1.Effects of PI3K/Akt Pathway in Wound Healing Process of Mice Skin.
Yan-ling GAO ; Chang-sheng LIU ; Rui ZHAO ; Lin-lin WANG ; Shan-shan LI ; Min LIU ; Miao ZHANG ; Shu-kun JIANG ; Zhi-ling TIAN ; Meng WANG ; Da-wei GUAN
Journal of Forensic Medicine 2016;32(1):7-12
OBJECTIVE:
To investigate the expressions and time-dependent changes of phosphatidylinositol-3-kinase (PI3K), phospho-PI3K (p-PI3K), protein kinase B (PKB/Akt) and phospho-Akt (p-Akt) during wound healing process of mice skin.
METHODS:
The changes of PI3K, p-PI3K, Akt and p-Akt expression in skin wound were detected by immunohistochemistry, Western blotting and real-time PCR.
RESULTS:
Immunohistochemistry showed the expression of PI3K and p-Akt were observed in mononuclear and fibroblast after skin wound, and reached peak in reconstruction. The positive bands of PI3K, p-PI3K, Akt and p-Akt were observed in all time points of the wound healing process by Western blotting. The expression peak of p-PI3K and p-Akt showed in inflammation and proliferation; the expression peak of PI3K and Akt in reconstruction. Real-time PCR showed the expression peak of PI3K mRNA in inflammation and reconstruction and the peak of Akt mRNA in reconstruction.
CONCLUSION
During the wound healing process, the expressions of PI3K, Akt, p-PI3K and p-Akt show different changes with significant correlation to wound time. The expression of PI3K/Akt may be a valuable marker for wound time estimation.
Animals
;
Blotting, Western
;
Class I Phosphatidylinositol 3-Kinases
;
Fibroblasts/metabolism*
;
Mice
;
Phosphatidylinositol 3-Kinase
;
Phosphatidylinositol 3-Kinases/metabolism*
;
Phosphorylation
;
Proto-Oncogene Proteins c-akt/metabolism*
;
Real-Time Polymerase Chain Reaction
;
Signal Transduction
;
Skin/injuries*
;
Wound Healing
2.Hexane Extract of Orthosiphon stamineus Induces Insulin Expression and Prevents Glucotoxicity in INS-1 Cells.
Hae Jung LEE ; Yoon Jung CHOI ; So Young PARK ; Jong Yeon KIM ; Kyu Chang WON ; Jong Keun SON ; Yong Woon KIM
Diabetes & Metabolism Journal 2015;39(1):51-58
BACKGROUND: Hyperglycemia, a characteristic feature of diabetes, induces glucotoxicity in pancreatic beta-cells, resulting in further impairment of insulin secretion and worsening glycemic control. Thus, preservation of insulin secretory capacity is essential for the management of type 2 diabetes. In this study, we evaluated the ability of an Orthosiphon stamineus (OS) extract to prevent glucotoxicity in insulin-producing cells. METHODS: We measured insulin mRNA expression and glucose-stimulated insulin secretion (GSIS) in OS-treated INS-1 cells after exposure to a high glucose (HG; 30 mM) concentration. RESULTS: The hexane extract of OS elevated mRNA expression of insulin as well as pancreatic and duodenal homeobox-1 of INS-1 cells in a dose-dependent manner. The hexane OS extract also increased the levels of phosphorylated phosphatidylinositol 3-kinase (PI3K) in a concentration-dependent manner. Additionally, Akt phosphorylation was elevated by treatment with 100 and 200 micromol of the hexane OS extract. Three days of HG exposure suppressed insulin mRNA expression and GSIS; these expressions were restored by treatment with the hexane OS extract. HG elevated peroxide levels in the INS-1 cells. These levels were unaffected by OS treatment under both normal and hyperglycemic conditions. CONCLUSION: Our results suggested that the hexane extract of OS elevates insulin mRNA expression and prevents glucotoxicity induced by a 3-day treatment with HG. This was associated with the activation of PI-3K and Akt.
Glucose
;
Hyperglycemia
;
Insulin*
;
Orthosiphon*
;
Phosphatidylinositol 3-Kinase
;
Phosphatidylinositol 3-Kinases
;
Phosphorylation
;
RNA, Messenger
3.The Effect of si-PKM2 on Proliferation and Apoptosis of Acute Leukemic Cells and Its Molecular Mechanism.
Li-Yuan LI ; Zi-Yuan NIE ; Xiao-Yan ZHANG ; Jian-Min LUO ; Lin YANG ; Qian WANG ; Xing-Zhe WANG
Journal of Experimental Hematology 2021;29(5):1394-1402
OBJECTIVE:
To investigate the effect of glycolytic enzyme pyruvate kinase type 2 (PKM2) on the proliferation and apoptosis of human leukemia HL-60 cells.
