OBJECTIVES: This study aimed to evaluate the diagnostic accuracy of Cyclin D1 as an adjunct immunomarker in CD99 positive small round cell neoplasms with primary consideration of PNET/EWS. MATERIALS AND METHODS: Tissue from 2017 to 2023 with a histopathologic diagnosis of CD99 positive small round blue cell tumors with primary consideration of Primitive Neuroectodermal Tumor (PNET)/Ewing Sarcoma were retrieved and Cyclin D1 immunohistochemical staining done. Diagnostic accuracy of Cyclin D1 immunostaining was determined by calculating the sensitivity, specificity, positive predictive value, and negative predictive value. RESULTS: Cyclin D1 immunohistochemical staining was performed in 19 specimens available, of which 13 yielded a positive result. Of these, 8 had a final histopathologic diagnosis of CD99 positive small round blue cell tumor with primary consideration of PNET/Ewing Sarcoma, resulting in sensitivity of 61.54%, specificity of 100%, positive predictive value of 100% and negative predictive value of 50.0%. The overall accuracy is 72.2%. CONCLUSION Cyclin D1 can be used as an adjunct immunomarker to aid in the diagnosis of CD99 positive round cell tumor with primary consideration of PNET/Ewing Sarcoma specifically in resource limited settings where molecular testing is not readily available. Given the high specificity of Cyclin D1 in such cases, it can be used to rule out other small round blue cell tumors that can also stain positive for CD99 such as Rhabdomyosarcoma. However, interpretation must be done in conjunction with the results of other immunohistochemical stains in order to increase its diagnostic accuracy.
Human ; Male,Female ; Cells ; Sarcoma, Ewing ; Sarcoma ; Neuroectodermal tumors, Primitive ; Cyclin D1

OBJECTIVE: To investigate the impacts of sulforaphane (SPN) on cell proliferation and apoptosis in acute promyelogenous leukemia by regulating the PI3K/Akt/mTOR signaling pathway. METHODS: NB4 cells were divided into 5 μmol/L SPN group, 10 μmol/L SPN group, 20 μmol/L SPN group, 740 Y-P (10 μmol/L) group and 20 μmol/L SPN+740 Y-P group, and the untreated NB4 cells were used as the control group. CCK-8, Hoechst 33342 staining, flow cytometry and monodansulfonylpentanediamine (MDC) were used to detect cell proliferation, apoptosis and autophagy, respectively. The expression levels of Bcl-2, Bax, cyclin D1 and LC3B mRNA were detected by qRT-PCR. Western blot was used to detect the expression levels of PI3K/Akt/mTOR pathway-related proteins in NB4 cells. RESULTS: Compared with the control group, the proliferation rate, Bcl-2, cyclin D1 mRNA expressions, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR ratio were greatly increased in the 740 Y-P group (P < 0.05), the apoptosis rate, percentage of MDC positive, Bax and LC3B mRNA expression levels were greatly decreased (P < 0.05). The proliferation rate, Bcl-2, cyclin D1 mRNA expression levels, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR ratio were greatly decreased in the 5 μmol/L SPN group, 10 μmol/L SPN group, and 20 μmol/L SPN group (P < 0.05), the apoptosis rate, percentage of MDC positive,Bax and LC3B mRNA expression levels were greatly increased, there were differences among different SPN treatment groups (P < 0.05). Compared with the 20 μmol/L SPN group, the proliferation rate, Bcl-2, cyclin D1 mRNA expression levels, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR ratio were greatly increased in the 20 μmol/L SPN+740 Y-P group(P < 0.05), the apoptosis rate, percentage of MDC positive, Bax and LC3B mRNA expression levels were greatly decreased (P < 0.05). Compared with the 740 Y-P group, the proliferation rate, Bcl-2, cyclin D1 mRNA expression levels, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR ratio in the 20 μmol/L SPN+740 Y-P group were greatly reduced (P < 0.05), the apoptosis rate, percentage of MDC positive, Bax and LC3B mRNA expression levels were greatly increased (P < 0.05). CONCLUSION SPN reduces the proliferation of acute promyelocytic leukemia cells and promotes cells apoptosis by inhibiting the PI3K/Akt/mTOR signaling pathway.
Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Humans ; TOR Serine-Threonine Kinases/metabolism* ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Isothiocyanates/pharmacology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Sulfoxides ; Cell Line, Tumor ; Cyclin D1/metabolism*

