Inhibited Cell Survival Research Articles

Apigenin Enhanced Radiation-Induced Apoptosis/Necrosis by Sensitization of LNCaP Prostate Cancer Cells to 6 MV Photon Beams.

Objective Whereas prostate cancer (PrCa) may be unresponsive or moderately responsive to radiation therapy (RT)- most common modality for treatment of PrCa- patients must receive a high dose of RT In order to achieve appropriate tumour control. However, this increase in radiation dose may lead to severe adverse effects in normal tissues. Sensitization of PrCa to radiation provides an alternate approach to improve the therapeutic efficacy of RT. This study aims to assess the radiosensitisation effect of apigenin (Api) on a prostate cancer cell line (LNCaP). Materials and Methods In this experimental study, LNCaP cells were treated with 0-80 µM Api to investigate its effect on LNCaP cell viability and determine its half-maximal inhibitory concentration (IC50). Next, the cells were divided into four groups: i. Control, ii. Cells treated with the IC50 concentration of Api, iii. Cells treated with 2 Gy ionizing radiation (IR), and cells co-treated with Api and IR. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, real-time polymerase chain reaction (PCR), and an Annexin V-FITC/PI assay were performed to assess cell survival, Bax and Bcl-2 expressions, and presence of apoptosis and necrosis. Results Api inhibited cell survival in a dose-dependent, but not time-dependent manner. Cells treated with Api had increased amounts of early apoptosis, late apoptosis, and secondary necrosis compared to the control group. This group also had decreased Bcl-2 gene expression and up-regulated Bax gene expression. Co-treatment with Api and IR significantly inhibited cell survival, and increased early apoptosis, late apoptosis and secondary necrosis compared to the other groups. There was a significant decrease in Bcl-2 gene expression along with up-regulation of Bax gene expression, and Bax/Bcl-2 ratio changes that favoured apoptosis. ConclusionApi inhibited PrCa cell survival and induced apoptosis as a single agent. In addition, Api significantly sensitized the LNCaP cells to IR and enhanced radiation-induced apoptosis.

Impact of growth hormone-releasing hormone antagonist on decidual stromal cell growth and apoptosis in vitro†.

Endometrial stromal cells remodeling is critical during human pregnancy. Growth hormone-releasing hormone and its functional receptor have been shown to be expressed in gynecological cancer cells and eutopic endometrial stromal cells. Recent studies have demonstrated the potential clinical uses of antagonists of growth hormone-releasing hormone as effective antitumor agents because of its directly antagonistic effect on the locally produced growth hormone-releasing hormone in gynecological tumors. However, the impact of growth hormone-releasing hormone antagonists on normal endometrial stromal cell growth remained to be elucidated. The aim of this study was to investigate the effect of a growth hormone-releasing hormone antagonist (JMR-132) on cell proliferation and apoptosis of human decidual stromal cells and the underlying molecular mechanisms. Our results showed that growth hormone-releasing hormone and the splice variant 1 of growth hormone-releasing hormone receptor are expressed in human decidual stromal cells isolated from the decidual tissues of early pregnant women receiving surgical abortion. In addition, treatment of stroma cells with JMR-132 induced cell apoptosis with increasing cleaved caspase-3 and caspase-9 activities and decrease cell viability in a time- and dose-dependent manner. Using a dual inhibition approach (pharmacological inhibitors and siRNA-mediated knockdown), we showed that JMR-132-induced activation of apoptotic signals are mediated by the activation of ERK1/2 and JNK signaling pathways and the subsequent upregulation of GADD45alpha. Taken together, JMR-132 suppresses cell survival of decidual stromal cells by inducing apoptosis through the activation of ERK1/2- and JNK-mediated upregulation of GADD45alpha in human endometrial stromal cells. Our findings provide new insights into the potential impact of growth hormone-releasing hormone antagonist on the decidual programming in humans.

Docosahexaenoic Acid Inhibits Cell Proliferation through a Suppression of c-Myc Protein in Pancreatic Ductal Adenocarcinoma Cells.

