OBJECTIVE

To explore the expression of CASP8 and its clinical significance in nasopharyngeal carcinoma (NPC).

METHODS

The differentially expressed genes between pooled NPC tissues and non-cancerous nasopharyngeal (NP) tissues were screened using 8 microarrays. Real-time PCR and immunohistochemistry were used to validate the detection results of CASP8 expression in NPC, and the correlation of CASP8 expression to the clinical characteristics was analyzed in the NPC cases.

RESULTS

Real-time PCR confirmed a reduced expression of CASP8 mRNA level in NPC tissues (P<0.0001), which was consistent with the microarray data. Immunohistochemistry indicated that CASP8 protein expression was also significantly down-regulated in NPC tissue compared to that in the non-cancerous nasopharyngeal tissues (P=0.02). The reduction of CASP8 expression was inversely correlated to lymph node metastasis (P=0.002) and the clinical stages (P=0.026) of NPC.

CONCLUSIONS

Decreased CASP8 expression is an unfavorable factor that promotes the development and progression of NPC.

Osteonecrosis of the jaw (ONJ) is a severe complication of bisphosphonate treatment.

OBJECTIVE

A detailed characterization of sampled peri-necrotic jawbone from bisphosphonate-treated patients was performed at tissue and cellular level (histological analyses and gene expression).

METHODS

Alveolar bone samples were collected from patients with (n = 5) and without ONJ (n = 5). Healthy patients served as controls (n = 10).

RESULTS

The histological analysis demonstrated low to moderate inflammation, displaying areas of inflammatory infiltrate in the bone marrow. Multinuclear giant cells and osteoclasts were found in both groups. Markers of bone formation (alkaline phosphatase, Col1a1, and osteocalcin), bone resorption (receptor activator of NF-kappaB ligand [RANKL], osteoprotegerin [OPG], tartrate-resistant acid phosphatase, and cathepsin K), inflammation (tumor necrosis factor-alpha, interleukin [IL]-1β, and IL-6), angiogenesis (vascular endothelial growth factor A), and apoptosis (Casp3, Casp8, p53, and Smac) were evaluated. Nonparametric statistical tests were used to identify differences between the groups. In patients with ONJ, the expression level of the proinflammatory marker IL-1β was strongly up-regulated compared with controls (p = .040).

CONCLUSIONS

A down-regulated expression of Casp8 compared with controls was observed (p = .014) in patients treated with bisphosphonates. The RANKL/OPG ratios were similar in the three groups. The results indicate a need to further investigate the molecular mechanisms involved in the course of ONJ related to antiresorptive treatment.

Guava Psidium guajava L (Pg) and bhumi amla Phyllanthus amarus Schum. et Thonn (Pa) are well-known plants in traditional medicine. However, the capacity of these plants for improving the immune system of aquatic species has received less attention so far. This study aimed to investigate the effects of single supply or mixture of Pg and Pa extracts on immune responses, disease resistance and liver proteome profiles in striped catfish Pangasianodon hypophthalmus. Fish were fed diets including basal diet 0% or one of three doses of each plant extract, either alone or in mixture, 0.08, 0.2, or 0.5% Pg, Pa or mixture (Pg:Pa, v/v) for 6 weeks. The immune parameters (respiratory burst activity (RBA); nitric oxide synthase (NOS), total immunoglobulin, lysozyme, and complement activities) were examined at W3, W6 post-feeding, and after challenge test. The growth parameters and the challenge test with Edwardsiella ictaluri were done at W6. The liver proteome profiles were analyzed in W6 at 0.08 and 0.5% of each extract. The results showed that extract-based diets significantly improved growth parameters in the Pg0.2 group compared to control. The cellular immune responses in spleen and the humoral immune responses in plasma were significantly improved in a dose and time-dependent manner. Diets supplemented with single Pg and Pa extracts, and to lesser extent to combined extracts, could significantly decrease the mortality of striped catfish following bacterial infection compared to control. The proteomic results indicated that some pathways related to immune responses, antioxidant and lipid metabolism were enriched in liver at W6. Several proteins (i.e., CD8B, HSP90AA1, HSP90AB1, PDIA3, CASP8, TUBA1C, CCKAR, GNAS, GRIN2D, PLCG1, PRKCA, SLC25A5, VDAC2, ACTN4, GNAI2, LCK, CARD9, NLRP12, and NLRP3) were synergistically upregulated in mixture of Pg and Pa-based diets compared to control and single dietary treatments. Taken together, the results revealed that single Pg and Pa extracts at 0.2 and 0.5% and their mixture at 0.08 and 0.5% have the potential to modulate the immune mechanisms and disease resistance of striped catfish. Moreover, the combination of Pg and Pa in diets suggested positive synergistic effects liver proteome profile related to immune system processes.

Keywords: Edwardsiella ictaluri; Phyllanthus amarus Schum et Thonn; Psidium guajava L; humoral immune response; liver proteome profile; plant extract-based diets; skin mucosal immune response; striped catfish.