Gastric cancer (GC) is characterized by high morbidity and mortality rates. Chinese agarwood comprises the resin-containing wood of Aquilaria sinensis (Lour.) Gilg., traditionally utilized for treating asthma, cardiac ischemia, and tumors. However, comprehensive research regarding its anti-GC effects and underlying mechanisms remains limited. In this study, Chinese agarwood petroleum ether extract (CAPEE) demonstrated potent cytotoxicity against human GC cells, with half maximal inhibitory concentration (IC₅₀) values for AGS, HGC27, and MGC803 cells of 2.89, 2.46, and 2.37 μg·mL^-1, respectively, at 48 h. CAPEE significantly induced apoptosis in these GC cells, with B-cell lymphoma-2 (BCL-2) associated X protein (BAX)/BCL-2 antagonist killer 1 (BAK) likely mediating CAPEE-induced apoptosis. Furthermore, CAPEE induced G₀/G₁ phase cell cycle arrest in human GC cells via activation of the deoxyribonucleic acid (DNA) damage-p21-cyclin D1/cyclin-dependent kinase 4 (CDK4) signaling axis, and increased Fe²⁺, lipid peroxides and reactive oxygen species (ROS) levels, thereby inducing ferroptosis. Ribonucleic acid (RNA) sequencing, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blotting analyses revealed CAPEE-mediated upregulation of heme oxygenase-1 (HO-1) in human GC cells. RNA interference studies demonstrated that HO-1 knockdown reduced CAPEE sensitivity and inhibited CAPEE-induced ferroptosis in human GC cells. Additionally, CAPEE administration exhibited robust in vivo anti-GC activity without significant toxicity in nude mice while inhibiting tumor cell growth and promoting apoptosis in tumor tissues. These findings indicate that CAPEE suppresses human GC cell growth through upregulation of the DNA damage-p21-cyclin D1/CDK4 signaling axis and HO-1-mediated ferroptosis, suggesting its potential as a candidate drug for GC treatment.
Animals ; Humans ; Mice ; Antineoplastic Agents, Phytogenic ; Apoptosis/drug effects* ; Cell Line, Tumor ; Cyclin D1/genetics* ; Cyclin-Dependent Kinase 4/genetics* ; DNA Damage/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Ferroptosis/drug effects* ; G1 Phase Cell Cycle Checkpoints/drug effects* ; Heme Oxygenase-1/genetics* ; Mice, Inbred BALB C ; Mice, Nude ; Plant Extracts/pharmacology* ; Stomach Neoplasms/physiopathology* ; Thymelaeaceae/chemistry* ; Up-Regulation/drug effects*

OBJECTIVES: This study aimed to evaluate the diagnostic accuracy of Cyclin D1 as an adjunct immunomarker in CD99 positive small round cell neoplasms with primary consideration of PNET/EWS.

MATERIALS AND METHODS: Tissue from 2017 to 2023 with a histopathologic diagnosis of CD99 positive small round blue cell tumors with primary consideration of Primitive Neuroectodermal Tumor (PNET)/Ewing Sarcoma were retrieved and Cyclin D1 immunohistochemical staining done. Diagnostic accuracy of Cyclin D1 immunostaining was determined by calculating the sensitivity, specificity, positive predictive value, and negative predictive value.

RESULTS: Cyclin D1 immunohistochemical staining was performed in 19 specimens available, of which 13 yielded a positive result. Of these, 8 had a final histopathologic diagnosis of CD99 positive small round blue cell tumor with primary consideration of PNET/Ewing Sarcoma, resulting in sensitivity of 61.54%, specificity of 100%, positive predictive value of 100% and negative predictive value of 50.0%. The overall accuracy is 72.2%.

CONCLUSION: Cyclin D1 can be used as an adjunct immunomarker to aid in the diagnosis of CD99 positive round cell tumor with primary consideration of PNET/Ewing Sarcoma specifically in resource limited settings where molecular testing is not readily available. Given the high specificity of Cyclin D1 in such cases, it can be used to rule out other small round blue cell tumors that can also stain positive for CD99 such as Rhabdomyosarcoma. However, interpretation must be done in conjunction with the results of other immunohistochemical stains in order to increase its diagnostic accuracy.

