MiR-34 by targeting p53 induces apoptosis and DNA damage in paclitaxel-resistant human oral squamous carcinoma cells

MicroRNA-34 (miR-34) is one the most important tumor suppressor miRNAs involving in the various aspects of oral cancer. The present study aimed to evaluate the effects of miR-34 restoration in OECM-1 oral cancer resistant to paclitaxel (OECM-1/PTX) and its underlying mechanisms through p53-mediated DNA damage and apoptosis. OECM-1 and OECM-1/PTX were transfected with miR-34 mimic and inhibitor. Cellular proliferation and apoptosis were evaluated through MTT assay and flow cytometry, respectively. The mRNA and protein expression levels of p53, p-glycoprotein (P-gp), ATM, ATR, CHK1, and CHK2 were assessed through qRT-PCR and western blotting. Rhodamin123 uptake assay was used to measure the P-gp activities. P53 expression was also suppressed by sing a siRNA transfection of cells. The expression levels of miR-34 were downregulated in OECM-1/PTX. Restoration of miR-34 led to increase in cytotoxic effects of paclitaxel in cells. In addition, the expression levels and activities of P-gp were reduced following miR-34 transfection. miR-34 transfection upregulated the p53, ATM, ATR, CHK1, and CHK2 expression levels in OECM-1/PTX cells. Furthermore, cells transfected with miR-34 showed higher levels of apoptosis. miR-34 restoration reverses paclitaxel resistance in OECM-1 oral cancer. The chemosensitive effects of miR-34 is mediated through increasing DNA damage and apoptosis in a p53 depended manner.     

Evaluation of in vivo genotoxic potential of fenofibrate in rats subjected to two-week repeated oral administration

Fenofibrate (FF), a peroxisome proliferator-activated receptor-alpha agonist, has been used as one of the hypolipidemic drugs in man and induces oxidative stress and promotes hepatocarcinogenesis in the liver of rodents. This chemical belongs to a class of non-genotoxic carcinogens, but DNA damage secondary to oxidative stress resulting from reactive oxygen species (ROS) generation is suspected in rodents given this chemical. To examine whether FF has genotoxic potential, partially hepatectomized F344 male rats were treated orally with 0, 1,000 or 2,000 mg/kg of FF for 2 weeks, followed by diet containing 0.15% 2 acetyl aminofluorene (2 AAF) for enhancement the tumor-promoting effect for 10 days and a single oral dose of carbon tetrachloride (CCl4) as the first experiment (liver initiation assay). As the second experiment, the in vivo liver comet assay was performed in hepatectomized rats, and the expression of some DNA repair genes was examined. In the liver initiation assay, the number and area of glutathione S-transferase placental form (GST-P)-positive single cells and foci did not increase in the FF treated groups. In the comet assay, positive results were obtained after 3 h of the last treatment of FF, and the expression of some DNA repair genes such as Apex1, Ogg1 and Mlh1 were upregulated in rats given the high dose of FF at 3 h after the treatment but not in 24 h after the treatment. The results of the present study suggest that FF causes some DNA damage in livers of rats, but is not a strong genotoxic substance leading to a DNA mutation since such DNA damage was repaired by the increased activity of some DNA repair genes.     

Diosmin abrogates chemically induced hepatocarcinogenesis via alleviation of oxidative stress,hyperproliferative and inflammatory markers in murine model

