Search

Copy number variations (CNVs) play a vital role in regulating genes expression and tumorigenesis. We explored the copy number alterations in early-stage lung adenocarcinoma using high-throughput sequencing and nucleic acid flight mass spectrometry technology, and found that 8q22.1-22.2 is frequently amplified in lung adenocarcinoma tissues. COX6C localizes on the region and its expression is notably enhanced that driven by amplification in lung adenocarcinoma. Knockdown of COX6C significantly inhibits the cell proliferation, and induces S-G2/M cell cycle arrest, mitosis deficiency and apoptosis. Moreover, COX6C depletion causes a deficiency in mitochondrial fusion, and impairment of oxidative phosphorylation. Mechanistically, COX6C-induced mitochondrial deficiency stimulates ROS accumulation and activates AMPK pathway, then leading to abnormality in spindle formation and chromosome segregation, activating spindle assemble checkpoint, causing mitotic arrest, and ultimately inducing cell apoptosis. Collectively, we suggested that copy amplification-mediated COX6C upregulation might serves as a prospective biomarker for prognosis and targeting therapy in patients with lung adenocarcinoma., (© 2024. The Author(s).)

Background: In recent years, gene expression-based analysis has been used for disease biomarker discovery, providing ways for better diagnosis, leading to improvement of clinical treatment efficacy. This study aimed to explore the role of miR-16-5p and ANLN in breast cancer (BC)., Methods: Cohort datasets of BC were obtained from the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) and analyzed by bioinformatics tools. qRT-PCR and western blotting were applied to validate ANLN and its protein expression. A dual-luciferase reporter assay was used to prove the regulatory relationship of miR-16-5p and ANLN. Finally, MTT, wound healing, Transwell invasion and flow cytometry analyses of the cell cycle and apoptosis were performed to assess cell proliferation, migration, invasion, cell cycle and apoptosis, respectively., Results: A total of 195 differentially expressed genes (DEGs) and 50 overlapping microRNAs (miRNAs) were identified. Among these DEGs and miRNAs, ANLN, associated with poor overall survival in BC, overlapped in the GSE29431, GSE42568, TCGA and GEPIA2 databases. Moreover, ANLN was highly expressed, while miR-16-5p was lower in BC cells than in breast epithelial cells. Then, we confirmed that ANLN was directly targeted by miR-16-5p in BC cells. Over-expression of miR-16-5p and knock-down of ANLN remarkably inhibited cell proliferation and migration as well as cell invasion, arrested the cells in G2/M phase and induced apoptosis in BC cells., Conclusions: These findings suggest that miR-16-5p restrains proliferation, migration and invasion while affecting cell cycle and promotes apoptosis by regulating ANLN, thereby providing novel candidate biomarkers for the diagnosis and treatment of BC., (© 2021. The Author(s).)

Previous studies reported the critical role of the brefeldin A-inhibited guanine nucleotide exchange protein 3-prohibitin 2 (BIG3-PHB2) complex in modulating estrogen signaling activation in breast cancer cells, yet its pathophysiological roles in osteosarcoma (OS) cells remain elusive. Here, we report a novel function of BIG3-PHB2 in OS malignancy. BIG3-PHB2 complexes were localized mainly in mitochondria in OS cells, unlike in estrogen-dependent breast cancer cells. Depletion of endogenous BIG3 expression by small interfering RNA (siRNA) treatment led to significant inhibition of OS cell growth. Disruption of BIG3-PHB2 complex formation by treatment with specific peptide inhibitor also resulted in significant dose-dependent suppression of OS cell growth, migration, and invasion resulting from G2/M-phase arrest and in PARP cleavage, ultimately leading to PARP-1/apoptosis-inducing factor (AIF) pathway activation-dependent apoptosis in OS cells. Subsequent proteomic and bioinformatic pathway analyses revealed that disruption of the BIG3-PHB2 complex might lead to downregulation of inner mitochondrial membrane protein complex activity. Our findings indicate that the mitochondrial BIG3-PHB2 complex might regulate PARP-1/AIF pathway-dependent apoptosis during OS cell proliferation and progression and that disruption of this complex may be a promising therapeutic strategy for OS., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Breast cancer (BC) threatens the life and health of women worldwide because of its high morbidity and mortality. The present study aimed to explore the biological functions and potential mechanism of BTNL9 in BC. RNA sequence and clinical data extracted from the Kaplan-Meier plotter database and The Cancer Genome Atlas (TCGA) were utilized to analyze the relationship between the expression level of BTNL9 in BC tissues and clinicopathological features and the effects of BTNL9 expression on the prognosis of BC. The diagnostic efficacy of BTNL9 expression was estimated by receiver operating characteristic (ROC) curve analysis. The mRNA and protein expression levels of BTNL9 in BC cell lines and in BC tissue were determined by quantitative real-time PCR (qPCR) and western blotting, respectively. The functions of BTNL9 were measured by colony formation, CCK-8, Transwell, flow cytometry and EdU assays. Western blotting analysis was also performed to explore the latent mechanism of BTNL9. The results showed that the expression of BTNL9 declined in BC tissues and cell lines. Low expression of BTNL9 was significantly associated with early progression of T stage, relapse-free survival (RFS), and poor overall survival (OS). Ectopic expression of BTNL9 inhibited cell proliferation, colony formation and metastasis and induced apoptosis in BC, while knockdown of BTNL9 had the opposite result. Furthermore, BTNL9 blocked BC cells in the G2/M phase via the P53/CDC25C and P53/GADD45 pathways. Our results suggest that BTNL9 may play a tumor-suppressive role in BC and has the potency to become a new biomarker for early BC diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: To investigate how EHD2 influences the development of colon cancer. Methods: Immunohistochemistry of 90 colon cancer tissue specimens were determined the expression of EHD2. The lentivirus-EHD2-transfected colon cancer cells were conducted to evaluate the biological behaviors. Results: EHD2 was closely associated with clinic pathological parameters ( p  < 0.001). EHD2 upregulation was relative with a longer overall survival. The results of the univariate and multivariate analyses indicated that EHD2 could be an independent prognosis marker. EHD2 overexpression suppressed cell invasion and proliferation, but enhanced cell apoptosis and cell cycle arrest. Conclusions: EHD2 might represent a therapeutic target of colon cancer.IMPACT STATEMENT What is already known on this subject? Membrane trafficking is crucial for cell proliferation, differentiation and apoptosis, especially tumorigenesis and development. EHD2 proteins play an important role in the regulation of membrane trafficking in endocytosis. EHD2 has been suggested to participate in the occurrence of some malignancies. What are the new findings? EHD2 could be an independent prognosis marker in colon cancer. EHD2 overexpression suppressed cell invasion and proliferation, but enhanced cell apoptosis and cell cycle arrest in vitro . EHD2 overexpression markedly increased the expression of EMT marker E-cadherin in colon cancer. How might it impact on clinical practice in the foreseeable future? EHD2 overexpression may inhibit tumorigenesis in colon cancer through the modulation of E-cadherin, the critical marker of EMT which is closely related to invasion and distant metastasis of tumor cells.

Candidate oncogene placenta specific 8 (PLAC8) has been identified to participate in different cellular process and human diseases. However, the effects of PLAC8 on cell proliferation and migration in human kidney cancer (KC) remained unclear. In current study, physiological effects of PLAC8 in immortalized human embryonic kidney cell line (HEK293T) were investigated in vitro. Two PLAC8 knockout (KO) cell lines were established via CRISPR/Cas9-mediated methods combined with fluorescence activated single cell sorting. To classify the characteristic of PLAC8 during cell proliferation and migration in HEK293T, cellular proliferative activity was analyzed by cell counting and colony formation assay. Cell cycle distribution was analyzed by flow cytometry. Cellular motile activity was analyzed by wound-healing and migration assay. Further underlying molecular mechanism was explored via western blot. With the KO cell lines, it was found that PLAC8 KO could decrease cell proliferation. Moreover, the inhibitory effects of PLAC8 KO on cell proliferation were associated with a G2/M arrest in cell cycle progression concomitant with a remarkable inhibition of Cyclin B1 and elevation of Cyclin A. The alteration of cell cycle proteins and E-cadherin might further associate with the enhancement of cell motility. Our study revealed a novel role for PLAC8 in cell proliferation and migration of HEK293T cells, which might shed light on further study of PLAC8 on human KC.

