| Background:Nasopharyngeal carcinoma is a malignant tumor that occurs in nasopharyngeal mucosa epithelial cells. Its occurrence, development, clinical manifestations and treatment is different from those of other head and neck cancers. There are about80,000new cases of nasopharyngeal carcinoma patients each year worldwide, with a male to female ratio of2.3:1. The situation in southern China and Southeast Asia is more severe, especially in southern China with20new cases every100000reported every year. It is one of the most common malignant tumors in southern china. The incidence age curve of nasopharyngeal carcinoma starts increasing from the age of20, peaking at the age group of50-60years old.The most common pathological type is low differentiated squamous carcinoma. It metastasizes mainly through the lymph node to the cervical lymph node, or through the blood stream to the bone, lung, and liver as well. Radiotherapy is the main therapeutic method that benefits mostly for the early, mid-term patients, with a survival rate of5year greater than80%.30%-40%patients will show distant metastasis and local recurrence with the development of lesions, though early radiotherapy can achieve satisfactory results. At the time patients are diagnosed, multiple cervical lymph node metastases are common a,pmg them because of the difficulties in early diagnosis. In the diagnosis of nasopharyngeal carcinoma patients, about70%patients have stage Ⅲ and Ⅳ cancers and have a poor prognosis. For those advanced nasopharyngeal carcinoma chemotherapy-based comprehensive therapy are administered. Although combination therapies alleviate patients, it confers radiotherapy and chemotherapy resistance, and side effects heavier. The overall5year survival rate is about50%. Therefore, we need to continue to study the pathogenesis of nasopharyngeal carcinoma in order to seek for better ways in the treatment of nasopharyngeal carcinoma.Most of the tumor treatments overcomes cancers by inducing tumor cell apoptosis. Apoptosis is a form of programmed cell death, that is, under certain internal and external signal transduction, relying on caspase participation, the chromatin and cytoplasm is condensed, nuclear fragmentation and the cytoplasm, cell shrinkage is decomposed into apoptotic body, be swallowed up by the neighboring cells or macrophages. Recent findings showed that autophagy, another form of programmed cell death, plays an important role in tumorigenesis and development. Not dependent on the involvement of caspase in cells, autophagy involves the formation of a double membrane around the organelle or cytosolic protein within the cell known as an autophagosome, which are then fused with lysosomes and degraded or recycled. Bcl-2family plays an important role in apoptosis. The Bcl-2family protein as the main proteins regulating apoptosis regulates cell apoptosis. Recent studies show that apoptosis and autophagy interact to promote cell death. It has been shown that, Bcl-2overexpression can inhibit autophagy. Bcl-2could induce apoptosis and influence autophagy. Bcl-2induced autophagy might be related to autophagy gene Beclinl. BH3analogues could compete with The Bel-2protein to bind BH3domain in beclinl, resulting in the releasing of Beclinl to promote autophagy. Therefore, studies on targeting bcl2, inducing tumor cell apoptosis and autophagy, inhibiting tumor proliferation, is a topic worthy of study.Cleary, etc. Studies have found that, EB virus has an open reading frame homologous to the Bel-2gene, called BHRF. Similar to the Bel-2, it can prevent the lymph cells of mice and human from apoptosis due to the lack of growth factor. EB virus can also mediate endogenous Bcl-2expression, thereby inhibiting cell apoptosis. It was confirmed that the virus contains additional genes that can indirectly cause the Bcl-2expression, and use Bcl-2protein to inhibit host cell apoptosis. In addition, Henderson and Sheng found that in EB virus latent infection period, expression of LNP1and BARF1can upregulate the expression of Bcl-2and MCL-1to protect host cells, preventing host cell apoptosis. Song et al found that the expression level of Bcl-2in nasopharyngeal carcinoma tissues was higher than that in adjacent non-cancerous tissues and inflammatory tissue. Liu Yangyun