Hepatocellular carcinoma (HCC) is a global health problem and is fourth leading cause of cancer related deaths. Now-a-days new strategies have been accounted for the chemoprevention of liver cancer due to ineffective traditional treatments against HCC. In the present study, we have shown that diosmin attenuates 2-AAF induced hepatic toxicity and early tumor promotion markers (ODC, PCNA and Ki67), its chemopreventive efficacy against DEN initiated and 2-AAF promoted hyper-proliferation and hepatocarcinogenesis in Wistar rats. Hepatocarcinogenesis has been characterized by the presence of apparent hepatic nodules, hepatic proliferation, elevation in the levels of proliferation markers (PCNA and Ki67), and inflammatory markers (COX-2 and iNOS) in DEN and 2-AAF administered rats. Protective efficacy of diosmin has been investigated in terms of its potential in reducing the percentage of visible hepatic nodules and the restoration of early tumor markers (PCNA, Ki67 and ODC), oxidative stress biomarkers, serum cytotoxicity markers (AST, ALT and LDH), cell necrosis markers (NF-kappa B and TNF-α) and inflammatory markers (COX-2 and iNos). Our study demonstrates that the inhibition of cell proliferation and down regulation of inflammatory markers may be, at least in part, the underlying mechanisms related to the liver tumor inhibition by diosmin. The present study allows us to conclude that diosmin being a dietary supplement, could be used as chemopreventive agent to prevent hepatocarcinogenesis.     

Different inhibitory effects in the early and late phase of treatment with KAT-681, a liver-selective thyromimetic, on rat hepatocarcinogenesis induced by 2-acetylaminofluorene and partial hepatectomy after diethylnitrosamine initiation.

We recently reported that short-term treatment with KAT-681 (KAT), a liver-selective thyromimetic, inhibits the development of preneoplastic lesions in rat livers and may be a candidate chemopreventive agent for hepatocarcinogenesis. In this study, time-course observations of hepatocellular proliferative lesions were carried out during short-term and long-term treatment with KAT to investigate its anti-hepatocarcinogenic effects. The hepatocellular proliferative lesions in male F344 rats were induced by the initiation treatment of diethylnitrosamine (DEN), followed by treatment with 2-acetylaminofluorene (2-AAF) and partial hepatectomy (PH). The rats were administered KAT orally at a dose of 0.25 mg/kg/day for 3 weeks (experiment 1) or 0.1 mg/kg/day for 20 weeks (experiment 2). In experiment 1, a serial reduction in the number of altered hepatocellular foci (AHF) with positive expression of glutathione S-transferase placental form (GST-P) was observed until day 14 of the treatment period. The proliferative index (PI) of hepatocytes in the AHF significantly increased in the KAT group throughout the treatment period, with a peak on day 2. KAT treatment showed no obvious effects on GST-P-positive hepatocellular adenomas (HCAs) at any time point. In contrast, long-term KAT treatment in experiment 2 revealed a reduction in the mean size of HCAs in addition to reductions in the number and mean size of AHF. The PIs within the lesions in KAT-treated rats were significantly lower than those in controls. The present study indicates that KAT has different inhibitory effects on hepatocarcinogenesis in the early and late phases of KAT treatment; there is a reduction in AHF with enhanced cell proliferation in the early phase and the inhibition of development of AHF and HCAs with suppression of cell proliferation in the late phase. These results may suggest further potential of KAT as a promising chemopreventive agent for hepatocarcinogenesis.     

肿瘤蛋白 53靶基因 1靶向微小 RNA-33a对胶质瘤细胞增殖、凋亡、迁移、侵袭的影响

目的 探讨肿瘤蛋白53靶基因1(TP53TG1)对胶质瘤细胞增殖、凋亡及迁移侵袭的影响和机制.方法 本研究起止时间为2019年1—6月,人正常神经胶质细胞(HA)和神经胶质瘤细胞U251购于上海斯信生物科技有限公司.实时定量PCR(qRT-PCR)检测HA和U251细胞中TP53TG1的表达水平.构建过表达TP53TG1的U251细胞株,噻唑蓝(MTT)法用于评估细胞增殖能力,流式细胞术分析细胞凋亡,Transwell法测定迁移和侵袭数,Western blotting检测细胞周期蛋白D1(Cyclin D1)、细胞周期蛋白激酶抑制剂(P21)、B细胞淋巴瘤-2(Bcl-2)、Bcl相关x蛋白(Bax)、基质金属蛋白酶2(MMP-2)、E钙黏蛋白(E-cad?herin)表达水平.双荧光素酶报告基因实验和Western blotting验证TP53TG1和微小RNA-33a(miR-33a)的靶向关系.结果 与人正常神经胶质细胞HA比较,神经胶质瘤细胞U251中TP53TG1的表达水平[(1.03±0.10)比(0.28±0.03)]显著降低.过表达TP53TG1能够显著抑制U251细胞活力,下调Cyclin D1、Bcl-2和MMP-2蛋白水平,减少细胞迁移数[(138±14.01)个比(72±7.26)个]和侵袭数[(126±12.54)个比(65±6.63)个],上调P21、Bax和E-cadherin蛋白表达,促进细胞凋亡[(8.16±0.86)％比(22.57±2.65)％].TP53TG1能够靶向负性调控miR-33a的表达.过表达miR-33a能够逆转miR-33a对U251细胞增殖、凋亡[(22.68±2.69)％比(11.36±1.20)％]、迁移[(70±7.05)个比(108±11.37)个]和侵袭[(70±7.05)个比(108±11.37)个]的影响.结论 TP53TG1通过靶向miR-33a抑制胶质瘤细胞增殖、迁移和侵袭,促进细胞凋亡.     

