Study About The Mechanism And Effect Of Hematoxylin, Chuanxiong On Lung Cancer Stem Like Cells In Vivo

BackgroundLung cancer is the most common cause of cancer death.In the past 10 years,the 5-year survival rate of lung cancer remains around 15%,because of drug resistance,relapse and metastasis of cancer. The reason of the phenomenon is that cancer stem cells (CSCs) closely related to tumor occurrence, development, transfer and resistance.At present,more and more experimental researchs have showed that tumor recurrence and metastasis has close relationship with the existence of cancer stem cells.Cancer stem cells whitch can effectively prevent cancer recurrence and metastasis, has been confirmed in many basic experiment,as therapeutic targets.Blood stasis syndrome is one of common syndromes in TCM, and it also occupied the main position in solid tumor such as lung cancer.Previous experiments have confirmed that the tumor patients with blood stasis is prevalence, its severity can be confirmed by the expression of adhesion molecules on he surface of cancer patients’platelets. In our previous study,we have confirmed blood activating herbs such as Chuanxiong, hematoxylin, Millettia and leech can inhibit the metastasisof lewis lung cancer. The results show that Chuangxiong and hematoxylin have more obvious inhibition in cancer metastasis. While in vivo, hematoxylin, Millettia and combination with cisplatin can arreste the tumor cells in G0/G1 phase, and may inhibit the expression of protein PCNA, HIF-laassociated with cell proliferation.In addition,they have effect on P16-Cyclin D1-CDK4-RB pathway.This study based on previous studies and combined with the CSCs theory will observe the effects of different doses of blood medicine hematoxylin, Chuanxiong on lung cancer stem-like cells and its mechanisms in vivo.Objective1.Separate the PG-BE1 stem like cells by Serum-free culture.Identify the PG-BE1 sphere cells’proliferation, anti-apoptosis, expression of cancer stem cell marker of tumor stem-like cells and other properties.2. Establish animal models with PG-BE1 stem like cells and observe the effects of different doses of hematoxylin, Chuanxiong on PG-BE1 stem-like cells in vivo by the expression of ABCG2 proteins.3. Explore the mechanisms of blood medicine for PG-BE1 stem like cells in vivo, which may be improved hypoxic microenvironment, inhibition of EMT of PG-BE1 stem like cells,by detecting the expression of HIF-1α, EMT-related marker proteins in vivo.Methods1. Separate the PG-BE1 sphere cells from PG-BE1 cells by Serum-free culture.Identify the PG-BE1 sphere cells biological behavior by flow cytometry, apoptosis detection, CCK-8 and western blot.2. Establish animal models with PG-BE1 stem like cells and detect the expression of ABCG2 protein treated with different doses of hematoxylin, Chuanxiong in vivo by laser scanning confocal, western blot, RT-PCR.3.Detect the expression of HIF-1 a protein, EMT-related marker proteins treated with different doses of hematoxylin, Chuanxiong in vivo by immunohistochemistry, western blot, RT-PCR.Results1.PG-BE1 cells cultured in serum-free culture began to form a small ball of cells in the third day,and the surface of cell ball was dark, the refractive index reduced until the sixth or seventh day.The third generation of PG-BE1 sphere cells were more regular. CCK-8 experiments suggest PG-BE1 sphere cells’OD value of the 3,4,5,6,7 days were significantly higher than PG-BE1 cells with statistically significant difference between them, P<0.01. PG-BE1 sphere cells and PG-BE1 cells’apoptosis rate was 0.47%,3.54% in early apoptosis,1.99%、3.12% in late apoptosis,and 2.46%、6.68% in total apoptosis, respectively.The apoptosis rate of PG-BE1 sphere cells were lower than PG-BE1 cells in in early apoptosis and total apoptosis, both with significant difference,P<0.05. Flow cytometry found that the proportion of expression of CD44+/CD24-markers in PG-BE1 sphere cells was 99.02%. Immunofluorescence suggested that the expression of ABCG2 protein was higher in PG-BE1 sphere cells,while the ABCG2 protein was low or even no expression in PG-BEl cells. Western blot suggested the expression of ABCG2,Oct-4, Nanog, SOX-2 protein in the PG-BEl sphere cells were significantly higher than PG-BEl cells,with significant differences,P<0.05 in Nanog group, ABCG2 group,and P<0.01 in Oct-4, SOX-2 two groups.2.The scanning confocal suggested Chuanxiong with different concentrations and DDP alone could inhibit the expression of ABCG2 protein, hematoxylin, Chuanxiong with DDP could enhance the inhibition of ABCG2 protein,especially in hematoxylin+ DDP group.Western blot found that low doses of Chuanxiong could inhibit the expression of ABCG2 protein significantly, there was a significant statistical difference compared with control,P<0.05. DDP alone could inhibit the expression of ABCG2 protein, with no statistically different campared with control, P> 0.05.Low dose Chuanxiong+DDP group had significantly