et al found expression rate of positive Bcl-2was as high as82%in nasopharyngeal carcinoma. Therefore targeted the Bcl-2gene therapy of nasopharyngeal carcinoma, also has important significance.Gossypol is a kind of yellow double naphthalene hydroxyl polyphenol aldehyde compounds extracted from the cotton stalk, leaf, seed and root. Gossypol acetate at first in the medical field as a male contraceptive clinical application for many years,and recent studies showed that as a small molecular model Bcl-2blocker, it exhibited significant antitumor activity in a variety of tumors. Apogossyplone (ApoG2), a new type of gossypol derivatives synthesized by removing two aldehyde, shows not only low toxic side effects, but also a wide variety of anti-tumor effect, showing a better prospect of application.ObjectiveThis experiment focus on high Bcl-2expression nasopharyngeal carcinoma cells CNE2as the research object, to study ApoG2’s inhibition and induction of cell apoptosis effect in CNE-2cells in vitro and in vivo, and preliminary discussion on the possible mechanism of action. At the same time, radiosensitizing effect of ApoG2in the CNE-2cell line by activating apoptosis and autophagy was observed, and its mechanism was investigated. To provide experimental basis for further study, and lay a theoretical foundation for ApoG2’s clinical application.Method1. to utilize CCK-8in vitro proliferation assay to measure effects of ApoG2on CNE-2cells after24h,48h and72h,and calculate the IC50; 2. To utilize CCK-8in vitro proliferation assay to measure effects of ApoG2combined with radiotherapy on CNE-2cells after48h and calculate the q value;3. To observe apoptotic morphological changes of ApoG2alone or combined with radiotherapy in CNE-2cells through Hoechst33258fluorescent staining4. To observe Morphological changes of autophagy of ApoG2monotherapy and combined with radiotherapy in nasopharyngeal carcinoma cell line induced by CNE-2by Acridine-orange (AO) staining;5. Transmission electron microscopy (TEM) were employed to observe the morphological alterations in autophagic cells after treatment of ApoG2cell ultrastructure in CNE-2cells.;6. Flow cytometry was used to detect the changes of apoptosis rate and cell autophagy rate for the ApoG2alone or in combination with radiotherapy effect in CNE-2cells;7. Western blot experiments detect changes of expression of the Bel-2protein and Beclinl protein after ApoG2single-agent and combined with radiotherapy on CNE-2cells;8. Copy model of CNE-2nasopharyngeal carcinoma in nude mice and observe tumor growth inhibition of ApoG2in vivo.Resultl.The CCK-8assay showed that the treatment resulted in dose-and time-dependent inhibition of cell proliferation when we treated CNE-2cells with5,10,20,40,60,and80μM ApoG2for24,48and72h. With increased drug concentration or prolonged time, the inhibitory action of ApoG2on CNE-2cells was accordingly enhanced (Fcencentration=1819.354, Pcencentration=0.000.Ftime=041.671, Ptime=0.000). And there is interaction between concentration and time (F=202.540, P=0.000). The50%inhibitory concentration (IC50)of ApoG2for72h was23.61μmol/L. The exposure to the combination ApoG2with radiation for48h,resulted in radiation dose-and concention-dependent inhibition on CNE2cells. ApoG2combined radiotherapy CNE-2cells48h, can see the comparison between different dose has significant difference (F=2726.28, P=0.000), ApoG2in comparison also has a significant difference between different drug concentrations (F=4316.28, P=0.000). The interaction effect between (F=18.54, P=0.000). Computing joint q value>1.15, ApoG2and radiation combined application with synergy.2. Compared with the control group, Hoechst-33258staining demonstrated the occurrence of apoptosis in the CNE-2cells treated with ApoG2or radiotherapy, or combination. However, the morphological changes in the nuclear condensation and fragmentationin in CNE-2cells treated by ApoG2combined with radiotherapy were most significant. ApoG2alone or in combination with radiotherapy group showed obvious pyknosis, fragmentation, chromatin condensation, formation of apoptotic bodies and apoptotic features, while in the control group cell chromatin evenly, morphological rules nuclear pale blue normal nucleus were shown. More apoptosis were observed