Reportedly, antiapoptotic Bcl-2 family proteins suppress apoptosis by binding to and inhibiting members of the CED-4 family of caspase activators. To explore this question, we used embryonic stem (ES) cells in which one (-/+) or both (-/-) copies of the gene encoding apoptotic protease activating factor 1 (Apaf-1), a CED-4 homologue, were disrupted by homologous recombination. Stable clones of heterozygous (-/+) and homozygous (-/-) Apaf-1 knockout ES cells that overexpressed Bcl-2 were generated. Withdrawal of serum growth factors or stimulation of heterozygous ES cells with staurosporine (STS), ultraviolet (UV)B irradiation, etoposide (VP16), or cisplatin induced apoptosis followed by cell death (determined by failure to exclude propidium iodide dye). These cell death stimuli also induced activation of several types of caspases and loss of mitochondrial membrane potential (DeltaPsi) in heterozygous (+/-) Apaf-1 knockout ES cells. In addition, overexpression of Bcl-2 protected against these events in Apaf-1-expressing ES cells. In contrast, STS, UVB, and VP16 induced little or no caspase activation and apoptosis in homozygous (-/-) Apaf-1 knockout ES cells. Nevertheless, Apaf-1-deficient ES cells subjected to these cell death stimuli or deprived of growth factors did eventually die through a nonapoptotic mechanism associated with loss of DeltaPsi. Moreover, Bcl-2 overprotection preserved DeltaPsi, reduced the percentage of Apaf-1(-/)- ES cells undergoing cell death, and increased clonigenic survival. The extent of Bcl-2-mediated cytoprotection was not significantly different for heterozygous (-/+) versus homozygous (-/-) Apaf-1 knockout cells. Furthermore, although Bcl-2 could be readily coimmunoprecipitated with Bax, associations with Apaf-1 were undetectable under conditions where Apaf-1 interactions with procaspase-9 were observed. We conclude that Bcl-2 has cytoprotective functions independent of Apaf-1, preserving mitochondrial function through a caspase-independent mechanism.

Apoptotic protease activating factor-1 (Apaf-1) is an adaptor molecule, essential for activating initiator caspase and downstream effector caspases, which directly cause apoptosis. In fruit flies, nematodes, and mammals, Apaf-1 has been extensively studied. However, the structure and function of Apaf-1 in Lepidoptera remain unclear. This study identified a novel Apaf-1 from Spodoptera litura, named Sl-Apaf-1. Sl-Apaf-1 contains three domains: a CARD domain, as well as NOD and WD motifs, and is very similar to mammalian Apaf-1. Interference of Sl-apaf-1 expression in SL-1 cells blocked apoptosis induced by actinomycin D. Overexpression of Sl-apaf-1 significantly enhances apoptosis induced by actinomycin D in Sf9/SL-1/U2OS cells, suggesting that the function of Sl-Apaf-1 is evolutionarily conserved. Furthermore, Sl-Apaf-1 could interact with Sl-caspase-5 (a homologue of mammalian caspase-9) and yielded a binding affinity of 1.37 × 106 M-1 according isothermal titration calorimetry assay. Initiator caspase (procaspase-5) of S. litura could be activated by Sl-Apaf-1 (without WD motif) in vitro, and the activated Sl-caspase-5 could cleave Sl-procaspase-1 (a homologue of caspase-3 in mammals), which directly caused apoptosis. This study demonstrates the key role of Sl-Apaf-1 in the apoptosis pathway, suggesting that the apoptosis pathway in Lepidopteran insects and mammals is conserved.

(2S,3S,4S,5R,6R)-6-(4-((4-guanidinobutoxy)carbonyl)-2,6-dihydroxyphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (ZYZ-488) was discovered as a novel inhibitor of apoptotic protease activating factor-1 (Apaf-1). In present work, a surface plasmon resonance (SPR) assay confirms the direct binding between ZYZ-488 and Apaf-1 and this interaction was found to be able to block the recruitment of procaspase-9 by Apaf-1. This study also shows that the treatment of MI (myocardial infarction) mice with this novel Apaf-1 inhibitor remarkably reduces the infarct size, improves cardiac functions, and attenuates the histopathology changes caused by MI. Meanwhile, here it is shown that ZYZ-488 decreases myocardial enzyme release, inhibits cardiomyocyte apoptosis, and suppresses the activation of the downstream cascade of caspases. Moreover, in silico prediction validated the drug-like properties of ZYZ-488. In conclusion, our findings present the first piece of evidence indicating the interaction between Apaf-1 and procaspase-9 as a novel therapeutic target in myocardial infarction and suggesting ZYZ-488 as a promising therapeutic option for myocardial infarction disease.

