Expression Of Apoptosis Regulators Research Articles

Abstract A05: Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies

Abstract Targeted therapies(TT) combining FLT3 inhibitors with hypomethylating agents (HMA) and Venetoclax (Ven) are highly effective against FLT3-ITD acute myeloid leukemia (AML) with high response rates. Signaling mutations along with emergence of rarer mutations are the key contributors to primary and secondary resistance to TT. We hypothesize that resistant leukemia clones have unique proteomic profiles that facilitate survival under therapy pressure(TP) and deciphering proteomic profiles at the single-cell level will delineate resistance mechanisms and adaptive responses driven by TP. We performed multiplexed single-cell proteomic analysis of serial PB and BM samples (n:162) from patients treated with FLT3i+HMA+/-VEN using CyTOF. Unsupervised analysis identified leukemia cells and non-malignant cellular elements of the leukemia compartment in an unbiased manner. Next, we interrogated the leukemia proteomic landscape to identify leukemia associated proteomic features. Since Ven targets BCL2 and FLT3i modulates the expression of anti-apoptotic molecules we assessed the expression of apoptosis regulators and found that leukemia cells with immature phenotype, including those with leukemia stem cell(LSC) phenotype, almost always expressed moderate-high levels of BCL2. Monocytic cells(MC) lacked BCL2 expression and had the highest levels of MCL1. Despite expressing moderate levels of MCL1 and BCL-XL, TT was effective in substantially eliminating CD34+ immature leukemia cells. We observed a relative enrichment of MCs, either benign or malignant, after TT. Since cells with MC phenotype were inherently resistant to TT we hypothesized that leukemia cell subsets having similar proteomic profiles to MCs will persist after TT. Phenotypic interrogation of leukemia cells (UMAP) revealed that LSCs generally clustered on the opposite pole distant from MCs and leukemia cells on poles facing MCs were more differentiated. Remarkably, FLT3-ITD mutation partners differentially altered the proteomic landscape. NPM1 mutant FLT3-ITD AML cells displayed a less diverse leukemia architectural organization with differentiation block. Contrarily, signaling mutations diversified the leukemia landscape into a diverse continuum of differentiation states. We also observed that RAS/MAPK mutations could overcome differentiation block and gave rise to a differentiation continuum encompassing less-differentiated, transitional and differentiated leukemia cells. The transitional CD34+ leukemia cells, mapped in close vicinity to MCs, and differentiated MCs that preferentially persisted at D28, had active signaling pathways and expressed CD36. The presence of CD34+ leukemia cells with persistent signaling at D28 was indicative of poor clinical outcomes. Conclusion: Multiplexed single-cell proteomic analysis identified a unique mode of resistance and proteomic features of surviving cells in with FLT3-ITD AML patients treated with TT, and elucidated how different mutation partners in FLT3-ITD AML differentially altered proteomic landscape to drive resistance to TT. Citation Format: Muharrem Muftuoglu, Musa Yilmaz, Mahesh Basyal, Li Li, Naval Daver, Michael Andreeff. Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A05.

In vitroSodium Fluoride Treatment Significantly Affects Apoptosis and Proliferation in the Liver of Embryonic Chickens

Sodium fluoride (NaF), although helpful in preventing dental decay, may negatively affect the body. The aim of this study was to examine the effects of a 6-hin vitrotreatment of livers isolated from 14-day-old chicken embryos with NaF at doses of 1.7 (D1), 3.5 (D2), 7.1 (D3) and 14.2 mM (D4), with regard to apoptosis, cell proliferation and tissue structure. The mRNA expression of the apoptosis regulatorsCYCS,APAF1,BCL2,CASP3,CASP9andTMBIM1was analysed by the qPCR method. Apoptotic cells were detected by a TUNEL assay. The tissue and DNA structure were also analysed by histological staining (H&E, Feulgen). The number of proliferating cells was determined and the apoptosis regulatory proteins were localised by the immunohistochemical staining of PCNA, CASP3 and APAF1. The results showed that the mRNA expression ofCYCS,BCL2,CASP3,CASP9andAPAF1increased significantly in the D1 group, as did that ofCASP9in the D3 group and ofBCL2andAPAF1in the D4 group. The number of apoptotic cells increased significantly in the D4 group, where they increased from 18% to 49%. On the other hand, the number of proliferating cells decreased gradually, in a dose-dependent manner, from 84% in the control group to 5.5% in the D4 group. The expression of apoptosis-regulating factors also increased: in the D3 and D4 groups, the CASP3 immunopositive reaction was more intensive in single cells in the embryonic livers, whereas that of APAF1 increased in the hepatocytes as well as in the hepatic blood vessel walls. The mechanism of the effect of NaF on apoptosis in the embryonic liver is very complex. In the groups exposed to higher doses of NaF, apoptosis was significantly stimulated, while proliferation was inhibited and the tissue structure was damaged. The expression of apoptosis regulators at the mRNA and protein levels increased, but the mRNA expression did not depend on the NaF dose. These results reveal that NaF, by changing the balance between apoptosis and the proliferation of hepatocytes, may disturb the development and function of the liver in embryonic chickens. Therefore, the risk of exposure to NaF should be considered when determining the standards for human and animal exposure to this compound.