Objective To establish U251 cells with inhibited expression of interferon-γ inducible protein 30 (IFI30), and to investigate the effect of IFI30 on cell biological function as well as its underlying mechanism. Methods Three knockdown sequences which target IFI30 were designed online and 3 small interfering RNAs (siRNA) were synthesized. After transfection, the inhibition efficiency was detected by real-time quantitative PCR. The siRNA sequence with the highest inhibition efficiency was selected to create short hairpin RNA (shRNA) plasmids. The recombinant plasmids and packaging plasmids were co-transfected into HEK293T cells to prepare lentivirus. The glioma U251 cells were transfected with lentivirus, and the positive cells were screened by puromycin. CCK-8 assay, 5-ethyl-2'-deoxyuridine (EdU) and colony formation assays were used to analyze cell proliferation; the flow cytometry was used to analyze cell cycle and apoptosis; the Transwell^TM assay was used to detect cell invasion; the wound-healing assay was employed to detect cell migration, and western blot analysis to detect the protein expresison of cyclin D1, B-cell lymphoma factor 2 (Bcl2), epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), signal transducer and activator of transcription 1 (STAT1). Results The sequence which effectively target IFI30 was screened and U251 cell line capable of inhibiting the IFI30 expression was successfully established. When IFI30 expression was knocked down, the proliferation of U251 cells was inhibited, along with increased ratio of cells in the phase G0/G1, the decreased phase S, the increased rate of cell apoptosis. The cell invasion and migration capabilities was also reduced. The decreased expression of cyclin D1, Bcl2 and N-cadherin were observed in U251 cells, and the expression of E-cadherin and the phosphorylation of STAT1 were found increased. Conclusion Knockdown of IFI30 inhibits the proliferation, invasion and migration of human glioma cell U251 and promotes its apoptosis by activating STAT1.
Humans ; Cyclin D1/genetics* ; HEK293 Cells ; Interferon-gamma ; RNA, Small Interfering ; Apoptosis/genetics* ; Cadherins ; Cell Proliferation/genetics* ; Glioma/genetics* ; Proto-Oncogene Proteins c-bcl-2 ; Oxidoreductases Acting on Sulfur Group Donors ; STAT1 Transcription Factor/genetics*

Objective:To investigate the effect of phosphorylated HSP27 on the proliferation and metastasis of nasopharyngeal carcinoma and its molecular mechanism. Methods:①Western blot assay was used to detect the expression levels of HSP27 and p-HSP27 in CNE1 and CNE2 cells of nasopharyngeal carcinoma. Inhibited the phosphorylation of HSP27, Transwell assay detected the metastasis ability of nasopharyngeal carcinoma cells. ②Nasopharyngeal carcinoma cell lines（CNE2-OE1 and CNE2-OE2） overexpressing phosphorylated and dephosphorylated mutants of HSP27 were synthesized, empty vector transfected CNE2 cells（CNE2-NC） were used as controls. The proliferation ability of the three groups of cells was detected by CCK8, and the expression levels of CyclinD1, Bax and Bcl-2 were detected by Western blot. Transwell was used to detect the migration and invasion ability of cells, and Western blot was used to detect the expression levels of E-cadherin, Vimentin, MMP2 and MMP9. Results:The expression level of HSP27 in CNE2 was higher than that of CNE1 cells, while the expression level of p-HSP27 was opposite. After inhibition of HSP27 phosphorylation, the invasion and migration ability of CNE1 cells decreased significantly, with no significant change in CNE2 cells. Compared with CNE2-NC, the growth rate of CNE2-OE1 decreased, and the growth rate of CNE2-OE2 increased. The expression level of CyclinD1 was down-regulated in CNE2-OE1 and higher in CNE2-OE2. The expression level of Bax in CNE2-OE1 was increased, while the expression of Bcl-2 was decreased. There was no significant change in the expression of Bax and Bcl-2 in CNE2-OE2. Compared with CNE2-NC, the migration ability of CNE2-OE1 was enhanced and the invasion ability was weakened, while the migration ability of CNE2-OE2 was weakened and the invasion ability was enhanced. There was no significant difference in the expression levels of E-cadherin was decreased in CNE2-OE1 and increased in CNE2-OE2. There was no significant difference in the expression levels of Vimentin among the three groups. The expression levels of MMP2 and MMP9 were up-regulated in CNE2-OE2, and slightly down-regulated in CNE2-OE1. Conclusion:HSP27 and p-HSP27 were differentially expressed in different nasopharyngeal carcinoma cells, and the metastasis ability of nasopharyngeal carcinoma was not only related to the expression level of HSP27, but also related to the level of p-HSP27. The p-HSP27 inhibited CNE 2 cell proliferation and promoted their apoptosis. As p-HSP27 plays different roles in different stages of nasopharyngeal carcinoma metastasis, it can increase the migration ability of CNE2 cells and reduce its invasion ability. p-HSP27 may induce EMT changes in CNE2 by down-regulating E-cadherin, thus promoting CNE2 migration, and may inhibit CNE2 invasion by down-regulating MMPs expression.
Humans ; Cell Proliferation ; Cell Line, Tumor ; Nasopharyngeal Carcinoma/pathology* ; Nasopharyngeal Neoplasms/pathology* ; Cell Movement ; HSP27 Heat-Shock Proteins/metabolism* ; Phosphorylation ; Neoplasm Metastasis ; Matrix Metalloproteinase 9/metabolism* ; Neoplasm Invasiveness ; Matrix Metalloproteinase 2/metabolism* ; Heat-Shock Proteins/metabolism* ; Cyclin D1/metabolism* ; Molecular Chaperones/metabolism* ; Cadherins/metabolism*

