Atractylenolide III induces apoptosis by regulating the Bax/Bcl-2 signaling pathway in human colorectal cancer HCT-116 Cells in vitro and in vivo.

Atractylodes is the dry root of atractylodes macrocephala koidz and has been commonly used as a traditional Chinese medicine (TCM). Atractylenolide III, a main component of atractylodes, has displayed significant effects on anti-inflammation and anticancer. However, the effects of atractylenolide III on growth inhibition and apoptosis induction in colon cancer remain unclear. The results showed that atractylenolide III significantly inhibited the cell growth and induce cellular apoptosis in HCT-116 cells in a concentration dependence manner in vitro. Mechanistic studies further showed that atractylenolide III could regulate the Bax/Bcl-2 apoptotic signaling pathway through promoting the expression of proapoptotic related gene/proteins Bax, caspase-9 and caspase-3 but inhibiting the expression of antiapoptotic related gene/protein Bcl-2 in HCT-116 cells. Furthermore, atractylenolide III also significantly inhibited the tumor growth of HCT-116 tumor xenografts bearing in nude mice through inducing apoptosis by upregulation of the expressions of Bax, cleaved caspase-3 and p53 but downregulation of the expressions of Bcl-2 in HCT-116 tumor tissues in vivo. The studies may provide the scientific rationale for the understanding of the anticancer effect of atractylenolide III. Therefore, atractylenolide III may have the potential to be developed as a promising novel anticancer agent for the treatment of colorectal cancer clinically.

The effects of L-carnitine on renal function and gene expression of caspase-9 and Bcl-2 in monosodium glutamate-induced rats.

BACKGROUND: Monosodium glutamate (MSG) is frequently consumed as a flavor enhancer or food additive. Possible damages induced by MSG effects on some organs have been stated in experimental animal models. The aim of the present study was to evaluate the protective effects of L-carnitine (L-ca) on the renal tissue in MSG-Induced Rats. METHODS: In this regard, 60 male rats were randomly divided into six groups (n = 10/each): 1 (Control); 2 (sham); 3 (L-carnitine 200 mg/kg b.w); 4 (MSG 3 g/kg b.w); 5 (MSG + L-carnitine 100 mg/kg); and 6 (MSG + L-carnitine 200 mg/kg). After 6 months, the rats were sacrificed, the blood sample collected and the kidneys harvested for evaluation of biochemical analytes, genes expression, and histopathological changes. RESULTS: MSG significantly increased the serum level of MDA, BUN, creatinine, uric acid and renal Caspase-9, NGAL and KIM-1 expression, but it decreased the serum activity also renal expression of SOD, catalase, GPX, and Bcl-2 expression compared to the control group. Treatment with L-ca significantly reduced the serum BUN, creatinine, uric acid and MDA level and increased catalase, GPX and SOD compared to the MSG group. However, only administration of L-ca 200 significantly decreased the caspase-9, NGAL and KIM-1; also, it increased the Bcl-2 expression in the kidney compared to the MSG group. CONCLUSIONS: Our findings indicated that L-carnitine had a major impact on the cell protection and might be an effective therapy in ameliorating the complications of the kidney induced by MSG via its antioxidant and anti-apoptotic properties.

Progesterone induces apoptosis by activation of caspase-8 and calcitriol via activation of caspase-9 pathways in ovarian and endometrial cancer cells in vitro.