Heat stress is a major concern in animal reproduction, as testicular temperature must be 3-5 °C below body core temperature for production of motile and fertile sperm in mammals. Although recent studies concluded that increased temperature per se was the underlying pathophysiology of testicular impairment, more studies are required to better understand the mechanisms. Therefore, our objective was to investigate the impacts of mild acute heat stress on sperm and testes, and based on mRNA, elucidate involvement of StAR, Trp53 and Trp53-dependent intrinsic and extrinsic apoptotic pathways in pathophysiology of testicular heat stress. Forty-eight C57 BCL6 elite male mice were equally allocated into six groups, anesthetized and the distal third of their body immersed in a water-bath at 40 or 30 °C (heat treatment and control, respectively) for 20 min. Intervals from heat exposure (Day 0) to euthanasia were: 8 and 24 h and 7, 14 and 21 d (plus a control group at 14 d). The epididymides were excised, minced and placed in Tyrode albumin lactate pyruvate hepes (TALPH) at 37 °C for 15 min to recover sperm. Based on computer assisted sperm analysis (CASA), heat treatment reduced total and progressive motility ∼40% (P < 0.05) on Days 14 and 21. Furthermore, percentage morphologically normal sperm was significantly decreased on Day 7, with greater reductions on Days 14 and 21, mostly due to increased midpiece defects. Acrosome integrity (FITC PSA) was decreased ∼35% at 8 h (P < 0.05) and reached a nadir on Day 14. There were decreases (P < 0.05) in seminiferous tubule diameter and testicular weight (relative to body weight) on Day 14. Testicular RNA was extracted, reverse-transcribed and cDNA used for PCR. Expression of genes Hspa1b (Hsp70) and Gpx1 had 7- and 10-fold increases (P < 0.001 for each) at 8 and 24 h, respectively, with Hspa1b remaining upregulated at 24 h, whereas StAR peaked at Day 14 (15-fold, P < 0.0001) and had returned to baseline on Day 21. Both Trp53 and Casp8 were upregulated (P < 0.05) on Day 14, whereas Bcl-2 was decreased (P < 0.05) on Days 7 and 14. In conclusion, acute mild heat stress severely reduced sperm quality and based on mRNA, there was upregulation of chaperone and antioxidant systems and Trp53-dependent intrinsic and extrinsic apoptotic pathways, with deleterious effects on sperm, spermatocytes and spermatids. These findings provided insights into the pathophysiology of heat stress and should contribute to development of evidence-based approaches to mitigate effects of testicular heating.

Keywords: Apoptosis; Apoptotic pathways; Mouse; Semen; Testes; Testicular thermoregulation; Trp53.

Caspase-8 (Casp8)-mediated signaling triggers extrinsic apoptosis while suppressing receptor-interacting protein kinase (RIPK) 3-dependent necroptosis. Although Casp8 is dispensable for the development of innate and adaptive immune compartments in mice, the importance of this proapoptotic protease in the orchestration of immune response to pathogens remains to be fully explored. In this study, Casp8-/-Ripk3-/- C57BL/6 mice show robust innate and adaptive immune responses to the natural mouse pathogen, murine CMV. When young, these mice lack lpr-like lymphoid hyperplasia and accumulation of either B220 + CD3+ or B220-CD3+CD4+ and CD8+ T cells with increased numbers of immature myeloid cells that are evident in older mice. Dendritic cell activation and cytokine production drive both NK and T cell responses to control viral infection in these mice, suggesting that Casp8 is dispensable to the generation of antiviral host defense. Curiously, NK and T cell expansion is amplified, with greater numbers observed by 7 d postinfection compared with either Casp8+/-Ripk3-/- or wild type (Casp8+/+Ripk3+/+ ) littermate controls. Casp8 and RIPK3 are natural targets of virus-encoded cell death suppressors that prevent infected cell apoptosis and necroptosis, respectively. It is clear from the current studies that the initiation of innate immunity and the execution of cytotoxic lymphocyte functions are all preserved despite the absence of Casp8 in responding cells. Thus, Casp8 and RIPK3 signaling is completely dispensable to the generation of immunity against this natural herpesvirus infection, although the pathways driven by these initiators serve as a crucial first line for host defense within virus-infected cells.

BACKGROUND

Folate deficiency is closely related to the development of neural tube defects (NTDs). However, the exact mechanism is not completely understood. This study aims to induce murine NTDs by inhibiting one of the folate metabolic pathways, de novo purine synthesis and preliminarily investigate the potential mechanisms. The key enzyme, glycinamide ribonucleotide formyl transferase (GARFT) was inhibited by a specific inhibitor, lometrexol (DDATHF) in the pregnant mice.

METHODS

Pregnant mice were intraperitoneally injected with various doses of DDATHF on gestational day 7.5 and embryos were examined for the presence of NTDs on gestational day 11.5. GARFT activity and levels of ATP, GTP, dATP and dGTP were detected in embryonic brain tissue. Proliferation and apoptosis was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemical assay and western blotting.

RESULTS

40 mg kg(-1) body weight (b/w) of DDATHF caused the highest incidence of NTDs (30.8 %) and therefore was selected as the optimal dose to establish murine NTDs. The GARFT activity and levels of ATP, GTP, dATP and dGTP in embryonic brain tissue were significantly decreased after DDATHF treatment. Furthermore, Levels of proliferation-related genes (Pcna, Foxg1 and Ptch1) were downregulated and apoptosis-related genes (Bax, Casp8 and Casp9) were upregulated. Expression of phosphohistone H3 was significantly decreased while expression of cleaved caspase-3 was greatly increased.

CONCLUSIONS

Results indicate that DDATHF induced murine NTDs by disturbing purine metabolism and further led to abnormal proliferation and apoptosis.

OBJECTIVE

To investigate the clinical significance of phosphorylated caspase-8 by Src in patients with operable non-small cell lung cancer.

