Network Pharmacology to Uncover the Molecular Mechanisms of Action of LeiGongTeng for the Treatment of Nasopharyngeal Carcinoma.

BACKGROUND Nasopharyngeal carcinoma (NPC) is a common head and neck cancer epidemic in southern China and southeast Asia. LeiGongTeng has been widely used for the treatment of cancers. The purpose of this study was to determine the pharmacological mechanism of action of LeiGongTeng in the treatment of NPC using a network pharmacological approach. MATERIAL AND METHODS The traditional Chinese medicine systems pharmacology (TCMSP) database was used to identify active ingredients and associated target proteins for LeiGongTeng. Cytoscape was utilized to create a drug-disease network and topology analysis was conducted to analyze the degree of each ingredient. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) online tool was applied for the construction and analysis of the protein-protein interaction (PPI) network, while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) functional analyses were utilized to determine drug-disease common genes. RESULTS 22 active ingredients including kaempferol, nobiletin, and beta-sitosterol, and 30 drug-disease common genes including VEGFA, CASP3, ESR1, and RELA were identified. GO analysis indicated that 94 biological processes, including RNA polymerase II, apoptotic process, response to drug, cell adhesion, and response to hypoxia, were found to be associated with NPC. The KEGG enrichment analysis showed that 58 pathways, including the PI3K-Akt signaling pathway, microRNAs in cancer, tumor necrosis factor (TNF) signaling pathway and pathways in cancer were found to be associated with NPC. CONCLUSIONS LeiGongTeng exerts its therapeutic effect through various biological processes and signaling pathways since it acts on several target genes. Systematic pharmacology can be used to predict the underlying function of LeiGongTeng and its mechanism of action in NPC.

Khz (fusion product of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis in human colon carcinoma HCT116 cells, accompanied by an increase in reactive oxygen species, activation of caspase 3, and increased intracellular Ca²âº.

Khz (a fusion mycelium of Ganoderma lucidum and Polyporus umbellatus mycelia) is isolated from ganoderic acid and P. umbellatus and it exerts antiproliferative effects against malignant cells. However, no previous study has reported the inhibitory effects of Khz on the growth of human colon cancer cells. In the present study, we found that Khz suppressed cell division and induced apoptosis in HCT116 cells. Khz cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Khz reduced cell viability and mitochondrial membrane potential levels and it also induced disruption of the mitochondrial membrane potential and increased calcium concentration and reactive oxygen species generation. Khz increased caspase 3, PARP, caspase 7, and caspase 9 levels, but reduced Bcl-2 protein levels. Flow cytometry showed that the percentage of HCT116 cells in the sub-G1 phase of the cell cycle increased in response to Khz treatment.

Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells.

BACKGROUND: Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells. METHODS: Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes. RESULTS: In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. CONCLUSIONS: In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the signaling mechanisms involve an increase in [Ca2+]i, P38 activation, and ROS generation via NADPH oxidase and mitochondria.

[Research on the effect of biyan qingdu granula drug-containing serum on inhibition of nasopharyngeal carcinoma cells growth in vitro].

OBJECTIVE: To investigate the effect of Biyan Qingdu Granula drug-containing serum (BQG-DS) on cell growth and apoptosis in nasopharyngeal carcinoma cell lines CNE1, CNE2, TWO3, C666-1, and explore the antineoplastic mechanism of Biyan Qingdu Granula. METHODS: SD rats were randomly divided into three groups: experimental (Biyan Qingdu Granula) group, positive control (cytoxan) group and negative control group. After administration of drug, the serum was collected from the treated animals. MTT assay was used to examine the effect of BQG-DS on the proliferation of CNE1, CNE2, TWO3, C666-1 cell, and flow cytometry was used to observe the cell cycle distribution. Apoptosis of CNE1, CNE2, TWO3, C666-1 cell was further investigated by inverted microscope. RESULTS: BQG-DS inhibited the proliferation of CNE1, CNE2, TWO3, C666-1 cell and the effects were in a time-and concentration-dependent manner. BQG-DS could also induce apoptosis while the G1 phase was arrested. CONCLUSION: BQG-DS inhibits proliferation of nasopharyngeal carcinoma cells via induction of apoptosis and arrest of cell cycle.

Long-term efficacy of microwave hyperthermia combined with chemoradiotherapy in treatment of nasopharyngeal carcinoma with cervical lymph node metastases.