Single-Cell Proteomic Interrogation of FLT3-ITD Leukemia Compartment Revealsdistinct Leukemia Cell Populations Resistant to Quizartinib, Decitabine and Venetoclax

Triplet therapy (TT) combining Quizartinib(Quiz), Decitabine(Dec) and Venetoclax(Ven) is highly effective against FLT3-ITD acute myeloid leukemia (AML) with high response rates (Yilmaz M, ASCO 2022).Signaling mutations, particularly those involving RAS/MAPK pathway, have emerged as key contributors to primary and secondary resistance to TT. Lack or loss of response to TT is not limited to presence or emergence of clones with signaling mutations and disease relapse occurs in FLT3-ITD AML patients with a variety of other mutations. We hypothesize that resistant leukemia clones have unique proteomic profiles that facilitate survival under therapy pressure and deciphering proteomic profiles at the single-cell level will delineate resistance mechanisms and adaptive responses driven by therapy pressure, and aid in identifying proteomic correlates of response and therapeutic vulnerabilities. To this end, we performed comprehensive single-cell proteomic analysis of serially collected samples (n:162) from patients treated with TT (n:14) and doublet (n:10, DEC+Quiz) therapy, using CyTOF and interrogated leukemia phenotypic profiles, apoptotic networks, signaling pathways, differentiation states and trajectories. First, we performed unsupervised analyses to identify leukemia cells and non-malignant cellular elements of the leukemia compartment in an unbiased manner, interrogated the leukemia proteomic landscape to identify leukemia associated proteomic features that may correlate with clinical response and assessed whether distinct mutation profiles are associated with specific proteomic profiles that may confer resistance to therapy. Since Ven targets BCL2 and FLT3i modulates the expression of anti-apoptotic molecules we assessed the expression of apoptosis regulators across leukemia landscapes and found that leukemia cells with immature phenotype, including those with leukemia stem cell(LSC) phenotype, almost always expressed moderate-high levels of BCL2. Monocytic cells(MC), generally situated in close proximity to leukemia cells in UMAPs, lacked BCL2 expression and had the highest levels of MCL1. Despite expressing moderate levels of MCL1 and BCL-XL, TT was effective in substantially eliminating CD34+ immature leukemia cells (Figure 1). Due to their contrasting apoptosis regulatory networks, we observed a relative enrichment of MCs, either benign or malignant, after TT. Since cells with MC phenotype were inherently resistant to TT we hypothesized that leukemia cell subsets having similar proteomic profiles to MCs will persist after TT. Of note, leukemia cell compartments harbor distinct differentiation hierarchies, the frequencies of which varied across patients. Phenotypic interrogation of leukemia cells (UMAP) revealed that LSCs generally clustered on the opposite pole distant from MCs and leukemia cells on poles facing MCs were more differentiated. Remarkably, FLT3-ITD mutation partners differentially altered and reshaped the proteomic landscape. NPM1 mutant FLT3-ITD AML cells displayed a less diverse leukemia architectural organization with differentiation block. Contrarily, signaling mutation diversified the leukemia landscape into a diverse continuum of differentiation states. Remarkably, we observed that RAS/MAPK mutations in FLT3-ITD AML overcame differentiation block and gave rise to a differentiation continuum encompassing less-differentiated, transitional and differentiated leukemia cells(Figure1). Importantly, we observed that transitional and CD34+ leukemia cells, mapped in close vicinity to MCs, and differentiated MCs preferentially persisted at D28, had active signaling pathways and expressed CD36, which indicates distinct metabolic programs could ensure survival under therapy pressure and also had lower levels of BCL2.The presence of CD34+ leukemia cells at D28, adapted under therapy pressure with persistent signaling at D28, both in CD34+ leukemia or MCs, was indicative of poor clinical outcomes. Conclusion: Single-cell proteomic assessment of FLT3-ITD AML treated with Quiz/Dec/Ven (TT) identifies a unique mode of resistance and proteomic features of surviving cells at D28, and elucidates how RAS/MAPK mutations diversify the leukemia proteomic landscape and modulate the architectural organization which eventually gives rise to emerging clones resistant to therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P344: THE DUAL BCL-2 AND BCL-XL INHIBITOR AZD4320 SHOWS ON-TARGET ACTIVITY IN ALL AND ACTS SYNERGISTICALLY WITH MCL-1 INHIBITION

