Anti-tumor Effects Of Iso-suillin From Suillus Flavus On The Human Lung Adenocarcinoma A549 Cells In Vitro And In Vivo

Objective: In recent years, with the environmental pollution, population aging and other factors, the global cancer patients and deaths continue to increase, and show a rapid growth trend year by year. In all the cancers, lung cancer is the most common and lethal malignant tumor, every year about 1.8million new cases of lung cancer occur all over the world, accounting for 13%of all cancer cases, and about 1.6 million people die of lung cancer,accounting for 20% of global cancer deaths. The five-year survival rate of lung cancer patients is less than 15%. In Chinese, the incidence and mortality of lung cancer have been ranked first, accounting for more than 1/3 of the globle lung cancers. At present, clinical effects is still in relatively low level,common chemotherapy drug efficiency is limited because of a series of side effects. Therefore, the searching or synthesizing more safe and effective antitumor drugs is still the urgent demand placed in front of the medical workers.Fungi are recorded in a long time ago as a drug. There are many fungal metabolites showing obvious anti-tumor, anti-oxidation, anti-infection,anti-inflammatory and analgesic effects. Some research data show that natural plants and fungi are rich in phenolic compounds, some of this show highly biological activity. Iso-suillin belongs to the prenylphenols, which is isolated from Suillus flavus, iso-suillin exerts strong biological activity compared with cisplatin and 5-fluorouracil, which can effectively inhibit the growth of tumor cells in vitro and induce cell cycle arrest and apoptosis, and no toxicity on normal human lymphocytes. The study is committed to further explore the anti-tumor mechanism of iso-suillin on human lung adenocarcinoma A549 cells in vitro and in vivo.Methods:1. Cell culture A549 cells were cultured in RPMI 1640 medium supplemented with 10%fetal bovine serum, penicillin 100 U/m L, and streptomycin 100 ?g/m L at37 ? in an incubator containing 5% CO₂.2. Cell viability detected by MTS assay The exponential growth phase of A549 cells were planted into 96-well multiplates and treated with series concentrations of iso-suillin, The cells were cultured for different time periods?6, 12, 24, 48, and 72 h?. 20 ?L MTS solution was added to each well. The cells were cultured for 1⁴ h, then the absorbance at 490 nm was measured using a microplate reader?Bio Tek, USA?.The 50% inhibitory concentration?IC50? was calculated.3. The distribution of cell cycle and apoptosis rate detected by flow cytometry?FCM?The cells were collected and washed one time in cold PBS, then stained using an Annexin V-FITC/PI kit according to the manufacturer's instruction.The cells were measured by flow cytometry and drew the histogram and calculate the percentage of G₀/G₁, S, G2/M phase and the apoptosis rate.4. Drug toxicity test of iso-suillin in nude mice Iso-suillin was administered subcutaneously on nude mice and observing the life state and body weight of nude mice for two weeks.5. Iso-suillin inhibits the proliferation of xenografte tumor The nude mice of transplanted tumor size with 90 110 mm3 were randomly divided into two groups, the control group adopt subcutaneous injection of saline and the treatment group use the subcutaneous injection administration adjacent to tumor every two days, then, observed two weeks and recorded for each mouse body weight and tumor size. The tumor volumes were calculated according to the following formula: tumor volume=length diameter×short diameter2/2.6. Embedding and slicing of transplanted tumor tissue The tumor tissue in nude mice fixed 48 h in 10% neutral formaldehyde,then, cutted into 3 mm thickness, conventional