Concurrent administration of ascorbic acid enhances liver tumor-promoting activity of kojic acid in rats

We previously found that administration of ascorbic acid (AA) enhances the liver tumor-promoting activity of kojic acid (KA) in mice. To examine the reproducibility of these results in rats and the underlying mechanism of this effect, we employed a two-stage liver carcinogenesis model using male F344 rats. Two weeks after initiation with diethylnitrosamine (DEN), the animals received a diet containing 2% KA and drinking water with or without 5,000 ppm AA for a period of 7 weeks. A DEN-alone group was also established as a control. One week after the commencement of the administration, the animals were subjected to two-thirds partial hepatectomy. At the end of the experiment, the livers were analyzed immunohistochemically, and the mRNA expression level and extent of lipid peroxidation were measured. AA treatment enhanced the KA-induced tumor-promoting activity in terms of the number and area of liver cell foci that were positive for glutathione-S-transferase placental form. AA coadministration increased the number of hepatocytes positive for proliferating cell nuclear antigen and inversely decreased the number of TUNEL-positive cells. However, the increased level of thiobarbituric acid reactive substances resulting from KA treatment was suppressed by coadministration of AA. Gene expression analyses using low-density microarrays and real-time RT-PCR showed that coadministration of AA resulted in upregulation of genes related to cell proliferation and downregulation of those involved in apoptosis and/or cell cycle arrest. These results indicate that the concerted effects of AA on cell proliferation and apoptosis/cell cycle arrest probably through its antioxidant activity are involved in this enhancement.     

Retinoid X receptor α in human liver is regulated by miR-34a

Retinoid X receptor α (RXRα) forms a heterodimer with numerous nuclear receptors to regulate drug- or lipid-metabolizing enzymes. In this study, we investigated whether human RXRα is regulated by microRNAs. Two potential recognition elements of miR-34a were identified in the RXRα mRNA: one in the coding region and the other in the 3′-untranslated region (3′-UTR). Luciferase assays revealed that miR-34a recognizes the element in the coding region. The overexpression of miR-34a in HepG2 cells significantly decreased the endogenous RXRα protein and mRNA levels. The stability of RXRα mRNA was decreased by the overexpression of miR-34a, indicating that miR-34a negatively regulates RXRα expression by facilitating mRNA degradation. We found that the miR-34a-dependent down-regulation of RXRα decreases the induction of CYP26 and the transactivity of CYP3A4. miR-34a has been reported to be up-regulated by p53, which has an ability to promote liver fibrosis. The p53 activation resulted in an increase of the miR-34a level and a decrease of the RXRα protein level. In addition, the miR-34a levels in eight fibrotic livers were higher than those in six normal livers, and the reverse trend was found for the RXRα protein levels. An inverse correlation was observed between the miR-34a and the RXRα protein levels in the 14 samples. Taken together, the data show that miR-34a negatively regulates RXRα expression in human liver, and affects the expression of its downstream genes. This miR-34a-dependent regulation might be the underlying mechanism responsible for the decreased expression of the RXRα protein in fibrotic livers.     