Background: Targeting anti-apoptotic BCL-2 family proteins by BH3-mimetics has become a promising treatment strategy in acute lymphoblastic leukemia (ALL). Heterogeneous activity of the selective BCL-2 inhibitor venetoclax has been observed, but other anti-apoptotic proteins including BCL-XL promote venetoclax insensitivity. In clinical trials, targeting BCL-XL has previously resulted in a dose-limiting decrease of platelets. AZD4320 was developed as a dual inhibitor of BCL-2 and BCL-XL, and its dendrimer conjugate (AZD0466) was recently reported to demonstrate anti-tumor activity in hematological cancer models, while showing only a transient thrombocytopenia. Aims: In this study, the anti-leukemia activities of the dual BCL-2 and BCL-XL inhibitor AZD4320 and of MCL-1-selective AZD5991 were evaluated and compared to the effects of other BH3-mimetics (BCL-2-selective venetoclax, BCL-XL-selective A-1331852 and MCL-1-selective S63845). The on-target activity of the inhibitors was functionally characterized and combination effects were analyzed. Methods: Cell viability assays were performed in ALL cell lines and patient-derived xenograft (PDX) samples analyzing half maximal effective concentrations (EC50). Protein complexes and expression of apoptosis regulators were analyzed by immunoprecipitation and immunoblotting. Dynamic BH3 profiling using synthetic BH3-peptides was performed to determine the dependency of ALL cells on BCL-2 family proteins. Combination effects were assessed by dose-response matrix analyses. Results: First, we determined the efficacy of the dual BCL-2 and BCL-XL inhibitor AZD4320 and of the MCL-1 inhibitor AZD5991 for cell death induction in seven B-cell precursor (BCP) ALL cell lines and in a series of 13 PDX samples, showing heterogeneous responses. Interestingly, sensitivities of individual samples to both compounds were not associated with each other. However, we found a significant correlation of sensitivity to the dual inhibitor (AZD4320) with BCL-2 inhibition (venetoclax; N=13; rs=0.56; p=0.049), but no association with BCL-XL inhibition (A-1331852). Analyzing activities of all five BH3-mimetics including venetoclax, we found lowest EC50 values for AZD4320, indicating particular sensitivity for this dual inhibitor as compared to the single inhibitors (p<0.001). Investigating dependencies of ALL cells on BCL-2 family proteins by dynamic BH3 profiling, we found a shift towards MCL-1-dependence upon exposure to AZD4320, while AZD5991 induced an increased combined dependence on BCL-2 and BCL-XL, indicating potential synergistic activity of triple inhibition. Using co-immunoprecipitation analyses, we found that the exposure of ALL cells to AZD4320 reduced binding of both BCL-2 and BCL-XL to BIM, confirming on-target activity. Moreover, AZD5991 reduced binding of BIM to MCL-1. Accordingly, combined treatment with both inhibitors results in the release of BIM and downstream apoptosis signaling. Assessing combinatorial treatment in a primary PDX sample using multi-dose matrix analyses of both inhibitors revealed a positive mean Bliss synergy score of +4.8 indicating synergistic activity. Importantly, the highest synergism was found at low concentrations of both inhibitors, suggesting efficacy at moderate concentrations, which could potentially be achieved in vivo. Summary/Conclusion: In summary, our study demonstrates sensitivity, on-target activity and synergism of the dual BCL-2 and BCL-XL inhibitor AZD4320 with inhibition of MCL-1, thereby providing strong evidence for further clinical evaluation in ALL.

Role of AMPK/mTOR-independent autophagy in clear cell renal cell carcinoma

We examined the status and role of autophagy, a process of lysosomal recycling of cellular material, in clear cell renal cell carcinoma (ccRCC). Paired samples of tumor and adjacent non-malignant...

Natural product-derived icaritin exerts anti-glioblastoma effects by positively modulating estrogen receptor β.

Glioblastoma is the most common malignancy of the central nervous system, and patients typically have a poor prognosis. Previous studies indicate a gender bias in the development of glioblastoma; women are at a lower risk compared with men, suggesting that estrogen may confer protective effects. Icaritin, a prenylflavonoid derivative from a Chinese herb of the Epimedium genus, selectively regulates the estrogen receptor (ER) and possesses anti-cancer properties. The aim of the present study was to investigate the protective effects of icaritin on glioblastoma and its underlying mechanisms, with a particular focus on its association with the ER. The results demonstrated that icaritin inhibited the growth of C6 and U87-MG glioblastoma cells in a dose- and time-dependent manner. At a concentration of 12.5 µM, icaritin induced apoptosis, which was characterized by the increased expression of the cleaved forms of caspases 3, 7, 8 and 9 and poly (ADP-ribose) polymerase, downregulation of BCL2 apoptosis regulator and upregulation of BCL2-associated X, apoptosis regulator expression. Additionally, icaritin inhibited the migration of C6 and U87-MG cells. The protein expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 were also downregulated following icaritin treatment. Furthermore, icaritin treatment increased the expression of estrogen receptor (ER)β and the phosphatase and tensin (PTEN) homolog oncoprotein, thus reducing the expression of downstream targets of PTEN; protein kinase B (Akt) and phosphorylated Akt. Subsequent experiments demonstrated that icaritin cooperates with 17β-estradiol to inhibit the growth of glioblastoma cells, and the inhibition of ERβ with the ERβ-specific antagonist ICI 182,780, attenuated the anti-glioblastoma effects of icaritin. In conclusion, the results of the present study demonstrate that the anti-glioblastoma effects of icaritin may be mediated by its modulation of ERβ.

