单启动子双表达载体pIRES-p14ARF-p53的构建及对骨肉瘤细胞增殖的抑制(英文)

背景:以往的研究表明,ADp14ARF转染p53阳性的肿瘤细胞系,可以发现明显的细胞增殖受阻现象。而转染p53阴性的肿瘤细胞系,尽管也可见肿瘤细胞增殖受阻,但在程度上明显轻于前者。同时转染p14ARF和p53两种基因,既增强p53表达又加强p53积累,能否更易促进肿瘤细胞凋亡?目的:利用基因工程技术构建双质粒表达载体pIRES-p14ARF-p53,并观察其对骨肉瘤细胞增殖生长的抑制作用。设计:随机对照观察。单位:华中科技大学同济医学院附属协和医院骨科。材料:实验于2005-01/2006-10于华中科技大学同济医学院基础医学院免疫教研室公共实验平台完成。人骨肉瘤MG-63细胞有华中科技大学同济医学院免疫教研室细胞室提供。含p53全长基因序列的质粒pIRES-p53和pIRES载体均购自武汉晶赛生物公司。方法:利用基因工程技术,将从培养的正常人肝细胞系L02细胞中扩增出的p14cDNA(0.5kb)亚克隆至pIRES载体中,通过PCR、限制性内切酶酶切鉴定重组质粒pIRES-p14ARF-p53。通过脂质体介导转染入骨肉瘤MG-63细胞中,并筛选出阳性克隆,将细胞分为3组:空白对照组(MG-63细胞),空载体对照组(稳定转染pIRES-neo细胞),p14ARF-p53组(稳定转染pIRES-p14ARF-p53细胞)。①采用流式细胞仪测定转染前后瘤细胞DNA含量和细胞周期。②逆转录PCR(RT-PCR)和Westernbolt对稳定转染后的瘤细胞p53、p14ARF蛋白的表达进行定性和半定量检测。③采用噻唑蓝比色法与细胞生长曲线观察细胞增殖情况。主要观察指标:①骨肉瘤细胞DNA含量和细胞周期。②瘤细胞p53、p14ARF蛋白的表达。③细胞增殖情况。结果:成功构建出双质粒表达载体pIRES-p14ARF-p53。①骨肉瘤细胞DNA含量和细胞周期:流式细胞仪检测发现转染后的瘤细胞多停滞于G1期。②蛋白表达检测结果:RT-PCR与Westernblot检测证实p14ARF、p53基因在靶细胞mRNA和蛋白水平分别有独立表达。③细胞生长情况:转染MG-63后24,48,72,96h瘤细胞生长抑制率分别为33.43%、69.37%、66.19%、75.26%,与空载体对照组差异显著(P<0.01)。结论:野生型p53和p14ARF可协同抑制骨肉瘤细胞的增殖促进瘤细胞的凋亡。

Upregulation of MiR-126 Delays the Senescence of Human Glomerular Mesangial Cells Induced by High Glucose via Telomere-p53-p21-Rb Signaling Pathway

Diabetic kidney disease (DKD)is a microvascular complication of type 2 diabetes.The study of DKD mechanisms is the most important target for the prevention of DKD.Renal senescence is one of the important pathogeneses for DKD,but the mechanism of renal and cellular senescence is unclear.Decreased expression of circulating miR-126 is associated with the development of DKD and may be a promising blood-based biomarker for DKD.This study is to probe the effect and mechanism of miR-126 on the aging of human glomerular mesangial cells (HGMCs)induced by high glucose.HGMCs were cultured with Roswell Park Memorial Institute (RPMI-1640)in vitro.The effect of high glucose on morphology of HGMCs was observed 72h after intervention.The cell cycle was examined by flow cytometry.The telomere length was measured by Southern blotting.The expression levels of p53,p21 and Rb proteins in p53-p21-Rb signaling pathway and p-statl,p-stat3 in JAK/STAT signaling pathway were detected by Western blotting respectively.The expression of miR-126 was examined by qRT-PCR.MiR-126 mimics was transfected into HGMCs.The effects of miR-126 mimics transfection on cell morphology,cell cycle,telomere length,p53,p21,Rb,p-stat1 and p-stat3 were observed. The results showed that high glucose not only arrested the cell cycle in G1phase but also shortened the telomere length.High glucose led to high expression of p53,p21,Rb,p-statl and p-stat3 and premature senescence of HGMCs by activating the telomere-p53-p21-Rb and JAK/STAT signaling pathways.Moreover,the miR-126 was decreased in HGMCs induced by high glucose.It was suggested that the transfection of miR-126 mimics could inhibit the telomere-p53-p21-Rb and JAK/STAT signaling pathway activity in vitro and delay the senescence of HGMCs.The results may serve as a new strategy for the treatment of DKD.

