Polyethyleneimine-polyethylene glycol copolymer targeted by anti-HER2 nanobody for specific delivery of transcriptionally targeted tBid containing construct.

The present research seeks to investigate the process of mixing targeted gene delivery and transcriptional targeting. We have conjugated Polyethylenimine polymers (PEI) and molecules of poly (ethylene glycol). The next step was covalent attachment of anti-HER2 variables domains of camelid heavy chains antibodies (VHHs) or nanobodies (Nbs) to the distal terminals of NHS-PEG3500 in PEI-PEG nanoparticles. The whole procedure yielded PEI-PEG-Nb immunoconjugates. Having determined the properties of polyplexes, steps were taken to investigate the most efficient ratio of PEI polymers to pDNA molecules (N/P) so that the greatest rate of transfection may be obtained. This immune targeted nano biopolymer could condense the gene constructs that coded a transcriptionally targeted truncated -Bid (tBid) killer gene which was controlled by the breast cancer-specific MUC1 promoter. The favourable physicochemical properties matching both the size and zeta potential were observed in engineered polyplexes. Elevated transfection efficiency in HER2 positive cell lines using Nb-modified polyplexes were shown by the results of flow cytometry as compared against non-modified particles. 1.6 and 4.8 fold higher transfection efficiencies were observed in in vitro gene expression researches which used PEI-PEG-Nb/pGL4.50 compared to the situation when native PEI polymers were utilized in both BT-474 and SK-BR-3, respectively. A 2.22 and 3.62 fold rise in the level of tBid gene expression in BT-474 and SK-BR-3 cell lines relative to unmodified PEI treated cells was the result of transfection with PEI-PEG-Nb/pMUC1-tBid, respectively. In those HER2-positive cells which were transfected by targeted polyplexes, higher levels of cell death were observed. This fact points not only to the effective targeted delivery, but it is also indicative of transcriptional targeting efficiency of tBid killer gene when its expression is controlled by MUC1 promoter.

Improved cytotoxicity of novel TRAIL variants produced as recombinant fusion proteins.

The TNF-Related Apoptosis Inducing Ligand (TRAIL) cytokine triggers apoptosis specifically in cancer cells. Susceptibility of a given cell to TRAIL depends on the activity of regulatory proteins, one of the most important of which is BID. The aim of this study was to increase the cytotoxic potential of TRAIL against cancer cells. TRAIL was fused to the BH3 domain of BID. Hence, TRAIL acted not only as an anticancer agent, but also as a specific carrier for the BID fragment. Two fusion protein variants were obtained by genetic engineering, harboring two different linker sequences. The short linker allowed both parts of the fusion protein to fold into their native structures. The long linker influenced the structure of the fused proteins but nonetheless resulted in their highest cytotoxic activity. Optimal buffer formulation was determined for all the analyzed TRAIL variants. Fusing the BH3 domain of BID to TRAIL improved the cytotoxic potential of TRAIL. Further, these findings may be useful for the optimization of other anticancer drugs based on TRAIL, since the appropriate formulation would secure their native structures during prolonged storage.

Dual targeting and enhanced cytotoxicity to HER2-overexpressing tumors by immunoapoptotin-armored mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are promising vehicles for the delivery of anticancer agents in cancer therapy. However, the tumor targeting of loaded therapeutics is essential. Here, we explored a dual-targeting strategy to incorporate tumor-tropic MSC delivery with HER2-specific killing by the immunoapoptotin e23sFv-Fdt-tBid generated in our previous studies. The MSC engineering allowed simultaneous immunoapoptotin secretion and bioluminescence detection of the modified MSCs. Systemic administration of the immunoapoptotin-engineered MSCs was investigated in human HER2-reconstituted syngeneic mouse models of orthotopic and metastatic breast cancer, as well as in a xenograft nude mouse model of orthotopic gastric cancer. In vivo dual tumor targeting was confirmed by local accumulation of the bioluminescence-imaged MSCs and persistence of His-immunostained immunoapoptotins in tumor sites. The added tumor preference of MSC-secreted immunoapoptotins resulted in a significantly stronger antitumor effect compared with purified immunoapoptotins and Jurkat-delivered immunoapoptotins. This immunoapoptotin-armored MSC strategy provides a rationale for its use in extended malignancies by combining MSC mobility with redirected immunoapoptotins against a given tumor antigen.

