Medires Publishers - Article

Abstract

In this study, the gene expression of some cancer-related genes was measured to differentiate between two types of therapeutic media in which hepatocellular carcinoma (HuH7) were harvested. The first was a conditional medium in which umbilical cord mesenchymal stem cells were harvested then diluted by DMEM media (1: 1) and the second was Co-cul- ture medium that mesenchymal stem cells and HuH7 cells (1:1) together were incubated for 3 days in a medium. The latter was called Co- culture medium. Comparison of the Results of gene expression of Surviving, PCNA, Beta-Catenin, Telo- merase and VEGF by using real- time PCR in several four-four-time intervals (48-72-96 hrs.) showed that in conditioned media and co-culture media, hepatoma cell line (HuH7) expressed a significant down-regulation in Surviving (p< 0.001), PCNA (p<0.001), VEGF(p<0.001) and Beta- Catenin(p=0.022) but not significant in Telomerase (p= 0.617). In our study, the measure of gene expression at different time (ALT), Aspartate aminotransferase (AST), Albumin, Alkaline phosphate, gamma GT, alpha- fetoprotein when compared regarding time Results in a significant in Alpha-fetoprotein (p < 0.001), ALT (p < 0.001) and AST (p < 0.001) while the results of Albumin (p = 0.650), Alkaline phosphatase (p = 0.329) and Gamma GT (p = 0.844) were non-significant.

Introduction

Hepatocellular carcinoma (HCC) represents the fifth most common malignant tumor and the third most common cause of mortality related to cancer in the world [1]. HCC is a cancer that has a high number of risk factors, such as viral infection by either virus B or C virus, alcohol, or prolonged aflatoxin exposure [2]. Umbilical cord mesenchymal stem cells retain their intrinsic stem cell potential while at the same time displaying high proliferation rates, low immunogenicity, and powerful capacity to differentiate into a wide range of lineages, including osteocytes, adipocytes, chondrocytes, and neural lineages [3]. So, UC-MSCs represent excellent can- engineering. Many preclinical studies on different disease models have shown that MSCs can accelerate wound recovery and suppress tumor progression. UC- MSCs possess a higher therapeutic potential than MSCs derived from other tissues. Increasing evidence suggests that the mechanism underlying UC-SMCs' efficacy depends mostly on cell secretions, in contrast to the early paradigm of codifferentiation capacity. They exhibit replacement and differentiation [4]. It has been shown that MSCs can produce a vast array of cytokines and extracellular molecules and express receptors and/or candidates for cell therapy in regenerative medicine and tissue er receptors both for cell–cell and cell–matrix interactions [5-6]. More recently, MSCs have become a topic of great focus in relation to cancer. Growing evidence suggests that MSCs home to not just injured tissues but tumors [7], where they have an important role in affecting the behavior of tumor cells. Thus, the ease of MSCs isolation and the fact that they can be home to wound or cancer sites make them a potential platform for cell-based targeting delivery. In response to liver injury or loss of liver mass, the proliferation of mature liver cells is the first-line defense to restore liver homeostasis [8]. Mesenchymal stem cells (MSCs) play several simultaneous roles to limit inflammation through releasing cytokines, and aiding healing by expression of growth factors [8]. The aim of this study was to investigate the effect of hMSCs derived from the umbilical cord on the growth of HepG2 to explore the anti-proliferative and the tumor suppressive effect of conditioning and co-culturing umbilical cord mesenchymal stem cells with hepatocellular carcinoma on human hepatoma cell line (HepG-2). The study also aimed to investigate the regressive changes in the ultrastructure of HepG2 cells using transmission electron microscopy.

Materials And Methods

Cell and Tissue Material Preparation:

This work was done in the Faculty of Medicine Ain Shams Research Institute (MASRI),Tissue Culture Unit. Mesenchy- mal Stem Cells from Human Umbilical cords Wharton’s Jelly (n = 12; gestational ages, 39- 40 weeks) were collected and processed within 2-3 hours after Caesarean births attending the Women's Hospital and Obstruction of Faculty of Medi- cine Ain Shams University.

Isolation of Mesenchymal stem cell from Umbilical Cord (UC)

UCs were washed with Dulbecco’s PBS several times, cut into small pieces, and filled with 0.1% collagenase type П for cell digestion (Sigma-Aldrich, St. Louis) in PBS and shaken in the water bath at 37°C for 60 min. Each UC was washed with proliferation medium, and the detached cells were harvested after gentle massage of the UC. Cells were centrifuged at 300 xg for 10 min, resuspended in proliferation medium, and seeded in 25- cm3 flasks (Nunclon, Germany) at a density of 5×105 cells per ml in complete media. After 72 hrs of incubation in a humidified atmosphere with 5% CO2, non-adherent cells were removed, and the culture medium was replaced every 3 days. On day 14, the adherent colonies of cells were almost confluent and removed by using trypsin EDTA, and counted. Cells were identified as being MSCs by their morphology, adherence, and their power to differentiate into osteocytes, neurocytes, and chondrocytes. Flow cytometry identification of UC- MSC surface biomarkers.UC-MSCs were used for cell identification, cells were washed with PBS and digested with 0.25% trypsin. After centrifuging at 300 x g for 5 min, the supernatant was removed and cells were washed twice with PBS. Cells were then incubated with FITC-conjugated primary antibodies against CD19, CD44 and CD105 for 30 min at room temperature in the dark. The expression of cell surface biomarkers was determined using flow cytometry analysis. Fluorescence acquisition was performed in a FACS Calibur II cytometer (Becton Dickinson, San Jose, CA, USA) [9].

Cell Culture of Human Hepatoma (HuH7) Cell Line:

Human Hepatoma (HuH7) cells in Egypt (Japanese male type collection) and grown in a sterile 75-cm2 tissue culture flask in a complete medium containing DMEM supplemented with 10% FBS, 1.5 %antibiotics (penicillin/streptomycin) and 0.05% of fungizone (antifungal) 95% air/5% CO2 at 37°C. Cells were cultured to 100% confluence.