Previously we have shown inhibition of endometrial cancer cell growth with progesterone and calcitriol. However, the mechanisms by which the two agents attenuate proliferation have not been well characterized yet. Herein, we investigated how progesterone and calcitriol induce apoptosis in cancer cells. DNA fragmentation was upregulated by progesterone and calcitriol in ovarian and endometrial cancer cells. Time-dependent treatment of ovarian cancer cells, ES-2, and TOV-21G with progesterone enhanced caspase -8 activity after 12 h, whereas OV-90, TOV-112D, HEC-1A, and HEC-59 cells showed increased activity after 24 h. Caspase 9 activity was increased in all cell lines after 24 h treatment with calcitriol. Pretreatment of cancer cells with a caspase-8 inhibitor (z-IETD-fmk) or caspase-9 inhibitor (Z-LEHD-fmk) significantly attenuated progesterone and calcitriol induced caspase-8 and caspase-9 expression, respectively. The expression of FasL, Fas, FAD, and pro-caspase-8, which constitute the death-inducing signaling complex (DISC), was upregulated in progesterone treated cancer cells. Knockdown of FAS or FADD with specific siRNAs significantly blocked progesterone-induced caspase-8. Cleavage of the BID was not affected by caspase-8 activation suggesting the absence of cross-talk between caspase-8 and caspase-9 pathways. Calcitriol treatment decreased mitochondrial membrane potential and increased the release of cancer cytochrome C. These findings indicate that progesterone induces apoptosis through activation of caspase-8 and calcitriol through caspase-9 activation in cancer cells. A combination of progesterone-calcitriol activates both extrinsic and intrinsic apoptotic pathways in cancer cells.

Thymol induces mitochondrial pathway-mediated apoptosis via ROS generation, macromolecular damage and SOD diminution in A549 cells.

BACKGROUND: Thymol is a monoterpene phenol found in thyme species plants. The present study was carried out to investigate the effect of thymol and its molecular mechanism on non-small lung cancer (A549) cells. METHODS: The cytotoxic effect of thymol on A549 cells was assessed via MTT assay. ROS production, macromolecular damage, apoptosis were determined using DCF-DA, PI, AO/EtBr stains, respectively. ROS-dependent effect of thymol was confirmed using NAC. The expression of caspase-9, Bcl-2, Bax and cell cycle profile was analyzed via western blot and FACS, respectively. RESULTS: The antiproliferative effect of thymol on A549 cells was found to be both dose and time dependent with IC50 values of 112 µg/ml (745 µM) at 24 h. Thymol treatment favored apoptotic cell death and caused G0/G1 cell cycle arrest. It mediated cellular and nuclear morphological changes, phosphatidylserine translocation, and mitochondrial membrane depolarization. Additionally, upregulation of Bax, downregulation of Bcl-2, and apoptotic fragmented DNA were also observed. Thymol induced ROS by reducing the SOD level which was confirmed via in vitro and in silico analysis. Furthermore, the levels of lipid peroxides and protein carbonyl content were elevated in thymol-treated groups. Notably, N-acetyl cysteine pretreatment reversed the efficacy of thymol on A549 cells. Moreover, thymol-treated human PBMC cells did not show any significant cytotoxicity. CONCLUSION: Overall, our results confirmed that thymol can act as a safe and potent therapeutic agent to treat NSCLC.

The soluble VEGF receptor sFlt-1 contributes to endothelial dysfunction in IgA nephropathy.

Endothelial injury is a common manifestation in IgA nephropathy (IgAN). After the previous identification of the upregulated soluble fms-like tyrosine kinase-1 (sFlt-1) correlated with endothelial injury in IgAN, in the present study, we further explored the role of sFlt-1 in endothelial injury in IgAN. We enrolled 72 patients with IgAN and detected the sFlt-1 levels. The polymeric IgA1 (pIgA1) complexes were isolated from the pooled plasma samples of another 10 patients with IgAN. Apoptosis proteins were detected in cultured human umbilical vein endothelial cells (HUVECs) with the stimulation of recombinant sFlt-1 or the caspase-9 inhibitor Z-LEHD-FMK. We identified there were positive correlations between sFlt-1 and IgA-IgG complex as well as vWF levels in patients with IgAN. The sFlt-1 levels in HUVECs were significantly upregulated by pIgA1 complex derived from IgAN patients in a concentration-dependent manner. The proliferation ability of HUVECs was damaged when stimulated with sFlt-1 protein in a time- and dose- dependent manner. And the apoptosis rate was up-regulated significantly as the stimulation concentrations of sFlt-1 increased. We found sFlt-1 challenge could significantly increase the expression of vWF. In addition, sFlt-1 increased the levels of caspase-9, caspase-3, Bax and mitochondrial membrane potential; facilitated the release of cytochrome C from mitochondria to cytoplasma. In contrast, Z-LEHD-FMK attenuated high sFlt-1-induced HUVECs apoptosis. In conclusion, our study demonstrated that sFlt-1 expression was up-regulated by the challenge of pIgA1 complex derived from patients with IgAN. Furthermore, increased sFlt-1 facilitated human umbilical vein endothelial cells apoptosis via the mitochondrial-dependent pathway.

