Licochalcone A induces cell cycle arrest in human lung squamous carcinoma cells via the PI3K/Akt signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of licochalcone A (LCA) on the proliferation and cell cycle of human lung squamous carcinoma cells and explore its possible molecular mechanism.@*METHODS@#MTT assay was used to detect the changes in proliferation of H226 cells after treatment with different concentrations of LCA for 48 h, and the IC50 of LCA was calculated. Flow cytometry was used to analyze cell cycle changes in H226 cells treated with 10, 20, and 40 μmol/L LCA, and the expressions of cyclin D1, cyclin-dependent kinase CDK2 and CDK4, and p-PI3K, PI3K, p-Akt, and Akt in the treated cells were detected using Western blotting. The effect of intraperitoneal injection of LCA for 24 days on tumor volume and weight was assessed in a BALB/c-nu mouse model bearing lung squamous carcinoma xenografts.@*RESULTS@#MTT assay showed that LCA significantly decreased the viability of H226 cells with an IC50 of 28.3 μmol/L at 48 h. Flow cytometry suggested that LCA treatment induced obvious cell cycle arrest at the G1 phase. LCA treatment also significantly decreased the expressions of cyclin D1, CDK2, and CDK4, and inhibited the phosphorylation of PI3K and Akt in H226 cells. In the tumor-bearing mice, LCA treatment for 24 days significantly reduced the tumor volume and weight.@*CONCLUSION@#LCA is capable of inhibiting the proliferation and inducing cell cycle arrest in lung squamous carcinoma cells possibility by regulating the PI3K/Akt singling pathway.

Overexpression of connexin 40 (Cx40) inhibits the proliferation of H9c2 cardiomyocytes in rats by cell cycle arrest / 细胞与分子免疫学杂志

Objective To establish a stable strain of H9c2 cardiomyocytes overexpressing Cx40 and preliminarily investigate the effect of lentiviral vector-mediated Cx40 protein overexpression on the proliferation of H9c2 cells and its related mechanisms. Methods The Cx40 gene fragment was cloned from H9c2 cells by PCR and linked with lentivirus vector pLVX-IRES-Puro to obtain the recombinant plasmid pLVX-Flag-Cx40. Recombinant lentiviral particles carrying Flag-Cx40 were obtained by cotransfection with packaging plasmids into HEK293T cells. A stable expression strain (H9c2-Flag-Cx40 cell) was screened from infected H9c2 cells by purinomycin. The expression of Cx40 protein was detected by Western blot analysis, and the effect of Cx40 on H9c2 cells proliferation was determined by CCK-8 assay; cell cycle changes were measured by flow cytometry; the expression of the cell cycle protein cyclin D1 was detected by qRT-PCR and Western blot analysis. Co-immunoprecipitation (Co-IP) immunoprecipitation and Western blot analysis were used to identify the binding of Cx40 and Yes associated protein (YAP) in H9c2 cells; cytoplasmic and cytosolic proteins were isolated to detect the effect of Cx40 on the localization of YAP using Western blot analysis. Results Sequencing results showed that the recombinant pLVX-Flag-Cx40 expression vector was successfully established. A stable transfected cell line containing recombinant Flag-Cx40 lentivirus (H9c2-Flag-Cx40 cell) was successfully constructed from H9c2 cells. Compared with the control group, overexpression of Cx40 significantly reduced the proliferation of H9c2 cells, arrested the cell cycle at G0/G1 and reduced cyclin D1 expression. A significant increase in YAP expression was observed in the cytoplasm of the H9c2-Flag-Cx40 stable cell line, while the expression in the nucleus was significantly reduced. Cx40 bound to YAP in the cytoplasm and prevented it from entering the nucleus to play the role of transcriptional coactivation. Conclusion Overexpression of Cx40 induces cell-cycle arrest at G0/G1 phase and inhibits the proliferation in H9c2 cells.

