Molecular Mechanisms Of Apoptosis Research Articles

Paclitaxel inhibits proliferation and promotes apoptosis through regulation ROS and endoplasmic reticulum stress in osteosarcoma cell

Paclitaxel is a chemotherapeutic drug for cancer, which is isolated from the Pacific yew tree. However, and the molecular mechanism and the antitumor effects of paclitaxel on osteosarcoma cell remain to be explored. The aim of our study was to explore the possible molecular mechanisms of apoptosis in osteosarcoma induced by paclitaxel. Paclitaxel can obviously decrease the proliferation of HOS-732 cells in a dose-dependent manner. Paclitaxel could induce the cell cycle arrest at the G2/M-phase and decreases the CDK5 and CCNE1 expression in HOS-732 cells. Paclitaxel promotes cell apoptosis in HOS-732 cells, which may be contacted to the decreasing of Bcl-2 protein expression. Further, the production of ROS in HOS-732 cells was remarkably increased with the increasing concentration of paclitaxel. Moreover, paclitaxel induces the ER-stress related gene and protein expression (GRP79, DDIT3 mRNA and GRP78, XBP-1 s, IRE1α protein expression) in osteosarcoma cells. Paclitaxel can inhibit the proliferation of HOS-732 cells and increase ROS and ER-stress response to promote cell apoptosis, suggesting that paclitaxel may represent a new therapeutic option for the treatment and prevention osteosarcoma.

Effect of Rehabilitation with Extremely Low Frequency Electromagnetic Field on Molecular Mechanism of Apoptosis in Post-Stroke Patients.

Apoptosis in acute stroke is associated with a negative prognosis and is correlated with the severity of the neurological deficit. However, there is no evidence that indicates that, in the subacute phase of the stroke, the apoptosis process might activate neuroplasticity. Therefore, in this study, we investigated the effect of an extremely low frequency electromagnetic field (ELF-EMF) on the molecular mechanism of apoptosis, as used in the rehabilitation of post-stroke patients. Patients with moderate stroke severity (n = 48), 3–4 weeks after incident, were enrolled in the analysis and divided into ELF-EMF and non-ELF-EMF group. The rehabilitation program in both groups involves the following: kinesiotherapy—30 min; psychological therapy—15 min; and neurophysiological routines—60 min. Additionally, the ELF-EMF group was exposed to an ELF-EMF (40 Hz, 5 mT). In order to assess the apoptosis gene expression level, we measured the mRNA expression of BAX, BCL-2, CASP8, TNFα, and TP53. We found that ELF-EMF significantly increased the expression of BAX, CASP8, TNFα, and TP53, whereas the BCL-2 mRNA expression after ELF-EMF exposition remained at a comparable level in both groups. Thus, we suggest that increasing the expression of pro-apoptotic genes in post-stroke patients promotes the activation of signaling pathways involved in brain plasticity processes. However, further research is needed to clarify this process.

The influence of selected gastrointestinal parasites on apoptosis in intestinal epithelial cells.

Studies on the parasite–host interaction may provide valuable information concerning the modulation of molecular mechanisms as well as of the host immune system during infection. To date, it has been demonstrated that intestinal parasites may affect, among others, the processes of digestion in the gastrointestinal system of the host, thus limiting the elimination of the parasite, the immune response as well as inflammation. However, the most recent studies suggest that intestinal parasites may also affect modulation of the apoptosis pathway of the host. The present paper presents the latest scientific information on the influence of intestinal parasite species (Blastocystis sp., Giardia sp., Cryptosporidium sp., Trichuris sp., Entamoeba histolytica, Nippostrongylus brasiliensis, Heligmosomoides polygyrus) on the molecular mechanisms of apoptosis in intestinal epithelial cells. This paper stresses that the interdependency between the intestinal parasite and the host results from the direct effect of the parasite and the host’s defense reactions, which lead to modulation of the apoptosis pathways (intrinsic and extrinsic). Moreover, the present paper presents the role of proteins involved in the mechanisms of apoptosis as well as the physiological role of apoptosis in the host’s intestinal epithelial cells.