Effect of MSCs on HuH7 cell growth:

HepG2 cells were treated with MSC-conditioned media or co-cultured with MSCs for 48, 72, and 96 hrs. to detect the inhibitory effect of MSCs on HepG2 cells.

Treatment of HuH7 with mesenchymal stem cell-conditioned media

Human MSCs were cultured as described above. At the proper time of the experiment (4th passage of MSCs) the media were collected from the flasks leaving the adherent MSCs. These media served as MSC-conditioned media (MSC-CM). HuH7 cells were treated with a mixture of complete medium containing DMEM supplemented with 10% FBS, antibiotics (penicillin/ streptomycin), fungizone, and hMSCs conditioned medium (1:1) for 0,48, 72, 96 hrs., and the culture medium were replaced every 24 hrs.

Treatment of HepG2 with co- culture conditioned media

(HepG2 with UC-MSC CM)HuH7 cells were treated with co-culture conditioned media for (48-72-96 hrs.), and the culture media were replaced every 24 hrs.

Co-culture conditioned media:

The growth medium of cultured HuH7 cells was removed and adherent cells were washed twice with 1x PBS and detached by incubation of cells with trypsin (2.5 g/L)/EDTA (1 g/L) for (4-5) min at 37°C. Cells were centrifuged at 1500-1000 rpm for 5-10 min, at 17°C. The cells were resuspended in DMEM and the cell suspension (HuH7) was added to the cultured MSCs. So, the co-culture medium was composed of DMEM (Lonza) supplemented with 10% fetal bovine USA) and the antibiotics penicillin/streptomycin and fungizone.

The ratio of UC-MSCs:

HuH7 cells was 1:1 and cells were co-cultured for 72hrs... At that time, the media were collected from the flasks and were used as hMSCs and HuH7 co-culture conditioned media.

Evaluation of Cell Viability

After 48, 72, or 96hrs. of HuH7 treated with MSC-conditioned media, HuH7 cells were collected and seeded onto a 96-well plate. Six wells were used for each treatment. Cell viability was determined by measuring the metabolism of a tetrazolium substrate, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). HuH7 treated with UC- MSC conditioned media and HepG2 treatedwith co-culture conditioned media in the concentration of 103-105 cells/well in a final volume of 100 µL of medium were cultured overnight in a flat bottom 96 well plate and incubated at 37 °C in a humidified air atmosphere enriched with 5% CO2. Then, 20 µl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5 mg/ml) was added to each well. After an additional 4 h of incubation at 37°C, the medium was removed and 100 µl dimethyl sulfoxide (DMSO) was added to each well to re-suspend the MTT metabolic product. The absorbance of the dissolved formazan was measured at 490 nm (A490) using a scanning microplate spectrophotometer (Bio-Rad). Cell proliferation was assessed as the percentage of inhibition compared to untreatedHuH7 as control cells. The proliferation inhibition rate was calculated using the following formula: Proliferation inhibition rate = (A490, Control-A490, Sample) / A490, Control × 100.

Metabolic Activity of HuH7 cells

The levels of albumin, serum liver enzymes such as alanine aminotransferase (GPT or ALT), aspartate aminotransferase (GOT or AST), ALP, and γ-GT were detected by a commercially available kit Roche Cobas Integra, USA. According to the manufacturer’s protocols.

Measurement of Tumor Markers

According to the manufacturer’s instructions. Alpha-fetoprotein (AFP) (ARCHITECT SYSTEM, from Abbott Laboratories, Ireland).

Gene Expression Analysis

RNA extraction and real-time PCR

Total RNA from treated and untreated HuH7 cells were har- vested and washed with PBS at 4°C. Total RNA from cell cul- tures was extracted using a total RNA extraction Kit supplied by Qiagen, according to the manufacturer’s instructions. All RNA samples were treated with DNase I at 37°C for 30 min for removal of any genomic DNA contamination. cDNA was generated from 500 ng of total RNA extracted with 1 μl (20 pmol) antisense primer and 0.8 μl superscript AMV reverse transcriptase at 25°C for 10 min, 37°C for 120 min, and 85°C for 5min and finally 4°C to infinity. The relative abundance of mRNA species was assessed using the SYBR® Green method on an ABI prism 5700 fast (Applied Biosystems, Foster City, CA, USA). The reverse transcription (RT) prod- uct was used to measure the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a positive house- keeping gene, the mRNA expression of PCNA, Survivin, β –catenin, VEGF, and Telomerase was done using real-time PCR (qPCR), and PCR primers were designed with Gene Runner Software (Hasting Software, Inc., Hasting, NY) for RNA sequences from GenBank (Table 1).

Table 1: Primers sequences used in this study (F, forward; R, reverse)

All primer sets had a calculated annealing temperature of 60°.Quantitative RT-PCR was performed in duplicate in a 20 µL reaction volume consisting of 2X SYBR Green PCR Master Mix (Applied Biosystems), 900nM of each primer, and 2- 3µL of cDNA. Amplification conditions denaturation at 95 °C for 15 min, followed by 45 cycles each cycle at 94°C for 15 sec, 60°Cfor 25 sec, and 72°C for 20 sec. Data from real-time assays were calculated using the v1·7 Sequence Detection Software from PE Biosystems. (Foster City,CA, USA)Relative expression of Surviving PCNA,β-Catenin, Telomerase, and VEGF mRNA was cal- culated using the comparative Ct method as previously de- scribed. All values were normalized to the glyceraldehyde-3- phosphate dehydrogenase (GAPDH)gene 2- ∆∆Ct method [∆Ct= Ct (gene) - Ct (GAPDH)] and reported as fold change (ΔΔCT) over background levels detected. Melting curves were performed to ensure that only a single product was am- plified.

Statistical analysis

The statistical analysis in this study was performed using the following programs: IBM SPSS 22.0.0.2. Python packages (Pandas 0.20.3 and SciPy 0.19.

Results

In the present study, the hepatoma cell line HuH7 was chosen as a model for liver cancer. The main objective of the study was to evaluate the effect of two different media, conditioned media (the medium in which umbilical cord mesenchymal cells were cultured) and co-culture medium (the medium in which mesenchymal and HuH7 cells were cultured) on different parameters in HuH7 cultured cells. Mesenchymal stem cells from cord (Wharton’s jelly) were also investigated at 0,3,10 and 14 days from culturing where the cells reached 95-100% confluence after 14 days (Figure 1).