Bryonia dioica Jacq. is a climbing perennial herb with tuberous roots which is widely used in traditional medicine in Algeria for the treatment of cancer. The present study aimed to evaluate the apoptogenic activity and phytochemical composition of the aqueous extract of B. dioica roots growing in Algeria. The cytotoxic effect of B. dioica aqueous decoction against breast cancer MDA‑MB‑231 cells was evaluated by an MTT assay. Apoptosis induction was assessed by an Annexin V‑fluorescien iosthiocyanate assay. Propidium iodide staining of cell DNA was used to assess the effects on the cell cycle. In addition to UV‑Visible (UV‑vis) analysis, the major compounds of the extracts were determined using liquid chromatography‑mass spectrometric analyses. Our results showed that the B. dioica aqueous extract induced cell death in a time‑dependent manner. The highest inhibitory effect was produced at concentrations of 50 µg/ml or higher after 72 h of treatment (91.15±0.71%). Furthermore, the extract induced apoptosis of MDA‑MB‑231 cells. At 250 µg/ml, 64.61% of the treated MDA‑MB‑231 cells were apoptotic. This was accompanied by cell cycle arrest at G2/M phase. The percentage of cells in G2/M increased from 15.7% (untreated cells) to 59.13% (50 µg/ml) and 58.51% (250 µg/ml). The UV‑vis absorption spectra of B. dioica aqueous extract showed two absorption bands characteristic of the flavonol skeleton; 350‑385 nm (Band I) and 250‑280 nm (Band II). Myricetin (2,5,7,3,4,5‑pentahydroxylflavonol) was found to be the major compound in the B. dioica aqueous extract. These findings suggest that B. dioica could be considered a promising source for developing novel therapeutics against breast cancer.

Though the advancement of chemotherapy drugs alleviates the progress of cancer, long-term therapy with anticancer agents gradually leads to acquired multidrug resistance (MDR), which limits the survival outcomes in patients. It was shown that dihydromyricetin (DMY) could partly reverse MDR by suppressing P-glycoprotein (P-gp) and soluble resistance-related calcium-binding protein (SORCIN) independently. To reverse MDR more effectively, a new strategy was raised, that is, circumventing MDR by the coadministration of DMY and ondansetron (OND), a common antiemetic drug, during cancer chemotherapy. Meanwhile, the interior relation between P-gp and SORCIN was also revealed. The combination of DMY and OND strongly enhanced antiproliferative efficiency of adriamycin (ADR) because of the increasing accumulation of ADR in K562/ADR-resistant cell line. DMY could downregulate the expression of SORCIN and P-gp via the ERK/Akt pathways, whereas OND could not. In addition, it was proved that SORCIN suppressed ERK and Akt to inhibit P-gp by the silence of SORCIN, however, not vice versa. Finally, the combination of DMY, OND, and ADR led to G2/M cell cycle arrest and apoptosis via resuming P53 function and restraining relevant proteins expression. These fundamental findings provided a promising approach for further treatment of MDR., (© 2018 Wiley Periodicals, Inc.)

Cytoplasmic dynein-1 is a large minus-end-directed microtubule motor complex involved in membrane trafficking, organelle positioning, and microtubule organization. The roles of dynein light intermediate chains (DLICs; DLIC1 and DLIC2) within the complex are, however, still largely undefined. In this study, we investigated the possible roles of DLICs in epithelial homeostasis and colon cancer development. Mutant clonal analysis of Drosophila Dlic in the follicular epithelium of Drosophila ovary showed defects in nuclear positioning, epithelial integrity, and apical cell polarity. Consistently, knockdown of human DLIC1 and DLIC2 in colon carcinoma cells resulted in damaged epithelial organization, disturbed lumen formation, and impaired apical polarity establishment in three-dimensional cell culture. Depletion of DLIC1 and DLIC2 led to reduced proliferation, enhanced apoptosis rates, disrupted mitotic spindle assembly, and induction of G2/M arrest in cell cycle progression. Moreover, reduced levels of DLIC1 in contrast to DLIC2 impaired the migratory ability. On the other hand, immunohistochemical examination of human colorectal tissue samples and further colorectal cancer dataset analysis showed a significant upregulation for DLIC1 in tumors, whereas DLIC2 expression was unchanged. In addition, the overexpression of DLIC1 caused increased proliferation, decreased apoptosis and enhanced migration, whereas DLIC2 overexpression did not result in any significant changes. Together, these results indicate that DLIC1 and DLIC2 contribute to the establishment and maintenance of epithelial homeostasis. Furthermore, these findings present the first evidence that DLIC1 and DLIC2 have distinct roles in colon cancer development and that DLIC1 may contribute to proliferative overgrowth and migratory characteristics., (© 2019 Federation of European Biochemical Societies.)

Objective: Forkhead box O1 (FoxO1) plays a crucial role in the development of many tumors. Cyclin D kinase (CDK) 1 could influence the nuclear export and activity of FoxO1 through phosphorylation of serine (S) 249 . However, the effects of S 249 phosphorylation in the development of glioma remain unclear. The aim of the present study is to assess the function of FoxO1:S 249 V mutant, which was converted S 249 phosphorylation site into valine (V) residues in the glioma development., Methods: FoxO1-knockdown U251 glioma cells (U251-KD cells) were established by infection of retrovirus particles with FoxO1 siRNA and FoxO1 restored cells (FoxO1:S 249 V) were obtained by re-introduction of FoxO1:S 249 V cDNA. We detected mRNA expression by real-time PCR, and cell cycle arrest and apoptosis by flow cytometric assay, and cell proliferation by BrdU assay and CCK-8 assay. The protective effects of FoxO1:S 249 V were detected by the xenograft tumor formation assay., Results: The FoxO1 mRNA expression was significantly decreased in the glioma specimens (n = 24). The U251-KD cells showed downregulation of p27 and Bim, while the phosphorylation of CDK1 was upregulated. FoxO1:S 249 V cells inhibited the phosphorylation of S 249 , and induced G2/M cell cycle arrest, following reduced cell growth and increased apoptosis. Moreover, FoxO1:S 249 V expression effectively inhibits the glioma growth., Conclusion: Our findings suggest that the forced FoxO1:S 249 V suppressed the cell growth through G2/M cell cycle arrests and increased apoptosis in glioma.

Overexpression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a key molecule of glucose metabolism in cytoplasm, has been found in various tumors. Emerging evidence has suggested that PFKFB3 is also located in the nucleus and apparent in regulatory functions other than glycolysis. In this study, we found that PFKFB3 expression is associated with hepatocellular carcinoma (HCC) growth and located mainly in the nucleus of tumor cells. PFKFB3 overexpression was associated with large tumor size (p = 0.04) and poor survival of patients with HCC (p = 0.027). Knockdown of PFKFB3 inhibited HCC growth, not only by reducing glucose consumption but also by damaging the DNA repair function, leading to G2/M phase arrest and apoptosis. In animal studies, overexpression of PFKFB3 is associated with increased tumor growth. Mechanistically, PFKFB3 silencing decreased AKT phosphorylation and reduced the expression of ERCC1, which is an important DNA repair protein. Moreover, PFK15, a selective PFKFB3 inhibitor, significantly inhibited tumor growth in a xenograft model of human HCC. PFKFB3 is a potential novel target in the treatment of HCC.

