Mcl-1 Research Articles

Dysregulation of apoptosis and autophagy gene expression in peripheral blood mononuclear cells of efficiently treated HIV-infected patients.

We measure the transcript levels of the proapoptotic GALIG, antiapoptotic MCL1 genes and those of the autophagy genes BECN1, MAP1LC3B, ATG9a, P62/SQSTM1, GABARAP, GABARAPL1 and GABARAPL2 to define if mRNA alteration can characterize HIV-infected patients effectively treated with combined antiretroviral therapy (cART). Monocentric pilot study conducted on peripheral blood mononuclear cell (PBMC) of 40 uninfected donors and 27 HIV-positive patients effectively treated by cART for at least 8.4 years. Transcripts of the various genes were quantified by reverse transcription (RT)-quantitative PCR (qPCR) and RT-droplet digital PCR and compared using the standard statistical Mann-Whitney U test and machine learning algorithms. A concomitant overexpression of GALIG and MCL1 is detected in PBMC of effectively cART-treated patients. Overexpression of MAP1LC3B and GABARAPL1 is also measured, whereas BECN1 is underexpressed. Finally, accurate classification (94.5%) of our PBMC samples as HIV-negative donors or HIV-positive cART-treated is obtained in three separate machine-learning algorithms with GABARAPL1 and ATG9a as input variables. cART-treated HIV patients display altered transcript levels for three genes of basal autophagy. Some of these alterations may appear contradictory: BECN1 and ATG9a, both key actors in the formation of mammalian autophagosome, exhibit decreased amount of transcripts, whereas mRNA from the ATG8 family increase. Given the known role of impaired basal autophagy in immune senescence and chronic inflammation, the functional significance of our findings should be explored in larger studies.

L-asparaginase induces intrinsic mitochondrial-mediated apoptosis in human gastric adenocarcinoma cells and impedes tumor progression

l-asparagine essentially regulates growth and proliferation of cancer cells. l-asparaginase is an anti-cancer enzyme that deprives the cancer cells of l-asparagine. The purpose of this study was to explore the mechanism of a novel l-asparaginase from Pseudomonas fluorescens on l-asparagine deprivation mediated anti-proliferation, apoptosis in human gastric adenocarcinoma cells and to evaluate inhibition of angiogenesis. We observed that, the presence of extracellular l-asparagine was essential for the growth of AGS cells. l-asparagine deprivation by l-asparaginase induced metabolic stress, cytotoxicity and apoptosis by G0 phase cell-cycle arrest, modulated the mitochondrial membrane integrity, accelerated caspase-3 activation and instigated DNA damage. The RT-PCR analysis of pro-apoptosis genes: bak1, bax, bbc3, bik, pmaip1, bnip3l, apaf1, casp3, casp7 and casp9 were significantly higher (P < 0.05), while anti-apoptotic markers xiap, bid, mcl1, and death receptor genes tnf and tradd were significantly down-regulated (P < 0.05). Additionally, higher protein expressions of p53, caspase-3 and TEM analysis showing modulations in mitochondria confirmed intrinsic apoptosis pathway. The enzyme impeded tumor progression through inhibition of cell migration and vascular remodelling of endothelial cells. Our findings suggests that the action of l-asparaginase alters mitochondrial membrane permeability and auxiliary activates intrinsic apoptosis. Therefore, this mechanistic approach might be considered as a targeted enzymotherapy against gastric adenocarcinoma.

7-Isopenthenyloxycoumarin, Arctigenin, and Hesperidin Modify Myeloid Cell Leukemia Type-1 (Mcl-1) Gene Expression by Hormesis in K562 Cell Line

Hormesis is a new concept in dose–response relationship. Despite of traditional dose–response curves, there is a low-dose stimulation and a high-dose inhibition in this case. Hormesis effect in apoptosis induction/inhibition by natural compounds is reported previously. Here, we searched this effect for myeloid cell leukemia type-1 (Mcl-1) gene expression by phytochemicals 7-isopenthenyloxycoumarin (7-IP), arctigenin (Arg), and hesperidin (Hsp). For this purpose, first we tested the cytotoxicity of various doses of these compounds against K562 leukemia cell lines for different times by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. After that we explored the effect of various doses of these phytochemicals on Mcl-1 gene expression for different times by real-time polymerase chain reaction method. We found that these phytochemicals have cytotoxicity against K562 cell line. Hesperidin is the most cytotoxic agent. We also found that these natural compounds have hormetic effect on Mcl-1 gene expression. The hormetic model in Mcl-1 gene expression is overcompensation stimulation. This phenomenon is reported for the first time. We conclude that 7-IP, Arg, and Hsp are cytotoxic against K562 cancerous cells and induce/inhibit Mcl-1 gene expression by hormesis dose–response relationship.

