Published online Feb 7, 2019. doi: 10.3748/wjg.v25.i5.567
Peer-review started: September 10, 2018
First decision: October 11, 2018
Revised: December 2, 2018
Accepted: December 6, 2018
Article in press: December 6, 2018
Published online: February 7, 2019
Hepatocellular carcinoma (HCC) is a malignant condition with a high incidence and no effective treatment. Mesenchymal stem cells (MSCs) secrete cytokines and growth factors known to have paracrine, trophic and immunomodulatory effects. Due to their paracrine and differentiation potential, adipose-derived SCs have proven therapeutic efficacy in many diseases. Their conditioned media (CM) has been shown to inhibit proliferation and increase apoptosis in HCC. However, many controversies have been noted concerning their role in cancer.
Many studies have demonstrated the effect of SCs or their CM on cancer, and some reports have shown that they suppress and inhibit tumor growth. Other studies have reported enhanced tumor growth and proliferation. There have not been any studies that reported the effect of adipose-derived MSCs (ADMSCs) on HCC proliferation and apoptosis. Thus, our aim was to investigate the therapeutic effects of adipose-derived SCs and their CM on HCC, specifically their effects on cancer cell marker expression, the proliferation and metastatic potential of cancer cells, and their effect on modulating cancer cell death.
By discovering that adipose-derived SCs and their CM modulate cancer marker expression and liver cancer cell proliferation and metastasis, we have opened a new path for research on the mechanism of action by which MSCs can affect cancer. If the results were to increase and stimulate cancer cells, then further investigations need to be pursued on two levels: (1) to study the behavior of SCs along with the factors contributing to the stimulatory effects; and (2) inhibition of the pathways leading to this progressive effect. In contrast, if the ADMSCs were to inhibit cancer and induce apoptosis, then ADMSCs could be a potential therapy for HCC, which currently has no cure. To achieve this goal, in vivo animal models and clinical studies need to be pursued.
In our study, the main objective was to investigate the role of ADMSCs and their CM in HCC using two cell lines, HepG2 and PLC-PRF-5. In particular, we wanted to study the effect of ADMSCs on alpha-fetoprotein (AFP) and Des-gamma-carboxyprothrombin (DCP) expression and the capacity of ADMSCs to modulate metastasis or proliferation of the above cancer cell lines. We studied TIMPs, P53, and RB. ADMSCs inhibited cell proliferation, decreased AFP and DCP expression and promoted apoptosis. In addition, ADMSCs decreased cancer cell migration and invasion by increasing TIMP expression. Our study has shed light on a novel apoptotic effect of MSCs on cancer. This will direct us and other researchers to further investigate the effect on other cell markers playing roles in cancer and the mechanisms by which ADMSCs exert their anti-cancer effects.
HCC cell lines purchased from ATCC were cultured in low glucose DMEM media. Adipose-derived MSCs isolated from lipoaspirates were cultured in DMEM nutrient mix F12. The isolation method of MSCs was modified and improved to obtain a high yield of living ADMSCs using a minimal quantity of fat and collagenases. Isolated ADMSCs were characterized to demonstrate their viability and capacity of multilineage differentiation.
The coculture conditions and treatment with ADMSC CM were extensively studied in order to determine the number of cells that should be used in all experiments. After co-culturing HCC cells with ADMSCs or stimulating with ADMSC CM, AFP and DCP protein and mRNA levels were detected using ELISA kits and real time PCR, respectively.
In addition, the proliferation level and apoptosis rate of HCC cells were measured using a WST-8 proliferation test and annexin V-FITC kit, respectively. Along with these tests, the mRNA levels of P53, RB, hTERT and c-Myc genes involved in the regulation of proliferation and apoptosis were quantified using real time PCR.
Furthermore, using wound healing assays and migration and invasion tests, we studied the effect of ADMSCs and their CM on HCC cell line metastasis. In parallel, TIMP mRNA levels were measured using real time PCR. TIMPs have been reported to play a major role in in inhibiting metastasis.
In all assays, the experiments were repeated at least three times in order to obtain statistically significant results.
In our study, ADMSCs inhibited cancer cell proliferation and increased cancer cell death when co-cultured with HepG2 and PLC-PRF-5. This effect was more significant in the case of direct co-culture, likely due to cell-cell interactions. The upregulation of the tumor suppressor genes P53 and RB and downregulation of c-Myc and hTERT might be the factors responsible for the mentioned findings. The mechanisms of these results should be further investigated.
We reported increased secretion of TIMP-1, -2, and -3, which may be partially responsible for the decreased HCC cell migration and invasion. Future studies should be performed to confirm this relation. In addition, further investigations are needed to study the involvement of the metalloproteinases MMP-2 and MMP-9 in the inhibition of metastasis. We also found that ADMSCs and ADMSC CM decreased HCC cell line migration and invasion.
We observed decreased AFP and DCP levels after coculturing HCC cells with ADMSCs or stimulating HCC cells with ADMSC CM. This might be an indication of an attempt by SCs to obliterate proliferation and tumor progression. These findings will be confirmed and used subsequently in an in vivo animal study.
This study reported many novel findings about the effects of ADMSCs on HCC. This is the first report to demonstrate a decrease in DCP expression in HCC cell lines. No other study has investigated the direct effect of ADMSCs on HCC proliferation and apoptosis. We reported novel molecules contributing to the effect of adipose-derived SCs on HCC, particularly TIMPs. We reported that coculture of ADMSCs with HepG2 or PLC-PRF-5 cell lines had an anti-cancer effect. This is explained by the inhibition of proliferation and cell death of the cancer cells. We also showed for the first time the effect of direct cell-cell interactions, which is a new mechanism by which ADMSCs might inhibit tumor cell proliferation. The indirect contact of ADMSCs with HCC cell lines inhibited their proliferation and metastasis and increased their apoptosis, though to a lesser extent than the direct coculture, suggesting that the paracrine effects of ADMSCs contribute to their antitumor effects.
These findings confirm that in certain types of cancer, MSCs could enhance tumor growth and in others, it can inhibit invasiveness and metastasis. This might be explained by the complexity of MSC sources, the malignant cell type involved, and the interaction between MSCs and tumor cells. The number of MSCs and the microenvironment might also influence tumor cell growth or inhibition.
Our findings will lead to many investigations: (1) the mechanism of cell-cell contact by which ADMSCs inhibit cancer cells (autophagy); (2) identification of the factors exerting these inhibitory effects. In particular, two candidates are STC-1 and DKK-1, which are known to have roles in suppressing the expression of genes involved in proliferation, migration and invasion and in the overexpression of apoptotic genes. These results should be demonstrated using WST-8 proliferation assays, apoptosis annexin/PI assays, and migration and invasion tests.
In vivo studies will be pursued to confirm our results, mainly the effect of ADMSCs and their CM on tumor growth, apoptosis and metastasis, as well as the paracrine effects of ADMSCs.
To understand and quantify the changes in hepatic cancer cell morphology when in direct contact with ADMSCs, a study will be conducted in collaboration with the Department of Physics at the American University of Beirut. This will also help us determine the mechanism by which ADMSCs induce HepG2 cell death.
In summary, ADMSCs are cells with complex mechanisms that have the capacity to interact with adjacent cells to exert trophic and paracrine effects, thus altering the microenvironment. Their role in each disease must be vigorously studied to elucidate their therapeutic effects. In this study, we determined the inhibitory effects of ADMSCs on cancer cell markers and on key factors known to play a major role in inflammation, invasion and metastasis. Our study has shed new light on the role of ADMSCs on HCC.