FBW7 is an E3 ubiquitin ligase and frequently mutated in various types of cancer. As a component of SCF ubiquitin ligase complex, FBW7 usually targets the substrates via K11 or K48-linked ubiquitylation and subsequent degradation of target proteins. Nevertheless, the role of FBW7 in mediating non-degradable ubiquitin signaling remains unknown in human cancers. In this study, we identified γ-catenin as a new binding protein of FBW7 by TAP-MS (tandem affinity purification-mass spectrum). Knockdown of FBW7 did not affect the stability of γ-catenin, but significantly reduced the K63-linked ubiquitin of γ-catenin, resulting in decreased expression of γ-catenin downstream gene 14-3-3σ. Rescue experiment revealed that γ-catenin promoted the expression of 14-3-3σ in a K63-linked ubiquitin signaling dependent manner. Furthermore, we showed that FBW7 cooperated with γ-catenin to inhibit G2/M cell cycle transition and cell proliferation. Taken together, our study uncovered a novel mechanism that FBW7 associated with γ-catenin and promoted its K63-linked ubiquitylation, providing new insights in understanding the role of FBW7 in inhibiting G2/M cell cycle transition and tumor cell proliferation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ubiquitin specific peptidase 39 (USP39) serves important roles in mRNA processing and is involved in tumorigenesis of multiple solid malignancies. However, the influence and underlying mechanism of USP39 on human renal cell carcinomas (RCC) remain to be elucidated. The current study investigated the functional roles of USP39 in human RCC cell lines. siRNA‑mediated RNA interference was used to downregulate USP39 in RCC cells. CCK‑8, wound healing and invasion assays were performed to assess the proliferative ability and metastatic potential. The cell cycle distribution and apoptosis were evaluated by flow cytometry. The activity of signaling pathways and the expression of cell cycle‑related proteins were detected by western blot analysis. The siRNA‑directed RNA interference targeting USP39 could effectively downregulate the expression level of USP39 in two RCC cell lines. Depletion of USP39 by siRNA significantly suppressed cell growth and decreased invasive capacity of RCC cells. Silencing of USP39 induced cell apoptosis and cell cycle arrest at G2/M phase. Additionally, the expression levels of apoptotic and G2/M phase‑related proteins were notably decreased following depletion of USP39. Mechanistically, downregulation of USP39 blocked the activation of Akt and extracellular signal regulated kinase signaling pathways in RCC cells. These findings indicate that USP39 may serve as an oncogenic factor in RCC and could be a potential therapeutic candidate for human RCCs.

Background: Dynamin-related protein 1 (Drp1) mediates mitochondrial fission. Recently, several studies have shown that Drp1 plays an important role in some cancers. However, little is known about Drp1 in cutaneous squamous cell carcinoma (SCC)., Objective: To investigate the role of Drp1 in the tumorigenesis of cutaneous SCCs., Methods and Results: We investigated cell proliferation, cell cycle, mitochondrial morphology, and MAPK signaling pathway using cutaneous SCC A431 and DJM1 cells that were transfected with shRNA vectors targeting Drp1. The Drp1 gene-knockdown SCC cells showed lower cell proliferation than scramble-control cells, as assessed by direct cell counting and clonogenic assays. DNA content analysis showed Drp1 knockdown to cause G2/M arrest. Morphologically, the depletion of Drp1 resulted in an elongated, hyper-fused mitochondrial network. The MEK inhibitor PD325901 suppressed cell proliferation, as well as inhibiting the phosphorylation of ERK 1/2 and Drp1 Ser616 . Also, PD325901 caused the dysregulation of the mitochondrial network. In tumor xenografts of DJM1 cells, the knockdown of Drp1 suppressed tumor growth in vivo, and clinically, the expression levels of Drp1 were higher in cutaneous SCCs than in normal epidermis, and correlated positively with the advanced clinical stages., Conclusion: Our results reveal a crucial function for Drp1 in regulating tumor growth, mitochondrial morphology, and cell cycle in cutaneous SCC, suggesting that Drp1 could be a novel target for skin tumor therapies., (Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Regulation of tumour cell proliferation by molecular chaperones is still a complex issue. Here, the role of the HSP90 molecular chaperone TRAP1 in cell cycle regulation was investigated in a wide range of human breast, colorectal, and lung carcinoma cell lines, and tumour specimens. TRAP1 modulates the expression and/or the ubiquitination of key cell cycle regulators through a dual mechanism: (i) transcriptional regulation of CDK1, CYCLIN B1, and MAD2, as suggested by gene expression profiling of TRAP1-silenced breast carcinoma cells; and (ii) post-transcriptional quality control of CDK1 and MAD2, being the ubiquitination of these two proteins enhanced upon TRAP1 down-regulation. Mechanistically, TRAP1 quality control on CDK1 is crucial for its regulation of mitotic entry, since TRAP1 interacts with CDK1 and prevents CDK1 ubiquitination in cooperation with the proteasome regulatory particle TBP7, this representing the limiting factor in TRAP1 regulation of the G2-M transition. Indeed, TRAP1 silencing results in enhanced CDK1 ubiquitination, lack of nuclear translocation of CDK1/cyclin B1 complex, and increased MAD2 degradation, whereas CDK1 forced up-regulation partially rescues low cyclin B1 and MAD2 levels and G2-M transit in a TRAP1-poor background. Consistently, the CDK1 inhibitor RO-3306 is less active in a TRAP1-high background. Finally, a significant correlation was observed between TRAP1 and Ki67, CDK1 and/or MAD2 expression in breast, colorectal, and lung human tumour specimens. This study represents the first evidence that TRAP1 is relevant in the control of the complex machinery that governs cell cycle progression and mitotic entry and provides a strong rationale to regard TRAP1 as a biomarker to select tumours with deregulated cell cycle progression and thus likely poorly responsive to novel cell cycle inhibitors. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Ubiquitin-like with plant homeodomain and ring finger domains, 1 (UHRF1) is overexpressed in a variety of tumor tissues and is negatively correlated with prognosis of patients with cancers, yet so far, a comprehensive study of UHRF1 in hepatocellular carcinoma (HCC) has not been conducted. The present study was designed to explore the expression of UHRF1, associated clinical implications, and its possible functions in HCC. Reverse transcription-polymerase chain reaction and immunohistochemical staining were used to detect UHRF1 expression in HCC specimens including cancerous and noncancerous tissues. Associations of UHRF1 expression with demographic and clinicopathologic features in HCC were analyzed, and the effects of RNA interference of UHRF1 on cell proliferation, cell cycle, apoptosis, and migration were investigated in vitro and in vivo. UHRF1 mRNA and protein expression were both upregulated and negatively correlated with prognosis in HCC patients. Furthermore, inhibition of proliferation, migration, invasion, and epithelial-mesenchymal transition progression were observed in vitro and in vivo after UHRF1 knockdown, moreover, G2/M arrest was detected in HCC cells. In conclusion, elevated UHRF1 expression contributes to poor prognosis by promoting cell proliferation and metastasis in HCC.

Previous reports have shown that expression of BARD1δ, a deletion-bearing isoform of BARD1, correlates with tumor aggressiveness and progression. We show that expression of BARD1δ induces cell cycle arrest in vitro and in vivo in non-malignant cells. We investigated the mechanism that leads to proliferation arrest and found that BARD1δ overexpression induced mitotic arrest with chromosome and telomere aberrations in cell cultures, in transgenic mice, and in cells from human breast and ovarian cancer patients with BARD1 mutations. BARD1δ binds more efficiently than BARD1 to telomere binding proteins and causes their depletion from telomeres, leading to telomere and chromosomal instability. While this induces cell cycle arrest, cancer cells lacking G2/M checkpoint controls might continue to proliferate despite the BARD1δ-induced chromosomal instability. These features of BARD1δ may make it a genome permutator and a driver of continuous uncontrolled proliferation of cancer cells.

