Study On Mechanisms Of β-Elemene And/or X-irradiation Against Lung Carcinoma

Lung cancer is a malignant tumor with the highest incidence and mortality in the world. There are over one million deaths due to malignant tumor of the lung and 1.2 million new cases of lung cancer occur worldwide annually, making it a main killer to human life. Surgical resection is the most successful option for cure for patients diagnosed as having lung cancer, whereas patients with more advanced disease have lost the opportunity for surgical treatment. Radiotherapy and chemotherapy remain the main treatment for most patients with lung cancer. Although therapies for lung cancer have improved mostly, the 5-year survival from this disease is only 17%. Obviously, developments of new medicine and new strategies to improve survival of patients with lung cancer are urgently needed.Elemene, a natural sesquiterpene extracted from the traditional Chinese medical herb Curcuma Wenyujin, is a mixture ofβ-,γ- andδ-elemene withβ-elemene as the main component, accounting for 60-72% of the three isoforms.This new anticancer drug presents many advantages, such as broad-spectrum, fewer and differential side effects and not easily directing multidrug resistance. The anti-tumor effect ofβ-elemene has been proved in treatment of carcinomas of lung, alimentary canal, brain and other superficial tumor. Apoptosis is the most studied program that plays an essential role in the antitumor activity ofβ-elemene and several studies have indicated thatβ-elemene can enhance radiosensitivity in several cancer cells. However, the precise molecular mechanisms by whichβ-elemene induces apoptosis and produces radiosensitization are not completely understood, needs further explored.In the present study, we investigeted the effects ofβ-elemene in combination with X-irradiation on DNA damage/repair and telomerase activity in lung adenocarcinoma cell line A549 and lung squamous carcinoma cell line H520. Meanwhile, we further explored the cellular mechanisms of apoptosis induced byβ-elemene in lung adenocarcinoma cell line A549. Thus it may provide some information for clinical application ofβ-elemene.Objective:1. Alkaline comet assay and neutral comet assay were taken to assess the effects ofβ-elemene in combination with X-irradiation on DNA damage and DNA repair in A549 cells and H520 cells.2. TRAP-PCR-silver assay was taken to assess the effects ofβ-elemene in combination with X-irradiation on telomarase activity in A549 cells and H520 cells.3. We studied the apoptosis induced byβ-elemene in A549 cells and explored the effects ofβ-elemene on lysosomal membrane permeabilization,mitochondrial membrane potential and oxidative stress in A549 cells. The overall object of the present study is to further explore the cellular mechanisms of apoptosis induced byβ-elemene in lung adenocarcinoma cell line A549.Methods:Part one:The effects ofβ-elemene and X-irradiation on DNA damage and DNA repair in lung cancer cell lines.1. Experimental groupings: A549 and H520 cells cultured in 25 cm2 flasks were divided into four groups: (1) Control: received RPMI 1640 medium. (2)β-Elemene: treated with different concentrations ofβ-elemene (final concentration: 5, 10 and 20μg/ml) for 24h. (3) Irradiation: irradiated using a linear accelerator 6MV-X. Radiation dose: 4 Gy at a rate of 3Gy min-1, the distance between X-ray source and specimen (SSD): 100. (4)β-Elemene and irradiation: after incubation withβ-elemene for 24h, the exponentially growing cells were irradiated with 4Gy X-irradiation.2. The alkaline comet assay and neutral comet assay were performed immediately after irradiation to assess DNA single strand breaks (SSB), double strand breaks (DSB) induced byβ-elemene and irradiation in A549 and H520 cells.3. The alkaline comet assay and neutral comet assay were performed at 2h, 6h and 24h postirradiation to assess the repair of SSB and DSB induced byβ-elemene and irradiation in A549 and H520 cells.Part two: The effects ofβ-elemene and X-irradiation on telomarase activity in lung cancer cell lines.1. Experimental groupings: A549 and H520 cells cultured in 25 cm2 flasks were divided into four groups: (1) Control: received