MicroRNA-378-3p/5p represses proliferation and induces apoptosis of oral squamous carcinoma cells via targeting KLK4

Oral squamous cell carcinoma (OSCC) is one of the most common types of head and neck neoplasm. Down-regulation of hsa-microRNA-378 (miR-378) has been proved in OSCC tissues, suggesting that miR-378 might play crucial roles in the progression of OSCC. The present study aimed to evaluate the effect of miR-378-3p/5p on the proliferation and apoptosis of OSCC in vitro and in vivo. According to the results, lentivirus-mediated overexpression of miR-378 lowered the colony formation efficiency, blocked cell cycle progression, and decreased the percentage of Ki-67 positive cells, whereas knockdown of miR-378-3p/5p led to the opposite results. Furthermore, the apoptosis of OSCC cells was induced by the overexpression of miR-378 as evidenced by decreasing Bcl-2/Bax ratio, increasing cleaved caspase-9, cleaved caspase-3, and cleaved PARP levels, and promoting the release of cytochrome c into the cytoplasm. However, the above results were reversed by miR-378-3p/5p silencing. In addition, the overexpression of miR-378 inhibited the activation of PI3K/AKT signalling pathway. Conversely, miR-378-3p/5p knockdown resulted in the inactivation of PI3K/AKT signalling pathway. Mechanically, we validated that miR-378-3p/5p could target kallikrein-related peptidase 4 (KLK4), and enforced overexpression of KLK4 counteracted miR-378 overexpression-induced apoptosis. Finally, tumourigenesis in nude mice was suppressed by the overexpression of miR-378, which was promoted by miR-378-3p/5p silencing. Taken together, these results suggest that miR-378 may be a potential target in the diagnoses and treatment of OSCC.     

MiR-199a-5p and miR-375 affect colon cancer cell sensitivity to cetuximab by targeting PHLPP1

Objectives: We aimed to analyze the differentially-expressed miRNAs in colon cancer cells in order to identify novel potential biomarkers involved in cancer cell resistance.

Design and methods: We investigated the miRNA expression profile of GEO human colon carcinoma cells, sensitive to the EGFR inhibitor Cetuximab (CTX) and their CTX-resistant counterpart (GEO CR) by using a miRNA chip.

Results: We found 27 upregulated and 10 downregulated miRNAs in GEO CR compared with GEO cells with a fold change ≥ 2. Among the upregulated miRNAs, we focused on miR-199a-5p and miR-375. We report that their enforced expression promotes CTX resistance, whereas their silencing sensitizes to the same drug. The ability of miR-199a-5p and miR-375 to target PHLPP1 (PH domain and leucine-rich repeat protein phosphatase 1), a tumor suppressor that negatively regulates the AKT pathway, accounts, at least in part, for their drug-resistance activity. Indeed, restoration of PHLPP1 increases sensitivity of the GEO cells to CTX and reverts the resistance-promoting effect of miR-199a-5p and miR-375.

Conclusion: This study proposes miR-199a-5p and miR-375 as contributors to CTX resistance in colon cancer and suggests a novel approach based on miRNAs as tools for the therapy of this tumor.     

Undecylprodigiosin selectively induces apoptosis in human breast carcinoma cells independent of p53

Undecylprodigiosin (UP) is a bacterial bioactive metabolite produced by Streptomyces and Serratia. In this study, we explored the anticancer effect of UP. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47D and one nonmalignant human breast epithelial cell line, MCF-10A, were tested in this study. We found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines in a dose- and time-dependent manner. In contrast, UP showed limited toxicity to MCF-10A cells, indicating UP's cytotoxic effect is selective for malignant cells. UP's cytotoxic effect was due to apoptosis, as confirmed by positive TUNEL signals, annexin V-binding, caspase 9 activation and PARP cleavage. Notably, UP-induced apoptosis was blocked by the pan-caspase inhibitor z-VAD.fmk, further indicating the involvement of caspase activity. Moreover, UP caused a marked decrease of the levels of antiapoptotic BCL-X(L), Survivin and XIAP while enhancing the levels of proapoptotic BIK, BIM, MCL-1S and NOXA, consequently favoring induction of apoptosis. Additionally, we found that cells with functional p53 (MCF-7, T47D) or mutant p53 (BT-20, MDA-MB-231) were both susceptible to UP's cytotoxicity. Importantly, UP was able to induce apoptosis in MCF-7 cells with p53 knockdown by RNA interference, confirming the dispensability of p53 in UP-induced apoptosis. Overall, our results establish that UP induces p53-independent apoptosis in breast carcinoma cells with no marked toxicity to nonmalignant cells, raising the possibility of its use as a new chemotherapeutic drug for breast cancer irrespective of p53 status.     

