Mechanism of Action of Periplogenin on Nasopharyngeal Carcinoma Based on Network Pharmacology and Experimental Study of Vitamin E Coupled with Periplogenin Self-Assembled Nano-Prodrug for Nasopharyngeal Carcinoma.

Periplogenin is a compound extracted from cortex periplocae. In the monomers' screening for inhibiting nasopharyngeal carcinoma, we found that periplogenin can inhibit nasopharyngeal carcinoma; however, its mechanism is still unclear. In this study, the chemical structure of periplogenin was uploaded to the PubChem database in order to obtain the target of periplogenin. The NPC's differential genes were obtained by analyzing the nasopharyngeal carcinoma data in the GEO database by R software. The common target of periplogenin and nasopharyngeal carcinoma was obtained through Cytoscape. Through R software analysis, ALB, epidermal growth factor receptor (EGFR), MAPK1, ESR1, MAPK8, SRC, CASP3, HSP90AA1, AR, MAPK14 may be the main targets of periplogenin in NPC. Through go enrichment analysis, it was found that periplogenin acted mainly on nasopharyngeal carcinoma through response to steroid metabolic process, cellular response to steroid hormone stimulus, hormone-mediated, and steroid hormone signaling pathway. After enrichment analysis on the Kyoto Encyclopedia of Genes and Genomes pathway, it was found that periplocan may inhibit NPC through the MAPK signaling pathway (the main signaling pathway), and the signaling pathway of proteoglycans in cancer, and the PI3K/AKT signaling pathway as well. In this study, we also carried out the experimental study of vitamin E together with periplogenin self-assembled nano-prodrugs in the treatment of NPC, and the results showed that tumor weight of PBS group was 0.531±0.039 g, while that of PPG group and MPSSV-NPs group was 0.258±0.059 g and 0.169±0.033 g, respectively, which was lower than PBS group. And the tumor inhibition rate of MPSSV-NPs was 69.41%, which was significantly higher than that of the PPG group (51.38%). This study demonstrated the mechanism of inhibition of nasopharyngeal carcinoma (NPC) by the monomer of periplogenin based on network pharmacology. We preliminarily confirmed that vitamin E coupled with a periplogenin self-assembled nano-prodrug has obvious effect in treating nasopharyngeal carcinoma.

[Influence of Nucleostemin Expression Down-Regulation on PI3K/AKT/mTOR Signal Pathway in HL-60 Cells].

OBJECTIVE: To explore the effect of nucleostemin(NS) RNAi on the expression of signal molecules in PI3K/AKT/mTOR pathway, a candidate of p53-independent signal pathway in the leukemia HL-60 cells. METHODS: The expression of NS was interfered by transfection of P53-deficient HL-60 cells with the recombinant lentivirus expression vector NS-RNAi-GV248. The exression of NS and signal molecules of PI3K/AKT/mTOR pathway were detected by Western blot. RESULTS: The fluorescence microscopy showed that the recombinant lentivirus vector NS-RNAi-GV248 transfected HL-60 cells successfully with a 80% transfection rate. Western blot showed that the expression of NS protein was inhibited obviously in HL-60 cells, and the expression levels of AKT, p-AKT, p70s6k and p-p70s6k were not statistically different(t1=2.31,P>0.05;t2=3.62,P>0.05;t3=1.60,P>0.05;t4=2.72,P>0.05) in comparison with control; the expression of GßL protein was statistically down-regnlated (t=15.01,P=0.002). CONCLUSION: The changes of GßL protein correlats with NS knockdown. The PI3K/AKT/mTOR pathway may be one of nucleostemin p53-independent signal pathways.

[Effect of Down-Regulatory Nucleostemin Expression on Autophagy Activities in p53 Null HL-60 Leukemia Cells].

