Expression of glucose transporter 3 in cutaneous squamous cell carcinoma and its effect on the proliferation, invasion and migration of A431 cells / 中华皮肤科杂志

Objective:To determine the expression of glucose transporter 3 (GLUT3) in cutaneous squamous cell carcinoma (cSCC), and to evaluate its effect on the cSCC cell line A431.Methods:From June 2016 to December 2020, 22 paraffin-embedded tissue specimens were collected from patients with pathologically confirmed cSCC in the Department of Dermatology, Peking University Third Hospital, and 20 discarded normal skin tissues after dermatological surgeries served as controls. Immunohistochemical assay was performed to determine the GLUT3 expression in cSCC tissues and normal skin tissues. Cultured A431 cells were divided into two groups: GLUT3 overexpression group transfected with a lentiviral vector carrying the SLC2A3 gene, and negative control group transfected with an empty lentiviral vector. Real-time fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of GLUT3 in A431 cells in different groups, the cell proliferation assay (MTS assay) was performed to estimate the cell proliferative activity, and the live-cell analysis system Incucyte S3 was used for real-time evaluation of the migratory and invasive abilities of A431 cells in different groups. The relative glucose consumption and lactic acid production in A431 cells at 48 hours were measured by using glucose and lactic acid assay kits, retrospectively. Two independent samples t-test was used for comparisons between two groups, and one-way analysis of variance was used for comparisons among multiple groups. Results:The GLUT3 expression was significantly higher in the cSCC tissues than in the normal skin tissues (immunohistochemical assay score: 9.39 ± 2.56 points vs. 2.30 ± 2.60 points; t = 8.91, P < 0.05). Compared with the negative control group, the mRNA and protein expression of GLUT3 markedly increased in the GLUT3 overexpression group. MTS assay showed significantly increased proliferative activity of A431 cells in the GLUT3 overexpression group compared with the negative control group after 24- and 96-hour treatment ( t = 2.49, 3.54, P = 0.048, 0.012, respectively); cell fusion rates in the scratched area were significantly higher in the GLUT3 overexpression group than in the negative control group in the cell migration assay at 6, 12 18, and 24 hours and cell invasion assay at 12, 18, and 24 hours (all P < 0.05). At 48 hours, the relative glucose consumption and lactic acid production in A431 cells were significantly higher in the GLUT3 overexpression group than in the negative control group ( t = 2.98, 2.20, P = 0.011, 0.038, respectively) . Conclusion:GLUT3 was highly expressed in the cSCC tissues, and may participate in the occurrence and development of cSCC by providing energy to cSCC cells via promoting glucose uptake in cells to enhance their proliferative, migratory and invasive abilities.

circ_0001821 regulates the proliferation, apoptosis, migration and invasion of cutaneous squamous cell carcinoma A431 cells by targeting miR-203 / 中国肿瘤生物治疗杂志

@#[摘 要] 目的： 探讨circ_0001821通过靶向miR-203调控皮肤鳞状细胞癌（cutaneous squamous cell carcinoma，CSCC）A431细胞增殖、凋亡、迁移及侵袭的分子机制。方法：选取2018年2月至2019年8月海南医学院第二附属医院收治的39例CSCC患者的癌及配对癌旁组织，以及人CSCC细胞系A431，用qPCR法检测CSCC癌和癌旁组织中circ_0001821的表达水平。利用脂质体转染技术，分别将si-circ_0001821、si-NC、miR-203 mimic、miR-NC、si-circ_0001821与anti-miR-NC、si-circ_0001821与anti-miR-203转染至A431细胞，用qPCR法检测转染细胞中circ_0001821和miR-203的表达水平，MTT法、流式细胞术和Transwell实验分别检测A431细胞的增殖、凋亡、迁移及侵袭能力，WB法检测细胞中增殖、凋亡、迁移及侵袭相关蛋白的表达水平。通过Circinteractome数据库预测circ_0001821与miR-203存在结合位点，双荧光素酶报告基因实验验证circ_0001821与miR-203的靶向关系。结果：CSCC组织中circ_0001821的表达水平显著高于癌旁组织（P<0.01）。转染si-circ_0001821可显著降低细胞中circ_0001821的表达水平（P<0.01），降低细胞增殖、迁移和侵袭能力（均P<0.01），提高细胞凋亡率（P<0.01）。双荧光素酶报告基因实验证实，circ_0001821可靶向结合miR-203。转染miR-203 mimic可显著降低A431细胞的增殖、迁移和侵袭能力（均P<0.01），提高细胞凋亡率（P<0.01）。共转染si-circ_0001821与anti-miR-203可明显逆转下调circ_0001821表达对A431细胞增殖、迁移、侵袭及凋亡的调控作用。结论：circ_0001821通过靶向miR-203调控CSCC细胞A431的增殖、迁移、侵袭能力及细胞凋亡。