A direct comparison of selective BH3-mimetics reveals BCL-XL, BCL-2 and MCL-1 as promising therapeutic targets in neuroblastoma

BackgroundDespite advances in the treatment of neuroblastoma, patients with high-risk disease still have dismal survival prognosis. Neuroblastoma cells display elevated expression of the antiapoptotic BCL-2 proteins, suggesting that BH3-mimetics may be a promising treatment option. Here, we investigated the role of BCL-2, BCL-XL and MCL-1 in neuroblastoma.MethodsA panel of neuroblastoma cell lines and primary patient-derived cells were exposed to BH3-mimetics targeting BCL-2 (ABT-199), BCL-XL (A1331852) or MCL-1 (S63845). In addition, protein expression and interaction patterns were analysed using Western blotting and immunoprecipitation.ResultsAll tested BH3-mimetics were able to induce apoptosis in neuroblastoma cell lines, indicating that not only BCL-2 but also BCL-XL and MCL-1 may be promising therapeutic targets. Primary patient-derived cells displayed highest sensitivity to A1331852, highlighting the important role of BCL-XL in neuroblastoma. Further analysis into the molecular mechanisms of apoptosis revealed that A1331852 and S63845 displaced proapoptotic proteins like BIM and BAK from their antiapoptotic targets, subsequently leading to the activation of BAX and BAK and caspase-dependent apoptosis.ConclusionsBy using selective BH3-mimetics, this study demonstrates that BCL-2, BCL-XL, and MCL-1 are all relevant therapeutic targets in neuroblastoma. A1331852 and S63845 induce rapid apoptosis that is initiated following a displacement of BAK from BCL-XL or MCL-1, respectively.

Molecular Mechanism of Apoptosis by Amyloid β-Protein Fibrils Formed on Neuronal Cells.

Aggregational states of amyloid β-protein (Aβ) are critical for its neurotoxicity, although they are not well-characterized, particularly after binding to the cell membranes. This is one reason why the mechanisms of Aβ neurotoxicity are controversial and elusive. In this study, the effects of toxic Aβ-(1-42) fibrils formed in the membrane on cellular processes were investigated using human neuroblastoma SH-SY5Y cells. Consistent with previous observations, fibrillar Aβs formed on the membranes induced activation of caspase-3, the effector caspase for apoptosis. Knockdown analyses of the initiator caspases, caspase-8 and caspase-9, indicated that the apoptosis was induced via activation of caspase-8, followed by activation of caspase-9 and caspase-3. We also found that inflammation signaling pathways including Toll-like receptors and inflammasomes NOD-, LRR-, and pyrin domain-containing protein 3 are involved in the initiation of apoptosis by the Aβ fibrils. These inflammation-related molecules are promising targets for the prevention of apoptotic cell death induced by Aβ.

Evaluation of some genes and proteins involved in apoptosis on human chronic myeloid leukemia cells (K562 cells) by datura innoxia leaves aqueous extract

Datura innoxia (D. innoxia) has an extensive usage in traditional medicine and can also be used for intervention therapy in order to treat cancer. Despite of accomplishing some researches on D. innoxia mechanism, still our knowledge is very little about exact D. innoxia apoptotic mechanism on human chronic myeloid leukemia cells (K562 cells). This study purpose was to clarify the molecular mechanism of apoptosis, which was mediated by D. innoxia leaves aqueous extract in K562 cells. MTT assay and flow cytometry was applied in order to assess the viability and apoptosis induction of K562 cells and normal human lymphoid B cells in the D. innoxia presence. Finally, the expression of the apoptotic related genes (p53, BAX, BCL2, Caspases 3, 6, 7 and 9) were evaluated using quantitative Real-Time PCR. Western blot analysis was applied for assessing the protein expression. MTT results indicated that D. innoxia could inhibit the viability of K562 cells in a dose- and time-dependent manner. In parallel, D. innoxia inhibitory effect on normal human lymphoid B cells was lower in comparison with its effect on K562 cells at the same concentrations and same incubation time. Apoptosis induction in K562 cells after D. innoxia exposure was determined by flow cytometry. Apoptosis was activated by D. innoxia in K562 cells throughout increasing the expression of P53, BAX/BCL2 ratio, caspase 9, 3, 6, 7. Western blot analysis demonstrated significant increase in cleaved PARP-1 and cleaved caspase 3 in treated K562 cells with high D. innoxia leaves aqueous extract concentration. D. innoxia leaves trigger apoptosis in K562 cells throughout intrinsic apoptotic pathway.Communicated by Ramaswamy H. Sarma

Microglia activation induces oxidative injury and decreases SIRT3 expression in dopaminergic neuronal cells.

