Skp2 inhibitor SKPin C1 decreased viability and proliferation of multiple myeloma cells and induced apoptosis

Multiple myeloma (MM) is a malignant neoplasm of plasma, and exhibits several harmful effects including osteolytic injuries, hypercalcemia, and immune dysfunction. Many patients with MM succumb to the underlying malignancy. An S-phase kinase-related protein 2 (Skp2) inhibitor, designated SKPin C1, has been developed and confirmed to have an inhibitory effect on metastatic melanoma cells. This study aimed to determine the effect of SKPin C1 on MM. Normal B lymphocytes, THP-1 cells, and MM U266 and RPMI 8226 cells were exposed to various dosages of SKPin C1 for 48 h. Cell proliferation was determined by MTT, EdU staining, and cell cycle assays. Western blot assays were performed to assess intracellular protein levels of Skp2, p27, and cleaved caspase-3. The amount of ubiquitin attached to p27 was determined using an immunoprecipitation assay. The viability of U266 and RPMI 8226 cells was significantly inhibited by 10 μM SKPin C1 and the inhibitory effect was enhanced with increasing doses of SKPin C1. In contrast, 50 μM SKPin C1 only marginally decreased viability of normal B lymphocytes in 12 h. Skp2 and p27 expression in U266 and RPMI 8226 cells was higher and lower, respectively, than that in the normal B lymphocytes. Treatment with SKPin C1 or Skp2 knockdown increased p27 protein levels in U266 and RPMI 8226 cells by preventing p27 from being ubiquitinated, which slowed the cell cycle, inhibited cell proliferation, and triggered apoptosis. Therefore, this study suggested SKPin C1 as a potent inhibitor against aberrant proliferation and immortalization of MM.

Inhibition of SKP2 Sensitizes Bromocriptine-Induced Apoptosis in Human Prolactinoma Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Prolactinoma (prolactin-secreting pituitary adenoma) is one of the most common estrogen-related functional pituitary tumors. As an agonist of the dopamine D2 receptor, bromocriptine is used widely to inhibit prolactinoma progression. On the other hand, it is not always effective in clinical application. Although a dopamine D2 receptor deficiency contributes to the impaired efficiency of bromocriptine therapy to some extent, it is unknown whether there some other underlying mechanisms leading to bromocriptine resistance in prolactinoma treatment. That is the main point addressed in this project. MATERIALS AND METHODS: Human prolactinoma samples were used to analyze the S-phase kinase associated protein 2 (SKP2) expression level. Nutlin-3/adriamycin/cisplatin-treated GH3 and MMQ cells were used to analyze apoptosis in SKP2 overexpression or knockdown cells. SKP2 expression and the interaction partners of SKP2 were also detected after a bromocriptine treatment in 293T. Apoptosis was analyzed in C25 and bromocriptine-treated GH3 cells. RESULTS: Compared to normal pituitary samples, most prolactinoma samples exhibit higher levels of SKP2 expression, which could inhibit apoptosis in a p53-dependent manner. In addition, the bromocriptine treatment prolonged the half-life of SKP2 and resulted in SKP2 overexpression to a greater extent, which in turn compromised its pro-apoptotic effect. As a result, the bromocriptine treatment combined with C25 (a SKP2 inhibitor) led to the maximal apoptosis of human prolactinoma cells. CONCLUSION: These findings indicated that SKP2 inhibition sensitized the prolactinoma cells to bromocriptine and helped promote apoptosis. Moreover, a combined treatment of bromocriptine and C25 may contribute to the maximal apoptosis of human prolactinoma cells.

Regulatory effect of Skp2 on the expression and transactivation of the androgen receptor in the progression of castration-resistant prostate cancer / 中华男科学杂志