Protective effects of HGF gene-expressing human mesenchymal stem cells in acetaminophen-treated hepatocytes.

Mesenchymal stem cells (MSC) secrete a great variety of cytokines that have beneficial paracrine actions. Hepatocyte growth factor (HGF) promotes proliferation in several cell types. The aim of the present study was to investigate the protective effect of HGF gene-transfected MSC (HGF-MSC) in acetaminophen (AAP)-treated hepatocytes. We transfected the HGF gene into MSCs and confirmed HGF expression by RT-PCR and western blot. The concentration of HGF in HGF-MSC conditioned media (HGFCM) was upregulated compared with that in control MSCCM samples. Cell viability was increased in HGFCM-treated hepatocytes. Expression of Mcl-1, an anti-apoptosis protein, was increased and expression of pro-apoptosis proteins (Bad, Bik and Bid) was decreased in HGFCM-treated hepatocytes. HGF-MSC had protective effects on AAP-induced hepatocyte damage by enhancing proliferation. These results suggest that HGF-expressing MSCs may provide regenerative potential for liver cell damage.

Sevoflurane post-conditioning protects primary rat cortical neurons against oxygen-glucose deprivation/resuscitation via down-regulation in mitochondrial apoptosis axis of Bid, Bim, Puma-Bax and Bak mediated by Erk1/2.

Temporal post-conditioning helps provide neuroprotection against brain injury secondary to ischemia-reperfusion and is considered an effective intervention, but the exact mechanism of sevoflurane post-conditioning is unclear. The essential axis involves activator Bid, Bim, Puma (BH3s), Bax, and Bak; activates the mitochondrial death program; and might be involved in a cell death signal. Extracellular signal-related kinases 1/2 (Erk1/2) play a pivotal role in cell growth and proliferation. We hypothesized that sevoflurane post-conditioning might inhibit Bid, Bim, Puma, Bax, and Bak expression and is activated by phosphor-Erk1/2 to decrease neuronal death. To test this hypothesis, we exposed primary cortical neuron cultures to oxygen-glucose deprivation for 1h, along with resuscitation for 24h (OGD/R). MTT assays, propidium iodide uptake (PI), JC-1 fluorescence, and Western blot indicated the following: decreased cell viability (P<0.05); increased cell death (P<0.05); decreased mitochondrial membrane potential (P<0.05); and decreased Bid, Bim, Puma, Bax, and Bak expression with OGD/R exposure. Inhibition of Erk1/2 phosphorylation could attenuate sevoflurane post-conditioning that mediated an increase in neuronal viability and mitochondrial membrane potential, as well as a decrease in cell death and Bid, Bim, Puma, Bax, and Bak expression after OGD/R treatment. The results demonstrated that sevoflurane post-conditioning caused a marked decrease in cortical neuronal death secondary to OGD/R exposure through the downregulation of the mitochondrial apoptosis axis involving Bid, Bim, Puma, Bax, and Bak that was mediated by the phosphorylation/activation of Erk1/2.

Inhibition of JNK3 promotes apoptosis induced by BH3 mimetic S1 in chemoresistant human ovarian cancer cells.

Previous studies have suggested that the novel BH3 mimetic S1 could induce apoptosis in diverse tumor cell lines through endoplasmic reticulum (ER) stress or mitochondrial cell death pathways. The activation of c-Jun N-terminal kinase (JNK) through inositol requiring enzyme-1 (IRE1) is closely connected to ER stress-induced apoptosis. However, the role of JNK is complex, as there are different JNK subtypes and the function of each subtype is still not entirely clear. Here we found that the mRNA expression of JNK3 was continuously high in S1-treated human ovarian cancer SKOV3/DDP cells using a human unfolded protein response (UPR) pathway PCR array. Pharmacological inhibition of JNK3 increased cell sensitivity to apoptosis induced by S1. Furthermore, inhibition of JNK3 induced accumulation of both acidic compartment and p62, and upregulated ROS production. Our results suggest that JNK3 plays a pro-survival role during ER stress through preventing the block of autophagic flux and reducing oxidative stress in SKOV3/DDP cells. Inhibition of JNK3 may be a potential method to enhance the killing effect of the Bcl-2 inhibitor S1.