METHODS:
si-PKM2 plasmid was transfected into HL-60 cells (set as si-PKM2 group), and blank vector transfected cells were set as control group (si-Ctl group). The expression levels of PKM2 mRNA and protein in si-Ctl group and si-PKM2 group were detected by RT-qPCR and Western blot. CCK-8 cell detection kit was used to detect the proliferation ability of the cells in the two groups. Flow cytometry was used to detect the changes of cell cycle and apoptosis. Western blot and RT-qPCR were used to detect the changes of p-Akt and p-mTOR protein levels in PI3K/Akt/mTOR signaling pathway and the changes of glycolysis-related mRNA levels of the cells in the two groups. The changes in glucose consumption and lactic acid production of the cells were assayed. Over expressed PKM2, HL-60 cells were treated with PI3K inhibitor LY294002 or galactose, the changes in cell proliferation ability, cell cycle and apoptosis, as well as changes in glucose consumption and lactic acid production were detected.
RESULTS:
Interfered by si-PKM2, mRNA and protein levels of PKM2 in si-PKM2 group significantly decreased, and proliferation ability of the cells was also reduced (P<0.05). After PKM2 knockdown, the cells were significantly blocked at G
CONCLUSION
PKM2 knockdown can inhibit the proliferation and induce apoptosis of HL-60 cells, and its molecular mechanism may be related to the PKM2-mediated PI3K/Akt/mTOR-glycolysis, which suggesting that PKM2 may serve as a molecular target for the prevention and treatment of leukemia.
Apoptosis
;
Cell Proliferation
;
Glycolysis
;
Humans
;
Phosphatidylinositol 3-Kinases/metabolism*
;
Pyruvate Kinase
4.Apoptotic Cell Death Induced by Disruption of Epithelial Cell and Extracellular Matrix Interactions in Mouse Inner Medullary Collection Duct Cells.
Jin Suk CHEON ; Mi Young PARK ; Yong Keun KIM ; Sang Ho LEE ; Jin Sup JUNG
Korean Journal of Nephrology 1999;18(1):37-45
Cell-matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation, and aspects of cell growth control. Upon detachment from the matrix epithelial cells enter into programmed cell death and this cell detachment-induced apoptosis has been referred to as "anoikis". This study was undertaken to determine whether apoptosis is induced by inhibition of contact with extracellular matrix in mouse inner medullary collecting duct cells (mIMCD-3), what are signaling mechanisms of the process and whether EGF protects detachment-induced apoptosis. Upon detachment from the extracellular matrix, MDCK and mIMCD-3, which were derived from inner medulla of SV40 transgenic mouse, entered into programmed cell death as a time-dependent manner. Apoptotic cell death induced by cell detachment increased in serum-free medium, which was partly protected by the addition of epidermal growth factor (EGF). Ly294002 and wortmannin, inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), negated the EGF effect, whereas PD98059, a MEK inhibitor, did not. The addition of SB203580, an inhibitor of p38 kinase, did not protect apoptosis in suspended mIMCD-3 cells. These results indicate that apoptosis is induced by inhibition of contact with extracellular matrix in mouse inner medullary collecting duct cells and that PI 3-kinase, not MAPK, is a key mediator of the EGF-induced survival of renal epithelial cells in the absence of attachment.
Animals
;
Apoptosis
;
Cell Death*
;
Epidermal Growth Factor
;
Epithelial Cells*
;
Extracellular Matrix*
;
Mice*
;
Mice, Transgenic
;
Phosphatidylinositol 3-Kinase
;
Phosphatidylinositol 3-Kinases
;
Phosphotransferases
5.Chitosan/PIK3CA siRNA nanoparticle-mediated PIK3CA gene interference decreases the invasive capacity of gastric cancer cells in vitro.
Xinke ZHOU ; Lu HE ; Min LIANG ; Jifang LIU
Journal of Southern Medical University 2014;34(10):1503-1506
OBJECTIVETo investigate the effect of PIK3CA/siRNA chitosan nanoparticle on the invasiveness of gastric carcinoma and the potential value of PIK3CA/siRNA chitosan nanoparticle in suppressing the metastasis of gastric carcinoma.
METHODSGastric cancer cells were treated with PIK3CA/siRNA nanoparticle (with a diameter of 350 nm), and the efficiency of PIK3CA gene interference was evaluated using Western blotting and real-time PCR. The changes of the invasive capacity of the treated cells was assessed with Transwell assay.