Effect of small interfering RNA targeting survivin gene on the expression of survivin and biological function of a human cutaneous squamous cell carcinoma cell line A431 / 中华皮肤科杂志

Objective To evaluate the effect of small interfering RNA (siRNA)targeting survivin gene on the expression of survivin and proliferation,apoptosis,migration and invasion of a human cutaneous squamous cell carcinoma cell line A431 in vitro.Methods A survivin-specific siRNA was designed and synthesized.Cultured A431 cells were divided into 3 groups to be transfected with 50.0 nmol/L liposome complexes containing survivin-specific siRNA (survivin siRNA group),50.0 nmol/L liposome complexes containing unrelated siRNA (negative control group) and 50.0 nmol/L prepared vesicles (blank control group).Real-time quantitative reverse transcription-PCR (RT-PCR) and Western blot analysis were performed to determine the mRNA and protein expression of survivin in A431 cells,respectively.Methyl thiazolyl tetrazolium (MTT) assay was conducted to evaluate cellular proliferative activity,flow cytometry using annexin-V/propidium iodide (PI) staining to detect cell apoptosis,Transwell assay to estimate migratory and invasive activities of A431 cells,and flow cytometry to detect cell cycle changes.Results At 48 hours after transfection,the mRNA and protein expression of survivin both significantly differed among the survivin siRNA group,negative control group and blank control group (mRNA:0.56 ± 0.15,0.88 ± 0.37,0.90 ± 0.43,F =276.67,P ＜ 0.001;protein:0.59 ± 0.04,0.86 ± 0.05,0.91 ± 0.07,F =243.61,P ＜ 0.001),the survivin siRNA group showed significantly lower mRNA and protein expression of survivin compared with the negative control group and blank control group (all P ＜ 0.05),and there were no significant differences between the negative control group and blank control group (both P ＞ 0.05).Repeated measures analysis of variance showed that the transfection with survivin siRNA could significantly inhibit the proliferation of A431 cells (F =13.19,P =0.004),the proliferation inhibition rate was significantly higher in the survivin siRNA group than in the negative control group and blank control group (both P ＜ 0.05),and no significant difference was observed between the negative control group and blank control group (P ＞ 0.05).At 24 hours after transfection,the apoptosis rate significantly differed among the 3 groups (F =83.97,P =0.002).The survivin siRNA group showed a significantly higher apoptosis rate compared with the negative control group and blank control group (both P ＜ 0.05),and there was no significant difference between the negative control group and blank control group (P ＞ 0.05).At 48 hours after transfection,the survivin siRNA group showed a significantly higher proportion of cells at G2/M phase,but lower number of migratory cells and invasive cells compared with the negative control group and blank control group (all P ＜ 0.05).Conclusion Survivin-specific siRNA can inhibit the expression of survivin gene and the proliferation of A431 cells,promote cell apoptosis,and suppress cell migration and invasion,indicating that survivin may serve as a genetic target for the treatment of cutaneous squamous cell carcinoma.

Effects of turmeric volatile oil combined with cisplatin on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431 and their mechanisms / 中华皮肤科杂志