Abstract 4836: Targeting transcriptional regulation in multiple myeloma with a covalent CDK7 inhibitor THZ1

Abstract Previous studies have shown that certain tumors such as MYCN-driven neuroblastoma and T-cell ALL may be sensitive to CDK7 inhibition. However, the impact of CDK7 disruption on the proliferation and survival of multiple myeloma (MM) cells, which are regulated, at least in part, by c-Myc, has not as yet been examined. To address this issue, the effects of THZ1, a selective covalent inhibitor of cyclin-dependent kinase CDK7, a component of the general transcription factor TFIIH, was examined in MM cells. Virtually all MM cell lines tested were sensitive to the growth-inhibitory and pro-apoptotic effects of very low (e.g., nM) THZ1 concentrations administered for 24 hr, including H929, OPM2, RPMI8226, and U266 cells, as well as bortezomib- or revlimid- resistant cells. THZ1 induced G2/M arrest and inhibited CDK7-mediated phosphorylation of CDK1/2 at T161 and T160 sites. Notably, THZ1 significantly inhibited general transcription by blocking the phosphorylation of the CTD (carboxy-terminal domain) of RNA Pol II at Ser5, Ser7, and Ser2 sites. Washout studies, in which cells were washed free of THZ1 and resuspended in drug-free medium, demonstrated thatTHZ1 acted irreversibly in blocking CTD phosphorylation. CDK7 inhibition was accompanied by down-regulation of MCL-1, c-Myc (L-myc in U266) and BCL-XL gene expression, all of which exert pro-survival effects. Concordant results were obtained when protein levels were monitored. Moreover, ectopic expression of c-Myc, MCL-1, or BCL-XL in MM cells markedly diminished THZ1 lethality, documenting the functional significance of down-regulation of these proteins in THZ1 activity. CRISPR/Cas9 editing of the CDK7 gene was them utilized to confirm the on-target impact of CDK7 disruption. Cells treated with constructs encoding two independent small guiding RNAs targeting CDK7 (sgCDK7) displayed markedly reduced CDK7 protein levels and sharply diminished H929 and OPM2 cell growth. Furthermore, co-culture with HS-5 cells failed to protect MM cells from THZ1, indicating that THZ1 can circumvent micro-environment forms of drug- resistance. Notably, THZ1 alone showed pronounced antitumor effects (e.g., inhibition of viability), and significantly enhanced proteasome inhibitor (e.g., bortezomib or carfilzomib) lethality in human primary CD138+ MM cells and primitive MM cells (CD138-/19+/20+/27+), but not normal hematopoietic cells (e.g., CD34+ cord blood cells). Finally, administration of THZ1 reduced tumor burden and significantly enhanced survival (P &amp;lt; 0.001) in flank and orthotopic xenograft mouse MM models with minimal toxicity. Collectively, these findings indicate that CDK7 is a potential therapeutic target in MM, and suggest that abrogating CDK7 activity by small molecules like THZ1 warrants further consideration as a therapeutic strategy in MM. Citation Format: Liang Zhou, Yu Zhang, Kanika Sharma, Jacquelyn McCarter, William Craun, Steven Grant. Targeting transcriptional regulation in multiple myeloma with a covalent CDK7 inhibitor THZ1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4836.

Abstract 2539: Antiapoptotic gene expression signature reveals a combined talk to prevent apoptosis: A model to choose the proper BH-3 mimetic drug

Abstract BACKGROUND: Apoptotic regulation involves several actors which can have a positive and negative effect and may represent a target for personalized therapy. Nowadays, BCL-2 inhibitors have successfully passed preliminary phases of clinical experimentation, however the involvement of other essential proteins could represent a resistance mechanism to BCL-2 inhibitors and reveal novel therapeutic targets in acute myeloid leukemia (AML). Our study aim to investigate the expression of anti-apoptotic genes in AML patients. METHODS: We performed Human Transcriptome Array 2.0 (Affymetrix) in a cohort of 59 newly diagnosed AML. K-means clustering has been used to categorize patients in different cluster. A pool of 7 healthy donors was used to normalize expression data. Survival analysis was conducted with Kaplan-Meyer method and differences in survival were assessed with Log-Rank test. RESULTS: BCL-2, MCL-1 and BCL2L1 gene expression values in 59 AML patients allowed us to establish 5 clusters (Fig. 1a): cluster 1 with the overexpression of MCL1, cluster 2 with low expression of all analyzed genes; cluster 3 with overexpression of BCL2L1, cluster 4 with overexpression of BCL-2 and cluster 5 with high values of expression of all 3 genes. Clusters 2 and 5 included the majority of patients (40/59, 68%). Furthermore, the 3-D Scatter Plot allowed us to observe how patients out of the central cloud (Clusters 2-5) always presented an overexpression of one out of the three anti-apoptotic genes. Moreover, when BCL-2 level is low (Cluster 1-2-3), MCL-1 and BCL2L1 levels are high, respectively. In term of overall survival (OS), there were no statistically significant difference between Cluster 1-3-4, where one different anti-apoptotic gene is always overexpressed. Finally, analyzing the impact of BCL-2 expression on OS in a cohort of 36 young patients treated with chemotherapy, those with high expression of the three antiapoptotic genes had worse outcome (p=0.07). CONCLUSION: BCL-2, MCL-1 and BCL2L1 expressions are quite balanced in AML patients evaluated. One out of the three genes is often overexpressed, confirming the key role of anti-apoptotic genes in leukemogenesis. Even though BCL-2 expression influence patients chemosensitivity, MCL-1 and BCL2L1 overexpression has been documented when BCL-2 is normally or downregulated. In conclusion, we suppose that BCL-2 inhibitors resistance may be caused by MCL-1 and BCL2L1 overexpression. Finally, a combined approach should be realized to switch-off leukemic anti-apoptotic mechanisms. Gene expression profile could provide a synthetic lethal instrument to choose the proper BH3-mimetic drug or combination. Supported by: ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project. Citation Format: Jacopo Nanni, Giorgia Simonetti, Samantha Bruno, Giovanni Marconi, Maria Chiara Fontana, Cristina Papayannidis, Maria Chiara Abbenante, Lorenzo Montanaro, Michele Cavo, Giovanni Martinelli. Antiapoptotic gene expression signature reveals a combined talk to prevent apoptosis: A model to choose the proper BH-3 mimetic drug [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2539.

