Tumor Biol. DOI 10.1007/s13277-014-2226-0


Down regulation of mammalian target of rapamycin decreases HIF-1α and survivin expression in anoxic lung adenocarcinoma A549 cell to elemene and/or irradiation Kun Zou & Enjuan Tong & Yinghui Xu & Xiaoqin Deng & Lijuan Zou

Received: 7 March 2014 / Accepted: 11 June 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract Mammalian target of rapamycin (mTOR) is associated with gene transcription, protein translation and initiation, the synthesis of ribosomes, and apoptosis. The down regulation of mTOR induces apoptosis in malignant tumor cells. Elemene, a sesquiterpene from the traditional Chinese medicinal herb Curcuma wenyujin, is active against a wide range of tumor types. In the present study, decreasing the expression of mTOR with mTOR small interfering RNA (siRNA) increased the toxicity of elemene and irradiation against hypoxic lung adenocarcinoma A549 cells. The results showed that transfecting mTOR siRNA into A549 cells significantly decreased the expression of hypoxia-inducible factor 1α (HIF-1α) and survivin. Compared to control cells, cells transfected with mTOR siRNA that were hypoxic exhibited increased apoptosis. Overall, the expression of HIF-1α and survivin proteins decreased following treatment with elemene and irradiation and after transfection with mTOR siRNA. Apoptosis was higher in transfected than in untransfected cells treated with elemene and/or irradiation.

Keywords Elemene . Irradiation . Hypoxia . siRNA

Kun Zou and Enjuan Tong are co-first authors. K. Zou : Y. Xu : X. Deng (*) First Affiliated Hospital of Dalian Medical University, 222 Zhong Shan Road, Dalian 116011, Liaoning, China e-mail: [email protected] E. Tong The Fifth People’s Hospital of Dalian Municipality, 890 Huang He Road, Dalian 116021, Liaoning, China L. Zou (*) Second Affiliated Hospital of Dalian Medical University, 467 Zhong Shan Road, Dalian 116023, Liaoning, China e-mail: [email protected]

Introduction Lung cancers are highly prevalent tumors causing over 1.3 million deaths each year [1]. Nonsmall cell lung cancer accounts for approximately 80 % of all cases of lung cancer [2]. Chemotherapy or radiotherapy remains the primary treatment after an initial diagnosis of lung cancer for patients with middle to advanced stages of this disease. However, the effectiveness of these treatments is limited, and the 5-year survival rate is poor [3]. An important factor in treatment failure is tumor resistance to these therapies. Thus, identifying new agents that can overcome chemoresistance or radio resistance is a potential means to improve the effectiveness of therapy and overall survival. Researchers have found that tumors contain greater numbers of hypoxic cells and that these hypoxic cells are resistant to radiation, thereby explaining the failure of tumor radiotherapy. To overcome hypoxia, attempts have been made to directly or indirectly increase the oxygen content in tumor tissue. However, the effectiveness of this approach was poor. An alternative approach has been to develop radio sensitizers to increase the radio sensitivity of hypoxic tumor cells. Elemene (1-methyl-1-vinyl-2, 4-diisopropenyl-cyclohexane), isolated from the traditional Chinese medicinal herb Curcuma wenyujin, is composed of β, γ, and δ isomers, with β-elemene being the active component. Many studies have demonstrated that elemene exhibits fewer side effects than other cytotoxic drugs and possesses activity against malignant tumors, including lung cancer, prostate cancer, melanoma, and glioma [4–9]. Elemene can enhance the radio sensitivity of human cancer cells both in vitro and in vivo. For example, combining elemene with irradiation increased the extent of apoptosis measured in a transplanted tumor in vivo and also in lung cancer cells in vitro [10, 11]. Elemene deregulates Bcl-2 and p53, increasing cell apoptosis and enhancing the sensitivity of the pulmonary adenocarcinoma A549 cell line to irradiation.

Tumor Biol.

The mechanism of this increased sensitivity may be related to blocking cell division at the G2/M stage by preventing the repair of the DNA damage induced by irradiation [12]. Mammalian target of rapamycin (mTOR) is a serine/ threonine protein kinase that regulates biological activities, including gene transcription, protein translation and initiation, ribosome synthesis, and apoptosis [13]. Most malignant tumors over-express mTOR, leading to abnormal signal regulation and tumorigenesis [14]. Thus, mTOR is considered to play important roles in tumor initiation and progression and is a critical factor in therapeutics and the patient’s prognosis. Human cancers expressing high levels of mTOR exhibit increased resistance to radiotherapy that results in poor overall survival [15]. Therefore, inhibiting the expression or activity of mTOR may be a promising means to enhance tumor radio sensitivity. Our preliminary studies showed that a sensitizing dose of elemene in combination with irradiation decreased the mTOR mRNA level of A549 cells in vivo. However, the underlying mechanisms of this sensitization remain unclear. In the current study, we investigated the antitumor activity of radiosensitizing doses of elemene on mTOR expression in hypoxic A549 cells. Underlying mechanisms were explored by decreasing mTOR expression using small interfering RNA (siRNA).