Figure 1: Photomicrographs showing growth of mesenchymal stem cells from the cord (Wharton’s jelly) (Inverted microscope, X400). In 0 time from culture, B) 3 days after changing media, C) 10 days from culture, D) 14 days showing 95-100% confluence of cells.

Culture of HuH7 cancer cells with hMSC- conditioned media

To determine whether the cultured hMSCs release soluble factors that can affect cancer cell growth, we cultured HuH7 in hMSCs-conditioned media. After 48hrs., the number of dead cells was increased and there were changes in shape that’s because conditioned media derived from hMSCs could inhibit cell growth relative to control cells (Figure 2).

Figure 2: Photomicrographs showing the effect of conditional media on hepatocellular carcinoma (HuH7) (Inverted mi- croscope, X400). A-Control (confluent HuH7), B- 48 hours after treatment, C- 72 hours after treatment, and D-96 hours after treatment. The results were generally in agreement with those obtained by the Trypan Blue assay, in which the conditioned media exerted an inhibitory effect on cell growth. After 72 hrs., the number of dead cells increased and cells were damaged while, after 96hrs. the larger number of dead cells was observed. After treatment of HuH7 cells with co-culture media (Figure 3), for 48hrs. a number of dead cells appeared and cells were damaged, after 72 hrs. the number of dead cells increased and cells were more dissociated while, after 96hrs. Most of the cells appeared dead and severely damaged.

Figure 3: Photomicrographs showing effect of co-culture media on hepatocellular carcinoma (HuH7) (Inverted microscope, X400). (A) Control (confluent HuH7), (B) 48 hours after treatment. (C) 72 hours after treatment, (D) 96 hours after treatment.

Cell Viability Assay (MTT)

By using the MTT assay, it was found that there were highly significant differences in the viability between the hepatoma cell line (HuH7) cultured in normal media, the hepatoma cell line (HuH7) cultured in the conditioned media (p 0.001). By comparing the cell viability at the four-four-four-time intervals regarding the non-normally distributed variables, there was a highly significant difference between the four groups (0 time (HuH7 as control), 48,72 and 96hrs.). The data in the two box plots (Fig. 4) showed that the cell viability was inversely proportional with increased time after 96 hrs. The cells showed the lowest value.

Figure 4: MTT box plot with time for conditioned media and Co-culture media. To compare the metabolic status of the HuH7 cells treated with UC-MSCs conditioned medium or the co-culture system, the albumin production, as well as the activity of the liver-specific enzymes AST, ALT, and GGT, were measured. There was a non-significant difference in albumin between the two groups (conditioned and co-culture groups) (p=0.054) but, in the conditioned media, comparing the four groups regarding the normally distributed variables, there was a significant difference in albumin between the four groups (0 times, 48, 72 and 96 hrs.) with a (p = 0.009). On the other hand, in the case of co-culture media, there was a non-significant difference (p = 0.650) between the four groups. Regarding αFP levels in HuH7 cultured in normal medium and those cultured in conditioned and co-culture media, there was an obvious change. There was a highly significant difference between groups at the different four-time intervals. By comparing αFP in cells cultured in normal media and those cultured in conditioned and co-culture media it was inversely proportional with decrease in αFP by increasing the time of incubation.

Table 1: showing normal variable data relation with time in conditioned and co-culture media As for liver enzymes (GOT & GPT) there was a highly significant difference between the four four-time intervals regarding GOT activity in the case of groups cultured in conditioned media (p<0.001) and those cultured in co-culture media (p 0.001). Unfortunately, there was no significant difference in GPT activity in corresponding of the four-time interval groups (0-time, 48, 72, and 96 hrs.) in conditioned media (p <0.674). In contrast, no significant difference was seen comparing the effect of UC-MSCs conditioned medium or the co-culture HuH7 cultures for both alkaline phosphatase and GGT regarding different four-hour intervals case of co-culture media there was a non-significant difference (p = 0.650) between the four groups. Regarding αFP levels in HuH7 cultured in normal medium and those cultured in conditioned and co-culture media, there was an obvious change. There was a highly significant difference between groups at the different four-four-time intervals. By comparing αFP in cells cultured in normal media and those cultured in conditioned and co-culture media it was inversely proportional with a decrease in αFP by increasing the time of incubation.

Table 2: Showing a comparison between the four groups regarding the non-normally distributed variables in two media

Gene Expression of Cancer Related Genes.

Survivin: by comparing the different time interval in both groups, there was highly significant difference between them (0- time (HuH7 as control), 48, 72 and 96 hrs.) in Survivn RQ (ΔΔCT) for Conditioned media (p<0.001) and Co-culture media (p<0.001)

PCNA: There was a high significant difference between the two groups (conditioned and co-culture groups) in PCNA (p=0.001). Comparing the four intervals of time. groups, there was significant difference between the four groups (0, 48, 72 and 96 hrs.) in PCNA for Conditioned media (p=0.001).

β-Catenin: By Comparing the two groups regarding the non- normally distributed variables, there was significant difference between the two groups (conditioned and co-culture groups) in β-Catenin C (p<0.001) and β-Catenin (p<0.022). Comparing the four groups regarding the non-normally distributed variables, there was significant difference between the four groups (0, 48, 72 and 96 hrs.) in β-Catenin for conditioned media (p<0.001) and in co-culture media (p <0.001).

Telomerase: By Comparing the two groups regarding the non-normally distributed variables, there was a nonsignificant difference between the two groups (conditioned and co-culture groups) in telomerase C (p=0.617 and p=0.611respectively). Comparing the four groups regarding the non-normally distributed variables, there was a significant difference between the four groups (0, 48, 72, and 96 hrs.) in telomerase for Conditioned media (p <0.001) and in co-culture media (p <0.001).

