Sphingosine-1-Phosphate Promotes Extravillous Trophoblast Cell Invasion by Activating MEK/ERK/MMP-2 Signaling Pathways via S1P/S1PR1 Axis Activation

Successful placentation depends on the proper invasion of extravillous trophoblast (EVT) cells into maternal tissues. Previous reports demonstrated that S1P receptors are expressed in the EVT cells and S1P could regulate migration and function of trophoblast cells via S1P receptors. However, little is known about roles of S1P in the invasion of EVT cells. Our study was performed to investigate S1P effect on the invasion of EVT cells. We used the extravillous trophoblast cell line HTR8/SVneo cells to evaluate the effect. In vitro invasion assay was employed to determine the invasion of HTR8/SVneo cells induced by S1P. MMP-2 enzyme activity and relative level in the supernatants of HTR8/SVneo was assessed by gelatin zymography and western blot. Based on the above, siRNA and specific inhibitors were used for the intervention and study of potential signal pathways, and Real-time qPCR and western blot were used to test the mRNA and protein level of potential signal targets. We found that S1P could promote HTR8/SVneo cell invasion and upregulates activity and level of MMP-2. The promotion requires activation of MEK-ERK and is dependent on the axis of S1P/S1PR1. Our investigation of S1P may provide new insights into the molecular mechanisms of EVT invasion.     

Repression of Kisspeptin1 weakens hydrogen peroxide‐caused injury in HTR8 cells via adjusting PI3K/AKT/mTOR pathway

Kisspeptin1 (KISS1) is a tumor metastatic suppressor, and its increased expression is validated in human placenta trophoblast cells. Nonetheless, the actions of KISS1 in hydrogen peroxide (H₂O₂)‐impaired human trophoblast HTR8 cells still remain imprecise. This research aims to uncover whether KISS1 can mitigate H₂O₂‐triggered cell injury. HTR8 cells were pretreated with 250 μM H₂O₂ for 4 hours; the autophagic markers (Beclin‐1 and LC3B), cell viability, invasion and apoptosis were appraised. Real‐time quantitative polymerase chain reaction and Western blot trials were enforced for the valuation of KISS1 mRNA and protein levels. After si‐KISS1 transfection and 3‐MA manipulation, the aforesaid biological processes were reassessed for ascertaining the influences of repressed KISS1 in H₂O₂‐impaired HTR8 cells. Phosphoinositide 3‐kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway was eventually estimated. H₂O₂ enhanced Beclin‐1 and LC3B expression, restricted cell viability, and invasion, and meanwhile caused apoptosis. The elevation of KISS1 evoked by H₂O₂ was observed in HTR8 cells. In addition, silencing KISS1 was distinctly annulled the function of H₂O₂ in HTR8 cells. Eventually, we observed that the repression of KISS1 triggered the activation of PI3K/AKT/mTOR in HTR8 cells under H₂O₂ management. The diverting research unveiled that KISS1 repression eased H₂O₂‐caused HTR8 cells injury via mediating PI3K/AKT/mTOR pathway.     

Anhuienoside C inhibits human ovarian cancer cell growth by inducing apoptosis,suppression of cell migration and invasion,and targeting PI3K/AKT/mTOR signaling pathway

The present study was initiated to examine the anticancer effects of Anhuienoside C (AC) against ovarian cancer and postulates the possible molecular mechanism of its action. 3-[4,5-Dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay was implemented for determination of the effects of AC on cell viability of the ovarian cancer OVACAR-3 cell line. To study cellular morphology, phase contrast microscopy was performed. Apoptosis was examined via acridine orange/ethidium bromide used staining assays. Flow cytometry was used to check the different phases of the cell cycle. Cell migration and invasion assays were performed via transwell chamber assay. The effects of AC on expression of phosphoinositide 3-kinases (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) protein in ovarian cell were assessed using western blotting assay. The results indicated that the cell proliferation rate lowered in AC-treated OVACAR-3 cells as compared to the untreated controls in a dose-dependent manner. Cell morphology changed substantially by the exposure to AC and remained dose dependent. These morphological changes were indicative of apoptotic cell death. Apoptosis analysis showed dose-dependent increase of apoptosis. The cell migration and invasion of OVACAR-3 cells was reduced to a minimum by AC in a dose-dependent manner. Finally, western blotting assay showed blocking of PI3K/AKT/mTOR signaling pathway with increasing AC doses. Taking all together, AC is a potential ovarian cancer inhibitor. It induces its anti-ovarian cancer effects via induction of apoptosis, delaying cell migration and invasion, and blocking PI3K/AKT/mTOR signaling pathway.