长链非编码前列腺癌相关转录因子 6靶向微小 RNA-139-3p对骨肉瘤细胞增殖和凋亡的影响

目的 探讨长链非编码RNA(LncRNA)前列腺癌相关转录因子6(PCAT6)对骨肉瘤细胞增殖和凋亡的影响和分子机制.方法 实时荧光定量PCR(RT-qPCR)检测20例骨肉瘤组织和与其对应的癌旁组织中PCAT6和微小RNA-139-3p(miR-139-3p)的表达水平.双荧光素酶报告基因实验和RT-qPCR验证PCAT6对miR-139-3p的靶向调控关系.将PCAT6小干扰RNA(si-PCAT6)、miR-139-3p模拟物(miR-139-3p mimics)分别转染骨肉瘤细胞SAOS2,细胞计数试剂盒(CCK-8)检测SAOS2细胞增殖活力,流式细胞术检测SAOS2细胞凋亡,蛋白质印迹法检测B细胞淋巴瘤/白血病-2(Bcl-2)、细胞周期素D1(Cyclin D1)、Bcl-2相关X蛋白(Bax)和P21、的表达水平.将si-PCAT6和miR-139-3p抑制物(anti-miR-139-3p)共转染至SAOS2细胞,采用上述方法检测细胞增殖、迁移侵袭能力以及凋亡变化.结果 与瘤旁组织相比,骨肉瘤组织中PCAT6的表达水平[(2.76±0.27)比(1.01±0.09)]显著升高,miR-139-3p的表达水平[(0.51±0.05)比(1.00±0.08)]显著降低(P<0.05).miR-139-3p是PCAT6的靶基因,PCAT6靶向负性调控miR-139-3p表达.抑制PCAT6表达或过表达miR-139-3p均可下调SAOS2细胞CyclinD1和Bcl-2蛋白表达,上调p21和Bax蛋白表达,降低细胞活力,促进细胞凋亡(P<0.05).抑制miR-139-3p表达可逆转si-PCAT6对骨肉瘤SAOS2细胞增殖抑制和凋亡的影响(P<0.05).结论 lncRNA PCAT6在骨肉瘤组织中表达上调,抑制miR-139-3p通过靶向miR-139-3p抑制骨肉瘤细胞增殖,诱导骨肉瘤细胞凋亡.     

High mobility group box associated with cell proliferation appears to play an important role in hepatocellular carcinogenesis in rats and humans

To identify genes important in hepatocellular carcinogenesis, especially processes involved in malignant transformation, we focused on differences in gene expression between adenomas and carcinomas by DNA microarray. Eighty-one genes for which expression was specific in carcinomas were analyzed using Ingenuity Pathway Analysis software and Gene Ontology, and found to be associated with TP53 and regulators of cell proliferation. In the genes associated with TP53, we selected high mobility group box (HMGB) for detailed analysis. Immunohistochemistry revealed expression of HMGBs in carcinomas to be significantly higher than in other lesions among both human and rat liver, and a positive correlation between HMGBs and TP53 was detected in rat carcinomas. Knock-down of HMGB 2 expression in a rat hepatocellular carcinoma cell line by RNAi resulted in inhibition of cell growth, although no effects on invasion were evident in vitro. These results suggest that acquisition of malignant potential in the liver requires specific signaling pathways related to high cell proliferation associated with TP53. In particular, HMGBs appear to have an important role for progression and cell proliferation associated with loss of TP53 function in rat and in human hepatocarcinogenesis.     