Microglia activation-mediated neuroinflammation plays an important role in the progression of Parkinson's disease (PD). However, effects of microglia activation on dopaminergic neuronal cell (DAC) fate are still poorly understood. The objective of this study was to explore the neurotoxic effects of microglia activation-mediated oxidative injury in DACs and its possible mechanisms. In the present study, microglia-DACs co-culture systems (murine BV-2 and MN9D cells, or primary microglia and mesencephalic neurons) were used to display the crosstalk between both cell types. The cytotoxicity of lipopolysaccharide-induced microglia activation led to the accumulation of intracellular reactive oxygen species, increased cell apoptosis rate, reduced number of DACs, concomitant to cell cycle arrest at G1 phase. Molecular mechanisms of apoptosis caused by microglia activation-induced oxidative injury included the increased opening of mitochondrial permeability transition pore and enhanced membrane potential depolarization in MN9D cells, down-regulation of Bcl-2 and up-regulation of Bax, caspase-3 expression in DACs. In addition, microglia activation made a significant reduction of SIRT3 and superoxide dismutase 2 gene expression in DACs. Taken together, these data imply that microglia activation promotes cell apoptosis through mitochondrial pathway and decreases SIRT3 expression in DACs, which may provide some support for PD progression promoted by neuroinflammation.

Molecular mechanisms of apoptosis and autophagy elicited by combined treatment with oridonin and cetuximab in laryngeal squamous cell carcinoma.

Combined oridonin (ORI), a natural and safe kaurene diterpenoid isolated from Rabdosia rubescens, and cetuximab (Cet), an anti-EGFR monoclonal antibody, have been reported to exert synergistic anti-tumor effects against laryngeal squamous cell carcinoma (LSCC) both in vitro and in vivo by our group. In the present study, we further found that ORI/Cet treatment not only resulted in apoptosis but also induced autophagy. AMPK/mTOR signaling pathway was found to be involved in the activation of autophagy in ORI/Cet-treated LSCC cells, which is independent of p53 status. Additionally, chromatin immunoprecipitation (ChIP) assay showed that ORI/Cet significantly increased the binding NF-κB family member p65 with the promotor of BECN 1, and p65-mediated up-regulation of BECN 1 caused by ORI/Cet is coupled to increased autophagy. On the other hand, we demonstrated that either Beclin 1 SiRNA or autophagy inhibitors could increase ORI/Cet induced-apoptosis, indicating that autophagy induced by combination of the two agents plays a cytoprotective role. Interestingly, 48h after the combined treatment, autophagy began to decrease but apoptosis was significantly elevated. Our findings suggest that autophagy might be strongly associated with the antitumor efficacy of ORI/Cet, which may be beneficial to the clinical application of ORI/Cet in LSCC treatment.

Molecular Mechanisms of Apoptosis in HepaRG Cell Line Induced by Polyphyllin VI via the Fas Death Pathway and Mitochondrial-Dependent Pathway.