Cutaneous squamous cell carcinoma (cSCC) accounts for 20% of non-melanoma skin cancer worldwide. MicroRNAs (miRNAs or miRs) are a subtype of non-coding RNA associated with the progression of various types of human cancer. MiR-186 has been demonstrated to act as an oncogene in human tumors. However, the role of miR-186 in cSCC remains unclear. The expression of miR-186 and apoptotic protease activating factor 1 (APAF1) was examined using reverse transcription-quantitative polymerase chain reaction, western blotting and immunofluorescence. The correlation between miR-186 and APAF1 was determined using a dual-luciferase assay. Mimics or inhibitors of miR-186 were transfected into A-431 cells to establish cell lines with overexpressed or knocked-down miR-186, respectively. EdU staining and colony formation assays were performed to detect cell proliferation. Transwell and wound-healing assays were performed to analyze cell invasion and migration, respectively. Hoechst staining and flow cytometry were performed to assess cell apoptosis and cell cycle distribution. MiR-186 expression was significantly increased, while APAF1 expression was significantly decreased in cSCC tissues compared with the controls. An miR-186 binding site was predicted in APAF1 and their expression was negatively correlated in cSCC tissues. Cell proliferation, invasion and migration were significantly enhanced in the miR-186-overexpressed A-431 cells and attenuated in miR-186 knockdown cells compared with the control. APAF1 expression was regulated by miR-186, while APAF1 knockdown significantly promoted cell invasion and inhibited cell apoptosis. In summary, the results of the present study indicate that miR-186 serves as an oncogene in cSCC by inhibiting APAF1.

How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the caspase activator apoptosis protease-activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2-like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model, we generated monoclonal antibodies to Apaf-1 and used them to determine its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-x(L) were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-x(L) precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-x(L) could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering Apaf-1 as proposed by the apoptosome model, Bcl-2-like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation.

Calcium hydroxide is a gold standard dental material generally used for pulpal and periapical therapy including regenerative endodontic procedures because of its positive properties. However, evaluation about this material on stem cells is limited. Human umbilical cord mesenchymal stem cells (HUCMSCs) are potential to be used in regenerative therapy. Regenerative therapy needs a sustainable cell supply to maintain its regenerative capacity. The aim of this study was to ascertain the apoptosis result of calcium hydroxide on HUCMSCs through the expression of apoptotic protease-activating factor-1 (APAF-1), caspase-3, and caspase-9.

Myocardial ischemia/hypoxia-reperfusion injury mediates the progression of multiple cardiovascular diseases. It has been reported that knockdown of adaptor protein containing a PH domain, PTB domain and leucine zipper motif 1 (APPL1) is a significant factor for the progression of myocardial injury. However, the role of APPL1 in myocardial ischemia remains unclear. Hence, the aim of the present study was to investigate the specific mechanism underlying the role of APPL1 in myocardial ischemia.In our study, the mRNA level of APPL1 was detected by quantitative real-time PCR (RT-qPCR). The expressions of APPL1, Apoptotic protease activating factor-1 (APAF-1), cleaved caspase9 and other inflammation- and apoptosis-related proteins were determined by western blotting. The secretion of inflammatory cytokines and lactate dehydrogenase (LDH) levels were measured by commercial assay kits. The H9C2 cell viability was analyzed by cell counting kit-8 (CCK-8) assay. The apoptosis rate of H9C2 cells was analyzed by TUNEL assay. The interaction between APPL1 and APAF-1/caspase9 was determined by Immunoprecipitation (IP).Our findings demonstrated that APPL1 was low expressed in myocardial ischemia tissues and cells. APPL1 knockdown suppressed the viability of myocardial ischemia cells and aggravated hypoxia/reperfusion-induced LDH hypersecretion, inflammation and apoptosis. In addition, the overexpression of APPL1 induced inactivation of APAF-1/Caspase9 signaling pathway. Significantly, APAF1 inhibitor reversed the effect of APPL1 knockdown on viability, LDH secretion, inflammation and apoptosis.We conclude that APPL1 inhibits myocardial ischemia/hypoxia-reperfusion injury via inactivation of APAF-1/Caspase9 signaling pathway. Hence, APPL1 may be a novel and effective target for the treatment of myocardial ischemia.