Cellular Composition and Expression of Cell Proliferation and Apoptosis Markers in Bone Marrow Cells after Heterotransplantation.

The effects of heterotransplantation on bone marrow structures were studied in experimental male mice. Suspension of the bone marrow from feline femur (1 ml bone marrow diluted in isotonic saline, 1:2) was injected into the caudal vein. Heterotransplantation led to an increase in the level of serotonin involved in cell differentiation. In 2 days, the counts of all cells except erythroid cells decreased. Immunohistochemical analysis of Ki-67 marker within 40 min after transplantation showed a significant increase of cell proliferative activity in comparison with that in intact mice. At later terms, the expression of Ki-67 in bone marrow cells decreased, while the expression of apoptosis regulator P-53 increased, which was associated with the presence of numerous dead cells.

Tyrosine kinase inhibitor imatinib modulates the viability and apoptosis of castrate-resistant prostate cancer cells dependently on the glycolytic environment

AimsThe tyrosine kinase inhibitor imatinib has been used in prostate cancer treatment with outcomes that did not follow the in vitro findings. The glycolytic environment has been shown to influence the efficacy of anti-cancer drugs. This study aimed to evaluate the effect of imatinib on cell viability, apoptosis, and metabolism in cell line models of castrate-resistant prostate cancer (CRPC) under hyperglycemic and hypoglycemic conditions. Main methodsDU145 and PC3 CRPC cell lines were exposed to 20 μM imatinib under 5 mM (hypoglycemia) or 30 mM glucose (hyperglycemia) for 48–72 h. Cell viability was assessed by the MTS assay. The expression of apoptosis regulators and glycolytic metabolism-related proteins was analysed by Western blot, and the activity of caspase-3 and lactate dehydrogenase (LDH) was determined spectrophotometrically. Glucose consumption and lactate production were determined using biochemical assays. Key findingsImatinib decreased CRPC cells viability, whereas increasing apoptosis; effects only observed in hyperglycemic conditions. Glucose consumption and lactate production were significantly increased in imatinib-treated DU145 and PC3 cells, and independently of glucose availability. Accordingly, LDH expression and activity were significantly increased in response to imatinib. SignificanceHigher glucose availability improved the effectiveness of imatinib suppressing survival and growth of CRPC cells. It was also shown that imatinib treatment stimulated the glycolytic metabolism of CRPC cells. This study first demonstrated that a glucose-enriched environment intensifies the effect of imatinib, which stimulates the interest for testing this compound into the clinical setting, namely in hyperglycemia conditions (diabetic patients) or in co-administration with inhibitors of glycolytic metabolism.

MiR-142-5p suppresses proliferation and promotes apoptosis of human osteosarcoma cell line, HOS, by targeting PLA2G16 through the ERK1/2 signaling pathway.

Previous studies have revealed that miR-142-5p serves a critical role in human cancer progression. However, the biological function of miR-142-5p in osteosarcoma (OS) development remains unclear. In the present study, the role of miR-142-5p in human OS HOS cells was determined, and the underlying mechanism involved was examined. Compared with the adjacent healthy tissues, the expression level of miR-142-5p was downregulated and the expression level of group XVI phospholipase A2 (PLA2G16) protein was upregulated in human OS tissues. The aforementioned results were also indicated in human OS HOS cells when compared with human fetal osteoblastic hFOB1.19 cells. Additionally, the results demonstrated that PLA2G16 was a direct target of miR-142-5p. miR-142-5p transfection upregulated the expression level of miR-142-5p and suppressed the expression level of PLA2G16 protein in HOS cells. MTT assays indicated a time-dependent decrease by miR-142-5p transfection in the proliferation of HOS cells. 5-bromo-2'-deoxyuridine incorporation assays confirmed that miR-142-5p transfection inhibited DNA synthesis in HOS cells. In addition, miR-142-5p transfection increased the Caspase-3 (CASP3) activity and apoptotic rate. Western blot analysis indicated that miR-142-5p transfection reduced BCL2, apoptosis regulator expression and upregulated the expression of CASP3 and BCL2 associated X, apoptosis regulator in HOS cells. Furthermore, miR-142-5p transfection decreased the expression levels of phosphorylated (p)-proto-oncogene, serine/threonine kinase, p-mitogen-activated protein kinase kinase, and p-extracellular signal-regulated kinase (ERK) 1/2 proteins in HOS cells. PLA2G16 overexpression restored the expression level of p-ERK 1/2 protein, which was reduced by miR-142-5p overexpression. MTT and CASP3 activity assays indicated that restoration of PLA2G16 reversed the tumour-suppressive role of miR-142-5p transfection in HOS cells. In conclusion, the results of the present study indicated that miR-142-5p suppressed proliferation and promoted apoptosis in human OS HOS cells by targeting PLA2G16 through ERK1/2 signaling pathway.

