Mechanism of modulation through PI3K-AKT pathway about Nepeta cataria L.'s extract in non-small cell lung cancer

Abstract

Non-small cell lung cancer (NSCLC) is regarded as one of the major intractable diseases, which was cured mainly by chemotherapeutics in the clinical treatment at present. But it is still a vital mission for the current medical and researchers that hunting a natural medicine which have little side effects and high-efficiency against the NSCLC on account of the shortcomings on current drugs. Nepeta cataria L. plays an important role in anti-cancer treatment according to the reports which was recorded in the Chinese Pharmacopoeia of version 2015 and belongs to one of the Traditional Chinese medicine (TCM). Microfluidic chip technology is widely used in scientific research field due to its high-throughput, high sensitivity and low cost with the continuous progress of science and technology. In this study, we investigate the effect of total flavonoid extracted from Nepeta cataria L. (TFS) through human lung cancer cell line A549 based on the microfluidic device and Flow Cytometry. So we detected the mRNA expression of MicroRNA-126 (miR-126), VEGF, PI3K, PTEN and proteins expression respectively to explore the partial PI3K-AKT pathway molecular mechanisms through Quantitative Real-time PCR (qRT-PCR) and Western Blot. The results showed that TFS can disturb the expression of miR-126 and regulate the PI3K-AKT signaling pathway to meet the effect of anti-cancer. Taking all these results into consideration we can draw a conclusion that TFS may be used as a novel therapeutic agent for NSCLC in the near future.

Keywords: A549 cell; MicroRNA-126; PI3K-AKT signaling pathway; inhibition of proliferation; non-small cell lung cancer.

Figure 1. The results of cell viability test (×40)

AO can across the intact cell membrane in order to combined with DNA emits intense green fluorescence; EB can across the broken cell membrane in order to combined with DNA emits intense orange-red fluorescence. All the cells were count by the Imageproplus (IPP, version 6.0) for calculating the Cell survival rate (%). The Cell survival rate (%) = (normal cells / the total number of A549 cells) ×100%.

Figure 2. The effect of TFS on the proliferation of A549 cells

The Hochest33342 and PI staining Assay used to detect the effect on the proliferation of A549 Cells. The apoptosis and necrosis rate was display in Figure 3A, *P<0.01vs CG. CG represents control group; PG represents positive group; LFG represents low total flavonoid extracted from Nepeta cataria L. group; MFG represents middle total flavonoid extracted from Nepeta cataria L. group; HFG represents high total flavonoid extracted from Nepeta cataria L. group; From the Figure 3B, we can see the variation tendency of the Apoptosis and necrosis rate.

Figure 3. The expression of miR-126 in human lung cancer cell line A549 (mean+/−SEM, n=3)

*P<0.01vs CG, P<0.05 as significant difference.

Figure 4. The pro-apoptotic effect of TFS on lung cancer cells (mean+/−SEM, n=3)

Cell apoptosis profiles by Annexin V-FITC/PI double fluorescent assay of A549 cells through flow cytometry. In Figure 5A, it is the histogram of Apoptosis and necrosis rate in each group. They all treated with TFS and Cisplatin in different concentrations for 36 h. In Figure 5B, I represents CG, II represents LFG, III represents MFG, IV represents HFG, V represents PG. *compared with CG, P<0.05 as significant difference.

Figure 5. The quantitative real-time polymerase chain reaction in A549 cells

The mRNA expressions of VEGF and PI3K was in the Figure 6A, The PTEN mRNA expressions was in Figure 6B, from the picture, we can see that VEGF and PI3K wassignificantly decreased while the PTEN was increased in admintration groups compared with CG. *compared with CG, P<0.05 as significant difference.

Figure 6. The protein expressions in PTEN/PI3K/AKT signaling pathway

CG represents control group; PG represents positive group; LFG represents low total flavonoid extracted from Nepeta cataria L. group; MFG represents middle total flavonoid extracted from Nepeta cataria L. group; HFG represents high total flavonoid extracted from Nepeta cataria L. group; *compared with CG, P<0.05 as significant difference.

Figure 7. The signaling pathways of TFS anti-NSCLC cancer

Different color shows different meanings: Red represents this protein upregulated, Green represents this protein downregulated, Yellow represents the proteins function.

Figure 8. The design of a cell culture microfluidic chip

(A) The schematic design of the microfluidic chip with VCL and cell culture chambers. The channel of blue was the VCL including GLs and LLs, the red channels was FCL, the function of the ellipses was for cells growth. (B) The pictorial diagram of the chip.