MCL1 gene co-expression module stratifies multiple myeloma and predicts response to proteasome inhibitor-based therapy.

Multiple myeloma (MM) is the second most common hematologic cancer, characterized by abnormal accumulation of plasma cells in the bone marrow. The extensive biological and clinical heterogeneity of MM hinders effective treatment and etiology research. Several molecular classification systems of prognostic impact have been proposed, but they do not predict the response to treatment nor do they correlate to plasma cell development pathways. Here we describe the classification of MM into two distinct subtypes based on the expression levels of a gene module coexpressed with MCL1 (MCL1-M), a regulator of plasma cell survival. The classification system enabled prediction of the prognosis and the response to bortezomib-based therapy. Moreover, the two MM subtypes were associated with two different plasma cell differentiation pathways (enrichment of a preplasmablast signature versus aberrant expression of B cell genes). 1q gain, harboring 63 of the 87 MCL1-M members including MCL1, was found in about 80% of the MM with upregulated MCL1-M expression. Clonal analysis showed that 1q gain tended to occur as an early clonal event. Members of MCL1-M captured both MM cell-intrinsically acting signals and the signals regulating the interaction between MM cells with bone marrow microenvironment. MCL1-M members were co-expressed in mouse germinal center B cells. Together, these findings indicate that MCL1-M may play previously inadequately recognized, initiating role in the pathogenesis of MM. Our findings suggest that MCL1-M signature-based molecular clustering of MM constitutes a solid framework toward understanding the etiology of this disease and establishing personalized care. Article Summary: A pathogenic mechanism-guided molecular classification would facilitate treatment decision and etiology research of multiple myeloma. On the basis of the expression levels of a gene module coexpressed with MCL1, we have established a classification scheme assigning multiple myeloma into two subtypes with distinct prognosis, treatment responses and pathogenic backgrounds.

Downregulation of MCL-1 by Diallyl Disulfide Induces G2/M Arrest in Human Leukemia K562 Cells and Its Mechanism

To investigate the inducing effect of down-regulation of MCL-1 by diallyl disulfide(DADS) on the G2/M arrest of human leukemia K562 cells and its mechanisms. CCK-8 was used to detect the effect of DADS on proliferation of K562 cells, flow cytometry was employed to observe the effect of cycle arrest by DADS and RNAi silencing MCL-1 gene in K562 cells. The expressions of MCL-1, PCNA and CDK1 in K562 cells treated with DADS were detected by Western blot. The amphigamy of MCL-1 with PCNA and CDK1 was detected by Coimmunoprecipitation. CCK-8 detection showed that the inhibition rates of K562 cells treated with 15, 30, 60, 120, 240 µmol/L DADS were 32.48%, 59.34%, 66.42%, 77.06%, 81.05% respectively (P<0.05). Flow cytometry analysis revealed that the perecentages of G2/M cells were increased to 18.6% and 34.4%, 17.5% and 28.5%, respectively at 24 and 48 h after treating K562 cells with 60 and 120 µmol/L DADS (P<0.05). And the perecentage of G2/M cells of silencing MCL-1 was significantly increased (P<0.05). Silencing effects of MCL-1+DADS on the cells were enhanced more significantly as compared with DADS or MCL-1 alone (P<0.05). Western blot exhibited that DADS could markedly downregulate the expression of MCL-1, PCNA and CDK1(P<0.05). Coimmunoprecipitation revealed that MCL-1 bound with PCNA and CDK1, then forming heterodimers, which were downregulated respectively more significantly than that in the control group after treating K562 cells with DADS for 8 h (P<0.05). DADS can inhibit the K562 cell proliferation and induce them arrest G2/M through downregulation of MCL-1, then decreasing the expression of PCNA and CDK1 in hunan leukemia K562 cells. Moreover, silencing MCL-1 can enhance the effect of DADS.