This study aimed to observe the effect of terpinen-4-ol(T4O) on the proliferation of vascular smooth muscle cells(VSMCs) exposed to high glucose(HG) and reveal the mechanism via the Krüppel-like factor 4(KLF4)/nuclear factor kappaB(NF-κB) signaling pathway. The VSMCs were first incubated with T4O for 2 h and then cultured with HG for 48 h to establish the model of inflammatory injury. The proliferation, cell cycle, and migration rate of VSMCs were examined by MTT method, flow cytometry, and wound healing assay, respectively. The content of inflammatory cytokines including interleukin(IL)-6 and tumor necrosis factor-alpha(TNF-α) in the supernatant of VSMCs was measured by enzyme-linked immunosorbent assay(ELISA). Western blot was employed to determine the protein levels of proliferating cell nuclear antigen(PCNA), Cyclin D1, KLF4, NF-κB p-p65/NF-κB p65, IL-1β, and IL-18. The KLF4 expression in VSMCs was silenced by the siRNA technology, and then the effects of T4O on the cell cycle and protein expression of the HG-induced VSMCs were observed. The results showed that different doses of T4O inhibited the HG-induced proliferation and migration of VSMCs, increased the percentage of cells in G_1 phase, and decreased the percentage of cells in S phase, and down-regulated the protein levels of PCNA and Cyclin D1. In addition, T4O reduced the HG-induced secretion and release of the inflammatory cytokines IL-6 and TNF-α and down-regulated the expression of KLF4, NF-κB p-p65/NF-κB p65, IL-1β, and IL-18. Compared with si-NC+HG, siKLF4+HG increased the percentage of cells in G_1 phase, decreased the percentage of cells in S phase, down-regulated the expression of PCNA, Cyclin D1, and KLF4, and inhibited the activation of NF-κB signaling pathway. Notably, the combination of silencing KLF4 with T4O treatment further promoted the changes in the above indicators. The results indicate that T4O may inhibit the HG-induced proliferation and migration of VSMCs by down-regulating the level of KLF4 and inhibiting the activation of NF-κB signaling pathway.
NF-kappa B/metabolism* ; Interleukin-18/metabolism* ; Proliferating Cell Nuclear Antigen/genetics* ; Cyclin D1/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Muscle, Smooth, Vascular ; Cell Proliferation ; Signal Transduction ; Cytokines/metabolism* ; Glucose/metabolism*