Combination of arginine deprivation with TRAIL treatment as a targeted-therapy for mesothelioma.

UNLABELLED: In the present study we present data to show that certain tumor cells including malignant pleural mesothelioma (MPM) cells do not express argininosuccinate synthetase (ASS), and thus are unable to synthesize arginine from citrulline. Exposure of these ASS-negative cells to the arginine degrading enzyme, arginine deiminase (ADI-PEG20), for 72 h results in significant increases in cleaved caspase-3. Importantly, this apoptotic signal is further strengthened by the addition of TNF-related apoptosis-inducing ligand (TRAIL). Using flow cytometry, we showed that the combination treatment (ADI-PEG20 at 50 ng/ml and TRAIL at 10 ng/ml) for 24 h resulted in profound cell death with 67% of cells positive for caspase-3 activity, while ADI-PEG20 alone or TRAIL alone resulted in only 10-15% cell death. This positive amplification loop is mediated through the cleavage of proapototic protein "BID". CONCLUSION: Our work represents a new strategy for treating patients with malignant pleural mesothelioma using targeted molecular therapeutics based on selected tumor markers, thus avoiding the use of potentially cytotoxic chemotherapy.

ALDOB acts as a novel HBsAg-binding protein and its coexistence inhibits cisplatin-induced HepG2 cell apoptosis.

Chronic infection with hepatitis B virus is a cause of end-stage liver disease and hepatocellular carcinoma (HCC). We previously screened fructose-bisphosphate aldolase B (ALDOB) as a candidate binding protein of hepatitis B surface antigen (HBsAg) using a yeast 2-hybrid assay. In this study we aimed to confirm ALDOB as a binding protein of the S region of the HbsAg (HBs) and to investigate the function and involved mechanism between its interactions during HCC development. Our results demonstrated that both of exogenous and endogenous ALDOB proteins bind to HBs and colocalize in the cytoplasm in vitro. The coexistence of HBs and ALDOB inhibit apoptosis of cisplatin-induced HepG2 cells. Furthermore, western blot analysis showed the coexistence of HBs and ALDOB enhance the phosphorylations of AKT and its downstream of GSK-3ß (phosphorylation); decreased expression of the pro-apoptotic proteins Bax, Bid, Bim, and Puma; and increased expression of the prosurvival proteins Bcl-2, Bcl-xl, and Mcl-1 in HepG2 cells. These findings suggest that interaction between HBs and ALDOB might be applied as a potential therapeutic target during the treatment of HBV-related hepatitis or HCC.

Induction of apoptosis by diphenyldifluoroketone in osteogenic sarcoma cells is associated with activation of caspases.

The aim of the present study was to investigate and compare the effects of diferuloylmethane (curcumin) and diphenyldifluoroketone (EF-24) on cell growth and apoptosis induction in human osteogenic sarcoma cells. This was examined by MTT assay, nuclear DAPI staining, caspase-activation assay, flow cytometry analysis and immunoblotting in Saos2 human osteogenic sarcoma cells. Curcumin and EF-24 inhibited the growth of Saos2 cells in a dose-dependent manner. The apparent potency of EF-24 was more than 3-fold higher that of curcumin. Treatment with curcumin or EF-24 resulted in nuclear condensation and fragmentation in the cells. The caspase-3/-7 activities were detected in living cells treated with curcumin or EF-24. Flow cytometry showed that the rate of apoptosis was increased by curcumin and EF-24 compared to the control. Curcumin and EF-24 promoted the proteolytic cleavages of procaspase-3/-7/-8/-9 with increases in the amount of cleaved caspase-3/-7/-8/-9. The curcumin- or EF-24-induced apoptosis in the Saos2 cells was mediated by the expression of Fas and activation of caspase-8, caspase-3 and poly(ADP-ribose) polymerase. Immunoblotting revealed the Bid and Bcl-2 proteins to be downregulated, and truncated-Bid, Bax and p53 proteins to be upregulated by curcumin and EF-24. Curcumin and EF-24 increased the Bax/Bcl-2 ratio significantly. These results suggest that the curcumin and EF-24 inhibit cell proliferation and induce apoptotic cell death in Saos2 human osteogenic sarcoma cells via both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway, and may have potential properties for anti-osteosarcoma drug discovery.

