miR-29a-3p orchestrates key signaling pathways for enhanced migration of human mesenchymal stem cells.

BACKGROUND: The homing of human mesenchymal stem cells (hMSCs) is crucial for their therapeutic efficacy and is characterized by the orchestrated regulation of multiple signaling modules. However, the principal upstream regulators that synchronize these signaling pathways and their mechanisms during cellular migration remain largely unexplored. METHODS: miR-29a-3p was exogenously expressed in either wild-type or DiGeorge syndrome critical region 8 (DGCR8) knockdown hMSCs. Multiple pathway components were analyzed using Western blotting, immunohistochemistry, and real-time quantitative PCR. hMSC migration was assessed both in vitro and in vivo through wound healing, Transwell, contraction, and in vivo migration assays. Extensive bioinformatic analyses using gene set enrichment analysis and Ingenuity pathway analysis identified enriched pathways, upstream regulators, and downstream targets. RESULTS: The global depletion of microRNAs (miRNAs) due to DGCR8 gene silencing, a critical component of miRNA biogenesis, significantly impaired hMSC migration. The bioinformatics analysis identified miR-29a-3p as a pivotal upstream regulator. Its overexpression in DGCR8-knockdown hMSCs markedly improved their migration capabilities. Our data demonstrate that miR-29a-3p enhances cell migration by directly inhibiting two key phosphatases: protein tyrosine phosphatase receptor type kappa (PTPRK) and phosphatase and tensin homolog (PTEN). The ectopic expression of miR-29a-3p stabilized the polarization of the Golgi apparatus and actin cytoskeleton during wound healing. It also altered actomyosin contractility and cellular traction forces by changing the distribution and phosphorylation of myosin light chain 2. Additionally, it regulated focal adhesions by modulating the levels of PTPRK and paxillin. In immunocompromised mice, the migration of hMSCs overexpressing miR-29a-3p toward a chemoattractant significantly increased. CONCLUSIONS: Our findings identify miR-29a-3p as a key upstream regulator that governs hMSC migration. Specifically, it was found to modulate principal signaling pathways, including polarization, actin cytoskeleton, contractility, and adhesion, both in vitro and in vivo, thereby reinforcing migration regulatory circuits.

Efficacy and Safety of Human Bone Marrow-Derived Mesenchymal Stem Cells according to Injection Route and Dose in a Chronic Kidney Disease Rat Model.

Background and Objectives: We compared the efficacy and safety of human bone marrow-derived mesenchymal stem cells (hBMSC), delivered at different doses and via different injection routes in an animal model of chronic kidney disease. Methods and Results: A total of ninety 12-week-old rats underwent 5/6 nephrectomy and randomized among nine groups: sham, renal artery control (RA-C), tail vein control (TV-C), renal artery low dose (RA-LD) (0.5×106 cells), renal artery moderate dose (RA-MD) (1.0×106 cells), renal artery high dose (RA-HD) (2.0×106 cells), tail vein low dose (TV-LD) (0.5×106 cells), tail vein moderate dose (TV-MD) (1.0×106 cells), and tail vein high dose (TV-HD) (2.0×106 cells). Renal function and mortality of rats were evaluated after hBMSC injection. Serum blood urea nitrogen was significantly lower in the TV-HD group at 2 weeks (p<0.01), 16 weeks (p<0.05), and 24 weeks (p<0.01) than in the TV-C group, as determined by one-way ANOVA. Serum creatinine was significantly lower in the TV-HD group at 24 weeks (p<0.05). At 8 weeks, creatinine clearance was significantly higher in the TV-MD and TV-HD groups (p<0.01, p<0.05) than in the TV-C group. In the safety evaluation, we observed no significant difference among the groups. Conclusions: Our findings confirm the efficacy and safety of high dose (2×106 cells) injection of hBMSC via the tail vein.

miR-96-5p targets PTEN to mediate sunitinib resistance in clear cell renal cell carcinoma.