METHODS

Src and caspase-8 expressions were tested using immunohistochemistry in non-small cell lung cancer tissues and control lung tissues. The expression of phosphorylated caspase-8 at 380 tyrosine by Src was detected using Western blotting. The disease-free survival (DFS) of patients positive and negative for phosphorylated caspase-8 was analyzed with Kaplan-Meire survival curve.

RESULTS

No significant difference was found in the positivity rate of caspase-8 or Src between the cancer tissues and control lung tissues (76.3% vs 83.3%, P>0.05; 70.1% vs 66.4%, P>0.05). All the patients with Casp8- and Src-positive cancers were positive for phosphoryalted caspase-8, whose expression rate was significantly higher in the cancer tissues than in the paired control lung tissues (52.4% vs 7.1%, P<0.05). The 2-year DFS was significantly higher in patients negative for phosphorylated caspase-8 than in the positive patients (32.0% vs 60.3%, P<0.05).

CONCLUSIONS

Phosphorylated caspase-8 may serve as a predictor for a poorer DFS in patients with operable non-small cell lung cancer.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used pharmaceuticals to treat pain, fever and inflammation. NSAIDs are also known to have many side effects including adverse effects on reproduction in both humans and animals. As NSAIDs usage is not regulated they are frequently detected at high concentrations in the environment. In order to understand the effect of NSAIDs on zebrafish sex differentiation, we used seven different NSAIDs which were either Cox-1 selective, Cox-1 biased, non-selective or COX-2 selective. We show that at higher concentration, NSAIDs are toxic to zebrafish embryo as they lead to mortality and hatching delay. Gene expression analysis following short term exposure of NSAIDs led to downregulation of female specific genes including zp2, vtg2 foxl2 and wnt4. Long term exposure of larvae to environmentally relevant concentrations of Cox-2 selective and non-selective NSAIDs resulted in male-biased sex ratio which confirmed the qRT-PCR analysis. However, the Cox-1 selective acetylsalicylic acid and the Cox-1 biased ketoprofen did not alter sex ratio. The observed male-biased sex ratio could also be due to induction of apoptosis process as the genes including p21 and casp8 were significantly upregulated following exposure to the Cox-2 selective and the non-selective NSAIDs. The present study indicates that NSAIDs alter sex differentiation in zebrafish, primarily through inhibition of Cox-2. This study clearly demonstrates that the use of NSAIDs and their release into the aquatic environment should be carefully monitored to avoid adverse effects to the aquatic organisms.

The association between apoptosis and neural tube defects (NTDs) is recognized as important, however, the precise link remains to be elucidated. Epigenetic modifications in human NTDs have been detected previously. In the present study, the occurrence of epigenetic modifications in apoptosis‑related genes was investigated in a retinoic acid (RA)‑induced mouse NTD model. Among 84 key genes involved in programmed cell death, 13 genes, including tumor necrosis factor (Tnf), annexin A5, apoptosis inhibitor 5, Bcl2‑associated athanogene 3, baculoviral IAP repeat‑containing 3, caspase (Casp)12, Casp4, Casp8, lymphotoxin β receptor, NLR family, apoptosis inhibitory protein 2, TNF receptor superfamily (Tnfrsf)1a, TNF superfamily (Tnfs)f10 and Tnfsf12, were downregulated, whereas nucleolar protein 3 was upregulated in the RA‑induced NTD mice. Chromatin immunoprecipitation assays revealed that the regulatory regions of these differentially expressed TNF‑related genes showed reduced histone H3K27 acetylation in NTDs, compared with control mice without NTDs. Reverse transcription‑quantitative polymerase chain reaction revealed that H3K27ac‑binding to the differentially regulated genes was markedly decreased in the NTD mice, whereas binding to the unchanged genes Casp3 and Nfkb1 was unaffected. In conclusion, certain TNF‑related genes appeared to be downregulated in NTDs, possibly as a result of abnormal histone H3K27 acetylation. These results shed new light on the epigenetic dysregulation of apoptosis‑related genes in NTDs.

The wound-healing process is a natural response to burn injury. Resveratrol (RES) may have potential as a therapy for wound healing, but how and whether RES regulates skin repair remains poorly understood. Human epidermal keratinocyte (HaCaT) cells were treated with lipopolysaccharide (LPS), and a mouse skin wound-healing model was established. Cell viability and apoptosis were analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide or flow cytometry. Cell proliferation was assessed by cell viability and colony-formation analyses. Cell migration was tested by wound-healing analysis. The microRNA-212 (miR-212) and caspase-8 (CASP8) levels were determined by quantitative reverse transcription polymerase chain reaction and western blotting. The correlation between miR-212 and CASP8 was analyzed by dual-luciferase reporter analysis. Skin wound healing in mice was assessed by measuring the wound area and gap after hematoxylin-eosin (HE) staining. RES reduced the LPS-induced reduction in viability and apoptosis in HaCaT cells. miR-212 expression was reduced by LPS and increased by exposure to RES. RES promoted cell proliferation and migration after LPS treatment by increasing miR-212 levels. CASP8 was a target of miR-212. CASP8 silencing promoted cell proliferation and migration, which was reversed by miR-212 knockdown in LPS-treated HaCaT cells. RES promoted skin wound healing in mice, which was reduced by miR-212 knockdown. Thus, RES facilitates cell proliferation and migration in LPS-treated HaCaT cells and promotes skin wound-healing in a mouse model by regulating the miR-212/CASP8 axis.