Objective To evaluate the effects of turmeric volatile oil (TVO) combined with cisplatin on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431,and to explore their mechanisms.Methods Some cultured A431 cells at exponential growth phase were divided into several groups to be treated with 5,10,20,40 and 80 mg/L TVO,as well as high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 1％ dimethyl sulfoxide (DMSO,control group),respectively.After 24-hour treatment,cell counting kit 8 (CCK8) assay was performed to estimate the proliferative activity of A431 cells in the above groups.Some other A431 cells were divided into 4 groups:control group treated with high-glucose DMEM containing 1％ DMSO,TVO group treated with 40 mg/LTVO,cisplatin group treated with 10 mg/L cisplatin,and TVO + cisplatin group treated with 40 mg/L TVO and 10 mg/L cisplatin.After 24-hour treatment,CCK8 assay was performed to estimate the cellular proliferative activity,inverted microscopy to observe changes in cell morphology,fluorescence microscopy to detect cell apoptosis after acridine orange (AO)/ethidium bromide (EB) double-staining,colorimetry to evaluate the activity of Caspase-3 and Caspase-9,and Western blot analysis to determine the protein expression of Caspase-3 and p-glycoprotein.Results After 24-hour treatment with 5,10,20,40 and 80 mg/L TVO,the cell proliferation rates were inhibited by (12.83 ± 6.4)％,(16.27 ± 11.4)％,(21.61 ± 9.1)％,(33.11 ± 2.0)％ and (46.00 ± 3.3)％ respectively,and the inhibition rates were all significantly higher in these groups than in the control group (4.03％ ± 1.4％,all P ＜ 0.05).The 50％ inhibitory concentration (IC50) of TVO at 24 hours was (61.66 ± 1.03) mg/L.Compared with the control group,the proliferation inhibition rates significantly increased in the TVO group,cisplatin group and TVO + cisplatin group (all P ＜ 0.05),suggesting that the combination of TVO and cisplatin showed synergistic inhibitory effects with a combination index of 1.366.Moreover,A431 cells turned round to different extents and became apoptotic in the TVO group and cisplatin group,and the TVO + cisplatin group showed obviously decreased number of cells and a large number of cell debris.The TVO + cisplatin group also showed significantly increased activity of Caspase-3 (1.520 ± 0.115) and Caspase-9 (2.760 ± 0.297) as well as protein expression of Caspase-3 (1.482 ± 0.016) compared with the TVO group (Caspase-3 activity:1.117 ± 0.095;Caspase-9 activity:1.259 ± 0.059;Caspase-3 protein expression:1.156 ± 0.006,all P ＜ 0.01) and cisplatin group (Caspase-3 activity:1.381 ± 0.089;Caspase-9 activity:1.829 ± 0.171;Caspase-3 protein expression:1.296 ± 0.021,all P ＜ 0.01),but significantly decreased p-glycoprotein expression (0.528 ± 0.014) compared with the TVO group (1.311 ± 0.011,P ＜ 0.01) and cisplatin group (1.169 ± 0.012,P ＜ 0.01).Conclusion TVO combined with cisplatin can synergistically inhibit the proliferation of A431 cells and induce cell apoptosis,which may be associated with activation of the caspase system and decreased expression of pglycoprotein.

Effect of aminolevulinic acid-based photodynamic therapy on the expression of protein kinase D1 and its phosphorylation sites in a cutaneous squamous cell carcinoma cell line A431 / 中华皮肤科杂志

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.

ABT-263 induced A431 cell apoptosis and its effect on AKT downstream signaling pathway / 中国职业医学

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.

The effect of near-infrared fluorescence conjugation on the anti-cancer potential of cetuximab / 한국실험동물학회지

This study investigated the anti-cancer potential of a near-infrared fluorescence (NIRF) molecule conjugated with Cetuximab (Cetuximab-NIRF) in six-week-old female BALB/c athymic (nu+/nu+) nude mice. A431 cells were cultured and injected into the animals to induce solid tumors. Paclitaxel (30 mg/kg body weight (BW)), Cetuximab (1 mg/kg BW), and Cetuximab-NIRF (0.25, 0.5 and 1.0 mg/kg BW) were intraperitoneally injected twice a week into the A431 cell xenografts of the nude mice. Changes in BW, tumor volume and weight, fat and lean mass, and diameter of the peri-tumoral blood vessel were determined after two weeks. Tumor volumes and weights were significantly decreased in the Cetuximab-NIRF (1 mg/kg BW) group compared with the control group (P < 0.001). Lean mass and total body water content were also conspicuously reduced in the Cetuximab-NIRF (1 mg/kg BW) group compared with the vehicle control group. Peri-tumoral blood vessel diameters were very thin in the Cetuximab-NIRF groups compared with those of the paclitaxel group. These results indicate that the conjugation of Cetuximab with NIRF does not affect the anti-cancer potential of Cetuximab and NIRF can be used for molecular imaging in cancer treatments.