Treatment of pancreatic cancer by inhibiting the aberrant activation of the survival signaling pathways has received considerable attention. We investigated the probable action of DHA on the suppression of cell proliferation in human pancreatic ductal adenocarcinoma (PDAC) cells. Our results demonstrated that DHA dose-dependently inhibited cell proliferation through an induction of cell cycle arrest in human PDAC cells. DHA suppressed the expression of phosphorylated-Rb (p-Rb), cyclin D1, cyclin E, cyclin A, E2F1 and c-Myc proteins. Blocking the activation of STAT3 signaling pathway led to an inactivation of CAMKII and increased phosphorylation of c-Myc (T58) protein accompanied with decreased expression of c-Myc protein. Treatment of DHA effectively inhibited cell survival through decreased phosphorylation levels of EGFR, STAT3 and CAMKII proteins. The mechanisms of action were associated with increased phosphorylation levels of c-Myc (T58) and instability of c-Myc proteins. DHA inhibited cell survival through an increased GSSG/GSH ratio and oxidative stress level in HPAF-II cells. DHA induced cell apoptosis through increased expression of Bax, c-caspase 3 and c-PARP proteins in HPAF-II cells. Moreover, treatment of DHA significantly inhibited nucleotide synthesis. In conclusion, DHA might significantly suppress the proliferation of PDAC cells and therefore have potential as an anti-cancer therapeutic agent.

Androgen receptor negatively regulates mitotic checkpoint signaling to induce docetaxel resistance in castration-resistant prostate cancer.

BackgroundDespite multiple treatment advances for castration‐resistant prostate cancer (CRPC), there are currently no curative therapies and patients ultimately to succumb to the disease. Docetaxel (DTX) is the standard first‐line chemotherapy for patients with metastatic CRPC; however, drug resistance is inevitable and often develops rapidly, leading to disease progression in nearly all patients. In contrast, when DTX is deployed with androgen deprivation therapy in castration‐sensitive disease, more durable responses and improved outcomes are observed, suggesting that aberrant androgen receptor (AR) signaling accelerates DTX resistance in CRPC. In this study, we demonstrate that AR dysregulates the mitotic checkpoint, a critical pathway involved in the anticancer action of DTX.MethodsAndrogen‐dependent and independent cell lines were used to evaluate the role of AR in DTX resistance. Impact of drug treatment on cell viability, survival, and cell‐cycle distribution were determined by plate‐based viability assay, clonogenic assay, and cell‐cycle analysis by flow cytometry, respectively. Mitotic checkpoint kinase signal transduction and apoptosis activation was evaluated by Western blotting. Pathway gene expression analysis was evaluated by RT‐PCR. A Bliss independence model was used to calculate synergy scores for drug combination studies.ResultsActivation of AR in hormone‐sensitive cells induces a rescue phenotype by increasing cell viability and survival and attenuating G2/M arrest in response to DTX. Analysis of mitotic checkpoint signaling shows that AR negatively regulates spindle checkpoint signaling, resulting in premature mitotic progression and evasion of apoptosis. This phenotype is characteristic of mitotic slippage and is also observed in CRPC cell lines where we demonstrate involvement of AR splice variant AR‐v7 in dysregulation of checkpoint signaling. Our findings suggest that DTX resistance is mediated through mechanisms that drive premature mitotic exit. Using pharmacologic inhibitors of anaphase‐promoting complex/cyclosome and polo‐like kinase 1, we show that blocking mitotic exit induces mitotic arrest, apoptosis, and synergistically inhibits cell survival in combination with DTX.ConclusionOur results suggest that targeting the mechanisms of dysregulated mitotic checkpoint signaling in AR‐reactivated tumors has significant clinical potential to extend treatment benefit with DTX and improve outcomes in patients with lethal prostate cancer.

HDAC/MIF dual inhibitor inhibits NSCLC cell survival and proliferation by blocking the AKT pathway

Non-small-cell lung carcinoma (NSCLC) is one of the most common forms of lung cancer, and a leading cause of cancer death among human beings. There is an urgent demand for novel therapeutics for the treatment of NSCLC to enhance the efficacy of the currently applied Tyrosine kinase inhibitors (TKIs) therapy and to overcome therapy-resistance. Here, we report a novel small-molecule inhibitor that simultaneously targets histone deacetylase (HDAC) and macrophage migration inhibitory factor (MIF). The HDAC/MIF dual inhibitor proved to be toxic for EGFR mutated (H1650, TKI-resistant) or knock out (A549 EGFR−/−) NSCLC cell lines. Further experiments showed that HDAC inhibition inhibits cell survival and proliferation, while MIF inhibition downregulates pAKT or AKT expression level, which both interfere with cell survival. Furthermore, the combination treatment of TKI and HDAC/MIF dual inhibitor showed that the dual inhibitor enhanced TKI inhibitory efficacy, highlighting the advantages of HDAC/MIF dual inhibitor for more effective treatment of NSCLC.