MicroRNAs are a class of small non-coding, endogenous RNAs involved in cancer development and progression. MicroRNA-221 (mir-221) has been reported to have both an oncogenic and tumor-suppressive role in human tumors, but the role of miR-221 in ovarian cancer is poorly understood. In the present study, the expression levels of miR-221 and the apoptosis protease activating factor 1 (APAF1) protein in 63 samples of ovarian cancer tissues and the cell lines, IOSE25, A2780, OVCAR3, SKOV3 and 3AO were detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. Cell proliferation was measured using cell counting kit-8 (CCK-8); cell migration and invasion were detected using a transwell assay; cell apoptosis was evaluated by flow cytometry and hoechst staining, and a luciferase assay was performed to verify a putative target site of miR-221 in the 3'-UTR of APAF1 mRNA. Expression of miR-221 was upregulated in ovarian cancer tissues. Patients with increased miR-221 expression levels had a reduced disease-free survival (P=0.0014) and overall survival (P=0.0058) compared with those with low miR-221 expression. Transfection of SKOV3 and A2780 cell lines with miR-221 inhibitor induced APAF1 protein expression, suppressed cell proliferation and migration and promoted tumor cell apoptosis. In conclusion, the APAF1 gene was confirmed as a direct target of miR-221 and overexpression of APAF1 suppressed ovarian cancer cell proliferation and induced cell apoptosis in vitro. These findings indicate that miR-221-APAF1 should be studied further as a potential new diagnostic or prognostic biomarker for ovarian cancer.

A proposed mechanism for the cardiotoxicity of doxorubicin (DOX) involves apoptosis in cardiomyocytes. In the study described here, we investigated the molecular basis for the differences in DOX-induced toxicity in adult rat cardiomyocytes (ARCM), neonatal rat cardiomyocytes (NRCM), and rat embryonic H9c2 cardiomyoblasts. Activation of caspase-9 and -3 was considerably lower in DOX-treated ARCM as compared with NRCM and H9c2 cardiomyoblasts. Addition of cytochrome c caused the activation of caspase-9 and -3 in permeabilized NRCM and H9c2 cardiomyoblasts but not in permeabilized ARCM. Expression of proapoptotic proteins, apoptotic protease activating factor-1 (Apaf1), and procaspase-9 was significantly lower, and abundance of antiapoptotic X-linked inhibitor of apoptosis protein (XIAP) was higher in ARCM, as compared with immature cardiac cells. Despite the abundance of XIAP in ARCM, its role in the inhibition of apoptosome function was dismissed, as second mitochondria-derived activator of caspases (Smac)-N7 peptide, had no effect on caspase activation in response to cytochrome c in these cells. Adenoviral expression of Apaf1 exacerbated the activation of caspase-9 and -3 in DOX-treated NRCM, but did not increase their activities in DOX-treated ARCM. This finding points to a major difference in the apoptotic signaling between immature and adult cardiomyocytes. The mitochondrial apoptotic pathway is limited in ARCM treated with DOX.

Seizures in puppies often present a diagnostic challenge in terms of identifying and treating the underlying cause. Dog breeds with mutations of the MDR1-gene are known to show adverse reactions to certain drugs, yet metabolic imbalance exacerbated by physiologically immature organs and other contributing pathologies require consideration before arriving at a diagnosis. This study analysed the brains of two male, 5-week-old Australian Shepherd siblings that died after displaying severe neurological symptoms upon administration of MilproVet® to treat severe intestinal helminth infection. Despite the initial symptoms being similar, their case histories varied in terms of the symptom duration, access to supportive therapy and post-mortem interval. Histopathology and immunohistochemistry were used to obtain more information about the phase of the pathological processes in the brain, employing protein markers associated with acute hypoxic damage (β-amyloid precursor protein/APP) and apoptosis (diacylglycerolkinase-ζ/DGK-ζ, apoptotic protease activating factor 1/Apaf1, and B-cell lymphoma related protein 2/Bcl-2). The results seem to reflect the course of the animals' clinical deterioration, implicating that the hypoxic damage to the brains was incompatible with life, and suggesting the usefulness of the mentioned immunohistochemical markers in clarifying the cause of death in animals with acute neurological deficits.