Impact of MAPK Cascade Pathway and P53 Pathway upon Liver Transplant

Summary： The change and the role of MAPK cascade pathway and P53 pathway after liver transplantation were explored. Thirty-four punctured donor liver specimens and 10 normal liver specimens were classified as group A （no rejection, n=10）, group B （mild/moderate acute rejection, n=10）, group C （serious acute rejection, n=8）, group D （chronic rejection/fibrosis, n=6） and group E （control, n= 10）. By using immunohistochemistry, the expression levels of mitogen activated protein kinase （MAPK）, Ras and P53 proteins, and by in situ hybridization, MAPK and ras mRNA expression levels were detected. The results showed that the expression levels of MAPK and Ras proteins were increased by turns in groups A, B and C, and decreased by turns in groups D and E. The protein expression of P53 was higher in the treated groups. The expression of Ras, HSP70 mRNA was identical as that of protein. It is suggested that the MAPK cascade pathway and P53 pathway can protect the hepatocytes by different mechanisms after liver transplantation. MAPKs cascade pathway repairs hepatocyte injury or accelerates hepatocytes into proliferation or differentiation. P53 pathway blocks cell cycle within G1 phase to make hepatocyte repair or apoptosis to reduce disorder differentiation.

The study on DNA methylation of p53-Bax mitochondrial apoptosis pathway in cholangiocarcinoma

Objective： To study the methylation status of several genes on p53-Bax mitochondrial apoptosis pathway and clinical significance in cholangiocarcinoma. Methods： Promoter hypermethylation of DAPK, p14 and ASC genes were detected by methylation-specific PCR. p53 gene status （exon 5-8） were examined by automated sequencing, combined with the clinical documents of patients by statistics analysis. Results： （1） We found 66.7% of 36 cases cholangiocarcinoma had methylation of at least one tumor suppressor gene. The frequency of tumor suppressor gene methylation in cholangiocarcinoma was： p14 （24%）, DAPK （30.6%）, TMSI/ASC （36.1%）. The frequency of tumor suppressor gene methylation in tissues near cancer was： DAPK （5.6%）, TMS1/ASC （8.3%）. （2） p53 gene mutations were found in 22 of 36 patients （61.1%）. （3） There were no statistically relationship among the methylation of DAPK, p14 and ASC genes. There were negative relationship differences between the methylation of p14 and p53 gene mutation （P 〈 0.05）. （4） p53 gene mutation combined with the methylation of tumor suppressor were 14 cases （38.9%）. There were statistically differences on extent of pathologic biology, differentiation and invasion （P 〈 0.05）. Conclusion： Our study indicated that methylation of p53-Bax mitochondrial apoptosis pathway in cholangiocarcinoma was a common epigenetic event. Although the methylation of ASC, DAPK genes was low, it might be significance for early diagnosis, p53 gene mutation combined with the methylation of tumor suppressor might be relationship with pathologic biology, it trended to more malignancy.

少阳主骨方介导p19^(Arf)-p53-p21^(Cip1)信号通路调控食蟹猴关节软骨退变的机制

目的根据"少阳主骨"理论,研究少阳主骨方介导p19Arf-p53-p21Cip1信号通路调控关节软骨退变的机制。方法通过X线选取13只膝关节退变的老年食蟹猴,随机选取1只进行病理学观察,其余食蟹猴随机分为少阳主骨方组、氨糖美辛组、生理盐水组,每组4只,连续灌胃8周后处死所有食蟹猴,取关节软骨进行病理学观察,RT-q PCR、Western blot检测关节软骨中p19Arf、p53、p21Cip1基因与蛋白的表达。结果老年食蟹猴KOA模型关节软骨符合KOA病理变化,3组食蟹猴Mankin评分少阳主骨方组7.38±0.52分、氨糖美辛组7.88±0.83分、生理盐水组8.38±0.74分,少阳主骨方组与生理盐水组间具有统计学差异(P<0.05);p19Arf、p53、p21Cip1基因与蛋白的表达少阳主骨方组<氨糖美辛组<生理盐水组,具有统计学差异(P<0.05)。结论少阳主骨方可以延缓关节软骨退变,调控p19Arf-p53-p21Cip1表达。

Growth arrest-specific gene 2 suppresses hepatocarcinogenesis by intervention of cell cycle and p53-dependent apoptosis