<p><b>OBJECTIVE</b>To determine the expression of Skp2 in different prostate cancer (PCa) cell lines and tissues, and explore its influence on the androgen receptor (AR) signaling pathway and development of castration-resistant prostate cancer (CRPC).</p><p><b>METHODS</b>The expression levels of Skp2 and AR in different PCa cell lines were detected by Western blot. After knockdown of Skp2 in the C4-2 and 22RV1 cells transfected with shRNA, the expressions of AR and P27 were determined and the activity of ARR3-Luc measured by dual-luciferase reporter gene assay following treatment with dihydrotestosterone (DHT). The expressions of AR and Skp2 in human naïve PCa or CRPC specimens were detected by immunohistochemical staining followed by analysis of their differences and correlation.</p><p><b>RESULTS</b>The Skp2 protein expression level was significantly higher in the C4-2 or 22RV1 cells than in the LNCaP cells. DHT treatment increased the expression of Skp2 in the C4-2 cells, but knock-down of Skp2 significantly up-regulated the expression of the well-known downstream protein P27 and down-regulated that of AR. Consistently, DHT treatment increased the activity of ARR3-Luc, while knockdown of Skp2 remarkably decreased it in the C4-2 and 22RV1 cells (P < 0.05). In addition, significantly higher expressions of Skp2 and AR were observed in the CRPC than in the naïve specimens (P < 0.05), with a positive correlation between the two proteins (r = 0.658 1, P < 0.05).</p><p><b>CONCLUSION</b>Skp2 can enhance the expression and transcription activity of the AR protein in CRPC cells or tissues and is promising to be a critical molecular therapeutic target.</p>

Reversion of multidrug resistance of human gastric cancer SGC7901/DDP cells by E2F-1 gene silencing / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of E2F-1 gene silencing on multidrug resistance of human gastric cancer SGC7901/DDP cells and its possible mechanisms.</p><p><b>METHODS</b>Gastric cancer SGC7901/DDP cells were seeded in 6 well plates and divided into three groups: the experimental group, blank control and the negative control groups. For the experimental group, the SGC7901/DDP cells were transfected with recombinant lentivirus vector (Lv-shRNA-E2F-1), while the negative control with an control lentiviral vector (Lv-shRNA-NC) and the blank control with no treatment. The E2F-1 protein level was analyzed by Western blot. MTT assay was used to detect the half maximal inhibitory concentration (IC50) of three chemotherapy drugs including adriamycin, 5-fluorouracil (5-Fu) and cisplatine (DDP) of the three cell groups. Flow cytometry (FCM) was used to detect the pump-out rate of adriamycin and apoptosis rate of the three cell groups. Semi-quantitative RT-PCR and Western blot were also used to detect the protein and mRNA levels of multidrug resistance-associated genes (MDR1, MRP) and apoptosis-related genes (c-Myc, Skp2, cyclinD1).</p><p><b>RESULTS</b>The expression of E2F-1 protein in the experimental group was significantly lower than that in the negative control and blank control groups (0.794 ± 0.033 vs. 1.487 ± 0.082 vs. 1.511 ± 0.084, P < 0.01). The IC50 of the three chemotherapy drugs (adriamycin, 5-Fu and cisplatine) in the experimental group was significantly lower than that of the negative control and blank control groups, respectively (P < 0.01). Compared with the negative control and blank control groups, the pump-out rate of adriamycin of the experimental group was significantly declined [(0.16 ± 0.01)% vs. (0.37 ± 0.01)% vs. (0.35 ± 0.02)%, P < 0.01]. However, the apoptosis rate of the experimental group was significantly higher than that of the negative control and blank control groups [(33.82 ± 1.26)% vs. (17.34 ± 0.81)% vs. (13.16 ± 1.06)%, P < 0.01]. The results of RT-PCR and Western blot assays showed that mRNA and protein expressions of five genes (MDR1, MRP, CyclinD1, c-Myc, Skp2) in the experimental group were significantly lower than that in the negative control and blank control groups, respectively (P < 0.01).</p><p><b>CONCLUSIONS</b>E2F-1 gene silencing enhances the chemosensitivity of gastric cancer SGC7901/DDP cells to the chemotherapeutic drugs, directly or indirectly downregulated the expression of MDR1 and MRP, and finally reverses the multidrug resistance of the gastric cancer cells. The mechanism may be associated with the suppression of cyclinD1, c-Myc and Skp2.</p>

Silencing of KIF14 interferes with cell cycle progression and cytokinesis by blocking the p27(Kip1) ubiquitination pathway in hepatocellular carcinoma

Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27Kip1 protein level specifically increased after KIF14 knockdown. The increase in p27Kip1 was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27Kip1 accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27Kip1 for degradation by the 26S proteasome, leading to accumulation of p27Kip1. The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.

