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Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.Mammalian cell death proceeds through a highly regulated program called apoptosis that is highly dependent on the mitochondria.1 Mitochondrial outer membrane (MOM) multiple apoptotic stresses permeabilize the MOM, resulting in the release of apoptogenic factors including cytochrome c, Smac, AIF, and endoG.2, 3, 4 Released cytochrome c activates Apaf-1, which assists in caspase activation. Then, activated caspases cleave cellular proteins and contribute to the morphological and biochemical changes associated with apoptosis. Bcl-2 family proteins control a crucial apoptosis checkpoint in the mitochondria.2, 5, 6, 7 Multidomain proapoptotic Bax and Bak are essential effectors responsible for the permeabilization of the MOM, whereas anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 preserve mitochondrial integrity and prevent cytochrome c efflux triggered by apoptotic stimuli. The third Bcl-2 subfamily of proteins, BH3-only molecules (BH3s), promotes apoptosis by either activating Bax/Bak or inactivating Bcl-2/Bcl-xL/Mcl-1.8, 9, 10, 11, 12 Upon apoptosis, the ‘activator'' BH3s, including truncated Bid (tBid), Bim, and Puma, activate Bax and Bak to mediate cytochrome c efflux, leading to caspase activation.8, 11, 12 Conversely, antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 sequester activator BH3s into inert complexes, which prevents Bax/Bak activation.8, 9 Although it has been proposed that Bax and Bak activation occurs by default as long as all of the anti-apoptotic Bcl-2 proteins are neutralized by BH3s,13 liposome studies clearly recapitulate the direct activation model in which tBid or BH3 domain peptides derived from Bid or Bim induce Bax or Bak oligomerization and membrane permeabilization.12, 14, 15Numerous studies have demonstrated a critical role for Bax in determining tumor cell sensitivity to drug induction and in tumor development. Bax has been reported to be mutated in colon16, 17 and prostate cancers,18, 19 contributing to tumor cell survival and promoting clonal expansion. Bax has been shown to restrain tumorigenesis20 and is necessary for tBid-induced cancer cell apoptosis.21 Loss of Bax has been reported to promote tumor development in animal models.22 Bax knockout (KO) renders HCT116 cells resistant to a series of apoptosis inducers.23, 24, 25 p53 has been reported to be a tumor suppressor,26 and its mutant can cause chemoresistance in cancer cells.27, 28, 29 Moreover, p53 is often inactivated in solid tumors via deletions or point mutations.30, 31 Thus, it is necessary to find an efficient approach or agent to overcome chemoresistance caused by Bax and/or p53 mutants.Few studies have focused on the role of Bak in tumor cell apoptosis and cancer development. Bak mutations have only been shown in gastric and colon cancer cells.32 Some studies have revealed that Bak is a determinant of cancer cell apoptosis.33, 34 Some studies have even demonstrated that Bak renders Bax KO cells sensitive to drug induction.33, 35 In this study, we are the first group to show that tBid induces Bak activation and the release of AIF and endoG in colon cancer cells, which causes cellular apoptosis independent of Bax/p53. We also found that caspase-3 is activated in apoptosis. Interestingly, downstream caspase-3 can strengthen Bak activation and the release of AIF and endoG during apoptosis via a feedback loop. Furthermore, we reveal that Akt upregulates apoptosis progression. These results will help us to better understand the function of mitochondrial apoptotic protein members in apoptosis and cancer therapies. Furthermore, our experiments may provide a theoretical basis for overcoming chemoresistance in cancer cells. 相似文献
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O Fortunato M Boeri M Moro C Verri M Mensah D Conte L Caleca L Roz U Pastorino G Sozzi 《Cell death & disease》2014,5(12):e1564