OBJECTIVE: The long-term efficacy of microwave hyperthermia combined with chemoradiotherapy in treating nasopharyngeal carcinoma (NPC) with metastatic foci in cervical lymph nodes was evaluated. METHODS: A total of 154 cases of N2 or N3 stage NPC were randomized into two groups: hyperthermia group (76 cases) and control group (78 cases). Both received cisplatin chemotherapy and radiotherapy. In addition, the hyperthermia group further received microwave hyperthermia to the metastatic cervical nodes with different patterns (before or after radiotherapy), heating temperatures (T90 < 43° and T90 ≥ 43°) and hyperthermia episodes (< 4 times, 4-10 times and > 10 times). RESULTS: The 3-month and 5-year complete response (CR) rates of cervical lymph nodes in the hyperthermia group were significantly higher than those in the control group. The 5-year disease-free survival (DFS) rate and the 3-year / 5-year overall survival rate in the hyperthermia group were also significantly higher. There was no significant difference in 5-year metastatic rates. In the hyperthermia group, the 3-month and 5-year CR rates of T90 < 43° treatment were significantly lower than with T90 ≥ 43° treatment. The CR rate was highest when the hyperthermia was performed 4-10 times. There were no significant differences in 3-month and 5-year CR rates between hyperthermia before or after radiotherapy treatment. CONCLUSION: Microwave hyperthermia combined with chemoradiotherapy can increase local control, DFS and 3, 5-year overall survival rates of patients with N2 ~ N3 stage NPC. The heating temperature should be over 43° with hyperthermia repeated 4-10 times.

Tea polyphenols protect against irradiation-induced injury in submandibular glands' cells: a preliminary study.

AIM: To study the protective effect of tea polyphenols (TPs) on submandibular glands affected by radiation injury. METHODS: Sixty rats were randomly divided into radiation group (R-group, N = 30) and TP-pre-treated-radiation group (TPR-group, N = 30). The rats were intragastrically administered with TP or normal sodium from 14 days before radiation, continuously daily, until the experiment. All the rats in both groups were irradiated with a single exposure dose of 15 Gy gamma rays that were delivered to the head and neck areas. Ten rats of each group were anatomised on the 3rd, 6th and 30th day after irradiation, respectively. The submandibular glands of the rats were removed for the study. The morphologic changes of the submandibular glands were observed by transmission electron microscopy (TEM). The terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labelling (TUNEL) method was used to detect apoptosis of the submandibular glands' cells. RESULTS: Electron microscope observation of the submandibular glands showed that the lesions of the TPR-group were mild. Change in apoptosis of the cells was not obvious compared with the R-group. The cell apotosis was typical after irradiation in the R-group. Apoptosis index that was detected in the cells of submandibular glands of the TPR-group was statistically significantly decreased compared with the R-group (P < 0.01) on the 3rd, 6th and 30th day after irradiation. CONCLUSION: TP could protect submandibular glands from radiation injuries, and the protection mechanism may be realised by anti-apoptosis.

[Scavenging effect of Sarcandra glabra powder on radiation-induced reactive oxygen species in the parotid gland of miniature pigs].

OBJECTIVE: To investigate the effect of Sarcandra glabra in scavenging reactive oxygen species (ROS) produced by γ-ray irradiation in the parotid gland of miniature pigs. METHODS: Forty-five male miniature pigs were randomly divided into control group, radiation group and radiation plus medication group, and each group contained 3 parallel groups (subgroups a, b and c). From 1 week before exposure of the parotid gland region to 15 Gy γ-ray irradiation (which was not administered in the control group), the miniature pigs in radiation plus medication group were given Sarcandra glabra powder, while those in the other groups received an equal amount of saline. Bilateral parotid glands were taken and weighed on the days 10, 40 and 90 following the exposure in subgroups a, b, and c, respectively, and ROS content in the parotid glands were determined by enzyme-linked immunosorbent assay. RESULTS: The content of ROS was significantly lower in radiation plus medication group than in the radiation group (P<0.01). In the radiation plus medication group, the ROS content showed no significant difference between subgroups a and b or between subgroups a and c (P>0.01), but differed significantly between subgroups b and c (P<0.01). Sarcandra glabra showed a strong ROS-scavenging effect 10 days after the irradiation, and the ROS content was similar with that in the control group (P>0.01); at 40 and 90 days, the ROS-scavenging effect of Sarcandra glabra was still observable, but the ROS content was significantly higher in the irradiation plus medication group than in the control group (P<0.01). CONCLUSION: Sarcandra glabra displays a ROS-scavenging effect in the parotid gland of miniature pigs against irradiation, especially at 10 days following the exposure, which may serve as the main mechanism for the protective effect of Sarcandra glabra against radiation injury in the parotid gland.