BACKGROUND Growth arrest-specific gene 2(GAS2)plays a role in modulating in reversible growth arrest cell cycle,apoptosis,and cell survival.GAS2 protein is universally expressed in most normal tissues,particularly in the liver,but is depleted in some tumor tissues.However,the functional mechanisms of GAS2 in hepatocellular carcinoma(HCC)are not fully defined.AIM To investigate the function and mechanism of GAS2 in HCC.METHODS GAS2 expression in clinic liver and HCC specimens was analyzed by real-time PCR and western blotting.Cell proliferation was analyzed by counting,MTS,and colony formation assays.Cell cycle analysis was performed by flow cytometry.Cell apoptosis was investigated by Annexin V apoptosis assay and western blotting.RESULTS GAS2 protein expression was lower in HCC than in normal tissues.Overexpression of GAS2 inhibited the proliferation of HCC cells with wide-type p53,while knockdown of GAS2 promoted the proliferation of hepatocytes(P<0.05).Furthermore,GAS2 overexpression impeded the G1-to-S cell cycle transition and arrested more G1 cells,particularly the elevation of sub G1(P<0.01).Apoptosis induced by GAS2 was dependent on p53,which was increased by etoposide addition.The expression of p53 and apoptosis markers was further enhanced when GAS2 was upregulated,but became diminished upon downregulation of GAS2.In the clinic specimen,GAS2 was downregulated in more than 60%of HCCs.The average fold changes of GAS2 expression in tumor tissues were significantly lower than those in paired non-tumor tissues(P<0.05).CONCLUSION GAS2 plays a vital role in HCC cell proliferation and apoptosis,possibly by regulating the cell cycle and p53-dependent apoptosis pathway.

DTL facilitates the Fanconi anemia pathway for ultraviolet-induced DNA repair in retinal pigment epithelial cells

The excessive energy of light,especially the invisible rays with lower wavelength,is basically absorbed by retinal pigment epithelium(RPE)and usually causes DNA damage.The molecular mechanism behind DNA damage repair response to this frequent stress in RPE is not clearly understood.In this study,we determined that the Fanconi anemia(FA)pathway was activated in human RPE ARPE-19 cells after ultraviolet(UV)B and C treatment.Moreover,immunoprecipitation(IP)of FANCD2 indicated that denticleless E3 ubiquitin protein ligase homolog(DTL)closely interacted with FANCD2.Knockdown of DTL weakened the activity of the FA pathway in ARPE-19 cells responding to UV treatment.Finally,the DTL promoter was incubated with a biotin-labeled probe and pulled down by streptavidin beads followed by the genomic DNA sonication.p53 was indicated by mass spectrum and further determined by chromatin IP assay.Taken together,our results demonstrated that DTL regulated by p53 could activate the FA pathway for UV-induced DNA damage repair in retinal pigment epithelial cells.

MAPK/ERK regulation of P53 in human epidermoid carcinoma cell line A431

Objective:To observe the impact of activation and inhibition of mitogen activated protein kinases(MAPK)/extracellular signalregulated protein kinase(ERK)signaling pathway on the proliferation and apoptosis of cutaneous squamous cell carcinoma(SCC).cells and investigate the interaction mechanism between MAPK/ERK signaling pathway and tumor suppressor gene P53 in SCC.Methods:Human A431 cells were cultured and divided into MAPK/ERK inhibition groups with low-,medium-and highconcentration of inhibitors(PD98059+DMSO),MAPK/ERK activation groups with low-,medium-and high-concentration of stimuli(IGF+PBS)and blank control group(DMSO).The cell proliferation in vitro was detected by MTT assay,with the cell apoptosis detected by flow cytometry(FCM)and the protein expression of P-ERK and P53 detected by western blot in each group.Results:The A431 cell proliferation was inhibited by different concentrations of PD98059 with a clear concentration-effect and time-effect relationship(p<.05);and the cell proliferation was promoted by the different concentrations of IGF with a clear concentration-effect and time-effect relationship(p<.05).The FCM results showed a significant increase in the apoptosis rate of A431 cells which were treated with PD98059,with a clear concentration-effect relationship(p<.05);while the apoptosis rate was decreased significantly after A431 cells were treated with IGF,also with a concentration-effect relationship(p<.05).The western blot results showed that the expression of P-ERK protein was decreased but the expression of P53 was increased after A431 cells were treated with PD98059.With the concentration of PD98059 going up,the decrease in P-ERK and the increase in P53 were more significant(p<.05);while the expression of P-ERK protein was increased but the expression of P53 was decreased after A431 cells were treated with IGF.With the concentration of IGF going up,the increase in P-ERK and the decrease in P53 were more significant(p<.05).According to Pearson correlation analysis,the expression of P53 was negatively correlated to that of P-ERK(p<.05).Conclusions:After MAPK/ERK signaling pathway was activated by IGF in A431 cells,the expression of pro-apoptotic factor P53 was decreased with the ability of cell proliferation enhanced and the ability of apoptosis reduced.However,after the inhibition of MAPK/ERK signaling pathway,the expression of pro-apoptotic factor P53 was increased with the ability of cell proliferation reduced and the ability of apoptosis increased.