Prostate apoptosis response-4 is involved in the apoptosis response to docetaxel in MCF-7 breast cancer cells.

Experimental evidence indicates that prostate apoptosis response-4 (Par-4, also known as PAWR) is a key regulator of cancer cell survival and may be a target for cancer-selective targeted therapeutics. Par-4 was first identified in prostate cancer cells undergoing apoptosis. Both intracellular and extracellular Par-4 have been implicated in apoptosis. Relatively little is known about the role of Par-4 in breast cancer cell apoptosis. In this study, we sought to investigate the effects of Par-4 expression on cell proliferation, apoptosis and drug sensitivity in breast cancer cells. MCF-7 cells were stably transfected with expression vectors for Par-4, or transiently transfected with siRNA for Par-4 knockdown. Cell proliferation assays were performed using MTT and apoptosis was evaluated using acridine orange staining, fluorescence microscopy and flow cytometry. Par-4 overexpression reduced MCF-7 proliferation rates. Conversely, Par-4 knockdown led to increased MCF-7 proliferation. Par-4 downregulation also led to increased BCL-2 and reduced BID expression. Par-4 overexpression did not affect the cell cycle profile. However, MCF-7 cells with increased Par-4 expression showed reduced ERK phosphorylation, suggesting that the inhibition of cell proliferation promoted by Par-4 may be mediated by the MAPK/ERK1/2 pathway. MCF-7 cells with increased Par-4 expression showed a marginal increase in early apoptotic cells. Importantly, we found that Par-4 expression modulates apoptosis in response to docetaxel in MCF7 breast cancer cells. Par-4 exerts growth inhibitory effects on breast cancer cells and chemosensitizes them to docetaxel.

Artemisinin induces A549 cell apoptosis dominantly via a reactive oxygen species-mediated amplification activation loop among caspase-9, -8 and -3.

This report is designed to explore the roles of caspase-8, -9 and -3 in artemisinin (ARTE)-induced apoptosis in non-small cell lung cancer cells (A549 cells). ARTE induced reactive oxygen species (ROS)-mediated apoptosis in dose- and time-dependent fashion. Although ARTE treatment did not induce Bid cleavage and significant loss of mitochondrial membrane potential, it induced release of Smac and AIF but not cytochrome c from mitochondria, and silencing of Bak but not Bax significantly prevented ARTE-induced cytotoxicity. Moreover, ARTE treatment induced ROS-dependent activation of caspase-9, -8 and -3. Of the utmost importance, silencing or inhibiting any one of caspase-8, -9 and -3 almost completely prevented ARTE-induced activation of all the three caspases and remarkably abrogated the cytotoxicity of ARTE, suggesting that ARTE triggered an amplification activation loop among caspase-9, -8 and -3. Collectively, our data demonstrate that ARTE induces a ROS-mediated amplification activation loop among caspase-9, -8 and -3 to dominantly mediate the apoptosis of A549 cells.

The roles of epigenetic modifications of proapoptotic BID and BIM genes in imatinib-resistant chronic myeloid leukemia cells.