Background: Radiation therapy plays a leading role in the treatment of prostate cancer, but the emergence of radioresistant forms of this disease dictates the need for a personalized ap-proach based on the data from genetic and epigenetic markers. Such markers include the copy number variation as well as gene and microRNA expression.

Purpose: The aim of the study was to validate the list of potential predictors of radioresistance of prostate tumor cells in a model experiment based on the determination of gene copy number variation, gene transcriptional activity and microRNA expression.

Material and methods: The study used a PC-3 prostate cancer cell culture. The determination of the relative copy number variation and expression of 32 genes (BRCA1, BRCA2, PTEN, CASP3, CASP8, BAX, BCL2, CASP9, P53, MDM2, AKT1, ATM, BRIP1, CDK1, CDKN1B, CCND1, CCND3, FGFR2, KU70, RAD50, RAP80, Rif1, RNF168, TopBP1, HIST, H2AX, EXO1, XRCC4, RBBP8, EP300, LIG4, C-FLIP), as well as 15 microRNAs (let-7, miR15a/&#8202;16, miR-17, miR-18a, miR-21, miR-24, miR-26b, miR-99a, miR-100, miR-101, miR-106a, miR-663a, miR-143, miR-145) was performed using the real-time quantitative polymerase chain reaction method. It was found that daily irradiation of PC-3 cells on a Novalis TX linear accelerator at doses of 6 and 7 Gy for 5 days leads to a significant decrease in the total number of cells and the number of viable cells. Nevertheless, after 5 days of irradiation, about 15% of the initial number of prostate tumor cells retained their viability, which is due to their special genetic and epigenetic characteristics: increased copy number and expression of genes BRCA2, CDK1, CDKN1B, H2AX, RAD50, XRCC4, RBBP8 and EP300 and reduced copy number and expression of CCND3, TP53, and BCL2 genes, as well as differential expression of six microRNAs (hsa-miR-18a-5p, hsa-miR-24-5p, hsa-miR-99a-5p, hsa-miR-100-5p, hsa-miR-145-5p, hsa-let-7a-3p).

Conclusion: This study enabled to identify genetic and epigenetic markers of prostate tumor cells resistance to radiation therapy.

Keywords: DNA repair; Radiation therapy; apoptosis; cell culture; copy number variation of genes; micro-RNA; prostate cancer; radiotherapy; transcriptional activity of genes.

Background: Lactucin, a naturally occurring active sesquiterpene lactone, is abundantly found in chicory and romaine lettuce. A recent study reported that lactucin could induce apoptosis in leukemia cells. However, its cytotoxicity and potential molecular mechanisms underlying cancer cell death remain unclear.

Objective: Therefore, in this study, we aimed to investigate the direct effect and underlying mechanism of action of lactucin on renal cancer cells.

Methods: MTT assay and flow cytometry were performed to evaluate the rate of cell proliferation and apoptosis, respectively. Western blotting, reverse transcription polymerase chain reaction, and protein stability analyses were performed to analyze the effect of lactucin on the expression of apoptosis-related proteins such as B-cell lymphoma 2 (BCL-2) and CFLAR (CASP8 and FADD like apoptosis regulator) long isoform (CFLAR_L) in Caki-1 human renal cancer cells. In addition, reactive oxygen species (ROS) generation was evaluated using flow cytometry.

Results: Lactucin treatment induced apoptosis in Caki-1 cells in a dose-dependent manner via activation of the caspase pathway. It downregulated BCL-2 and CFLAR_L expression levels by suppressing BCL-2 transcription and CFLAR_L protein stability, respectively. Pretreatment with N-acetyl-1-cysteine, a ROS scavenger, attenuated the lactucin-induced apoptosis and restored the BCL-2 and CFLAR_L expression to basal levels. Lactucin-facilitated BCL-2 downregulation was regulated at the transcriptional level through the inactivation of the NF-κB pathway.

Conclusions: Our study is the first to demonstrate that lactucin-induced apoptosis is mediated by ROS production, which in turn activates the caspase-dependent apoptotic pathway by inhibiting BCL-2 and CFLAR_L expression in Caki-1 cells.

Keywords: Apoptosis; BCL-2; CFLARL; Lactucin; ROS; Renal cancer.

Caspase-8 (CASP8) impacts antiviral immunity in expected as well as unexpected ways. Mice with combined deficiency in CASP8 and RIPK3 cannot support extrinsic apoptosis or RIPK3-dependent programmed necrosis, enabling studies of CASP8 function without complications of unleashed necroptosis. These extrinsic cell death pathways are naturally targeted by murine cytomegalovirus (MCMV)-encoded cell death suppressors, showing they are key to cell-autonomous host defense. Remarkably, Casp8^-/-Ripk3^-/-, Ripk1^-/-Casp8^-/-Ripk3^-/- and Casp8^-/-Ripk3^K51A/K51A mice mount robust antiviral T cell responses to control MCMV infection. Studies in Casp8^-/-Ripk3^-/- mice show that CASP8 restrains expansion of MCMV-specific natural killer (NK) and CD8 T cells without compromising contraction or immune memory. Infected Casp8^-/-Ripk3^-/- or Casp8^-/-Ripk3^K51A/K51A mice have higher levels of virus-specific NK cells and CD8 T cells compared to matched RIPK3-deficient littermates or WT mice. CASP8, likely acting downstream of Fas death receptor, dampens proliferation of CD8 T cells during expansion. Importantly, contraction proceeds unimpaired in the absence of extrinsic death pathways owing to intact Bim-dependent (intrinsic) apoptosis. CD8 T cell memory develops in Casp8^-/-Ripk3^-/- mice, but memory inflation characteristic of MCMV infection is not sustained in the absence of CASP8 function. Despite this, Casp8^-/-Ripk3^-/- mice are immune to secondary challenge. Interferon (IFN)γ is recognized as a key cytokine for adaptive immune control of MCMV. Ifngr^-/-Casp8^-/-Ripk3^-/- mice exhibit increased lifelong persistence in salivary glands as well as lungs compared to Ifngr^-/- and Casp8^-/-Ripk3^-/- mice. Thus, mice deficient in CASP8 and RIPK3 are more dependent on IFNγ mechanisms for sustained T cell immune control of MCMV. Overall, appropriate NK- and T cell immunity to MCMV is dependent on host CASP8 function independent of RIPK3-regulated pathways.

