Effects Of Human Mesenchymal Stem Cells On Proliferation, Metastasis, Apoptosis And Autophagy Of Lung Carcinoma Cells

IntroductionCurrently, lung cancer is the leading cause of cancer-related mortality throughout theworld. It became critical cancer-related mortality and is gradually rising through the world.It is difficult to diagnoses in the early stage and it couldn’t have complete surgicalresection.Although there have been significant advances in cancer treatments, thismalignancy remains poorly responsive to conventional therapy. Hence, it is urgent todetermine the survival mechanism of carcinoma cells to develop more efficient therapiesfor patients.Human mesenchymal stem cells (hMSCs) are pluripotent progenitor cells that residewithin the adult bone marrow. They have self-renewal capacity, long-term viability, and candifferentiate into the adipocytic, chondrocytic, or osteocytic lineages. Although hMSCsreside predominantly in the bone marrow, they are also distributed throughout many othertissues, where they are thought to function as local sources of dormant stem cells. Afterinjury or chronic inflammation, the wounded tissue would release specific endocrine signalsthat are then transmitted to the bone marrow, leading to the mobilization of multi-potenthMSCs and their subsequent recruitment to the damage site. Moreover, recent evidence hasindicated that hMSCs are recruited and incorporated within the connective tissue stroma oftumors. It possible to take advantage of hMSCs as carrier cells for tumor gene therapy.Therafoe, it is vital to define the role hMSCs in lung cancer cell growth for thereconstruction of the hMSCs mound for cancer gene therapy. However, the effects ofhMSCs on tumors are not clean, especially on lung carcinoma cells. We investigated theeffects on lung carcinoma cell in vitro and in vivo. Indirect culture system was used toinvestigate the effects of hMSCs on Proliferation, invasion, metastasis, of lung carcinomacells and try to exploration the possible mechanisms Nutrient deprivation and oxygen deficiency are representative characteristics of thesolid tumor microenvironment during cancer development. Autophagy is a well-establishedmechanism for degrading cytoplasmic proteins, macromolecules, and organelles to providea nutrient source to promote the survival of cells that are under metabolic stress. Starvationincreases the number and size of autophagosomes in many tissues, suggesting thatautophagy is a critical component of the body’s response to nutrient deprivation and aminoacid/fuel homeostasis. Autophagy has been implicated in a number of differentphysiological and pathological conditions, including development, differentiation,immunity, aging and cell death. In addition, accumulating evidence demonstratesinteresting links between autophagy and tumorigenesis, tumor progress, andchemoresistance. In particular, the regulation of autophagy in carcinoma cells is complexbecause it can enhance tumor cell survival in response to certain stresses. Because theeffects of hMSCs on tumors under stressful conditions have not been determined, weinvestigated the survival mechanisms used by stressed stromal cells on lung carcinoma cells.Indirect culture system was used to investigate the effects of hMSCs on viability andapoptosis in starved carcinoma cells and focused on the role of autophagy in regulating thesurvival of carcinoma cells.Methods:1. Isolation and identification of hMSCsIsolation and culture hMSCs from bone marrow adult healthy volunteers by densitygradient centrifugation, differential velocity adherent culture was used for purification ofhMSCs. HMSCs surface markers by flow cytometry were used to identify cell purity.HMSCs were cultured in osteogenic induction medium, adipogenic induction, chondrogenicinduction medium to identificated multiple differentiation capacity, determine osteogenicability by alizarin red staining, adipogenic ability by oil red O staining, chondrogenicability by alcian blue staining.2. The effects of hMSCs on Proliferation of lung carcinoma cells in vitro and in vivo.We used lung carcinoma cell line A549and SPC-1for investigation. Indirect culturesystem was used to investigate the effects of hMSCs on the change of A549and SPC-1incell viability, cell cycle, apoptosis, protein expression of PCNA、Cyclin-D1、VEGF. Meanwhile, we establish nude mouse model to observe the effects of hMSCs on tumorgrowth and to detect the change of CD34expression in the transplanted tumor. We try tounderstand the effect of hMSCs on proliferation and angiogenesis of lung cancer cells invitro and in vivo.3. The effects of hMSCs on invasion and metastasis of lung carcinoma cells in vitroand in vivo.We used lung carcinoma cell line A549and SPC-1for investigation. Indirect culturesystem was used to investigate the effects of hMSCs on the change of A549and SPC-1inthe ability of invasion and metastasis, the invasion-related protein expression of