Abstract Objective To investigate the Curcuma oil, Curcuma oil joint γ  ray irradiation of nasopharyngeal poorly differentiated squamous carcinoma cell line CNE  2 inhibition of proliferation and induction of apoptosis; analysis Curcuma oil Radiosensitization and research mechanism. Methods Cultured CNE  2 cells, Curcuma oil directly in vitro dosing role in CNE  2 cells using MTT, flow cytometry, electron microscopy and molecular biology methods, detection Curcuma oil alone or in combination with γ  rays on CNE  2 induction of apoptosis, radiosensitization effect. Results Curcuma oil used alone exact CNE  2 cells proliferation and induction of apoptosis, and conventional chemotherapy drug 5  Fu similar inhibitory effect (P> 0.05). Inhibition in a concentration-dependent drug Curcuma oil minimum onset concentration of 10μg/ml, the optimal duration of action for 48 hours; of 5μg/ml, 100μg/ml, 300μg/ml’s the Curcuma oil joint 100cGy γ  ray, 2 days after apoptosis from 0.5%, 2.15%, 10.2%, was significantly increased to 8.6%, 14% and 26% cell mortality are obvious synergies (P <0.01) less than 5%; combined effects of 96h The post-test group cells to death, apoptosis less; a 500μg/ml’s Curcuma oil major cause cell death, significant cytotoxicity. Flow cytometry results show higher concentrations the Curcuma oil CNE  2 cell cycle arrest in G1 phase. Electron microscopy of the ultrastructure of apoptosis apoptotic bodies can be seen. Conclusion Curcuma oil can inhibit the proliferation of nasopharyngeal carcinoma cell CNE  2 and induction of apoptosis; obvious sensitizing effect of the Curcuma oil and γ  ray 100cGy combined effects; their mechanism of action and the induction of tumor cell apoptosis and affect cell growth cycle.

Key words】 nasopharyngeal carcinoma; CNE  2; Curcuma oil; γ  ray; radiosensitizer

 Effect of Zedoary Oil for Apoptosis and Radiotherapy in Human Nasopharyngeal Carcinoma Cell Line CNE  2

    WU Dong  mei, YANG Rong  ning

    Department of Head and Neck Surgery, Cancer Hospital of Guangxi Medical University, Nanning 530021, China

    Correspondling Author: YANG Rong  ningAbstract: Objective To explore the Zedoary oil, Zedoary oil joint γ  ray irradiation on human nasopharyngeal carcinoma (NPC) cell lines CNE  2 inhibiting the proliferation and induction of apoptosis; analysis of whether the Zedoary oil enhancement of the role of radiotherapy.Methods MTT colorimetric assay and electronic microscope method were employed to examine the growth status and apoptosis of CNE  2 cells.Flow cytometry was employed to detect the effect of Zedoary oil, Zedoary oil and / or γ  radiation for growth 0.05) ;Zedoary oil drugs inhibit a concentrationdependent">inhibition in CNE  2 cells.Results Zedoary oil used alone, the CNE  2 cell proliferation is the exact effect, and all with conventional chemotherapy drug 5  Fu a similar effect (P> 0.05); Zedoary oil drugs inhibit a concentration  dependent , the minimum effect from the concentration of 10μg/ml, the best time of 48 hours; flow cytometry showed that 10μg/ml, 100μg/ml, 300μg/ml Zedoary oil  treated CNE  2 cells the apoptosis ratio of were 0.5%, 2.15%, 10.2% on 48 hours, respectively.In the group of treatment with 10μg/ml, 100μg/ml, 300μg/ml curcuma and 100cGy γ  radiation, the apoptosis ratio were 8.6%, 14%, 26% on 48 hours , respectively.And the average mortality rate of cells were below 5.0%. They has the synergistic action for apoptosis in CNE  2 cells (P <0.01). Combined 96 h after the test group of cells to death based, less apoptosis; cell death was induced by 500μg/ml curcuma.Zedoary oil is the CNE  2 cell block in G1 phase.Electron microscopy apoptosis ultrastructure can see that zedoary oil can be induced by CNE  2 cells apoptotic body, leading to apoptosis.Conclusion Zedoary oil to increase the in vitro method role in CNE  2 NPC cells can inhibit cell proliferation and induced apoptosis; Zedoary oil and small doses of γ  ray joint role obviously synergy; mechanism and its role in apoptosis induced by tumor cells and affect cell growth cycle.