Identification of biomarkers of chemically induced hepatocarcinogenesis in rasH2 mice by toxicogenomic analysis

Toxicogenomic approaches have been applied to chemical-induced heptocarcinogenesis rodent models for the identification of biomarkers of early-stage hepatocarcinogenesis and to help clarify the underlying carcinogenic mechanisms in the liver. In this study, we used toxiciogenomic methods to identify candidate biomarker genes associated with hepatocarcinogenesis in rasH2 mice. Blood chemical, histopathologic, and gene expression analyses of the livers of rasH2 mice were performed 7 and 91 days after the administration of the genotoxic hepatocarcinogens 2-acetylaminofluorene (AAF) and diethylnitrosoamine (DEN), the genotoxic carcinogen melphalan (Mel), and the nongenotoxic noncarcinogen 1-naphthylisothiocynate (ANIT). Histopathologic lesions and a rise in accompanying serum marker levels were found in the DEN-treated rasH2 mice, whereas no neoplastic lesions were observed in the rasH2 mice. However, biological functional analysis using Ingenuity Pathways Analysis (IPA) software revealed that genes with comparable molecular and cellular functions were similarly deregulated in the AAF- and DEN-treated rasH2 mice. We selected 68 significantly deregulated genes that represented a hepatocarcinogen-specific signature; these genes were commonly deregulated in both the AAF- and DEN-treated rasH2 mice on days 7 and 91. Hierarchical clustering analysis indicated that the expression patterns of the selected genes in the hepatocarcinogen (AAF and DEN) groups were distinctive from the patterns in the control, Mel, and ANIT groups. Biomarker filter analysis using IPA software suggested that 28 of the 68 signature genes represent promising candidate biomarkers of cancer. Quantitative real-time PCR analysis confirmed that the deregulated genes, which exhibited sustained up- and down-regulation up to day 91, are likely involved in early-stage hepatocarcinogenesis. In summary, the common and significant gene expression changes induced by AAF and DEN may reflect early molecular events associated with hepatocarcinogenesis, and these “signature” genes may be useful as biomarkers of hepatocarcinogenesis in mice.     

A comparison of gene expression changes in response to diethylstilbestrol treatment in wild-type and p53+/- hemizygous knockout mice using focussed arrays

We have investigated the hepatic response of female C57BL/6J wild-type and p53(+/-) hemizygous mice to genotoxic levels of diethylstilbestrol (DES) using cell cycle and apoptosis-focussed cDNA expression arrays. DES induced the expression of 12 genes (bad, bax, bcl-x, caspase-1, p53, cyclin D3, GADD45, p21, p15, p27, p57 and Skp1) and down-regulated the expression of eight genes (bcl-2, caspase-2, caspase-7, caspase-8, E124, iNOS, mdm2 and NFkappab1) at twofold or greater levels. Taken together, these changes were strongly reflective of the induction of apoptosis in the livers of DES-treated mice. Of those genes showing the greatest changes in response to DES, p53, p21 and p57 were expressed at 2.1, 1.7 and 1.6 times greater (respectively) in wild-type mice as compared with p53(+/-) hemizygous mice. Differences in p53, p21 and bax expression were confirmed by RT-PCR and we conclude that the compromised response of p53(+/-) mice is likely to play a central role in the earlier appearance of tumours in this model, following exposure to genotoxic carcinogens.     

Involvement of oxidative stress in hepatocellular tumor-promoting activity of oxfendazole in rats

The tumor-promoting effects of oxfendazole (OX), a benzimidazole anthelmintic, were investigated using a medium-term rat hepatocarcinogenesis model. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) and were given a powdered diet containing 0 or 500 ppm OX for 6 weeks from 2 weeks after DEN treatment. All animals were subjected to two-thirds partial hepatectomy 1 week after OX treatment. The numbers and areas of glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in the livers of rats treated with OX, with concomitantly increased cell proliferation, compared with those in the livers of the DEN alone group. Quantitative real-time RT-PCR analysis revealed that OX induced not only mRNA expression of phase I enzymes Cyp1a1, Cyp1a2, but also Nrf2-regulated phase II enzymes such as Gpx2, Nqo1, Yc2, Akr7a3 and Gstm1, presumably due to an adaptive response against OX-induced oxidative stress. Reactive oxygen species production increased in microsomes isolated from the livers of OX-treated rats. Furthermore, OX enhanced oxidative DNA damage (as assessed by 8-hydroxydeoxyguanosine; 8-OHdG) and lipid peroxidation (as assessed by thiobarbituric acid-reactive substances; TBARS). These results suggest that administration of OX at a high dose and for a long term enhances oxidative stress responses, which may contribute to its tumor-promoting potential in rats.     