Objective: Carfilzomib is a second-generation tetrapeptide epoxyketone proteasome inhibitor used in current clinical therapy of hematologic malignancies. The mechanism of proteasome inhibition in endometrial cancer is not very clear. Carfilzomib inhibition of type I endometrial carcinoma cell proliferation by inducing cell cycle arrest at the G2/M phase was investigated in our study., Materials and Methods: HEC-1-A and Ishikawa endometrial carcinoma cell lines and three tumor cell lines were treated by different concentrations of carfilzomib. Methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability. Flow cytometry was used to analyze the cell cycle. Western blot was used to detect proteins involved in cell cycle progression., Results: Carfilzomib impaired viability of myelogenous leukemia cell line K562, cervical cancer cell line HeLa, hepatocellular carcinoma cell line SMCC-7721, and endometrial carcinoma cell lines HEC-1-A and Ishikawa. The cell cycle was arrested at the G2/M phase in carfilzomib-treated HEC-1-A endometrial carcinoma cells, while it was arrested at both S and G2/M phases in carfilzomib-treated Ishikawa cells. Carfilzomib treatment significantly induced p21 Waf1/ Cip1 and p27, while substantially reduced cyclin D3 and cyclin-dependent kinase 1., Conclusion: This study showed that carfilzomib inhibited endometrial cancer proliferation by upregulating cyclin-dependent kinase inhibitors p21 Waf1/Cip1 and p27 Kip1 , and reducing cyclin-dependent kinase 1 to arrest the cell cycle at the G2/M phase., (Copyright © 2016. Published by Elsevier B.V.)

Galangin is an active pharmacological ingredient from propolis and Alpinia officinarum Hance, and has been reported to have anti-cancer and antioxidative properties. Berberine, a major component of Berberis vulgaris extract, exhibits potent anti-cancer activities through distinct molecular mechanisms. However, the anticancer effect of galangin in combination with berberine is still unknown. In the present study, we demonstrated that the combination of galangin with berberine synergistically resulted in cell growth inhibition, apoptosis and cell cycle arrest at G2/M phase with the increased intracellular reactive oxygen species (ROS) levels in oesophageal carcinoma cells. Pretreatment with ROS scavenger promoted the apoptosis dramatically induced by co-treatment with galangin and berberine. Treatment with galangin and berberine alone caused the decreased expressions of Wnt3a and β-catenin. Interestingly, combination of galangin with berberine could further suppress Wnt3a and β-catenin expression and induce apoptosis in cancer cells. Additionally, in nude mice with xenograft tumors, the combinational treatment of galangin and berberine significantly inhibited the tumor growth without obvious toxicity. Overall, galangin in combination with berberine presented outstanding synergistic anticancer role in vitro and in vivo, indicating that the beneficial combination of galangin and berberine might provide a promising treatment for patients with oesophageal carcinoma., (Copyright © 2016. Published by Elsevier Masson SAS.)

Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko)) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages., (© 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.)

The dietary compound phenethyl isothiocyanate (PEITC), an important tumoricidal component found in cruciferous vegetables, exhibits strong anticancer and chemopreventive effects in a variety of tumors. However, its role in human laryngeal cancer is unclear. The aim of the present study was to investigate whether PEITC exhibits anticancer properties in human laryngeal carcinoma Hep-2 cells in vitro and to identify the potential molecular mechanisms. The results showed that treatment of Hep-2 cells with PEITC significantly inhibited cell proliferation in a dose- and time-dependent manner, promoted apoptosis with concurrent G2/M cell cycle arrest and inhibited cell invasion in a dose-dependent manner. These effects were accompanied by significant alterations in the expression levels of key proteins associated with pro-survival signaling pathways, including PI3K, Akt, ERK, NF-κB, Bcl, Bax, cyclin B, CDK4 and CDK6. Importantly, these effects were not reflected in 16HBE normal human bronchial epithelial cells, suggesting a safe range of treatment concentrations between 0 and 10 µM PEITC. In summary, PEITC exhibited significant anticancer effects against human laryngeal cancer cells in vitro with low toxicological impact on normal bronchial epithelial cells. This was achieved through dysregulation of key proteins involved in the occurrence and development of tumors, thereby offering a valuable contribution to future strategies for the treatment and screening of patients with laryngocarcinoma.

LASP-1 is an actin-binding protein that regulates cytoskeletal dynamics and cell migration. LASP-1 was previously identified in a cDNA library from metastatic breast cancer samples. This protein has since been detected in multiple human cancers, including liver cancer, gastric cancer and pancreatic cancer. S100P is a small calcium-binding protein in the S100 protein family that regulates cellular, physiological and pathological processes in various cancers. However, the clinical significance of LASP-1 and S100P expression in gallbladder cancer (GBC) is not yet clear. In our study, we focused on the clinical significance, biological function and mechanism of LASP-1 in gallbladder cancer and detected LASP-1 and S100P overexpression in GBC tissues. The expression of LASP-1 was significantly correlated with poor prognosis in GBC patients (P < 0.05). Furthermore, down-regulation of LASP-1 expression resulted in the obvious inhibition of proliferation and migration and caused cell cycle arrest by down-regulating S100P via the PI3K/AKT pathway; in mice, tumor volume was significantly decreased. In conclusion, LASP-1 may act as an oncogene to regulate the expression of S100P to influence cellular functions in GBC. LASP-1 could serve as a genetic treatment target in GBC patients., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Endogenous bornavirus-like nucleoprotein elements (EBLNs) have been discovered in the genomes of various animals including humans, whose functions have been seldom studied. To explore the biological functions of human EBLNs, we constructed a lentiviral vector expressing a short-hairpin RNA against human EBLN1, which successfully inhibited EBLN1 expression by above 80% in infected human oligodendroglia cells (OL cells). We found that EBLN1 silencing suppressed cell proliferation, induced G2/M phase arrest, and promoted apoptosis in OL cells. Gene expression profiling demonstrated that 1067 genes were up-regulated, and 2004 were down-regulated after EBLN1 silencing. The top 10 most upregulated genes were PI3, RND3, BLZF1, SOD2, EPGN, SBSN, INSIG1, OSMR, CREB3L2, and MSMO1, and the top 10 most-downregulated genes were KRTAP2-4, FLRT2, DIDO1, FAT4, ESCO2, ZNF804A, SUV420H1, ZC3H4, YAE1D1, and NCOA5. Pathway analysis revealed that these differentially expressed genes were mainly involved in pathways related to the cell cycle, the mitogen-activated protein kinase pathway, p53 signaling, and apoptosis. The gene expression profiles were validated by using quantitative reverse transcription polymerase chain reaction (RT-PCR) for detecting these 20 most-changed genes. Three genes closely related to glioma, RND3, OSMR, and CREB3L2, were significantly upregulated and might be the key factors in EBLN1 regulating the proliferation and apoptosis of OL cells. This study provides evidence that EBLN1 plays a key role in regulating cell life and death, thereby opening several avenues of investigation regarding EBLN1 in the future.

