation between VDR gene promoter methylation and laboratory parameters in the different study groups
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The percentage of VDR gene promoter methylation was significantly higher in the HCC group compared to both CLD and control groups. The combined use of VDR gene promoter methylation with routinely used AFP improved the sensitivity. VDR gene promoter methylation can act as a potential prognostic tool amongst different HCC grades. The correlation indicated that VDR gene promoter methylation might be a potential biomarker in assessing high risk CLD patients with potential of carcinogenesis. Curcumin changed pattern of both VDR promoter methylation and VDR expression in HepG2 cells.
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Figure 1
Figure 2
Figure 3