Grape-seed proanthocyanidins ameliorate contact hypersensitivity induced by 2,4-dinitrofluorobenzene (DNFB) and inhibit T cell proliferation in vitro

Irinotecan (CPT-11) is topoisomerase I inhibitor used in the treatment of disseminated colorectal cancer. In colon cancer cells it induces DNA damage which leads to cytotoxicity with ensuing apoptosis or premature senescence. Despite its clinical use and efficiency in malignant colonocytes, its effects in normal colonic cells are relatively underexplored. In this work we report that CPT-11 induces dose-dependent cytotoxicity which results in apoptosis and premature senescence whose occurrence nevertheless varies in relation to the type of exposed cells. In normal colonic epithelial cells (NCM) the prevailing type of response is apoptosis whereas in normal colonic fibroblasts (NCF) it is premature senescence. Further analyses showed that CPT-11 induced in both types of cells DNA damage and activated stress response pathways including p53 and p16 but with varying activity of stress kinase p38 and selected stress-associated microRNAs. Epithelial cells upregulated the expression of p53, which was subsequently specifically phosphorylated, massively activated p38 and initiated mitochondrial, caspase-dependent apoptosis. These events occurred in the presence of moderately increased expression of miR-34a only. Conversely, in colonic fibroblasts p38 was only moderately activated, p53 as well as p16 expressions were upregulated in the presence of increased expression of miR-34a, miR-128a and miR-449a. Caspase-dependent apoptosis was found only in a minority of treated cells and the premature senescence phenotype was prevailing. Specific inhibition further proved that p53-dependent as well as independent mechanisms might be responsible for these cell type-specific differences.     

miR-34a通过调控雄激素受体基因抑制前列腺癌细胞系LNCaP增殖

目的:探究miR-34a对雄激素受体(AR)基因的调控作用及对前列腺癌(PCa)LNCaP细胞增殖、凋亡的影响。方法:收集2016年10月至2019年9月本院泌尿外科行前列腺穿刺活检确诊为PCa患者组织标本36例,另取同期行手术治疗切除的良性前列腺增生(BPH)组织标本41例。体外培养LNCaP细胞,分别转染miR-34a mimics(mimics组),miR-34a mimic NC(NC组),设正常生长细胞为空白对照组(BC组),另在mimics组基础上转染AR过表达(AR过表达组)载体及其对照(AR对照组),采用CCK-8试剂盒检测细胞活性,Annexin V-FITC/PI双染色流式细胞凋亡检测试剂盒检测细胞凋亡率,实时定量PCR(RT-qPCR)检测miR-34a及AR mRNA表达水平,免疫印迹法检测AR蛋白、细胞周期蛋白D1(cyclinD1)及原癌基因产物(c-Mcy)、细胞凋亡相关蛋白(Bcl-2、Bax、capase-3)的表达水平。结果:与BPH组织比较,PCa组织中miR-34a表达水平显著降低(P<0.05),AR mRNA及蛋白表达水平均显著增加(P<0.05);与BC、NC组比较,mimics组LNCap细胞miR-34a表达水平、细胞凋亡率、Bax蛋白表达水平显著增加(P<0.05),AR、Cyclin D1、Bcl-2、Bax蛋白表达水平显著降低(P<0.05),同一时间LNCap细胞活力均显著降低(P<0.05);双荧光素酶实验结果显示,miR-34a与AR可能存在一定的调控关系,过表达AR基因可逆转miR-34a mimics对LNCaP细胞增殖抑制,促进凋亡作用。结论:miR-34a可能通过调控AR抑制前列腺癌LNCaP细胞增殖,促进其凋亡。     

MicroRNA-497-5p down-regulation increases PD-L1 expression in clear cell renal cell carcinoma