Purpose: To determine whether LOX-1 regulates neutrophil apoptosis and fungal load in A. fumigatus keratitis.

Methods: Fas, FasL, CASP3, CASP8, CASP9 and BCL2 were tested in normal and infected corneas of C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of LOX-1 neutralizing antibody or inhibitor (Poly I). Clinical score was recored and HE staining was tested. Fungal load in mice corneas was observed by plate counting. Poly morphonuclear neutrophilic leukocytes (PMNs) were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of LOX-1 neutralizing antibody or Poly I. PCR, Western blot tested expression of Fas, FasL, CASP3, CASP8, CASP9, BCL2 and cleaved caspase-3. PMNs infiltration and TUNEL-positive cells were assessed by immunofluorescent staining. Flow cytometry assay tested the percentage of apoptosis neutrophils.

Results: Fas, Fas ligand, caspase-8, caspase-9 and caspase-3 mRNA levels were significantly higher in C57BL/6 mice corneas infected with A. fumigatus than normal corneas. Poly I treatment alleviated the severity and decreased clinical score at 3, 5 and 7 days post infecrion (p.i.). HE staining showed less infiltration in corneal tissue after LOX-1 inhibition. Plate counting experiment showed that number of viable fungus in corneas of Poly I treated group was significantly less than control group. LOX-1 neutralizing antibody or Poly I treatment significantly decreased neutrophil infiltration, the quantity of TUNEL-positive cells, the expression of Fas, Fas ligand, caspase-8, caspase-9, caspase-3, BCL2, cleaved caspase-3 and the percentage of apoptosis neutrophils compared with control corneas. LOX-1 neutralizing antibody treatment significantly decreased Fas, FasL, CASP3, CASP8, CASP9, BCL2 and cleaved caspase-3 expression in neutrophils.

Conclusion: LOX-1 inhibition decrease neutrophil apoptosis and fungal load in A. fumigatus keratitis.

Keywords: A. fumigatus keratitis; LOX-1; apoptosis; fungal load; neutrophil.

Tumor mass dormancy is the key intermediate step between immune surveillance and cancer progression, yet due to its transitory nature it has been difficult to capture and characterize. Little is understood of its prevalence across cancer types and of the mutational background that may favor such a state. While this balance is finely tuned internally by the equilibrium between cell proliferation and cell death, the main external factors contributing to tumor mass dormancy are immunological and angiogenic. To understand the genomic and cellular context in which tumor mass dormancy may develop, we comprehensively profiled signals of immune and angiogenic dormancy in 9,631 cancers from the Cancer Genome Atlas and linked them to tumor mutagenesis. We find evidence for immunological and angiogenic dormancy-like signals in 16.5% of bulk sequenced tumors, with a frequency of up to 33% in certain tissues. Mutations in the CASP8 and HRAS oncogenes were positively selected in dormant tumors, suggesting an evolutionary pressure for controlling cell growth/apoptosis signals. By surveying the mutational damage patterns left in the genome by known cancer risk factors, we found that aging-induced mutations were relatively depleted in these tumors, while patterns of smoking and defective base excision repair were linked with increased tumor mass dormancy. Furthermore, we identified a link between APOBEC mutagenesis and dormancy, which comes in conjunction with immune exhaustion and may partly depend on the expression of the angiogenesis regulator PLG as well as interferon and chemokine signals. Tumor mass dormancy also appeared to be impaired in hypoxic conditions in the majority of cancers. The microenvironment of dormant cancers was enriched in cytotoxic and regulatory T cells, as expected, but also in macrophages and showed a reduction in inflammatory Th17 signals. Finally, tumor mass dormancy was linked with improved patient survival outcomes. Our analysis sheds light onto the complex interplay between dormancy, exhaustion, APOBEC activity and hypoxia, and sets directions for future mechanistic explorations.

Keywords: APOBEC; angiogenesis; hypoxia; immunity; mutational signatures; tumor mass dormancy.

Objective: Acute promyelocytic leukemia (APL) is among the most threatening hematological malignant cancers. Defects in cell growth and apoptotic pathways lead to the pathogenesis of the disease as well as its resistance to therapy; therefore, it is a good model for examining pro-apoptotic agents. The present study compared the molecular mechanism induced by kaempferol and epigallocatechin gallate (EGCG) as well as all-trans retinoic acid (ATRA), in HL-60 leukemia cells during five days.

