Anti-neoplastic Activity Of Metformin In Lung Adenocarcinoma And The Mechanisms

Lung cancer is the most common cancer in the world both in terms of incidence and mortality of the total. Lung adenocarcinoma is a common histopathologic type and the percentage of patient with lung adenocarcinma is 30% of all the patients with lung cancer. In most adenocarcinoma patients the tumors grows close to pleura. Though the proliferation rate is not quite high, adenocarcinoma is very angio- and lymph-invasive. Most of the lung adenocarcinoma patients are in the late stage of the disease when first diagnosed and surgical resection is not applicable. Although combined-modality therapy with radiotherapy and chemotherapy has been applied, more than 85% of the patients die within 5 years. New therapeutic modalities are therefore necessary to improve the response to treatment.The biguanide metformin is one of the mostly used drugs for treatment of type 2 diabetes. By reducing hepatic gluconeogebesis and increasing glucose uptake in skeletal muscles, metformin can lower blood glucose levels while inducing no risk of hypoglycemia and very rarely causes lactic acidosis. Recent clinical studies have revealed that metformin treatment is associated with reduced cancer risk and improved prognosis. Evans et al did a pilot observational study reporting that among patients with type 2 diabetes, those treated with metformin have a lower incidence of cancer compared with those untreated. In an independent study, using administrative databases of a population-based cohort, Bowker et al reported that type 2 diabetes patients who use metformin have a lower cancer-related mortality compared with those that use sulfonylureas or insulin. There is also growing experimental evidence that metformin may impede the growth of human tumors, including prostate cancer, breast carcinoma, colon carcinoma, ovarian cancer, et al. However, until now, the relation between lung adenocarcinoma and metformin has not been studied and the precise anti-tumor mechanisms of the anti-neoplastic effects of metformin are not completely understood.In the present study, we evaluated the effects of metformin on the growth of lung adenocarcinoma in vitro and in vivo, and its relationship to AMPK activation, JNK/p38 MAPK--Caspase signaling pathways activation and growth arrest and DNA damage -inducible transcription factor (GADD) 153. Our results open new therapeutic possibilities for the use of metformin as an anti-neoplasm agent. Four parts are included in this study:Part one Effects of metformin on proliferation in lung adenocarcinoma cell lines A549 and NCI-H1299 in vitroObjective To study the effects of metformin on cell viability, cell cycle, apoptosis and the synergism with cispatin in lung adenocarcinoma cell lines A549 and NCI-H1299 in vitro. Methods and materials Cells were treated with metformin (0, 0.5, 2 or 8 mM) in vitro together with cisplatin (10ug/mL) or not. Cell growth was determined using Methylthiazolyldiphenyl-tetrazolium bromide (MTT) following the manufacturer's protocol at the time of 0, 24, 48 and 72 hours after treatment. After treatment with metformin for 48 hours, apoptosis and cell cycle of A549 cells were exemined by Flow cytometry. Apoptotic cells were analyzed by double staining withfluoresceinisothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI) . Cell cycle was analyzed by measuring the amount of PI-labeled DNA in ethanol-fixed cells. Results The cell viability started to decrease as early as 24 hours later after metformin treatment. Metformin induced significant proliferation inhibition on both A549 and NCI-H1299 cell lines in a dose- and time-dependent manner. When applied in combination with metformin, the cytotoxicity of cisplatin on both cells was stronger. After treatment for 48 hours, metformin at 2 and 8mM caused significant increases both in numbers of apoptotic (annexin+PI–) A549 cells and G1 phases fraction (P<0.05). While the apoptotic rate and cell-cycle progression of A549 cells treated with 0.5mM metformin did not change much compared to those of the control cells. Conclusions Metformin can inhibit the growth of lung adenocarcinoma cell lines significantly and potentiate cisplatin in a dose- and time-dependent manner in vitro. Cell-cycle progression is arrested in G1 stage and apoptosis is indued after metformin treatment.Part two Role of JNK/p38 MAPK--Caspase signaling pathways in metformin-induced apoptosis of lung adenocarcinoma cell line A549Objective To detect the effects of metformin on the phosphorylation of AMPK, JNK and p38 MAPK and the expression of cleaved Caspase-3, 8, 9 proteins in A549 cells, and investigate the correlation between JNK/p38 MAPK--Caspase signaling pathways and cell apoptosis. Methods and materials A549 cells were collected and lysed for total protein after treated with metformin (5 mM) for 0, 15, 30 and 60 minutes. Western blot analysis was performed to detect the expressions of pan- and pho- AMPK, JNK and p38 proteins. Cleaved Caspase-3, 8, 9 proteins were also detected. SP600125 and SB202190, the specific inhibitor for JNK and p38 MAPK signaling pathways respectively, were chosen to treat cells prior to metformin treatment and apoptotic rates of A549 cells were then analyzed by Flow cytometry.Results The expressions of pho-AMPK, pho-JNK and pho-p38 were detected after 15 min of metformin treatment. The levels of cleaved Caspase-8, 9 proteins elevated after 12 h of metformin treatment. After treatment for 30 min, the expression level of cleaved Caspase-3 protein also elevated. After pretreated with SP600125 or SB202190, the metformin-induced apoptotic rates of A549 cells