Table 4: Showing a comparison between the four time intervals in the two types of media. VEGF: By Comparing the two groups regarding the non-normally distributed variables, there was a significant difference between the two groups (conditioned and co-culture groups) VEGF (p<0.001). non-normally distributed variables, there was a significant difference between the four groups (0, 48, 72 and 96 hrs.) in VEGF for Conditioned media (p <0.001) and in co-culture media (p <0.001).

Discussion

MSCs have received increased attention for their ability to interact with cancer cells and make an impact on cancer cells’ behavior. However, their potential to exhibit promotion- al or inhibitive properties by providing the cancer cells with a microenvironment remains controversial. Some reports suggested that they exhibit a potent promotional effect by providing growth signals, while others suggested they have potential role in inhibition of the proliferation of cancer cells. The exact mechanism of their action in the regulation of can- cer cells has not been well elucidated yet. In the present study, the effect of conditioned media and co-culture media on cultured HuH7 cells was studied by measuring; morphological changes, cell viability, certain biochemical changes, expression of cancer- related genes. The results of this study showed morphological changes and dramatic decrease in cell viability which were all time depen- dent on the number of dead HuH7 cells which were cultured in conditioned media and co- culture media. El-nahrawy et al., showed morphological changes and possible beneficial effect of human umbilical cord MSCs- conditioned media on liver malignant cells HepG2 via the secretion of soluble factors or cytokines like IL-10 having anti- inflammatory and possible anti-tumor effects [11]. Ramasamy et al., studied the effect of MSC on the proliferative activity of malignant cells of different lineages. The tumor cell lines of hemato- poietic (BV173, K562, Jurkat, KG1a and wS9-B-LCL) and non-hematopoietic (UCH10 and CC3) origin were cultivated, at different ratios, in the presence of MSC and tested for their proliferative activity after 3 days of co-culture. MSC exhibit- ed a dose-dependent antiproliferative effect on all cell lines investigated [12]. Alpha-fetoprotein (αFP) is a good tumor marker that is ele- vated in 60–70% of patients with hepatocellular carcinoma. Normally, levels of αFP are below 10 ng/ml, but marginal elevations (10–100) are common in patients with chronic hepatitis. The progressive elevation of alpha-fetoprotein ≥7 ng/mL/month in patients with liver cirrhosis is useful for the diagnosis of hepatocellular carcinoma in patients that do not reach αFP levels ≥200 ng/mL [13]. Also, the serum concen- tration of αFP >400 ng/ml is considered a diagnostic marker for HCC. Although, less than half of HCC patients may gen- erate those high levels of αFP [14]. On the contrary, some investigations showed that αFP cannot be used in the ini- tial screening of HCC due to its low sensitivity at diagnostic values. In the present study αFP values were inversely pro- portional with the increase of time intervals of incubation of HuH7 cells whether in conditioned media or co-culture media as they showed highly significant decrease with the lowest values recorded after 96hrs. In clinical practices, liver functions have long been consid- ered to assume crucial status in the prognosis of many types of cancers such as gallbladder and colorectal cancers. Liv- er functions can be reflected by markers not only like ALB, GELO, and TP, indicators of the nutritional status, but also ALP, ALT, AST, γ– GT, LDH, TBIL, and DBIL, indices reflect- ing liver damage. All these liver function parameters were firstly evaluated for their effect on the overall survival in intra- hepatic cholangiocarcinoma (ICC) patients. Therefore, these liver enzymes may serve as valuable predictive markers in HCC patients [15]. To date; studies have shown those ab- normal changes of liver enzymes often lead to poor progno- sis in a multitude of cancers. Likewise, other liver function markers such as ALP and gamma glutamyl transpeptidase (γ-GT) were often Alpha-fetoprotein (αFP) is a good tumor marker that is elevated in 60– 70% of patients with hepato- cellular carcinoma. Normally, levels of αFP are below 10 ng/ ml, but marginal elevations (10–100) are common in patients with chronic hepatitis. The progressive elevation of alpha-fe- toprotein ≥7 ng/mL/month in patients with liver cirrhosis is useful for the diagnosis of hepatocellular carcinoma in patients that do not reach αFP levels ≥200 ng/mL [13]. Also, the serum concentration of αFP >400 ng/ml is considered a diagnostic marker for HCC. Although, less than half of HCC patients may generate those high levels of αFP [14]. On the contrary, some investigations showed that αFP cannot be used in the initial screening of HCC due to its low sensitivity at diagnostic values. In the present study αFP values were inversely proportion- al with the increase of time intervals of incubation of HuH7 cells whether in conditioned media or co-culture media as they showed highly significant decrease with the lowest val- ues recorded after 96hrs. In clinical practices, liver functions have long been considered to assume crucial status in the prognosis of many types of cancers such as gallbladder and colorectal cancers. Liver functions can be reflected by mark- ers not only like ALB, GELO, and TP, indicators of the nutri- tional status, but also ALP, ALT, AST, γ–GT, LDH, TBIL, and DBIL, indices reflecting liver damage. All these liver function parameters were first evaluated for their effect on the overall survival in intrahepatic cholangiocarcinoma (ICC) patients. Therefore, these liver enzymes may serve as valuable predictive markers in HCC patients [15]. To date; studies have shown those abnormal changes of liver enzymes often lead to poor prognosis in a multitude of cancers. Likewise, oth- er liver function markers such as ALP and gamma-glutamyl transpeptidase (γ-GT) were often risk factors in patients with some types of cancers [16]. In the present study it was found that ALP did not show any significant change in case of using co-culture media or con- ditioned media from 0 time until 96hrs. In tissue, ALP is well known as a membrane- bound ectoenzyme, used as an in- dicator to reflect hepatobiliary or bone diseases and that it is attached to the membrane via glycan phosphatidylinositol (GPI) anchor [17]. The data of supported the clinical observations recorded in numerous clinical studies that described the close relation- ship between the worsening of hepatocellular carcinoma with increased GT levels. The data of the present study showed highly significant decrease in the level of γGT with the in- crease of incubation time from 0 – 96 hrs. in case of using conditional media or co- culture media for treatment of HuH7 cells, which means according to the report of that these me- dia have antitumor effect [18]. The present data also showed lower levels of GOT and GPT after the incubation in condi- tional media or co- culture media at 48, 72, 96 hrs. This con- firms the conception that conditioned media and co-culture media have antitumor effect on HuH7 cell line. Angiogenesis is a complex multiple- step process, which involves endo- thelial cell proliferation, migration, tube formation, vascular network reorganization and stabilization [19]. Our findings demonstrate here that conditioned and co-culture medium of hMSCs has a suppressive effect on cell growth, depend- ing on time-response. This distinctive property of hMSCs is probably related to the competition of production of a combi- nation of cytokines with various effects in culture. Data from this study might provide some clues for the antitumor activity of conditioned and co-culture medium of hMSCs can avoid untoward effects of administered MSCs. Further functional investigation of the cellular and molecular events will enable the improved understanding of the mechanism of the action between cancer cells and hMSCs for development of effective therapy in the treatment of cancers. Analysis of propor- tion of cells in conditioned and co-culture medium of hMSCs showed that HepG2 cells were decreased in the percentage of cell population by time. The screen for the levels of cell expressing population in the mixture showed that decreased percentage of cancer cell population was apparently in pres- ence of hMSCs. This clearly indicated that cell proliferation could be regulated by hMSCs as a result of interaction be- tween hMSCs and cancer cells. Consistent with these re- sults, as revealed by RT-PCR