Expression Of Cyclin Research Articles

Interfering with pak4 Protein Expression Affects Osteosarcoma Cell Proliferation and Migration.

Purpose A number of studies have discovered various roles of PAK4 in human tumors, including osteosarcoma. However, the exact role of PAK4 in osteosarcoma and its mechanism have yet to be determined. Therefore, this study focused on interrogating the PAK4 effect on the proliferation and migration ability of osteosarcoma and its underlying mechanisms. Materials and Methods Western blot and QRT-PCR were utilized to quantify the PAK4 relative protein and mRNA levels. To measure cellular viability and mobility, the MTT and wound-healing assays were preferred. Results With the adenovirus-mediated overexpression of PAK4, the proliferation and migration of U2-OS and MG-63 osteosarcoma cells were stimulated. Furthermore, a liposome-mediated knockout of PAK4 will inhibit osteosarcoma cells from proliferating. In terms of mechanism, we observed the positive correlation of PAK4 expression with expression of P21, CyclinD1, CyclinE1, CDK2, and CDK6, which drives G0/G1 to the G2/M phase transition. PAK4 can also activate Erk expression in OS cells and induce EMT. Conclusion Interfering with PAK4 protein expression has been shown to affect osteosarcoma proliferation and migration.

LYAR promotes the proliferation of non-small cell lung cancer and is associated with poor prognosis.

The aim of the study was to investigate the clinical significance of Ly-1 antibody reactive clone (LYAR) in non-small-cell lung cancer (NSCLC). The expressions of LYAR at the protein level in representative paired NSCLC tumor tissues and adjacent non-tumor tissues were measured by Western blot and immunohistochemistry. Kaplan-Meier method was used to calculate the survival curve of patients with NSCLC. Cell Counting Kit-8 assay and flow cytometry were used to estimate the cell proliferation and cell cycle, respectively. Terminal-deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was performed to detect cell apoptosis. LYAR was dramatically overexpressed in NSCLC tissues which were closely related to the survival of patients with NSCLC. In clinical studies, the expression of LYAR was related to the clinical stage, histological differentiation, and Ki-67 expression. A positive correlation was found between LYAR and Ki-67 expression by Spearman's correlation test. After serum starvation for 72 h, serum re-addition significantly increased the expression of LYAR, PCNA, and Cyclin A and promoted the cell cycle progression. LYAR knockdown inhibited the proliferation and induced the G0/G1 cell cycle arrest and apoptosis of A549 cells. The present study revealed the clinical significance of LYAR in NSCLC. LYAR might serve as a tumor promoter in NSCLC progression by promoting the proliferation and inhibiting the apoptosis of NSCLC cells. Inhibiting the expression of LYAR was considered as a potential novel therapeutic strategy for NSCLC.

Laminaria japonica cyclic peptides exert anti-colorectal carcinoma effects through apoptosis induction in vitro and in vivo.

Laminaria japonica is consumed as a health food and used as a traditional medicine because of its biochemical and pharmacological abilities. However, the anti-tumor effect of L.japonica peptides has not been well explored. In the current study, three novel L. japonica cyclic peptides (LCPs) were isolated using an anti-cancer activity tracking method. LCP-3 [cyclo-(Trp-Leu-His-Val)] significantly induced apoptosis in Caco-2 cells in vitro. LCP-3 increased the Bax/Bcl-2 ratio, activated caspases, and regulated the p53/murine double minute 2 network. LCP-3 blocked Caco-2 cells in G0/G1 phase, which was accompanied by the inhibition of cyclin expression. Furthermore, LCP-3 inhibited colon cancer growth and induced cancer cell apoptosis in tumor-bearing mice. Notably, LCP-3 might be a potential agent for the prevention of colon cancer.