Endothelial activation of caspase-9 promotes neurovascular injury in retinal vein occlusion.

Central nervous system ischemic injury features neuronal dysfunction, inflammation and breakdown of vascular integrity. Here we show that activation of endothelial caspase-9 after hypoxia-ischemia is a critical event in subsequent dysfunction of the blood-retina barrier, using a panel of interrelated ophthalmic in vivo imaging measures in a mouse model of retinal vein occlusion (RVO). Rapid nonapoptotic activation of caspase-9 and its downstream effector caspase-7 in endothelial cells promotes capillary ischemia and retinal neurodegeneration. Topical eye-drop delivery of a highly selective caspase-9 inhibitor provides morphological and functional retinal protection. Inducible endothelial-specific caspase-9 deletion phenocopies this protection, with attenuated retinal edema, reduced inflammation and preserved neuroretinal morphology and function following RVO. These results reveal a non-apoptotic function of endothelial caspase-9 which regulates blood-retina barrier integrity and neuronal survival, and identify caspase-9 as a therapeutic target in neurovascular disease.

Action of Akt Pathway on La-Induced Hippocampal Neuron Apoptosis of Rats in the Growth Stage.

This study investigated the influences of lanthanum (La) exposure on learning and memory and the expression of apoptosis-related proteins in offspring rats. Wistar female rats were randomly divided into a control group (NC) and 0.25%, 0.5% and 1.0% LaCl3 treatment groups, with eight per group. La dye was transmitted to offspring rats through parental blood circulation and breast milk before delactation and through water drinking after delectation. Offspring rats were killed at 14, 28 and 42 days after birth. Hippocampal neurons were observed by microscope, and apoptosis and necrosis were tested. The expression levels of apoptosis-related proteins were detected by Western blot, and Morris water maze experiments were used to measure learning and memory abilities. LaCl3 groups showed longer escape latency periods and swimming distances than the NC group (p < 0.05). The 1.0% LaCl3 group passed across the target quadrants and platforms more times and stayed in the target quadrants for less time, than the NC group (p < 0.05). At 42 days, the apoptosis rate and necrosis in the hippocampus of the 1.0% LaCl3 group were significantly higher than those of other groups. There was a significant difference among LaCl3 groups in terms of protein expressions measured in the hippocampus. In LaCl3 groups, caspase-3 and caspase-9 were significantly higher than in the NC group (p < 0.05). Therefore, La exposure can promote neuronal apoptosis by regulating the protein expressions of Akt, Bcl-2, Bcl-xl, Bax, Bad, caspase-3 and caspase-9, thus damaging learning and memory and the hippocampal neurons of offspring rats.

α-Hederin Induces Apoptosis of Esophageal Squamous Cell Carcinoma via an Oxidative and Mitochondrial-Dependent Pathway.

