Effects Of Fullerene Derivatives On Fibroblasts In Tumor Microenvironment

As a global public health problem, tumor greatly endangers humanhealth. With the development of cancer pharmacology, improving tumormicroenvironment has become an important therapeutic strategy. Tumormicroenvironment consists of tumor cells, tumor infiltrated immune cells,interstitial cells and the active mediums secreted by these cells. Tumormicroenvironment plays an important role in cancer initiation, progressionand metastasis. The fibroblasts are most abundant in the microenvironmentof solid tumors. In the tumor microenvironment, fibroblasts can be activatedand obtain an activated phenotype, called activated fibroblasts orcancer-associated fibroblasts （CAFs）. CAFs can promote tumor growth andproliferation. By secreting several growth factors and chemotactic factors,CAFs can affect various aspects of the tumor cells. Therefore, fibroblastsreceived more and more attention as a new target for anti-cancer therapy.In recent years, fullerene derivatives with specific physicochemicalproperties have been developed for cancer diagnosis and therapy, such asphotodynamic therapy, photothermal treatment, radiotherapy andchemotherapeutics. However, the anti-tumor studies of fullerene derivativesmostly focused on the direct effect of the drugs on tumor cells, while littleattention has been paid to its impact on the tumor microenvironment. In thisstudy, the fullerene derivatives used are fullerenols (C₆₀（OH）₂₂) andGd@C₈₂（OH）₂₂(Gd@C₈₂).Part1: Synthesis of FullerenolsObjective: To synthetise the polyhydroxy fullerene derivatives--fullerenols, for subsequent experiments.Methods: Fullerenols were synthetised by the reaction of fullerene withNaOH using tetrabutyl ammonium hydroxide （TBAH） as a phase transfer catalyst, and purified by Sephadex G-25chromatography column. The shapeand particle size of fullerenols were characterized using transmissionelectron microscopy （TEM）.Results: We used90mg fullerene, ultimately get92mg fullerenols, withthe yield of67.27%. TEM characterization results showed that the diameterof fullerenol spherical nanoparticles was100-200nm in ultrapure water.Conclusions: Fullerenols were prepared with high yield and uniformparticle size by using TBAH as the catalyst.Part2: Effects of Fullerene Derivatives on Proliferation andSecretory Function of CAFs in Tumor Microenvironment andInvestigation of the MechanismsObjective: To study the effects of fullerenols and Gd@C₈₂on normalfibroblast--HFL-1cells and CAF--MCA205cells, and to explore themechanisms.Methods:1MCA205cells and HFL-1cells were cultured and treated with25μMor50μM of fullerenols or Gd@C₈₂for24h. The cell viability was measuredby CCK-8assay.2MCA205cells were treated with25μM fullerenols or Gd@C₈₂for24h.Then, the culture supernatant was mixed with normal medium （1:1） after thedrugs were filtered out. Lewis lung cancer （LLC） cells were cultured withculture supernatant of MCA205cells. Scratches assay was used to test themigration ability of LLC cells.3The cells were treated with25μM fullerenols or Gd@C₈₂for24h or72h. The concentration of TGF-β1, IL-6and VEGF in the culturalsupernatant was detected using ELISA.4Immunofluorescence technique was used to examine the expression ofTGF-β1protein in MCA205cells treated by25μM fullerenols or Gd@C₈₂for24h or72h.5Western blot was used to detect the expression of TGF-β1proteinafter MCA205cells were treated by25μM fullerenols or Gd@C₈₂for24h or 72h.6The mRNA expression levels of TGF-β1in MCA205cells treated by25μM fullerenols or Gd@C₈₂for24h or72h were determined by real-timePCR.Results:1The proliferation of MCA205cells was not significantly inhibited by25μM fullerenols or Gd@C₈₂. Gd@C₈₂50μM decreased proliferation ofMCA205cells. But25μM and50μM of fullerenols and Gd@C₈₂did notsignificantly affect proliferation of HFL-1cells.2LLC cells in control group migrated4h after scratch, while the cells inexperimental group did not.3The level of TGF-β1in the cultural supernatant of HFL-1cells wasnot significantly affected24h or72h after the cells were treated by25μMfullerenols or Gd@C₈₂. Fullerenols25μM or Gd@C₈₂25μM significantlydecreased the level of TGF-β1in the culture supernatant of MCA205cells. Atthe same time, fullerenols and Gd@C₈₂could significantly reduce content ofIL-6and VEGF in the culture supernatant of MCA205cells.4The results of immunofluorescence and Western blot showed thatfullerenols and Gd@C₈₂down-regulated the expression of TGF-β1protein inMCA205cells, and the effect was enhanced as time went on.5Real-time PCR results showed that mRNA expression of TGF-β1inMCA205cells treated by fullerenols or Gd@C₈₂was lower than that incontrol group.Conclusions:1The high concentration of Gd@C₈₂could suppress the proliferation ofMCA205cells, while no significant influence was induced by the lowconcentration of the fullerene derivatives. Neither high nor low concentrationof fullerene derivatives had significant effect on the proliferation of normalfibroblast--HFL-1cells.2Fullerene derivatives could inhibit the secretion of TGF-β1, IL-6andVEGF from MCA205cells and decrease the ability of promoting tumor cell migration of culture supernatant of MCA205cells.3Fullerene derivatives could inhibit TGF-β1gene and proteinexpressions in MCA205cells, resulting in a decline in its downstreamcytokine secretion.4The selective proliferation-inhibitory effect might be related tofullerenols and Gd@C₈₂selectively inhibiting MCA205cells to secretTGF-β1.