Lung cancer represents the leading cause of cancer-related death in developed countries. Despite the advances in diagnostic and therapeutic techniques, the 5-year survival rate remains low. The research for novel therapies directed to biological targets has modified the therapeutic approach, but the frequent engagement of resistance mechanisms and the substantial costs, limit the ability to reduce lung cancer mortality. MicroRNAs (miRNAs) are small noncoding RNAs with known regulatory functions in cancer initiation and progression. In this study we found that mir-660 expression is downregulated in lung tumors compared with adjacent normal tissues and in plasma samples of lung cancer patients with poor prognosis, suggesting a potential functional role of this miRNA in lung tumorigenesis. Transient and stable overexpression of mir-660 using miRNA mimics reduced migration, invasion, and proliferation properties and increased apoptosis in p53 wild-type lung cancer cells (NCI-H460, LT73, and A549). Furthermore, stable overexpression using lentiviral vectors in NCI-H460 and A549 cells inhibited tumor xenograft growth in immunodeficient mice (95 and 50% reduction compared with control, respectively), whereas the effects of mir-660 overexpression were absent in H1299, a lung cancer cell line lacking p53 locus, both in in vitro and in vivo assays. We identified and validated mouse double minute 2 (MDM2) gene, a key regulator of the expression and function of p53, as a new direct target of mir-660. In addition, mir-660 expression reduced both mRNA and protein expression of MDM2 in all cell lines and stabilized p53 protein levels resulting in an upregulation of p21WAF1/CIP1 in p53 wild-type cells. Our finding supports that mir-660 acts as a tumor suppressor miRNA and we suggest the replacement of mir-660 as a new therapeutic approach for p53 wild-type lung cancer treatment.Lung cancer is the leading cause of cancer death worldwide, resulting in >1.4 million deaths/year.1 Lung tumors are often discovered as locally advanced or metastatic disease, and despite improvements in molecular diagnosis and targeted therapies, the overall 5-year survival rate remains in the 10–20% range. Indeed, nonsmall cell lung cancer (NSCLC) is poorly chemosensitive to most of the available agents with response rates ranging from 10 to 25%.2 The discovery of recurrent mutations in the epidermal growth factor receptor (EGFR) kinase,3 as well as gene fusion products involving the anaplastic lymphoma kinase (ALK),4 has led to a marked change in the treatment of patients with lung adenocarcinoma, the most common type of lung cancer.5, 6 To date, patients with mutations in the EGFR gene, suitable for targeting by EGFR tyrosine kinase inhibitors, represent roughly 10%, whereas the subgroup of tumors with ALK rearrangements, targeted by ALK inhibitors, is only ~5%.7 Thus, the majority of lung tumors lack effective treatment and novel therapeutic strategies are still needed.MicroRNAs (miRNAs) are short noncoding RNAs, 20–24 nucleotides long, that have important roles in almost all biological pathways,8, 9, 10, 11 and influence cancer-relevant processes, such as proliferation,12 cell cycle,13 apoptosis,14 and migration.15 Many studies have reported the critical role of miRNAs in lung cancer pathogenesis and their potential as biomarkers for lung cancer risk stratification,16 outcome prediction,17 and classification of histological subtypes.18, 19 miRNAs are actively released by various cell types and can be detected in biological fluids, such as plasma and serum, making them suitable as circulating biomarkers in NSCLC.20, 21There is limited evidence of mir-660 deregulation in cancer and little is known about its role in lung tumorigenesis and its putative target genes. Mir-660 has been reported to be upregulated in chronic lymphocytic leukemia22, 23 and in leukemic cells after treatment with 4-hydroxynonenal, a compound that induces differentiation and blocks proliferation of leukemic cells.24 In a previous study we demonstrated that mir-660 was one of the 24 miRNAs deregulated in plasma samples of NSCLC patients identified in a low-dose computed tomography (LDCT) screening trial.20The p53 tumor suppressor protein is a key regulator of cell cycle G0/G1 checkpoint, senescence, and apoptosis in response to cellular stress signals.25, 26 Mouse double minute 2 (MDM2), a p53–E3 ubiquitin ligase,27 is the principal negative regulator of the expression level and function of p53.28, 29 Several studies have illustrated different mechanisms of p53 regulation by MDM2,30, 31 such as binding transactivation region of p53,32, 33 promoting nuclear export and cytoplasmic accumulation of p53 by monoubiquitination,34, 35 and inducing p53 proteosomal degradation by polyubiquitination.36 In addition, MDM2 gene has been reported to be amplified or overexpressed in a variety of human cancers, such as sarcoma,37 lymphoma,38 breast cancer,39 lung cancer,40 and testicular germ cell tumor.41 Several miRNAs targeting MDM2 have been identified, such as the mir-143/mir-145 cluster that can be induced by p53,42 as well as mir-25 and mir-32, known to inhibit tumor glioblastoma growth in mouse brain.43In this study, we report that mir-660 is downregulated in tissue and plasma samples of lung cancer patients and demonstrate that mir-660 replacement impairs the functionality of p53 wild-type (wt) lung cancer cells and inhibits in vitro and in vivo tumor growth. We showed that all the effects observed after mir-660 overexpression were absent in p53 ko cells, identified MDM2 as mir-660 direct target gene and indicate impairment of the MDM2/p53 interaction as the mechanism underlying tumor growth inhibition. 相似文献