BACKGROUND: α-Hederin has been shown promising anti-tumor potential against various cancer cell lines. However, reports about effects of α-hederin on esophageal squamous cell carcinoma (ESCC) are still unavailable. AIM: To investigate the inhibitory effects of α-hederin on ESCC and explore the underlying mechanism. METHODS: Human esophageal carcinoma cell line (Eca-109) was used for the experiment. Cell Counting Kit-8, flow cytometry, Hoechst 33258 staining, enhanced ATP assay kit, 2',7'-dichlorofluorescin diacetate, JC-1 kit, and Western bolt were used to assess the cell viability, cycle, apoptosis, cellular ATP content, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. Xenografted tumor model was constructed to evaluate the in vivo anti-tumor effects of α-hederin. RESULTS: Compared with control group, α-hederin significantly inhibited the proliferation, induced apoptosis of ESCC, and arrested the cell cycle in G1 phase (P < 0.05). α-Hederin induced the accumulation of ROS, decrement of ATP levels, and disruption of MMP (P < 0.05). The detection of mitochondrial and cytosol proteins showed that AIF, Apaf-1, and Cyt C were released and increased in cytoplasm, and then, caspase-3, caspase-9, and Bax were involved and increased, while Bcl-2 level was decreased (P < 0.05). Furthermore, the above changes were amplified in the group pretreated with L-buthionine sulfoximine, while N-acetyl-L-cysteine plays an opposite role (P < 0.05). Meanwhile, α-hederin significantly inhibited the growth of xenografted tumors with favorable safety. CONCLUSION: α-Hederin could inhibit the proliferation and induce apoptosis of ESCC via dissipation of the MMP with simultaneous ROS generation and activation of the mitochondrial pathway.

Murrayanine Induces Cell Cycle Arrest, Oxidative Stress, and Inhibition of Phosphorylated p38 Expression in A549 Lung Adenocarcinoma Cells.

BACKGROUND Murrayanine is a carbazole alkaloid derived from Murraya koenigii, which has been used in traditional Chinese medicine in the treatment of cancer. This study aimed to investigate the effects of murrayanine on human lung adenocarcinoma cells in vitro and to investigate the mechanisms of its action. MATERIAL AND METHODS A549 human lung adenocarcinoma cells and MRC-5 human lung fibroblasts were grown in culture, and an MTT assay determined cell viability. Cells were treated for 24 h with increasing doses of murrayanine (0, 9, 18, and 36 µM). Fluorescence, using 4', 6-diamidino-2-phenylindole (DAPI), acridine orange, ethidium bromide, and propidium iodide (PI), were used for the detection of apoptosis. The cell cycle was studied with fluorescence-activated cell sorting (FACS), and Western blot evaluated protein expression. RESULTS Murrayanine treatment resulted in significant dose-dependent inhibition of the growth of A549 cells (p<0.05), with an IC50 of 9 µM, and arrested the cells at the G2/M phase of the cell cycle, reduced the expression of cyclin D and E, CDK2, 4, and 6, and increased the expression of p21 and p27. Murrayanine treatment increased apoptosis of the A549 cells and increased cleaved of caspase-3 and caspase-9, and the Bax/Bcl-2 ratio. Murrayanine treatment increased levels of reactive oxygen species (ROS), disrupted the mitochondrial membrane potential, inhibited invasion, and inhibited phosphorylation of p38 mitogen-activated protein kinase (MAPK) of the A549 cells. CONCLUSIONS Murrayanine induced cell cycle arrest, oxidative stress, and inhibited the expression of phosphorylated p38 in A549 adenocarcinoma cells.

Radix Tetrastigma Hemsleyani Flavone Suppresses Cutaneous Squamous Cell Carcinoma A431 Cells via Proteasome Inhibition.