significantly decreased (P<0.05), though still higher than those of the cells treated with SP600125 or SB202190 only.Conclusions JNK/p38 MAPK--Caspase signaling pathways are involved in metformin-induced apoptosis in lung adenocarcinoma cell line A549.Part three GADD153 is involved in metformin-induced A549 cell apoptosisObjective To detect the effects of metformin on the expression of GADD153 mRNA and protein in A549 cells, and to investigate the role of GADD153 gene in metformin-induced A549 cell apoptosis.Methods and materials A549 cells were treated with metformin (5mM) for 0, 12, 24, 48 and 72 hours. The expression of GADD153 mRNA in A549 cells was detected by Realtime-PCR. GADD153 siRNA (50nM) or negative control siRNA (50nM) was transient transfected into human lung adenocarcinoma cell line A549 cells by INTERFERinTM siRNA transfection reagent. At 48h after transfection, cells were collected and the total RNA and protein were extracted. Realtime-PCR and western blot were performed to detect the level of GADD153 mRNA and protein respectively to assess the efficiency of GADD153 gene silencing. A549 cells were then divided into four groups: Negative control siRNA (siNEG) group, GADD153 siRNA (siGADD153) group, siNEG+metformin group and siGADD153+metformin group. The cells of the last two groups were treated with metformin (5mM) at 10h after siRNA transfection. PBS was used instead of metformin to the cells of siNEG group and siGADD153 group. After siRNA transfection and metformin or PBS treatment for another 48 hours, cell apoptotic rate of every group was detected by Flow cytometry.Results Realtime-PCR analysis showed that the expression of GADD153 mRNA in A549 cells increased significantly at 12h after metformin treatment (P<0.01) and kept increasing to a level as high as 14.12-fold of the original level after treatment for 72h. Realtime-PCR and western blot analysis showed that both GADD153 mRNA and protein expression were significantly decreased after transient transfected with specific GADD153 siRNA. The cell apoptitic rate of siGADD153+metformin group was significantly lower than that of siNEG+metformin group (P<0.05), though no significant difference was detected between siGADD153 group and siNEG group (P=0.591).Conclusions The apoptosis-inducing effect of metformin in lung adenocarcinoma cells is partly due to the up-regulation of GADD153 mRNA and protein in the cells under metformin treatment.Part four Repressive effects of metformin on the growth of xenograft of lung adenocarcinoma in nude miceObjective To study the suppressive effect of metformin on the growth of lung adenocarcinoma xenograft in nude mice and its security and to evaluate the potential value of metformin as a novel anti-neoclassic agent for the treatment of lung adenocarcinoma.Methods and materials A549 cells were harvested, washed twice in PBS, and resuspended in 200ul PBS before being inoculated subcutaneouly in 4- to 6-week-old females BALB/c nude mice on the right flank (2×107 cells per site). Treatment with metformin was started when the average tumor volumes reached 100mm3. Five mice were used per group. Metformin (200 mg or 40 mg×kg body weight) was dissolved in 200ul PBS and administered daily with i.p. injections. The control group received vehicle only (200ul PBS). Animal weight and tumor volume (mm3) were measured every 4 days and estimated from caliper measurements using the formulaπ/6×A×B2 (A is larger diameter, B is smaller diameter). After treatment for a month, the mice were all sacrified and paraffin sections of tumors were used for immunohistochemical analyses with hemotoxylin and eosin stain and proliferation with Ki-67 and apoptosis with TUNEL staining.Results No severe adverse effect was observed among the xenograft mice during metformin treatment. The growth of lung adenocarcinoma xenograft of met40 group and met200 group was significantly suppressed compared with that of the control group at every point of the treatment course. When the treatment ended after a month, the mice of met200 group had the smallest average tumor volume among the three groups, followed by those of met40 group. Compared with control group, the inhibition ratio of metformin at the concentration of 40 mg/kg/d and 200 mg/kg/d to the growth of tumor was 19.81%±6.25% and 40.70%±8.98%, respectively. The average positive rate of Ki-67 in tumors of the control, met40 and met200 group was 58.18%±12.83%, 38.80%±10.41% and 32.85%±10.14%, respectively. The average number of positive cells per HPF in tumors of the control, met40 and met200 group was 3.1±1.8, 13.6±3.41 and 16.35±3.07, respectively. Both the positive rate of Ki-67 and number of apoptotic cells in met40 and met200 group were significantly higher than those of the control group (P<0.01). Conclusions Metformin can inhibit the growth of lung adenocarcinoma xenograft in nude mice by attenuating the expression of the proliferation marker Ki-67 and inducing tumor cell apoptosis without any severe adverse effect.In summary, we found from this study that metformin can inhibit the proliferation of lung adenocarcinoma cell lines and potentiate cisplatin in a dose- and time-dependent manner in vitro. Both JNK/p38 MAPK--Caspase signaling pathways and GADD153 gene are involved in metformin-induced apoptosis. When applied to the mice with xenograft tumor, metformin can markedly inhibit the growth of the tumor while causing no severe adverse effect. Our results indicate that metformin has anti-neoplastic activity and may be used as an adjunctive agent for the treatment of lung adenocarcinoma. The detailed mechanisms of the anti-neoplastic activity of metformin should be further studied.