    Key words: NPC; CNE  2; Zedoary oil; γ  ray; Radiosensitivity

Nasopharyngeal carcinoma (nasopharyngeal carcinoma, NPC) is a malignant growth in the nasopharynx, Department of one of the human high incidence of head and neck cancer, 95% for poorly differentiated squamous cell carcinoma, and undifferentiated carcinoma [1], with a high transfer and high Recurrent. NPC etiology is not yet clear, may be related to smoking, EB virus infection, eating preserved foods, living environment related to contaminated.

    NPC diagnosis and treatment in recent years, a large number of basic and clinical research has made certain achievements, but the prognosis is still not ideal, high transfer and high recurrence makes radiation therapy of the disease there is a high rate of treatment failure 2  3], this end, the tumor discharge chemotherapy combination therapy and gene therapy has become the subject of research direction [4  5].

    Currently, clinical for nasopharyngeal carcinoma induction chemotherapy drugs mainly 5  Fu and platinum, its side effects; looking attenuated efficiency can play a drug clinical problems to be solved. Traditional Chinese medicine with natural medicine treatment of cancer has a wealth of clinical experience, Chinese herbal medicine mild in nature, relatively few of toxic side effects, not only with the chemotherapy drug applications can also be combined with radiotherapy integrated application, has played the role of radiosensitization. Can not tolerate high dose chemotherapy in patients with advanced nasopharyngeal some poor health, can improve their quality of life and prolong survival.

    Curcuma oil contains the volatile oil of Curcuma rhizomes, at home and abroad in recent years, through pharmacology studies found that it is a pharmacologically active, efficient and safe drugs [6], with a broad-spectrum anti-tumor, anti-bacterial, anti-viral, anti-clotting and antioxidant activity [7], antitumor main pharmacological effects of Curcuma oil. This has been confirmed in liver cancer, lung cancer, breast cancer, cervical cancer, but have not been reported in nasopharyngeal field.

    1 Materials and methods

    1.1 Materials

    CNE  2 cell line (provided by the Affiliated Cancer Hospital); The oil injection Ezhu (Heilongjiang Regal Pharmaceutical Co., Ltd.); RPMI1640 medium, trypsin (Gibcol BRL Company); newborn calf serum (holly); the rest main reagents were products of Sigma, USA. KGI the Annexin  FITC apoptosis detection kit (Nanjing KGI).

    1.2 The main instrument and equipment

    CO2 incubator, automatic microplate reader, flow cytometry, transmission electron microscopy.

    1.3 Experimental Methods

    1.3.1 Experimental groups The test set blank control group, 5  Fu control group, the experimental group (diluted spare concentration Curcuma oil).

    1.3.2 cell cultures will be in log phase growth in good condition CNE  2 cells at 37 ℃ digestion into single cell suspension; routine passage and incubated overnight at 37 ° C, 5% CO2 thermostat incubator The next cell morphology was observed and the medium was changed.

    1.3.3 γ  ray irradiation the 60CO machines irradiation plus 0.5cm standard filler from the Paper Source 80cm, depending on the test requires irradiation dose culture irradiation rear incubator.

    1.3.4 MTT assay the Curcuma oil CNE  2 cell proliferation inhibition take the logarithmic growth phase cells were seeded in a concentration of 1 × 104/ml six different 96-well cell culture plate, each well 100μl, leaving zero holes and to prevent edge effects. The conventional incubation 24h cell monolayer covered the bottom of the hole (96-well flat-bottom plate), the medium was changed; blank control group, 5  Fu control and experimental groups, each group of six wells, to continue in the incubator were cultured day, 1 to 6 days; take a 96-well plate, in the corresponding hole in the Add 20μl by MTT, culture was continued 4h after termination of culture, and suck out the supernatant, and each hole the same batch of DMSO 100μl, set the low-speed oscillation shaker; with full automatic microplate reader at the wavelength of 492nm colorimetric OD value of each well was measured average value, in accordance with the following formula to calculate the rate of cell growth inhibition. Cell growth inhibition rate = (absorbance values ​​of the control group, a test group absorbance value) / (control group, the absorbance values ​​ blank group absorbance value) × 100%.

    1.3.5 Flow cytometric analysis of the routine collection of the cell cycle after treatment of cells, the cell suspension was filtered, removing the adhesions cell population, low speed centrifugation 5min; supernatant, each tube PI dyebath 1ml, 4 ° C, dark 30min , set by flow cytometry, data collection, and analysis software used MultiCycle cell cycle analysis.

    1.3.6 Flow cytometry analysis of apoptosis rate, mortality conventional collected from each group for 48 hours after the CNE  2 cells, in strict accordance with instructions KGI Annexin V  FITC apoptosis detection kit, the treated cells Beckman Coulter flow cytometer for detection. Annexin V  FITC vs PI dual parameter plots created with the appropriate software analysis.