Targeted Inhibition of HK-II Reversed the Warburg Effect to Improve the Radiosensitivity of Laryngeal Carcinoma.

PurposeHexokinase-II (HK-II) is the key enzyme in the first rate-limiting step of glycolysis that catalyzes the conversion of glucose to glucose-6-phosphate. Here, we examined the association between HK-II expression and radioresistance in laryngeal carcinoma and whether the inhibition of HK-II expression can enhance the radiosensitivity of these tumors.MethodsThe effects of HK-II small interfering RNA (siRNA) on the radiosensitivity of Tu212 cells were examined in vitro and in vivo in a mouse model. Cells were irradiated using a 6-MV linear accelerator. The cell viability, cell survival, proliferation, apoptosis, and cell cycle of Tu212 cells were evaluated using trypan blue staining, colony formation assays, CCK-8 assays, and flow cytometry, respectively. Oxygen consumption, lactic acid production, glucose consumption, and the ATP level of Tu212 cells were also examined. The expression of glycolytic and regulatory enzymes involved in the tricarboxylic acid cycle was assessed using Western blotting.ResultsThe HK-II siRNA and X-ray combination treatment led to a significantly greater reduction of cell viability, inhibition of cell survival and proliferation, increased apoptosis, and increased G2 phase arrest compared to either treatment alone (all, P<0.01). HK-II siRNA increased the oxygen consumption rate of cells, significantly inhibited lactic acid production and glucose consumption, and significantly suppressed the upregulation of HK-II, pyruvate kinase M2 (PKM2), pyruvate dehydrogenase (PDH), phosphofructokinase platelet (PFKP), lactate dehydrogenase (LD), and citrate synthase (CS) (all, P<0.01).ConclusionThe inhibition of HK-II by siRNA enhances the radiosensitivity of laryngeal carcinoma Tu212 cells by inhibiting glycolysis and partially inhibiting oxidative phosphorylation.

Screening of anti-chronic nonbacterial prostatitis activity of different extractions of the aerial part of Glycyrrhiza uralensis, and network pharmacology research.

In the present study, anti-chronic nonbacterial prostatitis (CNP) pharmacological experiments using water and ethanol extraction of the aerial parts of Glycyrrhiza uralensis were performed to select the best active parts by comparing their efficacy in a CNP model established by injecting carrageenin into the ventral lobe of rat prostate. The anti-CNP activities and expression of serum inflammatory factors in rats were also analyzed. A Protein-Protein Interaction network was constructed, and core targets were screened using topology and analyzed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Water and ethanol extraction exhibited good inhibitory effect on the pathological changes of the prostate tissue, the expression of inflammatory factors and fibrosis factors in CNP rats, whereas no differences were observed compared with the positive control drug. Water extraction was more effective and significantly reduced PGE2 expression (P<0.05). Network pharmacology assays showed 15 active components in the aerial part of Glycyrrhiza uralensis, and 9 key CNP therapeutic targets of the aerial parts of Glycyrrhiza uralensis were identified. The effect of water exraction on chronic prostatitis rats was significant. The aerial part of Glycyrrhiza uralensis downregulated the levels of inflammatory factors and inhibited proinflammatory gene transcription, reduced oxidative stress response, inhibited cell survival pathways, regulated sex hormone levels, prevented immunostimulation and attenuated inflammation. This study provides a theoretical reference for the development of anti-CNP agents, and offers a novel methodology for identifying and clarifying the mechanisms underlying the efficacy of the anti-CNP components in the aerial part of Glycyrrhiza uralensis.

Network pharmacology and molecular docking reveal the effective substances and active mechanisms of Dalbergia Odoriferain protecting against ischemic stroke.