Novel mechanistic role of Kif26b in adipogenic differentiation of murine multipotent stromal cells

Emerging evidence delineates that obesity, a complex metabolic disorder, impairs the structure and function of stromal cells residing in various tissues. The exuberant adipose tissue mass observed in obesity is, in part, associated with hyperplasia of adipocytes resulting from recruitment of multipotent stromal cells within the stromal vascular fraction of adipose tissues. However, a clear understanding of the causal role of stromal cells and biological factors in obesity is lacking. In our quest to understanding the role of kinesin family member 26B (KIF26B), we found that KIF26B regulates osteogenic and chondrogenic differentiation of stromal/progenitor cells. In this study, we sought to examine the effects of Kif26b loss-of-function on adipogenic differentiation of murine C3H10T1/2 multipotent stromal cells. In vitro loss-of-function studies demonstrated that Kif26b knockdown by lentivirus mediated shRNA markedly dampened the differentiation potential of C3H10T1/2 cells to adipocytes and suppressed the expression of adipogenesis-related genes e.g., Pparg, C/ebpα, Fabp4 and Adipoq. Analysis of cell cycle revealed that Kif26b knockdown resulted in elevated expression of cyclins (Ccnd1, Ccnb1, Ccna2) along with rapid cell cycle progression from G0/G1 to S and G2 phases. Mechanistically, reduced adipogenic differentiation of Kif26b-deficient cells was partly dependent on PPARγ, a key transcription factor implicated in adipogenesis. This observation was experimentally supported as loss of adipogenesis was partially rescued by the addition of PPARγ agonist, rosiglitazone, in Kif26b-deficient cells. We further found that silencing of Kif26b lessened the protein levels of phospho-AKT(Ser473), phospho-S6(Ser235/236), and phospho-mTOR(Ser2448), the major component of AKT/mTOR complex 1 (mTORC1) signaling at the basal level. Together, these data define a novel role of Kif26b in regulating the commitment of C3H10T1/2 multipotent stromal cells to the adipocyte lineage and provide a practical framework for further experiments to establish its therapeutic potential for the treatment of problems associated with adipogenesis such as obesity at the cellular and molecular level.

Characterizations and the Mechanism Underlying Osteogenic Activity of Peptides from Enzymatic Hydrolysates of Stichopus japonicus.

Sea cucumber (Stichopus japonicus) is a kind of fishery product with high nutritional value. It exhibits a wide range of biological activity and has potential application in the food, pharmaceutical, and biomedical industries. However, there are no reports available on the effects of S. japonicus peptides (SJP) on bone mineral density regulations. The purpose of this work was to analyze the composition and osteogenic activity of SJP and explore its underlying mechanism. The results showed that SJP stimulated cell proliferation, differentiation, and mineralization in a dose-dependent manner. In addition, SJP could promote the proliferation of MC3T3-E1 cells by altering the cell cycle progression and regulating the expression of Cyclins. Besides, SJP activated the WNT/β-catenin pathway and increased the nuclear level of the active form β-catenin. Furthermore, SJP also induced the expression of bone morphogenetic protein (BMP-2) and increase the phosphorylation levels of p38, JNK, and ERK, suggesting that the osteogenic activity of SJP may be achieved through the activation of WNT/β-catenin and BMP/MAPK signal pathways. In vivo, SJP significantly inhibited the serum levels of RANKL, ALP, and TRAP, whereas it increased the levels of osteocalcin and osteoprotegerin in OVX-mice. These results indicate that SJP may have the potential to stimulate bone formation and regeneration, and may be used as a functional food or nutritional supplement to prevent osteoporosis.

Eight-Hit Evolutionary Pattern in ATM Gene of a Breast Carcinoma Patient: A Personalized Approach

Background: Different genetic variants in the ATM gene have been reported to be associated with breast carcinoma (BC). Purpose of the study was to consider the multi-insights experiments and explorations through the molecular, cellular and structural aspects of ATM.
 Methods and Results: D1853N polymorphism was traced in a proband with BC. DNA extracted from blood and tumor tissue was PCR-amplified and cloned to allelic localization of characterized variants. Protein expression and In silico analysis based on three different profiles involved in cell cycle were also performed to confirm mutational events. Findings revealed the molecular based sequential events as an eight-hit evolutionary pattern (8- Hit E.P) including D1853N as the first predisposing hit (inherited), pre-differentiation stage hits including IVS 36-8 T>C as the 2nd hit, V1833M as the 3rd hit, L1888L as the 4th, and somatic variants including IVS 36-46 C>T, L1842L, H1864H, and S1872R were considered as the 5-8th hits. Low protein expression of ATM in the majority of cells was observed, but the expression of cyclin E, CDC25A, P53, and Ki-67 was more diverse.
 Conclusions: Observations were reflective of the sequential molecular and cellular events through the entire patient’s life from the pre-differentiation embryonic stage and all through the post-birth periods. Mentioned hits seem to be effective on expression and function of ATM which confirmed by the expression and in silico analysis.