Polyphyllin VI, which is an active saponin, is mainly isolated from traditional medicinal plant Paris polyphylla, which causes liver damage in rats. In the present study, we aimed to explore the potential cytotoxicity of polyphyllin VI on the growth of HepaRG cells and to determine the molecular mechanism. The results revealed that polyphyllin VI changed cell morphology and induced apoptosis in HepaRG cells. Flow cytometric assay displayed that polyphyllin VI promoted the generation of reactive oxygen species (ROS), depolarized the mitochondrial membrane potential (MMP), and induced S phase cell cycle arrest by decreasing the expression of cyclin A2 and CDK2, while significantly increasing the expression of p21 protein. Polyphyllin VI induced the release of cytochrome c from the mitochondria to the cytosol and activated Fas, caspase-3, -8, -9, and PARP proteins. Pretreatment with NAC and Z-VAD-FMK (ROS scavenger and caspase inhibitor, respectively) on HepaRG cells increased the percentage of viable cells, which indicated that polyphyllin VI induced cell apoptosis through mitochondrial pathway by the generation of ROS and Fas death-dependent pathway. All of the effects are in dose- and time-dependent manners. Taken together, these findings emphasize the necessity of risk assessment to polyphyllin VI and offer an insight into polyphyllin VI-induced apoptosis of HepaRG cells.

Bioactive compound 1,8-Cineole selectively induces G2/M arrest in A431 cells through the upregulation of the p53 signaling pathway and molecular docking studies

BackgroundCallistemon citrinus has been traditionally known for its medicinal property. Recently, our research group identified 1,8-Cineole, as one of the predominant compound present in the hexane extract (HE-C), whose leaves have potent anticancer activity. Hypothesis/PurposeThe present study was designed to isolate 1,8-Cineole from Callistemon citrinus plant and to determine their role in anticancer effects in in vitro using skin carcinoma cells. Moreover, the molecular mechanism of apoptosis and molecular docking studies were also investigated. Study design/MethodsIn vitro cytotoxicity test was performed with HE-C fractionates 1F, 2F, and 3F against A431 and HaCaT cell lines. MTT and AB assay demonstrated that 1F was toxic to cancer cells with no adverse effect to non-malignant cells and it was subjected to 1H NMR, 13C NMR spectroscopy and further characterized by FTIR and GC–MS analysis. On the basis of spectroscopic data, the metabolite was confirmed as 1,8-Cineole. ResultsBased on the cytotoxicity results, the well-characterized metabolite 1,8-Cineole was investigated upon to understand the mechanism that caused cancer cell death. In this process, the changes in mitochondrial membrane potential (ΔΨm) were confirmed by Rh-123/DAPI staining; the ultra structure was observed by TEM and quantified by flow cytometric analysis. These results proved that the compound effectively induced the apoptosis and G2/M phase arrest in A431 cells by increasing the expression of p53 and that it was monitored by FACS. Further, the expression of apoptotic proteins, such as Bax/Bcl-2, Cyt-c, caspase-9, and caspase-3 was confirmed by western blot. The molecular docking simulations predicted the hydrophobic interaction between 1,8-cineole with Bcl-2 and PARP1 receptor. Conclusions1,8-Cineole is a potential candidate for skin carcinoma, which is possible by regulating the p53 apoptotic signaling pathway.

Inhibitory Effect of Histone Deacetylase Inhibitor SAHA on Proliferation of Mouse Multiple Myeloma Cell Line SP2/0 in vitro and in vivo

To explore the anti-myeloma effect of suberoylanilide hydroxamic acid (SAHA) and on mouse myeloma cell line SP2/0 in vitro and in vivo and its mechanism. The inhibitory effect of SAHA on SP2/0 cells was measured by CCK-8 assay,and the apoptosis and cell cycle were analyzed by flow cytometry FACS. The protein expression of Caspase-3 and p53 of SP2/0 cells treated with SAHA were examined by Western blot. Annexin V/7-AAD double staining was performed to detect the apoptosis of SP2/0 induced by SAHA in vitro. SP2/0 cells (1×106) resuspended in 200 µl PBS were inoculated subcutaneously and intravenously into BALB/c mice, so as to establish aggressive or non-aggressive myeloma-bearing mouse models respectively. On day 3 after modeling, mice received SAHA or vehicle control treatment by intraperitoneal injection. The dose of SAHA was 60 mg/kg·d, 5 times a week for 3 weeks. In SAHA-treated SP2/0 cells, the proliferation inhibition rate and apoptotic cells increased in a dose dependent manner. Also, SAHA significantly increased the ratio of cells in G2 phase and decreased in S phase. Molecular mechanisms of apoptosis and cell cycle arrest of SP2/0 induced by SAHA partly correlated with up-regulating the expression level of Caspase-3 and p53. In the non-aggressive myeloma-bearing mice, SP2/0 cells disappeared in peripheral blood after SAHA treatment. In the aggressive myeloma-bearing mice, inhibition of tumor growth and prolongation of the cell survival were observed after SAHA treatment. SAHA inhibited SP2/0 cell proliferation, this effect associates with inducing apoptosis and cell cycle arrest, the mechanism of SAHA ralates partly with activating Caspase-3 and p53 pathway.