Cetoacidosis diabética como debut de diabetes mellitus inmunomediada en paciente en tratamiento con inhibidores de checkpoint / Diabetic ketoacidosis as debut of immune-mediated diabetes mellitus in a patient in treatment with immune checkpoint inhibitors

Los inhibidores de checkpoint (ICP) son anticuerpos usados en inmunoterapia contra el cáncer. Uno de sus blancos de acción es el receptor de muerte celular programada-1 (PD-1), el cual es importante para mantener la tolerancia inmunitaria. Sin embargo, este mecanismo se asocia a riesgo de eventos adversos relacionados a la inmunidad que pueden afectar a múltiples órganos incluyendo el sistema endocrino. Se describe el caso inhabitual de un paciente que a los 18 meses de terapia con ICP debutó con cetoacidosis diabética (CAD).

Immune checkpoint inhibitors consist in antibodies used in immunotherapy against cancer. One of their targets is the programmed cell death-1 (PD-1) receptor, which is important in maintaining self-tolerance. However, this mechanism is associated with a risk for immune-related adverse events potentially affecting multiple organs, including the endocrine system. We describe the unusual case of a patient who, after 18 months of treatment with an immune checkpoint inhibitor, debuted with diabetic ketoacidosis

Cell cycle related long non-coding RNAs as the critical regulators of breast cancer progression and metastasis

Cell cycle is one of the main cellular mechanisms involved in tumor progression. Almost all of the active molecular pathways in tumor cells directly or indirectly target the cell cycle progression. Therefore, it is necessary to assess the molecular mechanisms involved in cell cycle regulation in tumor cells. Since, early diagnosis has pivotal role in better cancer management and treatment, it is required to introduce the non-invasive diagnostic markers. Long non-coding RNAs (LncRNAs) have higher stability in body fluids in comparison with mRNAs. Therefore, they can be used as efficient non-invasive markers for the early detection of breast cancer (BCa). In the present review we have summarized all of the reported lncRNAs involved in cell cycle regulation in BCa. It has been reported that lncRNAs mainly affect the cell cycle in G1/S transition through the CCND1/CDK4-6 complex. Present review paves the way of introducing the cell cycle related lncRNAs as efficient markers for the early detection of BCa.

Bioactive constituents of animal-derived traditional Chinese medicinal materials for breast cancer: opportunities and challenges / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Breast cancer is globally the most common invasive cancer in women and remains one of the leading causes of cancer-related deaths. Surgery, radiotherapy, chemotherapy, immunotherapy, and endocrine therapy are currently the main treatments for this cancer type. However, some breast cancer patients are prone to drug resistance related to chemotherapy or immunotherapy, resulting in limited treatment efficacy. Consequently, traditional Chinese medicinal materials (TCMMs) as natural products have become an attractive source of novel drugs. In this review, we summarized the current knowledge on the active components of animal-derived TCMMs, including Ophiocordycepssinensis-derived cordycepin, the aqueous and ethanolic extracts of O.sinensis, norcantharidin (NCTD), Chansu, bee venom, deer antlers, Ostreagigas, and scorpion venom, with reference to marked anti-breast cancer effects due to regulating cell cycle arrest, proliferation, apoptosis, metastasis, and drug resistance. In future studies, the underlying mechanisms for the antitumor effects of these components need to be further investigated by utilizing multi-omics technologies. Furthermore, large-scale clinical trials are necessary to validate the efficacy of bioactive constituents alone or in combination with chemotherapeutic drugs for breast cancer treatment.

Glucose-6 phosphatase catalytic subunit inhibits the proliferation of liver cancer cells by inducing cell cycle arrest / 中华肝脏病杂志

Objective: To investigate the effects of glucose-6-phosphatase catalytic subunit (G6PC) recombinant adenovirus on proliferation and cell cycle regulation of liver cancer cells. Methods: Recombinant adenovirus AdG6PC was constructed. Huh7 cells and SK-Hep1 cells were set as Mock, AdGFP and AdG6PC group. Cell proliferation and clone formation assay were used to observe the proliferation of liver cancer cells. Transwell and scratch assay were used to observe the invasion and migration of liver cancer cells. Cell cycle flow cytometry assay was used to analyze the effect of G6PC overexpression on the proliferation cycle of liver cancer cells. Western blot was used to detect the effect of G6PC overexpression on the cell-cycle protein expression in liver cancer cells. Results: The recombinant adenovirus AdG6PC was successfully constructed. Huh7 and SK-Hep1 cells proliferation assay showed that the number of proliferating cells in the AdG6PC group was significantly lower than the other two groups (P < 0.05). Clone formation assay showed that the number of clones was significantly lower in AdG6PC than the other two groups (P < 0.05), suggesting that G6PC overexpression could significantly inhibit the proliferation of liver cancer cells. Transwell assay showed that the number of cell migration was significantly lower in AdG6PC than the other two groups (P < 0.05). Scratch repair rate was significantly lower in AdG6PC than the other two groups (P < 0.05), suggesting that G6PC overexpression can significantly inhibit the invasion and migration of liver cancer cells. Cell cycle flow cytometry showed that G6PC overexpression had significantly inhibited the Huh7 cells G(1)/S phase transition. Western blot result showed that G6PC overexpression had down-regulated the proliferation in cell-cycle related proteins expression. Conclusion: G6PC inhibits the proliferation, cell-cycle related expression, and migration of liver cancer cells by inhibiting the G(1)/S phase transition.