MCL-1 expression of non-small cell lung cancer as a prognostic factor and MCL-1 as a promising target for gene therapy.

e24236 Background: MCL-1 is an important member in the pro-survival Bcl-2 family, plays a critical role in multidrug resistance. We investigated the significance for MCL-1 expression of non- small cell lung cancer (NSCLC) and the effect of inhibition of MCL-1 gene with siRNA (si-MCL-1) in NSCLC cells. Methods: Tumor tissues from 80 patients underwent R0 resection with NSCLC without any neoadjuvant therapy were analyzed. The intratumoral expressions of MCL-1 and Ki-67 were evaluated with immunohistochemistry in the tumor nucleus. Apoptotic index (AI) was defined as number of apoptotic cells, detected by TUNEL. Subsequently, we tested two MCL-1 overexpressing lung cancer cells, adenocarcinoma cell H358 and squamous carcinoma cell RERF, were used. Real-time RT-PCR was performed to evaluate gene expressions. The siRNA targeting MCL-1 (si-MCL-1) was transfected into cells. MTT assay was used to evaluate the cell viability and IC50. Results: The percentage of MCL-1 positive tumor nucleus varied greatly (median = 25.0%; mean±SD = 26.8±20.9%). A sample was classified as MCL-1 high tumor if > 25% of the nucleus in the tumor exhibited positive stainings. Among 80 patients with NSCLCs, 36(45.0%) were MCL-1 high status. The 5-year survival rate for patients with MCL-1 high tumors was significantly worse than that for patients with MCL-1 low tumors (68.3% vs. 93.1%, p = 0.0057). Regarding to the tumor proliferation, the Ki-67 index was significantly higher in the MCL-1 high tumor than that in MCL-1 low tumor (19.7±16.3% vs. 8.4±11.1%, p = 0.005). There was no significant relation between the AI and MCL-1 status. Si-MCL-1 effectively downregulated the MCL-1 expression (up to 80% at day 3) and significantly reduced the percentage of viable cells in these two cell lines as detected by MTT assay (p < 0.001, respectively). Multivariate analysis demonstrated MCl-1 expression to be a significant prognostic factor (hazard ratio, 4.069; p = 0.0405) Conclusions: MCL-1 expression is prognostic factor in NSCLC. The si-MCL-1 has an anti-proliferation effect against MCL-1-overexpressing NSCLC cells. MCL-1 appear to be a promising target for gene therapy.

Down-regulation of intracellular anti-apoptotic proteins, particularly c-FLIP by therapeutic agents; the novel view to overcome resistance to TRAIL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a member of the tumor necrosis factor (TNF) superfamily that induces apoptosis in different types of cancer cells via activation of caspase cascade. TRAIL interacts with its cognate receptors that placed on cancer cells surface, including TRAIL-R1 (death receptor 4, DR4), TRAIL-R2 (death receptor 5, DR5), TRAIL-R3 (decoy receptor 1, DcR1), TRAIL-R4 (decoy receptor 2, DcR2), and osteoprotegerin (OPG). Despite high apoptosis-inducing ability of TRAIL, various cancerous cells gain resistance to TRAIL gradually, and consequently TRAIL potential for apoptosis stimulation in these cells diminishes intensely. According to diverse ranges of examinations, intracellular anti-apoptotic proteins, such as cellular-FLICE inhibitory protein (c-FLIP), apoptosis inhibitors (IAPs), myeloid cell leukemia sequence 1 (MCL-1), BCL-2, BCL-XL, and survivin play key role in cancer cells resistance to TRAIL. These proteins attenuate cancer cells sensitivity to TRAIL via various functions, importantly through caspase cascade suppression. The c-FLIP avoids from caspase 8 activation by FADD via binding to caspase 8 cleavage of FADD. Moreover, it activates signaling pathways that involved in cancer cells survival and proliferation. Intriguingly, it appears that the down-regulation of intracellular anti-apoptotic proteins, particularly c-FLIP is effectiveness goal for TRAIL-resistant cancers therapy, because their up-regulation in association with poor prognosis has been observed in various types of TRAIL-resistant cancers. In this review, we tried to collect and examine investigations that researchers have been able to sensitize cancer cells to TRAIL through targeting of c-FLIP alone or with other intracellular anti-apoptotic proteins directly or indirectly. It seems that co-treatment of resistant cells by TRAIL with other therapeutic agents with the aim of intracellular anti-apoptotic proteins inhibition is hopeful and attractive approach to overcome various TRAIL-resistant cancers.