Oncogenic role of Skp2 and p27Kip1 in intraductal proliferative lesions of the breast / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate whether the connection of p27(Kip1) to S-phase kinase-associated protein 2 (Skp2) plays an oncogenic role in intraductal proliferative lesions of the breast.</p><p><b>METHODS</b>Here we investigated the mechanism involved in association of Skp2’s degradation of p27(Kip1) with the breast carcinogenesis by immunohistochemical method through detection of Skp2 and p27(Kip1) protein levels in 120 paraffin-embedded tissues of intraductal proliferative lesions including usual ductal hyperplasia (UDH, n=30), atypical ductal hyperplasia (n=30), flat epithelial atypia (FEA, n=30), and ductal carcinoma in situ (DCIS, n=30). Moreover, the expression status of Skp2 and p27(Kip1) in 30 cases of the normal breast paraffin-embedded tissues were explored.</p><p><b>RESULTS</b>The DCIS group was with the highest Skp2 level and the lowest p27(Kip1) level, and the UDH group was with the lowest Skp2 level and the highest p27(Kip1) level.Both Skp2 and p27(Kip1) levels in the DCIS group were significantly different from those in the UDH group (all P<0.01).The levels of Skp2 and p27(Kip1) in the FEA group were significantly different from both the DCIS and UDH groups (all P<0.05).p27(Kip1) was negatively correlated with Skp2 in both the UDH group (r=-0.629, P=0.026) and DCIS group (r=-0.893, P=0.000).</p><p><b>CONCLUSION</b>Overexpression of Skp2 might be the mechanism underlying p27(Kip1) over degradation.</p>

Novel roles of Skp2 E3 ligase in cellular senescence, cancer progression, and metastasis / 癌症

S-phase kinase-associated protein 2 (Skp2) belongs to the F-box protein family. It is a component of the SCF E3 ubiquitin ligase complex. Skp2 has been shown to regulate cellular proliferation by targeting several cell cycle-regulated proteins for ubiquitination and degradation, including cyclin-dependent kinase inhibitor p27. Skp2 has also been demonstrated to display an oncogenic function since its overexpression has been observed in many human cancers. This review discusses the recent discoveries on the novel roles of Skp2 in regulating cellular senescence, cancer progression, and metastasis, as well as the therapeutic potential of targeting Skp2 for human cancer treatment.

Correlation of Skp2 overexpression to prognosis of patients with nasopharyngeal carcinoma from South China / 癌症

S-phase kinase-associated protein 2 (Skp2), which plays a role in cell cycle regulation, is commonly overexpressed in a variety of human cancers and associated with poor prognosis. However, its role in nasopharyngeal carcinoma (NPC) is not well understood. In this study, we examined the clinical significance of Skp2, with a particular emphasis on overall survival (OS) and disease-free survival (DFS), in NPC cases in South China, where NPC is an epidemic. Additionally, we explored the function of Skp2 in maintaining a cancer stem cell-like phenotype in NPC cell lines. Skp2 expression was assessed for 127 NPC patients using tissue microarrays and immunohistochemistry and analyzed together with clinicopathologic features, OS, and DFS. Skp2 expression was detectable, or positive, in 75.6% of patients. Although there was no correlation between Skp2 and any clinicopathologic factor, Skp2 expression significantly portended inferior OS (P = 0.013) and DFS (P = 0.012). In the multivariate model, Skp2 expression remained significantly predictive of poor OS [P = 0.009, risk ratio (RR) = 4.06] and DFS (P = 0.008, RR = 3.56), and this was also true for clinical stage (P = 0.012 and RR=3.201 for OS; P = 0.002 and RR=1.94 for DFS) and sex (P = 0.016 and RR=0.31 for OS; P = 0.006 and RR = 0.27 for DFS). After Skp2 knockdown, a colony formation assay was used to evaluate the self-renewal property of stem-like cells in the NPC cell lines CNE-1 and CNE-2. The colony formation efficiency in CNE-1 and CNE-2 cells was decreased. In Skp2-transfected CNE-1 and CNE-2 cells, side population (SP) proportion was increased as detected by flow cytometry. Skp2 is an independent prognostic marker for OS and DFS in NPC. Skp2 may play a role in maintaining the cancer stem cell-like phenotype of NPC cell lines.

