ABSTRACT
This study aims to investigate the role of slient mating-type information regulation 2 homolog 1(SIRT1)/tuberous sclerosis complex 2(TSC2)/mammalian target of rapamycin(mTOR) signaling pathways in the Periplaneta americana extract CⅡ-3-induced senescence of human leukemia K562 cells. K562 cells were cultured in vitro and treated with 0(control), 5, 10, 20, 40, 80, and 160 μg·mL~(-1) of P. americana extract CⅡ-3. Cell counting kit-8(CCK-8) and flow cytometry were employed to examine the proliferation and cell cycle of the K562 cells. Senescence-associated β-galactosidase stain kit(SA-β-gal) was used to detect the positive rate of senescent cells. Mitochondrial membrane potential was detected by flow cytometry. The relative mRNA level of telomerase reverse transcriptase(TERT) was determined by fluorescence quantitative PCR. The mRNA and protein levels of SIRT1, TSC2, and mTOR were determined by fluorescence quantitative PCR and Western blot, respectively. The results showed that CⅡ-3 significantly inhibited the proliferation of K562 cells and the treatment with 80 μg·mL~(-1) CⅡ-3 for 72 h had the highest inhibition rate. Therefore, 80 μg·mL~(-1) CⅡ-3 treatment for 72 h was selected as the standard for subsequent experiments. Compared with the control group, CⅡ-3 increased the proportion of cells arrested in G_0/G_1 phase, decreased the proportion of cells in S phase, increased the positive rate of SA-β-Gal staining, elevated the mitochondrial membrane potential and down-regulated the mRNA expression of TERT. Furthermore, the mRNA expression of SIRT1 and TSC2 was down-regulated, while the mRNA expression of mTOR was up-regulated. The protein expression of SIRT1 and p-TSC2 was down-regulated, while the protein expression of p-mTOR was up-regulated. The results indicated that P. americana extract CⅡ-3 induced the senescence of K562 cells via the SIRT1/mTOR signaling pathway.
Subject(s)
Humans , Animals , Periplaneta , Sirtuin 1/genetics , K562 Cells , Signal Transduction , TOR Serine-Threonine Kinases/genetics , RNA, Messenger , MammalsABSTRACT
OBJECTIVE：To study the mechanism of Periplaneta americana extract degreasing cream and CⅡ-3（shorted for “degreasing cream ”and“CⅡ-3”）reversing the multi-drug resistance of human HepG 2/ADM cells. METHODS ：MTT assay was used to investigate the toxicity effects of different concentrations of sorafenib （positive control ），degreasing cream and C Ⅱ-3 on HepG2/ADM cells ，then IC 20 was calculated. The experiment was divided into sensitivity drug ，drug-resistance group ，sorafenib group，degreasing cream group and C Ⅱ-3 group. HepG 2 cells were included in sensitivity group ，and HepG 2/ADM cells were included in the latter 4 groups. Sensitivity group and drug-resistance group were treated with routine medium ，and other 3 groups were treated with relevant medicine （IC20 as drug concentration ）. The content of ADM in HepG 2/ADM cells was determined by Laser scanning confocal microscopy. The expression of apoptosis-related protein as Bcl- 2 and Cleaved-Caspase- 9 p37 were detected by Western blotting assay. RT-qPCR and immunocytochemistry were adopted to detect mRNA and protein expressions that related to multidrug resistance [P-gp （expression produce of MDR1 gene），LRP，BCRP] and that related to enzyme-mediated multidrug resistance pathway （GST-π and Topo Ⅱ）. RESULTS ：The IC 20 of degreasing cream ，CⅡ-3 and sorafenib were （2.40±0.16）， （200.44±27.52），（18.00±1.82）μg/mL，respectively. Compared with sensitivity group ，the protein expressions of Bcl- 2，P-gp， LRP，BCRP and Topo Ⅱ，the mRNA expressions of MDR 1， LRP，BCRP and GST-π were increased significantly in drug resistance group （P＜0.05 or P＜0.01）. Compared with @qq.com drug-resistance group ，the mRNA and protein expression of MDR1 mRNA and LRP ，BCRP，GST-π were significantly decreased in degreasing cream group and C Ⅱ-3 group（P＜ 0.05 or P＜0.01）；the protein expression of Bcl- 2 and the mRNA expression of Topo Ⅱ were significantly decreased （P＜0.01）， while the protein expression level of Cleaved-Caspase- 9 p37 was significantly increased in C Ⅱ -3 group （P＜0.05）. CONCLUSIONS：Degreasing cream and C Ⅱ-3 can reverse multidrug resistance of HepG 2/ADM cells by reducing drug efflux ， promoting cell apoptosis ，reducing the mRNA and protein expression of multi-drug resistance gene as well as gene in enzyme-mediated multi-drug resistance pathway. The effect of C Ⅱ-3 is better than that of degreasing cream.