Anti-Psoriatic Effects and IL-22 Targeting Mechanism of Indirubin by Suppressing Keratinocyte Inflammation and Proliferation

Indigo naturalis, which is extracted from the leaves and branches of Baphicacanthus cusia (Nees) Bremek, has traditionally been used to treat psoriasis. The current study aimed to examine a new mechanism of the components of indigo naturalis, including indirubin, indigo, and tryptanthrin. The anti-psoriatic effects were assessed by the proliferation biomarkers (Ki67, K16), cell cycle progression, ROS production, and interleukin profiling (ICAM-1, TNF-α, IL-6, and IL-8) in IL-22-treated HaCaT cells. Among the components, indirubin significantly decreased intracellular ROS production and lowered the production of ICAM-1, TNF-α, and IL-6 in IL-22-treated HaCaT cells. Indirubin, indigo, and tryptanthrin could decrease the proportion of Ki67-positive cells, but only indirubin decreased the proportion of cells entering the S phase and suppressed the expression of cyclin D1 and cyclin E1 in IL-22-treated HaCaT cells. Indirubin significantly suppressed the phosphorylation of STAT3 and ERK. In vivo, IL-22 was intradermally injected into mouse ears for six days and topically treated with 0.1% or 1% indirubin. In the IL-22-injected mice, treatment with indirubin inhibited epidermal hyperplasia. Immunohistochemistry and western blot analysis demonstrated the downregulation of K16 expression in psoriatic lesions. These results suggest that indirubin, which is a major component of indigo naturalis, may have therapeutic potential in an IL-22-induced psoriasis model.

Antitumor Effects of Scorpion Peptide Smp43 through Mitochondrial Dysfunction and Membrane Disruption on Hepatocellular Carcinoma.

Smp43, a cationic antimicrobial peptide identified from the venom gland of the Egyptian scorpion Scorpio maurus palmatus, shows cytotoxicity toward hepatoma cell line HepG2 by membrane disruption. However, its underlying detailed mechanisms still remain to be further clarified. In the present study, we evaluated the cellular internalization of Smp43 and explored its effects on cell viability, cell cycle, apoptosis, autophagy, necrosis, and factor expression related to these cellular processes in human HepG2. Smp43 was found to suppress the growth of HepG2, Huh7, and human primary hepatocellular carcinoma cells while showing low toxicity to normal LO2 cells. Furthermore, Smp43 could interact with the cell membrane and be internalized into HepG2 cells via endocytosis and pore formation, which caused a ROS production increase, mitochondrial membrane potential decline, cytoskeleton disorganization, dysregulation of cyclin expression, mitochondrial apoptotic pathway activation, and alteration of MAPK as well as PI3K/Akt/mTOR signaling pathways. Finally, Smp43 showed effective antitumor protection in the HepG2 xenograft mice model. Overall, these findings indicate that Smp43 significantly exerts antitumor effects via induction of apoptosis, autophagy, necrosis, and cell cycle arrest due to its induction of mitochondrial dysfunction and membrane disruption. This discovery will extend the antitumor mechanisms of antimicrobial peptides and contribute to the development of antitumor agents against hepatocellular carcinoma.

Centromere protein F promotes progression of hepatocellular carcinoma through ERK and cell cycle-associated pathways.