Recent advances in immunotherapy are raising hope to treat clear cell renal cell carcinoma (ccRCC) with PD-L1 inhibitors, but only a small portion of patients are PD-L1 positive. The heterogeneous expression pattern of PD-L1 in patient population suggests that PD-L1 expression is under the control of diverse regulatory mechanisms. Although recent studies have identified numerous novel PD-L1 regulators, reports on microRNAs which modulate PD-L1 expression are much scarce. In this study, we confirmed that PD-L1 expression was up-regulated in ccRCC compared to paired normal tissues. Using miRDB and miRTarBase, 11 microRNAs were predicted to target PD-L1. After measuring the microRNA panel with TaqMan assays, we found that microRNA-497-5p down-regulation was associated with PD-L1 up-regulation. In TCGA-KIRC dataset, microRNA-497-5p down-regulation was also associated with PD-L1 up-regulation as well as shorter survival. We further validated that PD-L1 was a direct target of microRNA-497-5p in two RCC cell lines. In addition, microRNA-497-5p inhibited cell proliferation, clone formation and migration, while promoted apoptosis in in-vitro assays. Our study reveals a novel regulatory mechanism of PD-L1 expression and the potential of miR-497-5p as therapeutic target and biomarker deserves further investigation.     

Inhibition of gap-junctional-intercellular communication in intact rat liver by nongenotoxic hepatocarcinogens

Many nongenotoxic hepatocarcinogens can induce cell proliferation, and inhibit apoptosis and gap-junctional-intercellular communication (GJIC). GJIC, the movement of small molecules (less than 1.2 kD) through membrane channels, is important in regulating cellular homeostasis and differentiation. The inhibition of hepatic GJIC can increase cell proliferation and possibly, inhibit apoptosis. In this study, the relationship between hepatic GJIC, proliferation, and apoptosis was examined in rats treated for 7 days with tumor-promoting doses of the nongenotoxic hepatocarcinogens phenobarbital (PB; 800 ppm), pregnenolone-16alpha-carbonitrile (PCN; 1000 ppm), and Aroclor 1254 (PCB; 100 ppm). In addition, 3-methylcholanthrene (3MC) was included as a negative control. PB, PCN, and PCB increased parenchymal-cell proliferation and inhibited hepatic apoptosis, while no alteration in these growth parameters was observed in 3MC-treated rats. GJIC, as measured by fluorescent-dye transfer through intact liver, was decreased nearly 50% by PB, PCN, and PCB, yet no effect on GJIC was observed in liver from 3MC-treated rats. These data indicate that compounds that inhibit GJIC in liver may be nongenotoxic hepatocarcinogens, which occurs simultaneously during increased cell proliferation and inhibited apoptosis.     

Cell cycle changes mediated by the p53/miR-34c axis are involved in the malignant transformation of human bronchial epithelial cells by benzo[a]pyrene

Characterization of aberrant microRNA (miRNA) expression during carcinogen-induced cell transformation will lead to a better understanding of the role of miRNAs in cancer development. In this investigation, we evaluated changes in p53 function and its downstream target miRNAs in benzo[a]pyrene (BaP)-induced transformation of human bronchial epithelial (HBE) cells. Chronic exposure to BaP induced malignant transformation of cells, in which there were increased levels of mutant p53 (mt-p53) and reduced expression of wild-type p53 (wt-p53) and phosphorylated p53 (p-p53). With acute (12 h) exposure to BaP, p-p53 was increased, and with increasing time of exposure (24 h), the increase in p-p53 at a concentration of 1 μM BaP was followed by a decline with increasing concentrations; wt-p53 and mt-p53 did not change. With prolonged exposure (48 h), p-p53 and wt-p53 decreased, but mt-p53 increased. At different exposure times, the levels of miR-34c were consistent with p-p53. Over-expression of miR-34c resulted in inhibition of the BaP-induced G1-to-S transition and diminished up-regulation of cyclin D. Further, up-regulation of miR-34c or silencing of cylin D prevented BaP-induced malignant transformation. Thus, changes in the cell cycle mediated by the p53/miR-34c axis are involved in the transformation cells induced by BaP.