A multiple receptor tyrosine kinase inhibitor, sunitinib, is a first-line therapy for clear cell renal cell carcinoma (CCRCC). Unfortunately, it has the major challenges of low initial response rate and resistance after about one year of treatment. Here we evaluated a microRNA (miRNA) and its target responsible for sunitinib resistance. Using miRNA profiling, we identified miR-96-5p upregulation in tumors from sunitinib-resistant CCRCC patients. By bioinformatic analysis, PTEN was selected as a potential target of miR-96-5p, which showed low levels in tumors from sunitinib-resistant CCRCC patients. Furthermore, PTEN and miR-96-5p levels were negatively correlated in a large The Cancer Genome Atlas kidney renal clear cell carcinoma cohort and high miR-96 and low PTEN represented poor prognosis in this cohort. Additionally, four-week sunitinib treatment increased miR-96-5p and decreased PTEN only in tumors from a sunitinib-resistant patient-derived xenograft model. We found a novel miR-96-5p binding site in the PTEN 3' UTR and confirmed direct repression by luciferase reporter assay. Furthermore, we demonstrated that repression of PTEN by miR-96-5p increased cell proliferation and migration in sunitinib-treated cell lines. These results highlight the direct suppression of PTEN by miR-96-5p and that high miR-96-5p and low PTEN are partially responsible for sunitinib resistance and poor prognosis in CCRCC.

Safety of autologous bone marrow-derived mesenchymal stem cells in erectile dysfunction: an open-label phase 1 clinical trial.

BACKGROUND AIMS: The efficacy of phosphodiesterase type 5 inhibitors (PDE5Is), which are commonly used to treat erectile dysfunction (ED), is not satisfactory in patients with denervation of the cavernous nerve due to pelvic surgeries and diabetes mellitus (DM). Pre-clinical studies using bone marrow-derived mesenchymal stem cells (BMSCs) to treat ED have shown promising results. The authors conducted a phase 1 clinical trial with autologous BMSCs in patients with ED due to radical prostatectomy or DM. METHODS: Ten patients (five with post-prostatectomy ED and five with DM-associated ED) who could not perform sexual activity despite taking the maximum dose of a PDE5I were enrolled. The brief clinical trial protocol was registered with the US National Institutes of Health on ClinicalTrials.gov (NCT02344849). The primary outcome was the safety of stem cell therapy, and the secondary outcome was the improvement of erectile function. RESULTS: Of the 13 patients screened, 10 were registered in the clinical trial and received autologous BMSCs and nine completed the clinical trial. One patient with post-prostatectomy ED experienced two treatment-emergent adverse events (TEAEs) (pyrexia and back pain), and two patients with DM-associated ED experienced a total of five TEAEs (one case each of viral upper respiratory tract infection, prostatitis and pruritus and two cases of hyperglycemia). Of these patients, one with DM-associated ED experienced two serious TEAEs (two instances of hyperglycemia). All TEAEs were considered not to be related to autologous BMSC therapy. In addition, no clinical significance was identified related to other safety measures, such as laboratory tests and vital signs. The mean International Index of Erectile Function score increased significantly at 1 month versus baseline (24.9 versus 18.1, P = 0.0222). CONCLUSIONS: This phase 1 clinical trial confirmed the safety and potential efficacy of autologous BMSC therapy in patients with ED. The authors' results need to be confirmed by a phase 2 clinical trial.

Epigenetic regulation of miR-29a/miR-30c/DNMT3A axis controls SOD2 and mitochondrial oxidative stress in human mesenchymal stem cells.