analysis, hMSCs treatment caused a marked higher or lower level of key molecules, such as Survivin, β – catenin, PCNA, Telomerase and VEGF. Survivin is an inhibitor of apoptosis protein, which is highly expressed in most cancers and associated with chemother- apy resistance, increased tumor recurrence, and shorter pa- tient survival, making antisurvivin therapy an attractive can- cer treatment strategy. Increased survivin expression was observed in the vast majority of cancers. These included esophageal, lung, ovarian, central nervous system, breast, colorectal, bladder, gastric, prostate, pancreatic laryngeal, uterine, hepatocellular, and renal cancers, as well as mela- noma and soft tissue sarcomas [20]. The current study sug- gests that UCUC-MSCs induces apoptosis of HuH7 cells, which is associated with downregulation of survivin mRNA expression. Survivin is a member of the inhibitor apopto- sis protein (IAP) family. These are bifunctional proteins that suppresses apoptosis and regulates cell division [21]. The expression of survivin is highly cancer-specific and is one of the top four transcripts uniformly upregulated in human cancers, but not in normal tissues [22]. The overexpression of survivin appears to correlate with aggressive tumor be- havior and poor prognosis in hepatocellular carcinoma [23]. So, survivin has become an ideal target for the diagnosis and treatment of cancer. Other studies have shown that overex- pression of survivin was closely related to chemo resistance, and inhibition of survivin improved the sensitivity of tumor to chemotherapy [24]. Survivin is essential for cell-cycle progression in hepatocellular carcinoma cells, and down-regulation of survivin expres- sion may lead to programmed cell death [25], indicating that survivin may be an appealing new target for novel therapies in hepatocellular carcinoma [26]. Another explanation was introduced by Dai et al., who indicated that the anti-apoptotic survivin molecule may promote S- phase entry, and cooperate with the G2/M checkpoint to regulate proper cell cycle progression? [27]. In the present study survivin gene is expressed at a very high rate at the beginning of experiment (0-time) when HuH7 cells were incubated in conditioned media and co-culture media survivin decreased significant- ly with the increase of time to 96 hrs. where it showed the lowest expression. Our results about survivin expression are also in agreement with Yang et al., who reported that UCUC- MSCs significantly inhibit the downregulating anti-apoptotic proteins Bcl- 2 and surviving [28]. Cell growth, the cell cycle and apoptosis are closely associ- ated with the genesis and development of liver cancer, and multiple factors are involved in their regulation, including surviving, caspase-3 and proliferating cell nuclear antigen (PCNA) which is associated with vital cellular processes [29-30]. The proliferating cell nuclear antigen (PCNA) is a nuclear protein which was independently discovered by Miyachi et al., as PCNA [31]. PCNA is the heart of many essential cel- lular processes such as DNA replication, repair of DNA dam- age, chromatin structure maintenance, chromosome segregation and cell-cycle progression and can be regarded as one of their common integrators [32]. Recent studies have reported that tumor cells express increased levels of PCNA, identifying it as a potential target for cancer therapy [33]. The increased expression of PCNA could have a predictive and prognostic value. However, the value of PCNA as a biomarker remains controversial [34] Li et al., who worked on HepG2 reported that, the expression of PCNA was significantly upregulated during HCC progres- sion when compared with the 0th week [35]. In the pres- ent study showed that PCNA might have anti proliferative effect, where we found a significant difference between the two groups (conditioned and co-culture) regarding PCNA (p=0.001). By comparing the time intervals groups (0- time, 48, 72 and 96 hrs., it was noticed that PCNA expression was inversely proportional to the increase in incubation time. we detected a down regulation of it after 48 hrs., which indicates a significant decrease in the proliferation of the HuH7 cells. Liver cancer is highly heterogeneous and involved deregulation of several signaling pathways. Wnt/β- catenin pathway is frequently upregulated in HCC and it is implicated in main- tenance of tumor initiating cells, drug resistance, tumor progression, and metastasis. A great target component of the β-catenin pathway with anticancer activity is underway [36]. Activation of the Wnt/b-catenin pathway has been observed in at least 1/3 of hepatocellular carcinomas (HCC) and a significant difference of these have mutations in the β-catenin gene. Therefore, effective inhibition of this pathway could provide a novel method to treat HCC [37]. Fifty percent of HCC that express c-myc or H-ras in the liver contain b-catenin mutations. [38] Suggested that b-catenin activation can cooperate with ras or myc in HCC progression. The present study showed a non- significant difference in β-catenin between the two groups of HuH7 cultured in con- ditioned and coculture media. There was a significant difference decrease between the four groups (0-time, 48, 72 and 96 hrs.) and the lowest value was seen after 96 hrs., which indicates the inhibition of β-catenin in HuH7 during treatment with media. Telomerase is a ribonucleoprotein enzyme that catalyzes the synthesis of telomeric DNA. It helps in the formation and pro- tection of telomere and also prevents cells from undergoing senescence [39]. Telomerase became an attractive poten- tial drug target in the fight against cancer owing to its low/ absent expression levels in normal somatic cells and high expression in cancer. Detection of telomerase activity has been proposed to be a useful tool in the diagnosis of pancre- atic cancer [40]. Recently abnormal activation of telomerase was found to occur in 85– 90% of all cancers and support the ability of cancer cells to bypass their proliferative limit, rendering them immortal [41] The present study showed that there was a highly significant difference according to time interval (0- time, 48, 72 and 96 hrs.) in telomerase level in case of using conditioned media (p 0.001) and in co– culture media was (p 0.001) and the general scope is gradual decrease with the increase of time indicating the ability of these media to be used as anticancer therapeutics. Although multiple proangiogenic factors have been identi- fied, among these factors, vascular endothelial growth factor (VEGF), which is expressed and secreted by tumor cells in hypoxia conditions, is considered to be the most potent an- giogenic factor in the process of tumor angiogenesis [42]. Hepatocellular carcinomas (HCCs) are characterized with abundant microvessel density and high levels of circulating VEGF, making anti-angiogenesis as an attractive therapeu- tic strategy [43]. Tumor growth inhibition mediated by UC- MSCs cells, in this study, was mainly through its antiangio- genic activity, rather than its direct cytotoxicity on tumor cells in HepG2; where VEGF secreted by HepG2 cells was sub- stantially decreased compared to the control. VEGFs mediate a plethora of biological processes in the en- dothelial cells such as cell proliferation, migration, survival, cell–cell communication, and differentiation. Some VEGFs also regulate vessel permeability. Signaling pathways ac- tivated by VEGFs play fundamental roles in the de novo formation of vessels from hematopoietic precursor cells, a process called vasculogenesis, and in angiogenesis, the for- mation of vessels from pre-existing vasculature [44]. Circulating VEGF concentrations also have been found to increase according to the HCC stage [44]. Nagy et al., inves- tigated the relationship between VEGF gene polymorphisms and the prognosis of HCC patients showing that VEGF poly- morphisms may be a significant genetic marker for HCC prognosis [44]. In the present study, VEGF expression levels in conditioned media and co-culture media were significantly different and there was an obvious decrease depending on the increase of incubation time. At 0-time VEGF expression level was high and then decreased gradually reaching the lowest level after 96 hrs. VEGF and its receptors have been found to be overexpressed in HCC in comparison with nor- mal liver tissue, and overexpression of VEGF was correlated with the poor prognosis [45]. Generally, VEGF secreted by tumor cells was an important initiator to provoke angiogenesis in tumor tissue, therefore, blocking VEGF secretion by tu- mor cells was also very important for angiogenesis inhibition

