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1.
Chinese Traditional and Herbal Drugs ; (24): 4208-4216, 2020.
Article in Chinese | WPRIM | ID: wpr-846234

ABSTRACT

Objective: To study the effect of rocaglaol from Aglaia odorata on HepG2 proliferation and to explore the potential anti-tumor mechanism. Methods: The MTT, colony formation, EdU incorporation, and CFDA-SE assays were used to determine the anti-proliferative activity of rocaglaol in HepG2 cells. Apoptosis and cell cycle distribution effect induced by rocaglaol were carried out by flow cytometry. The effect of rocaglaol on protein involved in the G2/M checkpoint and the MAPK pathway were performed by Western blotting analysis. Results: Rocaglaol significantly inhibited the viability of HepG2 cells in a dose-dependent and time-dependent manner. Rocaglaol was more effective than doxorubicin in the growth inhibition of HepG2 cells. However, rocaglaol-induced cytotoxicity in normal human hepatic cell line L02 was lower than that of doxorubicin. Treatment with different concentrations of rocaglaol at 48 h caused G2/M cell cycle progression inhibition, rather than apoptosis in HepG2 cells. Rocaglaol can significantly reduce the expression of G2/M cell cycle-regulating proteins cdc25C, cdc2, and cyclin B1 as well as increase the expression of ERK and JNK phosphorylation levels. Further study found that U0126 can partly abrogate the anti-proliferative activity in HepG2 cells, G2/M phase arrest and the reduction in the protein expression levels of cdc2 and cdc25C induced by rocaglaol. Conclusion: Our results demonstrated that rocaglaol was superior to doxorubicin in the inhibition of HepG2 cells proliferation and the selectivity of L02 cell activity. We provided evidence that the rocaglaol had the ability to continuously over-activate the ERK signaling in HepG2 cells, leading to the inhibition of cell proliferation through G2/M phase arrest.

2.
Military Medical Sciences ; (12): 359-362, 2017.
Article in Chinese | WPRIM | ID: wpr-612719

ABSTRACT

Objective To knockout the cell division cycle 25 homolog C(Cdc25C) gene in HeLa human cervical cancer cells and to construct HeLa Cdc25C gene knockout stable strains using clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 gene-editing system.Methods The sequence of the small guide RNA(sgRNA),which could specifically recognize the first exon of Cdc25C,was designed according to the target-designing rules of CRISPR/Cas9 for construction of eukaryotic recombinant expressional plasmids.After sequencing,the plasmid was transfected into HeLa cells.The stable Cdc25C-knocking out strains were screened through the stress of puromycin,and the knockout effect was detected by Western blotting.The cell cycle was analyzed by flow cytometry.Results The stable Cdc25C-knocking out strains were obtained.Moreover,the gene′s knockout obviously delayed the progression of G2/M phase.Conclusion The HeLa Cdc25C gene knockout stable strain is successfully built using CRISPR/Cas9 system,facilitating studies on the function of Cdc25C and the mechanism of carcinogenesis.

3.
Chinese Journal of Pathophysiology ; (12): 1611-1618, 2017.
Article in Chinese | WPRIM | ID: wpr-662744

ABSTRACT

AIM:To investigate the mechanism of the radiosensitizing effect of maximum non-cytotoxic doses of tetrandrine (Tet) on nasopharyngeal carcinoma cell lines CNE1 and CNE2.METHODS:The cells were treated with maximum non-cytotoxic doses of Tet (for CNE1 cells at 1.5 μmol/L and for CNE2 cells at 1.8 μmol/L),irradiation at 4 Gy,or combination of irradiation and maximum non-cytotoxic doses of Tet.The cell cycle distribution was analyzed by flow cytometry.The protein levels of γ-H2AX,cleaved caspase-3,p-CDC25C,CDK1,p-CDK1,cyclin B1,ERK and p-ERK were determined by Western blot.RESULTS:The expression of γ-H2AX was increased in CNE1 cells and CNE2 cells after combined treatment with irradiation and maximum non-cytotoxic doses of Tet.The percentages of CNE1 cells and CNE2 cells at G2/M phase in irradiation group were (18.09 ±0.42)% and (18.48 ± 1.32)%,respectively,which were decreased to (15.88 ± 1.04) % and (13.80 ± 0.82) % in combined treatment group,respectively (P < 0.05).Combined treatment enhanced the increase in the protein level of cleaved caspase-3 caused by irradiation.The protein levels of pCDC25C and p-CDK1 were increased in a dose-dependent manner by Tet treatment (P < 0.05),while the expression of CDK1 showed no difference among different doses of Tet treatments.The protein levels of p-CDC25C,p-CDK1 and CDK1 showed no difference after the treatment with maximum non-cytotoxic doses of Tet.The combined treatment with irradiation and the maximum non-cytotoxic doses of Tet decreased the protein levels of p-CDC25C and p-CDK1 (P <0.05),increased the expression of cyclin B1,and had no influence on the expression of CDK1 ( P <0.05).The combined treatment resulted in an increase in the protein level of p-ERK1 (P < 0.05).CONCLUSION:The maximum non-cytotoxic doses of Tet enhance the DNA damage and apoptosis in CNE1 cells and CNE2 cells caused by irradiation,and the mechanism might be associated with ending of G2/M arrest via activation of ERK/CDC25C/CDK1/cyclin B1 pathways.