Influence of 6-shogaol potentiated on 5-fluorouracil treatment of liver cancer by promoting apoptosis and cell cycle arrest by regulating AKT/mTOR/MRP1 signalling / 中国天然药物

Currently, chemoresistance seriously attenuates the curative outcome of liver cancer. The purpose of our work was to investigate the influence of 6-shogaol on the inhibition of 5-fluorouracil (5-FU) in liver cancer. The cell viability of cancer cells was determined by MTT assay. Liver cancer cell apoptosis and the cell cycle were examined utilizing flow cytometry. Moreover, qRT-PCR and western blotting was used to analyse the mRNA and protein expression levels, respectively. Immunohistochemistry assays were used to examine multidrug resistance protein 1 (MRP1) expression in tumour tissues. In liver cancer cells, we found that 6-shogaol-5-FU combination treatment inhibited cell viability, facilitated G0/G1 cell cycle arrest, and accelerated apoptosis compared with 6-shogaol or 5-FU treatment alone. In cancer cells cotreated with 6-shogaol and 5-FU, AKT/mTOR pathway- and cell cycle-related protein expression levels were inhibited, and MRP1 expression was downregulated. AKT activation or MRP1 increase reversed the influence of combination treatment on liver cancer cell viability, apoptosis and cell cycle arrest. The inhibition of AKT activation to the anticancer effect of 6-shogaol-5-FU could be reversed by MRP1 silencing. Moreover, our results showed that 6-shogaol-5-FU combination treatment notably inhibited tumour growth in vivo. In summary, our data demonstrated that 6-shogaol contributed to the curative outcome of 5-FU in liver cancer by inhibiting the AKT/mTOR/MRP1 signalling pathway.

Knockout of caspase-7 gene improves the expression of recombinant protein in CHO cell line through the cell cycle arrest in G2/M phase

BACKGROUND: Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. RESULTS: Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. CONCLUSIONS: These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.

Speckle type POZ adaptor protein (SPOP) and its role in cancer / La proteína adaptadora Speckle-type POZ (SPOP) y su papel en cáncer

Abstract Proteasomal degradation is an essential regulatory mechanism for cellular homeostasis maintenance. The speckle-type POZ adaptor protein (SPOP) is part of the ubiquitin ligase E3 cullin-3 RING-box1 complex, responsible for the ubiquitination and proteasomal degradation of biomolecules involved in cell cycle control, proliferation, response to DNA damage, epigenetic control, and hormone signaling, among others. Changes in SPOP have been associated with the development of different types of cancer, since it can act as a tumor suppressor mainly in prostate, breast, colorectal, lung cancer and liver cancer, due to point mutations and/or reduced expression, or as an oncogene in kidney cancer by protein overexpression. In endometrial cancer it has a dual role, since it can act as a tumor suppressor or as an oncogene. SPOP is a potential prognostic biomarker and a promising therapeutic target.

Resumen La degradación proteosómica es un mecanismo de regulación esencial para el mantenimiento de la homeostasis celular. La proteína adaptadora Speckle-type POZ (SPOP) hace parte del complejo ubiquitin ligasa E3 cullin-3 RING-box1, encargado de la ubiquitinación y degradación proteosomal de biomoléculas involucradas en el control del ciclo celular, proliferación, respuesta al daño de ADN, control epigenético, señalización hormonal, entre otros. Las alteraciones en SPOP han sido asociadas al desarrollo de diferentes tipos de cáncer, ya que puede actuar como supresor tumoral principalmente en cáncer de próstata, mama, colorrectal y pulmón, debido a mutaciones puntuales y/o expresión reducida o como oncogén en cáncer riñón por sobreexpresión de la proteína. En cáncer endometrial tiene un rol dual, ya que puede actuar como supresor tumoral o como oncogén. SPOP es considerado como un potencial biomarcador pronóstico y un objetivo terapéutico prometedor.