In chronic myeloid leukemia (CML), epigenetic modifications such as promoter hypermethylation and inactive histone modification are known mechanisms of drug resistance. In our study, we investigated the roles of promoter hypermethylation of BIM and BID genes and H3K27me3 histone modification on imatinib resistance. We detected higher expression levels of BIM and BID genes and lower expression levels of EZH2, EED2, SIRT1, and SUZ12 genes in imatinib-resistant K562/IMA-3 cells compared to imatinib-non-resistant K562 cells. While we determined the EZH2 and DNMT enzymes as bounded to the promoter of the BIM gene, we did not detect hypermethylation of this promoter. We also found the H3K27me3 histone modification promoter of BIM and BID genes in both cell lines. In conclusion, our results support the notion that DNA promoter methylation may be formed independently from EZH2-H3K27me3 and pro-apoptotic BIM and BID genes are not methyllated in the imatinib resistance of CML cells.

p38 MAPK/PP2Acα/TTP pathway on the connection of TNF-α and caspases activation on hydroquinone-induced apoptosis.

This study investigated tumor necrosis factor-α (TNF-α)-mediated death pathway contribution to hydroquinone (HQ) cytotoxicity in human leukemia U937 cells. HQ-induced apoptosis of human leukemia U937 cells was characterized by the increase in mitochondrial membrane depolarization, procaspase-8 degradation and tBid production. Downregulation of Fas-associated death domain protein (FADD) blocked HQ-induced procaspase-8 degradation and rescued the viability of HQ-treated cells, suggesting the involvement of a death receptor-mediated pathway in HQ-induced cell death. HQ induced increased TNF-α mRNA stability led to TNF-α protein expression upregulation, whereas HQ suppressed TNF-α-mediated NFκB pathway activation. HQ elicited protein phosphatase 2A catalytic subunit α (PP2Acα) upregulation via p38 mitogen-activated protein kinase (MAPK)-mediated CREB/c-Jun/ATF-2 phosphorylation, and PP2Acα upregulation was found to promote tristetraprolin (TTP) degradation. Suppression of p38 MAPK activation and protein phosphatase 2A (PP2A) activity abrogated TNF-α upregulation and procaspase degradation in HQ-treated cells. Overexpression of TTP suppressed HQ-induced TNF-α upregulation and restored the viability of HQ-treated cells. Moreover, TTP overexpression increased TNF-α mRNA decay in HQ-treated cells. Taken together, our data indicate that HQ elicits TNF-α upregulation via p38 MAPK/PP2A-mediated TTP downregulation, and suggest that the TNF-α-mediated death pathway is involved in HQ-induced U937 cell death. The same pathway was also proven to be involved in the HQ-induced death of human leukemia HL-60 and Jurkat cells.

Ethyl gallate induces apoptosis of HL-60 cells by promoting the expression of caspases-8, -9, -3, apoptosis-inducing factor and endonuclease G.

Many phytochemicals have been recognized to have potential therapeutic efficacy in cancer treatment. In this study, we investigated ethyl gallate (EG) for possible proapoptotic effects in the human promyelocytic leukemia cell line, HL-60. We examined cell viability, morphological changes, DNA content and fragmentation, and expression of apoptosis-related proteins for up to 48 h after EG treatment. The results showed that EG induced morphological changes and DNA fragmentation and reduced HL-60 cell viability in a dose-dependent and time-dependent manner. Western blotting analysis indicated that EG-mediated HL-60 apoptosis mainly occurred through the mitochondrial pathway, as shown by the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G), as well as the upregulation of Bcl-2-associated X protein (Bax). EG also activated the death receptor-dependent pathway of apoptosis by enhancing the expression of caspases-8, -9, and -3 and the Bcl-2 interacting domain (Bid). Collectively, our results showed that EG induces apoptosis in HL-60 via mitochondrial-mediated pathways.

Hexane extracts of garlic cloves induce apoptosis through the generation of reactive oxygen species in Hep3B human hepatocarcinoma cells.