Materials and methods: Cell viability was determined by resazurin assay following treatment with ATRA (10 µM), EGCG, and kaempferol (12.5-100 µM), and apoptosis was detected by the ANX V/PI kit. Moreover, the levels of genes involved in apoptosis (PI3K, AKT, BCL2, BAX, P21, PTEN, CASP3, CASP8, and CASP9) and multi-drug resistance (MDR, ABCB1 and ABCC1) were assessed by using real-time PCR test.

Results: Based on the findings, kaempferol decreased cell viability and increased apoptosis in HL60 cells more than EGCG. Apoptosis was induced via extrinsic and intrinsic pathways in HL60 cells by kaempferol and EGCG. In addition, kaempferol and EGCG increased apoptosis and inhibited MDR in a concentration- and time-dependent manner.

Conclusion: Kaempferol at high concentrations can be taken into consideration for treating patients with APL as compared with EGCG.

Keywords: APL; Apoptosis; EGCG; Kaempferol; MDR.

Along with early-onset cancers, multiple primary cancers (MPCs) are likely resulting from increased genetic susceptibility; however, the associated predisposition genes or prevalence of the pathogenic variants genes in MPC patients are often unknown. We screened 71 patients with MPC of the stomach, colorectal, and endometrium, sequencing 65 cancer predisposition genes. A subset of 19 patients with early-onset MPC of stomach and colorectum were further evaluated for variants in cancer related genes using both normal and tumor whole exome sequencing. Among 71 patients with MPCs, variants classified to be pathogenic were observed in 15 (21.1%) patients and affected Lynch Syndrome (LS) genes: MLH1 (n = 10), MSH6 (n = 2), PMS2 (n = 2), and MSH2 (n = 1). All carriers had tumors with high microsatellite instability and 13 of them (86.7%) were early-onset, consistent with LS. In 19 patients with early-onset MPCs, loss of function (LoF) variants in RECQL5 were more prevalent in non-LS MPC than in matched sporadic cancer patients (OR = 31.6, 2.73-1700.6, p = 0.001). Additionally, there were high-confidence LoF variants at FANCG and CASP8 in two patients accompanied by somatic loss of heterozygosity in tumor, respectively. The results suggest that genetic screening should be considered for synchronous cancers and metachronous MPCs of the LS tumor spectrum, particularly in early-onset. Susceptibility variants in non-LS genes for MPC patients may exist, but evidence for their role is more elusive than for LS patients.

Background: Suxiao Xintong dropping pills (SXXTDP), a traditional Chinese medicine, is widely applied for treating myocardial infarction (MI). However, its therapy mechanisms are still unclear. Therefore, this research is designed to explore the molecular mechanisms of SXXTDP in treating MI.

Methods: The active ingredients of SXXTDP and their corresponding genes of the active ingredients were retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. MI-related genes were identified via analyzing the expression profiling data (accession number: GSE97320). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to study the shared genes of drug and disease. Through protein-protein interaction (PPI) network and the Cytoscape plugin cytoHubba, the hub genes were screened out. The compounds and hub targets binding were simulated through molecular docking method.

Results: We obtained 21 active compounds and 253 corresponding target genes from TCMSP database. 1833 MI-related genes were identified according to P＜0.05 and |log2FC| ≥ 0.5. 27 overlapping genes between drug and disease were acquired. GO analysis indicated that overlapping genes were mainly enriched in MAP kinase activity and antioxidant activity. KEGG analysis indicated that overlapping genes were mainly enriched in IL-17 signaling pathway and TNF signaling pathway. We obtained 10 hub genes via cytoHubba plugin. Six of the 10 hub genes, including PTGS2, MAPK14, MMP9, MAPK1, NFKBIA, and CASP8, were acted on molecular docking verification with their corresponding compounds of SXXTDP.

Conclusion: SXXTDP may exert cardioprotection effect through regulating multiple targets and multiple pathways in MI.

Keywords: Medicine, Chinese Traditional; Molecular Docking Simulation; Myocardial Infarction, Pharmacology.

Objective: To evaluate role of molecular (endothelin-1, soluble Fas-L, NT-proBNP, TNF-α, interleukin-1β,) and genetic factors (NOS3 (rs1799983), EDNRA (C + 70G, rs5335), NADPH oxidase (C242T, rs4673), p53 protein (polymorphic marker-Arg72Pro exon 4, rs1042522), NOS3 (Glu298Asp, rs1799983), Caspase 8 (CASP8, rs3834129 and rs1045485), interleukin-1β gene (Il-1β, rs1143634), TNF-α gene (rs1800629), SOD2 (rs4880), GPX1 (rs1050450) in development of anthracycline-induced cardiotoxicity (AIC) in women without cardiovascular diseases.

Methods: A total of 176 women with breast cancer and without cardiovascular diseases who received anthracyclines were enrolled in the study. After the 12 months of chemotherapy (CT), all patients were divided into two groups: group 1 (n = 52) comprised patients with AIC, group 2 (n = 124) comprised those without it.

Results: Based on ROC-analysis, levels of endothelin-1 of ≥9.0 pg/mL (AUC of 0.699), sFas-L of ≥98.3 ng/mL (AUC of 0.990), and NT-proBNP of ≥71.5 pg/mL (AUC of 0.994;) were identified as a cut-off values predicting AIC during 12 months after CT. Whereas, NT-proBNP and sFas-L were more significant predictors than endothelin-1 (p < 0.001). The development of AIC was significantly related to Arg/Arg of p53 protein gene (OR = 2.972; p = 0.001), T/T of NOS3 gene (OR = 3.059, p = 0.018), T/T of NADPH oxidase gene (OR = 2.753, p = 0.008), and C/C of GPX1 (OR = 2.345; p = 0.007).