Dalbergia Odorifera (DO) has been widely used for the treatment of cardiovascular and cerebrovascular diseasesinclinical. However, the effective substances and possible mechanisms of DO are still unclear. In this study, network pharmacology and molecular docking were used toelucidate the effective substances and active mechanisms of DO in treating ischemic stroke (IS). 544 DO-related targets from 29 bioactive components and 344 IS-related targets were collected, among them, 71 overlapping common targets were got. Enrichment analysis showed that 12 components were the possible bioactive components in DO, which regulating 9 important signaling pathways in 3 biological processes including 'oxidative stress' (KEGG:04151, KEGG:04068, KEGG:04915), 'inflammatory response'(KEGG:04668, KEGG:04064) and 'vascular endothelial function regulation'(KEGG:04066, KEGG:04370). Among these, 5 bioactive components with degree≥20 among the 12 potential bioactive components were selected to be docked with the top5 core targets using AutodockVina software. According to the results of molecular docking, the binding sites of core target protein AKT1 and MOL002974, MOL002975, and MOL002914 were 9, 8, and 6, respectively, and they contained 2, 1, and 0 threonine residues, respectively. And some binding sites were consistent, which may be the reason for the similarities and differences between the docking results of the 3 core bioactive components. The results of in vitro experiments showed that OGD/R could inhibit cell survival and AKT phosphorylation which were reversed by the 3 core bioactive components. Among them, MOL002974 (butein) had a slightly better effect. Therefore, the protective effect of MOL002974 (butein) against cerebral ischemia was further evaluated in a rat model of middle cerebral artery occlusion (MCAO) by detecting neurological score, cerebral infarction volume and lactate dehydrogenase (LDH) level. The results indicated that MOL002974 (butein) could significantly improve the neurological score of rats, decrease cerebral infarction volume, and inhibit the level of LDH in the cerebral tissue and serum in a dose-dependent manner. In conclusion, network pharmacology and molecular docking predicate the possible effective substances and mechanisms of DO in treating IS. And the results are verified by the in vitro and in vivo experiments. This research reveals the possible effective substances from DO and its active mechanisms for treating IS and provides a new direction for the secondary development of DO for treating IS.

Intracellular Metabolism in Diabetic Embryopathy

Diabetes mellitus in early pregnancy can cause congenital birth defects in infants, a complication known as diabetic embryopathy. Formation of structural abnormalities, commonly seen in the central nervous and cardiovascular systems, is associated with increased programmed cell death (apoptosis) and decreased cell proliferation in the developing organs. Under maternal hyperglycemic conditions, influx of glucose into cells of the embryos disturbs intracellular metabolic homeostasis. Disturbed glycolysis generates factors through side pathways to perturb cell signaling and organelle functions. Perturbed phospholipid metabolism produces signaling metabolites and peroxidation products to suppress cell survival and induce apoptosis. Targeting the key processes and factors in the metabolism of glucose and phospholipids is a potential intervention strategy to prevent birth defects in diabetic pregnancies.

TMEM100 negatively regulated by microRNA‑106b facilitates cellular apoptosis by suppressing survivin expression in NSCLC.

Non‑small cell lung cancer (NSCLC) is a common malignant tumour. Nevertheless, the 5‑year survival rate of NSCLC patients remains poor. Thus, identifying critical factors involved in regulating the progression of NSCLC is important for providing potential treatment targets. In the present study, it was observed that transmembrane protein 100 (TMEM100) was significantly downregulated in NSCLC tissues compared with paired peritumoral tissues. Decreased TMEM100 expression was associated with poor clinical outcomes in NSCLC patients. Moreover, TMEM100 overexpression inhibited colony formation and facilitated apoptosis by suppressing survivin expression in NSCLC cells, whereas TMEM100 knockdown had the opposite effect. In addition, microRNA (miR)‑106b, a miR with controversial roles in different human cancers, was upregulated in NSCLC and directly downregulated TMEM100 expression. The roles of miR‑106b in cell survival were mitigated by the restoration of TMEM100. The aforementioned results indicated that TMEM100 induced cell apoptosis and inhibited cell survival by serving as a tumour suppressor and that miR‑106b‑mitigatedTMEM100 expression defined a potentially oncogenic pathway in NSCLC.

Knockdown of Myeloid Cell Leukemia-1 by MicroRNA-101 Increases Sensitivity of A549 Lung Cancer Cells to Etoposide.

Background: Studies have shown that myeloid cell leukemia-1 (Mcl-1) is the target gene for microRNA -101 (miRNA-101), and decreased levels of miRNA-101 are associated with elevated levels of Mcl-1 and lung cancer survival. The objective of the present study was to investigate the effect of miRNA-101 on the sensitivity of A549 lung cancer cells to etoposide. Methods: The study was conducted during 2018 and 2019 at Arak University of Medical Sciences, Arak, Iran. The effect of miRNA-101 on Mcl-1 expression was assessed using reverse transcription-quantitative polymerase chain reaction 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and trypan blue exclusion assays were performed to determine the effect of treatments on cell survival and proliferation, respectively. The interaction between miRNA-101 and etoposide was evaluated using the combination index analysis of Chou-Talalay. Apoptosis was quantified using ELISA cell death assay. ANOVA and Bonferroni’s tests were used to determine statistical differences between the groups (P<0.05). GraphPad Prism software (version 6.01) was used for data analysis.Results: The results showed that miRNA-101 clearly inhibited the expression of Mcl-1 and reduced the growth of A549 cells, relative to blank control and negative control miRNA (P<0.05). Transfection of miRNA-101 synergistically enhanced the sensitivity of the A549 cells to etoposide. Apoptosis assay data also showed that miRNA-101 triggered apoptosis and augmented the etoposide-mediated apoptosis. Conclusion: Up-regulation of miRNA-101 inhibited cell survival and proliferation, and sensitized A549 cells to etoposide by suppressing Mcl-1 expression. miRNA-101 replacement therapy can be considered as an effective therapeutic strategy in non-small cell lung cancer.