Progress of studies on anti-breast tumor mechanism of alkaloids / 中国中药杂志

Breast tumor has become one of the malignant tumors with the highest incidence, and is a serious threat to human health, especially to women. Chemotherapy is an important anti-breast tumor therapy, which can be used in almost every stage of breast tumor therapy alone or in the combination with surgery and radiation therapy. Alkaloids are a kind of ubiquitous natural products, and important active components of various medicinal plants. A large number of studies have shown that alkaloids could exert an anti-breast tumor effect by inhibiting proliferation, metastasis and angiogenesis, resisting mitosis, promoting apoptosis and autophagy, and triggering cell cycle arrest. The extensive anti-breast tumor effect makes alkaloids an important candidate drug source. This paper reviews the anti-breast tumor mechanism of natural products of alkaloids.

Juniperus communis extract induces cell cycle arrest and apoptosis of colorectal adenocarcinoma in vitro and in vivo

Juniperus communis (JCo) is a well-known traditional Chinese medicinal plant that has been used to treat wounds, fever, swelling, and rheumatism. However, the mechanism underlying the anticancer effect of JCo extract on colorectal cancer (CRC) has not yet been elucidated. This study investigated the anticancer effects of JCo extract in vitro and in vivo as well as the precise molecular mechanisms. Cell viability was evaluated using the MTT assay. Cell cycle distribution was examined by flow cytometry analysis, and cell apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Protein expression was analyzed using western blotting. The in vivo activity of the JCo extract was evaluated using a xenograft BALB/c mouse model. The tumors and organs were examined through hematoxylin-eosin (HE) staining and immunohistochemistry. The results showed that JCo extract exhibited higher cytotoxicity against CRC cells than against normal cells and showed synergistic effects when combined with 5-fluorouracil. JCo extract induced cell cycle arrest at the G0/G1 phase via regulation of p53/p21 and CDK4/cyclin D1 and induced cell apoptosis via the extrinsic (FasL/Fas/caspase-8) and intrinsic (Bax/Bcl-2/caspase-9) apoptotic pathways. In vivo studies revealed that JCo extract suppressed tumor growth through the inhibition of proliferation and induction of apoptosis. In addition, there was no obvious change in body weight or histological morphology of normal organs after treatment. JCo extract suppressed CRC progression by inducing cell cycle arrest and apoptosis in vitro and in vivo, suggesting the potential application of JCo extract in the treatment of CRC.

Palbociclib induces cell cycle arrest and senescence of human renal tubular epithelial cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of palbociclib on cell cycle progression and proliferation of human renal tubular epithelial cells.@*METHODS@#Human renal tubular epithelial cell line HK-2 was treated with 1, 5, 10, and 20 μmol/L of palbociclib, and the changes in cell proliferation and viability were examined by cell counting and CCK8 assay. EDU staining was used to assess the proliferation of HK-2 cells following palbiciclib treatment at different concentrations for 5 days. The effect of palbociclib on cell cycle distribution of HK-2 cells was evaluated using flow cytometry. SA-β-Gal staining and C12FDG senescence staining were used to detect senescence phenotypes of HK-2 cells after palbociclib treatment at different concentrations for 5 days. The relative mRNA expression levels of P16, P21, and P53 and the genes associated with senescence-related secretion phenotypes were detected by RT-PCR, and the protein expressions of P16, P21 and P53 were detected by Western blotting.@*RESULTS@#Palbociclib inhibited HK-2 cell proliferation and induced cell cycle arrest in G1 phase. Compared with the control cells, HK-2 cells treated with high-dose (10 μmol/L) palbociclib exhibited significantly suppressed cell proliferation activity, and the inhibitory effect was the most obvious on day 5 (@*CONCLUSIONS@#Palbociclib induces HK-2 cell senescence by causing cell growth arrest and delaying cell cycle progression.