4.
Chinese Journal of Pathophysiology ; (12): 1611-1618, 2017.
Article in Chinese | WPRIM | ID: wpr-660649

ABSTRACT

AIM:To investigate the mechanism of the radiosensitizing effect of maximum non-cytotoxic doses of tetrandrine (Tet) on nasopharyngeal carcinoma cell lines CNE1 and CNE2.METHODS:The cells were treated with maximum non-cytotoxic doses of Tet (for CNE1 cells at 1.5 μmol/L and for CNE2 cells at 1.8 μmol/L),irradiation at 4 Gy,or combination of irradiation and maximum non-cytotoxic doses of Tet.The cell cycle distribution was analyzed by flow cytometry.The protein levels of γ-H2AX,cleaved caspase-3,p-CDC25C,CDK1,p-CDK1,cyclin B1,ERK and p-ERK were determined by Western blot.RESULTS:The expression of γ-H2AX was increased in CNE1 cells and CNE2 cells after combined treatment with irradiation and maximum non-cytotoxic doses of Tet.The percentages of CNE1 cells and CNE2 cells at G2/M phase in irradiation group were (18.09 ±0.42)% and (18.48 ± 1.32)%,respectively,which were decreased to (15.88 ± 1.04) % and (13.80 ± 0.82) % in combined treatment group,respectively (P < 0.05).Combined treatment enhanced the increase in the protein level of cleaved caspase-3 caused by irradiation.The protein levels of pCDC25C and p-CDK1 were increased in a dose-dependent manner by Tet treatment (P < 0.05),while the expression of CDK1 showed no difference among different doses of Tet treatments.The protein levels of p-CDC25C,p-CDK1 and CDK1 showed no difference after the treatment with maximum non-cytotoxic doses of Tet.The combined treatment with irradiation and the maximum non-cytotoxic doses of Tet decreased the protein levels of p-CDC25C and p-CDK1 (P <0.05),increased the expression of cyclin B1,and had no influence on the expression of CDK1 ( P <0.05).The combined treatment resulted in an increase in the protein level of p-ERK1 (P < 0.05).CONCLUSION:The maximum non-cytotoxic doses of Tet enhance the DNA damage and apoptosis in CNE1 cells and CNE2 cells caused by irradiation,and the mechanism might be associated with ending of G2/M arrest via activation of ERK/CDC25C/CDK1/cyclin B1 pathways.

5.
Chinese Pharmacological Bulletin ; (12): 199-203,204, 2016.
Article in Chinese | WPRIM | ID: wpr-603582

ABSTRACT

Aim To investigate the effects of diallyl di-sulfide( DADS) on G2/M arrest in Chk1/MGC803 and Chk2/MGC803 cells so as to establish stable human gastric cancer MGC803 cells with overexpression of Chk1/2 gene. Methods The colony formation, flow cytometry, RT-PCR and Western blot were used to de-tect the proliferation, cell cycle, and expression of Chk1/2 mRNA and protein, p-Chk1/2, CDC25C and cyclinB1, respectively. Results The colony formation showed that the colony forming efficiency in Chk1/MGC803 and Chk2/MGC803 cells treated by 30 mg· L-1 DADS was lower than in control group and vector group ( P <0. 05 ) . Flow cytometry demonstrated that 41. 3%, 57. 4%, 68. 9% and 42. 9% of G2/M cells in Chk1/MGC803 were increased than in MGC803 and Chk2/MGC803 , respectively after treated by DADS in 12,24, 36 and 48 h(P <0. 05). At the same time, RT-PCR disclosed that expression of Chk1 and Chk2 mRNA had no marked change. Western blot showed that total proteins of Chk1 and Chk2 and p-Chk2 had invisible change, but expression of p-Chk1 was up-reg-ulated, and CDC25C and cyclinB1 were down-regula-ted time-dependently in Chk1/MGC803 cells ( P <0. 05 ) . Conclusion DADS arrests MGC803 cells at G2/M by increasing p-Chk1 expression to cause down-regulation of CDC25C and cyclinB1 simultaneously.