Hydrogen‑rich solution against myocardial injury and aquaporin expression via the PI3K/Akt signaling pathway during cardiopulmonary bypass in rats.

Myocardial ischemia, hypoxia and reperfusion injury are induced by aortic occlusion, cardiac arrest and resuscitation during cardiopulmonary bypass (CPB), which can severely affect cardiac function. The aim of the present study was to investigate the effects of hydrogen-rich solution (HRS) and aquaporin (AQP) on cardiopulmonary bypass (CPB)-induced myocardial injury, and determine the mechanism of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Sprague Dawley rats were divided into a sham operation group, a CPB surgery group and a HRS group. A CPB model was established, and the hemodynamic parameters were determined at the termination of CPB. The myocardial tissues were observed by hematoxylin and eosin, and Masson staining. The levels of myocardial injury markers [adult cardiac troponin I (cTnI), lactate dehydrogenase (LDH), creatine kinase MB (CK-MB) and brain natriuretic peptide (BNP)], inflammatory factors [interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α)] and oxidative stress indicators [superoxide dismutase (SOD), malondialdehyde (MDA) and myeloperoxidase (MPO)] were determined by ELISA. Furthermore, H9C2 cells were treated with HRS following hypoxia/reoxygenation. Cell viability and cell apoptosis were investigated. The expression of apoptosis regulator Bcl-2 (Bcl-2), apoptosis regulator Bax (Bax), caspase 3, AQP-1, AQP-4, phosphorylated (p)-Akt, heme oxygenase 1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) were investigated using western blotting and quantitative-polymerase chain reaction of tissues and cells. Following CPB, myocardial cell arrangement was disordered, myocardial injury markers (cTnI, LDH, CK-MB and BNP), inflammatory cytokines (IL-1β, IL-6 and TNF-α) and MDA levels were significantly increased compared with the sham group; whereas the SOD levels were significantly downregulated following CPB compared with the sham group. HRS attenuated myocardial injury, reduced the expression levels of cTnI, LDH, CK-MB, BNP, IL-1β, IL-6, TNF-α, MDA and MPO, and increased SOD release. Levels of Bcl-2, AQP-1, AQP-4, p-Akt, HO-1 and Nrf2 were significantly increased following HRS; whereas Bax and caspase-3 expression levels were significantly reduced following CPB. HRS treatment significantly increased the viability of myocardial cells, reduced the rate of myocardial cell apoptosis and the release of MDA and LDH compared with the CPB group. A PI3K inhibitor (LY294002) was revealed to reverse the protective effect of HRS treatment. HRS was demonstrated to attenuate CPB-induced myocardial injury, suppress AQP-1 and AQP-4 expression following CPB treatment and protect myocardial cells via the PI3K/Akt signaling pathway.

Paclitaxel suppresses proliferation and induces apoptosis through regulation of ROS and the AKT/MAPK signaling pathway in canine mammary gland tumor cells.

Paclitaxel is a diterpenoid compound, derived from the pacific yew (Taxus brevifolia) berry, which exhibits antineoplastic effects against various types of cancer. However, the antitumor effects and the molecular mechanisms of paclitaxel on canine CHMm cells remain to be elucidated. The aim of the present study was to investigate the antitumor effects of paclitaxel on CHMm cells and identify relevant signal transduction pathways modulated by paclitaxel using multiple methods including MTT assay, flow cytometry, acridine orange/ethidium bromide staining, transmission electron microscopy, determination of cellular reactive oxygen species (ROS), superoxide dismutase (SOD) and malondiadehyde (MDA) and western blotting, the data indicated that paclitaxel decreased cell viability, induced G2/M-phase cell cycle arrest, suppressed the expression of cyclin B1 and induced apoptosis in a dose-dependent manner. In addition, paclitaxel upregulated the expression of Bax and cytochrome c, but reduced expression of apoptosis regulator Bcl-2, resulting in activation of caspase-3, chromatin condensation, karyopyknosis, intracellular vacuolization, increased production of ROS and MDA, and decreased activity of SOD. However, these effects were inhibited when CHMm cells were treated with N-acetyl-L-cysteine. Furthermore, treatment with paclitaxel inhibited the level of of phospho (p)-RAC-α serine/threonine-protein kinase (AKT) and p-ribosomal protein S6 kinase proteins, and promoted phosphorylation of P38 mitogen-activated protein kinase (MAPK) and p-90 kDa ribosomal protein S6 kinase 1 proteins in CHMm cells. It was observed that paclitaxel in combination with pharmacological inhibitors of the P38 and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling pathways (SB203580 and LY294002, respectively) exerted synergistic inhibitory effects on the proliferation of the CHMm cells. The results of the present study demonstrated that paclitaxel inhibited tumor cell proliferation by increasing intrinsic apoptosis through inhibition of the PI3K/AKT signaling pathway and activation of MAPK signaling pathway in CHMm cells.