Vitamin K3 (menadione) Induces Cytotoxicity in Chronic Myeloid Leukemia Stem Cells By Upregulating DYRK2 through the Inhibition of SIAH2 Ubiquitin Ligase

Leukemic stem cells (LSCs) generated by the transformation of normal hematopoietic stem cells (HSCs) by BCR-ABL are elusive targets that can initiate and sustain leukemia owing to their unique capacity to regenerate themselves during cell division (self-renewal) and differentiate into a bulk of leukemia cells. Although chronic myeloid leukemia (CML) can be successfully managed using tyrosine kinase inhibitors (TKIs), drug discontinuation trials have shown that 30-60% of patients, depending on the drug used for first- and second-line treatment, can remain in treatment-free remission. Thus, discontinuation is still considered experimental and there are not safety guidelines to stop treatment. A better understanding of self-renewal mechanisms in LSCs will support the development of LSC-specific drugs to induce treatment-free remission in chronic-phase patients by eradicating BCR-ABL-positive LSCs. We recently discovered in mouse models that deletion of the Klf4 gene encoding for the transcription factor KLF4 severely abrogated progression of BCR-ABL(p210)-induced CML by impairing survival and self-renewal of LSCs but not normal hematopoietic stem cells (HSCs). Mechanistically, KLF4 represses the Dyrk2 gene and thus loss-of-KLF4 resulted in elevated DYRK2 protein levels, which was associated with p53-mediated apoptosis and inhibition of self-renewal via depletion of c-Myc protein in LSCs and not in normal HSCs. Here, we report that this mechanism is active in bone marrow cells from chronic-phase CML patients and that stabilization of DYRK2 protein by inhibition of the ubiquitin E3 ligase with vitamin K3 (VK3) has the potential therapeutic approach. Treatment of CML cell lines and bone marrow cells from CML patients with VK3 induced dose-dependent cytotoxicity and apoptosis. This was correlated with increased DYRK2 expression, depletion of c-Myc, and activation of p53-mediated apoptosis. Most importantly, VK3 induced DYRK2 expression and abrogated capacity of purified CD34+ CML stem cells, but not normal CD34+ cells, to generate colonies in methylcellulose assays. Cytotoxicity of VK3 largely depended on the expression of DYRK2, the reduced form menadiol inhibited cell viability similarly to menadione (VK3), and the presence of p53 enhanced cytotoxicity by inducing apoptosis in addition to inhibition of self-renewal. VK3 augmented cytotoxicity of Imatinib in CML cell lines and inhibited cell survival in Imatinib-resistant CML cells. The ability to target CML LSCs was confirmed by in vivo administration of VK3 in BCR-ABL induced mouse model followed by transplantation of leukemic bone marrow in secondary recipient mice. Altogether, our results suggest that stabilization of DYRK2 by inhibition of SIAH2 can induce apoptosis in bulk leukemia cells and LSCs by activating p53 and depleting c-Myc. DisclosuresNo relevant conflicts of interest to declare.

The curcumin analog (PAC) suppressed cell survival and induced apoptosis and autophagy in oral cancer cells

