Effects Of Fructus Viticis Total Flavonoids On Characteristics Of Lung Cancer Stem Cells Derived From NCI-H446Cell Line

Objective:To investigate the effects of Fructus Viticis total flavonoids(FVTF) on the characteristics including self-renewal, proliferation and invasion of lung cancer stem cells(LCSCs) derived from human small cell lung cancer NCI-H446cell line and its potential mechanism, which provide experiental basis on developing a candidate agent for radical curing lung cancer in the future.Methods:Human small cell lung cancer NCI-H446cell line was cultured in vitro. Then magnetic activated cell sorting system(MACS) and suspended culture with stem cell-conditioned medium were used to sort and amplify CD133+sphere-forming cells(SFCs). The CD133positive population was determined by flow cytometry (FCM) with PE-CD133antibody. Cell morphology was observed by optical microscope.3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT) assay was used to detected cell viability. The self-renewal capacity was exanined by tumor sphere formation assay. The invasion capacity was determined using Transwell assay and the tumorigenicity was tested using xenograft model in nude mouse. The protein expression levels of cancer stem cell markers(CD44and ALDH1), self-renewal associated transcription factors(Bmil, Nanog and OCT4) and invasion associated transcription factors(Twistl and Snail) were analyzed using western blot. Aforementioned the process was to identify whether CD133+SFCs of NCI-H446cell line possess the characteristics of LCSCs and examine the effects of indicated concentrations of FVTF (0.5,1.0,2.0μg/mL) on the characteristics of LCSCs. In addition, we will demonstrate the effects of indicated concentrations of FVTF (0.5,1.0,2.0μg/mL) on the protein expression of stem cell markers (CD133, CD44and ALDH1), p-Akt, self-renewal associated transcription factors(Bmil, Nanog and OCT4) and invasion associated transcription factors(Twist1and Snail) in LCSCs compared with the parent cells. We also determine the effects that PI3K specific inhibitor LY294002(5.0、10.0、20.0μM) alone or combined with FVTF regulate these protein expression and suppress the characteristics of LCSCs for investigating the possible mechanisms by which FVTF inhibit the function and characteristics of LCSCs through down-regulating the expression of p-Akt which lead to downregulation of CD133, CD44, ALDH1, Bmil, Nanog, OCT4, Twist and Snail protein expression levels.Result:1. CD133MACS sorted CD133+cells derived from human lung cancer NCI-H446cell line exhibit elevated CD133-positive percentage (CD133+cell population:91.85±2.17%vs CD133-cell population:0.03±0.01%, P <0.001). Both CD133+cell and parent cells can grow to non-adherence spheres namely lung cancer sphere in6-well ultra-low adherence plates with serum-free stem cell condition medium, yet CD133-cell can not. Compared with parent cells (64±11/1000cells), the number of sphere-forming cells of CD133+cell (239±26/1000cells) is higher. And compared with parent cells, the expression of stem cell markers(CD44and ALDHl), self-renewal associated transcription factors(Bmil, Nanog and OCT4) and invasion associated transcription factors(Twistl and Snail) is over-expressed in CD133+SFCs. CD133+SFCs possess the capability of forming secondary-passaged sphere and becoming a new lung cancer sphere from single cell. And they also have stronger capability of invasion (CD133+SFCs:384±43/5000cells vs parent cells:237±28/5000cells, P<0.05). As few as1×103cells from NCI-H446CD133+SFCs were able to grow into tumors (4/6) when subcutaneously injected into Blab/c-nu mice, while1×105parental cells were needed for tumor formation (3/6) in vivo. There was also a great difference in the time needed for tumor formation,18days for CD133+sphere cells compared to31days for the parental cells.2. The result of MTT assay show that FVTF relative selectly inhibited the proliferation of LCSCs (Value of IC50for CD133+SFCs:0.7μg/mL vs parent cells:33.7μg/mL, P<0.05). FVTF down-regulated the protein expression of stem cell markers (CD133, CD44and ALDH1), self-renewal associated transcription factors (Bmi1, Nanog and OCT4) and invasion associated transcription factors (Twist1and Snail1) in a dose-dependent manner. And FVTF not only significantly decreased the number of lung cancer spheres(P<0.05) and the size of spheres, but also suppressed the capability of forming secondary-passaged spheres and invasion of LCSCs in a dose-dependent manner (P<0.05).3. The result of western blot showed LY294002down-regulated the expression of p-Akt in a dose-dependent manner (P<0.05). Meanwhile, LY294002and FVTF synergistically down-regulated the expression of stem cell markers (CD133, CD44and ALDH1), self-renewal associated transcription factors(Bmil, Nanog and OCT4) and invasion associated transcription factors(Twistl and Snaill).4. LY294002suppressed sphere-forming capability in a dose-dependent manner(P<0.05). In addition, LY294002enhanced the inhibitory effects of FVTF on proliferation, sphere-forming and invasion in vitro of LCSCs.Conclusion:1. CD133+SFCs of NCI-H446cell line that were obtained by the comprehensive application of Magnetic activated cell sorting system(MACS) and suspended culture with serum-free stem cell-conditioned medium possess the characteristics of LCSCs.2. FVTF could inhibit the function and characteristics of LCSCs.3. The mechanism by which FVTF inhibits characteristics of LCSCs is associated with down-regulating expression of p-Akt in LCSCs, which lead to downregulation of the expressions of stem cell markers(CD133,CD44and ALDH1), self-renewal associated transcription factors(Bmil, Nanog and OCT4) and invasion associated transcription factors(Twistl and Snail1).