Expression of Bcl-2 Family Proteins in the Ovarian Follicular Apparatus in the Acute Period after Experimental Hyperthermia.

The expression of apoptosis regulators (proapoptotic protein Bad and anti-apoptotic protein Bcl-2) was analyzed and Bcl-2/Bad ratio in the follicular apparatus of the rat ovary was determined on day 3 after hyperthermia (rectal temperature 43.5°C). Hyperthermia in the catabolic phase leads to different degrees of activation of the molecular "switches" of apoptosis in cells of ovarian follicular epithelium. This was seen from increased intensity of immunohistochemical staining for Bad protein against the background of more pronounced expression of Bcl-2 protein. On day 3 after exposure to hyperthermia, Bcl-2/Bad ratio increased, which reflects antiapoptotic protection of cells and conditions for blockade of mitochondrial pathway of apoptosis in the follicular apparatus of the ovaries during the acute period after hyperthermia.

MicroRNA-124 suppresses growth and aggressiveness of osteosarcoma and inhibits TGF-β-mediated AKT/GSK-3β/SNAIL-1 signaling.

Osteosarcoma is one of the most common malignant tumors in adolescent populations and the prognosis remains incompletely understand. Previous reports have demonstrated that microRNA‑124 (miR‑124) has inhibitory effects on various human malignancies and is associated with tumor progression. However, the clinical significance and potential mechanisms of miR‑124 in the progression of osteosarcoma is not clearly understood. In this study, the potential molecular mechanism of miR‑124 in osteosarcoma tumorigenesis, growth and aggressiveness was investigated. The growth, proliferation, apoptosis, migration and invasion of osteosarcoma cells were investigated following miR‑124 transfection were determined by colony formation assay, western blotting, immunofluorescence, migration/invasion assays and reverse transcription‑quantitative polymerase chain reaction. Invivo anti‑cancer effects of miR‑124 were analyzed by a tumor growth assay, immunohistochemistry and survival rate observations. The results demonstrated that miR‑124 transfection significantly decreased integrin expression in osteosarcoma cells, and further inhibited growth, proliferation, migration and invasion of osteosarcoma cells. Flow cytometry assays indicated that miR‑124 transfection attenuated apoptosis resistance of osteosarcoma to tunicamycin, potentially via the downregulation of P53 and Bcl‑2 apoptosis regulator expression. Mechanistic assays demonstrated that miR‑124 transfection suppressed TGF‑β expression in osteosarcoma. An animal study revealed that tumor growth was reduced in tumor cells transfected with miR‑124 compared with control cells, and the survival rate was prolonged in mice with miR‑124 transfected xenografts compared with control tumors. In conclusion, these results indicate that miR‑124 transection inhibits the growth and aggressive of osteosarcoma, potentially via suppression of TGF‑β‑mediated AKT/GSK‑3β/snail family transcriptional repressor 1 (SNAIL‑1) signaling, suggesting miR‑124 may be a potential anti‑cancer agent/target for osteosarcoma therapy.

Molecular Grading of Oral Squamous Cell Carcinomas Infected with EBV

BackgroundSquamous Cell Carcinoma (SCC) is a type of cancer that is often found in oral cavity and areas of the head and neck. Viruses are major etiological factors through production of factors that can disturb proliferation and apoptosis regulators such as p53i, c-myc and bcl-2. This study aimed to determine the molecular grading of oral squamous cell carcinoma (OSCCs) infected with the Epstein-Barr Virus (EBV).MethodsTwenty-seven OSCC patients underwent biopsy to detect EBV infection through in situ hybridization for RNA EBV (EBER) and immunohistochemical analysis of latent membrane protein-1 (LMP-1) and EBV nuclear antigen-1 (EBNA-1). To assess molecular grades, cell proliferation and apoptosis regulator expression i.e. inactive p53 (p53i), c-myc and bcl-2, were immunohistochemically analysed.ResultsThe cases were divided into two groups; infected and non-infected by EBV. Regression analysis showed that only EBNA-1 expression could affect p53i expression. Based on regression equations molecular grading of OSCCs infected by EBV was divided into three: Grade I (low), EBNA-1 expression was 7.60, and p53i expression was 9.74-17.5; Grade II (medium), EBNA-1 expression was 7.61-19.7, and p53i 17.5-30.1; Grade III (high), EBNA-1 expression was 19.71, and p53i ≥ 30.1.ConclusionIn OSCC infected with EBV, only EBNA-1 expression can influence p53i expression.

Cyanidin ameliorates cisplatin-induced cardiotoxicity via inhibition of ROS-mediated apoptosis.