PAC (3,5-Bis (4-hydroxy-3-methoxybenzylidene)-N-methyl-4-piperidone), a novel bioactive curcumin analog, has been reported to have anticancer properties against various tumors. However, the anti-cancer effects of PAC on oral cavity squamous cell carcinoma were not studied yet. Our aim is to investigate the anti-oral cancer properties of PAC in vitro, and determine the molecular mechanisms underlying these effects. Viability assays including MTT and LDH were conducted to measure cell proliferation. Flow cytometry-based cytotoxicity assay was performed to detect autophagic cell death and oxidative stress markers. Western blotting was used for measuring protein expression/activation in apoptotic, autophagic and pro-carcinogenic cellular signaling pathways. We demonstrated that PAC preferentially and, in a dose, -dependent way kills oral cancer cells, but was not toxic to normal human gingival cells. PAC destabilizes cell-cycle distributions, inhibits the expression of oncogenes (cyclin D1) and that of cyclin-dependent kinase inhibitor (p21WAF1) is upregulated, increases the expression of p53 gene, and inhibits epithelial-mesenchymal transition markers in oral cancer cells. The PAC effect involve various signaling pathways including NF-κB, MAPK, Wnt, caspase-3/9 and PARP1. Finally, PAC demonstrated ability to induce autophagy, decrease production of reactive oxygen species, increase intracellular glutathione (GSH) activity, and reduce mitochondrial membrane potential in oral cancer cells. In conclusion, PAC inhibits the proliferation and increases the apoptosis and autophagy and oxidative stress of oral cancer cells. These effects involve ERK1/2, p38/JNK, NF-κB and Wnt cellular signaling pathways. Overall, our study suggests the potential use of PAC to treat oral cancer.

Calcitriol Combined With Platinum-based Chemotherapy Suppresses Growth and Expression of Vascular Endothelial Growth Factor of SKOV-3 Ovarian Cancer Cells.

To evaluate the effects of the combination of calcitriol and chemotherapy and to identify the molecular mechanisms underlying the effect of calcitriol on ovarian cancer cells. SKOV-3 cells were treated with calcitriol and cisplatin, and their effects alone and in combination in a dose-dependent manner were compared. Cell viability, cell proliferation, and apoptosis were assessed using the following assays: PrestoBlue, intracellular adenosine triphosphate, caspase-3/7 activity, annexin V, and immunoblotting, respectively. Calcitriol alone caused dose-dependent inhibition of cell survival and proliferation, and induced apoptotic cell death of SKOV-3 cells. We confirmed that the expression of vitamin D receptor was increased in a dose-dependent manner by calcitriol. Combination treatment using calcitriol at a physiological concentration of 10-100 nM plus cisplatin significantly suppressed cell survival and induced apoptosis. Furthermore, when calcitriol was administered alone, the activity of vascular endothelial growth factor decreased in a dose-dependent manner, and when combined with cisplatin, activity was more suppressed. In SKOV-3 ovarian cancer cells, calcitriol plus cisplatin exerted greater antiproliferative, apoptotic, and anti-angiogenic effects than cisplatin alone. Adding calcitriol to platinum-based chemotherapy might be beneficial to patients with ovarian cancer.

Curcumin-Galactomannoside Complex inhibits the Proliferation of Human Cervical Cancer Cells: Possible Role in Cell Cycle Arrest and Apoptosis.

Background:Cervical cancer is the most common cancer and has the highest morbidity rate of gynaecological malignancies in women worldwide. So, the development of effective anti-cancer agents to treat this condition is vital. Considering the recent interest in free (unconjugated) curcuminoids delivery, the present study investigated the efficacy of a novel food-grade, free-curcuminoids (curcumin-galactomannoside complex; CGM) on cervical cancer cells (HeLa) of human origin. In this study, we examined the anticancer potential of CGM as well as its effects on the cell cycle and the apoptosis of HeLa cancer cell. Methods:Determination of anti-proliferative and apoptosis validation of CGM on HeLa cells was performed by 3-(4,5-Dimethylthiazol-2-yl)-2, 5,-diphenyltetrazolium bromide (MTT), acridine orange/propidium iodide and annexin-V-fluorescein isothiocyanate assays. Measurement of Reactive Oxygen Species (ROS) production, Caspase activities and protein expression experiments were performed to investigate the potential mechanisms of action in the apoptotic process. Results:The cytotoxic assays revealed that the CGM showed inhibition of cell survival and exhibited high cytotoxic activity against HeLa cells at 25 μg/mL. Further studies on morphological changes were done in CGM-treated cervical cancer cells contributing to apoptosis. Flow cytometry analysis with Annexin V-FITC and PI staining precisely indicated that CGM induced apoptosis in HeLa cell lines at 25 μg/mL. By the supplementation of CGM showed an increase in Bax and cleaved caspase-8 protein in HeLa cells after 48 h exposure. Conclusion:The evidence obtained from this study suggests that CGM is a potent and promising natural formulation against cervical cancer cells via induction of apoptosis through ROS mediated mitochondrial damage in HeLa cells. Hence, CGM could be further explored as a potential lead in treating cancer.