Therapeutic targeting of the p53 pathway in cancer stem cells

Introduction: Cancer stem cells (CSCs) are a high profile drug target for cancer therapeutics due to their indispensable role in cancer progression, maintenance and therapeutic resistance. Restoring wild-type (WT) p53 function is an attractive new therapeutic approach for the treatment of cancer due to the well-described powerful tumor suppressor function of p53. As emerging evidence intimately links p53 and stem cell biology, this approach also provides an opportunity to target CSCs.

Areas covered: This review covers the therapeutic approaches to restore the function of WT p53, cancer and normal stem cell biology in relation to p53 and the downstream effects of p53 on CSCs.

Expert opinion: The restoration of WT p53 function by targeting p53 directly, its interacting proteins or its family members holds promise as a new class of cancer therapies. This review examines the impact that such therapies may have on normal and CSCs based on the current evidence linking p53 signaling with these populations.     

Cadmium induces ferroptosis and apoptosis by modulating miR-34a-5p/Sirt1axis in PC12 cells

Cadmium (Cd) is a potent neurotoxic metal present in the environment and food. In this study, CdCl₂ (2 or 4 μM) induced cytotoxicity and neurotoxicity in PC12 cells, causing decreases in cell viability and NEP protein expression and increase in p-tau protein expression. For the first time, CdCl₂-initiated injury was found to result from the induction of not only apoptosis but also ferroptosis, as evidenced by the increased iron content, ROS production, and mitochondrial membrane potential along with changes in the expressions of iron death-related genes (FTH1, GPX4, ASCL4, PTGS2, and NOX1) and levels of caspase9, Bax, and Bcl-2 proteins. The molecular mechanisms leading to apoptosis and ferroptosis at least included the participation of the miR-34a-5p/Sirt1 axis, in which miR-34a-5p promoted CdCl₂-induced neurotoxicity through targeting Sirt1. Knocking out miR-34a-5p attenuated CdCl₂-induced damage of PC12 cells, cytotoxicity and neurotoxicity. This research provides the underlying molecular mechanisms of CdCl₂-induced damage and asserts the role of miRNAs as critical regulators.     

p53在DNA损伤反应中的研究进展

p53基因是研究最广泛的抑癌基因之一,也是细胞内的一个强大的转录因子,在正常状态下呈低水平表达。在各种应激包括DNA损伤时,p53可以被不同的信号通路激活并稳定,通过增强其下游多种基因的转录而引起细胞周期阻滞、凋亡或衰老,保持细胞基因组的完整性并清除损伤细胞,这些生物学作用取决于不同的应激信号和细胞类型。p53通路是机体应对DNA损伤的天然防护屏障,对这一机制的深入研究可为肿瘤的发生发展和抗肿瘤药物的开发提供重要的信息。     

SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment.     

食管鳞状细胞癌中p53基因突变和蛋白表达研究

目的检测食管鳞状细胞癌(ESCC)中p53基因突变和蛋白表达及其与临床病理和预后的相关性。方法采用聚合酶链反应-单链构象多态性分析(PCR-SSCP)和免疫组织化学(SP)两种方法检测56例ESCC中p53基因突变和蛋白的表达。结果56例ESCC组织中p53蛋白阳性表达率为61%(34/56),它与患者的年龄、性别和家族史无关(P>0.05),有和无淋巴结转移的阳性率分别为81%(17/21),49%(17/35);生存率<3年组和>3年组的p53阳性表达率为74%(17/23),46%(13/28),差异有统计学意义。PCR-SSCP检测p53基因突变率为77%(43/56),突变位于第4外显子者5例,第5外显子者23例,第6外显子者1例,第7外显子者4例,第8外显子者7例,有3例在内显子。p53基因突变/过度表达率为46%(26/56),两种方法检测的符合率为55%(31/56)。结论p53基因突变/过度表达在食管鳞癌发生、发展中可能发挥重要作用,可作为判断食管鳞癌患者预后的参考指标之一。     

