ABSTRACT
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.
ABSTRACT
OBJECTIVE: To explore the changes of the serum immune cytokines in medical radiation workers exposure to low dose ionizing radiation. METHODS: Totally 244 medical professionals working with radiation(61 diagnosis radiology,51 nuclear medicine,74 radio therapeutics and 58 interventional radiology) from 7 hospitals of Guangdong Province were selected as study subjects by using the typical sampling method; 51 administration workers who did not expose to radiation were selected as control group. The radiation dose of these individuals was monitored by thermoluminescent measurement instrument for one year. Venous blood was collected and the levels of interferon γ(IFN-γ),interleukin 10(IL-10),transforming growth factor-β1(TGF-β1) in serum were examined by enzyme-linked immuno sorbent assay. RESULTS: The maximum annual average dose of radiation per person of the medical radiation workers was 0. 41 mSv/a. It was smaller than the occupational exposure limit(20. 00 mSv/a). The annual average dose of radiation per person in the group of nuclear medicine was significantly higher than those of diagnosis radiology,radio therapeutics and interventional radiology(P <0. 01). Among the male staffs,the expression of IL-10 in the diagnosis radiology group,radio therapeutics group and interventional radiology group was lower than that in the control group(P < 0. 05); the expression of IL-10 in radio therapeutics group was lower than those in nuclear medicine group and interventional radiology group(P < 0. 05); the ratio of IFN-γ/IL-10 in radio therapeutics group was higher than those in diagnosis radiology group,nuclear medicine group,interventional radiology group and control group(P < 0. 05). These individuals were divided into 3 different dose group(0. 03-,0. 06-and > 0. 15 m Sv/a) based on their average radiation dose. The expression of IL-10 in male staffs of these3 dose groups was lower than that of the male control group(P < 0. 05). CONCLUSION: Long-term low dose ionizing radiation may restrain the expression level of IL-10 in the male staffs.
ABSTRACT
Objective To explore the effects of ultraviolet on DNA damage in keratinocytes and to observe the protective role of resveratrol for the cells. Methods Comet assay was employed to evaluate the damage after radiation with different doses of UV rays (UVA, UVB and UVC) of 0, 10, 30, 50, 70 and 90 mJ/cm2, and the effects after pretreatment with various concentrations of resveratrol under irradiation with 30 mJ/cm2. Results UVA irradiation (0 ~ 90 mJ/cm2) had no significant effects on HaCaT cells. However, TailDNA%, TailLength, CometLength, TailMoment and OliveTailMoment showed both UVB and UVC induced DNA damage in a dose-de-pentent manner. UVC was more harmful than UVB at the same dose. Conclusions The DNA breakage induced by UVB and UVC is dose-dependent. As compared with UVB, UVC is more harmful to HaCaT cells. Resveratrol exerts a protective effect in HaCaT cells irradiated by UVB or UVC.
ABSTRACT
Objective To investigate the carcinogeic role of miR-365 in cuntanerous squamous cell carcinoma (cSCC).Methods Normal HaCaT cells were divided into control and irradiation groups,the later was exposed by UVB irradiation (50 J/m2).MicroRNA expression profiles of the two groups were analyzed by microRNA array.The expression variations of miR-365 in HaCaT,A431,Tca8113 and HSC-1 cells were validated by qRT-PCR analysis.The colony-forming and invasion capacities were dectected by colony forming assay and Transwell migration assay in vitro,respectively.HaCaTpre-miR365-2 highly expressing miR-365 was constructed by retroviral vector infection.Tumorigenicity evaluation was carried out by subcutaneously inject of the cells at the right back flank of nude mice.Results There were 30 microRNAs differentially expressed in HaCaT cells after UVB irradiation and miR-365 was one of the most sensitive miRNAs(as high 6.7 times as control).Expression of miR-365 in all the cSCC cell lines A431,Tca8113 and HSC-1 were significantly higher than that in HaCaT cell,in which the maximum was A431 (15.67 ±1.12 times,P ＜ 0.01),and the minimum was TcaS113 (4.72 ± 0.85 times,P ＜ 0.05).Knockdown of miR-365 in cSCC cell lines significantly inhibited the colony forming ability (t =13.68,P ＜ 0.05) and cell migration (t =19.98,P ＜ 0.05) in vitro.HaCaT cells overexpressing miR-365 by transient transfection significantly increased the ability of colony formation (t =7.11,P ＜ 0.05) and cell migration (t =22.03,P ＜0.05) in vitro.In addition,HaCaTpre-miR-365-2 cell line stably expressing miR-365 could successfully establish tumors in nude mice.Conclusions MiR-365 is an oncogene for cutaneous squamous cell carcinoma.
