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Objective To evaluate the effect of aminolevulinic acid-based photodynamic therapy (ALA-PDT) on the expression of protein kinase D1 (PKD1) in a cutaneous squamous cell carcinoma cell line A431,and to explore the mechanism underlying ALA-PDT-induced apoptosis of A431 ceils.Methods A431 cells were cultured in vitro,and cell counting kit-8 (CCK-8) assay was performed to select the optimal combination of ALA concentration and PDT dose with the strongest proliferation inhibitory effect.A431 ceils at exponential growth phase were randomly divided into 4 groups:control group receiving no treatment,ALA group treated with ALA solution alone,PDT group treated with PDT alone,and ALA-PDT group treated firstly with ALA solution and then with PDT.After 12-,24-,36-and 48-hour additional culture,CCK-8 assay was conducted to evaluate the cellular proliferation inhibition,and the apoptosis rate at the time point of the strongest proliferation inhibitory effect was measured by flow cytometry.RT-PCR was performed to determine the expression of protein kinase D1 gene (PRKD1) in A431 cells at different time points after the ALA-PDT treatment,and Western blot analysis to measure protein expression of PKD 1 and its phosphorylation at Tyr463 (pTyr463) and Ser916 (pSer916) in A431 cells.Results The combination of ALA at the concentration of 1.5 mmol/L with PDT at an irradiation dose of 2 J/cm2 was optimal due to its strongest proliferation inhibitory effect.After 12-,24-,36-and 48-hour additional culture,there were significant differences in the proliferation inhibition rate among the 4 groups (F =39.56,P ＜ 0.05).At 24 hours after the treatment,the ALA-PDT group showed significantly higher proliferation inhibition rate (46.26％ ± 1.25％) compared with the ALA group (14.65％ ± 0.33％,P ＜ 0.05),PDT group (14.96％ ± 0.68％,P ＜ 0.05) and control group (11.98％ ± 0.32％,P ＜ 0.05),as well as compared with that at 12 hours (P ＜ 0.05).At 24 hours after the treatment,the apoptosis rate significantly differed among the 4 groups (F =16.32,P ＜ 0.05),and the ALA-PDT group showed a significantly higher apoptosis rate (41.92％ ± 3.23％) compared with the control group (4.67％ ± 0.88％,P ＜ 0.05),ALA group (7.02％ ± 1.52％,P ＜ 0.05) and PDT group (8.37％ ± 0.59％,P ＜ 0.05).At 0,6,12,24,36 and 48 hours after the treatment,there were significant differences in the mRNA expression of PRKD 1 among the 4 groups (F =22.24,P ＜ 0.05),and the mRNA expression of PRKD1 at 24 hours was significantly lower than that at 0,6,12 hours (all P ＜ 0.05),but was not significantly different from that at 36 and 48 hours (both P ＞ 0.05).No significant difference in the Ser916-phosphorylated PKD1 expression was found among the 4 groups (F =1.53,P ＞ 0.05),while there were significant differences in the expression of PKD1 and Tyr463-phosphorylated PKD 1 among the 4 groups (F =10.04,8.27,both P ＜ 0.05).Additionally,the ALA-PDT group showed significantly lower expression of PKD 1 and Tyr463-phosphorylated PKD 1 compared with the control group,ALA group and PDT group (all P ＜ 0.05).Conclusion PKD1 may be involved in the photochemical process of A431 cell apoptosis induced by ALA-PDT,and may promote the occurrence of squamous cell carcinoma by Tyr463 phosphorylation.
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OBJECTIVE: To investigate the effect of ABT-263,an anti-apoptotic protein inhibitor,on human cutaneous squamous cell carcinoma A431 cells,and to explore its molecular mechanisms. METHODS: i) Total protein was extracted from human immortalized epidermal cells( Ha Ca T cells) and A431 cells in logarithmic growth phase. The protein expression of B-cell lymphoma-2( BCL-2) and BCL2-like 1( BCL-XL) was detected by Western blotting. ii) The A431 cells were treated with ABT-263( inhibitor group) and dimethyl sulfoxide( control group) at a concentration of 50 μmol/L for 4 and 9 hours. The morphological changes of the cells were examined by transmission electron microscopy. iii) The A431 cells were treated with 0,10,25,40,and 50 μmol/L of ABT-263 for 24 hours,and the cell viability was determined by CCK-8 assay. iv) The A431 cells were treated with different doses of ABT-263,and the expression of cleaved Caspase-3, cleaved poly( ADP-ribose) polymerase-1( PARP-1), phosphorylated protein kinase B [p AKT(ser473)],phosphorylated glycogen synthase kinase-3β(p GSK3β) and phosphorylated histone H2 AX(γH2 AX) was detected by Western blot. RESULTS: The relative expression of BCL-2 and BCL-XL in A431 cells were higher than those in Ha Ca T cells( P < 0. 01). Transmission electron microscopy results showed that A431 cells in inhibitor group gradually changed from normal morphology to apoptotic morphology,showing loss of microvilli,increased nuclear chromatin density and aggregation around the nuclear membrane,and nuclear fragmentation. The cell viability of A431 cells in 10,25,40 and 50 μmol/L groups were lower than those in control group( P < 0. 05). The relative expression of cleaved Caspase-3 and cleaved PARP-1 in A431 cells in 10,30 and 50 μmol/L groups were higher than those in control group( P < 0. 05).The relative expression of p AKT( ser473) and p GSK3β in A431 cells in 10,25,40 and 50 μmol/L groups were lower than those of the control group( P < 0. 05) and γH2 AX protein expression was higher than that of the control group( P <0. 05). A431 cell viability and p GSK3β protein expression decreased with the increase of inhibitor dosage( P < 0. 01).The relative expression of cleaved Caspase-3 and γH2 AX protein increased with the increase of inhibitor dosage( P <0. 01),showing dose-effect relationship. CONCLUSION: ABT-263 can induce apoptosis of A431 cells through mitochondria pathway and induce the inactivation of AKT/GSK3β pathway,which can promote the apoptosis of A431 cells with a doseeffect relationship.