Role of LINC00152 in non-small cell lung cancer / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer cases. The pathogenesis of NSCLC involves complex gene networks that include different types of non-coding RNAs, such as long non-coding RNAs (lncRNAs). The role of lncRNAs in NSCLC is gaining an increasing interest as their function is being explored in various human cancers. Recently, a new oncogenic lncRNA, LINC00152 (cytoskeleton regulator RNA (CYTOR)), has been identified in different tumor types. In NSCLC, the high expression of LINC00152 in tumor tissue and peripheral blood samples has been shown to be associated with worse prognoses of NSCLC patients. Overexpression of LINC00152 has been confirmed to promote the proliferation, invasion, and migration of NSCLC cells in vitro, as well as increase tumor growth in vivo. This review discusses the role of LINC00152 in NSCLC.

Carfilzomib inhibits the growth of lung adenocarcinoma via upregulation of Gadd45a expression / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Proteasome inhibitors have shown remarkable success in the treatment of hematologic neoplasm. There has been a lot of attention to applying these drugs for solid tumor treatment. Recent preclinical study has signified the effectiveness on cell proliferation inhibition in lung adenocarcinoma treated by carfilzomib (CFZ), a second generation proteasome inhibitor. However, no insight has been gained regarding the mechanism. In this study, we have systematically investigated the CFZ functions in cell proliferation and growth, cell cycle arrest, and apoptosis in lung adenocarcinoma cells. Flow cytometry experiments showed that CFZ significantly induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma. MTS and colony formation assays revealed that CFZ substantially inhibited survival of lung adenocarcinoma cells. All results were consistently correlated to the upregulation expression of Gadd45a, which is an important gene in modulating cell cycle arrest and apoptosis in response to physiologic and environmental stresses. Here, upregulation of Gadd45a expression was observed after CFZ treatment. Knocking down Gadd45a expression suppressed G2/M arrest and apoptosis in CFZ-treated cells, and reduced cytotoxicity of this drug. The protein expression analysis has further identified that the AKT/FOXO3a pathway is involved in Gadd45a upregulation after CFZ treatment. These findings unveil a novel mechanism of proteasome inhibitor in anti-solid tumor activity, and shed light on novel preferable therapeutic strategy for lung adenocarcinoma. We believe that Gadd45a expression can be a highly promising candidate predictor in evaluating the efficacy of proteasome inhibitors in solid tumor therapy.

Estudos funcionais e biológicos associados à interação de DUSP3 com a proteína HNRNPC1/C2 / Functional and biological studies associated to DUSP3 and HNRNPC1/C2 protein inte ractions

A regulação da fosforilação/desfosforilação das proteínas é o eixo central de muitas cascatas de sinalização. A fosfatase DUSP3, constituída apenas por um único domínio catalítico, desempenha papéis fundamentais na proliferação e senescência celular. Nas células HeLa, submetidas ao estresse genotóxico, o DUSP3 interage fisicamente com as proteínas HNRNPC, mas o efeito dessa função molecular ainda é desconhecido. Aqui demostramos que a ausência de DUPS3 mantem a proteína HNRNPC1/C2 num estado hiperfosforilado. Para entender melhor o envolvimento da interação DUSP3-HNRNPC nas funções biológicas da HNRNPC1/C2, foram estudadas células de fibroblasto deficientes de DUSP3. Foi analisado o efeito da deficiência de DUSP3 na biogênese dos ribossomos através do ensaio de perfil de polirribossomos e quantificação dos rRNAs com RT-qPCR. Os resultados mostraram que a deficiência de DUSP3 não afeta a maturação das subunidades ribossômicas, mas teria um impacto na transcrição dos pré-rRNAs e no acumulo das espécies 47S/45S. A expressado de genes contendo sequencias IRES foi analisado através do RT-qPCR e sua tradução ao longo do ciclo e em condições de estresse. Da expressão, não existe nenhuma diferença nos níveis de transcrição dos genes c-myc e xiap nas células normais e deficientes de DUSP3 em condições basais. Embora a síntese destas proteínas é maior nas células deficientes, mantendo um nível maior de tradução ao longo de todo o ciclo. Sob condições de estresse, esta duas proteínas sempre mantem uma maior expressão nas células Knockdown para DUSP3. Neste trabalho também foi estabelecido a presença de DUSP3 nos complexos da subunidade 40S, através do analise das frações obtidas do ensaio de polirribossomos e interação in vitro (Co-IP). A presença de DUSP3 nas subunidades 40S, os monossomas 80S e polissomos poderia ser através da interação direta com proteínas que possuem um domínio RRM e seria dependente dos complexos formados pelas proteínas e seus RNAs alvos. Aqui mostramos a interação in vitro de DUSP3 com a proteína PABP (com quatro domínios RRM), proteína que tem um papel importante na manutenção da taxa global de tradução, esta interação é enfraquecida na ausência de RNAs. A deficiência de DUSP3 também teria um impacto na interação das proteínas HNRNPC1/C2 e P53 in vitro. A ausência de DUSP3 diminui a interação HNRNPC-P53 através da hiperfosforilação da proteina HNRNPC1/C2. A perda desta interação, aumentaria os níveis da proteína P53 na célula deficiente de DUSP3 e poderia gerar parada no ciclo celular. Através de ensaios de imunofluorescência, se observo uma maior taxa de transcrição global na célula deficiente de DUSP3. Por fim, aqui demostramos que a interação direta de DUSP3 e HNRNPC1/C2 vai permitir a regulação das funções biológicas desta proteína, e a ausência de DUSP3 vai ter efeitos pleiotrópicos na homeostase da célula