Conclusion: Evaluation of polymorphisms genes of p53 (rs1042522), NOS3 (rs1799983), GPX1 (rs1050450), and NADPH oxidase (rs4673) can be recommended before CT for the risk assessment of AIC development. The serum levels of NT-proBNP and soluble Fas-L after CT may be considered as non-invasive biomarkers for prediction of AIC development during the 12 months.

Keywords: Anthracycline-induced cardiotoxicity; NT-proBNP; biomarkers; gene polymorphisms; prediction; soluble Fas-L.

Epigenetic modifications are considered of utmost significance for tumor ontogenesis and progression. Especially, it has been found that miRNA expression, as well as DNA methylation plays a significant role in central nervous system tumors during childhood. A total of 49 resected brain tumors from children were used for further analysis. DNA methylation was identified with methylation-specific MLPA and, in particular, for the tumor suppressor genes CASP8, RASSF1, MGMT, MSH6, GATA5, ATM1, TP53, and CADM1. miRNAs were identified with microarray screening, as well as selected samples, were tested for their mRNA expression levels. CASP8, RASSF1 were the most frequently methylated genes in all tumor samples. Simultaneous methylation of genes manifested significant results with respect to tumor staging, tumor type, and the differentiation of tumor and control samples. There was no significant dependence observed with the methylation of one gene promoter, rather with the simultaneous presence of all detected methylated genes' promoters. miRNA expression was found to be correlated to gene methylation. Epigenetic regulation appears to be of major importance in tumor progression and pathophysiology, making it an imperative field of study.

Keywords: childhood CNS tumors; mRNA; methylation; miRNA; microarray.

Loss of the 3p chromosome arm has previously been reported to be a biomarker of poorer outcome in both human papillomavirus (HPV)-positive and HPV-negative head and neck cancer. However, the precise operational measurement of 3p arm loss is unclear and the mutational profile associated with the event has not been thoroughly characterized. We downloaded the clinical, single nucleotide variation (SNV), copy number aberration (CNA), RNA sequencing, and reverse phase protein assay (RPPA) data from The Cancer Genome Atlas (TCGA) and The Cancer Proteome Atlas HNSCC cohorts. Survival data and hypoxia scores were downloaded from published studies. In addition, we report the inclusion of an independent Memorial Sloan Kettering cohort. We assessed the frequency of loci deletions across the 3p arm separately in HPV-positive and -negative disease. We found that deletions on chromosome 3p were almost exclusively an all or none event in the HPV-negative cohort; patients either had <1% or >97% of the arm deleted. 3p arm loss, defined as >97% deletion in HPV-positive patients and >50% in HPV-negative patients, had no impact on survival (p > 0.05). However, HPV-negative tumors with 3p arm loss presented at a higher N-category and overall stage and developed more distant metastases (p < 0.05). They were enriched for SNVs in TP53, and depleted for point mutations in CASP8, HRAS, HLA-A, HUWE1, HLA-B, and COL22A1 (false discovery rate, FDR < 0.05). 3p arm loss was associated with CNAs across the whole genome (FDR < 0.1), and pathway analysis revealed low lymphoid-non-lymphoid cell interactions and cytokine signaling (FDR < 0.1). In the tumor microenvironment, 3p arm lost tumors had low immune cell infiltration (FDR < 0.1) and elevated hypoxia (FDR < 0.1). 3p arm lost tumors had lower abundance of proteins phospho-HER3 and ANXA1, and higher abundance of miRNAs hsa-miR-548k and hsa-miR-421, which were all associated with survival. There were no molecular differences by 3p arm status in HPV-positive patients, at least at our statistical power level. 3p arm loss is largely an all or none phenomenon in HPV-negative disease and does not predict poorer survival from the time of diagnosis in TCGA cohort. However, it produces tumors with distinct molecular characteristics and may represent a clinically useful biomarker to guide treatment decisions for HPV-negative patients.

Keywords: chromosome loss; copy number alterations; genomics; head and neck cancer; mutational status.

Ent‑3α‑formylabieta‑8(14),13(15)‑dien‑16,12β‑olide (EFLDO) is a compound extracted from Euphorbia lunulata Bge exhibiting anti‑proliferative activity in vitro. In the present study, EFLDO was identified to sensitize HepG2 cells to tumor necrosis factor (TNF) superfamily member 10 (TNFSF10)‑induced apoptosis. Liver cancer cells were resistant to TNFSF10; however, EFLDO increased TNFSF10‑induced cancer cell viability inhibition and cell apoptosis induction as assessed by MTT assay and Annexin V‑fluorescein isothiocyanate (FITC)/propidium iodide assay, respectively. The western blotting results suggested that treatment with EFLDO increased TNFSF10‑induced upregulation of the protein expression levels of pro‑apoptotic proteins, including BCL2 associated agonist of cell death, BCL2 associated X, apoptosis regulator, caspase‑3 (CASP3) and CASP8. Furthermore, treatment with EFLDO increased TNFSF10‑mediated downregulation of the protein expression level of the anti‑apoptotic protein BCL2 apoptosis regulator. Notably, the increase in the activity of CASP3 was consistent with the western blotting results. Treatment with EFLDO sensitized liver cancer cells to TNFSF10, and apoptosis was induced via the upregulation of TNF receptor superfamily member 10a (TNFRSF10A) and TNFRSF10B in a tumor protein p53 (p53)‑dependent manner, as detected by reverse transcription‑quantitative polymerase chain reaction and western blot analyses. In addition, p53 was identified to be necessary for EFLDO‑induced sensitivity to TNFSF10, as assessed by western blotting and Annexin V‑FITC assay. Collectively, the present results suggested a novel mechanism underlying EFLDO function in liver cancer. Treatment with EFLDO was able to increase the antitumor effect of TNFSF10 in liver cancer cells in a p53‑dependent manner.