The use of human mesenchymal stem cells (hMSCs) in clinical applications requires large-scale cell expansion prior to administration. However, the prolonged culture of hMSCs results in cellular senescence, impairing their proliferation and therapeutic potentials. To understand the role of microRNAs (miRNAs) in regulating cellular senescence in hMSCs, we globally depleted miRNAs by silencing the DiGeorge syndrome critical region 8 (DGCR8) gene, an essential component of miRNA biogenesis. DGCR8 knockdown hMSCs exhibited severe proliferation defects and senescence-associated alterations, including increased levels of reactive oxygen species (ROS). Transcriptomic analysis revealed that the antioxidant gene superoxide dismutase 2 (SOD2) was significantly downregulated in DGCR8 knockdown hMSCs. Moreover, we found that DGCR8 silencing in hMSCs resulted in hypermethylation in CpG islands upstream of SOD2. 5-aza-2'-deoxycytidine treatment restored SOD2 expression and ROS levels. We also found that these effects were dependent on the epigenetic regulator DNA methyltransferase 3 alpha (DNMT3A). Using computational and experimental approaches, we demonstrated that DNMT3A expression was regulated by miR-29a-3p and miR-30c-5p. Overexpression of miR-29a-3p and/or miR-30c-5p reduced ROS levels in DGCR8 knockdown hMSCs and rescued proliferation defects, mitochondrial dysfunction, and premature senescence. Our findings provide novel insights into hMSCs senescence regulation by the miR-29a-3p/miR-30c-5p/DNMT3A/SOD2 axis.

Establishment of NOAEL for intracavernous injections of human bone marrow-derived mesenchymal stem cells in rats.

Purpose: To assess the possible negative health effects of human bone marrow-derived mesenchymal stem cells (hBMSCs) on fertility and early embryonic development following intracavernous injections in rats. Materials and Methods: A total of 88 Crl:CD(SD) male and female rats were equally divided into 4 groups in a random manner: control group (normal saline), low-dose group (2×105 hBMSCs), moderate-dose group (1×106 hBMSCs), and high-dose group (2×106 hBMSCs). hBMSCs or normal saline was injected into the penis of the rats 3 times at 2-week-intervals prior to mating. We compared each group with respect to parameters of reproduction and histopathology. Results: For male rats, various degrees of flushing and swelling were observed at the penile injection site in all the groups, although the severity increased in a dose-dependent manner in the hBMSC injection groups. There were no statistically significant differences in mean body weights and food consumption among all the groups of both sexes. There were no statistically significant differences in reproductive parameters among all the groups of both sexes. The absolute and relative organ weights did not significantly differ among the groups. At the time of necropsy, no remarkable findings were observed in gross examinations in all groups. On histopathological analysis, minimal mononuclear cell infiltration was observed in the right epididymis of each rat in the moderate- and high-dose groups. Conclusions: The non-toxic amount of hBMSCs for male fertility and early embryogenesis in rats under the test conditions was determined to be 2×106 cells/head.

Priming with Toll-like receptor 3 agonist or interferon-gamma enhances the therapeutic effects of human mesenchymal stem cells in a murine model of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease. Great efforts have been recently made to treat AD using mesenchymal stem cells (MSCs), which have immunomodulatory functions. However, the immunomodulatory effects of MSCs need to be enhanced for clinical application in the treatment of AD. OBJECTIVES: To evaluate and characterise the therapeutic effects of human Wharton's jelly-derived MSCs (WJ-MSCs) primed with the Toll-like receptor 3 agonist poly I:C or interferon-γ (IFN-γ) in a murine model of AD. METHODS: Mice were treated with Aspergillus fumigatus extract to induce AD and then subcutaneously injected with non-primed, poly I:C-primed or IFN-γ-primed WJ-MSCs. Clinical symptom scores, transepidermal water loss (TEWL), histological characteristics and cytokine levels were determined. Transcriptome profiling and pathway analyses of primed WJ-MSCs were conducted. RESULTS: The clinical symptom score and TEWL in skin lesions were reduced in mice administered non-primed and primed WJ-MSCs. Epidermal thickness and inflammatory cell infiltration in skin lesions were reduced more in mice administered primed WJ-MSCs than in mice administered non-primed WJ-MSCs. Secretion of interleukin-17 was significantly reduced in skin draining lymph nodes of mice administered primed WJ-MSCs. Genomics and bioinformatics analyses demonstrated the enrichment of certain pathways specifically in WJ-MSCs primed with poly I:C or IFN-γ. CONCLUSIONS: Priming with poly I:C- or IFN-γ improved the therapeutic effects of WJ-MSCs in a murine model of AD. This study suggests that priming with poly I:C or IFN-γ enhances the immunomodulatory functions of WJ-MSCs and can be used as a novel therapeutic approach for AD.