inglêsProtein phosphorylation/dephosphorylation regulation is a central axis of many signaling cascades. DUSP3 phosphatase, consisting only of a single catalytic domain, plays key roles in cell proliferation and senescence. In HeLa cells subjected to genotoxic stress, DUSP3 physically interacts with HNRNPC proteins, but the effect of this molecular function is still unknown. Here we demonstrate that the absence of DUPS3 keeps the HNRNPC1/C2 proteins in a hyperphosphorylated state. To better understand the involvement of DUSP3- HNRNPC interaction on the biological functions of HNRNPC1/C2, DUSP3 deficient fibroblast cells were studied. The effect of DUSP3 deficiency on ribosome biogenesis was analyzed by polyribosome profile assay and RT-qPCR for rRNA quantification. The results showed that DUSP3 deficiency does not affect ribosomal subunit maturation, but would have an impact on transcription of pre-rRNAs and accumulation of 47S / 45S species. The expression of genes containing IRES sequences was analyzed by RT-qPCR and their translation throughout the cycle and under stress conditions. From expression, there is no difference in transcriptional levels of c-myc and xiap genes in normal and DUSP3 deficient cells under basal conditions. Although, the synthesis of these proteins is higher in deficient cells and these maintain a higher level of translation throughout the cell cycle. Under stress conditions, these two proteins always maintain higher expression in Knockdown cells for DUSP3. In this work, the presence of DUSP3 in the 40S ribosomal subunit complexes was also established by analyzing the fractions obtained from the polyribosome assay and in vitro interaction (CoIP). The presence of DUSP3 in the 40S subunits, 80S monosomes and polysomes could be through direct interaction with proteins that have an RRM domain and would be dependent on the complexes formed by the proteins and their target RNAs. Here we show the in vitro interaction of DUSP3 with PABP protein (with four RRM domains), a protein that plays an important role in maintaining the overall translation rate, this interaction is weakened in the absence of RNAs. DUSP3 deficiency would also have an impact on the interaction of HNRNPC1/C2 and P53 proteins in vitro. The absence of DUSP3 decreases HNRNPC-P53 interaction through hyperphosphorylation of the HNRNPC1/C2 proteins. Loss of this interaction would increase P53 protein levels in the DUSP3 deficient cell and could lead to cell cycle arrest. Through immunofluorescence assays, a higher overall transcription rate is observed in the DUSP3 deficient cell. Finally, we demonstrate that the direct interaction of DUSP3 and HNRNPC1/C2 will allow the regulation of the biological functions of this protein, and the absence of DUSP3 will have pleiotropic effects on cell homeostasis

Genistein Inhibits Proliferation of BRCA1 Mutated Breast Cancer Cells: The GPR30-Akt Axis as a Potential Target

