| BackgroundsGliomas is the most common primary central nervous system (CNS) tumor which makes up about 30% of all central nervous system tumors and 80%of all malignant brain tumors. It has become an intracranial tumor the most difficult to be conquered as its high morbidity and mortality. Despite recent advances in the diagnosis, surgery, chemotherapy and radiation therapy, the prognosis for GBM remains very poor with the median survival time of only 12-15 months and the very low quality of life.Symptoms of gliomas depend on which part of the central nervous system is affected. A brain glioma can cause headaches, vomiting, seizures, and cranial nerve disorders as a result of increased intracranial pressure. Neurosurgery combined with radiotherapy and chemotherapy is the main therapy at present. Neurosurgery is very hard to remove the glioma completely, so the recurrence rate of glioma is very high after the surgery. In clinical, temozolomide (TMZ) is the first line chemotherapy drug for GBM. However, due to blood brain barrier (BBB), low O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation rate and multiple drug resistance of glioblastoma, the efficacy of TMZ-based radiochemotherapy is low. Thus, it is urgent to develop novel and effective treatment modalities including new chemotherapy drugs for the management of glioma.Interest in natural products as an alternative to synthetic drugs for cancer treatment is largely due to low cost, established historical use in traditional medicinal systems, easy availability and minimal or no toxicity. Phenethyl isothiocyanate (PEITC) is artificially synthesized from glucosinolates by cutting or chewing activated enzyme myrosinase and known to be one of the major bioactive compounds present in cruciferous vegetables such as watercress, broccoli and Brussels sprouts. Previous studies have revealed PEITC has broad spectrum and remarkable anti-cancer effects by inducing apoptosis and reversing chemotherapy-drug resistance. PEITC has been shown to selectively kill malignant cancer cells but not the corresponding normal cells through potent induction of reactive oxygen species (ROS) in malignant cancer cells but not in normal cells. Being a natural compound, PEITC is not expected to present any severe toxicity, unlike many traditional anti-cancer agents.we hypothesize whether PEITC may function as anti-cancer effects to malignant glioma cells.The anti-cancer activity of PEITC can be divided into chemopreventive and chemotherapeutic effects. Several epidemiological studies from different parts of the world provide a strong evidence for the reduced risk of cancer with higher intake of cruciferous vegetables.Although chemopreventive effects were identified through epidemiological evidence, chemical carcinogenesis models and transgenic mouse models, more extensive mechanistic studies were performed to evaluate its therapeutic potential in various pre-clinical models.In general, carcinogenesis occurs due to the bio-activation of carcinogens by oxidation, reduction or hydrolysis via phase I drug metabolizing enzymes. Hence, modulation of phase I enzymes can affect the carcinogen activation process. PEITC shows a dual activity on phase I enzymes. For example, PEITC on one hand causes induction of; however, it inhibits activity of certain CytP450 enzymes, such as CYP2E1, CYP3A4 and CYP2A3. PEITC have been shown to induce phase II detoxification enzymes, which can explain their chemopreventive activityTwo primary mechanisms that have been identified are cell cycle arrest and induction of apoptosis. PEITC-mediated generation of reactive oxygen species (ROS) is known to be a general mechanism of action leading to cytotoxic effects, especially specific to cancer cells. ROS generation by PEITC leads to mitochondrial deregulation and modulation of proteins like Bcl2, BID, BIM and BAX, causing the release of cytochrome c into cytosol leading to apoptosisAnother established mechanism to induce ROS toxicity is by the inhibition of ROS detoxification of the cell. Each cell has well-developed mechanisms to protect against ROS-induced damage. Primary ROS scavenging mechanisms include dismutation of superoxide anion to oxygen and H2O2. Further, hydrogen peroxide is converted into water by glutathione peroxidase(GPX), or to oxygen and water by the enzymatic action of catalase. Glutathione (GSH) is a substrate for GPX. PEITC binds to GSH and causes its depletion in cancer cells leading to ROS-induced cell damage. Interestingly, the sensitivity towards PEITC correlates with constitutive GSH levels present in the cells. The cells with higher levels of GSH were relatively more sensitive to PEITC, which may also explain the selectivity toward cancer cells compared with normal cells, since cancer cells have higher levels of GSH in normal