irradiation, cells were cultured in a hypoxia caddy that was purged with nitrogen gas for 30 min. Cells were cultured under normoxia condition first. Then put the cell culture board into impermeable anoxia box with sterile. Connect inlet duct of the box to nitrogen tank and open the valve of the nitrogen tank to fill nitrogen into anoxia box. Unclamp out gassing duct for 60 s and evacuate air in the anoxia box as far as possible. Then clip the out gassing duct by vessel clamp. Anoxia for 30 min later put the box into normoxia incubator [16].

siRNA transfection mTOR siRNA was purchased from Invitrogen (Grand Island, NY, USA). The specific sequences were 5′-AGGACGCUCA CAUUGCUAGAUGUGG-3′ and 5′-CCACAUCUAGCA AUGUGAGCGUCCU-3′. A549 cells were transfected with 80 nM of siRNA using Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturer’s protocol. Following transfection for 6 h, the cells were enriched with 10 % FBS, incubated overnight, treated as described above, and collected for analysis. Reagent Company had synthesized siRNAs for three strips each, and before the experiment, transfected the three strips of each siRNA into cells to select the best one.

Materials and methods Chemicals and materials Elemene was purchased from Jingang Pharmaceutical Company (Dalian, China). The human lung adenocarcinoma cell line, A549, was purchased from the Cell Center of the Chinese Academy of Medical Sciences. RPMI 1640 medium was obtained from Thermo Scientific (Logan, UT, USA). Fetal bovine serum was from TBD Bio (Tianjin, China). Antibiotics (penicillin, streptomycin), polyclonal anti-mTOR, hypoxiainducible factor 1α (HIF-1α), survivin, β-actin antibodies, and horseradish peroxidase (HRP)-conjugated secondary immunoglobulin G (IgG) antibodies were purchased from Abcam Biochemicals (Cambridge, UK). Cell culture and treatment Cells were cultured in RPMI 1640 medium, supplemented with 10 % fetal bovine serum (FBS) and antibiotics (100-IU/ ml penicillin and 100-μg/ml streptomycin) at 37 °C in a humidified atmosphere with 5 % CO2. Cells were allowed to attach for 24 h before treatment with 20 μg/ml elemene for an additional 24 h. The irradiated group then received 4 Gy X-ray irradiation at a rate of 2 Gy/min using a 6-mV linear accelerator (Varian; Palo Alto, CA, USA). The distance between the X-ray source and the specimen (SSD) was 100 mm. Before

Reverse transcription-polymerase chain reaction (RT-PCR) analysis Cells were plated in six-well plates at a density of 12×104 cells per well. Transfections with siRNA were performed as described above. Total RNA from each group was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The RT-PCR analysis was performed using an RT-PCR kit (Takara; Otsu, Japan). The specific primer sequences were as follows: for mTOR (5′-CGCTGT CATCCCTTTATCG-3′ and 5′-ATGCTCAAACACCTCC ACC-3′), for HIF-1α (5′-ATGAAGTGTACCCTAACTAG CCG-3′ and 5′-GCTTGAGTTTCAACCCAGACATA-3′), for survivin (5′-AGCCAGACTTGGCCCAGTGTTTC-3′ and 5′-GCACTTTCTCCGCAGTTTCCTCA-3′), and for GAPDH (5′-GCACCGTCAAGGCTGAGAAC-3′ and 5′ATGGTGGTGAAGACGCCAGT-3′). The RT-PCR protocol involved initial denaturation at 94 °C for 2 min, followed by 35 cycles at 94 °C for 30 s, annealing at 63 °C for 30 s, and extension at 72 °C for 1 min. The final extension was performed by an incubation step at 72 °C for 7 min. The PCR products were subjected to electrophoresis in agarose gel and were visualized with ethidium bromide. The bands were analyzed with Quantity One software (BioRad).

Tumor Biol. Fig. 1 a A549 cells transfected with or without mTOR siRNA were assessed for the expression of mTOR by RT-PCR. b mTOR mRNA levels were quantified by measuring the relative optical density (OD). The results are expressed as means ± SD of three independent experiments. *p>0.05, #p

or irradiation.

Mammalian target of rapamycin (mTOR) is associated with gene transcription, protein translation and initiation, the synthesis of ribosomes, and apopto...
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