    1.3.7 Transmission electron microscopy detection collected Curcuma oil half inhibition concentration (IC50) and the control group culture medium for 48 hours CNE  2 cells fixed by conventional methods, dehydration, embedding, repair block, producer, lead acetate uranium staining, transmission electron microscopy.

    1.4 statistical methods

    Take three times the average of all test results, application SPSS13.0 statistical software for analysis of variance, factorial analysis, P <0.05 or P <0.01 for the difference was statistically significant.

    2 Results

    2.1 MTT assay Curcuma oil CNE  2 cell proliferation inhibition

    Curcuma oil in the lower concentration (5μg/ml) of nasopharyngeal carcinoma cells in vitro CNE  2 growth promoting effect, and promote the growth effect between the concentration and the control group, the difference was statistically significant (P = 0.000) ; higher concentrations of nasopharyngeal carcinoma cell growth inhibition, and with the increasing concentration inhibition enhanced; compared with the control group, the difference was statistically significant (P <0.01); detected within six days 48h, the inhibition rate of the highest number (48.78%); each experimental group no obvious rule extended inhibition rate changes over time; six days pairwise comparisons showed no significant difference (P> 0.05), Figure 1; Curcuma oil group and the corresponding concentration of 5  Fu CNE  2 cell proliferation inhibition there was no statistically significant difference (P> 0.05). In this analysis only at 48h curve, as shown in Figure 2.

    2.2 Curcuma oil induced CNE  2 cells 48 hours after the apoptosis rate, mortality

    Significant concentration-dependent inhibition of the growth of the oil of Curcuma CNE  2 cells, to the role of the 48h largest drug suppression 5μg / ml, 10μg / ml group cell growth inhibition with the control group showed no significant difference, 300μg/ml group after 48h significantly inhibited and induced apoptosis (P <0.01), the cell death rate more than 500μg / ml group is greater than the rate of apoptosis, significant cytotoxicity, as shown in Table 1.

    2.3 Curcuma oil and γ  ray combined CNE-2 cells apoptosis rate, mortality

    2.3.1 5μg / ml, 100μg / ml, 300 μg / ml Curcuma oil joint 50cGy γ  ray effect 1,2,4 days no obvious radiotherapy synergies.

    2.3.2 5μg / ml, and 100 μg / ml, 300μg / ml Curcuma oil Table 1 Curcuma oil induced CNE  2 cell apoptosis rate, mortality 48h after joint 100cGy γ  ray one day no significant effect on cell , two days after the combined effects of group apoptosis rate of 0.5%, 2.15%, 10.2%, was significantly increased to 8.6%, 14% and 26%, as shown in Figure 3. Cell mortality rates are below 5%, the combined effects of 4 days after cell death-based and apoptosis fewer.

    2.3.3 5μg/ml, 100μg/ml, 300μg/ml Curcuma oil joint 200cGy the role of γ  ray 1,2,4 days after the death, the apoptosis rate decreased mainly the inhibition of tumor.

    2.4 Flow cytometric analysis of cell cycle results

    Γ  ray of observation Curcuma oil, Curcuma oil +100 cGy of CNE  2 cells role 24h, 48h, 96h three time points of the cell cycle changes were found in cell cycle arrest in G1 phase, S phase, G2 / M phase cells were decreased; especially for 48h, the cells accumulate arrest in G1 phase, S phase, G2 / M phase cell ratio was significantly decreased, 48h only detailed results are shown in Table 2. Table 2 drugs for 48h CNE  2 cell cycle

    2.5 TEM test results

    Visible nucleus, nuclear membrane integrity, nuclear pulp imbalance, the nucleolus is a clearer, more complete organelles in the cytoplasm, the cell surface microvilli control group, showing significant phenomenon of cell division. The experimental group showed reduced microvilli, nuclear condensation, chromatin dense set of edges, can see a large number of apoptotic body formation, as shown in Figure 4 to 6.