RESULTSPIK3CA/siRNA chitosan nanoparticle efficiently lowered the expression level of PIK3CA and significantly decreased the invasion of BGC823 cells.
CONCLUSIONPIK3CA gene interference mediated by PIK3CA/siRNA chitosan nanoparticle can decrease the invasive capacity of gastric cancer cells in vitro.
Cell Line, Tumor ; Cell Proliferation ; Chitosan ; Class I Phosphatidylinositol 3-Kinases ; Humans ; Nanoparticles ; Phosphatidylinositol 3-Kinases ; genetics ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; Stomach Neoplasms ; pathology
6.Multiple oncogenic mutations related to targeted therapy in nasopharyngeal carcinoma.
Jian-Wei ZHANG ; ; Tao QIN ; ; Shao-Dong HONG ; ; Jing ZHANG ; ; Wen-Feng FANG ; ; Yuan-Yuan ZHAO ; ; Yun-Peng YANG ; ; Cong XUE ; ; Yan HUANG ; ; Hong-Yuan ZHAO ; ; Yu-Xiang MA ; ; Zhi-Huang HU ; ; Pei-Yu HUANG ; ; Li ZHANG ;
Chinese Journal of Cancer 2015;34(4):177-183
INTRODUCTIONAn increasing number of targeted drugs have been tested for the treatment of nasopharyngeal carcinoma (NPC). However, targeted therapy-related oncogenic mutations have not been fully evaluated. This study aimed to detect targeted therapy-related oncogenic mutations in NPC and to determine which targeted therapy might be potentially effective in treating NPC.
METHODSBy using the SNaPshot assay, a rapid detection method, 19 mutation hotspots in 6 targeted therapy-related oncogenes were examined in 70 NPC patients. The associations between oncogenic mutations and clinicopathologic factors were analyzed.
RESULTSAmong 70 patients, 12 (17.1%) had mutations in 5 oncogenes: 7 (10.0%) had v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) mutation, 2 (2.8%) had epidermal growth factor receptor (EGFR) mutation, 1 (1.4%) had phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutation, 1 (1.4%) had Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, and 1 (1.4%) had simultaneous EGFR and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations. No significant differences were observed between oncogenic mutations and clinicopathologic characteristics. Additionally, these oncogenic mutations were not associated with tumor recurrence and metastasis.
CONCLUSIONSOncogenic mutations are present in NPC patients. The efficacy of targeted drugs on patients with the related oncogenic mutations requires further validation.
Carcinoma ; Class I Phosphatidylinositol 3-Kinases ; Humans ; Mutation ; Nasopharyngeal Neoplasms ; Neoplasm Recurrence, Local ; Oncogenes ; Pharmacogenetics ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins B-raf ; Receptor, Epidermal Growth Factor
7.Effects of class I( phosphatidylinositol-3-kinases inhibitor on gastric cancer cell xenografts in nude mice.
Ru-Lu LIU ; Kui ZHAO ; Jia-Lei SUN ; Li-Yan YU ; Bao-Song ZHU ; Xiao-Dong YANG ; Chun-Gen XING
Chinese Journal of Gastrointestinal Surgery 2013;16(5):484-488
OBJECTIVETo investigate the effect of recombinant adenovirus (phosphatidylinositol-3-kinases(PI3K)(I()-RNAi-AD which blocks the class I( PI3K signaling pathway on gastric carcinoma cells xenografts in nude mice.
METHODSSubcutaneous tumor models of nude mice were established with SGC7901 cells and randomly divided into PI3K(I()-RNAi-AD group, NC-RNAi-GFP-AD group and control group. The tumor size and the inhibitory rate of tumor growth on days 3, 6, and 9 after cell transplantation were measured. The expression of TNF-α, COX2, P53, PCNA, E-cadherin and nm23/DNPK in tumor tissues were detected by immunohistochemistry.
RESULTSTumor growth was significantly inhibited in the PI3K(I()-RNAi-AD group(14.2%, 21.0%, and 28.1%) on days 3, 6, 9 compared with NC-RNAi-GFP-AD group(1.3%, 1.9%, and 2.0%, all P<0.05). The expressions of TNF-α, P53, E-cadherin and nm23/DNPK were up-regulated, and the expressions of COX2 and PCNA were down-regulated in the PI3K(I()-RNAi-AD group by immunohistochemical staining(all P<0.05).
CONCLUSIONSPI3K(I()-RNAi-AD can inhibit the growth of SGC7901 cell transplantation tumor in vivo in nude mice by inhibiting cell growth, reducing the capacity of tumor invasion and inhibiting tumor angiogenesis.