Norcantharidin induces apoptosis in human glioma cell via mitochondrial pathway

Objective To elucidate the molecular mechanism of norcantharidin (NCTD) in inducing apoptosis of glioma cells. Methods Glioma C6 cells were treated with various dosages of NCTD (25, 50, 100, 200 μmol/L) for 24, 48 h. The cell growth inhibition was measured by methyl thiazol tetrazolium (MTT) assay. The apoptosis of cells were analyzed by Annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) staining. The signaling pathway changes were detected by Western blotting. Results NCTD effectively inhibited cell proliferation of C6 cells in a time and dose-dependent manner. The 24 h and 48 h half maximal inhibitory concentration (IC50) were 96.5 and 48.7 μmol/L. It had a significant inhibition of cell proliferation at 24 h and 48 h (F=78.202, 897.714, P=0.000). Compared with the control group (0.9±3.1)%, after 10 h treatment, the cell apoptosis rates induced by 10, 30, 50 μmol/L NCTD were (2.0±2.9)%, (8.4±4.1)% and (19.7±5.7)%, respectively, which indicated that NCTD could induce apoptosis of cells in a dose-dependent manner (F=76.246, P=0.000). Meanwhile, the expression levels anti-apoptotic proteins B cell lymphoma/leukemia-2 (bcl-2) and Mcl-1 were significantly reduced. Conclusion NCTD inhibits glioma cell growth and induces cell apoptosis, and the anticancer activity of NCTD may be induced through targeting bcl-2 anti-apoptotic family members. Key words: Glioma; Norcantharidin; Apoptosis; B cell lymphoma/leukemia-2 gene

LINC00152 down-regulated miR-193a-3p to enhance MCL1 expression and promote gastric cancer cells proliferation.

The present work aimed to probe into the effect of long non-coding RNA (lncRNA) LINC00152 on gastric cancer (GC) cells proliferation by regulating miR-193a-3p and its target gene MCL1. Transfected si-LINC00152 was used to down-regulate LINC00152, and cells proliferation was measured by the cell counting kit-8 (CCK-8) assay. Cell apoptosis and cell cycle were analyzed by flow cytometry (FCM). Besides, we also detected the potential functional effects of differential expression of LINC00152 in vivo using nude mouse xenograft model. We overexpressed and downexpressed miR-193a-3p to study the in vitro effect of miR-193a-3p on GC cells proliferation and vitality. And MCL1 was silenced by shRNA to investigate the effect of MCL1 on proliferation of GC cells. In this research, LINC00152 was proven to have a higher expression level in GC tissues than in the adjacent normal tissues. GC cells proliferation was inhibited after LINC00152 was down-regulated. LINC00152 inhibited the expression of miR-193a-3p, which negatively regulated MCL1. In addition, GC cells proliferation was inhibited by cell transfection with shRNA-MCL1, and enhanced by transfection with miR-193a-3p mimics. Our study suggested that LINC00152 was overexpressed in GC tissues, and it down-regulated miR-193a-3p to enhance MCL1 expression thereby promoting GC cells proliferation.

Expression Profile of Transcription Factor ELK-1 and ELK-1 Target Genes on Lymphoma-Leukemia Cell Lines

Prognostic molecular markers identified in leukemia are becoming increasingly important especially in risk stratification and to determine therapy. In this study, we investigate the role of ELK-1 transcription factor and its potential target genes in four cell lines; Daudi, Jurkat, K-562 and HL-60. To evaluate ELK-1, MCPIP, MCL-1, BCL-10, CEBPB and SRF genes expression profiles we have performed a Real-time PCR analysis on Daudi, Jurkat, K-562 and HL-60 cell lines. ELK-1 over expression concomitant with SRF overexpression was detected only in Daudi cell line while only SRF overexpression was detected in jurkat cells. Expression of MCPIP, MCL-1, BCL-10 and CEBPB genes were decreased in all cell lines. Protein levels or phosphorylation status of ELK-1, BCL-10, CEBPB, MCL-1, MCPIP and SRF, moreover, changes that may occur when ELK-1 continuous overexpression is provided or completely silenced in these cell lines have not been evaluated. These questions are suggestions for future investigations.

Effect of celecoxib on anoikis resistance in non-small cell lung cancer induced by surgical stress