Hepatocellular carcinoma (HCC) is one of the deadliest cancer types worldwide. The centromere proteins (CENPs) are critical for the mitosis-related protein complex and are involved in kinetochore assembly and spindle checkpoint signaling during mitosis. However, the clinical significance of CENPs in the recurrence and progression of HCC remains poorly understood. Here, we examined the expression of all CENPs and their association with recurrence and survival of HCC patients using the global gene expression profile dataset established in our laboratory. The effect of silencing CENPF on cell viability, migration, and epithelial-mesenchymal transition (EMT) were detected using CCK-8, transwell, and western blot, respectively. RT-qPCR and western blot were performed to confirm the silencing of CENPF and the relationship between STAT5A and CENPF, while tumorigenesis was tested using the HCC Huh7 xenograft mouse model. Most of the CENPs is overexpressed in HCC, and overexpression of CENPF was significantly associated with the poor survival of HCC patients. CENPF promoted HCC cell lines migration and EMT progression. Knockdown CENPF inhibited cell growth activity against human HCC cells in vitro and xenograft tumors in vivo. Bioinformatics analysis revealed that CENPF genes are enriched in the cell cycle. Silencing CENPF arrested cell cycle at the G2/M phase and inhibited Cyclin B1 and Cyclin E1 expressions. Meanwhile, silencing CENPF prohibited phosphorylation of ERK and the expression of NEK2. Additionally, we found that STAT5A down-regulated CENPF expression and inhibited cancer cell growth viability. In conclusion, our data suggested that CENPF could be potentially developed into a theranostic biomarker to tackle HCC progression.

Polyphenols from Hippophae rhamnoides suppressed colon cancer growth by regulating miRNA-mediated cell cycle arrest and apoptosis in vitro and in vivo

• HPs60 treatment result in alteration of miRNA expression profile, among which, 29 up-regulated and 9 down-regulated. • Hsa-miR-195-5p, hsa-miR-497-5p and hsa-miR-1247-3p are the reliable targets of HPs60. • HPs60 up-regulate miR-195-5p/497-5p expression, suppresses that of cyclin D1/2/2 and cyclin E1, then leads to G1 arrest. • HPs60 promotes miR-1247-3p-mediated expression of caspase 2, and inhibits that of miR-195-5p/497-5p-mediated Bcl-2, ultimately induces apoptosis. Colorectal cancer (CRC) is the most common gastrointestinal cancer. Hippophae rhamnoides are reported to possess a variety of active substances. MiRNAs are new therapeutic targets. Here, it found that Hippophae rhamnoides L. polyphenols (named as HPs60) exhibited significant anti-colon cancer activity in vitro and in vivo . And the main components were identified by LC-MS as kaempferol and its derivatives. Subsequently, by RNA-sequencing and searching GEO and TCGA database, three reliable miRNAs including hsa-miR-195-5p, hsa-miR-497-5p and hsa-miR-1247-3p were eventually screened. As expected, hsa-miR-195-5p and hsa-miR-497-5p were upregulated upon HPs60 treatment, while hsa-miR-1247-3p was downregulated. Furthermore, HPs60 remarkably suppressed the expression of cyclinD1, cyclinD2, cyclinD3, cyclinE1 and Bcl-2, which are the targets of miR-195-5p and miR-497-5p. Correspondingly, HPs60 promoted expression of caspase-2, a target gene of miR-1247-3p. All of these results suggest that HPs60 may explore as natural bioactive ingredients and reveal the potential therapeutic targets responsible for HPs60 against CRC.

Activity tracking isolation of Gelsemium elegans alkaloids and evaluation of their antihuman gastric cancer activity in vivo