BACKGROUND: BRCA1 mutated breast cancer cells exhibit the elevated cell proliferation and the higher metastatic potential. G protein-coupled receptor 30 (GPR30) has been shown to regulate growth of hormonally responsive cancers, such as ovarian and breast cancers, and high expression of GPR30 is found in estrogen receptor (ER)-negative breast cancer cells. ER-negative breast cancer patients often have a mutation in the tumor suppressor gene, BRCA1. This study explored antiproliferative effects of genistein, a chemopreventive isoflavone present in legumes, and underlying molecular mechanisms in triple negative breast cancer cells with or without functionally active BRCA1.METHODS: Expression of BRCA1, GPR30 and Nrf2 was measured by Western blot analysis. Reactive oxygen species (ROS) accumulation was monitored by using the fluorescence-generating probe, 2’,7’-dichlorofluorescein diacetate. The effects of genistein on breast cancer cell viability and proliferation were assessed by the MTT, migration and clonogenic assays.RESULTS: The expression of GPR30 was dramatically elevated at both transcriptional and translational levels in BRCA1 mutated breast cancer cells compared to cells with wild-type BRCA1. Notably, there was diminished Akt phosporylation in GPR30 silenced cells. Treatment of BRCA1 silenced breast cancer cells with genistein resulted in the down-regulation of GPR30 expression and the inhibition of Akt phosphorylation as well as the reduced cell viability, migration and colony formation. Genistein caused cell cycle arrest at the G₂/M phase in BRCA1-mutant cells through down-regulation of cyclin B1 expression. Furthermore, BRCA1-mutant breast cancer cells exhibited higher levels of intracellular ROS than those in the wild-type cells. Genistein treatment lowered the ROS levels through up-regulation of Nrf2 expression.CONCLUSIONS: Lack of functional BRCA1 activates GPR30 signaling, thereby stimulating Akt phosphorylation and cell proliferation. Genistein induces G2/M phase arrest by down-regulating cyclin B1 expression, which is attributable to its suppression of GPR30 activation and Akt phosphorylation in BRCA1 impaired breast cancer cells.

Overexpression of Tumor Protein p53-regulated Apoptosis-inducing Protein 1 Regulates Proliferation and Apoptosis of Breast Cancer Cells through the PI3K/Akt Pathway / 한국유방암학회지

PURPOSE: Tumor protein p53-regulated apoptosis-inducing protein 1 (TP53AIP1) functions in various cancers. We studied the effect and molecular mechanism of TP53AIP1 in breast cancer. METHODS: The degree of correlation between TP53AIP1 expression and overall survival in patients with breast cancer was obtained from the online The Cancer Genome Atlas database. Six of the TP53AIP1 levels in the tumor and adjacent non-tumor tissues randomly selected from 38 breast cancer patients were determined. Transgenic technology was used to enhance the expression of TP53AIP1 in breast cancer cell lines, MDA-MB-415 and MDA-MB-468, and to observe the effects of gene overexpression on the proliferation, cell cycle, and apoptosis of breast cancer cells. The molecular mechanism of association between cell cycle- and apoptosis-related factors and the phosphoinositide 3-kinases/protein kinase B (PI3K/Akt) pathway was also studied. RESULTS: The messenger RNA and protein expression levels of TP53AIP1 in cancer tissues were significantly lower than those in the control group. TP53AIP1 overexpression inhibits cell viability. The mechanism of TP53AIP1 inhibition of proliferation and growth of breast cancer cells includes cell cycle arrest, apoptosis promotion (p < 0.01), promotion of the expression of cleaved-caspase-3 (p < 0.01), cleaved-caspase-9 (p < 0.01), B cell lymphoma/leukemia-2 (Bcl-2)-associated X protein, and p53 (p < 0.01), and the inhibition of Bcl-2, Ki67, and PI3K/Akt pathways (p < 0.01). CONCLUSION: TP53AIP1 may be a novel tumor suppressor gene in breast cancer and can potentially be used as an effective target gene for the treatment of breast cancer.