Melatonin: A regulator of the interplay between FoxO1, miR96, and miR215 signaling to diminish the growth, survival, and metastasis of murine adenocarcinoma.

Melatonin (Mel.), also known as the magic hormone, is a nocturnally secreted hormone orchestrates the clearance of free radicals that have been built up and cumulated during day. This study aims to detect the impact of pineal gland removal on the incidence of tumor development and to assess the signaling pathways via which exogenous melatonin counteract cancer growth. This goal has been achieved by novel approach for pineal destruction using dental micromotor which validated by melatonin downregulation in blood plasma. Mice were injected sub-cutenously with Ehrlich cells to develop solid tumor as a murine model of breast cancer. The increase at tumor markers carcino embryonic antigen, TNFα, and nuclear factor kappa-light-chain-enhancer of activated B cells was over countered by exogenous melatonin supplementation (20 mg/kg) daily for 1month. The anticancer effects of melatonin were significantly mediated by scavenging H2 O2 and NO and diminishing of lipid peroxidation marker malondialdehyde. The real-time polymerase chain Rx analyses indicated a significant effect of Melatonin in upregulating the expression of miR215, fork head box protein O1 (foxO1), and downregulation of miR96. Flowcytometric analyses indicated a significant effect of melatonin on induction of cell cycle arrest at G1 phase which was further confirmed by Ki67 downregulation. Immunohistochemical analyses indicated the role of melatonin in upregulating P53-dependent apoptosis and downregulating CD44 signaling for survivin, matrix metallo-protein kinase 2, and vascular endothelial growth factor to inhibit cell survival and metastasis. In conclusion, this study sheds the light on M./P53/miR215/CD44 with an emphasis on M./miR96//foxO1 signaling cascades, as a novel pathway of melatonin signaling in adenocarcinoma to diminish cancer cell growth, survival and metastasis.

Novel Primary Human Cancer Stem-Like Cell Populations from Non-Small Cell Lung Cancer: Inhibition of Cell Survival by Targeting NF-κB and MYC Signaling.

There is growing evidence that cancer stem cells (CSCs), a small subpopulation of self-renewal cancer cells, are responsible for tumor growth, treatment resistance, and cancer relapse and are thus of enormous clinical interest. Here, we aimed to isolate new CSC-like cells derived from human primary non-small cell lung cancer (NSCLC) specimens and to analyze the influence of different inhibitors of NF-κB and MYC signaling on cell survival. CSC-like cells were established from three squamous cell carcinomas (SCC) and three adenocarcinomas (AC) of the lung and were shown to express common CSC markers such as Prominin-1, CD44-antigen, and Nestin. Further, cells gave rise to spherical cancer organoids. Inhibition of MYC and NF-κB signaling using KJ-Pyr-9, dexamethasone, and pyrrolidinedithiocarbamate resulted in significant reductions in cell survival for SCC- and AC-derived cells. However, inhibition of the protein–protein interaction of MYC/NMYC proto-oncogenes with Myc-associated factor X (MAX) using KJ-Pyr-9 revealed the most promising survival-decreasing effects. Next to the establishment of six novel in vitro models for studying NSCLC-derived CSC-like populations, the presented investigations might provide new insights into potential novel therapies targeting NF-κB/MYC to improve clinical outcomes in NSCLC patients. Nevertheless, the full picture of downstream signaling still remains elusive.

Gossypol decreased cell viability and down-regulated the expression of a number of genes in human colon cancer cells

Plant polyphenol gossypol has anticancer activities. This may increase cottonseed value by using gossypol as a health intervention agent. It is necessary to understand its molecular mechanisms before human consumption. The aim was to uncover the effects of gossypol on cell viability and gene expression in cancer cells. In this study, human colon cancer cells (COLO 225) were treated with gossypol. MTT assay showed significant inhibitory effect under high concentration and longtime treatment. We analyzed the expression of 55 genes at the mRNA level in the cells; many of them are regulated by gossypol or ZFP36/TTP in cancer cells. BCL2 mRNA was the most stable among the 55 mRNAs analyzed in human colon cancer cells. GAPDH and RPL32 mRNAs were not good qPCR references for the colon cancer cells. Gossypol decreased the mRNA levels of DGAT, GLUT, TTP, IL families and a number of previously reported genes. In particular, gossypol suppressed the expression of genes coding for CLAUDIN1, ELK1, FAS, GAPDH, IL2, IL8 and ZFAND5 mRNAs, but enhanced the expression of the gene coding for GLUT3 mRNA. The results showed that gossypol inhibited cell survival with decreased expression of a number of genes in the colon cancer cells.