Pien Tze Huang Inhibits Proliferation of Colorectal Cancer Cells through Suppressing PNO1 Expression and Activating p53/p21 Signaling Pathway

ObjectiveTo explore the regulatory effect of Pien Tze Huang (PZH) on targeting partner of NOB1 (PNO1) and it’s down-stream mediators in colorectal cancer (CRC) cells.MethodsQuantitative polymerase chain reaction was performed to determine mRNA levels of PNO1, TP53, and CDKN1A. Western blotting was performed to determine protein levels of PNO1, p53, and p21. HCT-8 cells were transduced with a lentivirus over-expressing PNO1. Colony formation assay was used to detect cell survival in PNO1 overexpression of HCT-8 cells after PZH treatment. Cell-cycle distribution, cell viability and cell apoptosis were performed to identify the effect of PNO1 overexpression on cell proliferation and apoptosis of HCT-8 cells after PZH treatment. Xenograft BALB/c nude mice bearing HCT116 cells transduced with sh-PNO1 or sh-Ctrl lentivirus were evaluated. Western blot assay was performed to detect PNO1, p53, p21 and PCNA expression in tumor sections. Terminal deoxynucleotidyl transferase dUTP nick end labling (TUNEL) assay was used to determine the apoptotic cells in tissues.ResultsPZH treatment decreased cell viability, down-regulated PNO1 expression, and up-regulated p53 and p21 expressions in HCT-8 cells (P<0.05). PNO1 overexpression attenuated the effects of PZH treatment, including the expression of p53 and p21, cell growth, cell viability, cell cycle arrest and cell apoptosis in vitro (P<0.05). PNO1 knockdown eliminated the effects of PZH treatment on tumor growth, inhibiting cell proliferation inhibition and apoptosis induction in vivo (P<0.05). Similarly, PNO1 knockdown attenuated the effects of PZH treatment on the down-regulation of PNO1 and up-regulation of p53 and p21 in vivo (P<0.05).ConclusionThe mechanism by which PZH induces its CRC anti-proliferative effect is at least in part by regulating the expression of PNO1 and its downstream targets p53 and p21.

A comprehensive view on the fisetin impact on colorectal cancer in animal models: Focusing on cellular and molecular mechanisms

Flavonoids, including fisetin, have been linked to a reduced risk of colorectal cancer(CRC) and have potential therapeutic applications for the condition. Fisetin, a natural flavonoid found in various fruits and vegetables, has shown promise in managing CRC due to its diverse biological activities. It has been found to influence key cell signaling pathways related to inflammation, angiogenesis, apoptosis, and transcription factors.The results of this study demonstrate that fisetin induces colon cancer cell apoptosis through multiple mechanisms. It impacts the p53 pathway, leading to increased levels of p53 and decreased levels of murine double minute 2, contributing to apoptosis induction. Fisetin also triggers the release of important components in the apoptotic process, such as second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI and cytochrome c. Furthermore, fisetin inhibits the cyclooxygenase-2 and wingless-related integration site(Wnt)/epidermal growth factor receptor/nuclear factor kappa B signaling pathways, reducing Wnt target gene expression and hindering colony formation. It achieves this by regulating the activities of cyclin-dependent kinase 2 and cyclin-dependent kinase 4, reducing retinoblastoma protein phosphorylation, decreasing cyclin E levels, and increasing p21 levels, ultimately influencing E2 promoter binding factor 1 and cell division cycle 2(CDC2) protein levels. Additionally, fisetin exhibits various effects on CRC cells, including inhibiting the phosphorylation of Y-box binding protein 1 and ribosomal S6 kinase, promoting the phosphorylation of extracellular signal-regulated kinase 1/2, and disrupting the repair process of DNA double-strand breaks. Moreover, fisetin serves as an adjunct therapy for the prevention and treatment of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α(PIK3CA)-mutant CRC, resulting in a reduction in phosphatidylinositol-3 kinase(PI3K) expression, Ak strain transforming phosphorylation,m TOR activity, and downstream target proteins in CRC cells with a PIK3CA mutation.These findings highlight the multifaceted potential of fisetin in managing CRC and position it as a promising candidate for future therapy development.