MMP-2and MMP-9. Meanwhile, experimental metastasis was examined using abdominal cavitytumor xenografts metastasis models plus regular hMSCs intraperitoneal injection.wedetects the change of MMP-2and MMP-9expression in the transplanted tumor. We try tounderstand the effect of hMSCs on invasion and metastasis of lung cancer cells in vitro andin vivo.4. Effects of hMSCs on apoptosis and autophagy of lung carcinoma cellsWe investigated the survival mechanisms used by stressed stromal cells on lungcarcinoma cell line A549and SPC-1in vitro and in vivo. Indirect culture system was usedto investigate the effects of hMSCs on viability and apoptosis in starved carcinoma cellsand focused on the role of autophagy in regulating the survival of carcinoma cells. Then,we observed the change of apoptosis and autophagy while the ion of autophagy wasinhibited by the autophagic inhibitor-3-MA.also,we detected the apoptosis and autophagyrelated protein expression of Bcl-2，Beclin-1. We try to understand the regulation of hMSCson apoptosis and autophagy of lung cancer cells in vitro and in vivo.Results:1. Succeeded isolation and identified hMSCsWe have succeeded isolation and culture highly homogeneous and adherent growthhMSCs from bone marrow adult healthy volunteers by density gradient centrifugation.HMSCs surface markers by flow cytometry were negative expression (<5%)of CD45,CD34, CD14, CD19, HLA-DR, and positive expression of CD73, CD90, CD105(>95%).And hMSCs could differentiate to osteoblasts, adipocytes and chondroblasts in vitro. hMSCs could succeeded stain by alizarin red, oil red O, alcian blue. All of aboveaccordding with minimal criteria to define hMSCs minimal criteria。2. HMSCs could inhibit tumor proliferation in vitro, but benefit to tumor proliferationin vivo.We observed hMSCs inhibition tumor proliferation by indirect culture system in vitro.hMSCs could regulate cell cycle progression of lung carcinoma cells. It could make themost of cell arrest in G0/G1phase and decrease S phase cells.the lower expression ofPCNA和Cyclin-D1lung carcinoma cells point out the change of cell cycle progressionmay be the reason. However, hMSCs could promote the proliferation of tumor, higherCD34expression in the transplanted tumor and VEGF expression in lung carcinoma cellsmeans better tumor angiogenesis by hMSCs plays important roles on the inhibition.3. HMSCs could inhibit invasion and metastasis of lung carcinoma cells in vivo and invitroWe observed hMSCs inhibition lung carcinoma cells invasion and metastasis bytranswell chamber in vitro. We succeeded establish experimental metastasis usingsubcutaneouly tumor xenograft models and abdominal cavity tumor xenografts metastasismodels. As the same as that in vitro, hMSCs also inhibition lung carcinoma cells metastasis.Lower MMP-2and MMP-9expression may play an important role on the inhibition.4. HMSCs protected against apoptosis by enhancing autophagy in lung carcinoma cellsduring serum deprivation，A549and SPC-1cells had higher viability when co-culturedwith hMSCs and that this was mainly attributed to decreased apoptosis. Autophagosomeswere analyzed using GFP-LC3and electron microscopy, which showed that autophagy wassignificantly activated in the starved co-culture groups. However, the inhibition ofautophagy by the autophagic inhibitor-3-MA significantly abrogated the apoptosisreduction in either single groups or co-culture groups under serum deprivation. We foundlung carcinoma cells have the lower expression of Bcl-2and higher expression of Beclin-1when co-cultured with. It point out could hMSCs trigger autophagy by Beclin-1without theinhibition of Bcl-2.We also observed that hMSCs promoted tumor initiation and growth invivo, and observed more autophagy happened in co-cultured tumors.hMSCs could. Inconclusion, our study demonstrates that hMSCs can protect carcinoma cells from nutrientdeprivation-induced apoptosis and promote tumor initiation and growth. Also more interestingly, autophagy plays an important role in the survival of cancer cells.Conclusion:1.We have succeeded isolation and culture hMSCs，HMSCs surface markers analysisand multiple differentiation capacity identification satisfy with minimal criteria to definehMSCs minimal criteria.2. HMSCs play dual roles on proliferation of lung carcinoma cells in vitro and in vivo.hMSCs could inhibit PCNA and Cyclin-D1expression to regulate cell cycle progression oflung carcinoma cells to inhibition tumor proliferation in vitro; however better tumorangiogenesis bringed by hMSCs is beneficial to tumor proliferation in vivo, VEGF may bethe reason..3. HMSCs could inhibit invasion and metastasis of lung carcinoma cells; inhibition ofMMP-2and MMP-9expressions play important roles on the inhibition.4. HMSCs protected against apoptosis by enhancing autophagy in lung carcinomacells.HMSCs trigger autophagy by Beclin-1without the inhibition of Bcl-2plays animportant role in the survival of cancer cells...