BACKGROUND Radix Tetrastigma Hemsleyani Flavone (RTHF) has detoxification and anti- inflammation activity and is widely used. Here, we report that RTHF inhibits cell proliferation and induces apoptosis in cutaneous squamous cell carcinoma A431 cells and is a potential strategy for cancer therapy. MATERIAL AND METHODS A431 cells were cultured in different concentrations of RTHF. The inhibition of cell proliferation was assessed by MTT assay, cell apoptosis was shown through FCM, and cell invasion was assessed by Transwell methods. Enzyme proteasome assay was used to detect the activity of proteasome and DUB. Expression of apoptosis-related and ubiquitin proteasome pathway-associated proteins were assessed by PCR and Western blot. RESULTS RTHF obviously suppressed the proliferation and induced apoptosis of A431 cells in a dose-dependent manner. Transwell assay showed that RTHF inhibited the cell metastasis significantly. Enzyme proteasome assay show that the RTHF treatment of activity of proteasome and DUB was significantly lower than in control. RTHF increased the expression of Bax and inhibited Bcl-2, pro-caspase3, and pro-caspase9 activity. The expression of USP14, UCHL5, and POH1 decreased and ub-prs increased significantly in the treatment group. CONCLUSIONS Our study reveals that RTHF-mediated inhibition of DUBs and proteasome may provide a potential strategy for cancer therapy.

Piperine functions as a tumor suppressor for human ovarian tumor growth via activation of JNK/p38 MAPK-mediated intrinsic apoptotic pathway.

Piperine, a kind of natural alkaloid found in the fruit of black (Piper nigrum Linn) and long (Piper longum Linn), has shown antitumor activities toward various cancer cell lines. However, the antitumor effects of Piperine on ovarian cancer and the underlying mechanism are not fully elucidated. Our result showed that Piperine reduced the cell viability of A2780 cells in a concentration and time-dependent manner, but has not any effect on normal ovarian cells. Flow cytometric analysis revealed that Piperine suppressed cells proliferation via induction of apoptosis, which was followed by release of mitochondrial cytochrome c to cytosol, activation of caspase-3 and -9, as well as cleaved PARP. Moreover, Western blot results confirmed that Piperine (8, 16, and 20 µM) decreased phosphorylation of JNK and p38 MAPK in A2780 cells. In addition, caspase-3 inhibitor (Z-DEVD-FMK), caspase-9 inhibitor (Z-LEDH-FMK), JNK-inhibitor (SP600125), or p38 MAPK inhibitor (SB203580) could abate the apoptosis induced by Piperine (20 µM) treatment, while caspase-8 inhibitor (Z-IETD- FMK) exhibited no inhibitory effect on the induction of apoptosis in A2780 cells. These results provide the first evidence for the anticancer potential of Piperine in ovarian cancer cells, partially via JNK/p38 MAPK-mediated intrinsic apoptotic pathway.

MSP-4, an Antimicrobial Peptide, Induces Apoptosis via Activation of Extrinsic Fas/FasL- and Intrinsic Mitochondria-Mediated Pathways in One Osteosarcoma Cell Line.

Osteosarcoma (OS) is a common malignant bone cancer. The relatively high density of a person's bone structure means low permeability for drugs, and so finding drugs that can be more effective is important and should not be delayed. MSPs are marine antimicrobial peptides (AMP) and natural compounds extracted from Nile tilapia (Oreochromis niloticus). MSP-4 is a part of the AMPs series, with the advantage of having a molecular weight of about 2.7-kDa and anticancer effects, although the responsible anticancer mechanism is not very clear. The goal of this study is to determine the workings of the mechanism associated with apoptosis resulting from MSP-4 in osteosarcoma MG63 cells. The study showed that MSP-4 significantly induced apoptosis in MG63 cells, with Western blot indicating that MSP-4 induced this apoptosis through an intrinsic pathway and an extrinsic pathway. Thus, a pretreatment system with a particular inhibitor of Z-IETD-FMK (caspase-8 inhibitor) and Z-LEHD-FMK (caspase-9 inhibitor) significantly attenuated the cleavage of caspase-3 and prevented apoptosis. These observations indicate that low concentrations of MSP-4 can help induce the apoptosis of MG63 through a Fas/FasL- and mitochondria-mediated pathway and suggest a potentially innovative alternative to the treatment of human osteosarcoma.

[Role of green tea polyphenols in noise-induced hearing loss].