Luteolin protects against myocardial ischemia/reperfusion injury by reducing oxidative stress and apoptosis through the p53 pathway

Objective:Myocardial ischemia/reperfusion injury(MIRI)is an obstacle to the success of cardiac reperfusion therapy.This study explores whether luteolin can mitigate MIRI by regulating the p53 signaling pathway.Methods:Model mice were subjected to a temporary surgical ligation of the left anterior descending coronary artery,and administered luteolin.The myocardial infarct size,myocardial enzyme levels,and cardiac function were measured.Latent targets and signaling pathways were screened using network pharmacology and molecular docking.Then,proteins related to the p53 signaling pathway,apoptosis and oxidative stress were measured.Hypoxia/reoxygenation(HR)-incubated HL1 cells were used to validate the effects of luteolin in vitro.In addition,a p53 agonist and an inhibitor were used to investigate the mechanism.Results:Luteolin reduced the myocardial infarcted size and myocardial enzymes,and restored cardiac function in MIRI mice.Network pharmacology identified p53 as a hub target.The bioinformatic analyses showed that luteolin had anti-apoptotic and anti-oxidative properties.Additionally,luteolin halted the activation of p53,and prevented both apoptosis and oxidative stress in myocardial tissue in vivo.Furthermore,luteolin inhibited cell apoptosis,JC-1 monomer formation,and reactive oxygen species elevation in HR-incubated HL1 cells in vitro.Finally,the p53 agonist NSC319726 downregulated the protective attributes of luteolin in the MIRI mouse model,and both luteolin and the p53 inhibitor pifithrin-a demonstrated a similar therapeutic effect in the MIRI mice.Conclusion:Luteolin effectively treats MIRI and may ameliorate myocardial damage by regulating apoptosis and oxidative stress through its targeting of the p53 signaling pathway.Please cite this article as:Zhai P,Ouyang XH,Yang ML,Lin L,Li JY,Li YM,Cheng X,Zhu R,Hu DS.Luteolin protects against myocardial ischemia/reperfusion injury by reducing oxidative stress and apoptosis through the p53 pathway.J Integr Med.2024;22(6):652–664.

Emodin induces apoptosis in human prostate cancer cell LNCaP

Aim： To elucidate effects and mechanisms of emodin in prostate cancer cells. Methods： Viability of emodin-treated LNCaP cells and PC-3 cells was measured by MTT assay. Following emodin treatments, DNA fragmentation was assayed by agarose gel electrophoresis. Apoptosis rate and the expression of Fas and FasL were assayed by flow cytometric analysis. The mRNA expression levels of androgen receptor （AR）, prostate-specific antigen （PSA）, p53, p21, Bcl-2, Bax, caspase-3, -8, -9 and Fas were detected by RT-PCR, and the protein expression levels of AR, p53 and p21 were detected by Western blot analysis. Results： In contrast to PC-3, emodin caused a marked increase in apoptosis and a decrease in cell proliferation in LNCaP cells. The expression of AR and PSA was decreased and the expression of p53 and p21 was increased as the emodin concentrations were increased. In the same time, emodin induced apoptosis of LNCaP cells through the upregulation of caspase-3 and -9, as well as the increase of Bax/Bcl-2 ratio. However, it did not involve modulation of Fas or caspase-8 protein expression. Conclusion： In prostate cancer cell line, LNCaP, emodin inhibites the proliferation by AR and p53-p21 pathways, and induces apoptosis via the mitochondrial pathway.

p53 promotes AKT and SP1-dependent metabolism through the pentose phosphate pathway that inhibits apoptosis in response to Nutlin-3a

Nutlin-3a is a MDM2 antagonist and preclinical drug that activates p53. Cells with MDM2 gene amplification are especially prone to Nutlin-3a-induced apoptosis, though the basis for this is unclear. Glucose metabolism can inhibit apoptosis in response to Nutlin-3a through mechanisms that are incompletely understood. Glucose metabolism through the pentose phosphate pathway （PPP） produces NADPH that can protect cells from potentially lethal reactive oxygen species （ROS）. We compared apoptosis and glucose metabolism in cancer cells with and without MDM2 gene amplification treated with Nutlin-3a. Apoptosis in MDM2-amplified cells was associated with a reduction in glycolysis and the PPP, reduced NADPH, increased ROS, and depletion of the transcription factor SP1, which normally promotes PPP gene expression. In contrast, glycolysis and the PPP were maintained or increased in MDM2 non-amplified cells treated with Nutlin-3a. This was dependent on p53-mediated AKT activation and was associated with maintenance of SP1 and continued expression of PPP genes. Knockdown or inhibition of AKT, SP1, or the PPP sensitized MDM2-non-amplified cells to apoptosis. The data indicate that p53 promotes AKT and SP1-dependent activation of the PPP that protects cells from Nutlin-3a-induced apoptosis. These findings provide insight into how glucose metabolism reduces Nutlin-3a-induced apoptosis, and also provide a mechanism for the heightened sensitivity of MDM2-amplified cells to apoptosis in response to Nutlin-3a.