Oxidative stress and apoptosis serve an essential role in cisplatin-induced cardiotoxicity, which limits its clinical use, and increases the risk of cardiovascular disease. As a natural drug, the antioxidant and antitumor effects of cyanidin have been recognized, but its protective effect on cisplatin-induced cardiomyocyte cytotoxicity remains unclear. H9c2 cells were treated with cisplatin (1–40 µM) in the presence or absence of cyanidin (40–80 µM), subsequently; oxidative stress, apoptosis and mitochondrial function were assessed using several techniques. The results demonstrated that cyanidin was able to dose-dependently reverse cisplatin-induced cell damage and apoptosis, attenuate the accumulation of reactive oxygen species (ROS), and mitochondrial membrane potential depolarization, downregulate the expression of Bcl-2 homologous antagonist/killer, upregulate the expression of apoptosis regulator Bcl-2, and reduce the activation of caspase 3, caspase 9, but not caspase 8. Furthermore, the results revealed that the translocation of apoptosis regulator Bax (Bax) from the cytoplasm to the mitochondrial membrane serves an essential role in cisplatin-induced apoptosis. Cyanidin was able to block the translocation of Bax and reduce the release of cytochrome c from cytoplasm. These data indicate that cyanidin attenuates cisplatin-induced cardiotoxicity by inhibiting ROS-mediated apoptosis, while the mitochondrial and extracellular regulated kinase signaling pathways may also serve important roles.

MicroRNA‑206 contributes to the progression of steroid‑induced avascular necrosis of the femoral head by inducing osteoblast apoptosis by suppressing programmed cell death 4.

The expression of microRNA-206 (miR-206) is aberrantly induced in steroid-induced avascular necrosis of femoral head (SANFH). Therefore, investigating the function of miR-206 in SANFH and uncovering the functional mechanism associated with the condition will promote the understanding and treatment of the disease. The purpose of the present study was to investigate the pro-osteoclasteogenic effect of miR-206 that occurs through regulation of programmed cell death 4 (PDCD4). The expression of miR-206 and PDCD4 was analyzed in the clinical SANFH specimens. The level of miR-206 and PDCD4 was regulated in human osteoblast lineage hFOB1.19 and the effect of different treatments on cell viability, proliferation, apoptosis and differentiation potential of osteoblasts were analyzed with a Cell Counting kit-8, 5-ethynyl-2′-deoxyuridine staining, flow cytometry and Hoechst staining. The expression of miR-206 was upregulated while PDCD4 was downregulated in the SANFH specimens. Induced expression of miR-206 decreased cell viability and proliferation, while apoptosis was induced. At the molecular level, overexpression of miR-206 inhibited the expression of PDCD4, alkaline phosphatase (ALP) and B-cell lymphoma 2 (Bcl-2), and increased the expression of apoptosis regulator Bcl2-X-associated protein (Bax). Inhibiting the expression of miR-206 increased cell viability and proliferation but had no effect on cell apoptosis, as detected by flow cytometry and Hoechst staining. However, at the molecular level, inhibiting the expression of miR-206 induced expression of PDCD4, ALP and Bcl-2, while it decreased the expression of Bax. Additionally, knockdown of PDCD4 blocked the effect of miR-206 inhibition on hFOB1.19 cells, representing a PDCD4-dependent manner of miR-206 in inducing apoptosis of osteoblasts. Therefore, miR-206 promoted the onset of SANFH by inducing apoptosis and suppressed the proliferation of osteoblasts, which was dependent on the inhibition of PDCD4.

Danshensu attenuates aldosterone-induced cardiomyocytes injury through interfering p53 pathway.

Heart failure, characterized by impaired systolic and/or diastolic function, is a common cardiovascular disease. The loss of cardiomyocytes due to various factors, including through necrosis or apoptosis can result in heart failure. Previous studies have indicated that excessive aldosterone (ALD) serves an essential role in the process of heart failure, and the heart is also one of the direct targets of ALD, which can provoke hypertrophy and the apoptosis of cardiomyocytes. The aim of the present study was to investigate the protective effect of danshensu (DSS) on ALD‑induced cardiomyocytes injury. The present results demonstrated that DSS increased cell viability and decreased the leakage of lactate dehydrogenase in cardiomyocytes exposed to ALD. In addition, DSS decreased the apoptotic rate of ALD‑stimulated cells. Further research indicated that DSS‑ and cellular tumor antigen p53 (p53)‑alone or combination treatment was able to decrease the expression levels of apoptosis regulator BAX and caspase‑3, and increase the expression of apoptosis regulator B‑cell lymphoma (Bcl)‑2 in ALD‑stimulated cardiomyocytes. Taken together, the results of the present study suggest that DSS inhibits the harmful effects of ALD on cardiomyocytes via interfering with the p53 signaling pathway. These results provide novel evidence for the potential protective effects of DSS.

Consecutive stimulation of HBsAg promotes the viability of the human B lymphoblastoid cell line IM-9 through regulating the SIRT1-NF-κB pathway.