inhibited the expression of ABCG2 protein, but compared to the single low-dose Chuanxiong, there were no statistically different, P> 0.05. RT-PCR showed low dose of Chuanxiong significantly decreased the expression of ABCG2 mRNA, with significant statistical difference, P<0.05; low dose of Chuanxiong with DDP had no use in inhibition expression of ABCG2 mRNA.3.Immunohistochemistry showed all the groups could inhibit the expression of HIF-la,except the low-dose hematoxylin,with statistically significant difference, P <0.05. DDP alone significantly inhibited the expression of HIF-la, which could enhance the inhibition with low-dose hematoxylin, Chuanxiong used in combination,compared with the single low-dose hematoxylin, Chuanxiong,with significant difference, P<0.05. Western blot found that low-dose Chuanxiong could inhibit the expression of HIF-la compared with control, with significant difference, P <0.05, and DDP alone could inhibit the expression of HIF-la, while there was no difference between Chuanxiong and Chuanxiong with DDP.RT-PCR testing found that low doses of hematoxylin, Chuanxiong could inhibit the expression of HIF-lamRNA compared with control group,with statistically different, P<0.05.4. Immunohistochemical found that all of the E-cadherin protein expression showed a promoting effect with statistically significant difference,P<0.05,in addition to low-dose hematoxyl. DDP could significantly promote the E-cadherin protein expression with significant difference,P<0.05. But hematoxylin, Chuanxiong with DDP did not significantly facilitate the expression of E-cadherin protein.High and low dose of hematoxylin did not inhibit expression of N-cadherin protein,the rest of them expressed N-cadherin protein lower than the control group, P<0.05.DDP could reduce the expression of N-cadherin, and combine with hematoxylin, Chuanxiong could enhance the inhibition of the expression of N-cadherin protein with significant difference,P<0.05.Low dose Chuanxiong inhibited the expression of Vimentin protein with significantly different, P<0.05. DDP alone could inhibit the expression of Vimentin, but there was no difference,P>0.05.DDP combination with hematoxylin and Chuanxiong did not significantly enhance its inhibition. Western blot showed the groups were able to promote the expression of E-cadherin protein, in addition to the low-dose group hematoxylin and DDP. Low-dose blood medicine with DDP promoted the expression of E-cadherin protein significantly with statistically difference, P<0.05. Except hematoxylin group, other groups could inhibit the expression of N-cadherin protein, with significant difference,P<0.05.DDP combination with high dose of hematoxylin and Chuanxiong inhited the expression of N-cadherin with significantdifference,P<0.05.Low dose blood medicine could inhibit the expression of Vimentin, with significant difference,P<0.05.DDP expressed Vimentin protein did not have any intervention, while low-dose Chuanxiong + DDP group significantly inhibited the expression of Vimentin protein,compared with control,P<0.05 and compared with low dose Chuanxiong,P>0.05. Chuanxiong did can inhibit the expression of Snail transcription factor, P<0.05. DDP alone had no effect on expression of Snail,and DDP combination with hematoxylin, Chuanxiong could not improve the expression of Snail. RT-PCR showed low-dose Chuanxiong could decrease the level of N-cadherin, Vimentin, Snail mRNA, having astatistically significant difference compared with the control group,P<0.05. DDP had no significant effect on N-cadherin, Vimentin, Snail mRNA expression.hematoxylin, and Chuanxiong combination with DDP did not promote the mRNA expression levels of the three, with significant difference,compared with the control group, P<0.05.Conclusion1. The PG-BE1 sphere cells obtained from serum free culture has strong proliferation, anti-apoptosis ability, high expression of ABCG2, Nanog, Oct4, SOX2 tumor markers.These abilities prove that PG-BE1 sphere cells have cancer stem cells’ biological behaviors.2. Low doses of Chuanxiong could inhibite the growing of PG-BE1 stem like cells.And combination with DDP, its inhibition does not increase. low dose of Chuanxiong inhibition expression of ABCG2 protein maybe related to inhibition of ABCG2 mRNA. Therefore we believe that the killing effect of low doses of Chuanxiong on PG-BE1stem like cells may be reached through inhibition expression of ABCG2 mRNA.3.Low doses of Chuanxiong can inhibit the expression of HIF-1α protein, and has no significant effect on mRNA expression levels. Low dose of Chuanxiong inhibiting PG-BE1 stem cell like cells in vitro maybe relate to inhibition expression of HIF-1α protein.4. Low dose of Chuanxiong possibly inhibite the expression of E-cadherin protein, N-cadherin protein, Vimentin protein, Snail by inhibiting the mRNA expression of EMT-related markers.And achieve the aim to inhibition the occurrence of EMT,to inhibit proliferation of cancer stem like cells.