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Objective To estimate the effect of berberine on the proliferation of and expressions of apoptosisrelated factors Bax and Bcl-2 in a human skin squamous cell carcinoma cell line A431.Methods A431 cells were cultured in vitro,and classified into various groups to be treated with berberine at different concentrations (12.5,25,5,100 mg/L) or cisplatin at 250 mg/L (positive control group) for different durations (12,24,48 and 72 hours).The A431 cells remaining untreated served as the negative control group.Subsequently,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cell growth,and inverted microscopy to observe cell morphology.Real time quantitative reverse transcription-PCR and an immunofluorescence assay were conducted to measure the mRNA and protein expressions of Bax and Bcl-2 respectively.Statistical analysis was done by multi-way analysis of variance (ANOVA) using the software SPSS 13.0.Results MTr assay showed that berberine inhibited the growth of A431 cells,and the inhibitory effect increased with the increase in concentration (F =1118.312,P ＜ 0.001) and treatment duration (F =510.927,P ＜ 0.001) of berberine.Moreover,there was a significant interaction between the concentration and treatment duration of berberine (F =70.239,P ＜ 0.001).Inverted microscopy revealed that when the concentration of berberine increased,cell density was reduced,and cell morphology changed from polygonal to round with cell body shrinkage.The ratio of bax to Bcl-2 mRNA was elevated with the increase in treatment duration and concentration of berberine,and there were significant differences in the mRNA ratio among cells treated with berberine for different time durations at same concentrations (F =226.231,1300.636,4325.139 for berberine at 25,50 and 100 mg/L respectively,all P＜ 0.001).Immunofluorescence staining indicated that the fluorescence intensity of Bax was enhanced,while that of Bcl-2 was weakened after berberine treatment.Conclusions Berberine inhibits the growth of A431 cells in a dose-and timedependent manner,and may induce the apoptosis of A431 cells via regulating the expressions of Bax and Bcl-2.
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Aim To investigate the effect of baicalin on cell proliferation and cell migration in human skin SCC A431 cell line. Methods The A431 cells were incu- bated with 50 mg·L-1 baicalin. The protein level of cofilin-1 was assayed by Western blot. Cofilin-1 specific siRNA fragment was designed , synthesized and trans- fected into A431 cells. The proliferative activity and migration ability of cells were assessed by CCK8 assay and scratch wound healing assay separately. ResultsWestern blot results showed that baicalin treatment in-hibited the cofilin-1 protein expression to 49.3% com-pared with the control group. Single baicalin treatment and cofilin-1 silencing could drease the A431 cell growth and migration. And cofilin-1 silencing signifi- cantly enhanced the efficacy of baicalin. Conclusions Baicalin could significantly inhibit the tumor cell's growth and migration in the A431 cell line. And cofi-lin-1 might become the potential target gene to enhance the effect of anticancer drugs.
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PURPOSE: We performed this study to evaluate the process of radiation induced apoptosis in A431 skin epithelial cancer cell line. MATERIALS AND METHODS: Low to high dose radiation (0, 2, 5, 10, 25 Gy) was given to A431 cells by Cs-137 cell irradiator. Apoptosis was evaluated by cell morphology, dye exclusion test, and DNA laddering. RESULTS: Cell viability decreased as the radiation dose increased. Number of apoptotic bodies increased as radiation dose increased. It increased most significantly at 12 hours after irradiation. Lactate dehydrogenase activity in culture medium increased according to radiation dose and time after irradiation. CONCLUSION:: Radiation-induced apoptosis which was the main course of cell death in A431 cells could be analyzed quantitatively by counting apoptotic bodies under microscope. Apoptosis increased as radiation dose increased.