Clinical significance of SKP2 and MRP-1/CD9 expression in glottic carcinoma and adjacent tissues / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To study the expression of SKP2 and MRP-1/CD9 protein in glottic cancer and adjacent tissues, and to analyze its significance for a safe surgical margin.@*METHOD@#Thirty-eight cases of glottic squamous cell carcinoma were studied for its cancer tissue, tissue 2 mm, 5 mm, and 10 mm away from cancer, and 10 cases of vocal cord polyp were served as control. SKP2 and MRP-1/CD9 protein were examined by immunohistochemical method.@*RESULT@#The positive expression of SKP2 proteins decreased in sequence of polyp mucosa, those adjacent to carcinoma (10 mm, 5 mm, 2 mm ) and cancer tissue, and there was significant difference between them (P < 0.05); On the contrary, the positive expression of the MRP-1/CD9 proteins increased in sequence of polypous mucosa, those adjacent to carcinoma (10 mm, 5 mm, 2 mm) and cancer tissue,and there was significant difference between them (P < 0.05).@*CONCLUSION@#SKP2 and MRP-1/CD may act as the reference index for judging the biological specialty of LSCC. It is appropriate to regard 5 mm or above 5 mm away from tumors as a safe margin for surgical treatment of glottic carcinoma.

The Effect of Rosiglitazone on the Cell Proliferation and the Expressions of p27 and Skp2 in Helicobacter pylori Infected Human Gastric Epithelial Cells / 대한소화기학회지

BACKGROUND/AIMS: Ligands for peroxisome proliferator-activated receptor gamma (PPAR gamma), a member of the ligand-activated nuclear receptor superfamily, exhibit anti-tumoral effects and are associated with de novo synthesis of proteins involved in regulating the cell cycle and cell survival/death. Helicobacter pylori (H. pylori) is an etiologic agent for gastric adenocarcinoma, and raises the cell turnover of gastric epithelium. The aim of this study was to investigate the effect of PPAR gamma ligand rosiglitazone on the cell proliferation and the expressions of p27 and Skp2 protein in H. pylori infected gastric epithelial cells. METHODS: We examined the expression of PPAR gamma by Western blot in H. pylori infected AGS human gastric epithelial cells. The effect of rosiglitazone on the survival of H. pylori infected AGS cells was assessed by cell viability assay. After the treatment of rosiglitazone in H. pylori infected AGS cells, the expressions of p27 and Skp2 were assessed by Western blot. RESULTS: The expression of PPAR gamma protein was increased in H. pylori infected AGS cells. Cell growth was inhibited and decreased in dose- and time- dependent manner in H. pylori infected AGS cells treated with rosiglitazone. A decrease in Skp2 expression and a reciprocal increase in p27 expression were found in dose- and time-dependent manner in H. pylori infected AGS cells treated with rosiglitazone. CONCLUSIONS: Rosiglitazone inhibited the growth of H. pylori infected AGS cells. Rosiglitazone attenuated Skp2 expression, thereby promoting p27 accumulation in H. pylori infected human gastric epithelial cells. Further studies will be needed to find the effects of accumulation on cell turnover in H. pylori infection and the role in the H. pylori-associated gastric carcinogenesis.

The role of Skp2 in extranodal NK/T-cell lymphoma / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>Expression of Skp2 was related with the prognosis of several tumors. However, there was no intensive study on the relationship between Skp2 and extranodal NK/T cell lymphoma. This study was to explore the role of Skp2 in extranodal NK/T cell lymphoma.</p><p><b>METHODS</b>The clinicopathological data of 39 patients with extranodal NK/T cell lymphoma were analyzed. The expression of Skp2 was examined by immunohistochemistry on formalin fixed, paraffin embedded tissue sections.</p><p><b>RESULTS</b>Among the patients with high expression of Skp2, complete remission (CR) rate was only 14.3% (2/14). However, CR rate among the patients with low expression of Skp2 was 68.0% (17/25). Significant difference was shown between these two groups (P < 0.001). In the group of low expression, the median overall survival (OS) was 85.59 months (95% CI: 35.83 135.34 months), the 1 and 2 year OS rates were 81% and 71%, respectively. However, in the group of high expression, the median OS was only 9.73 months (95% CI: 2.05-17.40 months), the 1 and 2 year OS rates were 42% and 14%, respectively. There was statistical difference between these two groups (P < 0.001). Multivariate analysis showed that Skp2 expression (P <0.001), LDH (P = 0.026) and ECOG PS (P = 0.003) were dependent prognostic factors of extranodal NK/T cell lymphoma.</p><p><b>CONCLUSION</b>High expression of Skp2 is an independent unfavorite adverse prognostic factor of extranodal NK/T cell lymphoma.</p>