Adenoviridae ; Animals ; Cell Line, Tumor ; Cell Proliferation ; Class I Phosphatidylinositol 3-Kinases ; Heterografts ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Phosphatidylinositol 3-Kinases ; Phosphatidylinositols ; Stomach Neoplasms
8.Expert consensus on the clinical application of PI3K/AKT/mTOR inhibitors in the treatment of advanced breast cancer.
Chinese Journal of Oncology 2022;44(7):673-692
Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway (PAM pathway) plays an important role in the development of breast cancer and are closely associated with the resistance to endocrine therapy in advanced breast cancer. Therefore, anti-cancer treatment targeting key molecules in this signaling pathway has become research hot-spot in recent years. Randomized clinical trials have demonstrated that PI3K/AKT/mTOR inhibitors bring significant clinical benefit to patients with advanced breast cancer, especially to those with hormone receptor (HR)-positive, human epidermal growth factor receptor (HER) 2-negative advanced breast cancer. Alpelisib, a PI3K inhibitor, and everolimus, an mTOR inhibitor, have been approved by Food and Drug Administration. Based on their high efficacy and relatively good safety profile, expanded indication of everolimus in breast cancer have been approved by National Medical Products Administration. Alpelisib is expected to be approved in China in the near future. The members of the consensus expert panel reached this consensus to comprehensively define the role of PI3K/AKT/mTOR signaling pathway in breast cancer, efficacy and clinical applications of PI3K/AKT/mTOR inhibitors, management of adverse reactions, and PIK3CA mutation detection, in order to promote the understanding of PI3K/AKT/mTOR inhibitors for Chinese oncologists, improve clinical decision-making, and prolong the survival of target patient population.
Breast Neoplasms/metabolism*
;
Consensus
;
Everolimus/therapeutic use*
;
Female
;
Humans
;
MTOR Inhibitors
;
Phosphatidylinositol 3-Kinase/metabolism*
;
Phosphatidylinositol 3-Kinases/metabolism*
;
Phosphoinositide-3 Kinase Inhibitors
;
Proto-Oncogene Proteins c-akt/metabolism*
;
Sirolimus/therapeutic use*
;
TOR Serine-Threonine Kinases/metabolism*
9.PIK3CA gene mutations in Chinese women with HR+/HER2- breast cancer.
Sha Fei WU ; Xue Fei WANG ; Yuan Yuan LIU ; Chuan XIA ; Zhi Yong LIANG ; Xuan ZENG
Chinese Journal of Pathology 2022;51(12):1246-1250
Objective: To investigate the spectrum of PIK3CA gene mutations in Chinese women with hormone receptor positive and HER2 negative (HR+/HER2-) breast cancer, to provide the genetic evidence for identifying potential beneficiaries from specific PI3K isoform inhibitors in Chinese women with breast cancer and to develop detection strategies. Methods: A total of 365 breast cancer specimens archived at the Peking Union Medical College Hospital, Beijing, China from January 2017 to October 2017 were screened. Among these patients, 186 HR+/HER2- women with invasive breast cancer were collected. PIK3CA gene mutations were detected using next generation sequencing technology. The gene variant features were then analyzed and compared with reported data. Results: Among the 186 HR+/HER2- breast cancer cases, 40 (21.5%,40/186) cases harbored PIK3CA gene mutations. Exons 9 and 20 of PIK3CA mutations occurred in 92.5%(37/40)of the tumors, which included E545K, E545G, Q546K, E542K, Q546R, P539R, E547D, H1047R, H1047L, H1047Q and N1044Y. Only one case harbored the exon 7 C420R mutation. Additionally, exons 1 (F83C) and 5 (G364R) uncommon mutations were discovered respectively in 2 cases. Based on the finding, 85.0% (34/40) of cases with known mutations could be detected using companion diagnostic methods. Moreover, 25.0% (10/40) of patients had two or three variants, which were composed of E726K/N345K, H1047Q/N345K, H1047R/G364R, H1047R/E453K, E545G/E726K, E542K/E726K, E542K/H1047R, E545K/H1047R/H1047L and E545K/E547D. The lymph node positive rate in these patients with PIK3CA mutation was remarkably higher than those without (i.e., wild type, <i>Pi><0.05). Conclusions: In this group of HR+/HER2- breast cancer patients, common PIK3CA gene mutations account for the vast majority of the mutations. New rare variants in PIK3CA are also identified while their clinical significance needs to be further studied in a large cohort and/or multi-center study.
Humans
;
Female
;
Breast Neoplasms/genetics*
;
East Asian People
;
China
;
Class I Phosphatidylinositol 3-Kinases/genetics*