• Forty-two Gelsemium elegans alkaloids (GEAs) were isolated in an antineoplastic activity tracking method and structure identified. • GEA-9 induced mitochondria-mediated apoptosis by increasing Bax/Bcl-2 and p53/MDM2 expression ratio in MKN-45 cells. • GEA-9 inhibited the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. • GEA-9 inhibited the growth of MKN-45 cells xenograft tumors. Gastric cancer (GC) is a fatal form of tumor that has no effective therapeutic agents. In this study, an anticancer tracking method was applied to ioslate Gelsemium elegans alkaloids (GEAs) in vitro . GEA-9 (11-methoxy- N 1 -demethoxygelsemamide) exhibited the highest antiproliferative activity in MKN-45 cells and induced mitochondrion-mediated apoptosis, It decreased the mitochondrial potential and increased the Bax/Bcl-2 and p53/MDM2 expression ratio. It also induced cell cycle arrest in MKN-45 cells by regulating the cyclin expression. It Inhibited the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway and the growth of MKN-45 cell xenograft tumors, which were associated with light chain 3-II activation and mTOR inhibition. In summary, we first demonstrated the antineoplastic effect of GEA-9 in vitro and in vivo . We also elucidated that the underlying antineoplastic mechanisms of GEA-9 involved the crosstalk between apoptosis and autophagy by inhibiting of the PI3K/Akt/mTOR pathway. This study might provide a rationale for future clinical applications of GEAs as a chemotherapeutic agents for GC. GEAs were isolated in a bio-assay guided method, and GEA-9 involve cross talk between apoptosis and autophagy via inhibition of the PI3K/Akt/mTOR pathway in gastric cancer MKN-45 cells.

Rosmarinic Acid Decreases the Malignancy of Pancreatic Cancer Through Inhibiting Gli1 Signaling

BackgroundRosmarinic acid (RA) has been shown to exert anti-tumor effects on various types of cancer. However, its roles in the treatment of pancreatic ductal adenocarcinoma (PDAC) and the underlying mechanisms remain elusive. PurposeThe present study aimed to investigate the therapeutic effects of RA on PDAC as well as the underlying mechanisms. Study designEvaluation of the effects of RA on PDAC malignancy both in vitro and in vivo. MethodsCell counting kit 8 (CCK8) assay, colony formation assay, 5-Ethynyl-2′-deoxyuridine (EDU) incorporation assay, cell cycle analysis, and apoptosis assay were conducted to assess the inhibitory effect of RA on PDAC cell proliferation. Meanwhile, western blotting and RT-qPCR assay were performed to detect the target gene expression at protein and mRNA levels, respectively. Moreover, the in vivo anti-tumor activities of RA were assayed in an xenograft mouse model of PDAC. ResultsRA dramatically down-regulated Gli1 and its downstream targets. Further studies showed that RA prevents the nuclear translocation of Gli1, while promoting the degradation of cytosolic Gli1 via the proteasome pathway. Moreover, we observed that RA induced G1/S cell cycle arrest and apoptosis in the PDAC cells through regulating the expression of P21, P27, CDK2, Cyclin E, Bax, and Bcl-2, it inhibited the PDAC cell migration and invasion via E-cadherin and MMP-9. Notably, Gli1 overexpression markedly reversed the above RA-induced effects on PDAC cells, whereas Gli1 knockdown enhanced the effects. Additionally, the in vivo assays demonstrated that RA suppresses the tumor growth of PDAC presumably by inhibiting Gli1. ConclusionWe provided evidence that RA restrained the nuclear translocation of Gli1 and facilitates Gli1 degradation via proteasome pathway, reducing the malignancy of PDAC cells. These findings implicated RA as a therapeutic agent for PDAC.

Trehalose inhibits proliferation while activates apoptosis and autophagy in rat airway smooth muscle cells

Trehalose is a disaccharide with multiple important biological activities. In many cell types, Trehalose regulates the physiological behaviors of proliferation, apoptosis and autophagy. But the effects of trehalose on ASMCs have never been reported. Here, we showed that trehalose activated autophagy of ASMCs at low dose, inhibited proliferation and induced apoptosis of ASMCs at high dose. Further study, we found the cell cycle was arrested in S and G2\\M phases, the expression of CyclinA1 and CyclinB1 decreased. Then, we investigated the ratio of Bcl-2/Bax was drastically reduced. Next, we detected an important transcription factor TFEB, which is closely related to autophagy. We found TFEB was highly activated with trehalose treatment. And many downstream autophagy-related genes of TFEB were also up-regulated. In summary, trehalose plays an important role on the regulation of proliferation, apoptosis and autophagy of ASMCs.

Comparative Study on Inhibition of Pancreatic Cancer Cells by Resveratrol Gold Nanoparticles and a Resveratrol Nanoemulsion Prepared from Grape Skin.