ABSTRACT
OBJECTIVE：To preliminarily study the antitumor mechanism of Periplaneta americana extract C Ⅱ-3 on MFC tumor-bearing mice. METHODS ：Balb/c mice were randomly divided into model group （normal saline 20 mL/kg）and C Ⅱ-3 group （200 mg/kg），with 6 mice in each group. MFC cell suspension （0.2 mL）was injected under the right armpit of mice. On the next day，mice were given relevant medicine intragastrically ，once a day ，for consecutive 10 d. 24 h after the last administration ，Based on the measurement of tumor size ， 1H-NMR technology combined with unsupervised PCA ，supervised PLS-DA and OPLS-DA were used to compare metabolic spectrum of liver tissue from tumor-bearing mice of 2 groups，to analyze differential metabolites and to explore the potential antitumor mechanis m of C Ⅱ -3. RESULTS ：Compared with model group ，the tumor body was significantly reduced in tumor-bearing mice of C Ⅱ-3 group. There were differences in 1H-NMR spectra between the 2 No.81960712）； groups. According to unsupervised PCA ，supervised PLS-DA and OPLS-DA ，totally six potential differential metabolites ，as glycogen （increased），pyruvate （decreased），arginine （de- creased），hydroxyproline （increased），inosine （increased） and niacinamide （increased），were identified in the liver tissue，which were mainly attributed to the metabolism of arginine ，energy and nucleic acid. CONCLUSIONS：The anti tumor effect of C Ⅱ-3 may be related to the regulation of arginine metabolism ，energy metabolism and nucleic acid metabolism.
ABSTRACT
OBJECTIVE： To investigate anti-tumor effects of Periplaneta americana polypeptide PAP-2 on H22 tumor-bearing mice. METHODS： The mice tumor-bearing model was established by subcutaneous injection of ascites of H22 hepatocellular carcinoma mice via axilla. 70 mice were randomly divided into model group （normal saline）， 5-FU group （positive drug control， 20 mg/kg）， P. americana extract skimmed cream group （200 mg/kg， calculated by extract）， CⅡ-3 group （polypeptide isolated from skimmed cream as main active ingredient， 200 mg/kg， calculated by extract） and polypeptide PAP-2 high-dose， medium-dose and low-dose groups （isolated from CⅡ-3， 200， 100， 50 mg/kg， calculated by monomer）， with 10 mice in each group. The mice in the 5-FU group were given intraperitoneal injection once every other day， while the mice in the other groups were given intragastric administration once a day， the administration cycle was 10 d. After medication， the changes of tumor were observed and the organs （spleen， thymus and liver） index were measured. Histopathological changes of tumor tissue were observed after HE staining. The contents of VEGF， IL-1β and IL-4 in serum were determined by ELISA. RESULTS： Skimmed cream， CⅡ-3 and different doses of PAP-2 could inhibit the growth of tumor in tumor-bearing mice to different extent and increase organ index， and PAP-2 showed a dose-effect relationship. The tumor inhibition rate （38.95％） of PAP-2 high dose group was significantly higher than those of skimmed cream group and CⅡ-3 group （P＜0.05）， which was close to that （40.87％） of 5-FU group （P＞0.05）. Spleen index， thymus index and liver index of mice in PAP-2 high dose group were significantly those of model group and CⅡ-3 group （P＜0.05）； and the liver index of mice in PAP-2 high dose group was significantly higher than that of skimmed cream group （P＜0.05）. In addition， PAP-2 could decrease the serum contents of VEGF and IL-4， and increased serum content of IL-1β， with high dose group showed significant difference compared with model group （P＜0.05）； the serum content of IL-1β of mice in PAP-2 high dose group was significantly higher the that of skimmed cream group and CⅡ-3 group （P＜0.05）， serum contnet of IL-4 in PAP-2 high dose group was significantly lower the that of skimmed cream group and CⅡ-3 group （P＜0.05）， but the serum content in which was significantly lower than that of skimmed cream group and CⅡ-3 group（P＜0.05）. CONCLU- SIONS： P. americana polypeptide PAP-2 it has a certern anti-tumor effects on H22 tumor-bearing mice， and its can increase the index of organs of H22 tumor-bearing mice， decrease the contents of VEGF and IL-4 in serum， increase the content of IL-1β in serum.