The radiosensitizing effect of Paeonol on lung adenocarcinoma by augmentation of radiation-induced apoptosis and inhibition of the PI3K/Akt pathway.

PURPOSE: To investigate the radiosensitizing effect and mechanism of action by the natural product Paeonol on lung adenocarcinoma both in vitro and in vivo. MATERIALS AND METHODS: Two lung adenocarcinoma cell lines (human lung adenocarcinoma cell line A549 and mouse Lewis lung carcinoma (LLC) cell line) were chosen for this research. In order to select the experimental concentrations of Paeonol, cytotoxicity was determined using a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide) assay. A clonogenic assay was performed to measure the radiosensitizing effects. Apoptosis was determined by the Tunel (terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling) assay and flow cytometry. Protein expression was analyzed by Western blotting. To test the radiosensitizing effect in vivo, a transplanted tumor model was established. RESULTS: The MTT assay showed that Paeonol inhibited proliferation of cells. Paeonol concentration ranged from an IC5 (5% inhibiting concentration) to an IC20 and was used at non-toxic concentrations for subsequent experiments. The clonogenic assay showed that Paeonol enhanced the radiosensitivity of cells. Data from the Tunel assay and flow cytometry verified that Paeonol enhanced radiation-induced apoptosis. Paeonol inhibited the activation of the PI3K/AKT (Phosphatidylinositol 3-kinase/ Protein Kinase B) pathway and down-regulated the expression of COX-2 (Cyclooxygenase-2) and Survivin. Paeonol (1718 mg/kg) combined with 10 Gy irradiation inhibited the growth of a transplanted tumor model in vivo, resulting in the longest tumor growth time, tumor growth delay and the highest inhibition ratio when compared with the radiotherapy alone group. CONCLUSIONS: It is reported for the first time that Paeonol has a radiosensitizing effect on lung adenocarcinoma both in vitro and in vivo. This effect could be related to the augmentation of radiation-induced apoptosis and the inhibition of the PI3K/Akt signalling pathway and its downstream proteins: COX-2 and Survivin.

Changes in intracellular redox status influence multidrug resistance in gastric adenocarcinoma cells.

Multidrug resistance (MDR) to chemotherapeutic agents is a major obstacle for the treatment of various types of cancers. The exact mechanism of MDR has not yet been fully clarified, although it has been frequently associated with the variation of intracellular redox status. The levels of intracellular glutathione (GSH) are considered to play a vital role in the regulation of the intracellular redox status. In our study, we investigated the effects of buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis, and NAC, a cysteine source for GSH synthesis, on sensitive gastric adenocarcinoma cells (SGC7901) and cisplatin-resistant SGC7901/DDP cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The two cell lines were pretreated with various non-toxic concentrations of BSO for 24 h and combined with fluorouracil (5-FU) or mitomycin (MMC) in the presence or absence of NAC before culturing further. After various treatments, the IC(50) values of MMC and 5-FU were calculated and intracellular GSH levels were measured using the glutathione reductase/5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) recycling assay without anticancer drug stimulation under the same microenvironments. The study demonstrated that BSO increased the sensitivity of the cells to chemotherapeutics while NAC exhibited the reverse effect, particularly in drug-resistant cells. It is, therefore, possible that changes in intracellular GSH levels affect the chemosensitivity of the resistant cells to a greater extent than that of their parent cells. This study indicates that variation in the intracellular redox status may be closely correlated with MDR and may provide a valuable basic strategy for anticancer therapy.

Regulation of hypoxia inducible factor-1α expression by the alteration of redox status in HepG2 cells.

Hypoxia inducible factor-1 (HIF-1) has been considered as a critical transcriptional factor in response to hypoxia. It can increase P-glycoprotein (P-Gp) thus generating the resistant effect to chemotherapy. At present, the mechanism regulating HIF-1α is still not fully clear in hypoxic tumor cells. Intracellular redox status is closely correlated with hypoxic micro-environment, so we investigate whether alterations in the cellular redox status lead to the changes of HIF-1α expression. HepG2 cells were exposed to Buthionine sulphoximine (BSO) for 12 h prior to hypoxia treatment. The level of HIF-1α expression was measured by Western blot and immunocytochemistry assays. Reduce glutathione (GSH) concentrations in hypoxic cells were determined using glutathione reductase/5,5'-dithiobis-(2-nitrob-enzoic acid) (DTNB) recycling assay. To further confirm the effect of intracellular redox status on HIF-1α expression, N-acetylcysteine (NAC) was added to culture cells for 8 h before the hypoxia treatment. The levels of multidrug resistance gene-1 (MDR-1) and erythropoietin (EPO) mRNA targeted by HIF-1α in hypoxic cells were further determined with RT-PCR, and then the expression of P-Gp protein was observed by Western blotting. The results showed that BSO pretreatment down-regulated HIF-1α and the effect was concentration-dependent, on the other hand, the increases of intracellular GSH contents by NAC could partly elevate the levels of HIF-1α expression. The levels of P-Gp (MDR-1) and EPO were concomitant with the trend of HIF-1α expression. Therefore, our data indicate that the changes of redox status in hypoxic cells may regulate HIF-1α expression and provide valuable information on tumor chemotherapy.