RPMI 1640 medium. (2)β-Elemene: treated with different concentrations ofβ-elemene (final concentration: 20μg/ml) for 24h. (3) Irradiation: irradiated using a linear accelerator 6MV-X. Radiation dose: 4 Gy at a rate of 3Gy min-1, the distance between X-ray source and specimen (SSD): 100. (4)β-Elemene and irradiation: after incubation with 20μg/mlβ-elemene for 24h, the exponentially growing cells were irradiated with 4Gy X-irradiation.2. The TRAP-PCR-silver assay was performed at 24h postirradiation to assess telomarase activity induced byβ-elemene and irradiation in A549 and H520 cells.Part three: The cellular mechanisms of apoptosis induced byβ-elemene in A549 cells1. The MTT assay was performed to evaluate the inhibitory rate induced byβ-elemene in A549 and H520 cells.2. A549 cells were treated with various concentrations ofβ-elemene (final concentration: 5, 10 and 20μg/ml) for as long as 36h (12h, 24h and 36h). DNA damage was assessed by comet assay. Apoptosis was assessed by examining cell morphology, flow cytometry and caspase-3 activation.3. Lysosomal membrane permeabilization induce byβ-elemene at different times was determined by staining with Aericine Orange (AO) using a fluorescence spectrophotometer and a fluorescence microscope. Cathepsin D expression in cytosolic was assessed by Western bolts analysis to validate the lysosomal membrane damage. Mitochondrial membrane potential induce byβ-elemene at different times was determined by staining with Rhodamine 123 using a fluorescence spectrophotometer.4. We used the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) to monitor the levels of reactive oxygen species (ROS) and glutathione (GSH) to elucidate the oxidative stress induced byβ-elemene.5. We investigated whether pepstatin A, an inhibitor of cathepsin D, inhibited DNA damage, mitochondrial membrane permeabilization and apoptosis followingβ-elemene exposure. It is possible to infer that cathepsin D may play an important role in the induction of apoptosis byβ-elemene exposure.Results:Part one:1.β-Elemene alone could cause SSB and DSB in A549 and H520 cells, and the degrees of SSB and DSB were augmented as the concentrations ofβ-elemene increased (P<0.05).2. The alkaline comet assay and neutral comet assay were performed immediately after irradiation. A549 and H520 cells exposed toβ-elemene and then to irradiation showed a higher number of DNA strand breaks for SSB and DSB than those exposed to irradiation alone orβ-elemene alone (P<0.01), indicating thatβ-elemene in combination with X-irradiation can enhance DNA damage.3. The alkaline comet assay and neutral comet assay were performed at different times postirrdiation, By 2h postirradiation, irradiation alone group had returned to baseline in A549 cells (P>0.05). But the DNA repair in irradiation alone group in H520 cells needs 6h, it indicates that the DNA damage induced by 4 Gy irradiation is easier to repair in A549 cells than in H520 cells.4. The alkaline comet assay and neutral comet assay were performed at 2h, 6h and 24h postirradiation, there was a significant increase in the amounts of SSB and DSB remaining compared with cells receiving only irradiation orβ-elemene in A549 cells (P<0.01). In H520 cells,β-elemene and irradiation can significantly inhibite the repair of DSB. The SSB induced by combination group is not significantly different withβ-elemene alone group in H520 cells at 6h and 24h postirradiation, This data suggested thatβ-elemene can not inhibite the repair of SSB in H520 cells.Part two:1. Compared with control group, the band colour ofβ-elemene alone group lightened, indicating thatβ-elemene can inhibite telomarase activity in A549 and H520 cells.2. Compared with control group, the band colour of irradiation group in A549 cells blackened, but in H520 cells the band colour of irradiation group lightened, this indicating that 4Gy-radiation can up-regulate the telomarase activity of A549 cells but suppress the telomarase activity of H520 cell.3. Compared withβ-elemene group or irradiation group, the band colour of combination group lightened significantly, this indicating thatβ-elemene and irradiation can suppress the telomarase activity in A549 cells and H520 cells.Part three:1.