Garlic (Allium sativum) compounds have recently received increasing attention due to their cancer chemopreventive properties, and their anticancer activities are extensively reported in many cancer cell lines. However, the anticancer activity and the signaling pathway associated with the induction of apoptosis by extracts of garlic cloves have not been elucidated. In this study, we examined the effects of hexane extracts of garlic cloves (HEGCs) on reactive oxygen species (ROS) production and the association of these effects with apoptotic cell death, using a Hep3B human hepatocarcinoma cell line in vitro. The results demonstrated that HEGCs mediate ROS production, and that this mediation is followed by a collapse of mitochondrial membrane potential (MMP, ΔΨm), the downregulation of anti-apoptotic Bcl-2 and Bcl-xL and the activation of caspase-9 and -3. HEGCs also promoted the activation of caspase-8 and the downregulation of Bid, a BH3-only pro-apoptotic member of the Bcl-2. However, the apoptotic phenomena displayed by HEGCs were significantly diminished by the presence of z-VAD-fmk (non-selective caspase inhibitor). Moreover, N-acetyl-L-cysteine (NAC), a widely used ROS scavenger, effectively blocked the HEGC-induced apoptotic effects via the inhibition of ROS production and MMP collapse. These observations clearly indicate that HEGC-induced ROS are key mediators of MMP collapse, which leads to the induction of apoptosis, followed by caspase activation.

The flavonoid resveratrol suppresses growth of human malignant pleural mesothelioma cells through direct inhibition of specificity protein 1.

Resveratrol (Res), from the skin of red grapes, induces apoptosis in some malignant cells, but there are no reports on the apoptotic effect of Res on human malignant pleural mesothelioma. We found that Res interacts with specificity protein 1 (Sp1). The IC50 for Res was 17 µM in MSTO-211H cells. Cell viability was decreased and apoptotic cell death was increased by Res (0-60 µM). Res increased the Sub-G1 population in MSTO-211H cells and significantly suppressed Sp1 protein levels, but not Sp1 mRNA levels. Res modulated the expression of Sp1 regulatory proteins including p21, p27, cyclin D1, Mcl-1 and survivin in mesothelioma cells. After treatment with Res, apoptosis signaling cascades were activated by the activation of Bid, Bim, caspase-3 and PARP, upregulation of Bax and downregulation of Bcl-xL. Res (20 mg/kg daily for 4 weeks) effectively suppressed tumor growth in vivo in BALB/c athymic (nu+/nu+) mice injected with MSTO-211H cells, an effect that was mediated by inhibition of Sp1 expression and induction of apoptotic cell death. Our results strongly suggest that Sp1 is a novel molecular target of Res in human malignant pleural mesothelioma.

Significance of decreased apoptosis, role of Bid expression and Bcl-Xl/Bid ratio in human jejunal stromal tumors.

Given that the studies on human small intestinal stromal tumors are very limited, our study was designed to determine the significance of apoptosis, the role of some apoptosis-related proteins, including Bax, Bad, Bid, Bcl-2, Bcl-Xl, and Bcl-W, in jejunal stromal tumors (JST). For this purpose, 52 samples were examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunohistochemistry, including 40 cases from normal jejunum and 12 cases from the JST. The results showed that a significantly decreased apoptosis was observed in JST compared with normal jejunum (median of apoptotic index, 0.13% vs 15.17%, p < 0.001). In univariate analysis, statistically significant differences were shown in percentage of positive-expression of Bid (0.0% vs 77.5%, p < 0.001), Bcl-2 (91.7% vs 42.5%, p = 0.003), and Bcl-Xl (75.0% vs 35.0%, p = 0.014), but not in Bax, Bad, and Bcl-W. Moreover, Bcl-Xl was an independent parameter associated with JST by multivariate Logistic analysis. For the role of ratios of anti-apoptotic proteins to pro-apoptotic proteins, Bcl-Xl/Bid ratio was the most preferable index. In conclusion, decreased apoptosis and expression of Bid, increased level of Bcl-Xl may play some important roles in human JST, and Bcl-Xl/Bid ratio may be as a new potentially associated index, although they should be validated in more studies.

Effect of RNA interference of BID and BAX mRNAs on apoptosis in granulosa cell-derived KGN cells.