cells.Objective:Tumor development process is very complex, involving multiple genes and multiple steps, has aroused great attention of the international community. Although the tumor prevention and treatment research has made great progress in recent years, the mortality rate caused by malignant tumor is still very high. The prevention and treatment of tumor is still a difficult problem faced by the medical research community. Therefore, it is important to improve the cure rate of tumor or reduce tumor mortality. Minimal invasive treatment and targeted anti-tumor drug therapy is the development trend of the treatment of tumors, especially the development of highly efficient and low toxicity of targeted drugs. In recent years, chemical prevention of cancer get more and more people's attention. Studies found that people's daily consumption of plants or fruits are rich in anti-tumor components, such as grapes contain lacquer flavonoids, green tea contains tea polyphenols, enzymes contained in cruciferous and isothiocyanates, etc. Epidemiological studies have shown that cruciferous plants, such as radish, cabbage, broccoli, mustard have large amounts of glucosinolates. When the plant tissue is destroyed, the glucosinolates can be hydrolyzed to produce different thiocyanate. Therefore, increasing the intake of cruciferous plants will greatly reduce the incidence of cancer. Isothiocyanate has a strong irritating odor. It is volatile and soluble in ether, ethanol and other organic solvents and pH and temperature have great impact on its stability. Common isothiocyanates includes phenethyl isothiocyanate (PEITC), allyl isothiocyanate, sulforaphane and rosin. Comparing the biological activities of isothiocyanates and nitriles, it was found that isothiocyanates can induce the production of type ? detoxification enzymes, whereas nitriles cannot.Glioma is one of the most common malignant tumors of the central nervous system originating from the human neuroepithelium. It has the characteristics of high morbidity, high recurrence rate, high mortality, rapid invasion and difficult to cure. Glioma has cell heterogeneity according to the different degree of differentiation. Glioma accounts for 40% to 60% of primary brain tumors, including astrocytoma, anaplastic astrocytoma, glioblastoma, medulloblastoma, ependymoma, and oligodendrocytes. The 1-year survival rate of glioblastoma patients with medium and low differentiation is< 5%. Because of its poorly differentiated, aggressive and fast proliferation characteristics, the current surgical treatment methods used cannot completely cute tumor, although radiation and chemotherapy is taken after surgery, but the tolerance of tumor cells to radiation leading to the recurrence of residual lesions. In addition, due to tumor drug resistance and adverse reactions after individual treatment, tumor tissue and the surrounding tissue, high interstitial hydrostatic pressure and other factors have impacts on the effect of chemotherapy, leading to an average survival time is only 9-15 months in patients with glioma. Therefore, the complete removal of glioma cells still faces great challenges. In order to overcome these difficulties, it is important to find new methods for the treatment of glioma. Therefore, inhibition of abnormal activation of tumor signaling pathway is one of the effective pathways to kill tumor cells, inhibit cell proliferation, induce cell differentiation, and then cure glioma.In recent years, with the in-depth study of isothiocyanates (ITCs), its remarkable anti-cancer activity has been paid close attention to by investigators and has become one of the most potential natural functional factors to prevent the occurrence and development of cancer. At present, twenty kinds of isothiocyanate with anticancer activity have been found. Among them, sulforaphane (SFN) can block the progression of esophageal cancer, gastric cancer, liver cancer and bladder cancer in mice, while benzylisothiocyanate (BITC) and PEITC can inhibit mouse liver cancer, lung cancer, intestinal cancer and esophageal cancer induced by carcinogens. Moreover, PEITC has a significant inhibitory effect on rats with lung cancer and esophageal cancer caused by smoking. At present, the anticancer mechanism of isothiocyanates is to decrease the activity of phase I enzyme or to increase the activity of detoxification enzyme of stage II, thereby enhancing the metabolic ability of the carcinogen and detoxification and detoxification. In addition, the occurrence and development of cancer is also related to oxidative damage to the body, such as the increase of free radicals and related changes in oxidized system. Phenylethyl isothiocyanate (PEITC) is one of the members of the ITC family. It is one of the most widely studied substances because of its remarkable chemical