Patients with chronic HBV infection have been reported to suffer a significantly increased risk of NHL, but the underlying mechanisms remain to be clearly explained. The aim of the present study was to clarify the relationship between chronic HBV infection and NHL development. Fluorescence-activated cell sorting, Annexin V/7-aminoactinomycin D staining and MTS assay were used to analyze the rate of apoptosis and cell viability. In addition, western blotting was used to detect protein expression. The effects of the activator of SIRT1, SRT1720, and the inhibitor of SIRT1, nicotinamide, were also analyzed. The expression levels cytokines and chemokines were determined by multiplex assay. Hepatitis B surface antigen (HBsAg) was demonstrated to increase the viability of the human peripheral B lymphoblastoid cell line, IM-9, in a dose- and time-dependent manner. HBsAg also decreased histone H3 acetylation and p21 expression at the molecular level. HBsAg upregulated the expression of anti-apoptotic B-cell lymphoma-extra-large and B-cell lymphoma 2 proteins, and inactivated the intrinsic apoptosis pathway by reducing BCL2 associated X, apoptosis regulator expression and increasing the expression of sirtuin 1 (SIRT1) and nuclear factor-κB (NF-κB). HBsAg also altered the levels of certain chemokines and cytokines, including interleukin (IL)-4, -10 and -12, C-X-C motif chemokine 10 and C-C motif chemokine ligand 5. Inhibition of SIRT1 suppressed the effects induced by HBsAg. The anti-apoptotic effect of HBsAg in IM-9 cell lines occurred via the promotion of cell viability, inhibition of apoptosis, regulation of chemokines and cytokines, acetylation of histone H3 and alteration of SIRT1 and NF-κB expression. In conclusion, chronic stimulation with HBsAg promoted the viability of the human B lymphoblastoid cell line, IM-9, through regulation of the SIRT1-NF-κB pathway. This may be an underlying mechanism of HBV-associated NHL.

MiR-101 Enhances Cisplatin-Induced DNA Damage Through Decreasing Nicotinamide Adenine Dinucleotide Phosphate Levels by Directly Repressing Tp53-Induced Glycolysis and Apoptosis Regulator Expression in Prostate Cancer Cells.

Tp53-induced glycolysis and apoptosis regulator (TIGAR) enhances the pentose phosphate pathway, thereby contributing directly to DNA repair due to generation of nicotinamide adenine dinucleotide phosphate (NADPH) and ribose-5-phosphate, two key precursors of DNA synthesis and repair. Targetscan database showed that miR-101 was predicted to potentially target TIGAR. Therefore, we speculated that miR-101 could enhance cisplatin-induced DNA damage by directly repressing TIGAR expression in prostate cancer cells. We found that upregulation of miR-101 inhibited viability, induced apoptosis, increased glycolysis rate and fructose-2,6-bisphosphate levels, decreased glucose-6-phosphate dehydrogenase expression and NADPH levels, and enhanced cisplatin-induced DNA damage in prostate cancer cells. We also demonstrated that TIGAR was a direct target of miR-101 by using luciferase activity assay. Furthermore, this study revealed that the roles of knockdown of TIGAR were similar to miR-101 upregulation in prostate cancer cells. Taken together, miR-101 inhibited viability, induced apoptosis, reprogramed glucose metabolism, and enhanced cisplatin-induced DNA damage through decreasing NADPH levels by directly suppressing the expression of TIGAR in prostate cancer cells.

Neuroprotective effects of polydatin against mitochondrial-dependent apoptosis in the rat cerebral cortex following ischemia/reperfusion injury.

The neuroprotective effect of polydatin (PD) against hemorrhagic shock-induced mitochondrial injury has been described previously, and mitochondrial dysfunction and apoptosis were reportedly involved in ischemic stroke. In the present study the neuroprotective effect of PD in preventing apoptosis was evaluated following induction of focal cerebral ischemia by middle cerebral artery occlusion (MCAO) in rats. PD (30 mg/kg) was administered by caudal vein injection 10 min prior to ischemia/reperfusion (I/R) injury. 24 h following I/R injury, ameliorated modified neurological severity scores (mNSS) and reduced infarct volume were observed in the PD treated group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and Annexin V/propidium iodide assays demonstrated the anti-apoptotic effect of PD in the ischemic cortex. In addition, PD improved I/R injury-induced mitochondrial dysfunction, reflected by morphological observations and measurements of mitochondrial membrane potential and intracellular ATP measurement. Western blot analysis revealed an increase in B-cell lymphoma 2 apoptosis regulator (Bcl-2) expression, and a decrease in Bcl-2-associated protein X apoptosis regulator expression in the PD group in comparison with the vehicle treated group. PD treatment also prevented the release of cytochrome c from mitochondria into the cytoplasm, and blunted the activities of caspase-9 and caspase-3. Furthermore, PD treatment decreased the levels of reactive oxygen species in neurons isolated from the ischemic cortex. The findings of this study, therefore, suggest that PD has a dual effect, ameliorating both oxidative stress and mitochondria-dependent apoptosis, making it a promising new therapy for the treatment of ischemic stroke.