References

1. Ziogas, Ioannis A., and Georgios Tsoulfas. "Advances and challenges in laparoscopic surgery in the manage- ment of hepatocellular carcinoma." World journal of gas- trointestinal surgery 9, no. 12 (2017): 233.
   View at Publisher | View at Google Scholar
2. Flores-Téllez, Teresita NJ, Saúl Villa-Treviño, and Caro- lina Piña-Vázquez. "Road to stemness in hepatocellular carcinoma." World journal of gastroenterology 23, no. 37 (2017): 6750.
   View at Publisher | View at Google Scholar
3. Wei, Li, Jing Zhang, Xiu-Bin Xiao, Hai-Xing Mai, Ke Zheng, Wan-Liang Sun, Lei Wang et al. "Multiple in- jections of human umbilical cord-derived mesenchymal stromal cells through the tail vein improve microcircula- tion and the microenvironment in a rat model of radia- tion myelopathy." Journal of Translational Medicine 12 (2014): 1-12.
   View at Publisher | View at Google Scholar
4. Bai, Lipeng, Danting Li, Jie Li, Zhengzhong Luo, Shumin Yu, Suizhong Cao, Liuhong Shen, Zhicai Zuo, and Xia- oping Ma. "Bioactive molecules derived from umbilical cord mesenchymal stem cells." Acta Histochemica 118, no. 8 (2016): 761-769.
   View at Publisher | View at Google Scholar
5. Lee, Hyun Jung, Byung Hyune Choi, Byoung-Hyun Min, Young Sook Son, and So Ra Park. "Low-intensity ultra- sound stimulation enhances chondrogenic differentiation in alginate culture of mesenchymal stem cells." Artificial organs 30, no. 9 (2006): 707-715.
   View at Publisher | View at Google Scholar
6. Mueller, Ingo, Marion Vaegler, Christina Holzwarth, Nilo- lay Tzaribatchev, Stefan M. Pfister, B. Schütt, P. Reize, Johann Greil, Rupert Handgretinger, and Maximillian Rudert. "Secretion of angiogenic proteins by human multipotent mesenchymal stromal cells and their clinical potential in the treatment of avascular osteonecrosis." Leukemia 22, no. 11 (2008): 2054-2061.
   View at Publisher | View at Google Scholar
7. Yang, Chang-Ching, Yang-Hsin Shih, Miau-Hwa Ko, Shao-Yun Hsu, Henrich Cheng, and Yu-Show Fu. "Transplantation of human umbilical mesenchymal stem cells from Wharton's jelly after complete transection of the rat spinal cord." PloS one 3, no. 10 (2008): e3336.
   View at Publisher | View at Google Scholar
8. Phinney, Donald G., and Mark F. Pittenger. "Concise review: MSC-derived exosomes for cell-free therapy." Stem cells 35, no. 4 (2017): 851-858.
   View at Publisher | View at Google Scholar
9. Lech, Wioletta, Anna Figiel-Dabrowska, Anna Sarnows- ka, Katarzyna Drela, Patrycja Obtulowicz, Bartlomiej Henryk Noszczyk, Leonora Buzanska, and Krystyna Domanska-Janik. "Phenotypic, functional, and safety control at preimplantation phase of MSC-based therapy." Stem cells international 2016, no. 1 (2016): 2514917.
   View at Publisher | View at Google Scholar
10. Rödel, Claus, Joachim Haas, Anke Groth, Gerhard G. Grabenbauer, Rolf Sauer, and Franz Rödel. "Sponta- neous and radiation-induced apoptosis in colorectal car- cinoma cells with different intrinsic radiosensitivities: sur- vivin as a radioresistance factor." International Journal of Radiation Oncology* Biology* Physics 55, no. 5 (2003): 1341-1347.
    View at Publisher | View at Google Scholar
11. EL-nahrawy HH, Mohamed ME, EL-desoky MA et al. (2016). Immunomodulatory effect of human umbilical cord blood-derived mesenchymal stem cells on hepato- cellular carcinoma cell lines (HepG2). Med. J., 84 (2): 33-37. 7.
    View at Publisher | View at Google Scholar
12. Ramasamy, Rajesh, E. WF Lam, I. Soeiro, Veronica Ti- sato, D. Bonnet, and F. Dazzi. "Mesenchymal stem cells inhibit proliferation and apoptosis of tumor cells: impact on in vivo tumor growth." Leukemia 21, no. 2 (2007): 304-310.
    View at Publisher | View at Google Scholar
13. Arrieta, Oscar, Bernardo Cacho, Daniela Morales-Espi- nosa, Ana Ruelas-Villavicencio, Diana Flores-Estrada, and Norma Hernández-Pedro. "The progressive eleva- tion of alpha fetoprotein for the diagnosis of hepatocellu- lar carcinoma in patients with liver cirrhosis." BMC can- cer 7 (2007): 1-9.
    View at Publisher | View at Google Scholar
14. Colombo, Massimo. "Screening for cancer in viral hep- atitis." Clinics in liver disease 5, no. 1 (2001): 109-122.
    View at Publisher | View at Google Scholar