Objective To investigate the effect and its molecular mechanism of celecoxib on anoikis resistance in non-small cell lung cancer cell line A549 induced by lung cancer surgical stress. Methods A549 cells were cultured using cell culture plate covering poly-(2-hydroxyethyl methacrylate) HEMA. The control group, prostaglandin E2 (PGE2) treatment group, and PGE2 combined celecoxib treatment group were established. Anoikis of cells was assessed by flow cytometry after treatment for 48 h. Western blotting was used to detect the activation of mitogen extracellular kinase (MEK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway and the expression of apoptosis related proteins, Mcl-1 and Bim, in each group. BALB/c mice were used to simulate the surgical stress, and A549 cells were injected into the tail vein to establish the animal model of lung metastasis. Mice were divided into control group, surgery group and surgery plus celecoxib group (completely randomized grouping), the mice were killed after 4 weeks and lung metastasis was observed, and the expression of Mcl-1 and Bim in pulmonary nodules was detected by immunohistochemical method. Results Flow cytometry results showed that apoptosis rate of A549 cells cultured in poly-HEMA was (41.0±2.4)%, that in PGE2 treated group was (22.8±3.2)% (as compared with the control group, P=0.012), and that in the PGE2 combined with celecoxib treatment group was (36.6±2.4)% (as compared with the PGE2 group, P=0.034). The results of Western blotting showed that PGE2 could activate the MEK/ERK1/2 pathway, up-regulate the phosphorylation level of ERK1/2, and down-regulate the expression of Bim, while celecoxib could significantly inhibit the effects of PGE2. In addition, PGE2 had no obvious effect on PI3K/Akt pathway, but celecoxib could inhibit the pathway, and down-regulate the phosphorylation level of Akt and inhibit the expression of Mcl-1. Immunohistochemically, the expression level of Mcl-1 and Bim protein in the pulmonary nodules was consistent with that of experiments in vitro. Conclusion PGE2 is the main cytokine production in the surgical stress. This study shows that PGE2 has obvious effect in vitro on anoikis resistance, and celecoxib inhibits the MEK/ERK1/2 pathway and the PI3K/Akt pathway to increase the expression of pro-apoptotic protein Bim and decrease the expression of anti-apoptotic protein Mcl-1 in vitro and in vivo, which can promote anoikis to reduce lung cancer cells metastasis. Key words: Celecoxib; Non-small cell lung cancer; Surgical stress; Anoikis

Extract of Stellerachamaejasme L(ESC) inhibits growth and metastasis of human hepatocellular carcinoma via regulating microRNA expression

BackgroundMicroRNAs(miRNAs)are involved in the initiation and progression of hepatocellular carcinoma. ESC, an extract of Stellerachamaejasme L, had been confirmed as a potential anti-tumor extract of Traditional Chinese Medicine. In light of the important role of miRNAs in hepatocellular carcinoma, we questioned whether the inhibitory effects of ESC on hepatocellular carcinoma (HCC) were associated with miRNAs.MethodsThe proliferation inhibition of ESC on HCC cells was measured with MTT assay. The migration inhibition of ESC on HCC cells was measured with transwell assay. The influences of ESC on growth and metastasis inhibition were evaluated with xenograft tumor model of HCC. Protein expressions were measured with western blot and immunofluorescence methods and miRNA profiles were detected with miRNA array. Differential miRNA and target mRNAs were verified with real-time PCR.ResultsThe results showed that ESC could inhibit proliferation and epithelial mesenchymal transition (EMT) in HCC cells in vitro and tumor growth and metastasis in xenograft models in vivo. miRNA array results showed that 69 differential miRNAs in total of 429 ones were obtained in MHCC97H cells treated by ESC. hsa-miR-107, hsa-miR-638, hsa-miR-106b-5p were selected to be validated with real-time PCR method in HepG2 and MHCC97H cells. Expressions of hsa-miR-107 and hsa-miR-638 increased obviously in HCC cells treated by ESC. Target genes of three miRNAs were also validated with real-time PCR. Interestingly, only target genes of hsa-miR-107 changed greatly. ESC downregulated the MCL1, SALL4 and BCL2 gene expressions significantly but did not influence the expression of CACNA2D1.ConclusionThe findings suggested ESC regressed growth and metastasis of human hepatocellular carcinoma via regulating microRNAs expression and their corresponding target genes.

Therapeutic potential of Bcl-xL/Mcl-1 synthetic inhibitor JY-1-106 and retinoids for human triple-negative breast cancer treatment.

Overexpression of anti-apoptotic proteins belonging to the B cell lymphoma (Bcl)-2 family is observed in numerous cancer types and has been postulated to promote cancer cell survival and chemotherapy resistance. Bcl-extra large (xL)/myeloid cell leukemia sequence (Mcl)-1 was demonstrated to be expressed at relatively high levels in clinically aggressive basal-like cancers and inhibiting Bcl-xL overexpression could potentially provoke cell death. A molecule able to target Bcl-xL/Mcl-1, JY-1-106, is herein under investigation. It is also known that vitamin A-derived compounds exhibit antitumor activity in a variety of in vitro experimental models, promoting their effects via nuclear receptor isoforms including retinoic acid receptors (RARs). Pre-clinical observation highlighted that triple negative (estrogen receptor/progesterone receptor/human epidermal growth factor receptor)-breast cancer cells displayed resistance to retinoids due to the RARγ high expression profile. The present study used the triple-negative human breast cancer cell line, MDA-MB-231, to analyze the effects of the Bcl-xL/Mcl-1 synthetic inhibitor, JY-1-106, alone or in combination with retinoids on cell viability. The results revealed a synergistic effect in reducing cell viability primarily by using JY-1-106 with the selective RARγ antagonist SR11253, which induces massive autophagy and necrosis. Furthermore, the results highlighted that JY-1-106 alone is able to positively influence the gene expression profile of p53 and RARα, providing a therapeutic advantage in human triple-negative breast cancer treatment.