    3 Discussion

    Curcuma oil resistant tumor’s experiments confirmed [8  11]: Curcuma oil have significant anti-tumor, such a role in the mechanism to some extent, is the induction of apoptosis; also been reported that the Curcuma oil have direct cellular cytotoxicity caused by degeneration and necrosis of the tumor cells; By to build NPC vitro model, we Annexin and PI double staining and cell cycle [12] study, explore the inhibitory effect of the of Curcuma oil on poorly differentiated squamous cell carcinoma CNE  2 cells also get the above conclusions. The Curcuma oil anti CNE  2 cells in a concentration-dependent manner, the apparent inhibition and induction of apoptosis (P <0.01) in the the 300μg/ml role 48h after, 500μg/ml more cell death rate is greater than the rate of apoptosis, has obvious cytotoxicity; inhibition concentration and the corresponding concentration of 5  Fu group CNE  2 cells showed no significant difference (P> 0.05); and each experimental group no significant law extended inhibition rate changes over time, to the role of 48h inhibition rate highest  2 anti-cancer effect of the oil of Curcuma CNE, a dose-dependent reference value in the practical application of more than a time-dependent manner, that Sheng, Zhao, Tan, Wu and Zhao [13  17] reported in lung cancer, stomach cancer, liver cancer, human endometrial cancer. Shen Hung et al [18] found in the study, Curcuma by inhibition of COX  2 and its downstream PGE2 expression of VEGF downregulation and inhibition of tumor. Our experiments show that: induced by Ezhu oil CNE  2 cells to produce the highest rate of apoptosis 48h point in time, and the dynamic observation of turmeric oil, Curcuma oil +100 cGy γ  ray  2 cells and on the CNE 24,48,96 h three The point in time of the cell cycle changes were found The cells pile arrest in G1 phase, S phase, G2 / M phase cell ratio was significantly decreased, especially for 48h, cells arrest in G1 phase, which showed that Curcuma The oil can block cancer cell migration from G0/G1 to S and G2 / M phase transition. Therefore, we believe: that the induction of cell cycle arrest, inhibition of DNA synthesis and proliferative activity of the tumor cells may be an important aspect of Curcuma oil resistant nasopharyngeal mechanism.

    Curcuma oil and γ  ray combined effects of display: 5μg / ml, γ  ray of 100μg / ml, 300μg / ml of Curcuma oil joint 100cGy the two days after the apoptosis rate of 0.5%, 2.15%, 10.2% significantly with improve to 8.6% , 14% and 26%, and cell death are in less than 5%; curcuma aromatica oil with small doses of γ  ray joint role of the CNE  2 cells have significant radiosensitization role [19] (P <0.01); Mi Fushun [20] in the lung cancer study also confirmed the the Curcuma oil can play radiosensitization characteristics. But no relevant reports in the field of head and neck tumors. Sensitizing effect of traditional Chinese medicine, mostly blood circulation drugs, sensitizing mechanism is unknown, may be related to lower blood viscosity, expansion of peripheral blood vessels to improve the blood supply of the tumor bed to improve local tumor oxygen content induced telomerase activity etc., to enhance the sensitivity of tumor cells to radiation [21  24], pending further study.

    In addition, experiments confirmed [25], Curcuma can significantly improve immune function in mice, Curcuma decoction has a protective effect on the of irradiated mice’s 300cGy hematopoietic, immune and microcirculation; animal pre Note Ezhu oil, enhance animal anti-ray irradiation capacity; ointment coated with 1% Curcuma prevention ray irradiation damage to the skin. Curcuma also can significantly reduce cancer of VEGF content of PGE and reduce the rate of cancer metastasis.

    Electron microscopy results show: the experimental group showed reduced microvilli, nuclear condensation, chromatin dense set of edges, you can see the formation of apoptotic bodies; ultrastructure of cancer cells in the control group visible cells well-developed large nuclear nuclear membrane integrity, nuclear cytoplasm ratio, Ren clearer, more complete organelles in the cytoplasm, the cell surface microvilli, obviously mitosis. Will naturally lose its malignant proliferation of tumor cell apoptosis activity [26]. In our experiments this has also been confirmed again.

    In summary, we see a clear, Curcuma oil can inhibit and induce the poorly differentiated squamous cell carcinoma CNE  2 apoptosis, inhibition was no significant difference compared with traditional chemotherapy drug 5  Fu, and be able to increased ray radiosensitivity of nasopharyngeal carcinoma cells, the destruction rate of radiation on the tumor cells, significantly enhanced the radiation effect. Curcuma oil could become a low toxicity, high efficiency, can improve the immunity of cancer patients, chemotherapy or Chemotherapy drugs; radiotherapy is also certain body protection role, promising radiation sensitizer and there are good prospects for the treatment of metastatic recurrent nasopharyngeal carcinoma, but deep-seated anti-tumor effect also requires further development, such as apoptosis-related genes in the gene microarray cDNA, cell cycle regulation of gene expression changes, clear anti-NPC mechanism of Curcuma oil, lay a theoretical foundation for the research and development of gene drug. Also induce immune protective effect from Curcuma oil start-depth study of the mechanism of tumor vaccine active immunization, developed a vaccine specific killing NPC – Curcuma oil vaccines.