ABSTRACT
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.
ABSTRACT
Objective To study the function of let-7 and miR-24 in UVB-induced apoptosis.Methods After NIH3T3 cells were irradiated with UVB.Hoechest33342/PI staining was used to study the cell apoptosis and RT-PCR was used to uralyzc the expression level of let-7 and miR-24.In addition,potential target genes of these miRNAs in PicTar were classified into different function categories through GOstat software.Results Compared to the control,the NIH3T3 cells exposure to UVB appeared typical apoptotic and necrotic ceils by fluorescence microscope.The exprossion level of let-7 and miR-24 in NIH3T3 cells after UVB irradiation was higher than that of the control.Among the target genes,casp3,bc1212,map3kl and cdk5 were also involved in UVB-induced apoptosis mechanism.Conclusion Let-7 and miR-24 play a role in UVB-induced apoptosis.
ABSTRACT
The infection of HBV may cause acute and chronic hepatitis B,and potentially lead to hepatocirrhosis and liver cancer.As a defense mechanism of organism to resist external infection,RNA interference(RNAi) has become a powerful tool for us to study its effects on antiviral infection and gene therapy in recent years.In this article we summarize the mechanisms of RNA interference and the progress on anti-HBV infection studies by RNAi.
ABSTRACT
Objective: To observe the expression of Smad in diabetic rat’s kidney and the effect of BaoYuan II to it.Methods: SD rats were randomly divided into four groups: control group,model group,Benazepril-treatment group and BaoYuan II-treatment group.The latter three groups were induced to be diabetic nephropathy by intraperitoneal injection with streptozotocin and then the drugs were treated respectively.After eight weeks,the plasma albumin,blood glucose,urinary protein,urinary?2-M were determined and expression of mRNA of TGF-?1,Smad2,Smad3,Smad7 was detected by RT-PCR.Results: Compared with diabetic rats group,the level of plasma albumin was increased and the level of blood glucose,urinary protein,urinary?2-M was depressed in BaoYuan II-treatment group.The expression of TGF-?1 and Smad2,Smad3 were down-regulated and Smad7 was up-regulated in model group.The effects of BaoYuan II were similar to Benazepril.Conclusion: BaoYuan II had protected effect to diabetic nephropathy rats.It acted by regulating TGF-?1 and Smad2,Smad3,Smad7 possibly.
ABSTRACT
Objective To investigate the pro-apoptotic effects of an ?1-adrenergic inhibitor,doxazosin,in HeLa cells and the role of transcription factor activator protein-2? (AP-2?) in the process.Methods The HeLa cells were treated with doxazosin (0,10,40 or 60 ?mol/L) for 16 h,and cell apoptosis was detected by flow cytometric analysis.After doxazosin (60 ?mol/L) treated the HaLa cells,the cells transfected with AP-2? overexpressing constructs or an antisense oligonucleotide against AP-2? for 16 h,the apoptosis was analyzed with FCM.The expression of AP-2? and caspase-3 in above 3 groups of cells was detected by relative quantitative RT-PCR and Western blot analysis,respectively.Results Doxazosin increased the apoptotic rate and total cell death rate of the HeLa cells,and upregulated the expressions of AP-2? and caspase-3 in a dose-dependent manner.Overexpressing AP-2? improved the increased apoptotic rate and total cell death rate induced by doxazosin,and enhanced the increased expression of caspase-3.Whereas doxazosin-induced apoptosis and the total cell death in HeLa cells were decreased by antisense AP-2?,and antisense AP-2? in part abolished the increased effects of doxazosin on caspase-3 expression.Conclusion Doxazosin induces apoptosis in HeLa cells in a dose-dependent manner,and transcription factor AP-2? is functionally involved in this process.