Cache Domain Containing 1 Is a Novel Marker of Non-Alcoholic Steatohepatitis-Associated Hepatocarcinogenesis.

Simple SummaryThe aim of the present study was to discover novel early molecular biomarkers of liver neoplasms which arise in non-alcoholic steatohepatitis (NASH) Stelic Animal Model (STAM) mice. Significant increase of lipid deposits, hepatocyte ballooning, fibrosis, and incidences and multiplicities of hepatocellular adenomas and carcinomas were detected in the livers of 18-week-old STAM mice. From the results of proteome analysis of STAM mice hepatocellular carcinomas, significant elevation of a novel protein, cache domain-containing 1 (CACHD1) was found. Furthermore, we observed CACHD1-positive foci in STAM mice livers, which number, area, and cell proliferation index within the foci were significantly elevated. Results of immunohistochemical and in vitro functional analysis indicated that CACHD1 may become a useful early biomarker and potential molecular target in NASH-associated hepatocarcinogenesis, which is involved in control of cell proliferation, autophagy and apoptosis.In the present study, potential molecular biomarkers of NASH hepatocarcinogenesis were investigated using the STAM mice NASH model, characterized by impaired insulin secretion and development of insulin resistance. In this model, 2-days-old C57BL/6N mice were subjected to a single subcutaneous (s.c.) injection of 200 μg streptozotocin (STZ) to induce diabetes mellitus (DM). Four weeks later, mice were administered high-fat diet (HFD) HFD-60 for 14 weeks (STAM group), or fed control diet (STZ group). Eighteen-week-old mice were euthanized to allow macroscopic, microscopic, histopathological, immunohistochemical and proteome analyses. The administration of HFD to STZ-treated mice induced significant fat accumulation and fibrosis development in the liver, which progressed to NASH, and rise of hepatocellular adenomas (HCAs) and carcinomas (HCCs). In 18-week-old animals, a significant increase in the incidence and multiplicity of HCAs and HCCs was found. On the basis of results of proteome analysis of STAM mice HCCs, a novel highly elevated protein in HCCs, cache domain-containing 1 (CACHD1), was chosen as a potential NASH-HCC biomarker candidate. Immunohistochemical assessment demonstrated that STAM mice liver basophilic, eosinophilic and mixed-type altered foci, HCAs and HCCs were strongly positive for CACHD1. The number and area of CACHD1-positive foci, and cell proliferation index in the area of foci in mice of the STAM group were significantly increased compared to that of STZ group. In vitro siRNA knockdown of CACHD1 in human Huh7 and HepG2 liver cancer cell lines resulted in significant inhibition of cell survival and proliferation. Analysis of the proteome of knockdown cells indicated that apoptosis and autophagy processes could be activated. From these results, CACHD1 is an early NASH-associated biomarker of liver preneoplastic and neoplastic lesions, and a potential target protein in DM/NASH-associated hepatocarcinogenesis.

Constitutive GLI1 expression in chondrosarcoma is regulated by major vault protein via mTOR/S6K1 signaling cascade

Hedgehog signaling plays a pivotal role in embryonic pattern formation and diverse aspects of the postnatal biological process. Perturbation of the hedgehog pathway and overexpression of GLI1, a downstream transcription factor in the hedgehog pathway, are highly relevant to several malignancies including chondrosarcoma (CS). We previously found that knocking down expression of GLI1 attenuates the disrupted Indian hedgehog (IHH) signal pathway and suppresses cell survival in human CS cells. However, the underlying mechanisms regulating the expression of GLI1 are still unknown. Here, we demonstrated the implication of GLI1 in SMO-independent pathways in CS cells. A GLI1 binding protein, major vault protein (MVP), was identified using the affinity purification method. MVP promoted the nuclear transport and stabilization of GLI1 by compromising the binding affinity of GLI1 with suppressor of fused homolog (SUFU) and increased GLI1 expression via mTOR/S6K1 signaling cascade. Functionally, knockdown of MVP suppressed cell growth and induced apoptosis. Simultaneous inhibition of MVP and GLI1 strongly inhibits the growth of CS in vitro and in vivo. Moreover, IHC results showed that MVP, GLI1, and P-p70S6K1 were highly expressed and positively correlated with each other in 71 human CS tissues. Overall, our findings revealed a novel regulating mechanism for HH-independent GLI1 expression and provide a rationale for combination therapy in patients with advanced CS.