     

Bromodomain and extraterminal domain inhibitor enhances the antitumor effect of imatinib in gastrointestinal stromal tumours

In gastrointestinal stromal tumours (GISTs), the function of bromodomain‐containing 4 (BRD4) remains underexplored. BRD4 mRNA abundance was quantified in GISTs. In the current study, we investigated the role of BRD4 in GISTs. Our results show a significant enhancement in BRD4 mRNA and a shift from very low‐risk/low‐risk to high‐risk levels as per NCCN specifications. Overexpression of BRD4 correlated with unfavourable genotype, nongastric location, enhanced risk and decreased disease‐free survival, which were predicted independently. Knockout of BRD4 in vitro suppressed KIT expression, which led to inactivation of the KIT/PI3K/AKT/mTOR pathway, impeded migration and cell growth and made the resistant GIST cells sensitive to imatinib. The expression of KIT was repressed by a BRD4 inhibitor JQ1, which also induced myristoylated‐AKT‐suppressible caspases 3 and 9 activities, induced LC3‐II, exhibited dose‐dependent therapeutic synergy with imatinib and attenuated the activation of the PI3K/AKT/mTOR pathway. In comparison with their single therapy, the combination of JQ1/imatinib more efficiently suppressed the growth of xenografts and exhibited a reduction in KIT phosphorylation, a decrease in Ki‐67 and in the levels of phosphorylated PI3K/AKT/mTOR and enhanced TUNEL staining. Thus, we characterized the biological, prognostic and therapeutic implications of overexpressed BRD4 in GIST and observed that JQ1 suppresses KIT transactivation and nullifies the activation of PI3K/AKT/mTOR, providing a potential strategy for treating imatinib‐resistant GIST through dual blockade of KIT and BRD4.     

SIRT3 deficiency is resistant to autophagy-dependent ferroptosis by inhibiting the AMPK/mTOR pathway and promoting GPX4 levels

Ferroptosis, an autophagy-dependent cell death, is characterized by lipid peroxidation and iron accumulation, closely associated with pathogenesis of gestational diabetes mellitus (GDM). Sirtuin 3 (SIRT3) has positive regulation on phosphorylation of activated protein kinase (AMPK), related to maintenance of cellular redox homeostasis. However, whether SIRT3 can confer autophagy by activating the AMPK-mTOR pathway and consequently promote induction of ferroptosis is unknown. We used human trophoblastic cell line HTR8/SVneo and porcine trophoblastic cell line pTr2 to deterimine the mechanism of SIRT3 on autophagy and ferroptosis. The expression of SIRT3 protein was significantly elevated in trophoblastic cells exposed to high concentrations of glucose and ferroptosis-inducing compounds. Increased SIRT3 expression contributed to classical ferroptotic events and autophagy activation, whereas SIRT3 silencing led to resistance against both ferroptosis and autophagy. In addition, autophagy inhibition impaired SIRT3-enhanced ferroptosis. On the contrary, autophagy induction had a synergistic effect with SIRT3. Based on mechanistic investigations, SIRT3 depletion inhibited activation of the AMPK-mTOR pathway and enhanced glutathione peroxidase 4 (GPX4) level, thereby suppressing autophagy and ferroptosis. Furthermore, depletion of AMPK blocked induction of ferroptosis in trophoblasts. We concluded that upregulated SIRT3-enhanced autophagy activation by promoting AMPK-mTOR pathway and decreasing GPX4 level to induce ferroptosis in trophoblastic cells. SIRT3 deficiency was resistant to high glucose- and erastin-induced autophagy-dependent ferroptosis and is, therefore, a potential therapeutic approach for treating GDM.     