Effect and mechanism of curcumin on proliferation and apoptosis of human hepatoma HCC-LM3 cells

Objective To investigate the effects of curcumin on the growth and apoptosis of human hepatoma HCC-LM3 cells and explore the molecular mechanisms. Methods The human hepatoma HCC-LM3 cells were treated with different concentrations of curcumin and the cell growth inhibition rate was detected by CCK-8. The effect of curcumin on human hepatoma HCC-LM3 cells was observed. Refer to the relevant literature, the human hepatoma HCC-LM3 cells were treated with the concentration of 2.5, 5.0, 10.0, 15.0, 20.0, 40.0, 60.0 μmol/L of curcumin for 48 hours, taking the 0 μmol/L curcumin as control group, and the cell growth inhibition rate was detected by CCK-8. According to the results of CCK-8, selecting the concentration of 0 μmol/L as control group and the concentration of 10.0, 20.0, 40.0 μmol/L as experimental groups, which has significant difference on growth inhibition rates. Cell cloning assay was used to detect cell cloning ability, Flow cytometry was used to detect apoptosis, and Western blotting to detect the protein expression levels of Mcl-1, Bax, Bcl-2 and Bcl-xL. The measurement data were expressed in (±s), and the single factor analysis of variance was used for comparison between groups. Results CCK-8 assay showed that with treated by the concentration of 2.5, 5.0, 10.0, 15.0, 20.0, 40.0, 60.0 μmol/L, the growth inhibition rates were(6.71±3.45)%, (12.33±5.02)%, (20.07±5.60)%, (57.80±7.34)%, (78.37±6.53)%, (91.73±6.14)% and (96.18±3.45)%, suggesting that curcumin could inhibit the growth of human hepatoma HCC-LM3 cells in a dose-dependent manner. Cell clone formation experiment showed that curcumin could inhibit the clone of the human hepatoma HCC-LM3 cells, and the clone of the cells was inhibited significantly when the concentration of the curcumin was over 20.0μmol/L. The result of Annexin V-FITC/PI double staining analysis showed that the apoptotic rates of experimental groups and control groups were (5.20±1.44)%, (9.90±3.31)%, (55.67±5.29)%, (79.63±4.71)%, with all the apoptotic rates of experimental group over the control groups (P<0.05), suggesting curcumin could induce the apoptosis of human hepatoma HCC-LM3 cells.The Westen blotting showed that curcumin increased the expression of Bax protein while decreasing expression of Mcl-1 protein significantly in concentration-dependent manner (P<0.05), but have no effect on the expression of Bcl-2 and Bcl-xL proteins. Conclusion Curcumin could inhibit the proliferation and clone of human hepatoma HCC-LM3 cells, and induce apoptosis in a dose dependent manner. Key words: Curcumin; Carcinoma, hepatocellular; Cell proliferation; Apoptosis; HCC-LM3

Abstract P1-03-06: GNBP2 suppresses tumor growth and cancer stem cell load of triple negative breast cancer by controlling STAT3 pathway

Abstract Background: Gametogenetin binding protein 2 (GGNBP2) is encoded in human chromosome 17q12-q23, a region known as a breast and ovarian cancer susceptibility locus. GGNBP2 has a single C2H2 zinc finger and a consensus LxxLL nuclear receptor (NR) binding motif. We have reported that GGNBP2 suppresses ERα-positive breast tumorigenesis by acting as a nuclear receptor co-repressor to restrain ERα activity. However, the detailed molecular mechanisms of GGNBP2 and its role in triple negative breast cancer (TNBC) remain largely unclear. Methods: A human breast cancer tissue array containing 138 human breast tumor tissues were utilized to examine GGNBP2 expression in breast cancer samples by IHC. To address the potential anti-breast tumor activity of GGNBP2 in vitro, we expressed exogenous GGNBP2 in TNBC cells, including MDA-MB-231 and Cal51 cell lines. Cell proliferation and cell cycle were assessed by cell growth curve/EdU assays and flow cytometry after propidium Iodide staining. Apoptosis was determined by flow cytometry after annexin V staining, by caspase 3/7 and caspase 9 activity assays. Cancer stem cell properties were determined by expression of CD44/CD24/ALDH1 markers. The levels of phosphorylated STAT3 and total STAT3 were determined by western blot. Quantitative PCR and Western blot were carried out to evaluate the effects of GGNBP2 overexpression on STAT3 target genes, CCND1, Mcl-1, survivin, bax and bim expression. Results: GGNBP2 expression is down-regulated in TNBC cells and patient tumors and it is associated with poor patient survival. Overexpression of GGNBP2 significanly induces cell cycle G0/G1 phase arrest and apoptosis in TNBC cell lines. Expression of cancer stem cell markers also decreased in GGNBP2-overexpressed TNBC cells. GGNBP2 reduces the expression levels of CCND1, Mcl-1 and survivin, promotes the expression levels of bax and bim proteins. Importantly, overexpression of GGNBP2 inhibits STAT3 phosphorylation and STAT3 downstream garget gene expression, including CCND1, Mcl-1 and survivin. Conclusion: GGNBP2 serves as a critical nuclear negative regulator of STAT3-mediated gene expression and tumorigenesis. Citation Format: Zhang J. GNBP2 suppresses tumor growth and cancer stem cell load of triple negative breast cancer by controlling STAT3 pathway [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-03-06.