embedding and slicing, andH&E staining and observed under the microscope.7. The apoptosis station of tumor tissue detected by TUNEL assay The tissue sections were stained by TUNEL assay after dewaxing and observed under fluorescence microscope camera.8. Observation of morphologic changes A549 cells were seeded into 24-well plates and exposed to the indicated concentration of iso-suillin for 24 h. The cells were stained with DAPI.Cellular morphology was observed under a fluorescence microscope?Nikon,Tokyo, Japan?.9. Analysis of the mitochondrial membrane potential A549 cells in the logarithmic growth phase were treated with different concentrations of iso-suillin, then, observed under the fluorescence microscope after JC-1 staining.10. The related protein expression detected by Western Blot assay The cell total protein was abstracted from A549 cells. Protein concentration was determined using BCA assay kit. The relate proteins of DNA damage, cell cycle, apoptosis were detected by Western Blot assay.11. Statistical analyses The results were expressed as mean ± standard deviation and evaluated with SPSS 13.0 by analysis of variance?One-Way ANOVA?. P<0.05 was considered statistically significant.Results:1. The effect of iso-suillin on the proliferation activity of A549 cells A549 cells were treated with different concentrations of iso-suillin for 6, 12,24, 48, or 72 h, and MTS assay showed the IC50 values were 80.86, 39.40,11.56, 1.43, 0.43 ?M. The proliferation activity of A549 cells decreased in a concentration-dependent and the time-dependent manner.2. The effect of iso-suillin on the growth of transplanted tumor in nude mice There were no abnormalities in nude mice after subcutaneous injection of different concentrations of iso-suillin and no significant difference in the weight and diet of nude mice campared with the control. The rate of tumorformation of A549 cells was 100%, and the tumor volumes were about 100mm3 in the fourteenth days later after inoculation. A total of 12 mice with transplanted tumor volume between 90¹10 mm3 were randomly divided into control and experimental group, the treatment group were subcutaneous injected iso-suillin adjacent to the tumor, in the 1, 5, 9, 13 and 17 d, the nude mice body weight of control group were 16.98±0.35, 17.13±0.30, 17.16±0.54,18.29±0.55 and 19.18±1.21; the tumor volumes of control group were89.38±15.30, 125.68±8.83, 220.38±48.63, 311.16±93.67 and 600.91±133.0;the body weight of treatment were 17.12±0.36, 17.13±0.38, 17.58±0.61,18.46±1.15 and 19.62±0.83; the tumor volumes of treatment were93.06±17.79, 128.89±12.97, 205.70±34.75, 310.57±87.93 and 383.36±107.11.On the 17 day, the tumor volume had significant difference compared with the control group?P<0.05? and there was no significant difference in body weight of nude mice?P>0.05?.3. The effect of iso-suillin on cell cycle and apoptosis rate of A549 cells A549 cells were treated with different concentrations iso-suillin for 24 h The G₀/G₁ phase cells respectively are 44.8±2.17 %, 58.5±1.98 %,59.3±1.76 % and 55.1±2.62 %; S phase are 46.9±2.23 %, 28.1±1.74 %,27.8±3.02% and 31.7±2.38%; the G2/M phase are 8.3±3.12%, 13.4±2.56%,15.9±2.48% and 13.2±1.56%. The proportion of G₀/G₁ phase cells increased compared with control?P<0.05?. The early apoptosis rates are 3.16±0.08%,9.71±1.25%, 40.88±5.26% and 39.35±4.56%; the later apoptosis rates are0.65±0.09%, 3.07±1.32%, 14.83±1.25% and 18.43±2.35%, there are both significant in A549 cells compared with the control group?P<0.05?.4. The effect of iso-suillin on the morphology of A549 cells A549 cells were treated with different concentrations of iso-suillin for 24 h,cells in the control group were more, firmly adherent, full individual state, and showed fusiform or polygon, the cell