Curcumin stimulates angiogenesis through VEGF and expression of HLA‐G in first‐trimester human placental trophoblasts

Curcumin has a protective role in placental diseases like preeclampsia and preterm birth. Very little is known about its functional effects on growth, angiogenesis, and epigenetic activities of human first trimester placenta. HTR8/SVneo trophoblasts cells were used as model for human first trimester placenta. Effects of curcumin (≥80%) in these cells were investigated using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), radioactive thymidine uptake, quantitative real‐time polymerase chain reaction (qRT‐PCR), promoter DNA methylation, qRT‐PCR array, tube formation, wound healing, and immunoblot assays. PC3 (prostate cancer), JEG‐3 (trophoblast), and HMEC‐1 (endothelial) cells were used as control in various experiments. Unlike in PC3 cells, curcumin stimulated growth, proliferation, and viability in HTR8/SVneo cells. Curcumin increased tube formation, and messenger RNA (mRNA) expression of angiogenic factors such as vascular endothelial growth factor A (VEGFA) and protein expression of proangiogenic factor VEGF receptor‐2 and fatty acid‐binding protein‐4 (FABP4) in these cells. Curcumin‐stimulated tube formation was associated with an increased expression of VEGFR2 and FABP4. The stimulatory effects of curcumin were inhibited by VEGFR2 (SU5416) and FABP4 (BMS309403) inhibitors. Curcumin also significantly increased both mRNA and protein expression of HLA‐G in HTR8/SVneo cells. Curcumin increased mRNA expression of DNMT3A and NOTCH signaling system whereas down‐regulated mRNA expression of HSD11β2. Curcumin enhanced hypomethylation of gene promoters against oxidative stress and DNA damage pathway mediators. Curcumin promotes cell growth, migration, and thus angiogenic potential of these cells. Increased expression of HLA‐G by curcumin, hitherto unknown, is a novel finding since HLA‐G not only favors the immune environment for invasive trophoblasts but also positively modulates angiogenesis.     

Histone Deacetylase Inhibitors Inhibit the Proliferation of Gallbladder Carcinoma Cells by Suppressing AKT/mTOR Signaling

Gallbladder carcinoma is an aggressive malignancy with high mortality mainly due to the limited potential for curative resection and its resistance to chemotherapeutic agents. Here, we show that the histone deacetylase inhibitors (HDACIs) trichostatin-A (TSA) and suberoylanilide hydroxamic acid (SAHA) reduce the proliferation and induce apoptosis of gallbladder carcinoma cells by suppressing the AKT/mammalian target of rapamycin (mTOR) signaling. Gallbladder carcinoma SGC-996 cells were treated with different concentrations of TSA and SAHA for different lengths of time. Cell proliferation and morphology were assessed with MTT assay and microscopy, respectively. Cell cycle distribution and cell apoptosis were analyzed with flow cytometry. Western blotting was used to detect the proteins related to apoptosis, cell cycle, and the AKT/mTOR signaling pathway. Our data showed that TSA and SAHA reduced SGC-996 cell viability and arrested cell cycle at the G1 phase in a dose- and time-dependent manner. TSA and SAHA promoted apoptosis of SGC-996 cells, down-regulated the expression of cyclin D1, c-Myc and Bmi1, and decreased the phosphorylation of AKT, mTOR p70S6K1, S6 and 4E-BP1. Additionally, the mTOR inhibitor rapamycin further reduced the cell viability of TSA- and SAHA-treated SGC-996 cells and the phosphorylation of mTOR, whereas the mTOR activator 1,2-dioctanoyl-sn-glycero-3-phosphate (C8-PA) exerted the opposite influence. Our results demonstrate that histone deacetylase inhibitors (HDACIs) suppress the proliferation of gallbladder carcinoma cell via inhibition of AKT/mTOR signaling. These findings offer a mechanistic rationale for the application of HDACIs in gallbladder carcinoma treatment.     

Bone morphogenetic protein 2 induces the activation of WNT/β-catenin signaling and human trophoblast invasion through up-regulating BAMBI