A mouse model of neuropathic pain consisting of chronic constriction injury (CCI) of the sciatic nerve was used to examine the involvement of reactive oxygen species (ROS) in early spinal cord pro-apoptotic gene over-expression during the development of neuropathic pain. RT-PCR analysis showed increased expression of bax, apoptotic protease-activating factor-1 (apaf-1), and caspase-9 in the dorsal horn spinal cord 3 days after chronic constriction injury of sciatic nerve. Consistent with biomolecular data, a marked increase in TUNEL-positive and caspase-3 active form was observed by 3 days CCI. Administration of phenyl-N-tert-butylnitrone (PBN), a potent ROS scavenger, reduced the development of thermal hyperalgesia and mechanical allodynia at 1 and 3 days post-CCI, and decreased the mRNA levels of bax, apaf-1, and caspase-9. PBN also reduced apoptotic and active Caspase-3 positive profiles in the superficial laminae (I-III) of the spinal cord. This study provides evidence that PBN inhibits over-expression of pro-apoptotic genes and neural apoptosis in the spinal cord dorsal horn induced by early-CCI of the sciatic nerve. These findings suggest that ROS regulate expression of some apoptotic genes which might play a role in the onset of neuropathic pain.

Acute liver injury is characterized by fibrosis, inflammation and apoptosis, leading to liver failure, cirrhosis or cancer and affecting the clinical outcome in the long term. However, no effective therapeutic strategy is currently available. Breviscapine, a mixture of flavonoid glycosides, has been reported to have multiple biological functions. The present study aimed to investigate the effects of breviscapine on acute liver injury induced by CCl4 in mice. C57BL/6 mice were subjected to intraperitoneal injection with CCl4 for 8 weeks with or without breviscapine (15 or 30 mg/kg). Mice treated with CCl4 developed acute liver injury, as evidenced by histological analysis, Masson trichrome and Sirius Red staining, accompanied with elevated levels of alanine aminotransferase and aspartate aminotransferase. Furthermore, increases in pro‑inflammatory cytokines, chemokines and apoptotic factors, including caspase‑3 and poly(ADP ribose) polymerase‑2 (PARP‑2), were observed. Breviscapine treatment significantly and dose‑dependently reduced collagen deposition and the fibrotic area. Inflammatory cytokines were downregulated by breviscapine through inactivating Toll‑like receptor 4/nuclear factor-κB signaling pathways. In addition, co‑administration of breviscapine with CCl4 decreased the apoptotic response by enhancing B‑cell lymphoma-2 (Bcl‑2) levels, while reducing Bcl‑2‑associated X protein, apoptotic protease activating factor 1, caspase‑3 and PARP activity. Furthermore, CCl4‑induced oxidative stress was blocked by breviscapine through improving anti‑oxidants and impeding mitogen‑activated protein kinase pathways. The present study highlighted that breviscapine exhibited liver‑protective effects against acute hepatic injury induced by CCl4 via suppressing inflammation and apoptosis.

To examine the significance of the methylation level of the p53 target and tumour suppressor genes apoptotic protease activating factor-1 (APAF-1) and death-associated protein kinase-1 (DAPK-1) in 80 microdissected tumour samples from transitional cell carcinoma (TCC) of the bladder and 80 tumour samples from clear-cell renal cell carcinoma (RCC) as well as from non-tumourous bladder and kidney tissue. Growth-inhibitory effects of the demethylating agents 5-Aza-2'-deoxycytidine (5-Aza-CdR) and zebularine were investigated in TCC and RCC cell lines. The methylation frequency of APAF-1 (DAPK-1) was 100% (77%) in TCC and 100% (33%) in RCC. The methylation levels of APAF-1 could differentiate between the individual tumour stages in TCC as well as in RCC. The APAF-1 methylation levels in RCC were significantly higher in tumours larger than 4 cm and in high-grade tumours. The methylation frequencies in normal tissue for APAF-1 (DAPK-1) were 11% (8%) in bladder tissue and 9% (5%) in kidney tissue. The growth-inhibitory effect of the demethylating agents in TCC (RT4, T24) and RCC (A498, ClearCa-5) cell lines resulted in a 17-132% prolongation of the doubling time (DT). In RCC cell lines, zebularine was superior to 5-Aza-CdR in achieving a DT prolongation. Quantitative real time RT-PCR detected a re-expression of mRNA transcripts of APAF-1 or DAPK-1. In conclusion, demethylating agents effectively retard growth of TCC and RCC cell lines. Methylation level analysis of specific genes has the potential for further tumour characterisation in TCC and RCC.