15. Zhang, Chenyue, Haiyong Wang, Zhouyu Ning, Litao Xu, Liping Zhuang, Peng Wang, and Zhiqiang Meng. "Serum liver enzymes serve as prognostic factors in pa- tients with intrahepatic cholangiocarcinoma." OncoTar- gets and therapy (2017): 1441-1449.
    View at Publisher | View at Google Scholar
16. Ma, Hui, Lan Zhang, Bei Tang, Yan Wang, Rongxin Chen, Boheng Zhang, Yi Chen et al. "γ-Glutamyltrans- peptidase is a prognostic marker of survival and recur- rence in radiofrequency-ablation treatment of hepatocel- lular carcinoma." Annals of surgical oncology 21 (2014): 3084-3089.
    View at Publisher | View at Google Scholar
17. Moss, D. W. "Perspectives in alkaline phosphatase re- search." Clinical chemistry 38, no. 12 (1992): 2486-2492.
    View at Publisher | View at Google Scholar
18. Moreira, Andrea Janz, Graziella Ramos Rodrigues, Sil- via Bona, Leila Xavier Sinigaglia Fratta, Giovana Regina Weber, Jaqueline Nascimento Picada, Jorge Luiz Dos Santos, Carlos Thadeu Cerski, Claudio Augusto Mar- roni, and Norma Possa Marroni. "Ductular reaction, cy- tokeratin 7 positivity, and gamma-glutamyl transferase in multistage hepatocarcinogenesis in rats." Protoplasma 254 (2017): 911-920.
    View at Publisher | View at Google Scholar
19. Domigan, Courtney K., and M. Luisa Iruela-Arispe. "Re- cent advances in vascular development." Current opin- ion in hematology 19, no. 3 (2012): 176-183.
    View at Publisher | View at Google Scholar
20. Fukuda, Seiji, and Louis M. Pelus. "Survivin, a cancer target with an emerging role in normal adult tissues." Mo- lecular cancer therapeutics 5, no. 5 (2006): 1087-1098.
    View at Publisher | View at Google Scholar
21. Wall, Nathan R., Daniel S. O’Connor, Janet Plescia, Yves Pommier, and Dario C. Altieri. "Suppression of sur- vivin phosphorylation on Thr34 by flavopiridol enhances tumor cell apoptosis." Cancer research 63, no. 1 (2003): 230-235.
    View at Publisher | View at Google Scholar
22. Velculescu, Victor E., Stephen L. Madden, Lin Zhang, Alex E. Lash, Jian Yu, Carlo Rago, Anita Lal et al. "Anal- ysis of human transcriptomes." Nature genetics 23, no. 4 (1999): 387-388.
    View at Publisher | View at Google Scholar
23. Bao, Shi-Ting, Shui-Qing Gui, and Mu-Sheng Lin. "Rela- tionship between expression of Smac and Survivin and apoptosis of primary hepatocellular carcinoma." Hepato- biliary & Pancreatic Diseases International: HBPD INT 5, no. 4 (2006): 580-583.
    View at Publisher | View at Google Scholar
24. Liang, Sheng-ran, Guang-rui Hu, Li-juan Fang, Su-jing Huang, Jin-song Li, Ming-yi Zhao, and Min-jie Meng. "CpG oligodeoxynucleotides enhance chemosensitivity of 5-fluorouracil in HepG2 human hepatoma cells via downregulation of the antiapoptotic factors survivin and livin." Cancer Cell International 13 (2013): 1-10.
    View at Publisher | View at Google Scholar
25. Ito, Takeshi, Katsuya Shiraki, Kazushi Sugimoto, Take- nari Yamanaka, Katsuhiko Fujikawa, Masaaki Ito, Kou- jiro Takase et al. "Survivin promotes cell proliferation in human hepatocellular carcinoma." Hepatology 31, no. 5 (2000): 1080-1085.
    View at Publisher | View at Google Scholar
26. Dai, Dejian, Yunjia Liang, Zhihua Xie, Jun Fu, Yuhao Zhang, and Zhijin Zhang. "Survivin deficiency induces apoptosis and cell cycle arrest in HepG2 hepatocellu- lar carcinoma cells." Oncology reports 27, no. 3 (2012): 621-627.
    View at Publisher | View at Google Scholar
27. Yang, Chao, Deqiang Lei, Weixiang Ouyang, Jinghua Ren, Huiyu Li, Jingqiong Hu, and Shiang Huang. "Con- ditioned media from human adipose tissue-derived mes- enchymal stem cells and umbilical cord-derived mesen- chymal stem cells efficiently induced the apoptosis and differentiation in human glioma cell lines in vitro." BioMed research international 2014, no. 1 (2014): 109389.
    View at Publisher | View at Google Scholar
28. Strzalka, Wojciech, and Alicja Ziemienowicz. "Prolifer- ating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation." Annals of botany 107, no. 7 (2011): 1127-1140.
    View at Publisher | View at Google Scholar
29. Vogl, Thomas J., Elsie Oppermann, Jun Qian, Ulli Imlau, Andreas Tran, Yousef Hamidavi, Huedayi Korkusuz et al. "Transarterial chemoembolization of hepatocellular car- cinoma in a rat model: the effect of additional injection of survivin siRNA to the treatment protocol." BMC cancer 16 (2016): 1-8.
    View at Publisher | View at Google Scholar