MicroRNA controls of cellular senescence.

Cellular senescence is a state of permanent cell-cycle arrest triggered by different internal and external stimuli. This phenomenon is considered to be both beneficial and detrimental depending on the cell types and biological contexts. During normal embryonic development and after tissue injury, cellular senescence is critical for tissue remodeling. In addition, this process is useful for arresting growth of tumor cells, particularly during early onset of tumorigenesis. However, accumulation of senescent cells decreases tissue regenerative capabilities and induces inflammation, which is responsible for cancer and organismal aging. Therefore cellular senescence has to be tightly regulated, and dysregulation might lead to the aging and human diseases. Among many regulators of cellular senescence, in this review, I will focus on microRNAs, small non-coding RNAs playing critical roles in diverse biological events including cellular senescence. [BMB Reports 2018; 51(10): 494-500].

Antioxidant effects of selenocysteine on replicative senescence in human adipose-derived mesenchymal stem cells.

In most clinical applications, human mesenchymal stem cells (hMSCs) are expanded in large scale before their administration. Prolonged culture in vitro results in cellular senescenceassociated phenotypes, including accumulation of reactive oxygen species (ROS) and decreased cell viabilities. Profiling of stem cell-related genes during in vitro expansion revealed that numerous canonical pathways were significantly changed. To determine the effect of selenocysteine (Sec), a rare amino acid found in several antioxidant enzymes, on the replicative senescence in hMSCs, we treated senescent hMSCs with Sec. Supplementation of Sec in the culture medium in late-passage hMSCs reduced ROS levels and improved the survival of hMSCs. In addition, a subset of key antioxidant genes and Sec-containing selenoproteins showed increased mRNA levels after Sec treatment. Furthermore, ROS metabolism and inflammation pathways were predicted to be downregulated. Taken together, our results suggest that Sec has antioxidant effects on the replicative senescence of hMSCs. [BMB Reports 2017; 50(11): 572-577].

Bone marrow-derived mesenchymal stromal cell therapy in a rat model of cavernous nerve injury: Preclinical study for approval.

BACKGROUND AIMS: Although clinical studies using stem cells to treat erectile dysfunction have been performed or are ongoing, there is little consensus on the optimal protocol. We aimed to develop a protocol optimizing human bone marrow-derived mesenchymal stromal cell (hBMSC) therapy in a rat model of cavernous nerve injury. METHODS: We performed, in order, a dose-finding study, a toxicokinetic study of hBMSCs, and a study to determine the timing and number of cell injections. RESULTS: From the dose-finding study, 1 × 10(6) cells were selected as the dose per hBMSC injection. From the toxicokinetic study, 14 days was selected as the interval between repeat treatments. In the final study, the ratio of maximal intracavernous pressure to mean arterial pressure was significantly lower in the control group than in the sham group (23.4% vs. 55.1%, P <0.001). An immediate single injection of hBMSCs significantly improved erectile function compared with the control group (39.8%, P = 0.035), whereas a delayed single injection showed improvement with a marginal trend (38.1%, P = 0.079). All histomorphometric changes were significantly more improved in the immediate or delayed single injection groups than in the control group. Repeat treatments did not provide any benefit for the recovery of erectile function and histomorphometric changes. CONCLUSIONS: Intracavernous injection of 1 × 10(6) hBMSCs results in a recovery of penile erection and histomorphometric changes in a rat model of cavernous nerve injury, even when treatment was delayed until 4 weeks after cavernous nerve injury.

Inhibition of EHMT2/G9a epigenetically increases the transcription of Beclin-1 via an increase in ROS and activation of NF-κB.