Mechanism of the Inhibition of Decitabine in A431 Cells / 中国医科大学学报

Objective To investigate the effect of decitabine on the biological function of A431 cells and examine its mechanism.Methods Different concentrations (5,10,and 20 μmol/L) of decitabine were used to treat A431 cells.The effect of decitabine on cell proliferation was observed on MTT.Hoechst 33258 staining was used to detect A431 cell apoptosis after the administration of decitabine.The metastasis of A431 cells was detected via transwell assay.The expressions of the proteins related to cell proliferation,apoptosis,invasion,and metastasis were detected using Western blotting and real-time polymerase chain reaction (PCR).Results The MTT assay revealed that after treatment with 5,10,and 20 μmol/L decitabine,the inhibition rates of cell proliferation were 43.81％ ± 1.53％,48.64％ ± 4.65％,and 50.69％ ± 4.99％.With the increase in drug concentration,the rate of inhibition of A431 cell proliferation increased significantly (P ＜ 0.05).Hoechst 33258 staining showed that this treatment could promote apoptosis significantly.The transwell experiments showed that decitabine significantly inhibited the metastasis of A431 cells.Western blotting and real-time PCR showed that the expressions of Cyclin B1,CDC2,Bax,Bcl-2 and MMP2 protein and mRNA in A431 cells were significantly affected by decitabine (P ＜ 0.05).Conclusion Decitabine has a strong inhibitory effect on the biological function of vulvar squamous cell carcinoma and can be used as a chemotherapeutic agent for vulvar squamous cell carcinoma.

The effect of PKCζinhibitor on the proliferation and invasion of skin squamous carcinoma cell line A-431 / 天津医药

Objective To investigate the effect of protein kinase C (PKC)ζinhibitor T5450996 on the proliferation and invasion of skin squamous carcinoma cell line A-431. Methods PKCζinhibitor T5450996 was screened through Z′-LYTE?kit. Cell proliferation assay and cell cycle analysis were used to observe the effects of T5450996 on the proliferation of skin squamous carcinoma A-431 cells. Scratch assay and invasion assay were used to explore the effects of T5450996 on the migration and invasion of skin squamous carcinoma A-431 cells. Results The PKCζinhibitor T5450996 can inhibit the activity of PKCζkinase, and the IC50 value of T5450996 was about 35μmol/L. Compared to the control group, 35μmol/L and 70 μmol/L concentrations of T5450996 significantly suppressed the proliferation of A-431 cells and blocked the cell cycle of A-431 cells. The results of scratch assay and invasion assay indicated that the migration and invasion capacities of A-431 cells were markedly impaired after the treatments with 35μmol/L and 70μmol/L concentrations of T5450996 (P0.05). Conclusion PKCζinhibitor T5450996 significantly inhibits the proliferation and invasion capacities of skin squamous carcinoma cell line A-431, and which may be a small molecular inhibitor with potential applications in the future.

Apoptosis-inducing effect of 5-fluorouracil combined with resveratrol on tumor cells in vitro and in vivo / 中国药学杂志

OBJECTIVE: To study the effects of 5-Fu combined with resveratrol (Res) on the growth and apoptosis of A431 and TE-1 cell lines and the underlying mechanism. To study the therapeutic effects of 5-Fu/Res combination on mouse skin papilloma chemically induced by DMBA/TPA. METHODS: The effects of 5-Fu/Res combination on the viability of cancer cells were evaluated by MTT assay, growth curves assay and LDH releasing assay. The inhibitory effect of combination of the two drugs was analyzed by the method of Chou and Talalay. Apoptoses of A431 and TE-1 were determined by inverted microscope and gel electrophoresis of DNA fragment analysis. Intracellular Ca2+ concentration was determined to study the underlying mechanisms of 5-Fu and Res on the apoptosis of cancer cells. The mouse skin papilloma model was established by DMBA and TPA, the expression of actived-caspase-3 in mouse skin was examined by immunohistochemistry. RESULTS: Combination of 5-Fu and Res decreased the median inhibitory concentration (IC50) to cancer cells remarkably. 5-Fu/Res combination showed a synergistic effect on apoptosis of A431 and TE-1 cells. Much more typical morphological changes of apoptosis and amount of fragmented DNA were observed in the cells treated with 5-Fu and resveratrol in combination than that in the cells treated with the agents alone. The increase of [Ca2+]i induced by 5-Fu/Res combination might be involved in the apoptotic induction. There was a significant decrease of the number of tumors after treatment with 5-Fu and Res in the tumor-bearing mice model. Active caspase-3 in the cells of mice skin was generated by 5-Fu in combination resveratrol was more effective than either alone. CONCLUSION: The 5-Fu/Res combination shows synergistic anti-tumor effects both in vitro and in vivo.