β-Elemene had a half-maximal (IC50) inhibitory effect on A549 cells group and H520 cells group at 152.64μg/ml and 72.86μg/ml at 24h, respectively.2. DNA damage was induced in A549 cells from 24h treatment ofβ-elemene, there was no increase in apoptotic cells above background levels at 24 h. At 36 h, cell viability was significantly reduced following exposure toβ-elemene, morphological evaluation of apoptosis using Hoechst 33342 reinforced these observations. A striking increase in caspase-3 activity was also observed in A549 cells after cells were incubated for 36 h withβ-elemene.3. As early as 12 h following exposure toβ-elemene, there were increased AO fluorescence and cathepsin D expression in A549 cells, indicating lysosomal membrane permeabilization occured following 12hβ-elemene exposure. A549 cells treated withβ-elemene for 12 h exhibited mitochondrial membrane potential similar to control cells (P>0.05). After exposure toβ-elemene for 24 h, the decrease in mitochondrial membrane potential was detected. These results showed that lysosomal membrane permeabilization and decrease of mitochondrial membrane potential occured with lysosome changes preceding mitochondrial membrane potential changes.4. A significant increase of DCF fluorescence intensity was observed in cells treated withβ-elemene for 24h (P<0.01), indicating it can induce the formation of intracellular ROS in A549 cells. We also determined GSH level under the same experimental conditions, after cells were incubated withβ-elemene for 24 h, a striking decrease of intracellular GSH was also observed.5. The cathepsin D inhibitor pepstatin A prevented subsequent DNA damage, decrease of mitochondrial membrane potential and apoptosis induced byβ-elemene. Conclusion:Part one:1.β-Elemene alone induces SSB and DSB in A549 cells and H520 cells.2. The combination ofβ-elemene and irradiation resulted in significantly higher SSB and DSB than each treatment alone in A549 cells and H520 cells. Therefore, our study indicates thatβ-elemene radiosensitization correlates with an increase in radiation-induced DNA damage.3. The repair of SSB and DSB by 4Gy-irradiation needs 2h in A549 cells and 6h in H520 cells, the DNA damage is easier to repair in A549 cells than in H520 cells. It may be the cause of radioresistant in lung adenocarcinoma.4.β-Elemene in combination with irradiation can significantly inhibite the repair of SSB and DSB in A549 and H520 cells, Thus, we conclude thatβ-elemene produces radiosensitization through inhibiting the repair of radiation-induced damage.Part two:1.β-Elemene alone can suppress the telomarase activity of A549 and H520 cell.2. 4Gy-radiation can up-regulate the telomarase activity of A549 cells but suppress the telomarase activity of H520 cells. Up-regulating of the telomarase activity may be the cause of radioresistant in lung adenocarcinoma.3.β-Elemene in combination with irradiation can suppress the telomarase activity in A549 cells and H520 cells. We conclude thatβ-elemene produces radiosensitization through inhibiting the telomarase activity.Part three:1. There were different inhibitory rates byβ-elemene in A549 cells and H520 cells. These data indicate different sensitivities of tumor cells towardβ-elemene.2. DNA damage was induced in A549 cells from 24h treatment ofβ-elemene, there was no increase in apoptotic cells above background levels at 24 h.β-Elemene induces apoptosis following 36h exposure.3. The lysosomal membrane permeabilization occured following 12hβ-elemene exposure. After exposure toβ-elemene for 24 h, the decrease in mitochondrial membrane potential was detected. Lysosomal membrane permeabilization was found to precede any effect on mitochondrial (24h), and apoptosis (36h).4. We found a dose-dependent ROS production and depletion of cellular GSH following exposure of A549 cells toβ-elemene.β-Elemene can induced oxidative stress in A549 cells.5. The cathepsin D inhibitor pepstatin A prevented subsequent DNA damage, decrease of mitochondrial membrane potential and apoptosis, which suggests that the release of cathepsin D from disrupted lysosomes plays an important role inβ-elemene-induced apoptosis in A549 cells.