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Recent studies have shown that induced pluripotent stem cells (iPSCs) retain a memory of their origin and exhibit biased differentiation potential. This finding reveals a severe limitation in the application of iPSCs to cell-based therapy because it means that certain cell types are not available for reprogramming for patients. Here we show that the iPSC differentiation process is accompanied by profound gene expression and epigenetic modifications that reflect cells'' origins. Under typical conditions for mammary differentiation, iPSCs reprogrammed from tail-tip fibroblasts (TF-iPSCs) activated a fibroblast-specific signature that was not compatible with mammary differentiation. Strikingly, under optimized conditions, including coculture with iPSCs derived from the mammary epithelium or in the presence of pregnancy hormones, the fibroblast-specific signature of TF-iPSCs obtained during differentiation was erased and cells displayed a mammary-specific signature with a markedly enhanced ability for mammary differentiation. These findings provide new insights into the precise control of differentiation conditions that may have applications in personalized cell-based therapy.The mammary gland is a primary target for carcinogenesis. Breast cancer occurs at a high rate and affects one in eight women in Western countries during their lifetime.1, 2 In the United States alone, 232 340 new invasive breast cancer cases were reported for women in 2013 and 39 620 patients died.3 Regenerative therapy of the damaged mammary gland tissues is the best way to restore breast functions; therefore, the creation of stem cells that are capable of developing into fully functional mammary glands is desirable. There are two distinct types of pluripotent stem cells that may be used for this purpose. The first is embryonic stem cells (ESCs) derived from the inner cell mass of embryonic blastocysts,4 and the second is induced pluripotent stem cells (iPSCs) obtained by reprogramming somatic cells.5 Although, in theory, both ESCs and iPSCs can be differentiated into any type of mature cell, use of the latter is more desirable because it does not require the killing of embryos, and the cells can be derived from virtually any type of tissue. In addition, because iPSCs can be generated from the same patient, the use of iPSCs avoids the immunosuppressive reactions that have long hampered organ and tissue transplantation.6, 7, 8 However, recent studies have shown that some iPSCs seem to retain a memory of their origin and exhibit skewed potential during differentiation for tissue/organ formation.9, 10, 11, 12, 13, 14 This feature may represent a limitation if certain cell types from diseased tissues or organs are not available for reprogramming.Numerous studies about the use of ESCs have indicated that, although these cells have the potential to generate all cell types, their differentiation depends critically on many factors.14, 15, 16 Precise conditions are required for driving cells into specific pathways leading to new lineage formation (reviewed in Murry and Keller17 and Cahan and Daley18). Based on these observations, we hypothesized that the skewed differentiation of iPSCs could be overcome by providing favorable conditions for differentiation. To test this hypothesis, we have generated iPSCs from mouse mammary epithelial cells (ME-iPSCs) and mouse-tail fibroblasts (TF-iPSCs), and have studied the gene expression profiles and epigenetic modifications during differentiation. We found that, although these iPSCs activate distinct signature memories that are reflective of their origins during the differentiation process, the fate of iPSCs could be redirected under optimized conditions in favor of the formation of a desired tissue/organ. 相似文献
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