Expression of S-phase kinase associated protein 2 in laryngeal carcinoma and its molecular mechanisms involved in regulation of proliferation and apoptosis of Hep-2 cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the expression of S-phase kinase associated protein 2 (Skp2) in human laryngeal squamous cell carcinomas and to explore the effect of silencing of Skp2 by RNAi on the proliferation and apoptosis and the expressing of p27 protein as well as change of cell cycle in laryngeal carcinoma cell line Hep-2 cell.</p><p><b>METHODS</b>The expression of Skp2 in laryngeal carcinoma tissues of different differentiation grade was detected by immunohistochemistry. According to the encoding sequence of mRNA of Skp2, two pieces of oligonucleotide sequences were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pGPU6/Neo siRNA expression vector. The recombinant plasmids pGPU6Skp2 were transfected into Hep-2 cells induced by lipofectamine(TM) 2000. The expression level of Skp2 and p27 were examined by flow cytometry. The cell proliferation was examined by MTT assay. Flow cytometry was performed to analyze apoptosis and cell cycle.</p><p><b>RESULTS</b>Positive expression of Skp2 was detected in all 52 cases of laryngeal carcinoma tissues. The positive rate of expression of Skp2 in well-differentiated laryngeal carcinoma group was lower than that in middle-poorly differentiated group (chi(2) = 7.33, P < 0.05). DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pGPU6 vector, and Skp2 protein expression in the transfected cells were inhibited significantly. The fluorescence index of Skp2 protein expression in pGPU6-Skp2 group was significantly inhibited compared with that in blank pGPU6 group (t = 19.42, P < 0.01). The inhibition ratio of cell proliferation in pGPU6-Skp2 group was 26.93% which was strikingly higher than that of blank pGPU6 group 2.47% (t = 15.23, P < 0.01). The apoptosis ratio in pGPU6-Skp2 group was 11.71% which was increased significantly compared with that of their blank pGPU6 group 1.93% (t = 17.92, P < 0.01). In cell cycle study the percentage of S phase cells in pGPU6-Skp2 group was significantly higher than that in blank pGPU6 group (t = 7.73, P < 0.05). The fluorescence index of p27 protein level was significantly higher than that in blank pGPU6 group (t = 2.86, P < 0.05).</p><p><b>CONCLUSIONS</b>The high level expression of Skp2 in laryngeal carcinoma is significantly related to the differentiation of laryngeal carcinoma. Silencing of Skp2 expression could inhibit cell proliferation and increase cell apoptosis in Hep-2 cells which might be related to the were of p27 protein level and S phase cell cycle arrest of Hep-2 cell.</p>

Detection of Skp2 mRNA gene expression using fluorogenic probe quantitative RT-PCR method in laryngeal squamous cell carcinoma / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#The current study was designed to examine the expression of Skp2 gene in laryngeal squamous cell carcinoma (LSCC) and to investigate the role of Skp2 gene in tumorigenesis and progression of LSCC.@*METHOD@#FQ-PCR method was used to examined the expression of Skp2 gene in 40 LSCC and 10 normal laryngeal mucosa tissues, and relationship between its expression and clinical biological factors of patients with LSCC was analyzed.@*RESULT@#The median copy number of Skp2 mRNA expression in LSCC was 6622.54 copy/microg RNA, the median copy number of Skp2 mRNA expression in normal laryngeal mucosa tissues was 0 copy/microg RNA, there was a very significant difference between them (P < 0.01); The positive rate of Skp2 mRNA expression in LSCC and adjacent normal laryngeal tissue were 50%, 0, respectively (P < 0.01). The median copy number of Skp2 RNA expression in LSCC with cervical lymph node metastasis was 617138.4 copy/microg RNA, the median copy number of Skp2 mRNA expression in LSCC without cervical lymph node metastasis was 0 copy/microg RNA, there was a very significant difference between them (P < 0.05); The positive rate of Skp2 mRNA expression in LSCC with and without cervical lymph node metastasis were 100.00%, 35.48%, respectively (P < 0.01).@*CONCLUSION@#Skp2 gene might have relation with the cervical lymph node metastasis of LSCC. FQ-PCR is an accurate assay to detecting expression of Skp2 mRNA in patient with LSCC. The level of Skp2 mRNA expression might be a new and more accurate marker, and it can be used to predict cervical lymph node metastasis of LSCC.