Resveratrol, a phenolic compound possessing vital biological activities such as anti-cancer, is present abundantly in grape skin, a waste produced during the processing of grape juice. The objectives of this study were to prepare resveratrol-gold nanoparticles and a resveratrol nanoemulsion from grape skin and study their inhibition effects on pancreatic cancer cells BxPC-3. The spherical-shaped citrate gold nanoparticles (GNPs) and resveratrol-gold nanoparticles (R-GNPs) were, respectively, prepared with a surface plasmon resonance peak at 528 and 538 nm, mean particle size of 20.8 and 11.9 nm, and zeta-potential at −32.7 and −66.7 mV, by controlling an appropriate concentration of citrate/resveratrol and gold chloride as well as stirring time and temperature. The resveratrol nanoemulsion, composed of soybean oil, Tween 80, and sucrose fatty acid ester in glycerol and water, possessed a high storage stability with a mean particle size of 14.1 nm, zeta-potential of −49.7 mV, and encapsulation efficiency of 95.5%. An antiproliferation study revealed that both R-GNPs and resveratrol nanoemulsion could effectively inhibit the growth of pancreatic cancer cells BxPC-3, with the latter showing a higher inhibition effect. Western blot analysis implied that both can down-regulate expressions of cyclin A, cyclin B, CDK1, and CDK2 and up-regulate expressions of p53 and p21, accompanied by enhancing cytochrome C expression, decreasing BcL-2 expression, increasing Bax expression, and leading to the elevation of caspase-8, caspase-9, and caspase-3 activities for cell apoptosis execution. Future research is needed to study the inhibition of pancreatic tumors in vivo by R-GNPs and resveratrol nanoemulsions.

Zinc is an essential element for the maintenance of redox homeostasis and cell cycle in murine auditory hair cells

A nutrition deficiency is one of the various causes of hearing loss. Zinc is an essential element for cell proliferation, antioxidant reactions, and the maintenance of hearing ability. Our previous studies have reported that the auditory brainstem response (ABR) threshold is increased in mice fed with zinc-deficient diets. However, the molecular mechanism of zinc involved in auditory system remains to be elucidated. In the present study, we examined the detrimental effects of zinc deficiency on cell cycle progression in murine auditory cells (HEI-OC1). The treatment of HEI-OC1 cells with 0.5 μM TPEN (N,N,N′,N′-Tetrakis (2-pyridylmethyl) ethylenediamine) for 24 h inhibited cell proliferation, accumulation of reactive oxygen species (ROS), and induction of apoptosis. The cell proliferation block was caused by a G1/S phase arrest. Supplementation of the cell growth medium with 5 μM ZnCl2 after exposure to TPEN attenuated ROS accumulation and the arrest caused by the zinc deficiency. The ABR threshold was elevated in mice fed with a zinc-deficient diet. Additionally, we observed an increased expression of p21 and decreased expression of cyclin E and pRb in the spiral ganglion (SG), the organ of Corti (OC), Limbus (L), and stria vascularis (SV) in the zinc-deficient mouse cochlea. These results indicated that zinc is an essential nutrient for proliferation via the cell cycle and that a dysregulation of the cell cycle may cause hearing loss.

Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats.

