| Objectives To establish the pemetrexed-resistant human lung adenocarcinoma cancer cell line A549/PEM with repeatedly exposing lung adenocarcinoma cancer cell line A549 in high-level concentration of pemetrexed. To research the expression and significance of PTEN, m TOR and AICARFT in the human lung adenocarcinoma cancer cell line A549 pemetrexed resistance.Methods 1 Established the pemetrexed-resistant human lung adenocarcinoma cancer cell line A549/PEM by repeatedly exposing lung adenocarcinoma cancer cell line A549 with high-level concentration of pemetrexed. Firstly, applied different concentration of pemetrexed to A549, MTT method was used to make sure of the appropriate drug concentration of pemetrexed to induce A549/PEM. According to the inhibition rate and cell morphology change after exposed to the pemetrexed with different intervention concentration to choose the concentration of pemetrexed. After high concentrations of pemetrexed intervention, the procesures included repeatedly frozen storaging,recoverying, observing the stability and detecting the resistance of A549/PEM by MTT method. Repeated the reduction culture of A549/PEM aimed to to achieve the required resistance index, named A549/PEM. 2 Observed the cell morphology of A549 and A549/PEM with themicroscope. Drawed cell growth curve and calculated double erythroplasia time of tumor cell by MTT. The distribution of A549 and A549/PEM cell cycle was evaluated by flow cytometry assay. 3 Detected the resistance of A549/PEM for DDP(cisplatin), taxol(paclitaxel), VP- 16(etoposide), VCR(vincristine) by MTT.Extracted the m RNA and proteins of A549 and A549/PEM. The m RNA and protein expressions of PTEN, m TOR and AICARFT in A549 and A549/PEM were detected by RT-PCR and Western Blot method.Results 1 According to the inhibition rate and cell morphology change of A549 after exposure to the drug, selected the final concentration of pemetrexd is 500 ng/ml.Undergone pemetrexed repeated and intermittent exposure for 4 months, successfully established pemetrexed drug-resistant cell line A549/PEM with human lung adenocarcinoma A549. The value of resisitant index of A549/PEM was elevated less than27 times, the number was(26.87±1.81). 2 The A549/PEM cell line is much more irregular than the A549 in cell morphology. The grow rate of A549 and A549/PEM is close, doubling time is(63.26 ± 0.65)h and(62.45±0.50)h(P = 0.162). 3 The ratio of A549 is(57.71±0.77)% in G1 phase, A549/PEM is(56.74±0.47)% in G1 phase(P=0.137). A549 is(12.22±2.08)% in G2/M phase, A549/PEM is(7.93±1.80)% in G2/M phase(P=0.55). A549 and A549/PEM in phase G1 and G2/M phase cells ratio had no obviously changed(P>0.05). Flow cytometry analysis show S arrest in A549/PEM was higher than that of A549((35.32±2.21)%VS(30.06±1.61)%(P=0.029). The same as resistantance of A549/PEM were showed to DDP(cisplatin) and VP-16(eoposide), RI is(1.93±0.02) and(8.26±1.55); As for sensitivity to taxol(paclitaxel) and VCR(vincristine), RI is(0.89 ±0.10) and(0.95 ±0.11). There was no statistically significant difference of A549 and A549/PEM(P>0.05). 5 Over-expression of m RNA of PTEN,m TOR and AICARFT in A549/PEM were detected by RT-PCR(P<0.01). The relative expression quantity of m RNA of PTEN, m TOR, AICARFT in A549 were 0.99±0.01,0.99±0.02, 0.99±0.02, versus A549 were 2.24±0.16, 2.18±0.04, 2.14±0.04. 6 Overexpression protein of PTEN, m TOR and AICARFT in A549/PEM were detected by Westren-blot(P<0.01). The grey value of PTEN, m TOR, AICARFT of A549 were396.76±28.66, 404.94±53.47, 412.21±96.48 and relative expression(GAPDH) is0.29±0.02, 0.30±0.04, 0.31±0.07, while A549/PEM were 815.43±73.92, 861.40±169.52,894.18±128.73 and relative expression(GAPDH) is 0.65±0.32, 0.71±0.24, 0.72±0.20.Conclusions 1 Established successfully the pemetrexed-resistant human lung adenocarcinoma cancer cell line A549/PEM by repeatedly exposing lung adenocarcinoma cancer cell line A549 to high-level concentration of pemetrexed. 2A549/PEM is also has the resistance to DDP(cisplatin) and VP-16(eoposide) while has the sensitivity to taxol(paclitaxel) and VCR(vincristine), provided reference for the drug choice after pemetrexed resistant. 3 Acquired pemetrexed drug resistance in lung adenocarcinoma appear over-express of the PTEN, m TOR and AICARFT. |
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