Effects of sirolimus and starved culture on autophagy of human A431 cells / 中华皮肤科杂志

Objective To evaluate effects of sirolimus(a classic autophagy inducer)and starved culture on autophagy of a human cutaneous squamous cell carcinoma cell line A431. Methods Cultured A431 cells and HeLa (a human cervical carcinoma cell line)cells were both classified into 5 groups to be treated with DMEM alone(control group), 0.1%dimethyl sulfoxide alone(DMSO group), 20 nmol/L sirolimus(20?nmol/L sirolimus group), 80 nmol/L sirolimus(80?nmol/L sirolimus group), and Earle′s balanced salt solution(EBSS group)respectively. After 4?hour treatment, Western blot analysis was performed to measure the expressions of autophagy?related markers microtubule?associated protein 1 light chain 3A/3B (LC3A/B) and recombinant gamma?aminobutyric acid receptor associated protein(GABARAP), and acridine orange staining to determine autophagy levels in these cells. Results As Western blot analysis showed, the ratio of LC3A/B?Ⅱto LC3A/B?Ⅰin A431 cells was similar between the control group and DMSO group(P > 0.05), but significantly higher in the 20?nmol/L sirolimus group, 80?nmol/L sirolimus group and EBSS group than in the control group(all P < 0.05). Western blot results from HeLa cells were similar to those from A431 cells. Bivariate correlation analysis revealed that the protein expression of GABARAP was positively correlated with that of LC3A/B ?Ⅰ in both HeLa cells(r = 0.869, 95% CI: 0.807 - 0.999, P = 0.051)and A431 cells(r = 0.837, 95% CI: -0.173 - 0.989, P = 0.037), but negatively correlated with that of LC3A/B?Ⅱ in both HeLa cells(r = -0.742, 95% CI: -0.982 - 0.406, P = 0.042)and A431 cells(r = - 0.684, 95% CI: -0.977 - 0.500, P = 0.047). Acridine orange staining showed that the percentages of autophagosome?positive A431 cells and HeLa cells were significantly increased in both the 80?nmol/L sirolimus group(23.750% ± 0.260% and 33.307% ± 0.715% respectively)and EBSS group(32.450% ± 0.488% and 66.097% ± 1.141% respectively) compared with the control group(15.987% ± 0.242% and 14.117% ± 0.295%, respectively, all P < 0.05). Conclusion The classic autophagy inducer sirolimus and starved culture can upregulate the autophagy level of A431 cells, and GABARAP may be highly correlated with LC3A/B.

Effects of inhibition of glucose-6-phosphate dehydrogenase expression on proliferation and cell cycle distribution of A431 cells / 中华皮肤科杂志

Objective To evaluate effects of downregulation of glucose?6?phosphate dehydrogenase(G6PD) expression on proliferation and cell cycle distribution of cutaneous squamous cell carcinoma(CSCC)cells. Methods Western blot analysis was performed to measure the protein expression of G6PD in normally cultured human HaCaT keratinocytes, SCL?1 and A431 CSCC cells. When A431 cells grew to 85%-90%confluence, a small interfering RNA (siRNA)targeting G6PD(G6PD?siRNA group)and a negative control siRNA(siRNA control group)were transfected into them separately, and untransfected A431 cells served as the untransfected group. CCK?8 assay was performed to evaluate proliferative activity of the A431 cells on days 0, 1, 2, 3 and 4 after transfection, Western blot analysis to measure G6PD, cyclin D1 and CDK4 protein expressions in A431 cells, and flow cytometry to analyze cell cycle distribution in A431 cells after 48 hours of additional culture. Results The protein expression of G6PD was significantly higher in normally cultured SCL?1 cells(0.308 ± 0.023)and A431 cells(0.643 ± 0.046)than in HaCaT cells(0.100 ± 0.019, both P 0.05). Compared with the untransfected group and siRNA control group, the G6PD?siRNA group showed significantly higher proportions of A431 cells in G0/G1 phase(both P < 0.001), but significantly lower proportions of A431 cells in S phase(both P<0.001). Conclusion G6PD may play important roles in the regulation of proliferation and cell cycle distribution of CSCC cells.