6.
Chinese Pharmacological Bulletin ; (12): 221-226, 2015.
Article in Chinese | WPRIM | ID: wpr-473704

ABSTRACT

Aim To study the effects of cycle arrest and molecular mechanism in human leukemia HL-60 cells induced by diallyl disulfide ( DADS ) . Methods Cell count, colony formation in soft agar experiments and flow cytometry analysis were employed to observe the DADS-induced cell growth inhibition and the effect of cycle arrest in HL-60 cells. The expressions of Chk1/2 and its downstream element in HL-60 cells were detected by Western blot. Results Cell count revealed that population doubling time increased to 35. 03 h and 71. 82 h, respectively, from 19. 14 h in HL-60 cells treated with 60 and 120 μmol·L-1 DADS ( P0. 05). The expression of Cdc25C, CyclinB1 and CDK1 decreased after treated with 60 μmol·L-1 DADS in a time-dependent manner ( P0. 05 ) . Conclusion DADS can in-hibit the proliferation of HL-60 cells, and induce G2/M arrest through Chk1/Cdc25 C/CyclinB1/CDK1 path-way.

7.
Chinese Pharmacological Bulletin ; (12): 222-225, 2010.
Article in Chinese | WPRIM | ID: wpr-403991

ABSTRACT

Aim To explore the effects of the Melittin on growth and cell cycle of SGC-7901 cells.Methods Growth inhibition effect of Melittin was evaluated using SRB in SGC-7901 cells in vitro;Melittin induced cell cycle arrest was investigated using flow cytometry assay;reverse transcription PCR(RT-PCR)was used to detect the associated protein mRNA of cell cycle.Results Proliferation activity of SGC-7901 cells was inhibited after treatment with Melittin(1,2,4,8,16,32×10~(-3) μg·L~(-1))(P<0.05 or P<0.01)for 24 h;Flowcytometry analysis revealed that SGC-7901 cells accumulated in the G_2/M phase after treatment with Melittin(4,8×10~(-3) μg·L~(-1))for 24 h;the expression of CylinB1,CDK1 and Cdc25c mRNA were decreased.Conclutions Proliferation activity of SGC-7901 cells was inhibited by Melittin,which may be related to the inhibitory effect of Melittin on associated protein transcription in the G_2/M stage of SGC-7901 cells.

8.
Korean Journal of Obstetrics and Gynecology ; : 2156-2165, 2006.
Article in Korean | WPRIM | ID: wpr-16773

ABSTRACT

OBJECTIVE: This study was undertaken to quantitatively detect Cdc25A, Cdc25B and Cdc25C in cervical carcinoma and determine the relationship between the expression of mRNA and protein of cell division cycle (Cdc)25 phosphatase and various clinicopathologic prognostic factors of cervical carcinoma. METHODS: 39 patients diagnosed with cervical carcinoma between February 2000 to March 2005 and 10 patients with benign gynecologic disease were enrolled in this study. A reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis were used to analyze the expression of Cdc25 phosphatase mRNA and protein in fresh invasive cervical cancer tissue and normal cervix tissue. RESULTS: The mRNA expressions of Cdc25A, Cdc25B and Cdc25C in the cancer tissues were significantly greater than in the control (p=0.02, 0.01, 0.02), respectively. A Western blot analysis yielded same results (p=0.01, 0.02, 0.01). There were also significant relationships between the age and the Cdc25B mRNA expression (p=0.03), between the cell type and the Cdc25C mRNA expression (p=0.04). However, other clinicopathologic prognostic factors including stage, subtype, SCC Ag level, DNA flow cytometry, lymph node metastasis, lymphovascular space invasion and HPV positivity were not statistically significant. CONCLUSION: Our results show that Cdc25A, Cdc25B and Cdc25C expression levels were significantly greater in cervical cancer patient group than in those of control group. Thus Cdc25 phosphatase might play an important role in carcinogenesis of cervical carcinoma. Further studies based on the correlation between Cdc25 phosphatase and survival rate would be need to support Cdc25 phosphatase as a prognostic factor of cervical carcinoma.


Subject(s)
Female , Humans , Blotting, Western , Carcinogenesis , cdc25 Phosphatases , Cell Cycle , Cervix Uteri , DNA , Flow Cytometry , Genital Diseases, Female , Lymph Nodes , Neoplasm Metastasis , RNA, Messenger , Survival Rate , Uterine Cervical Neoplasms
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