Molecular Mechanism of Apoptosis Signal‐Regulating Kinase I Oligomerization and Auto‐activation

Apoptosis Signal-Regulating Kinase I (ASK1) is a member of the Mitogen Activated Protein Kinase (MAPK) pathway. Responding to various stimulations, ASK1 activates four MAPK kinase (MKK) substrates including MKK4/7 and MKK3/7, which phosphorylate the c – Jun N terminal Kinase (JNK) and p38, respectively. The dysfunction and misregulation of ASK1 lead to various diseases such as cancers, cardiovascular diseases, neurodegenerative disorders, inflammatory diseases and diabetes. The activation of ASK1 is highly regulated by many regulatory factors. It is widely believed that the oligomerization of ASK1 is essential for its activation. However, the molecular mechanism of ASK1 activation largely remains unclear. We recently found out that the purified ASK1 is capable of auto-activating itself and it exists as a huge oligomer instead of a homodimer as previous studies suggested. To explore the mechanism of ASK1 oligomerization and auto-activation, we have generated a series of ASK1 truncates and hoping to identify the responsible domains/motifs for its oligomerization and auto-activation. We have purified two ASK1 truncates - ASK1-NK (ASK1 protein expressing N terminal and Kinase domains) and ASK1-K (ASK1 protein expressing Kinase domain only). After performing gel filtration, it was found out that ASK1-NK forms monomer and ASK1-K forms dimer in native state. We are currently developing protocols of kinase assay for different ASK1 truncates to analyze its auto-activation mechanism. In conclusion, this study will help us to unveil the detailed mechanism of ASK1 oligomerization which will surely enrich our knowledge in understanding the activation process of ASK1. Support or Funding Information This project is supported by Faculty Research Grant to Xuanzhi Zhan and Student Research Grant to Adua Rahman awarded by Tennessee Technological University. ASK1 mediated pathway Purification of ASK1 truncates. (A) ASK1-K domain was purified using Heparin and Q-sepharose column chromatography. The purified ASK1-K domain (~2μg) is shown in SDS-PAGE; (B) ASK1-NK domain was purified using Q – Sepharose and Phenyl – Sepharose column chromatography. The purified ASK1-NK domain (~2μg) is shown in SDS-PAGE. Sephacryl gel filtration chromatography of ASK1 truncates. (A) ASK1-NK is observed as monomer (116KDa). (B) Predominant form of ASK1-K is dimer (77 KDa). (C) The chromatogram of gel filtration standard and log of molecular weight vs. elution volume plot is generated to determine the molecular weights of the ASK1 truncates. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

English

Preeclampsia is one of the main causes of maternal and perinatal mortality and morbidity. The pathogenesis of preeclampsia remains unclear until now. It is believed that regulation of apoptosis in trophoblast cells plays an important role in the pathophysiology of preeclampsia. Failure of spiral arteries remodeling will eventually lead to placental hypoxia lead to excessive trophoblast apoptosis. The molecular mechanism of apoptosis is very complicated involving many signaling molecules included Bcl-2 proteins. The Bcl- 2 protein group consists of proapoptosis proteins (Bax) and apoptosis inhibitor proteins (Bcl-2 and Bcl-xL). The aimed of this stuty was to compare the expression of Bcl-xL protein in placental trophoblast cells of pregnancy complicated by severe preeclampsia with that normotensive pregnancy. This study was an observational study with cross sectional design involving 43 pregnancy patients with severe preeclampsia and 38 normotensive pregnancy who treated in Dr. Sardjito General Hospital, Yogyakarta from October 2011 until March 2012. Placenta samples were obtained from all subjects for Bcl-xL protein expression analysis using immunohistochemistry technique. Data were analyzed using independent t-test, chi-square test, and logistic regression. A p value <0.05 was considered significant. Significant difference in Bcl-xL protein expression in trophoblast cells of pregnancy complicated by severe preeclampsia (1.29 ± 0.12) compared to that normotensive pregnancy (1.71 ± 0.14) was reported (p = 0.00). In addition, logistic regression test showed that diagnosis of severe preeclampsia had a statistically significant role in Bcl-xL protein expression (p= 0.000). In conclusion, the expression of Bcl-xL protein is lower in pregnancy complicated by severe preeclampsia compared to normotensive pregnancy.