Both bone morphogenetic protein 2 (BMP2) and WNT/β-catenin signaling promote human trophoblast cell invasion. BMP2 has been shown to up-regulate bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) in granulosa cells. Besides, studies indicate BAMBI is a positive regulator for WNT/β-catenin signaling. However, whether BMP2 can increase BAMBI expression to induce WNT/β-catenin signaling for trophoblast cell invasion is still unknown. To study the roles of BAMBI in BMP2-induced activation of WNT/β-catenin signaling and human trophoblast invasion, we used immortalized human extravillous trophoblast (EVT) cell line (HTR8/SVneo) and primary human EVT cells as study models. Messenger RNA and protein levels of target genes were checked with RT-qPCR and Western blot assay respectively. The function of target proteins was studied via small interfering RNA (siRNA)-mediated knockdown. In addition, trophoblast cell invasiveness was examined by matrigel-coated transwell assays. Our results demonstrate that BMP2 treatment increased BAMBI mRNA levels and the activation of WNT/β-catenin signaling including the raised phosphorylation of GSK3β, the up-regulation of active (non-phosphorylated) β-catenin as well as its downstream target molecule cyclin D1, all of which were totally blocked by the activin receptor-like kinases (ALK) 2/3 inhibitor DMH1 or siRNA-mediated knockdown of BAMBI in HTR8/SVneo cells. Consistently, in primary human EVT cells, BMP2 also induced the up-regulation of BAMBI and the activation of WNT/β-catenin signaling indicated by the increased levels of active β-catenin and cyclin D1, which were completely blocked by BAMBI knockdown. In conclusion, BMP2 promotes the activation of canonical WNT/β-catenin signaling and human trophoblast cell invasion by up-regulating BAMBI.     

Comparative proteomic analysis of trophoblast cell models reveals their differential phenotypes,potential uses,and limitations

Trophoblastic cell lines are widely used in in vitro studies of placental function as a surrogate for primary trophoblasts. To date, no reference proteomics dataset exists to directly compare the shared and unique characteristics of these cells. Here, we performed comparative proteomic profiling of the BeWo and HTR8/SVneo cell lines using label‐free quantitative MS. A total of 1557 proteins were identified, which included 338 uniquely attributed to BeWo cells, and a further 304 specifically identified in HTR8/SVneo cells. Raw data are available via ProteomeXchange, identifier PDX005045. Of the 915 proteins expressed by both cell lines, 105 were of higher abundance in BeWo cells, while 199 proteins had a significantly higher expression in HTR8/SVneo cells. Comparative GO of unique and upregulated proteins revealed principal differences in cell junction/adhesion, catenin complex, spindle and microtubule associated complex, as well as cell differentiation. Our data indicate that BeWo cells express an epithelial proteome more characteristic of villous trophoblasts, whereas HTR8/SVneo cells embrace a mesenchymal phenotype, more characteristic of extravillous trophoblasts. This novel comparative proteomic profiling of these trophoblastic cell lines provides a useful platform for future investigations of placental function.     

ATL1 inhibits the proliferation and invasion of trophoblast cells via inhibition of the mTOR signaling pathway

Pre-eclampsia (PE) is a major cause of hypertension in maternal and fetal. Atlastin-1 (ATL1), one regulator of endoplasmic reticulum (ER) morphology, participates in tubular ER formation and protein synthesis. The objective of this study is to investigate the role and molecular mechanism of ATL1 in PE. GEO databases showed that ATL1 was upregulated in PE patients. Our data also found that ATL1 was highly expressed in PE placental tissues. The cell viability, proliferation, migration, and invasion of HTR-8/SVneo cells increased/decreased after the downregulation/upregulation of ATL1. The mTOR pathway is the downstream pathway of ATL1. The levels of p-p70S6K and p-mTOR were increased/decreased after the downregulation/upregulation of ATL1. Moreover, rapamycin, an inhibitor of mTOR pathway, reversed the promotive effect of siATL1 on proliferation, migration, and invasion in HTR-8/SVneo cells. In conclusion, ATL1 inhibits the proliferation and invasion of trophoblast cells via the inhibition of the mTOR signaling pathway in HTR-8/SVneo cells.     

Downregulation of SPARC Expression Inhibits the Invasion of Human Trophoblast Cells In Vitro

Successful pregnancy depends on the precise regulation of extravilloustrophoblast (EVT) invasion into the uterine decidua. SPARC (secreted protein acidic and rich in cysteine) is a matricellular glycoprotein that plays critical roles in the pathologies associated with obesity and diabetes, as well as tumorigenesis. The objective of this study was to investigate the role of SPARC in the process of trophoblast invasion which shares many similarities with tumor cell invasion. By Western blot, higher expression of SPARC was observed in mouse brain, ovary and uterus compared to other mouse tissues. Immunohistochemistry analysis revealed a spatio-temporal expression of SPARC in mouse uterus in the periimplantation period. At the implantation site of d8 pregnancy, SPARC mainly accumulated in the secondary decidua zone (SDZ), trophoblast cells and blastocyst. The expression of SPARC was also detected in human placental villi and trophoblast cell lines. In a Matrigel invasion assay, we found SPARC-specific RNA interference significantly reduced the invasion of human extravilloustrophoblast HTR8/SVneo cells. Microarray analysis revealed that SPARC depletion upregulated the expression of interleukin 11 (IL11), KISS1, insulin-like growth factor binding protein 4 (IGFBP4), collagen type I alpha 1 (COLIA1), matrix metallopeptidase 9 (MMP9), and downregulated the expression of the alpha polypeptide of chorionic gonadotropin (CGA), MMP1, gap junction protein alpha 1 (GJA1), et al. The gene array result was further validated by qRT-PCR and Western blot. The present data indicate that SPARC may play an important role in the regulation of normal placentation by promoting the invasion of trophoblast cells into the uterine decidua.     