Objective: To assess the feasibility of nuclear score combined with cyclin D1 immunocytochemistry in classifying indeterminate thyroid nodules with fine-needle aspiration (FNA) cytological diagnosis of Bethesda category Ⅲ-Ⅴ. Methods: A consecutive cohort of 118 thyroid FNA specimens with indeterminate diagnosis (TBSRTC category Ⅲ-Ⅴ) and available histopathologic follow-up data were collected between December 2018 and April 2022 at the Department of Pathology, Beijing Hospital, China. These cases were subjected to cytological evaluation and cyclin D1 immunocytochemistry. The optimal cut-off points of a simplified nuclear score and the percentage of cyclin D1-positive cells for the diagnosis of malignancy or low-risk neoplasm were determined using the receiver operating characteristic (ROC) curves and area under the ROC curve (AUC). The specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) of nuclear score and cyclin D1 immunostaining were evaluated from the crosstabs based on cut-off points. The diagnostic accuracy of simplified nuclear score combined with cyclin D1 immunostaining was estimated using ROC curve analysis. Results: Nuclear grooves, intra-nuclear inclusions and chromatin clearing were more commonly found in malignancy/low-risk neoplasms than benign lesions (P=0.001, P=0.012 and P=0.001 respectively). A cut-off point of≥2 for the simplified nuclear score was sensitive for defining malignancy/low-risk neoplasm, and its PPV, NPV, sensitivity and specificity were 93.6%, 87.5%, 99.0% and 50.0% respectively. A positive cut-off point of 10% positive thyroid cells in cyclin D1 immunostaining demonstrated sensitivity of 88.5%, specificity of 100%, PPV of 100% and NPV of 53.8% for correctly detecting thyroid malignancy or low-risk neoplasm. The sensitivity and PPV of simplified nuclear score combined with cyclin D1 immunostaining were 93.3% and 100%, respectively. Both specificity and NPV were maintained at high levels (100% and 66.7%, respectively). The diagnostic accuracy of simplified nuclear score combined with cyclin D1 immunostaining in detecting thyroid malignancy/low-risk neoplasm was increased to 94.1% compared to using either of them alone. Conclusions: Combing simplified nuclear score and cyclin D1 immunostaining on FNA cytology specimens can increase the diagnostic accuracy in classifying thyroid nodules of indeterminate cytological categories. Thus, this supplementary approach provides a simple, accurate, and convenient diagnostic method for cytopathologists so that may reduce unnecessary thyroidectomies.
Humans ; Thyroid Nodule/pathology* ; Biopsy, Fine-Needle ; Cyclin D1 ; Immunohistochemistry ; Thyroid Neoplasms/pathology* ; Retrospective Studies

Humans ; Cyclin D1 ; Melanoma ; Skin Neoplasms ; Cyclin-Dependent Kinase 4

OBJECTIVE: To investigate the effect of Zuogui Jiangtang Jieyu Decoction (ZJJ) on Shh signaling and self-renewal of neural stem cells in the hippocampal dentate gyrus of diabetic rats with depression. METHODS: Diabetic rat models with depression were randomly divided into model group, positive drug (metformin + fluoxetine) group, and low-, medium-, and high-dose ZJJ groups (n=16), with normal SD rats as the control group. The positive drugs and ZJJ were administered by gavage, and the rats in the control and model groups were given distilled water. After the treatment, blood glucose level was detected using test strips, and behavioral changes of the rats were assessed by forced swimming test and water maze test. ELISA was used to examine the serum level of leptin; The expressions of nestin and Brdu proteins in the dentate gyrus of the rats were detected using immunofluorescence assay, and the expressions of self-renewal marker proteins and Shh signaling proteins were detected using Western blotting. RESULTS: The diabetic rats with depression showed significantly increased levels of blood glucose and leptin (P < 0.01) and prolonged immobility time in forced swimming test (P < 0.01) and increased stage climbing time with reduced stage seeking time and stage crossings in water maze test (P < 0.01). The expressions of nestin and Brdu in the dentate gyrus, the expressions of cyclin D1, SOX2, Shh, Ptch1, Smo in the hippocampus and the nuclear expression of Gli-1 were decreased (P < 0.01) while hippocampal Gli-3 expression was increased significantly (P < 0.01) in the rat models. Treatment of rat models with high-dose ZJJ significantly reduced the blood glucose (P < 0.01) and leptin level (P < 0.05) and improved their performance in behavioral tests (P < 0.01). The treatment also obviously increased the expressions of nestin, Brdu, cyclin D1, SOX2, Shh, Ptch1, and Smo and the nuclear expression of Gli-1 in the dentate gyrus (P < 0.01) and reduced hippocampal expression of Gli-3 (P < 0.05) in the rat models. CONCLUSION ZJJ can significantly improve the self-renewal ability of neural stem cells and activate Shh signaling in dentate gyrus of diabetic rats with depression.
Animals ; Rats ; Blood Glucose ; Bromodeoxyuridine ; Cell Self Renewal ; Cyclin D1 ; Dentate Gyrus ; Depression ; Diabetes Mellitus, Experimental ; Hippocampus ; Leptin ; Nestin ; Rats, Sprague-Dawley