Fungal 7-epi-10-deacetyltaxol produced by an endophytic Pestalotiopsis microspora induces apoptosis in human hepatocellular carcinoma cell line (HepG2)

BackgroundPaclitaxel (taxol) is a potent anticancer drug that is used in the treatment of a wide variety of cancerous. In the present study, we identified a taxol derivative named 7-epi-10-deacetyltaxol (EDT) from the culture of an endophytic fungus Pestalotiopsis microspora isolated from the bark of Taxodium mucronatum. This study was carried out to investigate the effects of fungal EDT on cell proliferation, the induction of apoptosis and the molecular mechanisms of apoptosis in human hepatoma HepG2 cells in vitro.MethodsThe endophytic fungus was identified by traditional and molecular taxonomical characterization and the fungal EDT was purified using column chromatography and confirmed by various spectroscopic and chromatographic comparisons with authentic paclitaxel. We studied the in vitro effects of EDT on HepG2 cells for parameters such as cell cycle distribution, DNA fragmentation, reactive oxygen species (ROS) generation and nuclear morphology. Further, western blot analysis was used to evaluate Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), p38-mitogen activated protein kinase (MAPK) and poly [ADP-ribose] polymerase (PARP) expression.ResultsWe demonstrate that the fungal EDT exhibited significant in vitro cytotoxicity in HepG2 cells. We investigated cytotoxicity mechanism of EDT in HepG2 cells. The results showed nuclear condensation and DNA fragmentation were observed in cells treated with fungal EDT. Besides, the fungal EDT arrested HepG2 cells at G2/M phase of cell cycle. Furthermore, fungal EDT induced apoptosis in HepG2 cells in a dose-dependent manner associated with ROS generation and increased Bax/Bcl-2 ratio, p38 MAPKs and PARP cleavage.ConclusionsOur data show that EDT induced apoptotic cell death in HepG2 cells occurs through intrinsic pathway by generation of ROS mediated and activation of MAPK pathway. This is the first report for 7-epi-10-deacetyltaxol (EDT) isolated from a microbial source.

Procaspase-activating compound-1 induces apoptosis in Trypanosoma cruzi.

Some therapeutics for parasitic, cardiac and neurological diseases activate apoptosis. Therefore, the study of apoptotic proteins in pathogenic organisms is relevant. However, the molecular mechanism of apoptosis in unicellular organisms remain elusive, despite morphological evidence of its occurrence. In Trypanosoma cruzi, the causative agent of Chagas disease, metacaspase 3 (TcMCA3), seems to have a key role in parasite apoptosis. Accordingly, this work provides data concerning TcMCA3 regulation through its interaction with procaspase-activating compound 1 (PAC-1), a procaspase 3 activator. Indeed, PAC-1 reduced T. cruzi epimastigote viability with an IC50 of 14.12µM and induced loss of mitochondrial potential and exposure of phosphatidylserine, features of the apoptotic process. Notwithstanding, those PAC-1-inducible effects were not conserved in metacyclic trypomastigotes. Moreover, PAC-1 reduced the viability of mammalian cells with a greater IC50 (25.70µM) compared to T. cruzi epimastigotes, indicating distinct modes of binding between caspases and metacaspases. To shed light on the selectivity of metacaspases and caspases, we determined the structural features related to the PAC-1 binding sites in both types of proteins. These data are important for improving the understanding of the apoptosis pathway in T. cruzi so that TcMCA3 could be better targeted with future pharmaceuticals.