Objective: To investigate the role and mechanism of action of green tea polyphenols in noise-induced hearing loss. Methods: Male specific pathogen-free guinea pigs were randomly divided into normal control group with 9 guinea pigs, noise exposure group with 36 guinea pigs, and green tea polyphenol intervention group with 36 guinea pigs. Auditory brainstem response (ABR) threshold shift was examined before noise exposure and at 1, 3, 7, and 14 days of noise exposure. The surface preparation of cochlear basilar membrane was used for hair cell count and the morphology of hair cells was also observed. Western blot was used to observe the expression of cysteinyl aspartate-specific protease-9 (caspase-9) and cysteinyl aspartate-specific protease-3 (caspase-3) in cochlear tissue. Results: Both the noise exposure group and the green tea polyphenol intervention group had an increase in ABR threshold after noise exposure, and the green tea polyphenol intervention group had a significantly lower ABR threshold shift than the noise exposure group at all time points (P<0.05). Both groups had enlargement, atrophy, or loss of hair cells after noise exposure, and at 7 and 14 days of noise exposure, the noise exposure group had a significantly higher rate of abnormal hair cells than the green tea polyphenol intervention group (P<0.05). Both groups had an increase in the expression of caspase-9 and caspase-3 after noise exposure, and the noise exposure group had a significantly greater increase than the green tea polyphenol intervention group (P<0.05). Conclusion: Green tea polyphenols can reduce noise-induced hearing loss and hair cell injury, possibly by regulating the expression of caspase-9 and caspase-3.

Apoptotic and nonapoptotic function of caspase 7 in spermatogenesis.

Recent studies have reported that caspase 7 has an apoptotic and nonapoptotic function. However, the relationship between caspase 7 and spermatogenesis remains unknown. This study aimed to investigate the possible function of caspase 7 during normal and abnormal spermatogenesis. The cleaved form of caspase 7 was detected in testis tissues at different postpartum times (5-14 weeks) by qRT-PCR, Western blot and immunohistochemistry (IHC). Then, the mice models of spermatogenic dysfunction were obtained by busulfan (30 mg kg-1 to further evaluate the potential function and mechanism of caspase 7. qRT-PCR and Western blot results showed that caspase 7 expression was gradually elevated from 5 to 14 weeks, which was not connected with apoptosis. IHC results revealed that caspase 7 was mainly located in spermatogenic cells and Leydig cells. In addition, spermatogenic dysfunction induced by busulfan gradually enhanced the apoptosis and elevated the expression of caspase 3, caspase 6, and caspase 9, but decreased the expression of caspase 7 in spermatogenic cells. However, when spermatogenic cells were mostly disappeared at the fourth week after busulfan treatment, caspase 7 expression in Leydig cells was significantly increased and positively correlated with the expression of caspase 3, caspase 6, and caspase 9. Therefore, these results indicate that caspase 7 has a nonapoptic function that participates in normal spermatogenesis, but also displays apoptotic function in spermatogenic dysfunction.

Anticancer activity of Ashwagandha against human head and neck cancer cell lines.

BACKGROUND: The aim of this study was to determine the apoptotic activity of methanol extract of Ashwagandha (MEAG) and in human head and neck squamous cell carcinoma (HNSCC) cells and to investigate the underlying mechanisms. METHODS: We investigated the effects of MEAG on programmed cell death in HNSCC cells using a Live/Dead assay, detection of nuclear morphologic changes, Mitotracker, siRNA knockdown, and RT-PCR. RESULTS: Treatment with MEAG showed dose-dependent growth-inhibitory activity that attribute to caspase-dependent apoptosis. Loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase 9 suggested that MEAG leads to activation of mitochondria-mediated apoptosis. MEAG selectively upregulated the expression of Bim protein at the transcriptional level and induced the translocation of Bim into the mitochondria. Knockdown of Bim by siRNA partially blocked MEAG-mediated apoptosis. MEAG also caused an increase in truncated Bid (t-Bid), cleaved caspase-8, and death receptor 5 (DR5). Interestingly, withaferin A (WA), a bioactive component of MEAG, clearly induced apoptosis accompanied by upregulation of Bim, t-Bid, caspase-8, and DR5 similar to the effects of MEAG. CONCLUSIONS: These suggest that MEAG and WA may be potential natural materials for the treatment of HNSCC.