MiR-34a通过调控Fra-1影响人胃癌细胞侵袭转移

目的 探讨过表达miR-34a对胃癌细胞侵袭转移增殖能力的影响,以及其可能的作用机制.方法 通过转染miR-34amimics上调其表达,利用划痕迁移实验、transwell侵袭实验和CCK8实验检测过表达miR-34a对SGC-7901,BCG-823细胞的迁移、侵袭和增殖能力的影响.采用生物信息学预测及双荧光素酶实验考察miR-34a对靶基因Fra-1的靶向调控机制.结果 转染miR-34amimics过表达miR-34a能显著抑制胃癌细胞SGC-7901,BCG-823的迁移、侵袭和增殖能力.miR-34a能够靶向负性调控Fra-1的表达,影响下游侵袭转移相关蛋白分子MMP9、Cyclin D1的表达.结论 过表达胃癌细胞系中miR-34a可靶向抑制Fra-1的表达,从而抑制胃癌细胞的侵袭转移增殖能力,影响胃癌进展.     

p53/miR-34a通路介导羽扇豆醇的抑制人膀胱癌T24细胞增殖作用

摘 要 目的：探讨羽扇豆醇对人膀胱癌T24细胞增殖的影响及对p53/miR-34a通路调控机制。方法: 运用CCK-8法检测不同浓度羽扇豆醇（5~120 μmol·L^-1）分别作用24 h和48 h对T24细胞增殖的影响;运用CCK-8法结合Caspase抑制药确证参与羽扇豆醇诱导细胞死亡的Caspase亚型;分别运用荧光定量PCR（qPCR）和蛋白免疫印记评价羽扇豆醇对miR-34a及p53蛋白表达的影响;运用qPCR评价羽扇豆醇对miR-34a下游靶基因Bcl-2、CD44、c-Myc的mRNA表达的影响。结果: T24细胞经羽扇豆醇处理后,其细胞增殖受到明显抑制,且呈一定的剂量依赖性;药物作用24 h和48 h时羽扇豆醇的半抑制浓度（IC₅₀）分别为（77.23±6.78）,（64.58±4.23）μmol·L^-1。与对照组相比,羽扇豆醇能够使T24细胞中p53蛋白表达上调,还能够增加miR-34a表达水平,差异具有统计学意义（P<0.01）;羽扇豆醇处理后细胞内Bcl-2、CD44、c-Myc的mRNA表达量下调,差异具有统计学意义（P<0.01）。结论:羽扇豆醇具有抑制膀胱癌T24细胞增殖能力,其作用机制与调控p53/miR-34a通路有关。     

Denbinobin induces apoptosis by apoptosis-inducing factor releasing and DNA damage in human colorectal cancer HCT-116 cells

Denbinobin is a phenanthraquinone derivative present in the stems of Ephemerantha lonchophylla. We showed that denbinobin induces apoptosis in human colorectal cancer cells (HCT-116) in a concentration-dependent manner. The addition of a pan-caspase inhibitor (zVAD-fmk) did not suppress the denbinobin-induced apoptotic effect, and denbinobin-induced apoptosis was not accompanied by processing of procaspase-3, -6, -7, -9, and -8. However, denbinobin triggered the translocation of the apoptosis-inducing factor (AIF) from the mitochondria into the nucleus. Small interfering RNA targeting of AIF effectively protected HCT-116 cells against denbinobin-induced apoptosis. Denbinobin treatment also caused DNA damage, activation of the p53 tumor suppressor gene, and upregulation of numerous downstream effectors (p21^WAF1/CIP1, Bax, PUMA, and NOXA). A HCT-116 xenograft model demonstrated the in vivo efficacy and low toxicity of denbinobin. Taken together, our findings suggest that denbinobin induces apoptosis of human colorectal cancer HCT-116 cells via DNA damage and an AIF-mediated pathway. These results indicate that denbinobin has potential as a novel anticancer agent. Shiow-Lin Pan and Che-Ming Teng contributed equally to this work.     

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.     