Enhanced Anti-Cancer Effects of Conditioned Medium from Hypoxic Human Umbilical Cord–Derived Mesenchymal Stem Cells

BACKGROUND AND OBJECTIVES: There have been contradictory reports on the pro-cancer or anti-cancer effects of mesenchymal stem cells. In this study, we investigated whether conditioned medium (CM) from hypoxic human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) (H-CM) showed enhanced anti-cancer effects compared with CM from normoxic hUC-MSCs (N-CM). METHODS AND RESULTS: Compared with N-CM, H-CM not only strongly reduced cell viability and increased apoptosis of human cervical cancer cells (HeLa cells), but also increased caspase-3/7 activity, decreased mitochondrial membrane potential (MMP), and induced cell cycle arrest. In contrast, cell viability, apoptosis, MMP, and cell cycle of human dermal fibroblast (hDFs) were not significantly changed by either CM whereas caspase-3/7 activity was decreased by H-CM. Protein antibody array showed that activin A, Beta IG-H3, TIMP-2, RET, and IGFBP-3 were upregulated in H-CM compared with N-CM. Intracellular proteins that were upregulated by H-CM in HeLa cells were represented by apoptosis and cell cycle arrest terms of biological processes of Gene Ontology (GO), and by cell cycle of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In hDFs, negative regulation of apoptosis in biological process of GO and PI3K-Akt signaling pathway of KEGG pathways were represented. CONCLUSIONS: H-CM showed enhanced anti-cancer effects on HeLa cells but did not influence cell viability or apoptosis of hDFs and these different effects were supported by profiling of secretory proteins in both kinds of CM and intracellular signaling of HeLa cells and hDFs.

Paip1 Indicated Poor Prognosis in Cervical Cancer and Promoted Cervical Carcinogenesis / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: This study was aimed to investigate the role of poly(A)-binding protein-interacting protein 1 (Paip1) in cervical carcinogenesis. MATERIALS AND METHODS: The expression of Paip1 in normal cervical epithelial tissues and cervical cancer (CC) tissues were detected by immunohistochemistry. In vivo and in vitro assays were performed to validate effect of Paip1 on CC progression. RESULTS: Paip1 was found to be up-regulated in CC, which was linked with shorter survival. Knockdown of Paip1 inhibited cell growth, induced apoptosis and cell cycle arrest in CC cells, whereas its overexpression reversed these effects. The in vivo tumor model confirmed the pro-tumor role of Paip1 in CC growth. CONCLUSION: Altogether, the investigation demonstrated the clinical significance of Paip1 expression, which prompted that the up-regulated of Paip1 can presumably be a potential prognostic and progression marker for CC.

Therapeutic Targeting of the DNA Damage Response Using an ATR Inhibitor in Biliary Tract Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The DNA damage response (DDR) is a multi-complex network of signaling pathways involved in DNA damage repair, cell cycle checkpoints, and apoptosis. In the case of biliary tract cancer (BTC), the strategy of DDR targeting has not been evaluated, even though many patients have DNA repair pathway alterations. The purpose of this study was to test the DDR-targeting strategy in BTC using an ataxia-telangiectasia and Rad3-related (ATR) inhibitor. MATERIALS AND METHODS: A total of nine human BTC cell lines were used for evaluating anti-tumor effect of AZD6738 (ATR inhibitor) alone or combination with cytotoxic chemotherapeutic agents through MTT assay, colony-forming assays, cell cycle analyses, and comet assays. We established SNU478-mouse model for in vivo experiments to confirm our findings. RESULTS: Among nine human BTC cell lines, SNU478 and SNU869 were the most sensitive to AZD6738, and showed low expression of both ataxia-telangiectasia mutated (ATM) and p53. AZD6738 blocked p-Chk1 and p-glycoprotein and increased γH2AX, a marker of DNA damage, in sensitive cells. AZD6738 significantly increased apoptosis, G2/M arrest and p21, and decreased CDC2. Combinations of AZD6738 and cytotoxic chemotherapeutic agents exerted synergistic effects in colony-forming assays, cell cycle analyses, and comet assays. In our mouse models, AZD6738 monotherapy decreased tumor growth and the combination with cisplatin showed more potent effects on growth inhibition, decreased Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling than monotherapy with each drug. CONCLUSION: In BTC, DDR targeting strategy using ATR inhibitor demonstrated promising antitumor activity alone or in combination with cytotoxic chemotherapeutic agents. This supports further clinical development of DDR targeting strategy in BTC.