Pro-survival members of the BCL-2 family, including MCL-1, are emerging as important proteins during the development and therapeutic response of solid tumors. Notably, high levels of MCL-1 occur in breast cancer, where functional dependency has been demonstrated using cell lines and mouse models. The utility of restoring apoptosis in cancer cells through inhibition of pro-survival BCL-2 proteins has been realized in the clinic, where the first specific inhibitor of BCL-2 is approved for use in leukemia. A variety of MCL-1 inhibitors are now undergoing clinical trials for blood cancer treatment and application of this new class of drugs is also being tested in solid cancers. On-target compounds specific to MCL-1 have demonstrated promising efficacy in preclinical models of breast cancer and show potential to enhance the anti-tumor effect of conventional therapies. Taken together, this makes MCL-1 an extremely attractive target for clinical evaluation in the context of breast cancer.Abbreviations: ADC (antibody-drug conjugate); AML (Acute myeloid leukemia); APAF1 (apoptotic protease activating factor 1); bCAFs (breast cancer associated fibroblasts); BCL-2 (B-cell lymphoma 2); BH (BCL-2 homology); CLL (chronic lymphocytic leukemia); EGF (epidermal growth factor); EMT (epithelial to mesenchymal transition); ER (estrogen receptor); FDA (food and drug administration); GEMM (genetically engineered mouse model); HER2 (human epidermal growth factor 2); IL6 (interleukin 6); IMM (inner mitochondrial membrane); IMS (intermembrane space); MCL-1 (myeloid cell leukemia-1); MOMP (mitochondrial outer membrane permeabilisation); MM (multiple myeloma); PDX (patient-derived xenograft); OMM (outer mitochondrial membrane); PROTAC (proteolysis-targeting chimeras) TNBC (triple negative breast cancer); UPS (ubiquitin mediated proteolysis system).

Apoptotic protease-activating factor 1 (Apaf-1) is a component of apoptosome, which regulates caspase-9 activity. In addition to apoptosis, Apaf-1 plays critical roles in the intra-S-phase checkpoint; therefore, impaired expression of Apaf-1 has been demonstrated in chemotherapy-resistant malignant melanoma and nuclear translocation of Apaf-1 has represented a favorable prognosis of patients with non-small cell lung cancer. In contrast, increased levels of Apaf-1 protein are observed in the brain in Huntington's disease. The regulation of Apaf-1 protein is not yet fully understood. In this study, we show that etoposide triggers the interaction of Apaf-1 with Cullin-4B, resulting in enhanced Apaf-1 ubiquitination. Ubiquitinated Apaf-1, which was degraded in healthy cells, binds p62 and forms aggregates in the cytosol. This complex of ubiquitinated Apaf-1 and p62 induces caspase-9 activation following MG132 treatment of HEK293T cells that stably express bcl-xl. These results show that ubiquitinated Apaf-1 may activate caspase-9 under conditions of proteasome impairment.

The Chinese herbal compound Heshouwuyin has been shown to downregulate the apoptotic rate of testicular tissue cells in Wistar naturally aging rats, and this effect might be related to the mitochondrial pathway [15]. Apoptotic protease activating factor-1 (Apaf-1) is a major component of the apoptotic complex, which is a key element of the mitochondrial endogenous apoptotic pathway [13]. To further clarify the mechanism of Heshouwuyin in the mitochondrial apoptotic pathway, this study used Apaf-1 as a target to explore the mechanism by which Heshouwuyin inhibits the Apaf-1 pathway of spermatogenic cell apoptosis.

