The purpose of this study was to explore the mechanism of Solanine disrupting energy metabolism in human renal cancer ACHN cells and to clarify its target. The specific method was to culture human renal cancer ACHN cell lines, and to intervene with Solanine of high, medium and low concentrations. The content of ATP in cells was measured by ELISA method. The expression of HIF-1α protein and the expression of PI3K, AKT, p-PI3K, p-AKT in PI3K/AKT pathway were detected by Western blotting. The results showed that compared with the control group, the relative expression of p-PI3K and p-AKT showed a downward trend with the increase of Solanine concentration (P 0.05). In addition, the relative expression of HIF-1α also showed a downward trend (P < 0.05). According to the above results, it is suggested that Solanine can significantly inhibit the energy metabolism of renal cancer cells, the main mechanism of which is the down-regulation of HI-1αf downstream of the PI3K/Akt pathway by inhibiting the phosphorylation process of PI3K/p-PI3K and Akt/p-Akt.
Objective
To observe the effects of Yiqi Jiedu prescription drug serum on the apoptosis of human renal cell carcinoma ACHN cells and Notch1 gene in Notch signal pathway.
Methods
The healthy SD female rats were selected, which were divided into 4 groups, 8 rats in each group: the rats were fed with the normal saline 10 ml·kg-1·d-1 (the blank serum group); the rats were fed with the Yiqi Jiedu prescription decoctum of 50 g·kg-1·d-1 (the high-concentration of Yiqi Jiedu prescription serum group); the rats were fed with the Yiqi Jiedu prescription decoctum of 25 g·kg-1·d-1 (the medium-concentration of Yiqi Jiedu prescription serum group); the rats were fed with the Yiqi Jiedu prescription decoctum of 12.5 g·kg-1·d-1 (the low-concentration medicated of Yiqi Jiedu prescription serum group); then the serum would be extracted 10 days later in each group. ACHN cells at exponential phase were cultured in the above 4 groups. The proliferation of ACHN cells in each group was observed by using CCK-8 method. The apoptosis of ACHN cells was detected by using flow cytometry (FCM). The expression levels of Notch1 mRNA of ACHN cells in each groups were detected by using real time fluorescence quantitative polymerase chain reaction (RT-PCR).
Results
The inhibition rates of ACHN cells in the high-concentration group, the medium-concentration group, the low-concentration group of Yiqi Jiedu prescription and the blank serum group 24 h later were (12.34±4.25)%, (7.47±1.40)%, (2.52±0.62)%, (1.05±0.31)%, respectively (F=15.04, P < 0.01); after 48 h, the inhibition rates were (24.20±2.41)%, (14.23±1.56)%, (5.08±1.34)%, (1.16±0.14)%, respectively (F=227.36, P < 0.01); after 72 h, the inhibition rates were (32.16±2.45)%, (25.05±3.69)%, (10.29±2.76)%, (1.07±0.71)%, respectively (F=110.51, P < 0.01). The results showed that Yiqi Jiedu prescription drug serum could significantly inhibit the proliferation of ACHN cells in a dose-dependent manner. Besides, the inhibition rate differences at all time points of the high-concentration serum group (F=31.44, P < 0.01), the medium-concentration serum group (F=49.61, P < 0.01) and the low-concentration serum group (F=68.78, P < 0.01) were all statistically significant, and they were in a time-dependent manner. The apoptosis rate of cells in the high-concentration group, the medium-concentration group, the low-concentration group of Yiqi Jiedu prescription and the control serum group was (34.5±1.5)%, (24.4±1.5)% and (13.1±0.5)%, (5.2±0.3)%, respectively, and the differences were statistically significant (F=1 153.36, P < 0.01). The relative expression level of Notch1 mRNA of cells in the high-concentration group, the medium-concentration group, the low-concentration serum group of Yiqi Jiedu prescription and the control serum group was 0.213±0.032, 0.432±0.049, 0.781±0.018, 1.013±0.047, respectively, and the differences were statistically significant (F=270.60, P < 0.01).
Conclusion
Yiqi Jiedu prescription drug serum can induce apoptosis of ACHN cells, and its mechanism may be related to the down-regulation of the expression level of Notch1 receptors.
Key words:
Kidney neoplasms; Yiqi Jiedu prescription; Apoptosis; Drug serum; Notch1 receptors
Objective
To isolate and identify stem-like cells (CSCs) from human ACHN renal cell carcinoma cell line.
Methods
ACHN cancer stem cells CSCs were enriched by serum-free culture condition. Methyl thiazolyl tetrazolium (MTT) method was performed to draw the growth curve. Flow cytometry was performed to evaluate the expressions of two CSCs markers, CD133 and CD44. Real-time polymerase chain reaction (RT-PCR) was performed to evaluate the expression of stem-related genes (Oct3/4, Bmi, Nanog, and β-catenin). The tumorigenicity in vivo was also examined.
Results
ACHN CSCs were formed by sphere-forming culturing. The data demonstrated that there were stronger capacities of proliferation in ACHN tumor spheres than that of ACHN cells. And the expressions of CD133 and CD44 were also significantly enhanced in tumor spheres. The data showed that four genes (Oct3/4, Bmi, Nanog, and β-catenin) were upregulated in sphere-forming cells. Sphere-forming cells had a higher tumorigenic potential in vivo than incubated cells.
Conclusions
Human ACHN renal cell carcinoma cell line sphere-forming cells can be enriched by serum-free culture condition and exhibits several characteristic cancer stem cells properties.
Key words:
Kidney neoplasms; Neoplastic stem cells
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