Glycogen synthase kinase 3β inhibition reduces mitochondrial oxidative stress in chronic myocardial ischemia

ObjectivesGlycogen synthase kinase 3β (GSK-3β) inhibition has been reported to increase microvascular density and improve myocardial blood flow in a porcine model of chronic myocardial ischemia and metabolic syndrome. Inhibition of GSK-3β can also be cardioprotective by modulating fibrosis signaling and mitochondrial-induced apoptosis. We hypothesized GSK-3β inhibition would have a beneficial effect on myocardial fibrosis and oxidative stress in a porcine model of chronic myocardial ischemia and metabolic syndrome. MethodsPigs were fed a high fat diet for 4 weeks followed by placement of an ameroid constrictor to the left circumflex coronary artery. Three weeks later animals received either no drug or a GSK-3β inhibitor. The diets and placebo/GSK-3β inhibition were continued for an additional 5 weeks, the pigs were then euthanized, and the myocardial tissue was harvested. Collagen expression was analyzed via Picrosirius staining. Oxidative stress was analyzed via Oxyblot analysis. Protein expression was analyzed via Western blot. ResultsGSK-3β inhibition was associated with decreased collagen expression and oxidative stress in the ischemic and nonischemic myocardial tissue compared with control. There was a decrease in profibrotic proteins transforming growth factor-β, p-SMAD2/3, and matrix metalloproteinase-9, and in proapoptotic and oxidative stress proteins, apoptosis inducing factor, the cleaved caspase 3/caspase 3 protein ratio and phosphorylated myeloid cell leukemia sequence-1 in the GSK-3β inhibited group compared with the control. ConclusionsIn the setting of metabolic syndrome and chronic myocardial ischemia, inhibition of GSK-3β decreases collagen formation and oxidative stress in myocardial tissue. GSK-3β inhibition might be having this beneficial effect by downregulating transforming growth factor-β/SMAD2/3 signaling and decreasing mitochondrial induced cellular stress.

Expressions and significance of Mcl-1 and Caspase-3 and theirs correlation in tissues of prostate cancer

Objective To study the relationship of the expressions of Mcl-1 and Caspase-3 in tissues of prostate cancer(PCa) and benign prostatic hyperplasia (BPH), investigate the correlation of Mcl-l and Caspase-3. Methods The expressions of Mcl-1 and Caspase-3 were observed separately in 30 of BPH, and 35 of PCa by means of immunohistochemical methods. Results Mcl-1 positive rate in BPH was 13.33%(4/30), and in PCa was 82.86%(29/35). Caspase-3 positive rate in BPH was 73.33%(22/30), and in PCa was 25.71%(9/35). Expression of Mcl-1 and Caspase-3 in PCa was unrelated with preperative PSA, distant metastases, Gleason score and clincal stage(P>0.05). There was a negative relationship between the expression of Mcl-1 and Caspase-3 in tissues of PCa(rs=-0.748, P<0.05). Conclusions The expression of Mcl-1 is raised while the expression of Caspase-3 is low in PCa tissue, showing that they may have a common mechanism in the process of apoptosis. Key words: Prostatic Neoplasms; Prostatic Hyperplasia; Caspase 3

Quercetin manipulates the expression of genes involved in the reactive oxygen species (ROS) process in chicken heterophils.

Chicken heterophils generate reactive oxygen species (ROS) molecules to defend against invading pathogens. The present study examined effects of quercetin on chicken heterophils. Heterophils were stimulated with PBS, 50 µM quercetin (QH), PMA or Escherichia coli (EC) and the resulting intracellular ROS molecules were determined. Flow cytometry results showed that cells stimulated with QH, PMA and EC had a higher ROS production. Increases in intracellular ROS molecules were identified in all treatment groups by fluorescence microscopy. Determination of the ability of quercetin to manipulate mRNA expression of ROS subunits was assessed using real-time RT-PCR. Quercetin and other stimulants up-regulated the majority of genes involved in ROS production: CYBB (NOX2), NCF1 (p47phox), NCF2 (p67phox), NOX1 and RAC2. The antioxidant property of QH was explored by measuring mRNA expression of CAT and SOD1. The data indicate increased levels of CAT with all treatments; however, only QH attenuated the expression of the SOD1 gene. To further investigate the effects of ROS-driven inflammation or cell death, IL6, CASP8 and MCL1 genes were preferentially tested. The inflammatory gene (IL6) was profoundly down-regulated in the QH- and PMA-treated groups while EC induced a strikingly high IL6 expression level. Investigation of the known apoptotic (CASP8) and anti-apoptotic (MCL1) genes found down-regulation of CASP8 in the QH- and PMA-treated groups which were contradicted to the MCL1 gene. In conclusion, quercetin can enhance ROS production by regulating the expression of genes involved in ROS production as well as in subsequent processes.