Poly (ADP-ribose) polymerase1 (PARP1) has been reported as a possible target for chemotherapy in many cancer types. However, its action mechanisms and clinical implications for gastric cancer survival are not yet fully understood. Here, we investigated the effect of PARP1 inhibition in the growth of gastric cancer cells. PARP1 inhibition by Olaparib or PARP1 siRNA could significantly attenuate growth and colony formation of gastric cancer cells, and which were mediated through induction of G2/M cell cycle arrest but not apoptosis. FOXO3A expression was induced by PARP1 inhibition, suggesting that FOXO3A might be one of downstream target of the PARP1 effect on gastric cancer cell growth. In addition, by performing tissue microarrays on the 166 cases of gastric cancer patients, we could observe that the expression status of PARP1 and FOXO3A were significantly associated with overall survival (OS) and relapse-free survival (RFS). Strikingly, combined expression status of PARP1 and FOXO3A showed better prediction for patient's clinical outcomes. The patient group with PARP1+/FOXO3A- expression had the worst prognosis while the patient group with PARP1-/FOXO3A+ had the most favorable prognosis (OS: P = 6.0 × 10(-9), RFS: P = 2.2 × 10(-8)). In conclusion, we suggest that PARP1 and FOXO3A play critical roles in gastric cancer progression, and might have therapeutic and/or diagnostic potential in clinic.

Aim: To determine the expression of microRNA-210 (miR-210) in hepatocellular carcinoma (HCC) and to examine its role using HCC cells., Methods: The expression of miR-210 was determined in 21 pairs of HCC samples and the corresponding surrounding non-tumor tissues. The effects of miR-210 on proliferation and cell cycle progression were examined using HepG2 and HuH7 cells. Over-expression and inhibition of miR-210 was achieved by transfection of the cells with miR-210 mimic or inhibitor. Luciferase reporter constructs were used to identify the miR-210 interacting site on Yes1. Yes1 expression was examined after miR-210 transfection, as well as in the HCC samples., Results: miR-210 was significantly up-regulated by 3.4 fold (P < 0.01) in the tumor samples. The over-expression of miR-210 significantly reduced cell proliferation compared to the mock-treated cells (68.9% ± 7.4% and 53.6% ± 5.0%, P < 0.05 for the HepG2 and HuH7 cells respectively). Analysis of the HuH7 cells transfected with miR-210 mimic by flow cytometry showed that the cells took a longer time to reach the G2/M phase. The interaction between miR-210 and the 3'UTR of the Yes1 transcript was confirmed using a luciferase reporter assay. Over-expression of miR-210 reduced the expression of Yes1 protein in both HuH7 and HepG2 cells. Tumors with a greater than four-fold increase in the expression of miR-210 showed consistently lower expressions of Yes1 in the tumors. In nocodazole-treated cells with a significant G2/M cell population, Yes1 protein was significantly reduced and pre-inhibition of miR-210 in HuH7 cells was able to prevent the reduction of Yes1 protein expression. Knock-down of Yes1 by siRNA also led to reduced cell proliferation (70.8% ± 7.5%, P < 0.05 in the HuH7 cells)., Conclusion: Up-regulation of miR-210 inhibits cell proliferation. Yes1 is a target of miR-210 and affects cell proliferation in HCC.

Primary squamous cell carcinoma of the thyroid is a rare and aggressive type of neoplasm, which is routinely treated with surgery; however, despite this, survival time is not commonly more than six months. Thus, the aim of the present study was to determine the efficacy of pseudolaric acid (PAB) as a therapeutic agent. PAB is an antitubulin agent, and in the present study, inhibition of the SW579 thyroid squamous cell carcinoma cell line by PAB was investigated. PAB was found to inhibit SW579 cell growth in a time‑ and dose‑dependent manner via interference in α‑tubulin polymerization. However, the inhibitory role of PAB in SW579 cells was not predominantly due to apoptosis, but was due to the cytostatic status resulting from cell cycle arrest. The present study proposes that this is the underlying mechanism of the antitumor properties of PAB. During cytostatis, autophagy was activated to sustain cell survival and SW579 cell migration was inhibited. Nuclear p53 expression was observed to be reduced, however the role of reduced p53 requires further investigation. Therefore, PAB induced cytostasis, which inhibited SW579 cell growth and therefore may function as an antitubulin therapeutic agent.

Breast cancer is the most common cause of death among women. KIF3C, a member of kinesin superfamily, functions as a motor protein involved in axonal transport in neuronal cells. To explore the expression, regulation and mechanism of KIF3C in breast cancer, 4 breast cancer cell lines and 93 cases of primary breast cancer and paired adjacent tissues were examined. Immunohistochemistry, Real Time Polymerase Chain Reaction (RT-PCR), Western blot, flow cytometry, short hairpin RNA (shRNA) interference, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation techniques and xenograft mice model were used. We found that KIF3C was over-expressed in breast cancer tissues and such high KIF3C expression was also associated with tumor recurrence and lymph node metastasis. Silencing of KIF3C by shRNA inhibited epithelial-mesenchymal transition and metastasis by inhibiting TGF-β signaling and suppressed breast cancer cell proliferation through inducing G2/M phase arrest. The tumor size was smaller and the number of lung metastatic nodules was less in KIF3C depletion MDA-MB-231 cell xenograft mice than in negative control group. These results suggested that high expression of KIF3C in breast cancer may be associated with the tumor progression and metastasis., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The coiled coil is a superhelical structural protein motif involved in a diverse array of biological functions, and the abnormal expression of the coiled-coil domain containing proteins has a direct link with the phenotype of tumor cell migration, invasion and metastasis. The aim of this study was to investigate the critical role of Coiled-coil domain-containing protein 34 (CCDC34) in bladder carcinogenesis, which has never been reported to date. Here, we found CCDC34 expression was elevated in bladder cancer tissues and cell lines. The knockdown of CCDC34 via lentivirus-mediated siRNA significantly suppressed bladder cancer cells proliferation and migration, and induced cell cycle arrest at G2/M phase and increased apoptosis in vitro. In addition, CCDC34 knockdown suppressed bladder tumor growth in nude mice. Moreover, CCDC34 silencing decreased the phosphorylation of MEK, ERK1/2, JNK, p38 and Akt, and the expressions of c-Raf and c-Jun, indicating MAPK and AKT pathways (ERK/MAPK, p38/MAPK, JNK/MAPK and PI3K/Akt) might be involved in CCDC34 regulation of bladder cancer cell proliferation and migration. Our findings revealed for the first time a potential oncogenic role for CCDC34 in bladder carcinoma pathogenesis and it may serve as a biomarker or even a therapeutic target for bladder cancer.

MiR-125a has been characterized as a tumor suppressor in several cancers. However, the role of miR-125a in cervical cancer is unknown. In this study, we found the expression of miR-125a was downregulated in cervical cancer patients, and negatively correlated with the tumor size, FIGO stage, and preoperative metastasis. Kaplan-Meier analysis showed that miR-125a expression predicted favorable outcome for cervical cancer patients. Dual luciferase assays identified the STAT3 gene as a novel direct target of miR-125a. Functional studies showed that miR-125a overexpression significantly suppressed the growth, invasion and epithelial-mesenchymal transition (EMT) of cervical cancer cells both in vitro and in vivo via decreasing STAT3 expression. Moreover, miR-125a conferred to G2/M cell cycle arrest, accompanied by inhibition of several G2/M checkpoint proteins. Mechanistically, inactivation of miR-125a during cervical carcinogenesis was caused by HPV suppression of p53 expression. Clinically, STAT3, the expression of which, predicted poorer outcome, was inversely correlated with miR-125a in cervical cancer. These data highlight the importance of miR-125a in the cell proliferation and progression of cervical cancer, and indicate that miR-125a may be a useful therapeutic target for cervical cancer.

2-oxogluatrate and Fe(II)-dependent oxygenase domain-containing protein 1 (OGFOD1) was recently revealed to be a proline hydroxylase of RPS23 for translational termination. However, OGFOD1 is nuclear, whereas translational termination occurs in the cytoplasm, raising the possibility of another function of OGFOD1 in the nucleus. In this study, we demonstrate that OGFOD1 is involved in cell cycle regulation. OGFOD1 knockdown in MDA-MB-231 breast cancer cells significantly impeded cell proliferation and resulted in the accumulation of G1 and G2/M cells by decreasing the mRNA levels of G1/S transition- and G2/M-related transcription factors and their target genes. We also confirmed that OGFOD1 is highly expressed in breast cancer tissues by bioinformatic analysis and immunohistochemistry. Thus, we propose that OGFOD1 is required for breast cancer cell proliferation and is associated with poor prognosis in breast cancer.

