ABSTRACT
Objective: Marsdenia tenacissima extract (MTE) is a traditional Chinese herbal medicine with anti-cancer activity. In some previous studies, different mechanism actions of the anti-cancer effect of MTE have been revealed. In this study, we first observed that MTE exhibited G2/M cell cycle arrest on two different human breast cancer cell lines, MDA-MB-231 and MCF-7 by mediating 14-3-3σ and c-myc. Methods: The effect of MTE on G2/M cell cycle arrest was evaluated in MDA-MB-231 and MCF-7 cell lines. MTT assay was done for evaluation of cell viability. Flow cytometry was employed for cell cycle analysis. Western blotting analysis and immunohistochemistry were performed to analyze the expression of G2/M cell cycle-related key protein in cells and tissue samples. Animal studies have been conducted to elucidate the anti-tumor effect of MTE. Results: Cell cycle is the backbone for developing cancer. Cell cycle proteins play a major role in the progression of cell cycle and cell proliferation. However, some key protein directly or indirectly modulate the action of cell cycle protein that highly affect cell cycle regulation. In order to investigate cellular proliferation of cancer, we observed that MTE induced the upregulation of 14-3-3σ and downregulation of c-myc, and then reduced the expression of G2/M cell cycle associated key protein, leading to the inhibition of cellular entry into mitosis phase. We also confirmed that MTE exerted a significant antitumor effect on the MDA-MB-231 xenograft model in vivo. Conclusion: G2/M cell cycle arrest occurred by the action of MTE, mediated by the upregulation of 14-3-3σ as well as downregulation of c-myc in MDA-MB-231 and MCF-7 cell lines.
ABSTRACT
Filamin A and 14-3-3-σ are closely associated with the development of breast cancer.However,the exact relationship between them is still unknown.The present study aimed to examine the interaction of filamin A with 14-3-3-σ in the invasion and migration of breast cancer.RNA interference technology was employed to silence filamin A in MDA-MB-231 cells.Real-time PCR and Western blotting were used to detect the expression of filamin A and 14-3-3-σ at mRNA and protein levels,respectively.Double immunofluorescence was applied to show their colocalization morphologically.Wound healing assay and Trans-well assay were used to testify the migration and invasion of MDA-MB-231 cells in filamin A-silenced cells.The results showed that silencing filamin A significantly increased the mRNA and protein levels of 14-3-3σ.In addition,double immunofluorescence displayed that filamin A and 14-3-3σ were predominantly colocalized in the cytoplasm of MDA-MB-231 cells.Silencing filamin A led to the enhanced fluorescence of 14-3-3σ.Furthermore,cell functional experiments showed that silencing filamin A inhibited the migration and invasion of MDA-MB-231 cells in vitro.In conclusion,silencing filamin A may inhibit the invasion and migration of breast cancer cells by upregulating 14-3-3σ.
ABSTRACT
Filamin A and 14-3-3-σ are closely associated with the development of breast cancer.However,the exact relationship between them is still unknown.The present study aimed to examine the interaction of filamin A with 14-3-3-σ in the invasion and migration of breast cancer.RNA interference technology was employed to silence filamin A in MDA-MB-231 cells.Real-time PCR and Western blotting were used to detect the expression of filamin A and 14-3-3-σ at mRNA and protein levels,respectively.Double immunofluorescence was applied to show their colocalization morphologically.Wound healing assay and Trans-well assay were used to testify the migration and invasion of MDA-MB-231 cells in filamin A-silenced cells.The results showed that silencing filamin A significantly increased the mRNA and protein levels of 14-3-3σ.In addition,double immunofluorescence displayed that filamin A and 14-3-3σ were predominantly colocalized in the cytoplasm of MDA-MB-231 cells.Silencing filamin A led to the enhanced fluorescence of 14-3-3σ.Furthermore,cell functional experiments showed that silencing filamin A inhibited the migration and invasion of MDA-MB-231 cells in vitro.In conclusion,silencing filamin A may inhibit the invasion and migration of breast cancer cells by upregulating 14-3-3σ.