OBJECTIVE: Based on previous microarry and bioinformatic analysis results, to investigate the effect of nucleostemin(NS) expression down-regulation on autophagy activity in p53 null HL-60 leukemia cells, so as to provide evidence for studying mechanisms of p53-independent signal pathway of NS in details. METHODS: The autophagy activity of HL-60 cells after down-regulation of NS expression was detected with acidine orange staining, Western blot and transmission electron mcrioscope technique. RESULTS: The expression level of NS in test groups was lower than that in blank control and negative control groups after HL-60 cells were readily transinfected by lentivirus. The result of acidine orange staining showed that the number of acid vesicular organelle in test groups(22.4±0.76)% was higher than that in blank control groups(3.1±0.28)% and negative control groups(6.2±0.64)% (P<0.05). Western blot showed that the ratio of LC3II/LC3I in test groups(1.537±0.072) was higher than that in blank control and negative control groups (1.010±0.039) and (0.608±0.008). The result of transmission electron mcrioscopy also showed that the number of autophagosomes in test group(8.7±3.1) was higher than that in the blank control and negative control groups(4.2±1.2) and (2.3±0.5). CONCLUSION: Autophagy activty can be enhanced after the level of NS was down regulated. The change indicates the signaling transductions screened by bioinformatic analysis may be one of p53-independent pathway of NS, which lays a foundation for contineously studying key points of p53-independent signal pathway of NS.

Gene expression profiling of NB4 cells following knockdown of nucleostemin using DNA microarrays.

Nucleostemin (NS) is mainly expressed in stem and tumor cells, and is necessary for the maintenance of their self-renewal and proliferation. Originally, NS was thought to exert its effects through inhibiting p53, while recent studies have revealed that NS is also able to function independently of p53. The present study performed a gene expression profiling analysis of p53­mutant NB4 leukeima cells following knockdown of NS in order to elucidate the p53­independent NS pathway. NS expression was silenced using lentivirus­mediated RNA interference technology, and gene expression profiling of NB4 cells was performed by DNA microarray analysis. A total of 1,953 genes were identified to be differentially expressed (fold change ≥2 or ≤0.5) following knockdown of NS expression. Furthermore, reverse­transcription quantitative polymerase chain reaction analysis was used to detect the expression of certain candidate genes, and the results were in agreement with the micaroarray data. Pathway analysis indicated that aberrant genes were enhanced in endoplasmic, c­Jun N­terminal kinase and mineral absorption pathways. The present study shed light on the mechanisms of the p54­independent NS pathway in NB4 cells and provided a foundation for the discovery of promising targets for the treatment of p53-mutant leukemia.

[Expression of CD71 on cell proliferation in hematologic malignancy and its correlation with Ki-67].

OBJECTIVE: This study was to explore the expression of CD71, as a proliferation indicator, on cell proliferaration in hematologic malignancy and its correlation with Ki-67, so as to assess the feasibility of CD71 instead of Ki-67 for assaying cell proliferation by flow cytometry (FCM). METHODS: (1) Compared with mature B lymphoctyes during stationary phase in peripheral blood from healthy people, the cell cycle and the expression of CD71 and Ki-67 of cell lines from patients with leukemia and lymphoma were examined, the correlation among CD71, S-phase cell fraction (SPF) and Ki-67 were analyzed; (2) Compared with mature B lymphoctyes in bone marrow from non-hematologic disease patients, the expression and correlation of CD71 and Ki-67 of all kinds of leukemic cells and myeloma cells from bone marrow were analyzed by using Ki-67/CD71/CD45/CD123, Ki-67/CD71/CD45/CD20 or Ki-67/CD71/CD45/CD138. RESULTS: (1) in respect to the expression rate of CD71 on tumor cell lines, the expression rate of CD71 on HL-60 cells was (99.77 ± 0.064)%, the expression rate of CD71 on NB4 cells was (99.23 ± 0.12)%, the expression rate on THP-1 cells was (98.90 ± 0.30)% and the expression rate on K562 cells was (97.03 ± 0.15)% in myelogenous leukemia cell lines, the expression rate of CD71 on Raji cells was (99.35 ± 0.21)% and the expression rate on Mino cell was (96.95 ± 0.42)% in lymphoma cell lines, which were also obviously higher than that on cells of the control group (P < 0.05); (2) in respect to the expression rate of CD71 on tumor cells in bone marrow, the expression rate of CD71 on poorly differentiated AML(M1 and M2) cells was (51.50 ± 19.31)%, the expression rate of CD71 on acute promyelocytic leukemia (AML-M3) cells was (35.71 ± 14.02) %, the expression rate of CD71 on acute monocytic leukemia (AML-M5) cells was (30.54 ± 14.38)%, the expression rate of CD71 on acute T lymphoblastic leukemia cells was (68.40 ± 20.83)%, the expression rate of CD71 on acute B lymphoblastic leukemia was (39.67 ± 18.27)%, the expression rate of CD71 on multiple myeloma (MM) cells was (55.49 ± 18.15%), the expression rate of CD71 on chronic lymphocytic leukemia(CLL) was (1.32 ± 0.33%), which were also higher than that on cells in the control group(P < 0.05) except for CLL cells (P > 0.05); (3) CD71 had a positive linear corrlation with SPF in cell lines (r = 0.914, P < 0.05), and also had a positive linear corrlation with Ki-67 in cell lines and carcinoma cells from bone marrow (r = 0.894,r = 0.904, P < 0.05). CONCLUSION: The CD71 can take the place of Ki-67 as an indicator of cell proliferation activity of hematologic malignancies and the determination CD71 by FCM is simpler and better than that of Ki-67 in respest of methodology.