Potentilla anserina polysaccharide alleviates cadmium-induced oxidative stress and apoptosis of H9c2 cells by regulating the MG53-mediated RISK pathway

Oxidative stress plays a crucial role in cadmium(Cd)-induced myocardial injury.Mitsugumin 53(MG53)and its mediated reperfusion injury salvage kinase(RISK)pathway have been demonstrated to be closely related to myocardial oxidative damage.Potentilla anserina L.polysaccharide(PAP)is a polysaccharide with antioxidant capacity,which exerts protective effect on Cd-induced damage.However,it remains unknown whether PAP can prevent and treat Cd-induced cardiomyocyte damages.The present study was desgined to explore the effect of PAP on Cd-induced damage in H9c2 cells based on MG53 and the mediated RISK pathway.For in vitro evaluation,cell viability and apoptosis rate were analyzed by CCK-8 assay and flow cytometry,respectively.Furthermore,oxidative stress was assessed by 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)staining and using superoxide dismutase(SOD),catalase(CAT),and glutathione/oxidized glutathione(GSH/GSSG)kits.The mitochondrial function was measured by JC-10 staining and ATP detection assay.Western blot was performed to detect the expression of proteins related to MG53,the RISK pathway,and apoptosis.The results indicated that Cd increased the levels of reactive oxygen species(ROS)in H9c2 cells.Cd decreased the activities of SOD and CAT and the ratio of GSH/GSSG,resulting in decreases in cell viability and increases in apoptosis.Interestingly,PAP reversed Cd-induced oxidative stress and cell apoptosis.Meanwhile,Cd reduced the expression of MG53 in H9c2 clls and inhibited the RISK pathway,which was mediated by decreasing the ratio of p-Akt^(Ser473)/Akt,p-GSK3β^(Ser9)/GSK3β and p ERK1/2/ERK1/2.In addition,Cd impaired mitochondrial function,which involved a reduction in ATP content and mitochondrial membrane potential(MMP),and an increase in the ratio of Bax/Bcl-2,cytoplasmic cytochrome c/mitochondrial cytochrome c,and Cleaved-Caspase 3/Pro-Caspase 3.Importantly,PAP alleviated Cd-induced MG53 reduction,activated the RISK pathway,and reduced mitochondrial damage.Interestingly,knockdown of MG53 or inhibition of the RISK pathway attenuated the protective effect of PAP in Cd-induced H9c2 cells.In sum,PAP reduces Cd-induced damage in H9c2 cells,which is mediated by increasing MG53 expression and activating the RISK pathway.

Extract from Zanthoxylum piperitum Induces Apoptosis of AGS Gastric Cancer Cells through Akt/MDM2/p53 Signaling Pathway

Objective:To determine the effect of Zanthoxylum piperitum extracet(ZPE)on apoptosis and analyze anticancer substances in ZPE,changes in proteins related to apoptosis,and pathological changes in tumors in mouse.Methods:Fifteen 4-week-old female BALB/c nu/nu mice were divided into 3 groups depending on ZPE dose,with 5 in each group.AGS gastric carcinoma cells(1 x 10^(6) cells/200 jxL)were subcutaneously injected into the flank of each mouse.One week after the injection of AGS cells,ZPE was administered to the skin tissue[10 or 50 mg/(kg-d)]in the low-and high-dose groups,respectively for 20 days.Control animals were injected with vehicle only.After 3 weeks,the tumor was extracted and carried out for immunohistochemistry,the tendency of apoptosis and p53 in the body was checked using TdT-mediated dUTP nick-end labeling(TUNEL)assay.For 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,annexin V dead cell staining,cell cycle arrest and Western blotting,AGS gastric carcinoma cells were incubated with various concentrations of ZPE for 24 h.Cell survival rates were analyzed by MTT assays.Apoptosis was analyzed using annexin V dead cell staining and cell cycle arrest and measured using Muse cell analyzer.Results:High performance liquid chromatography(HPLC)analysis showed that ZPE contained organic sulfur compounds such as alliin and S-allylcysteine.MTT assay results revealed that ZPE(10-85»xg/mL)could effectively inhibit the growth of AGS gastric cancer cells at higher concentrations(P<0.05,P<0.01).The annexin V&dead cell staining assay and cell cycle arrest assay confirmed a dose-dependent increase in the apoptosis rate and G!phase in ZPE(10-70 jig/mL)groups.ZPE decreased the expression of anti-apoptotic proteins(p-Akt,p-MDM2,Bcl-2),while increased pro-apoptotic proteins(cleaved PARP,p53,pro-Caspase 3,Bax).TUNEL assays revealed an increase in cell apoptosis.Immunohistochemistry staining confirmed the involvement of p53.Conclusion:ZPE decreases AGS cell proliferation and induces apoptosis by inhibiting Akt and MDM2 expression.