The Effects of Silica Nanoparticles on Apoptosis and Autophagy of Glioblastoma Cell Lines.

Silica nanoparticles (SiNPs) are one of the most commonly used nanomaterials in various medical applications. However, possible mechanisms of the toxicity caused by SiNPs remain unclear. The study presented here provides novel information on molecular and cellular effects of SiNPs in glioblastoma LBC3 and LN-18 cells. It has been demonstrated that SiNPs of 7 nm, 5–15 nm and 10–20 nm induce time- and dose-dependent cytotoxicity in LBC3 and LN-18 cell lines. In contrast to glioblastoma cells, we observed only weak reduction in viability of normal skin fibroblasts treated with SiNPs. Furthermore, in LBC3 cells treated with 5–15 nm SiNPs we noticed induction of apoptosis and necrosis, while in LN-18 cells only necrosis. The 5–15 nm SiNPs were also found to cause oxidative stress, a loss in mitochondrial membrane potential, and changes in the ultrastructure of the mitochondria in LBC3 cells. Quantitative real-time PCR results showed that in LBC3 cells the mRNA levels of pro-apoptotic genes Bim, Bax, Puma, and Noxa were significantly upregulated. An increase in activity of caspase-9 in these cells was also observed. Moreover, the activation of SiNP-induced autophagy was demonstrated in LBC3 cells as shown by an increase in LC3-II/LC3-I ratio, the upregulation of Atg5 gene and an increase in AVOs-positive cells. In conclusion, this research provides novel information concerning molecular mechanisms of apoptosis and autophagy in LBC3 cells.

Molecular interplays in hepatic stellate cells: apoptosis, senescence, and phenotype reversion as cellular connections that modulate liver fibrosis.

Liver fibrosis is a pathophysiological process correlated with intense repair and cicatrization mechanisms in injured liver, and over the past few years, the characterization of the fine-tuning of molecular interconnections that support the development of liver fibrosis has been investigated. In this cellular process, the hepatic stellate cells (HSCs) support the organ fibrogenesis. The HSCs are found in two distinct morpho-physiological states: quiescent and activated. In normal liver, most HSCs are found in quiescent state, presenting a considerable amount of lipid droplets in the cytoplasm, while in injured liver, the activated phenotype of HSCs is a myofibroblast, that secrete extracellular matrix elements and contribute to the establishment of the fibrotic process. Studies on the molecular mechanisms by which HSCs try to restore their quiescent state have been performed; however, no effective treatment to reverse fibrosis has been so far prescribed. Therefore, the elucidation of the cellular and molecular mechanisms of apoptosis, senescence, and the cell reversion phenotype process from activate to quiescent state will certainly contribute to the development of effective therapies to treat hepatic fibrosis. In this context, this review aimed to address central elements of apoptosis, senescence, and reversal of HSC phenotype in the control of hepatic fibrogenesis, as a guide to future development of therapeutic strategies.

Abstract 2327: Apoptosis of melanoma induced by non-thermal atmospheric pressure bio-compatible plasma activated media