Ropivacaine- and bupivacaine-induced death of rabbit annulus fibrosus cells in vitro: involvement of the mitochondrial apoptotic pathway.

OBJECTIVE: The purposes of this study were to assess whether local anesthetics (LAs), such as ropivacaine and bupivacaine, could induce apoptosis of rabbit annulus fibrosus (AF) cells in vitro and further to explore the possible underlying mechanism. METHODS: Rabbit AF cells at second passage were treated with saline solution and various concentrations of LAs. Apoptosis of AF cells were examined by cell counting kit-8 (CCK-8), Annexin V assays, Hoechst 33342 staining, and Caspase-3, -9 activity assays. The expression of apoptosis-related markers was detected by real-time PCR (RT-PCR) and Western Blot. The JC-1 staining was used to evaluate the change of mitochondrial membrane potential (MMP). Moreover, the levels of reactive oxygen species (ROS) were determined with fluorescent probe DCFH-DA. RESULTS: The results of flow cytometry indicated that LAs could induce apoptosis of rabbit AF cells in a dose-dependent manner. Apoptosis was confirmed by cell morphology, condensed nuclei and activation of Caspase-3 and -9. In addition, the molecular data showed that LAs could significantly up-regulate the expression of Bax, accompanied by a significant down-regulation of Bcl-2 expression. Furthermore, we also observed that LAs resulted in alteration of MMP and accumulation of intracellular ROS in AF cells. Blockade of ROS production by N-acetyl-l-cysteine (NAC) inhibited LAs-induced apoptosis. CONCLUSIONS: These findings suggest that LAs in clinically relevant concentrations could induce apoptosis of rabbit AF cells in vitro, and the mitochondrial pathway was, at least in part, involved in the LAs-mediated apoptosis. Further investigations focusing on the potential cytotoxicity of LAs on IVD cells are needed.

Functional annotation of native enhancers with a Cas9-histone demethylase fusion.

Understanding of mammalian enhancers is limited by the lack of a technology to rapidly and thoroughly test the cell type-specific function. Here, we use a nuclease-deficient Cas9 (dCas9)-histone demethylase fusion to functionally characterize previously described and new enhancer elements for their roles in the embryonic stem cell state. Further, we distinguish the mechanism of action of dCas9-LSD1 at enhancers from previous dCas9-effectors.

Lantana camara Induces Apoptosis by Bcl-2 Family and Caspases Activation.

Breast cancer is one of the most common cancers worldwide, and the second most fatal cancer in women after lung cancer. Because there are instances of cancer resistance to existing therapies, studies focused on the identification of novel therapeutic drugs are very important. In this study, we identified a natural anticancer agent from Lantana camara, a flowering plant species of the genus Verbena. The extract obtained from the L. camara exhibited cell death properties in the human breast cancer cell line, MCF-7. We found that the apoptosis induced by treatment with the L. camara extract was regulated by the Bcl-2 family. Bid and Bax was increased and Bcl-2 was decreased by L. camara extract. L. camara extract modulated cleavage of caspase-8, and caspase-9, as well as poly (ADP-ribose) polymerase (PARP). Our results support the potential use of the L. camara extract as an anti-breast cancer drug.

FoxO transcription factors support oxidative stress resistance in human chondrocytes.