Oncogenic potential of IDH1R132C mutant incholangiocarcinoma development in mice

AIM: To investigate whether IDH1R132 C mutant in combination with loss of p53 and activated Notch signaling promotes intrahepatic cholangiocarcinoma(ICC) development.METHODS: We applied hydrodynamic injection and sleeping beauty mediated somatic integration to induce loss of p53(via sh P53), activation of Notch [via intracellular domain of Notch1(NICD)] and/or overexpression of IDH1R132 C mutant together with the sleeping beauty transposase into the mouse liver. Specifically, we co-expressed sh P53 and NICD(sh P53/NICD, n = 4), sh P53 and IDH1R132C(sh P53/IDH1R132 C, n = 3), NICD and IDH1R132C(NICD/IDH1R132 C, n = 4), as well as NICD, sh P53 and IDH1R132C(NICD/sh P53/IDH1R132 C, n = 9) in mice. Mice were monitored for liver tumor development and euthanized at various time points. Liver histology was analyzed by hematoxylin and eosin staining. Molecular features of NICD/sh P53/IDH1R132 C ICC tumor cells were characterized by Myc tag, Flag tag, Ki-67, p-Erk and p-AKT immunohistochemical staining. Desmoplastic reaction in tumor tissues was studied by Picro-Sirius red staining.RESULTS: We found that co-expression of sh P53/NICD, sh P53/IDH1R132 C or NICD/IDH1R132 C did not lead to liver tumor formation. In striking contrast, coexpression of NICD/sh P53/IDH1R132 C resulted in ICC development in mice(P < 0.01). The tumors could be identified as early as 12 wk post hydrodynamic injection. Tumors rapidly progressed, and by 18 wk post hydrodynamic injection, multiple cystic lesions could be identified on the liver surface. NICD/sh P53/IDH1R132 C liver tumors shared multiple histological features of human ICCs, including hyperplasia of irregular glands. Importantly, all tumor cells were positive for the biliary epithelial cell marker cytokeratin 19. Extensive collagen fibers could be visualized in tumor tissues using Sirus red staining, duplicating the desmoplastic reaction observed in human ICC. Tumors were highly proliferative and expressed ectopically injected genes. Together these studies supported that NICD/sh P53/IDH1R132 C liver tumors were indeed ICCs. Finally, no p-AKT or p-ERK positive staining was observed, suggesting that NICD/sh P53/IDH1R132 C driven ICC development was independent of AKT/m TOR and Ras/MAPK signaling cascades. CONCLUSION: We have generated a simple, nongermline murine ICC model with activated Notch, loss of p53 and IDH1R132 C mutant. The study supported the oncogenic potential of IDH1R132 C.

Huanglian decoction suppresses the growth of hepatocellular carcinoma cells by reducing CCNB1 expression

BACKGROUND Hepatocellular carcinoma(HCC)is one of the most prevalent cancers in human populations worldwide.Huanglian decoction is one of the most important Chinese medicine formulas,with the potential to treat cancer.AIM To investigate the role and mechanism of Huanglian decoction on HCC cells.METHODS To identify differentially expressed genes(DEGs),we downloaded gene expression profile data from The Cancer Genome Atlas Liver Hepatocellular Carcinoma and Gene Expression Omnibus(GSE45436)databases.We obtained phytochemicals of the four herbs of Huanglian decoction from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform.We also established a regulatory network of DEGs and drug target genes and subsequently analyzed key genes using bioinformatics approaches.Furthermore,we conducted in vitro experiments to explore the effect of Huanglian decoction and to verify the predictions.In particular,the CCNB1 gene was knocked down to verify the primary target of this decoction.Through the identification of the expression levels of key proteins,we determined the primary mechanism of Huanglian decoction in HCC.RESULTS Based on the results of the network pharmacological analysis,we revealed 5 bioactive compounds in Huanglian decoction that act on HCC.In addition,a protein-protein interaction network analysis of the target genes of these five compounds as well as expression and prognosis analyses were performed in tumors.CCNB1 was confirmed to be the primary gene that may be highly expressed in tumors and was significantly associated with a worse prognosis.We also noted that CCNB1 may serve as an independent prognostic indicator in HCC.Moreover,in vitro experiments demonstrated that Huanglian decoction significantly inhibited the growth,migration,and invasiveness of HCC cells and induced cell apoptosis and G2/M phase arrest.Further analysis showed that the decoction may inhibit the growth of HCC cells by downregulating the CCNB1 expression level.After Huanglian decoction treatment,the expression levels of Bax,caspase 3,caspase 9,p21 and p53 in HCC cells were increased,while the expression of CDK1 and CCNB1 was significantly decreased.The p53 signaling pathway was also found to play an important role in this process.CONCLUSION Huanglian decoction has a significant inhibitory effect on HCC cells.CCNB1 is a potential therapeutic target in HCC.Further analysis showed that Huanglian decoction can inhibit HCC cell growth by downregulating the expression of CCNB1 to activate the p53 signaling pathway.