We previously reported that BIX-01294 (BIX), a small molecular inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2/G9a), induces reactive oxygen species (ROS)-dependent autophagy in MCF-7 cells. Herein, we analyzed the epigenetic mechanism that regulates the transcription of Beclin-1, a tumor suppressor and an autophagy-related gene (ATG). Inhibition of EHMT2 reduced dimethylation of lysine 9 on histone H3 (H3K9me2) and dissociated EHMT2 and H3K9me2 from the promoter of Beclin-1. To this promoter, RNA polymerase II and nuclear factor kappa B (NF-κB) were recruited in a ROS-dependent manner, resulting in transcriptional activation. Moreover, treatment with BIX reversed the suppression of Beclin-1 by the cooperative action of EHMT2 and DNA methyltransferase 1 (DNMT1). Accordingly, a combination treatment with BIX and 5-Aza-2'-deoxycytidine (5-Aza-Cd), a DNMT1 inhibitor, exerted a synergistic effect on Beclin-1 expression. Importantly, high levels of EHMT2 expression showed a significant association with low levels of Beclin-1 expression, which was related to a poor prognosis. These findings suggest that EHMT2 can directly repress Beclin-1 and that the inhibition of EHMT2 may be a useful therapeutic approach for cancer prevention by activating autophagy.

Novel peptides functionally targeting in vivo human lung cancer discovered by in vivo peptide displayed phage screening.

Discovery of the cancer-specific peptidic ligands have been emphasized for active targeting drug delivery system and non-invasive imaging. For the discovery of useful and applicable peptidic ligands, in vivo peptide-displayed phage screening has been performed in this study using a xenograft mouse model as a mimic microenvironment to tumor. To seek human lung cancer-specific peptides, M13 phage library displaying 2.9 × 10(9) random peptides was intravenously injected into mouse model bearing A549-derived xenograft tumor through the tail vein. Then the phages emerged from a course of four rounds of biopanning in the xenograft tumor tissue. Novel peptides were categorized into four groups according to a sequence-homology phylogenicity, and in vivo tumor-targeting capacity of these peptides was validated by whole body imaging with Cy5.5-labeled phages in various cancer types. The result revealed that novel peptides accumulated only in adenocarcinoma lung cancer cell-derived xenograft tissue. For further confirmation of the specific targeting ability, in vitro cell-binding assay and immunohistochemistry in vivo tumor tissue were performed with a selected peptide. The peptide was found to bind intensely to lung cancer cells both in vitro and in vivo, which was efficiently compromised with unlabeled phages in an in vitro competition assay. In conclusion, the peptides specifically targeting human lung cancer were discovered in this study, which is warranted to provide substantive feasibilities for drug delivery and imaging in terms of a novel targeted therapeutics and diagnostics.

Raloxifene induces autophagy-dependent cell death in breast cancer cells via the activation of AMP-activated protein kinase.

Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death. Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.

Ibulocydine sensitizes human cancers to radiotherapy by induction of mitochondria-mediated apoptosis.

BACKGROUND AND PURPOSE: Ibulocydine (IB), a novel prodrug of CDK inhibitor, has been reported to have anti-cancer effect in human hepatoma cells. In order to address its feasibility as a radiosensitizer to improve radiotherapeutic efficacy for human cancers, this study was designed. MATERIAL AND METHODS: Human cancer cells of lung and colon were treated with IB and/or radiotherapy (RT). The cellular effects were assessed by CCK-8, clonogenic, flow cytometric, and western blotting assays. In vivo radiotherapeutic efficacy was evaluated using the xenograft mouse model. RESULTS: Combined treatment of IB and RT significantly reduced viability and survival fraction of the cells. Apoptotic cell death accompanied with activation of caspases, decrease in Bcl-2/Bax expression, loss of mitochondrial membrane potential (MMP) leading to release of cytochrome c into cytosol was observed. Recovery of Bcl-2 expression level by introducing Bcl-2 expressing plasmid DNA compromised the loss of MMP and apoptosis induced by IB and RT. In vivo therapeutic efficacy of combined treatment was verified in the xenograft mouse model, in which tumor growth was markedly delayed by RT with IB. CONCLUSIONS: IB demonstrated the property of sensitizing human cancer cells to RT by induction of mitochondria-mediated apoptosis, suggesting that IB deserves to be applied for chemoradiotherapy.