Abstract Melanoma is the most dangerous type of skin cancer, which mostly related with the exposure of ultraviolet (UV), and especially happened to those who have low skin pigment levels. Epidemiological studies showed Europeans and North Americans have a high incidence of melanoma, while it is less common in Asia and Africa. Therefore, the new and more effective methods to treat melanoma are still necessarily needed. Non-thermal atmospheric pressure bio-compatible plasma (NBP) is defined as a partially ionized gas with electrically charged particle, which has been reported have cytotoxicity on various cancer cells induce DNA damage and apoptosis other than normal cells. Reactive oxygen and/or nitrogen species (RONS) were proved to play the most important role during this process, by inducing oxidative stress and depolarization of mitochondria membrane potential with the consequence of various cancer cells apoptosis. The plasma activated media (PAM), which the media treated directly by NBP with different time, containing mostly long-live secondary species such as hydrogen peroxide (H2O2) which have been confirmed anti-microorganism and cytotoxic activity. Researches already investigated that PAM also effective in cancer therapy via inducing apoptosis. However, the underlying molecular mechanisms of apoptosis are remaining elusive. The purpose of this study was to evaluate the potential of PAM as an effective tool to induce apoptosis in melanoma cells. Our results showed that PAM has a killing effect to melanoma cells in a time-dependent manner, for 3minutes treatment could induce almost 40% cells entry death, after treated by 5 minutes more than 60%, and 10 minutes treatment reduced the cell viability to approximately 30%. Annexin-V/PI staining demonstrated that PAM kills these cells via apoptosis pathway. We also found that with a manner of treatment time PAM significantly increased the concentration of intracellular NO and H2O2, reflecting an influx of extracellular RONS may result in melanoma cells apoptosis. Besides, western blot assay showed that P53 and caspase 3 increased after PAM treatment. Taken together, PAM is effective to induce the apoptosis in melanoma cells in vitro, further in vivo experiments will be performed to investigate the functional effects in the animals. Citation Format: Ihn Han, Ying Li, Eun Ha Choi. Apoptosis of melanoma induced by non-thermal atmospheric pressure bio-compatible plasma activated media [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2327. doi:10.1158/1538-7445.AM2017-2327

P 119 - ER stress related lipid accumulation and apoptotic cell death in nonalcoholic fatty liver diesease

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease that ranges from benign steatosis to nonalcoholic steatohepatitis (NASH) . The role of cholesterol alterations in NAFLD progression have been shown in various studies. Additionally, activation of ER stress, induces inflammation, insulin resistance and apoptotic cell death which results in NAFLD/NASH transition. UPR is mediated by the activation of three ER proteins: IRE1, PERK and ATF-6 . These proteins are inactivated by binding to the ER chaperone GRP78 under normal conditions. Following disassociation of GRP78, each of these sensor proteins (PERK, ATF6 and IRE1) use a unique mechanism to induce transcription factors. If the stress is too severe and excess the capacity of defense mechanisms, cells switch to apoptotic cell death pathways which is mainly driven by CHOP. The aim of this study is to identify the effect of ER stress on the molecular mechanisms of apoptosis in hypercholesterolemia induced NAFLD. In this direction, lipid accumulation and protein expressions of GRP78, GRP94, IRE1, PERK, Bax, CHOP, Caspase 3 has been investigated in the liver tissues of NAFLD rabbit model. Our model demostrated that high cholesterol diet increase ER stress, that triggers apoptosis in the progresion of NAFLD.

Molecular mechanisms of apoptosis in hepatocellular carcinoma cells induced by ethanol extracts of Solanum lyratum Thumb through the mitochondrial pathway.

AIMTo explore the induction effects and mechanism of Solanum lyratum Thumb (ST) on human hepatocellular carcinoma SMMC-7721 cells through the mitochondrial pathway.METHODSThe experiments were conducted on three groups: an experimental group (with ST ethanol extracts’ concentration being 2.5, 5 and 10 mg/L), a negative control group (with only nutrient solution, 0 mg/L ST ethanol extracts), and a positive control group (2.5 mg/L DDP). The inhibition rate of cell proliferation was checked by using the methyl thiazolyl tetrazolium method, and cell apoptosis was tested by TUNEL method. Furthermore, RT-PCR was used to examine mRNA expression of Fas, FasL, caspase-8, caspase-3, p53 and Bcl-2 genes.RESULTSCompared with the negative control group, the inhibition and apoptosis rates of the experimental group with different concentrations of ST extracts on human hepatocellular carcinoma SMMC-7721 cells significantly increased (P < 0.05). Besides, the mRNA expression of FasL and Bcl-2 significantly decreased (P < 0.05) while the mRNA expression of Fas, caspase-8, caspase-3 and p53 increased significantly. When compared with the positive control group, the experimental groups with 5 mg/L ST ethanol extracts showed effects similar to the positive control group.CONCLUSIONST ethanol extracts induced the apoptosis of hepatocellular carcinoma SMMC-7721 cells through up-regulated Fas, caspase-8, caspse-3 and p53, and down-regulated FasL and Bcl-2 in the mitochondrial pathway.