BACKGROUND

Dehydroleucodine, a natural sesquiterpene lactone from Artemisia douglassiana Besser (Argentine) and Gynoxys verrucosa (Ecuador).

OBJECTIVE

To define the molecular mechanisms underlying the effect of dehydroleucodine on the human glioblastoma cells.

METHODS

Various techniques (cDNA expression array, real-time quantitative PCR, chromatin immunprecipitation, luciferase reporter assay, use of phosphospecific antibodies, immunoprecipitation, immunoblotting, apoptosis and autophagy assays) were employed to define and validate multiple molecular gene targets affected in human glioblastoma cells upon dehydroleucodine exposure.

RESULTS

Dehydroleucodine exposure upregulated the total and phosphorylated (p-Y99) levels of TP73 in U87- MG glioblastoma cells. We found that TP73 silencing led to a partial rescue of U87-MG cells from the cell death induced by dehydroleucodine. Upon the dehydroleucodine exposure numerous gene targets were upregulated and downregulated through a TP73-dependent transcriptional mechanism. Some of these gene targets are known to be involved in cell cycle arrest, apoptosis, autophagy and necroptosis. Dehydroleucodine induced the TP73 binding to the specific genes promoters (CDKN1A, BAX, TP53AIP1, CYLD, RIPK1, and APG5L). Moreover, the exposure of U87-MG cells to dehydroleucodine upregulated the protein levels of CDKN1A, BAX, TP53AIP1, CYLD, RIPK1, APG5L, and downregulated the CASP8 level. The formation of RIPK1 protein complexes and phosphorylation of MLKL were induced by dehydroleucodine supporting the notion of multiple cell death mechanisms implicated in the tumor cell response to dehydroleucodine.

CONCLUSIONS

This multifaceted study led to a conclusion that dehydroleucodine induces the phosphorylation of tumor protein TP73 and in turn activates numerous TP73-target genes regulating apoptosis, autophagy and necroptosis in human glioblastoma cells..

Ethnopharmacological relevance: Clerodendrum cyrtophyllum Turcz has been used in traditional medicine for the treatment of various diseases. In spite of its therapeutic applications, research on its toxicity and teratogenicity is still limited.

Aim of the study: The study aimed to investigate the developmental toxicity of the ethanol extract of C. cyrtophyllum (EE) in zebrafish embryo model.

Material and methods: Major compounds from crude ethanol extract of Clerodendron cyrtophyllum Turcz leaves were determined using HPLC-DAD-Orbitrap-MS analysis. The developmental toxicity of EE were investigated using zebrafish embryo model. Zebrafish embryos at 6 h post-fertilization (hpf) were treated with EE at different concentrations. Egg coagulation, mortality, hatching, yolk sac oedema, pericardial oedema and teratogenicity were recorded each day for during a 5-day exposure. At time point 120 hpf, body length, pericardial area, heartbeat and yolk sac area were assessed. In order to elucidate molecular mechanisms for the developmental toxicity of EE, we further evaluated the effects of the EE on the expression of genes involved on signaling pathways affecting fish embryo's development such as heart development (gata5, myl7, myh6, has2, hand2, nkx 2.5), oxidative stress (cat, sod1, gpx4, gstp2), wnt pathway (β-catenin, wnt3a, wnt5, wnt8a, wnt11), or cell apoptosis (p53, bax, bcl2, casp3, casp8, casp9, apaf-1, gadd45bb) using qRT-PCR analysis.

Results: Our results demonstrated that three major components including acteoside, cirsilineol and cirsilineol-4'-O-β-D-glucopyranoside were identified from EE. EE exposure during 6- 96 h post-fertilization (hpf) at doses ranging from 80 - 200 μg/ml increased embryo mortality and reduced hatching rate. EE exposure at 20 and 40 μg/ml until 72 - 120 hpf induced a series of malformations, including yolk sac oedema, pericardial oedema, spine deformation, shorter body length. Based on two prediction models using a teratogenic index (TI), a 25% lethality concentration (LD25) and the no observed-adverse-effect level (NOAEL), EE is considered as teratogenic for zebrafish embryos with TI (LC50/EC50) and LD25/NOAEC values at 96 hpf reaching 3.87 and 15.73 respectively. The mRNA expression levels of p53, casp8, bax/bcl2, gstp2, nkx2.5, wnt3a, wnt11, gadd45bb and gata5 were significantly upregulated by EE exposure at 20 and 40 μg/ml while the expression of wnt5, hand2 and bcl2 were downregulated.

Conclusions: These results provide evidence for toxicity effects of EE to embryo stages and provide an insight into the potential toxicity mechanisms on embryonic development.

Keywords: Teratogenicity; apoptosis; embryonic development; oxidative stress; wnt pathway.