30. Miyachi, Kiyomitsu, Marvin J. Fritzler, and Eng M. Tan. "Autoantibody to a nuclear antigen in proliferating cells." The Journal of Immunology 121, no. 6 (1978): 2228- 2234.
    View at Publisher | View at Google Scholar
31. Stoimenov, Ivaylo, and Thomas Helleday. "PCNA on the crossroad of cancer." (2009): 605-613.
    View at Publisher | View at Google Scholar
32. Dillehay, Kelsey L., William L. Seibel, Daoli Zhao, Shan Lu, and Zhongyun Dong. "Target validation and struc- ture–activity analysis of a series of novel PCNA inhibi- tors." Pharmacology Research & Perspectives 3, no. 2 (2015): e00115.
    View at Publisher | View at Google Scholar
33. Lindström, Mikael S., and Keng-Ling Wallin. "Prognostic role of proliferating cell nuclear antigen (PCNA) in can- cer and other diseases." Proliferating Cell Nuclear Anti- gen 81 (2006): 96-9.
    View at Publisher | View at Google Scholar
34. Li, San-Qiang, Zhi-Hong Hu, Sha Zhu, Dong-Mei Wang, Hong-Mei Han, and Hua-Jie Lu. "The effect of ADAM8 on the proliferation and apoptosis of hepatocytes and hepatoma carcinoma cells." Journal of Biochemical and Molecular Toxicology 29, no. 9 (2015): 440-448.
    View at Publisher | View at Google Scholar
35. Li, San-Qiang, Zhi-Hong Hu, Sha Zhu, Dong-Mei Wang, Hong-Mei Han, and Hua-Jie Lu. "The effect of ADAM8 on the proliferation and apoptosis of hepatocytes and hepatoma carcinoma cells." Journal of Biochemical and Molecular Toxicology 29, no. 9 (2015): 440-448.
    View at Publisher | View at Google Scholar
36. Vilchez, Valery, Lilia Turcios, Francesc Marti, and Rober- to Gedaly. "Targeting Wnt/β-catenin pathway in hepato- cellular carcinoma treatment." World journal of gastroen- terology 22, no. 2 (2016): 823.
    View at Publisher | View at Google Scholar
37. Gedaly, Roberto, Roberto Galuppo, Michael F. Daily, Ma- lay Shah, Erin Maynard, Changguo Chen, Xiping Zhang et al. "Targeting the Wnt/β-catenin signaling pathway in liver cancer stem cells and hepatocellular carcinoma cell lines with FH535." PloS one 9, no. 6 (2014): e99272.
    View at Publisher | View at Google Scholar
38. Coste, Alix de La, Béatrice Romagnolo, Pierre Billuart, Claire-Angélique Renard, Marie-Annick Buendia, Olivier Soubrane, Monique Fabre et al. "Somatic mutations of the β-catenin gene are frequent in mouse and human hepatocellular carcinomas." Proceedings of the National Academy of Sciences 95, no. 15 (1998): 8847-8851.
    View at Publisher | View at Google Scholar
39. Zisuh, Anutebeh Verdo, Tian-Quan Han, and Shen-Dao Zhan. "Expression of telomerase & its significance in the diagnosis of pancreatic cancer." Indian Journal of Medi- cal Research 135, no. 1 (2012): 26-30.
    View at Publisher | View at Google Scholar
40. Ohuchida, Kenoki, Kazuhiro Mizumoto, Yasuhiro Ogura, Nami Ishikawa, Eishi Nagai, Koji Yamaguchi, and Masao Tanaka. "Quantitative assessment of telomerase activity and human telomerase reverse transcriptase messen- ger RNA levels in pancreatic juice samples for the diag- nosis of pancreatic cancer." Clinical cancer research 11, no. 6 (2005): 2285-2292.
    View at Publisher | View at Google Scholar
41. Armstrong, Christine A., and Kazunori Tomita. "Funda- mental mechanisms of telomerase action in yeasts and mammals: understanding telomeres and telomerase in cancer cells." Open biology 7, no. 3 (2017): 160338.
    View at Publisher | View at Google Scholar
42. Ferrara, Napoleone. "VEGF and the quest for tumour angiogenesis factors." Nature Reviews Cancer 2, no. 10 (2002): 795-803.
    View at Publisher | View at Google Scholar
43. Cebe-Suarez, S., A. Zehnder-Fjällman, and K. Ball- mer-Hofer. "The role of VEGF receptors in angiogene- sis; complex partnerships." Cellular and molecular life sciences 63 (2006): 601-615.
    View at Publisher | View at Google Scholar
44. Nagy, Janice A., Laura Benjamin, Huiyan Zeng, Ann M. Dvorak, and Harold F. Dvorak. "Vascular permeability, vascular hyperpermeability and angiogenesis." Angio- genesis 11 (2008): 109-119.
    View at Publisher | View at Google Scholar
45. Huang, Jianfei, Xialing Zhang, Qi Tang, Feng Zhang, Yuhua Li, Zhenqing Feng, and Jin Zhu. "Prognostic sig- nificance and potential therapeutic target of VEGFR2 in hepatocellular carcinoma." Journal of clinical pathology 64, no. 4 (2011): 343-348.
    View at Publisher | View at Google Scholar