Protein expression of Skp2 in osteosarcoma and its relation with prognosis / 中南大学学报(医学版)

OBJECTIVE@#To investigate the expression of Skp2 and its relation with P27 expression, clinic pathologic features, and prognostic indicator in osteosarcoma.@*METHODS@#We collected osteosarcoma specimen from 52 patients (29 males and 23 females), who were all treated by radical resection of tumor. The expression of Skp2 and P27 was determined by SP immunohistochemistry. Forty-four patients were followed up for 4 to approximately 84(mean = 31.2)months, while the other 8 patients were lost. Twenty of them survived over 5 years and 24 died.@*RESULTS@#In osteosarcoma, Skp2 highly expressed (mean value was 1.74). Expression intensity of Skp2 at the stage III was obviously higher than that of the stage II(IIa and IIb) (P 0.05). The expressions of skp2 and P27 were negative correlation in osteosarcoma (r = -0.907, P < 0.05).@*CONCLUSION@#Skp2 plays an important role in the occurrence and development of osteocarcoma by causing the degradation of P27, and can be an important prognostic indicator in osteosarcoma.

Expression of Skp2 and PTEN and its correlation in glottic carcinoma / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the expression and clinical significance of Skp2 and PTEN in glottic carcinoma and the relationship between the two genes.@*METHOD@#Formalin-fixed and paraffin-embedded tissues, which came from 42 cases of glottic carcinoma and 16 cases of atypical hyperplasia of vocal fold and 27 cases of vocal cord polyp, were detected for the expression of Skp2, PTEN by SP immunohistochemistry, then we analyzed the result statistically.@*RESULT@#The expression rates of Skp2 protein in vocal cord polyp, atypical hyperplasia of vocal cord and glottic carcinoma were 11.11%, 37.50%, 40.48% respectively. There was significant difference among them (P < 0.01); the expression rates of PTEN protein in vocal cord polyp, atypical hyperplasia of vocal cord and glottic carcinoma were 100.00%, 75.00%, 52.38% respectively. There was significant difference among them (P < 0.05), the expressions of Skp2 and PTEN in glottic carcinoma were associated with clinical stage, lymph nodal metastases and prognosis (P < 0.05); there was a negative correlation between the expression of Skp2 and PTEN, and their correlation coefficient was r= -0.4301 (P < 0.01).@*CONCLUSION@#The expressions of Skp2 and PTEN may play an important roles in the tumorigenesis, metastases and poor prognosis of glottic carcinoma. These changes may be the early molecular event of the carcinogenesis. The high expression of Skp2 was negative correlation with the lower PTEN in glottic carcinoma.

Construction siRNA expressing vector of Skp2 and the inhibitory effects on the expression of Skp2 in laryngeal carcinoma cell line Hep2 cell / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To construct the siRNA expression vector of Skp2 and inhibit the expression of Skp2 through RNA interference in laryngeal carcinoma cell line Hep2 cell.@*METHOD@#According to the encoding sequence of mRNA of Skp2, two pieces of oligonucleotide sequences were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pGPU6/Neo siRNA expression vector, and the recombinant plasmid was transformed into strain DH5a. The plasmid identified by restriction enzyme was used for sequencing. After being identified by sequencing, the recombinant plasmids pGPU6Skp2 were transfected into Hep2 cells. Skp2 expression in the transfected cells was assayed by flow cytometry.@*RESULT@#DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pGPU6 vector, and Skp2 expression in the transfected cells was down-regulated significantly by pGPU6Skp2 at the protein level.@*CONCLUSION@#The siRNA expression vector of Skp2 was successfully constructed and could inhibit Skp2 expression in Hep2 cells. This result will facilitate further studies of Skp2 in gene therapy for tumors.

Detection of Skp2 and p27kip1 expression in human renal cell carcinoma using tissue chip technique / 南方医科大学学报

<p><b>OBJECTIVE</b>To detect the expression of skp2 and p27kip1 in human renal cell carcinoma (RCC) using tissue chip technique, and evaluate the relationship between the proteins and the biological behavior of RCC.</p><p><b>METHODS</b>Tissue chip technique and immunohistochemical SP method was used to detect the expression of skp2 and p27kip1 in normal and tumor tissues.</p><p><b>RESULTS</b>The positivity rate of Skp2 in RCC was significantly higher than that in normal renal tissues (P=0.025). The positivity rate of Skp2 expression in RCC was significantly correlated to poor differentiation of the tumor (P=0.002), and was not associated with the patients gender, age, tumor size, lymph node metastasis and stages of RCC (P>0.05). The positivity rate of p27kip1 in RCC was significantly lower than that in normal renal tissues (P=0.007). The positivity rate of p27kip1 expression was inversely correlated to the malignancy and stage of RCC (P<0.05), but not with the patients' age, gender, lymph node metastasis and tumor size (P>0.05). An inverse correlation was noted between Skp2 and p27kip1 expressions (r= -0.273, P=0.014).</p><p><b>CONCLUSION</b>Overexpression of Skp2 protein may lead to decreased p27kip1 level in RCC, indicating its involvement in the carcinogenesis and development of RCC.</p>