Robust mechanisms to control cell proliferation have evolved to maintain the integrity of organ architecture. Here, we investigated how two critical proliferative pathways, Myc and E2f, are integrated to control cell cycles in normal and Rb-deficient cells using a murine intestinal model. We show that Myc and E2f1-3 have little impact on normal G1-S transitions. Instead, they synergistically control an S-G2 transcriptional program required for normal cell divisions and maintaining crypt-villus integrity. Surprisingly, Rb deficiency results in the Myc-dependent accumulation of E2f3 protein and chromatin repositioning of both Myc and E2f3, leading to the 'super activation' of a G1-S transcriptional program, ectopic S phase entry and rampant cell proliferation. These findings reveal that Rb-deficient cells hijack and redeploy Myc and E2f3 from an S-G2 program essential for normal cell cycles to a G1-S program that re-engages ectopic cell cycles, exposing an unanticipated addiction of Rb-null cells on Myc.

Adrenal carcinoma (ACC) is a rare neoplasm with a poor outcome. Aberrant expression of β-catenin has been found in approximatively 30% of ACC. We herein studied its effects on the growth of the human ACC cell line H295R. The cells were infected with short hairpin RNA (shRNA)-mediated silencing β-catenin. Two shRNAs used induced down-regulation of β-catenin protein levels. The expression of these shRNAs decreased cell growth and increased H295R cells in S and G2/M phases. This cytostatic effect is due to a decrease of phosphorylated MAPK and to an up-regulation expression of the cyclin-dependent kinase inhibitors p57(KIP2), p21(CIP) and p27(KIP1). In addition, the knockdown of β-catenin decreased phosphorylated Akt and increased apoptosis. Finally, loss of β-catenin was sufficient to induce the reversal of the epithelial-to-mesenchymal transition. We then transplanted these genetically modified H295R cells in Scid mice. Tumor growth suppression was achieved by the two shRNAs showing in vitro efficacy. Proliferation was not reduced in silenced tumors. In contrast, p57, p27 and p21 proteins were found expressed at high levels in silenced tumors along with an increase in apoptotic cells. These findings indicate that β-catenin loss in H295R cells inhibits tumor growth by inducing transcriptional and functional changes.

Culture of mouse spermatogonial stem cells (mSSCs) contributes to understanding the mechanisms of mammalian spermatogenesis. Several key growth factors such as GDNF and FGF2 have been known to be essential for the proliferation of cultured mSSCs. However, additional factors regulating SSC proliferation remain to be identified. In this study, we report that IGF-1R signaling is required for the proliferation of cultured mSSCs by promoting the G2/M progression of the cell cycle. IGF-1 and its receptor IGF-1R are expressed in cultured mSSCs as well as in isolated Sertoli cells and interstitial cells. Blockage of IGF-1R signaling either by knockdown of IGF-1R or by the IGF-1R-specific inhibitor picropodophyllin (PPP) significantly reduced the proliferation of mSSCs, increased their apoptosis, and impaired their stem cell activity in an insulin-independent manner. PPP treatment of mSSCs blocked the G2/M progression. In contrast, both GDNF withdrawal and FGF2 signaling blockade decreased the entry of mSSCs into their S phases. Consistently, IGF-1 promoted the G2/M progression of thymidine-treated mSSCs, which were arrested at G1/S boundary synchronously; while GDNF and/or FGF2 stimulated their entry into the S phase. Moreover, IGF-1 activated the phosphorylation of AKT but not that of ERK1/2 in mSSCs. These results indicate that IGF-1R signaling stimulates the proliferation of mSSCs using a distinct mechanism from those by GDNF and FGF2, and will contribute to the establishment of a chemically defined culture system.

To avoid cell cycle arrest or apoptosis, rapidly proliferating cancer cells have to promote DNA double strand break (DSB) repair to fix replication stress induced DSBs. Therefore, developing drugs blocking homologous recombination (HR) and nonhomologous end joining (NHEJ) - 2 major DSB repair pathways - holds great potential for cancer therapy. Over the last few decades, much attention has been paid to explore drugs targeting DSB repair pathways for cancer therapy. Here, using 2 well-established reporters for analyzing HR and NHEJ efficiency, we found that both HR and NHEJ are elevated in hepatoma cell lines Hep3B and HuH7 compared with normal liver cell lines Chang liver and QSG-7701. Our further study found that Harmine, a natural compound, negatively regulates HR but not NHEJ by interfering Rad51 recruitment, resulting in severe cytotoxicity in hepatoma cells. Furthermore, NHEJ inhibitor Nu7441 markedly sensitizes Hep3B cells to the anti-proliferative effects of Harmine. Taken together, our study suggested that Harmine holds great promise as an oncologic drug and combination of Harmine with a NHEJ inhibitor might be an effective strategy for anti-cancer treatment.

Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of human cancer. Ellagic acid (EA) has been known for its chemopreventive activity against various cancers and numerous investigations have shown its apoptotic activity both in vivo and in vitro. The present study was focused to elucidate the anticancerous effect and the mode of action of EA against HCT-15 colon adenocarcinoma cells. Cell viability was assessed using trypan blue assay at different concentrations. EA also promoted cell cycle arrest substantially at G2/M phase in HCT-15 cells. The activities of alkaline phosphatase and lactate dehydrogenase were decreased upon EA treatment, which shows the antiproliferative and the cytotoxic effects, respectively. The production of reactive oxygen intermediates, which were examined by 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA), increased with time, after treatment with EA. In further studies, EA inhibited proliferation-associated markers proliferating cell nuclear antigen and cyclin D1. The induction of apoptosis was accompanied by a strong inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway by EA. The expression of PI3K and pAkt was down-regulated in EA-treated cells, compared to normal cells. Further, EA promoted the expression of Bax, caspase-3, and cytochrome c, and suppression of Bcl-2 activity in HCT-15 cells that was determined by western blot analysis. Increased annexin V apoptotic cells and DNA fragmentation also accompanied EA-induced apoptosis. In conclusion, EA increased the production of ROS, decreased cell proliferation, and induced apoptosis in HCT-15 cells, and thus can be used as an agent against colon cancer.

Background/aims: The mitotic kinesin superfamily protein KIF14 is essential for cytokinesis and chromosome segregation, and increased KIF14 expression is related to a variety of human cancers. However, the role of KIF14 in the development and malignant progression of astrocytomas and the underlying mechanisms remain unclear. The present study examined the relation between KIF14 and the pathogenesis of malignant astrocytoma., Methods and Results: The role of KIF14 in astrocytoma development and progression was investigated by analyzing KIF14 expression using SYBR Green quantitative real-time RT-PCR, western blotting and immunohistochemistry in human astrocytoma and normal brain tissues. KIF14 expression was higher in astrocytoma samples, and was positively correlated with pathological grade and proliferative activity indicated by Ki-67 staining. SiRNA knockdown of KIF14 inhibited tumor growth in vitro and in vivo, attenuated anchorage-independent growth, and induced G2/M phase arrest, cytokinesis failure and apoptosis in glioblastoma cell lines in association with decreased AKT phosphorylation and activity., Conclusions: The upregulation of KIF14 in astrocytoma is associated with disease severity, and suppression of KIF14 inhibits cell proliferation and induces apoptosis through a mechanism involving the inactivation of AKT signaling, suggesting that KIF14 plays an important role in astrocytoma tumorigenesis and could be a promising molecular target for anticancer therapy., (© 2015 The Author(s) Published by S. Karger AG, Basel.)