in combined treatment group.3. AO staining revealed more bright red acidic vesicular organelles in the combination group. Therapeutic effects of ApoG2monotherapy and combined with radiotherapy on CNE-2cells48h, cell nucleus and cytoplasm in control cells showed bright green fluorescence, ApoG2and combined radiotherapy group cytoplasm or nucleus of acidic autophagosome was dyed bright red fluorescence, combined radiotherapy group role in autophagy phenomenon more obvious.4.An increase in the number of large vacuoles and double-layered membrane structure was observed under TEM in the combination group.It was observed that the therapeutic effects of ApoG2monotherapy and combined with radiotherapy on CNE-2cells in48h by Transmission electron microscope.that drug group (ApoG240u mol/L) after treatment with48h in CNE-2cells, large vacuoles increased,and multiple scattered in the membrane bilayer structure and other fine autophagy phenomenon appears,while the control group of CNE-2cells have good morphology.5. Flow cytometry (FCM) to detect apoptosis rate showed that ApoG2single-agent and combined radiotherapy effects on CNE-2cells48h, the control group the apoptosis rate was (3.90+0.34)%, ApoG2group is (19.52+1.18)%, the difference was statistically significant (F=485.294, P=0.000); Control group, ApoG2single medicine group, the simple radiotherapy group and combined radiation treatment group, the apoptosis rate statistically significant mean differences between groups (F=149.511, P=0.000), the joint action group compared with the other three groups were statistically significant difference (P<0.05), the two drugs in combination with the apoptosis rate is higher.6. Examination of the FCM showed that autophagy fluorescence intensity for the control group (0.92+0.31)%,(28.24+7.35)%for40apog2group, the difference was statistically significant (F=41.354, P=0.003), ApoG2group is higher than the control group.7. Western blot results showed that CNE-2cells treated with40umol/L of ApoG2resulted in reduced the Bcl2protein expression than the control group(differences statistically significant (F=68.909, P=0.001)); Beclinl protein expression increased, compared with the control group(differences statistically significant (F=497.906, P=0.000)). In combined radiotherapy experiments, the Bel-2protein expression relative to the amount, statistically significant mean differences between groups (F=69.248, P=0.000), and combined with other three groups was statistically significant (P<0.05), the joint group of Bel-2protein expression of the quantity is lower. Beclinl protein relative expression, statistically significant mean differences between groups (F=111.591, P=0.000), the joint action group compared with the other three groups were statistically significant difference (P<0.05), the joint group of Beclinl protein expression of the higher quantity.8. Transplantation tumor experiment in vivo showed that after being administered with the drug12days, stripping tumor, after weighing the tumor, the tumor inhibition rate was65.49%, compared with control group, tumor weight had significant differences after treatment, P<0.05). No adverse reactions occur in nude mice during whole experiment period. According to the evaluation criteria:the tumor growth inhibition rate<40%, ineffective; The tumor growth inhibition rate of40%or higher, and P<0.05is effective. We believe that ApoG2could significantly inhibit the proliferation of CNE-2cells in vivo.ConclusionsApoG2can inhibit the proliferation of CNE-2cells and induce apoptosis and autophagic programmed cell death in vitro and in vivo. ApoG2can also increase CNE-2cells’ sensitivity to radiation therapy, promote the occurrence of apoptosis and autophagy, and synergistically inhibit tumor cell proliferation. The combined application of ApoG2and ceramide at lower concentrations promotes apoptosis and autophagy, and synergistically inhibits the proliferation of human nasopharyngeal carcinoma cells. Such effects may be related to the down-regulation of Bcl-2expression and the up-regulation of Beclinl expression. Meantime, a decrease in Bcl-2expression disturbed its binding with beclinl, resulting in more liberated beclinl protein and promoting auphagy. |
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