Cathepsin D (CTSD), the major lysosomal aspartic protease that is widely expressed in different tissues, potentially regulates the biological behaviors of various cells. Follicular granulosa cells are responsive to the increase of ovulation number, hence indirectly influencing litter size. However, the mechanism underlying the effect of CTSD on the behaviors of goat granulosa cells has not been fully elucidated. This study used immunohistochemistry to analyze CTSD localization in goat ovarian tissues. Moreover, western blotting was applied to examine the differential expression of CTSD in the ovarian tissues of monotocous and polytocous goats. Subsequently, the effects of CTSD knockdown on cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific traits, including bone morphogenetic protein receptor IB (BMPR-IB), follicle-stimulating hormone (FSHR), and inhibin α (INHA), were determined in granulosa cells. Results showed that CTSD was expressed in corpus luteum, follicle, and granulosa cells. Notably, CTSD expression in the monotocous group was significantly higher than that in the polytocous group. In addition, CTSD knockdown could improve granulosa cell proliferation, inhibit cell apoptosis, and significantly elevate the expression of proliferating cell nuclear antigen (PCNA) and B cell lymphoma 2 (Bcl-2), but it lowered the expression of Bcl-2-associated X (Bax) and caspase-3. Furthermore, CTSD knockdown significantly reduced the ratios of cells in the G0/G1 and G2/M phases but substantially increased the ratio of cells in the S phase. The expression levels of cyclin D2 and cyclin E were elevated followed by the obvious decline of cyclin A1 expression. However, the expression levels of BMPR-IB, FSHR, and INHA clearly increased as a result of CTSD knockdown. Hence, our findings demonstrate that CTSD is an important factor affecting the litter size trait in goats by regulating the granulosa cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific trait.

LncRNA ASAP1-IT1 enhances cancer cell stemness via regulating miR-509-3p/YAP1 axis in NSCLC

BackgroundNon-small cell lung cancer (NSCLC) is a major cause of cancer-related death worldwide, and cancer stem cell is responsible for the poor clinical outcome of NSCLC. Previous reports indicated that long noncoding RNAs (lncRNAs) play important roles in maintaining cancer stemness, however, the underlying mechanisms remain unclear. This study investigates the role of ASAP1 Intronic Transcript 1 (ASAP1-IT1) in cancer cell stemness of NSCLC.MethodsThe expression of ASAP1-IT1, microRNA-509-3p (miR-509-3p) and apoptosis-/stemness-related genes was analyzed by qRT-PCR in NSCLC tissues, cancer cells and spheres of cancer stem cells. Knockdown of ASAP1-IT1 or overexpression of miR-509-3p in NSCLC cells by infection or transfection of respective plasmids. Sphere formation and colony formation were used to detect NSCLC stem cell-like properties and tumor growth in vitro. Luciferase reporter assays, RNA immunoprecitation (RIP) and qRT-PCR assays were used to analyze the interaction between lncRNA and miRNA. The expression of expression of regulated genes of ASAP1-IT1/miR-509-3p axis was evaluated by qRT-PCR and Western blot. The NSCLC xenograft mouse model was used to validate the role of ASAP1-IT1 in NSCLC stemness and tumor growth in vivo.ResultsASAP1-IT1 was up-regulated in NSCLC tissues, cancer cells, and in spheres of A549-derived cancer stem cells. Downregulation of ASAP1-IT1 or overexpression of miR-509-3p significantly decreased cell colony formation and stem cell-like properties of A549-dereived stem cells with decreased expression of stem cell biomarkers SOX2, CD34, and CD133, and suppressing the expression of cell growth-related genes, Cyclin A1, Cyclin B1, and PCNA. Furthermore, knockdown of ASAP1-IT1 or overexpression of miR-509-3p repressed tumor growth in nude mice via reducing expression of tumorigenic genes. ASAP1-IT1 was found to interact with miR-509-3p. Moreover, overexpression of ASAP1-IT1 blocked the inhibition by miR-509-3p on stem cell-like properties and cell growth of A549-dereived stem cells both in vitro and in vivo. Finally, the level of YAP1 was regulated by ASAP1-IT1 and miR-509-3p.ConclusionsYAP1-involved ASAP1-IT1/miR-509-3p axis promoted NSCLC progression by regulating cancer cell stemness, and targeting this signaling pathway could be is a promising therapeutic strategy to overcome NSCLC stemness.