membrane was integrity. The number of cells in experimental group was significantly decreased, the cells began to detach from the bottom wall and shrink into a round. Cell membrane bubbled,cell lysis into many apoptosic bodies scattered. After DAPI staining,iso-suillin induced cell morphological changes and cell numbers decreased.Cells began to shrink and became smaller, and showed nuclear fragmentation compared with control. The results indicated that iso-suillin has the ability to induce apoptosis and cell proliferate inhibition.5. The effect of iso-suillin on the morphology and apoptosis of A549 cells in nude mice transplanted tumor The tumor tissue sections were observed under the microscope after H&E staining. In the control group, the cell morphology was complete, the nucleus was clearly visible, and the color was uniform; in the treatment group, the cytoplasm of the transplanted tumor tissue was concentrated, the nucleus was contracted, the staining was deeper, the cell membrane was not complete, and the tumor necrosis was seen. Tissue sections were observed under the fluorescence microscope after TUNEL assay and DAPI staining, the green fluorescence of experiment group was very strong compared with control,indicating that there was DNA breaks in tumor tissue. The results showed that iso-suillin can inhibit cell proliferation and induce cell necrosis or apoptosis in nude mice transplanted tumor.6. The effect of iso-suillin on mitochondrial membrane potential A549 cells were treated with different concentrations of iso-suillin for 24 h,then the cells were stained with JC-1 and analyzed under the fluorescence microscope, according to the ratio of green and red fluorescence, the results showed that the decreasing of mitochondrial memberane potential was relationship with drug concentration.7. The effect of iso-suillin on A549 cell cycle, apoptosis and p53 signal pathway related proteins A549 cells were treated with different concentrations of iso-suillin for 24 h,the ratios of IOD between caspase-8 and ?-actin were 0.17±0.04, 0.21±0.04,0.30±0.05 and 0.45±0.07; the ratio of Caspase-9 were 0.14±0.03, 0.37±0.05,0.41±0.07 and 0.64±0.06; the ratio of pro-caspase-3 were 0.48±0.06,0.46±0.08, 0.44±0.05 and 0.38±0.06; the ratio of cleaved caspase-3 were0.02±0.01, 0.03±0.01, 0.08±0.02 and 0.15±0.04; the ratio of Bax were0.43±0.05, 0.57±0.03, 1.56±0.04 and 0.83±0.14; the ratio of Bcl-2 were0.16±0.03, 0.14±0.04, 0.09±0.02 and 0.07±0.02; the ratio of p27 were0.13±0.04, 0.42±0.09, 0.87±0.12 and 0.93±0.10; the ratio of cyclin D1 were1.42±0.11, 0.87±0.09, 0.43±0.04 and 0.08±0.03; the ratio of CDK4 were0.43±0.05, 0.37±0.06, 0.38±0.02 and 0.29±0.06; the ratio of E2F-1 were0.27±0.04, 0.24±0.03, 0.04±0.07 and 0.02±0.02; the ratio of ?-H2 AX were0.04±0.02, 0.08±0.03, 0.25±0.04 and 0.56±0.09; the ratio of p53?phosphor S20? were 0.02±0.01, 0.03±0.01, 0.09±0.04 and 0.20±0.08; the ratio of p53?phosphor S15? were 0.04±0.02, 0.09±0.04?0.16±0.05 ? 0.34±0.09; the ratio of wild p53 were 0.48±0.07, 0.35±0.08, 0.12±0.04 ? 0.03±0.01; the results showed that cleaved caspase-3,-8,-9, Bax, p27, ?-H2 AX, p53?phospho S20?, p53?phospho S15?protein expression was up-regulated;Bcl-2, E2F-1, p53, cyclin D1 protein expression was down-regulated. There was significance between experimental and control group?P<0.05?.Conclusions:1 Iso-suillin can significantly suppress A549 cells proliferation and viability and induce cell apoptosis in vitro.2 Iso-suillin can suppress the growth of nude mice xenograft tumors and induce tumor apoptosis, but has no toxicity on nude mice.3 Iso-suillin can induce G₀/G₁ cell cycle arrest in A549 cells.4 Iso-suillin can induce cell DNA damage in A549 cells.5 Iso-suillin may induce G₀/G₁ cell cycle arrest and apoptosis by p53-dependent pathway.