Effect of turmeric volatile oil on proliferation and apoptosis of human skin SCC A431 cells / 中国中药杂志

To investigate the effect of turmeric volatile oil (TVO) on the apoptosis and proliferation of human skin SCC A431 cells, A431 cells were incubated with different concentrations (5-80 mg•L⁻¹) of TVO in vitro．The proliferation and cell cycle were assessed by CCK8 assay. The change of morphology was observed with inverted microscope. Apoptosis was evaluated with AO/EB double staining and flow cytometry (FCM); cell cycle was analyzed with FCM ．Western blot method was used to detect caspase-3 and caspase-9 protein expression. Results indicated that TVO has significant inhibitory effects on the growth of A431 cells in a dose dependent relationship, the difference between groups has statistically significant (P<0.05). TVO group compared with control group, concentrations in cells shrivel and broken phenomenon, cell apoptosis rate increased, and a dose dependent and increased the expression of caspase-3 and caspase-9. The experiment results suggested that TVO could restrain skin squamous carcinoma A431 cells proliferation, and induce its apoptosis. The mechanism may be related to increase the expression of caspase-3 and caspase-9.

Effect of berberine on the proliferation and apoptosis of a human skin squamous cell carcinoma cell line A431 / 中华皮肤科杂志

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.

Effect of baicalin on proliferation and migration activity in human skin SCC cells / 中国药理学通报

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.

Expression of adenosine triphosphate-binding cassette transporter 2 in the tissue and side population of a cell line (A431) of human cutaneous squamous cell carcinoma and its significance / 中华皮肤科杂志

ObjectiveTo investigate the expression of stem cell marker adenosine triphosphate (ATP)-binding cassette transporter 2 (ABCG2) in the tissue and side population (SP) of a cell line A431 of human cutaneous squamous cell carcinoma(SCC).MethodsSP cells were separated by flow cytometry from cultured A431 cells.Methyl thiazolyl tetrazolium(MTT) assay was used to evaluate the proliferative ability of,reverse transcription-PCR to determine the expression of ABCG2 in,SP and non-SP cells.Immunohistochemistry (MaxVision method) was carried out to detect the expression of ABCG2 protein in tissue specimens from 10 patients with SCC.ResultsSP cells existed in cultured A431 cells,and accounted for about 1.1％ of A431 cells.The SP cells had a stronger growth and colony-forming ability than non-SP cells did.The number of cell clones formed by SP cells and non-SP cells was 114.8 ± 4.95 and 44.5 ± 3.67,respectively,per well in a 24-well plate( t =27.92,P ＜ 0.01 ).The expression level of ABCG2 mRNA was significantly higher in SP cells than in non-SP cells(t =5.22,P＜ 0.01).There existed a small number of ABCG2 positive cells in SCC tissue,and ABCG2 was mainly expressed in the cytoplasm and membrane of tumor cells.ConclusionsSP cells exist in A431 cells,which have characteristics of stem cells and highly express ABCG2.ABCG2 may be a potential stem cell surface marker of SCC.

Differential expression of miR-21 in UVB irradiated HaCaT cells and A431 cells / 中华放射医学与防护杂志

Objective To study the expression level of miR-21 in UVB irradiated HaCaT cells and A431 cells. Methods Real-time qPCR was used to examine the expression level of miR-21 in HaCaT cells and A431 cells after 50 J/m2UVB radiation. The possible target genes were predicted by PicTar and performed function categories with Gostat analysis. Results Compared with the HaCaT cells, miR-21 the expression level in A431 cells increased over 4 times. At 2h and 4h after UVB irradiation, the expression level of miR-21 in HaCaT cells were up regulated, and it lowered 2 times at 8 h compared with the control.There was no further change in the expression level of miR-21 after 12 h. While miR-21 expression levels in A431 cells were not changed signifcantly. The results of target prediction and Gostat analysis suggested that PIK3R1, BCL2 and E2F3 were involved in the cell differentiation and cell process. Conclusion miR-21 possibly involved in the pathogenesis of epidermal squamous cell carcinoma and the mechanism of UVB-induced injury.

Studies on the Radiation Induced Apoptosis by Morphological and Biochemical Analysis in A431 Cells / 대한핵의학회잡지

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.