prophylactic effect. PEITC is one of the best studied members of isothiocyanates (ITC), a variety of edible cruciferous vegetables including broccoli, watercress, and cabbage, and have generated particular interest because of its remarkable chemopreventive activity. Many literature reports proved that phenethyl isothiocyanate exhibited significant anti-cancer chemopreventive effects such as lung and leukemia cancer. Therefore, PEITC will be one of the most promising drugs for cancer prevention and treatment. Currently, the United States and Anderson Cancer Center is using PEITC for the treatment of lung cancer and breast cancer in the phase I clinical trial (patent number:NCICN-55120). At the same time, the Minnesota State University, Masonic Cancer Research Center combined the National Cancer Center are also carrying out PEITC treatment of lung cancer stage II clinical study. In this study, we explored the inhibitory effects as well as mechanisms of PEITC on human glioma LN229 cells, with the hope to provide evidence for the application of PEITC to the prevention and treatment of human glioma.Methods:In this study, human glioma LN229 cells were selected as the study object. The effect of PEITC on the growth and proliferation of tumor cells was studied by MTT. MTT is a yellow dye. Mitochondria in living cells contain succinate dehydrogenase, which can reduce MTT, and then produce blue-violet under the action of cytochrome c. Thus, the number of viable cells was estimated by the microplate reader 490 nm. The effect of PEITC on the cell cycle was analyzed by Annexin-V-FITC/PI flow cytometry. Annexin-V is a Ca2+-dependent phospholipid-binding protein. Annexin-V binds to phosphatidylserine on the cell surface in the presence of Ca2+, whereas the early event of cell apoptosis occurs when phosphatidylserine is exposed to the cell membrane; PI is a DNA dye, when the cells into the late apoptosis, the permeability of the cell membrane changes, PI can enter the cells and the DNA and cells, therefore, by Annexin-V/PI double staining can detect cells Apoptosis. The expression levels of reactive oxygen species (ROS) and glutathione (GSH) in tumor cells were detected by DCFH-DA and enzyme-linked immunosorbent assay (ELISA), respectively. The DCFH-DA probe can pass through the cell membrane and is catalyzed by esterase to produce DCFH, which can transform non-fluorescent DCFH into fluorescent DCF. Moreover, the activities of superoxide dismutase (SOD) and cysteinyl aspartate specific proteinase-3 (Caspase-3) were detected after PEITC treatment.Results:The results showed that cell growth was inhibited after PEITC was applied to glioma LN229 cells for 72 h, and the inhibitory effect was PEITC concentration-dependent (IC50= 20 ?M). PEITC had higher inhibition to the cell growth when the concentrations were 10 ?M and 20 ?M, and showed no toxicity or toxicity to normal cells. Therefore, the PEITC concentrations selected in the following studies were 10 ?M and 20 ?M, respectively. The effect of PEITC on the apoptosis of glioma LN229 cells was analyzed by flow cytometry. The results showed that the apoptosis rate of LN229 cells treated with 10 ?M and 20 ?M PEITC for 24 hours were 30.3% and 64.9% respectively (P< 0.05). At the same time, the percentage of cells in G2/M phase was significantly increased, and the proportion of cells in G0/G1 phase was significantly decreased (P< 0.05). The level of ROS in 10 ?M PEITC group was 4 times higher than that in control group, and that in 20 ?M PEITC group was 6 times higher than that in control group, and the statistical significance was statistically significant (P< 0.05). In addition, treatment of LN229 cells with 10 ?M and 20 ?M PEITC for 24 h resulted in a significant decrease in the expression of GSH and SOD in tumor cells. The expression of GSH in 10 ?M PEITC group and 20 ?M PEITC group were 69.3% and 42.4% compared with the control group (P< 0.05), and in 10?M PEITC group and 20 uM PEITC group, the expression of SOD were 60.7% and 20.6% compared with the control group (P< 0.05). Moreover, the activity of Caspase-3 was significantly higher in the tumor cells treated with PEITC than those in the control group (P< 0.05).Conclusion:PEITC could inhibit the growth of glioma LN229 cell line, promote apoptosis and block the cell cycle. Furthermore, PEITC significantly increased the ROS level in tumor cells and inhibited the expression of SOD and GSH, leading to degradation or activation of caspase-3, and ultimately accelerate the death of tumor cells. In addition, since PEITC is mainly derived from vegetables, so it is weak or non-toxic for normal cells. Therefore, PEITC will be a safe and effective targeted anti-tumor drug, and will have a wide range of market applications. |
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