Heterogeneous Expression Of AFP In Different Subpopulations Of Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. While many therapies still attempt to attack cancer tumors as homogenous tissue, recent evidence suggests that a newer approach in viewing the cancerous tissue as composed of heterogeneous cells that may include cancer stem cells, may be more effective. Alpha-fetoprotein (AFP) is highly expressed during the fetal life, essentially in the liver and the visceral endoderm of the yolk sac. At birth, its expression is shut down to a very low level. Meanwhile, the expression of AFP can be observed in the adult liver in some physio- pathological cases such as liver regeneration or hepatoma development. As we know, over expression of AFP is observed in about 2/3 HCC patients. However, the expressions of AFP in HCC are different from each other, the expression of well- differentiated HCC tissues is higher than moderately and poorly differentiated tissues. In another word, AFP is one of characteristics of cells with capability of regeneration or primitive cells such as stem cells, progenitor cells and so on. What's more, more and more evidence show that AFP has play an important role in human immunity system. It can inhibit functions of antigen present cells such as DC, et al. It also can inhibit the functions of T lymph cells. AFP has some relationship with the recidivation and metabasis of cancer.Recently, more and more studies show that there is increasing evidence of finding a small side population (SP) both from established cancer cell lines and primary tumors.SP cells were identified having stem like characteristics, the capacity for self-renewal to allow the maintenance of an undifferentiated stem cell population and the ability to undergo differentiation. It is also reported that SP cells that function as cancer stem cells were detected in some HCC cell lines . Whether SP cells are contained in MHCC97 and HepG2, and whether AFP expression are heterogeneous in the same cell lines can do help to explain the existence of heterogeneity of cancer cells.Object:This study is to explore the expression characteristics of alpha- fetoprotein(AFP) in different subpopulations of MHCC97 and HepG2 cell lines of human hepatocellular carcinoma.Methods:1 Flow Cytometry Analysis for Hoechst Efflux and Cells Sorting Cells were stained with 5 g/mL of Hoechst 33342 in 5 mL of DMEM containing 2% bovine serum albumin (BSA) at 37°C for 90 min, with a concentration of 106 cells/ mL. After incubation, the cells were washed with Hanks'balanced salt solution. In addition, 2 g/mL of propidium odide (PI) was added for dead cell discrimination. Verapamil was also added to a parallel set of samples for 10 min before Hoechst staining to analyze the effect of inhibiting.Hoechst efflux. Hoechst efflux were analyzed using a fluorescence- activated cell sorter with CELLQuest software. After that, the SP cells and Non-SP cells were sorted respectively.2 ImmunocytochemistrySP cells and Non-SP cells were grown on coverslips respectively for 4 h, fixed with 4% paraformaldehyde , then cells were exposed to 0.3% H2O2 in absolute methanol at room temperature for 15 minutes to inhibit endogenous peroxidase activity. Immunocytochemical staining was performed with commercially available primary antibody and other reagents completed according to the manufacturer's protocol. Essentially, coverslips were incubated in the 4% normal horse serum for murine monoclonal antibodies blocking serum for 30 minutes at room temperature followed by incubation at 4C overnight with the mouse monoclonal anti-AFP antibody in the dilution of 1:100. After washing in Tris buffer, species-specific secondary antibody was added and incubated for 30 minutes at room temperature. Coverslips were then stained with 3,3′-diaminobenzidine (DAB) for 10 minutes at room temperature, rinsed in running water, and dehydrated with xylene, and mounted in DPX (BDH). Negative controls were obtained by omitting the primary antibody. Lg IOD(integrated optical density) was used for measurement of the expression of AFP, results are presented as means±SD. Two-tailed Student t test was used for comparison of Lg IOD of different subpopulations. A probability of less than 5% (P < 0.05) was considered statistically significant.3 Reverse transcription–polymerase chain reaction (RT-PCR)Single-step RNA isolation was performed using TRIzol Reagent, according to the manufacturer's protocol. We synthesized cDNAs from 2.0 g of total RNA using random primers with reverse transcriptase. With 15% of the cDNA, PCR was performed for 22 cycles with gene-specific primers (conditions per cycle: 30 seconds at 95°C, 30 seconds at 56°C, and 90 seconds at 72°C) using a Eppendorf PCR System. Housekeeping gene glyceraldehyde- 3-phosphate dehydrogenase (GAPDH)was used as internal control. PCR products were separated by electrophoresis through a 1.2% agarose gel and stained with 0.2% ethidium bromide. The intensity of each band was determined under ultraviolet illumination. LgOD(AFP/GAPDH) was used for measurement of the expression of AFP mRNA, results are presented as means±SD. Two-tailed Student t test was used for comparison of LgOD(AFP/GAPDH) of different subpopulations. A probability of less than 5% (P < 0.05) was considered statistically significant. ResultsHere we show that the SP cells were detected in MHCC97 and HepG2 cell lines with flow cytometry analysis by the ability of these cells to extrude the Hoechst 33342 dye, but it only took little part. Immunocytochemical staining was performed to show the presence of AFP in SP and Non- SP cells, and AFP is much more expressed in SP cells on the protein level. The different of AFP mRNA between subpopulations was shown by RT-PCR. Our findings reveal that the HCC has the characteristic of heterogeneous. SP cells may be a source of cancer stem cells in the HCC, which need to be targeted for effective cancer therapy.ConclusionStudies show that cancer cells are heterogeneous in the capacity for self-renewal to allow the maintenance of an undifferentiated stem cell population and the ability to undergo differentiation. Certain subpopulations from acute myeloid leukemia and breast carcinoma can recreate the tumor when injected into immune deficient mice. Kondo has detected SP cells in C6 glioma cell lines that also form tumors in nude mice, whereas non SP cells did not form tumors. In addition, they have found SP cells in four of six cancer cell lines tested. These studies of SP cells with stem cell like properties are of utmost importance as the presence of these cancer stem cells could lead to an explanation of recurrence of cancer even after extensive treatment. cancer stem cells in the new paradigm may be critical players in drug resistance and metastases, elimination of SP cells may be a vital mechanism to control cancer.SP cells have been found in several kinds of HCC cell lines. Our studies suggest that in MHCC97 and HepG2 cell lines, SP cells are also detected, and the percentage of it is very low. AFP is one of the characteristics of cells with capability of regeneration or primitive cells such as stem cells, progenitor cell. Immunocytochemistry and reverse transcription–polymerase chain reaction results show the significant difference between SP cells and Non-SP cells reveal that the AFP expression is varied in the same cell lines, which enrich the evidence of heterogeneity of cancer cells. Furthermore, AFP expression can reflect the cell differentiation in some extent, the high expression of AFP in SP cells reveals this subpopulation are more primitive than the other one. Although SP cells express more AFP, it only take little part of the whole cells. It do help to explain why serum AFP concentration is low or negative in part of HCC patients.