OBJECTIVE: A major signaling pathway that regulates cellular aging is the insulin/insulin-like growth factor 1 (IGF-1)/phosphatidylinositol 3-kinase (PI3K)/Akt/FoxO transcription factor axis. We previously observed that FoxO transcription factors are dysregulated in aged and OA cartilage. The objective of this study was to investigate the impact of down-regulated FoxO transcription factors on chondrocytes. METHODS: Small interfering RNAs (siRNAs) targeting FOXO1 (siFOXO1) and FOXO3 (siFOXO3) were transfected into human articular chondrocytes. Cell viability following treatment with the oxidant tert-butyl-hydroperoxide (tBHP) was measured by MTT assay. Caspase 3/7 activation and apoptotic cells were examined. Gene and protein expression of antioxidant proteins and autophagy-related proteins and changes in inflammatory mediators following treatment with interleukin-1ß were assessed. Cells transfected with FOXO plasmids were also analyzed. RESULTS: Cell viability was significantly reduced by siFOXO after treatment with tBHP. Apoptosis accompanied by caspase activation was significantly increased in siFOXO-transfected chondrocytes. Knockdown of FOXO1 and FOXO1+3 resulted in significant reductions in levels of glutathione peroxidase 1 (GPX-1), catalase, light chain 3 (LC3), Beclin1, and sirtuin 1 (SIRT-1) proteins following treatment with tBHP. In contrast, the constitutive active form of FOXO3 increased cell viability while inducing GPX-1, Beclin1, and LC3 in response to tBHP. Expression and production of ADAMTS-4 and chemerin were significantly increased in siFOXO-transfected chondrocytes. CONCLUSION: Reduced expression of FoxO transcription factors in chondrocytes increased susceptibility to cell death induced by oxidative stress. This was associated with reduced levels of antioxidant proteins and autophagy-related proteins. Our data provide evidence for a key role of FoxO transcription factors as regulators of chondrocyte oxidative stress resistance and tissue homeostasis.

Rice bran phytic acid induced apoptosis through regulation of Bcl-2/Bax and p53 genes in HepG2 human hepatocellular carcinoma cells.

Phytic acid (PA) has been reported to have positive nutritional benefits and prevent cancer formation. This study investigated the anticancer activity of rice bran PA against hepatocellular carcinoma (HepG2) cells. Cytotoxicty of PA (0.5 to 4mM) was examined by MTT and LDH assays after 24 and 48 h treatment. Apoptotic activity was evaluated by expression analysis of apoptosis-regulatory genes [i.e. p53, Bcl-2, Bax, Caspase-3 and -9] by reverse transcriptase-PCR and DNA fragmentation assay. The results showed antioxidant activity of PA in Fe3+ reducing power assay (p ≤ 0.03). PA inhibited the growth of HepG2 cells in a concentration dependent manner (p ≤ 0.04). After 48h treatment, cell viability was recorded 84.7, 74.4, 65.6, 49.6, 36.0 and 23.8% in MTT assay and 92.6, 77.0%, 66.8%, 51.2, 40.3 and 32.3% in LDH assay at concentrations of 1, 1.5, 2.0, 2.5, 3.0, and 3.5mM, respectively. Hence, treatment of PA for 24h, recorded viability of cells 93.5, 88.6, 55.5, 34.6 and 24.4% in MTT assay and 94.2, 86.1%, 59.7%, 42.3 and 31.6%, in LDH assay at concentrations of 1, 2.2, 3.0, 3.6 and 4.0mM, respectively. PA treated HepG2 cells showed up-regulation of p53, Bax, Caspase-3 and -9, and down- regulation of Bcl-2 gene (p ≤ 0.01). At the IC50 (2.49 mM) of PA, the p53, Bax, Caspase-3 and-9 genes were up- regulated by 6.03, 7.37, 19.7 and 14.5 fold respectively. Also, the fragmented genomic DNA in PA treated cells provided evidence of apoptosis. Our study confirmed the biological activity of PA and demonstrated growth inhibition and induction of apoptosis in HepG2 cells with modulation of the expression of apoptosis-regulatory genes.