Pancreatic cancer is one of the most aggressive solid malignancies with a dismal survival rate. Recent studies have shown that high expression levels of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript-1 (MALAT-1) correlate with several solid tumors. However, the underlying molecular mechanisms and its clinical significance in pancreatic cancer remain to be elucidated. In the present study, our results showed that MALAT-1 expression levels were upregulated in pancreatic cancer tissues compared with adjacent noncancerous controls. Consistently, higher expression level of MALAT-1 was found in all seven pancreatic cancer cell lines relative to the human pancreatic ductal epithelial cell. Further function analysis revealed that downregulation of MALAT-1 could inhibit tumor cell proliferation and decrease cell migration and invasion in vitro. The underlying mechanisms are possibly involved in inducing G2/M cell cycle arrest, promoting cell apoptosis, suppressing epithelial-mesenchymal transition and reducing cancer stem-like properties. In conclusion, this study indicated that MALAT-1 may serve as an oncogenic lncRNA that is involved in malignancy phenotypes of pancreatic cancer. Therefore, it may be used as a potential therapeutic target.

In prostate cancer (PCa), the functional synergy between androgen receptor (AR) and nuclear factor-κ B (NF-κB) escalates the resistance to therapeutic regimens and promotes aggressive tumor growth. Although the underlying mechanisms are less clear, gene regulatory abilities of coactivators can bridge the transcription functions of AR and NF-κB. The present study shows that MYST1 (MOZ, YBF2 and SAS2, and TIP60 protein 1) costimulates AR and NF-κB functions in PCa cells. We demonstrate that activation of NF-κB promotes deacetylation of MYST1 by sirtuin 1. Further, the mutually exclusive interactions of MYST1 with sirtuin 1 vs AR regulate the acetylation of lysine 16 on histone H4. Notably, in AR-lacking PC3 cells and in AR-depleted LNCaP cells, diminution of MYST1 activates the cleavage of poly(ADP-ribose) polymerase and caspase 3 that leads to apoptosis. In contrast, in AR-transformed PC3 cells (PC3-AR), depletion of MYST1 induces cyclin-dependent kinase (CDK) N1A/p21, which results in G2M arrest. Concomitantly, the levels of phospho-retinoblastoma, E2F1, CDK4, and CDK6 are reduced. Finally, the expression of tumor protein D52 (TPD52) was unequivocally affected in PC3, PC3-AR, and LNCaP cells. Taken together, the results of this study reveal that the functional interactions of MYST1 with AR and NF-κB are critical for PCa progression.

Histamine (HA) is a neurotransmitter synthesized in most mammalian tissues exclusively by histidine decarboxylase enzyme. Among the plethora of actions mediated by HA, the modulatory effects on steroidogenesis and proliferation in Leydig cells (LCs) have been described recently. To determine whether the effects on LCs reported could be extrapolated to all steroidogenic systems, in this study, we assessed the effect of this amine on adrenal proliferation and steroidogenesis, using two adrenocortical cell lines as experimental models, murine Y1 cells and human NCI-H295R cells. Even when steroidogenesis was not modified by HA in adrenocortical cells, the biogenic amine inhibited the proliferation of H295R cells. This action was mediated by the activation of HRH1 subtype and an increase in the production of inositol phosphates as second messengers, causing cell-cycle arrest in the G2/M phase. These results indicate a new role for HA in the proliferation of human adrenocortical cells that could contribute to a better understanding of tumor pathology as well as to the development of new therapeutic agents.

B7-H1, a member of the B7 family of proteins, is hypothesised to play an important role in the immune escape of tumours through its binding to the PD-1 receptor on activated T and B cells. By inducing T lymphocyte apoptosis, tumour cells can suppress an effective antitumour immune response. Although the immunosuppressive effect of B7-H1 has been studied in many tumours, its other biological functions remain unclear. We previously demonstrated a high expression level of this molecule in pancreatic cancer patient samples and its antiapoptotic effect in pancreatic ductal adenocarcinoma (PDA) cells. The aim of the present study was to investigate the possible role of B7-H1 in the proliferation of PDA cells. Functional studies were performed using pancreatic cell lines that were genetically engineered to express high or low levels of B7-H1, and we found that the overexpression of B7-H1 through plasmid transfection in PDA cells promoted cell proliferation. Conversely, the short-hairpin RNA (shRNA) knockdown of B7-H1 inhibited PDA cell proliferation. Further analyses of the cell cycle and cell cycle-related molecules confirmed this result. Taken together, our results indicate that the upregulation of B7-H1 in pancreatic cancer cells promotes proliferation and accelerates carcinogenesis; these data, therefore, provide insights into the effects of B7-H1 overexpression on pancreatic tumour cells.

While cell proliferation is an essential part of embryonic development, cells within an embryo cannot proliferate freely. Instead, they must balance proliferation and other cellular events such as differentiation and morphogenesis throughout embryonic growth. Although the G1 phase has been a major focus of study in cell cycle control, it is becoming increasingly clear that G2 regulation also plays an essential role during embryonic development. Here we discuss the role of Cdc25, a key regulator of mitotic entry, with a focus on several recent examples that show how the precise control of Cdc25 activity and the G2/M transition are critical for different aspects of embryogenesis. We finish by discussing a promising technology that allows easy visualization of embryonic and adult cells potentially regulated at mitotic entry, permitting the rapid identification of other instances where the exit from G2 plays an essential role in development and tissue homeostasis.

Centromere protein A (CENP-A) is a homolog of histone H3 that epigenetically marks the heterochromatin of chromosomes. CENP-A is a critical component of the cell cycle machinery that is necessary for proper assembly of the mitotic spindle. However, the role of CENP-A in the heart and cardiac progenitor cells (CPCs) has not been previously studied. This study shows that CENP-A is expressed in CPCs and declines with age. Silencing CENP-A results in a decreased CPC growth rate, reduced cell number in phase G 2/M of the cell cycle, and increased senescence associated β-galactosidase activity. Lineage commitment is not affected by CENP-A silencing, suggesting that cell cycle arrest induced by loss of CENP-A is a consequence of senescence and not differentiation. CENP-A knockdown does not exacerbate cell death in undifferentiated CPCs, but increases apoptosis upon lineage commitment. Taken together, these results indicate that CPCs maintain relatively high levels of CENP-A early in life, which is necessary for sustaining proliferation, inhibiting senescence, and promoting survival following differentiation of CPCs.

Hematopoietic pre-B-cell leukemia transcription factor (PBX)-interacting protein (HPIP) has been shown to play a role in cancer development and progression. However, the detailed role of HPIP in cancer cell growth and the exact mechanism by which HPIP regulates cancer cell proliferation remains unclear. Here, we report that HPIP is overexpressed in most of 328 liver cancer patients and regulates hepatoma cell proliferation through G2/M checkpoint activation. HPIP increased anchorage-dependent and -independent growth of human liver cancer cell lines. The amino acid region 531-631 of HPIP was important for its modulation of liver cancer cell growth. The increased effects of HPIP on liver cancer cell proliferation were associated with activation of the G2/M cell-cycle concomitant with a marked increase of cyclin B1 and the inhibition of the negative G2/M phase regulator GADD45α. HPIP knockdown dramatically suppressed the growth of HepG2 liver cancer cells in nude mice. These data highlight the important role of HPIP in liver cancer cell growth and suggest that HPIP may be a good target for liver cancer therapy., (© 2013 International Union of Biochemistry and Molecular Biology.)

