Nicotine-enhanced stemness and epithelial-mesenchymal transition of human umbilical cord mesenchymal stem cells promote tumor formation and growth in nude mice
Cigarette smoking is a well-known risk factor in the development and progression of malignant diseases. Nicotine, the major constituent in cigarette smoke, has also shown negative effects on stem cells. Mesenchymal stem cells (MSCs) have been widely demonstrated to migrate into tumors and play key roles in cancer progression. However, the mechanisms by which nicotine impacts MSCs and tumorigenesis of lung cancer are still undetermined. In this study we investigated the effects of nicotine on human umbilical cord mesenchymal stem cells (hUC-MSCs) and the impacts of nicotine-treated hUC-MSCs on tumor formation and progression. We found that nicotine has a toxic effect on hUC-MSCs and changes the morphology, inhibits proliferation and promotes apoptosis of hUC-MSCs in a dose-dependent manner. Nicotine-treated hUC-MSCs produce higher level of IL-6. Moreover, nicotine promotes migration, stemness and epithelial-mesenchymal transition (EMT) of hUC-MSCs by inhibiting E-cadherin expression and upregulating mesenchymal markers such as N-cadherin and Vimentin, leading to the induction of stem cell markers Sox2, Nanog, Sall4, Oct4 and CD44. Migration and proliferation of non-small cell lung cancer A549 cells and breast cancer MCF-7 cells are promoted after their coculture with nicotine-treated hUC-MSCs in a cell-cell contact-independent manner. Furthermore, nicotine-treated hUC-MSCs promote tumor formation and growth of A549 cells in nude mice. These studies demonstrated that the enhanced stemness and EMT of hUC-MSCs induced by nicotine are critical for the development of tobacco-related cancers.After 10 days of culture, the cells displayed a polygonal, spindly and fibroblast-like morphology and began to form colonies (Figure
1A). Endothelial progenitor cells were gradually eliminated after multiple medium replacements and PBS washing. Consistent with known MSC phenotypes, passage 3 cells highly expressed MSCs markers CD29 (99.7%), CD90 (99.6%), and CD105 (99.8%), while low expressed B lymphocyte surface markers CD19 (0.1%) as shown in Figure 1B, 1C. After 2 or 3 weeks in culture in the specific medium, the cells were capable of differentiating into osteocytes and adipocytes, as shown by positive staining of ALP and Oil Red O (Figure
1D), strongly suggesting that the cells have the multilineage differentiation potential. To further confirm this, expression of osteogenic and adipocyte markers were examined. BMP-3 mRNA level was significantly higher and PPARγ-2 mRNA level was significantly lower in osteogenic group compared to adipogenic group (Figure
1E). These data indicated that we efficiently generated hUC-MSCs which were used in the following studies.In our previous study, we had successfully isolated MSCs from the human umbilical cord [69, 70]. Fresh umbilical cords were collected from healthy donors with informed consents at the First People’s Hospital of Zhenjiang, China. We used those hUC-MSCs for the current set of experiments. The cells of passages 3 to 5 were used in the experiments. HUC-MSCs were plated at a density of 1 × 105 cells per 25 cm2 flask (Coning, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium with low glucose (L-DMEM) containing 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) and 1% antibiotics (100 U/ml penicillin and 100 U/ml streptomycin; Beyotime, Haimen, China) in a humidified atmosphere with 5% CO2 at 37°C, and the medium was changed every 3 days thereafter. When the adherent cells reached nearly 80% to 90% confluence, the cells were digested by 0.25% trypsin-EDTA (Invitrogen, USA) and subcultured in a new flask for further expansion.We would like to thank the staff at our laboratory for the assistance they have provided in this study.CONFLICTS OF INTERESTThe authors declare that they have no competing interests.FUNDINGThis work was supported by the Fund for Jiangsu Specially-Appointed Professor (2014JSTPJS-53) and the National Science Foundation for Young Scientists of China (81500351).