CGP74514A enhances TRAIL-induced apoptosis in breast cancer cells by reducing X-linked inhibitor of apoptosis protein.

BACKGROUND: Despite the selectivity of Tumor necrosis factor Related Apoptosis-Inducing Ligand (TRAIL) for cancer cell killing activity, breast cancer cells are resistant to TRAIL-induced apoptosis for various reasons. MATERIALS AND METHODS: From a functionally-characterized small-molecule dataset, CGP74514A was identified as a TRAIL sensitizer in MCF-7 breast cancer cells. Combination of sub-toxic dose of TRAIL with CGP74514A was evaluated in three TRAIL-resistant breast cancer cells, MCF-7, T47D and SK-BR-3. RESULTS: In all tested cells, CGP74514A enhanced TRAIL sensitivity. Combination treatment triggered apoptotic events faster than single treatment. Regarding its mechanism of action, CGP74514A reduced cytosolic X-linked inhibitor of apoptosis protein (XIAP). Small interfering RNA-mediated knockdown experiments showed that reduction of XIAP is the reason of sensitization. CONCLUSION: CGP74514A sensitized breast cancer cells to TRAIL via reduction of XIAP expression level.

miR-140-5p suppresses BMP2-mediated osteogenesis in undifferentiated human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have self-renewal and differentiation capabilities but the regulatory mechanisms of MSC fate determination remain poorly understood. Here, we aimed to identify microRNAs enriched in hMSCs that modulate differentiation commitments. Microarray analysis revealed that miR-140-5p is commonly enriched in undifferentiated hMSCs from various tissue sources. Moreover, bioinformatic analysis and luciferase reporter assay validated that miR-140-5p directly represses bone morphogenic protein 2 (BMP2). Furthermore, blocking miR-140-5p in hMSCs increased the expression of BMP signaling components and critical regulators of osteogenic differentiation. We propose that miR-140-5p functionally inhibits osteogenic lineage commitment in undifferentiated hMSCs.

Global knockdown of microRNAs affects the expression of growth factors and cytokines in human adipose-derived mesenchymal stem cells.

Cell therapies utilizing mesenchymal stem cells (MSCs) have a great potential in many research and clinical settings. The mechanisms underlying the therapeutic effects of MSCs have been studied previously and the paracrine effects elicited by their production of various growth factors and cytokines were recognized as being crucial. However, the molecular controls that govern these paracrine effects remain poorly understood. To elucidate the molecular regulators of this process, we performed a global knockdown of microRNAs (miRNAs) in human adipose-derived mesenchymal stem cells (hADSCs) by inhibiting DGCR8, a key protein in miRNA biogenesis. Global disruption of miRNA biogenesis in hADSCs caused dramatic changes in the expression of subsets of growth factors and cytokines. By performing an extensive bioinformatic analysis, we were able to associate numerous putative miRNAs with these genes. Taken together, our results strongly suggest that miRNAs are essential for the production of growth factors and cytokines in hADSCs.

Sodium meta-arsenite induces reactive oxygen species-dependent apoptosis, necrosis, and autophagy in both androgen-sensitive and androgen-insensitive prostate cancer cells.