In the Western world, colorectal cancer (CRC) is the third most common cancer with poor prognosis. To identify the proteins and to elucidate the possible mechanisms involved in colorectal carcinogenesis, 2-DE coupled with MS/MS analysis were employed to compare the global protein profile between CRC and individual matched normal tissues from 8 CRC patients. Of 36 proteins identified, carbonic anhydrase II (CA II) was one of most significantly altered and its downregulation in CRC tissues was verified by RT-PCR, western blotting and immunohistochemistry methods, suggesting that CA II may serve as a potential biomarker for CRC diagnosis. To investigate the function and mechanisms of CA II in CRC, a stable SW480 colorectal cancer cell line overexpressing CA II was established. It was shown that overexpression of CA II remarkably suppressed tumor cell growth both in vitro and in vivo, which was in part interpreted by cell cycle arrest at G0/G1 and G2 phase. Further mechanism analysis revealed that the sensitivity of colorectal cancer cells to chemotherapy drugs could be increased by CA II overexpression. Taken together, these data suggest that CA II may be a potential biomarker for early diagnosis of CRC and the results may contribute to a better understanding of the molecular mechanism of CRC and colorectal cancer treatment.

Growing evidence indicates that some tumor suppressive miRNAs are subject to epigenetic modifications during carcinogenesis. Here, we found that a large miRNA cluster of C19MC was upregulated in HCC cells after combined treatment with DNA methylation inhibitor and histone deacetylase inhibitor. MiR-517a and miR-517c were strikingly different from the remaining 41 miRNAs in C19MC. Ectopic expression of MiR-517a and miR-517c inhibited cell proliferation by blocking G2/M transition, whereas down-regulation of miR-517a and miR-517c facilitated cell growth. We further showed Pyk2 is a target of miR-517a and miR-517c and both the miRNAs are downregulated in HCC samples. These data collectively suggest that down-regulation of both miR-517a and miR-517c contribute to HCC development through regulating Pyk2., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

TTRAP is a multi-functional protein that is involved in multiple aspects of cellular functions including cell proliferation, apoptosis and the repair of DNA damage. Here, we demonstrated that the lentivirus-mediated overexpression of TTRAP significantly inhibited cell growth and induced apoptosis in osteosarcoma cells. The ectopic TTRAP suppressed the growth and colony formation capacity of two osteosarcoma cell lines, U2OS and Saos-2. Cell apoptosis was induced in U2OS cells and the cell cycle was arrested at G2/M phase in Saos-2 cells. Exogenous expression of TTRAP in serum-starved U2OS and Saos-2 cells induced an increase in caspase-3/-7 activity and a decrease in cyclin B1 expression. In comparison with wild-type TTRAP, mutations in the 5'-tyrosyl-DNA phosphodiesterase activity of TTRAP, in particular TTRAP(E152A), showed decreased inhibitory activity on cell growth. These results may aid in clarifying the physiological functions of TTRAP, especially its roles in the regulation of cell growth and tumorigenesis.

The SAM and SH3 domain containing 1 (SASH1) gene was originally identified as a potential tumor suppressor gene in breast cancer, mapped on chromosome 6q24.3. The expression of SASH1 plays a prognostic role in human colon cancer. Its expression is frequently downregulated in several human malignancies. However, the biological function of SASH1 in melanoma cells is yet to be determined. In this study, in order to investigate the tumor suppressive effects of the SASH1 gene, an A-375 stable melanoma cell line was established, overexpressing the SASH1 gene. The stable cell line was examined using proliferation assay, apoptosis assay, cell cycle analysis and real-time PCR. The results indicated that the tumor suppressive activity of SASH1 derived from G2/M arrest in A-375 cells, and that the phosphorylation of Cdc2 or the disruption of cyclin B-Cdc2 binding may be responsible for the G2/M arrest.

ZNF403, also known as GGNBP2 (gametogenetin binding protein 2), is a highly conserved gene implicated in spermatogenesis. However, the exact biological function of ZNF403 is not clear. In this study, we identified the role of ZNF403 in cell proliferation and cell-cycle regulation by utilizing short hairpin RNA (shRNA)-mediated knockdown. ZNF403-specific shRNA expressing helper-dependent adenoviral vector (HD-Ad-ZNF403-shRNA) was constructed and transduced human cell lines. ZNF403 mRNA and protein expression levels were inhibited as evidenced by real-time PCR and western blot analyses. Noticeably, we found that knockdown of ZNF403 expression suppressed cell proliferation compared to the non-target shRNA and vector controls. Furthermore, cell-cycle analysis demonstrated that downregulation of ZNF403 promoted G2/M cell-cycle arrest in a dose-dependent manner. Moreover, human cell-cycle real-time PCR array revealed that ZNF403 knockdown influenced the expression profile of genes in cell-cycle regulation. Among these genes, western blot analysis confirmed the protein up-regulation of p21 and down-regulation of MCM2 in response to the ZNF403 knockdown. Additionally, knockdown of ZNF403 also showed an anti-carcinogenetic effect on anchorage-independent growth by colony formation assay and tumor cell migration by wound-healing assay with human laryngeal cancer cell line Hep-2 cells. Altogether, our findings suggest an essential role of ZNF403 in cell proliferation and provide a new insight into the function of ZNF403 in regulating the G2/M cell-cycle transition.

The proliferative effect of some compounds that are aryl hydrocarbon (Ah) receptor ligands was studied on hepatoma 27 cells with absent expression of Ah receptor. Compounds of the polycyclic aromatic hydrocarbon (PAH) class benzo/a/pyrene, 3-methylcholanthrene, 7,12-dimethylbenz/a/anthracene, and benzo/e/pyrene as well as β-naphthoflavone (β-NF) and chlorinated hydrocarbon Aroclor 1254 were studied. It was found that carcinogenic PAH and β-NF stimulate cell proliferation both under conditions of standard serum content and in a medium with low serum content. More efficient stimulation of proliferation was observed in the case of low serum content. Aroclor 1254 and benzo/e/pyrene did not stimulate cell proliferation. Stimulation of proliferation was accompanied by activation of the ERK1/2-dependent MAP-kinase cascade. Benzo/a/pyrene caused a decrease in the number of cells in G1 phase of the cell cycle and increase in number of cells in G2/M phases under conditions of cell growth in media with low serum content. Carcinogenic PAH and β-NF activated transcription factor AP-1, and in this case activation was more pronounced in cells grown in medium with low serum content. A possible mechanism of activation of proliferation by an Ah receptor-independent pathway is discussed.

Background & Aims: Nucleoplasmic Ca(2+) regulates cell growth in the liver, but the proteins through which this occurs are unknown., Methods: We used Rapid Subtraction Hybridization (RaSH) to subtract genes in SKHep1 liver cells expressing the Ca(2+) buffer protein parvalbumin (PV) targeted to the nucleus, from genes in cells expressing a mutated form of nuclear-targeted PV which has one of two Ca(2+)-binding sites inactivated. The subtraction permitted the selection of genes whose expression was affected by a small alteration in nuclear Ca(2+) concentration., Results: The asparaginyl endopeptidase legumain (LGMN) was identified in this screening. When Ca(2+) was buffered in the nucleus of SKHep1 cells, LGMN mRNA was decreased by 97%, in part by a transcriptional mechanism, and decreased expression at the protein level was observed by immunoblot and immunofluorescence. Treatment with hepatocyte growth factor increased LGMN expression. Knockdown of LGMN by siRNA decreased proliferation of SKHep1 cells by ∼50% as measured both by BrdU uptake and mitotic index, although an inhibitor of LGMN activity did not affect BrdU incorporation. A significant reduction in the fraction of cells in G2/M phase was seen as well. This was associated with increases in the expression of cyclins A and E. Furthermore, LGMN expression was increased in hepatocellular carcinoma cells relative to normal hepatocytes in the same specimens., Conclusions: These findings suggest a new role for LGMN and provide evidence that nuclear Ca(2+) signals regulate cell proliferation in part through the modulation of LGMN expression. Increased expression of LGMN may be involved in liver carcinogenesis., (Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)