Gene expression profiling of HL-60 cells following knockdown of nucleostemin using DNA microarrays.

Nucleostemin (NS) plays an important role in tumorigenesis and progression. Most studies consider that NS plays its role through combining with p53 and inhibiting it, however our previous studies revealed that NS could also function without the existence of p53. To date, few studies have focused on the p53-independent pathway of NS, and its molecular mechanism remains unknown. The aim of the present study was to investigate the p53-independent pathway of NS in the human acute myeloid leukemia cell line HL-60 which was p53-null by using the DNA microarray technique. Lentivirus-mediated RNA interference technique was used to knock down NS expression in HL-60 cells, and then DNA microarray and bioinformatics were used to analyze the gene expression profiling changes. The microarray data showed that after knocking down NS in HL-60 cells, 2,628 differentially expressed genes were identified through ≥ 2 or ≤ 0.5-fold-change, in which 818 genes were upregulated and 1,810 genes were downregulated. Real-time quantitative polymerase chain reaction (qPCR) validated the reliability of DNA microarray data. Pathway analysis showed extensive signal pathways in HL-60 cells were influenced by inhibiting NS expression. In particular, the inhibition of PI3K-AKT pathway, JAK-STAT pathway, RAS-RAF-MEK-ERK1/2 pathway and activation of JNK pathway, p38 MAPK pathway may associate with the apoptosis of HL-60 cells after knocking down NS. The findings of this study provide insight to further explore the specific molecular mechanism of NS function in p53-null leukemia and they also lay the foundations for exploring new therapeutic targets for p53-null leukemia and even p53-null tumors.

[Down-regulatory effect of Nucleostemin expression on signal molecule of PI3K/AKT/mTOR pathway in HL-60 cells].

This study was purpose to explore the down-regulatory effect of nucleostemin (NS) expression on signal molecules of PI3K/AKT/mTOR pathway belonged to candidate ways of p53-independent signal pathway in the leukemia cells. The expression of NS was interfered by using recombinant lentivirus expression vector NS-RNAi-GV248 to transfect HL-60 cells of p53 deficiency. The expression of NS and signal molecules of PI3K/AKT/mTOR pathway were detected by using Real-time PCR. The results of showed that the HL-60 cells were transfected by recombinant lentivirus vector NS-RNAi-GV248 successfully and with transfection rate up to 80%. According to results of Real-time PCR detection, the inhibition rate of NS gene was 56.5% in HL-60 cells. And the expression levels of PI3K,AKT and GßL mRNA (0.491 ± 0.084,0.398 ± 0.164, 0.472 ± 0.097 respectively) were obviously down-regulated by silencing NS, and showed statistical difference (P < 0.05) in comparison with control (1.002 ± 0.171, 1.000 ± 0.411, 1.001 ± 0.206 respectively) . It is concluded that the changes of signal molecules of PI3K/AKT/mTOR pathway positively correlate with NS down-regulation, which provides evidence for confirming PI3K/AKT/mTOR signal pathway possible as a type of NS p53-independent pathway.