ABSTRACT
Objective To explore the role of 14-3-3σ in cell cycle arrest,proliferation inhibition of HaCaT cells after UVB exposure.Methods Crystal violet assay was used to determine the viable density of HaCaT,HaCaTKD cells after being irradiated with UVB of different doses (10,20,30,40,50,60 and 80 mJ/cm2) for 48 h.After HaCaT and HaCaTKD being treated with 30 mJ/cm2irradiation,cell growth and cell cycle distribution were detected by CCK-8 assay and PI staining combined with flow cytometry,respectively.Western blot was used to evaluate the protein expression of 14-3-3σ,Cdc2,Cdc25c and Cyclin B1.Results After 48 h,the survival rate of both HaCaT and HaCaTKD decreased in a dosedependent manner.Especially,HaCaTKD cells had drastically low proliferation rate compared with normal HaCaT at 10 mJ/cm2 (t =8.83-49.63,P ＜ 0.05).The proliferation rate of HaCaTKD cells was significantly lower than that of HaCaT cells (F =32.89,P ＜ 0.05).After treatment with 30 mJ/cm2 UV irradiation,the ability of proliferation in normal HaCaT cells was recovered after 24 h while there was no proliferation in HaCaTKD cells within 72 h after the same treatment.The distribution of cell cycle has little change in HaCaTKD (P ＞ 0.05).UVB treatment led to cell cycle arrest from 6 to 18 h in HaCaT cells(t =7.41,9.22,9.16,P ＜0.05)while no cell cycle arrest could be observed in the HaCaTKID cell.Western blot detection indicated that the expression of 14-3-3σ in HaCaT cells was upregulated(t =5.42-9.57,P ＜0.05)while the Cdc25c and Cyclin B1 proteins were downregulated in both HaCaT and HaCaTKD cells (t =3.95-11.21,P ＜0.05).Specifically,Cdc2 protein decreased in HaCaT cells(t =4.93-5.37,P ＜ 0.05)but there was no change in HaCaT~ cells.Conclusions 14-3-3σ protein affects the proliferation and cell cycle of HaCaT cells after UVB irradiation.14-3-3oσ co-activates the expression of Cdc2,Cdc25cand Cyclin B1 to mediate UVB-induced G2/M arrest in HaCaT cells.
ABSTRACT
OBJECTIVE: To explore the role of 14-3-3σ and TPD52L1 in apoptosis,proliferation and cell cycle in HaCaT cells treated with low dose ultraviolet B( UVB). METHODS: i) HaCaT cells in logarithmic growth phase were exposed to UVB irradiation( cumulative exposure dose was 2. 97 ×10~(-2)J /cm~2) and were harvested after culture for 6-48 hours.HaCaT cells with no UVB irradiation were set as the control( pseudo-irradiation). The apoptosis of cells was detected by flow cytometry. The cells were divided into the control group and the UVB group exposed to UVB irradiation. They were harvested after being cultured for 0-72 hours,and monotetrazolium assay was used to detect the proliferation ability of cells.ii) HaCaT cells were randomly divided into control group( given pseudo-irradiation) and UVB group. Cells were cultured for 0-24 hours and then harvested. Flow cytometry was used to detect the proportion of G2 / M phase of cells. iii) After HaCaT cells were exposed to low dose of UVB irradiation and cultured for 3-24 or 3-30 hours,they were harvested. In addition,the control was treated with pseudo-irradiation. Real-time fluorescence quantitative polymerase chain reaction was used to detect the mRNA relative expression levels of 14-3-3σ and TPD52L1. The Western blotting was used to detect the protein relative expression of 14-3-3σ and TPD52L1 in cells. RESULTS: i) With 24 hours after UVB irradiation as the cell cycle observation time,the apoptosis of HaCaT cells reached to the maximum value and the proliferation ability to the minimum value( P < 0. 05). ii) After irradiation,the proportion of G2 / M phase in the control group showed a periodic change that it maintained at the highest levels after 6-12 hours of exposure to pseudo-irradiation,and at the time point of 18 hours it was decreased to the lowest value,the proportion of G2 / M phase in the UVB group was higher than that in control group at time points of 6,12 and 18 hours after irradiation( P < 0. 05),and sustained at the highest value,which resulted in significant G2 / M phase block. iii) After UVB treatment,the relative expression of mRNA and protein of 14-3-3σ first increased and then decreased. The relative expression level of mRNA of 14-3-3σ reached the highest level in 3 hours after irradiation( P < 0. 05),and maintained at the peak level at time points of 3-12 hours( P < 0. 05),and then gradually decreased until it returned to normal level 24 hours after irradiation; the relative expression of 14-3-3σ protein began to rise at time point of 6 hours after irradiation( P < 0. 05),and reached the peak value at time point of 18 hours( P < 0. 05),and then gradually decreased,but it did not recover to normal level after irradiation for 30 hours. The relative expression of mRNA of TPD52L1 first decreased and then increased. It reached the lowest level in 12 hours after irradiation( P <0. 05),and then increased gradually and reached the peak value after 24 hours( P < 0. 05). The relative expression of TPD52L1 protein first increased and then decreased. It got to the peak 24 hours after irradiation( P < 0. 05),and 30 hours after irradiation it returned to normal level( P < 0. 05). CONCLUSION: 14-3-3σ and TPD52L1 might jointly play an important role in cell apoptosis,proliferation and G2 / M phase block in HaCaT cells induced by low dose UVB irradiation.