Sodium meta-arsenite (NaAsO2), a novel compound synthesized by Komipham International Co. Ltd, is an orally bioavailable, water-soluble trivalent arsenical that has shown potent cytotoxic activity in human solid cancer cells in vitro and in vivo, and is currently undergoing phase I/II clinical trials for the treatment of prostate cancer. In this study, mechanisms of cell death induced by sodium meta-arsenite were investigated. Sodium meta-arsenite reduced cell viability and increased the sub-G1 population in cell cycle analysis in both androgen-sensitive LNCaP and androgen-insensitive CWR22RV1 cells. The apoptosis induced by sodium meta-arsenite was associated with cleavage of caspases 3, 8, and 9, and poly (ADP-ribose) polymerase (PARP) and increased annexin V-positive cells, and was inhibited by the pan-caspase inhibitor Z-VAD-fmk. Sodium meta-arsenite also increased the level of the autophagy marker microtubule-associated protein 1 light chain 3 (LC3)-II and the number of autophagic vacuoles as shown by electron microscopy. Both the autophagy inhibitor 3-methyladenine and the necrosis inhibitor necrostatin-1 blocked cell death induced by sodium meta-arsenite. Moreover, sodium meta-arsenite led to the accumulation of intracellular reactive oxygen species (ROS) and N-acetyl-L-cysteine (NAC), a ROS scavenger, decreased sodium meta-arsenite-induced levels of cleaved PARP and LC3-II. Propidium iodide (PI) staining also showed that NAC restored membrane integrity, damaged by sodium meta-arsenite. Therefore, these results suggest that sodium meta-arsenite induces apoptotic, necrotic, and autophagic cell death through intracellular ROS accumulation in both androgen-sensitive and androgen-insensitive prostate cancer cells and may be used as a new anticancer drug for the treatment of prostate cancer.

Curcumin enhances TRAIL-induced apoptosis of breast cancer cells by regulating apoptosis-related proteins.

The TNF-related apoptosis inducing ligand (TRAIL) has promising anti-cancer therapeutic activity, although significant percentage of primary tumors resistant to TRAIL-induced apoptosis remains an obstacle to the extensive use of TRAIL-based mono-therapies. Natural compound curcumin could potentially sensitize resistant cancer cells to TRAIL. We found that the combination of TRAIL with curcumin can synergistically induces apoptosis in three TRAIL-resistant breast cancer cell lines. The mechanism behind this synergistic cell death was investigated by examining an effect of curcumin on the expression and activation of TRAIL-associated cell death proteins. Immunoblotting, RNA interference, and use of chemical inhibitors of TRAIL-activate signaling revealed differential effects of curcumin on the expression of Mcl-1 and activities of ERK and Akt. Curcumin-induced production of reactive oxygen species did not affect total expression of DR5 but it enhanced mobilization of DR5 to the plasma membrane. In these breast cancer cells curcumin also induced downregulation of IAP proteins. Taken together, our data suggest that a combination of TRAIL and curcumin is a potentially promising treatment for breast cancer, although the specific mechanisms involved in this sensitization could differ even among breast cancer cells of different origins.

Conservation and variability in the structure and function of the Cas5d endoribonuclease in the CRISPR-mediated microbial immune system.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form an RNA-mediated microbial immune system against invading foreign genetic elements. Cas5 proteins constitute one of the most prevalent Cas protein families in CRISPR-Cas systems and are predicted to have RNA recognition motif (RRM) domains. Cas5d is a subtype I-C-specific Cas5 protein that can be divided into two distinct subgroups, one of which has extra C-terminal residues while the other contains a longer insertion in the middle of its N-terminal RRM domain. Here, we report crystal structures of Cas5d from Streptococcus pyogenes and Xanthomonas oryzae, which respectively represent the two Cas5d subgroups. Despite a common domain architecture consisting of an N-terminal RRM domain and a C-terminal ß-sheet domain, the structural differences between the two Cas5d proteins are highlighted by the presence of a unique extended helical region protruding from the N-terminal RRM domain of X. oryzae Cas5d. We also demonstrate that Cas5d proteins possess not only specific endoribonuclease activity for CRISPR RNAs but also nonspecific double-stranded DNA binding affinity. These findings suggest that Cas5d may play multiple roles in CRISPR-mediated immunity. Furthermore, the specific RNA processing was also observed between S. pyogenes Cas5d protein and X. oryzae CRISPR RNA and vice versa. This cross-species activity of Cas5d provides a special opportunity for elucidating conserved features of the CRISPR RNA processing event.