Apoptotic protease-activating factor 1 (APAF-1) is a central component of the intrinsic pathway of apoptosis. Our study aims at searching the role of APAF-1 in iron overload myelodysplastic syndrome (MDS). Erythroid apoptosis rate, mRNA expression levels of APAF-1, and caspase-9 activity were determined by flow cytometry, quantitative real-time PCR, and colorimetric assay in MDS patients, respectively. In addition, K562 and MDS-L cell lines were incubated with different concentrations of ferric ammonium citrate (FAC) or ferric ammonium citrate + desferrioxamine (FAC + DFO) in vitro to observe the alteration in erythrocyte apoptosis rate, APAF-1 mRNA, and protein expression levels. Moreover, as control, erythroid apoptosis rate and APAF-1 mRNA expression were detected after silencing APAF-1 expression by endoribonuclease-prepared small interfering RNAs (esiRNAs) in K562 and MDS-L cell lines. Both erythroid apoptosis rate and APAF-1 mRNA expression of the iron overload (IO) group were significantly higher than those of the non-IO group (P < 0.001 and P < 0.001). There is a significant difference of caspase-9 activity between the IO group and the non-IO group (P < 0.001). Erythroid apoptosis rate and APAF-1 mRNA expression of K562 and MDS-L cell lines significantly elevated after FAC incubation in different concentrations (P < 0.001 and P < 0.001 for K562; P < 0.001 and P < 0.001 for MDS-L), while erythroid apoptosis rate and APAF-1 mRNA expression in the FAC + DFO group declined (P < 0.001 and P < 0.001 for K562; P < 0.001 and P < 0.001 for MDS-L). After silencing of APAF-1 expression with specific esiRNAs, erythroid apoptosis rate and APAF-1 mRNA expression of K562 and MDS-L cell lines markedly decreased (P < 0.001 and P < 0.001 for K562; P < 0.001 and P < 0.001 for MDS-L). APAF-1 plays an important role in iron-induced erythroid apoptosis increase in MDS.

The apoptotic protease-activating factor 1 (Apaf-1) controls the onset of many known forms of intrinsic apoptosis in mammals. Apaf-1 exists in normal cells as an autoinhibited monomer. Upon binding to cytochrome c and dATP, Apaf-1 oligomerizes into a heptameric complex known as the apoptosome, which recruits and activates cell-killing caspases. Here we present an atomic structure of an intact mammalian apoptosome at 3.8 Å resolution, determined by single-particle, cryo-electron microscopy (cryo-EM). Structural analysis, together with structure-guided biochemical characterization, uncovered how cytochrome c releases the autoinhibition of Apaf-1 through specific interactions with the WD40 repeats. Structural comparison with autoinhibited Apaf-1 revealed how dATP binding triggers a set of conformational changes that results in the formation of the apoptosome. Together, these results constitute the molecular mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.

Binding of cytochrome c, released from the damaged mitochondria, to the apoptotic protease activating factor 1 (Apaf-1) is a key event in the apoptotic signaling cascade. The binding triggers a major domain rearrangement in Apaf-1, which leads to oligomerization of Apaf-1/cytochrome c complexes into an apoptosome. Despite the availability of crystal structures of cytochrome c and Apaf-1 and cryo-electron microscopy models of the entire apoptosome, the binding mode of cytochrome c to Apaf-1, as well as the nature of the amino acid residues of Apaf-1 involved remain obscure.

MicroRNAs have been proved to participate in multiple biological processes in cancers. For developing resistance to cytotoxic drug, cancer cells, especially the cancer stem cells, usually change their microRNA expression profile to survive in hostile environments. In the present study, we found that expression of microRNA-27a was increased in colorectal cancer stem cells. High level of microRNA-27a was indicated to induce the resistance to TNF-related apoptosis-inducing ligand (TRAIL). Knockdown of microRNA-27a resensitized colorectal cancer stem cells to TRAIL-induced cell death. Mechanically, the gene of Apaf-1, which is associated with the mitochondrial apoptosis, was demonstrated to be the target of microRNA-27a in colorectal cancer stem cells. Knockdown of microRNA-27a increased the expression level of Apaf-1, thus enhancing the formation of Apaf-1-caspase-9 complex and subsequently promoting the TRAIL-induced apoptosis in colorectal cancer stem cells. These findings suggested that knockdown of microRNA-27a in colorectal cancer stem cells by the specific antioligonucleotides was potential to reverse the chemoresistance to TRAIL. It may represent a novel therapeutic strategy for treating the colorectal cancer more effectively.