In mitochondrion-dependent type II apoptosis, BH3-interacting domain death agonist (BID) and BCL-2-associated X protein (BAX) promote death ligand and receptor-mediated cell death. In porcine ovaries, the levels of BID and BAX increase in follicular granulosa cells during atresia. In the present study, to confirm the pro-apoptotic activity of BID and BAX in granulosa cells, we examined the effect of RNA interference of BID or BAX on apoptosis using a human ovarian granulosa tumor cell line, KGN. By reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, expression of BID and BAX was detected in KGN cells. Then, we suppressed BID and BAX mRNA expression in KGN cells using small interfering RNA (siRNA). When BID or BAX was suppressed, a significant decrease in the apoptotic cell rate was noted. In granulosa-derived cells, BID and BAX showed pro-apoptotic activity. These results suggest that BID and BAX act as signal-transducing factors in mitochondrion-dependent type II apoptosis.

Gene expression profiling indicate role of ER stress in miR-23a~27a~24-2 cluster induced apoptosis in HEK293T cells.

Previously, we had reported that overexpression of miR-23a~27a~24-2 cluster induces caspase-dependent and -independent apoptosis via JNK in human embryonic kidney (HEK293T) cells. Herein, we describe the molecular mechanism(s) responsible for miR-23a~27a~24-2 cluster induced apoptosis. Gene expression profiling was used to characterize the transcriptional response to miR-23a~27a~24-2 cluster overexpression in HEK293T cells. The microarray analysis gave 1,025 differentially expressed genes and analysis of the gene expression data with Ingenuity Pathway Analysis (IPA) software revealed p53 signaling, oxidative stress response and mitochondrial dysfunction among the top processes being affected. This data substantiates our previous study where we had reported increase of ROS and the release of proapoptotic factors such as cytochrome c (cyt c) and apoptosis-inducing factor (AIF) from the mitochondria to cytosol. Additionally, components of ER stress-mediated apoptosis pathway i.e., C/EBP homologous protein (CHOP/DDIT3/GADD153) and TRIB3, an Akt inhibitor were found to be significantly enriched. Also, the enhanced expression of ATF3 and ATF4 was observed at RNA as well as protein level in miR-23a~27a~24-2 cluster overexpressed HEK293T cells. Induction of BIM appeared to be specific, because ER stress caused only a minor change in the expression of the related BH3-only proteins BID or PUMA. The fact that miR-23a~27a~24-2 cluster triggered endoplasmic reticulum stress (ER stress) was further established by the increase in cytosolic calcium levels after overexpression of this cluster in HEK293T cells. These findings were also supported by PANTHER analysis wherein biological process categories of apoptosis and stress response were enriched. Taken together, this work underlines the role of ER stress in miR-23a~27a~24-2 cluster mediated apoptosis in HEK293T cells. Since the detailed knowledge of this cluster induced apoptosis has now been elucidated, the in vivo study of this cluster would help evaluate the prospect of its use as a therapeutic in diseases known to occur because of deregulation of apoptosis.

5-azacytidine, a chemotherapeutic drug, induces TRAIL-mediated apoptosis in mouse thymocytes in vivo.

5-azacytidine (5AzC) is a cytidine analogue with two main effects on cellular conditions; DNA damage, resulting in apoptosis, and DNA hypomethylation, restoring normal growth control and differentiation. However, the molecular mechanism of 5AzC-induced apoptosis is not fully understood. The aim of the present study is to clarify this mechanism in mouse thymocytes in vivo. Ten-week-old, male C57BL/6J mice were injected with 5AzC (100 mg/kg) intraperitoneally, and thymuses were examined for apoptotic changes. In the 5AzC-treated thymus, increases of TUNEL-positive thymocytes and cleaved caspase-3 protein, both biochemical features of apoptosis, were detected. 5AzC-induced apoptosis was observed even in the thymuses of mice deficient in p53, a critical factor in the intrinsic apoptotic pathway, and mice with mutated Fas, a death receptor. Furthermore, levels of p53 and Fas proteins were unchanged in the thymus following 5AzC-treatment in wild-type mice. In the 5AzC-treated thymus, the level of cleaved caspase-8 protein, an initiator of the extrinsic apoptotic pathway, increased with the cleavage of its target protein, Bid. Moreover, the level of TRAIL protein, which induces apoptosis through the cleavage of caspase-8, robustly increased in the thymus treated with 5AzC. In conclusion, the 5AzC-induced apoptosis of thymocytes in vivo is implemented through the extrinsic pathway with the activation of TRAIL.