Expression variation and significance of Skp2 and p27(kip1) during the proliferation of Jurkat cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression variation and significance of Skp2 and p27(kip1) during the proliferation of lymphoma cell line Jurkat cells.</p><p><b>METHODS</b>The binding of p27(kip1) and Skp2 in Jurkat cells were detected by immunoprecipitation. Jurkat cells were treated with serum starvation and release synchronization. The expression variation and subcellular localization of p27(kip1) and Skp2 were detected by subcellular fractionation, Western blot and double immunofluorescence labelling.</p><p><b>RESULTS</b>The results of immunoprecipitation suggested that p27(kip1) and Skp2 could bind each other in Jurkat cells. During the proliferation of Jurkat cells, the protein expression of p27(kip1) decreased and intranuclear p27(kip1) decreased significantly, while the Skp2 protein increased and cytoplasmic Skp2 increased significantly.</p><p><b>CONCLUSION</b>During the proliferation of Jurkat cells, the increased cytoplasmic synthesis of Skp2 may speed up p27(kip1) degradation via the ubiquitin-proteasome pathway, then intranuclear p27(kip1) decreases significantly, leading to an increased cell cycling activity.</p>

Effect of Skp2 antisense oligodeoxynucleotide on growth and proliferation of gastric carcinoma SGC-7901 cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effect of S-phase kinase-associated protein 2 antisense oligodeoxynucleotide (Skp2 ASODN) on the growth and proliferation of gastric carcinoma SGC-7901 cells and its mechanism.</p><p><b>METHODS</b>The Skp2 oligodeoxynucleotides (ODNs) were embedded in cationic liposome Lipofectamine 2000 reagent and transfected into SGC-7901 cells. The cell growth and proliferation were observed with light microscopy and MTT assay. Cell cycle was measured by flow cytometry. The expression levels of Skp2 and p27 mRNA were detected by reverse transcription-polymerase chain reaction. The expression levels of Skp2 protein and its substrate p27 protein were detected by Western blot.</p><p><b>RESULT</b>After treatment with Skp2 ASODN, the growth and proliferation of SGC-7901 cells were inhibited in a dose-dependent manner with a peak value at 48 h. The inhibition rate of 200 nmol/L group at 48 h was 42.4 % (P<0.01). In cell cycle study the percentage of S phase cells in 200 nmol/L group was significantly higher than that in normal control group (P<0.05). Both Skp2 mRNA and its protein levels in 200 nmol/L group were significantly lower than those in control group and in Skp2 nonsense oligodeoxynucleotide (Skp2 NSODN) group (P<0.05). However, p27 mRNA level remained unchanged although its protein level was significantly higher than that in control group and NSODN group (P<0.05).</p><p><b>CONCLUSION</b>Skp2 ASODN can inhibit the growth and proliferation of SGC-7901 cells, which may be mediated by interfering with ubiquitin-proteosome pathway and cell cycle regulation.</p>

Growth inhibition of human leukemia cell line U937 by all-trans retinoic acid and its mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effect of all-trans retinoic acid (ATRA) on U937 cell growth and its mechanism.</p><p><b>METHODS</b>Cell cycle was detected by flow cytometry (FCM), expressions of cell cycle associated protein and the p27 related protein were detected by Western blot. The binding of P27 and Skp2 was detected by immunoprecipitation.</p><p><b>RESULTS</b>FCM displayed that ATRA could inhibit the proliferation of U937 cells. At 72 h on 1 micromol/L ATRA treatment, 72% of the cells were arrested at G0/G1 phase. Western blot displayed that ATRA could decrease the expression of cyclin A, up-regulate the expression of p21 and p27, and down-regulate the expression of p27 related proteins Skp2. p27 could bind with Skp2 in U937 cells as detected by immunoprecipitation.</p><p><b>CONCLUSION</b>ATRA may arrest the proliferation of U937 cells through the reduction of Skp2 expression, and finally the induction of the accumulation of p27.</p>