Aberrant calcium signalling downstream of mutations in TP53 and the PI3K/AKT pathway genes promotes disease progression and therapy resistance in triple negative breast cancer

Triple-negative breast cancer(TNBC)is characterized as an aggressive form of breast cancer(BC)associated with poor patient outcomes.For the majority of patients,there is a lack of approved targeted therapies.Therefore,chemotherapy remains a key treatment option for these patients,but significant issues around acquired resistance limit its efficacy.Thus,TNBC has an unmet need for new targeted personalized medicine approaches.Calcium(Ca^(2+))is a ubiquitous second messenger that is known to control a range of key cellular processes by mediating signalling transduction and gene transcription.Changes in Ca^(2+)through altered calcium channel expression or activity are known to promote tumorigenesis and treatment resistance in a range of cancers including BC.Emerging evidence shows that this is mediated by Ca^(2+)modulation,supporting the function of tumour suppressor genes(TSGs)and oncogenes.This review provides insight into the underlying alterations in calcium signalling and how itplays a key role in promoting disease progression and therapy resistance in TNBC which harbours mutations intumour protein p53(TP53)and the PI3K/AKT pathway.

Network pharmacology analysis combined with experimental verification of the molecular mechanism of Xihuang pill in treating liver cancer

Background:Xihuang pill is a kind of traditional Chinese medicine,which has been widely used in the treatment of kinds of cancer.However,there is still a lack of systematic understanding of the molecular mechanism of Xihuang pill in the treatment of liver cancer.In this work,we aim to explore the molecular mechanism of Xihuang pill in treating liver cancer.Methods:The functional components in Xihuang pill were collected from Traditional Chinese Medicine Database and Analysis Platform.The target genes of these components were also collected using Traditional Chinese Medicine Database and Analysis Platform.The target genes of liver cancer were predicted using GeneCards database.The intersecting genes were then analyzed with Venn diagrams.Kyoto Encyclopedia of Genes and Genomes and Database for Annotation,Visualization,and Integrated Discovery were used to analyze the pathway.Then,cell counting kit-8 was used to measure the half-maximal inhibitory concentration of Xihuang pills.The living dead cell staining method was used to observe the survival of cells.HepG2 cell apoptosis was tested by flow cytometry with fluorescein isothiocyanate/propidium iodide double staining method,and then the mitochondrial damage was also detected by flow cytometry.The expression of target genes was detected by quantitative real-time polymerase chain reaction.Results:A total of 130 compounds and 198 genes were identified as potential active ingredients and putative liver cancer‑related targets.We obtained 1,899 disease targets and 297 transcriptome targets from the database.Six drug-disease intersecting genes,CCNB1,BIRC5,TOP2A,ESR1,IGF2 and IGFBP3 were obtained.They are enrichment in apoptosis,PI3K-AKT signaling pathway,MAPK signaling pathway,pathways in cancer and p53 signaling pathway.Besides,it was found that the apoptosis rate of the HepG2 cells in Xihuang pill treated group was significantly higher than that of the control group.And the apoptosis rate gradually increased in a dose dependent manner of Xihuang pill treatment.Xihuang pill also induced the mitochondrial membrane potential damage.Compared with the control group,the expression level of CCNB1 and BIRC5 was induced,while the expression level of IGF2 was reduced after Xihuang pill treatment.Conclusion:Xihuang pill may act on six proteins(CCNB1,BIRC5,TOP2A,ESR1,IGF2 and IGFBP3)and cover multiple pathways to form a therapeutic network to treat liver cancer.