Synthetic Tryptanthrin Derivatives Induce Cell Cycle Arrest and Apoptosis via Akt and MAPKs in Human Hepatocellular Carcinoma Cells

Trytanthrin, found in Ban-Lan-Gen, is a natural product containing an indoloquinazoline moiety and has been shown to possess anti-inflammatory and anti-viral activities. Chronic inflammation and hepatitis B are known to be associated with the progression of hepatocellular carcinoma (HCC). In this study, a series of tryptanthrin derivatives were synthesized to generate potent anti-tumor agents against HCC. This effort yielded two compounds, A1 and A6, that exhibited multi-fold higher cytotoxicity in HCC cells than the parent compound. Flow cytometric analysis demonstrated that A1 and A6 caused S-phase arrest and downregulated the expression of cyclin A1, B1, CDK2, and p-CDC2. In addition to inducing caspase-dependent apoptosis, A1 and A6 exhibited similar regulation of the phosphorylation or expression of multiple signaling targets, including Akt, NF-κB, and mitogen-activated protein kinases. The anti-tumor activities of A1 and A6 were also attributable to the generation of reactive oxygen species, accompanied by an increase in p-p53 levels. Therefore, A1 and A6 have potential clinical applications since they target diverse aspects of cancer cell growth in HCC.

Primary high-grade serous ovarian cancer cells are sensitive to senescence induced by carboplatin and paclitaxel in vitro

BackgroundVarious types of normal and cancer cells undergo senescence in response to carboplatin and paclitaxel, which are considered the gold standard treatments in ovarian cancer management. Surprisingly, the effect of these drugs on ovarian cancer cell senescence remained unknown.MethodsThe experiments were conducted on primary high-grade serous ovarian cancer cells. Molecular markers of senescence were evaluated using cytochemistry and immunofluorescence. Cell cycle distribution was analyzed using flow cytometry. Expression of cyclins and signaling pathways was tested using western blot. Telomere length and telomerase activity were measured using qPCR, and the colocalization of telomeres with DNA damage foci using immuno-FISH. Oxidative stress-related parameters were quantified using appropriate fluorescence probes. Production of cancerogenic agents was analyzed using qPCR and ELISA.ResultsCarboplatin applied with paclitaxel induces senescence of ovarian cancer cells in vitro. This activity was reflected by permanent G2/M growth arrest, a high fraction of cells expressing senescence biomarkers (SA-β-Gal and γ-H2A.X), upregulated expression of p16, p21, and p53 cell cycle inhibitors, and decreased expression of cyclin B1. Neither telomere length nor telomerase activity changed in the senescent cells, and the majority of DNA damage was localized outside telomeres. Moreover, drug-treated cancer cells exhibited increased production of STAT3 protein, overproduced superoxide and peroxides, and increased mitochondrial mass. They were also characterized by upregulated ANG1, CCL11, IL-6, PDGF-D, TIMP-3, TSP-1, and TGF-β1 at the mRNA and/or protein level.ConclusionsOur findings imply that conventional chemotherapy may elicit senescence in ovarian cancer cells, which may translate to the development of a cancer-promoting phenotype, despite the inability of these cells to divide.

CDCA7 Facilitates Tumor Progression by Directly Regulating CCNA2 Expression in Esophageal Squamous Cell Carcinoma.

Background CDCA7 is a copy number amplified gene identified not only in esophageal squamous cell carcinoma (ESCC) but also in various cancer types. Its clinical relevance and underlying mechanisms in ESCC have remained unknown.MethodsTissue microarray data was used to analyze its expression in 179 ESCC samples. The effects of CDCA7 on proliferation, colony formation, and cell cycle were tested in ESCC cells. Real-time PCR and Western blot were used to detect the expression of its target genes. Correlation of CDCA7 with its target genes in ESCC and various SCC types was analyzed using GSE53625 and TCGA data. The mechanism of CDCA7 was studied by chromatin immunoprecipitation (ChIP), luciferase reporter assays, and rescue assay.ResultsThe overexpression of CDCA7 promoted proliferation, colony formation, and cell cycle in ESCC cells. CDCA7 affected the expression of cyclins in different cell phases. GSE53625 and TCGA data showed CCNA2 expression was positively correlated with CDCA7. The knockdown of CCNA2 reversed the malignant phenotype induced by CDCA7 overexpression. Furthermore, CDCA7 was found to directly bind to CCNA2, thus promoting its expression.ConclusionsOur results reveal a novel mechanism of CDCA7 that it may act as an oncogene by directly upregulating CCNA2 to facilitate tumor progression in ESCC.