食管鳞癌组织中hPOT1、hTERT及p53的表达

目的 探讨食管鳞癌组织中端粒保护蛋白1(hPOT1)、端粒酶催化亚基(hTERT)及p53的表达及其意义.方法 采用免疫组织化学技术检测82例食管鳞癌组织中hPOT1、hTERT及p53的表达情况.结果 82例鳞癌组织中,hPOT1蛋白阳性表达率为78.05%(64/82),hTERT蛋白阳性表达率为68.29%(56/82),p53蛋白的阳性表达率为65.85%(54/82),它们与淋巴结转移、病理分期密切相关(P＜0.05).结论 联合检测hPOT1、hTERT及p53蛋白的表达对判断食管鳞癌的预后有一定的指导意义.     

miR-34b通过靶向AGR2调控口腔癌糖脂代谢的相关性研究

目的 探索miR-34b在口腔癌中的作用机制。方法 检测口腔癌细胞系中miR-34b和AGR2的表达情况。在口腔癌细胞中过表达miR-34b或敲降AGR2后,采用MTT法检测口腔癌细胞的增殖;油红O染色法检测口腔癌细胞中脂肪和葡萄糖含量;双荧光酶报告基因验证miR-34b与AGR2的关系。结果 miR-34b在口腔癌细胞系中低表达,而AGR2在口腔癌细胞系中高表达。过表达miR-34b或敲降AGR2可抑制口腔癌细胞增殖,并降低细胞中脂肪和葡萄糖含量。结论 miR-34b可通过靶向AGR2调控口腔癌的糖脂代谢,对口腔癌的预防和治疗具有重要意义。     

Eriocalyxin B induces apoptosis and cell cycle arrest in pancreatic adenocarcinoma cells through caspase- and p53-dependent pathways

Pancreatic cancer is difficult to detect early and responds poorly to chemotherapy. A breakthrough in the development of new therapeutic agents is urgently needed. Eriocalyxin B (EriB), isolated from the Isodon eriocalyx plant, is an ent-kaurane diterpenoid with promise as a broad-spectrum anti-cancer agent. The anti-leukemic activity of EriB, including the underlying mechanisms involved, has been particularly well documented. In this study, we demonstrated for the first time EriB's potent cytotoxicity against four pancreatic adenocarcinoma cell lines, namely PANC-1, SW1990, CAPAN-1, and CAPAN-2. The effects were comparable to that of the chemotherapeutic camptothecin (CAM), but with much lower toxicity against normal human liver WRL68 cells. EriB's cytoxicity against CAPAN-2 cells was found to involve caspase-dependent apoptosis and cell cycle arrest at the G2/M phase. Moreover, the p53 pathway was found to be activated by EriB in these cells. Furthermore, in vivo studies showed that EriB inhibited the growth of human pancreatic tumor xenografts in BALB/c nude mice without significant secondary adverse effects. These results suggest that EriB should be considered a candidate for pancreatic cancer treatment.     

Diepoxybutane induces caspase and p53-mediated apoptosis in human lymphoblasts

Diepoxybutane (DEB) is the most potent metabolite of the environmental chemical 1,3-butadiene (BD), which is prevalent in petrochemical industrial areas. BD is a known mutagen and human carcinogen, and possesses multiorgan systems toxicity that includes bone marrow depletion, spleen, and thymus atrophy. Toxic effects of BD are mediated through its epoxy metabolites. In working towards elucidating the cellular and molecular mechanisms of BD toxicity, we investigated the ability of DEB to induce apoptosis in human lymphoblasts. DEB induced a concentration and exposure time-dependent apoptosis, which accounted for the DEB-induced loss of cell viability observed in TK6 lymphoblasts. The DEB-induced apoptosis was inhibited by inhibitors of caspases 3 and 9. The role of p53 in mediating the DEB-induced apoptosis was also investigated. DEB induced elevated p53 levels in direct correlation to the extent of DEB-induced apoptosis, as the concentration of DEB increased up to 5 microM. The extent of DEB-induced apoptosis was dramatically higher in TK6 lymphoblasts as compared to the genetically paired p53-deficient NH32 lymphoblasts under the same experimental conditions. Our results confirm and extend observations on the occurrence of apoptosis in DEB exposed cells, and demonstrate for the first time the elevation of p53 levels in human lymphoblasts in response to DEB exposure. In addition, our results demonstrate for the first time that DEB-induced apoptosis is mediated by caspases 3 and 9, as well as the p53 protein. It is possible that DEB-induced apoptosis may explain BD-induced bone marrow depletion, spleen and thymus atrophy in BD-exposed animals.