Declination of long noncoding RNA paternally expressed gene 10 inhibits A375 cells proliferation,migration, and invasion via mediating microRNA-33a

The importance of long noncoding RNAs (lncRNAs) has been certified in malignant melanoma. Nonetheless, the functions of lncRNA paternally expressed gene 10 (PEG10) in malignant melanoma remain uninvestigated. This research discloses the influence of PEG10 in the biological actions of malignant melanoma cells. The sh-PEG10 plasmid was transfected into A375 cells; meanwhile, the effects of declined PEG10 on cell viability, apoptosis, migration, invasion, and the correlative protein levels were probed. The miR-33a expression in sh-PEG10-transfected cells was examined, and the above biological processes were studied again in miR-33a inhibitor-transfected A375 cells. Phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and mechanistic target of rapamycin (mTOR) pathways were delved via Western blot. We found that the enhancement of PEG10 was discovered in melanoma tissues compared to related nonmelanoma tissues. Declination of PEG10 frustrated cell viability, repressed cyclinD1 and CDK4 expression, and triggered apoptosis, as well as suppressed migration and invasion in A375 cells. A negative correction between PEG10 and miR-33a was confirmed, and repressed miR-33a inverted the functions of PEG10 repression in A375 cells. In addition, PEG10 repression discouraged the activation of PI3K/AKT and mTOR pathways via elevation of miR-33a. These results indicated that declination of PEG10 restrained A375 cell growth, migration, and invasion via adjusting miR-33a and PI3K/AKT and mTOR pathways.     

Retracted: Long noncoding RNA MEG3 is a tumor suppressor in choriocarcinoma by upregulation of microRNA-211

Tumor suppressor long noncoding RNA maternally expressed gene 3 (lncRNA MEG3) exists in various cancers. Nonetheless, the functions of lncRNA MEG3 in choriocarcinoma (CC) are still not well studied. We explored the effects of lncRNA MEG3 on human CC JEG-3 and BeWo cells. lncRNA MEG3 was overexpressed, and the effects of lncRNA MEG3 on cell viability, proliferation, apoptosis, migration, and invasion were assessed by the cell counting kit-8 assay, western blot analysis, flow cytometry (plus western blot analysis), and transwell assay (plus western blot analysis), respectively. Then, the expression level of miR-211 was detected by real-time quantitative polymerase chain reaction. After that, the effects of dysregulated microRNA-211 (miR-211) with overexpressing lncRNA MEG3 on JEG-3 cells and BeWo cells were testified. Western blot analysis was used to study the involvements of the signaling pathways in the lncRNA MEG3-associated modulation. We found that lncRNA MEG3 upregulation reduced cell viability, inhibited proliferation, migration and invasion, and promoted apoptosis. Expression of miR-211 was upregulated after lncRNA MEG3 overexpression. Effects of lncRNA MEG3 overexpression were augmented by miR-211 overexpression, while they were declined by miR-211 silencing. Phosphorylated levels of PI3K, AKT, and AMP-activated protein kinase (AMPK) were decreased by lncRNA MEG3 overexpression via regulation of miR-211. To sum up, lncRNA MEG3 could repress proliferation, migration and invasion, and promote apoptosis of JEG-3 and BeWo cells through upregulating miR-211. The PI3K/AKT and AMPK pathways were inhibited by lncRNA MEG3 overexpression via regulation of miR-211.     

Plac8‐mediated autophagy regulates nasopharyngeal carcinoma cell function via AKT/mTOR pathway

To explore the relationship between autophagy and cell function, we investigated how PLAC8‐mediated autophagy influences proliferation, apoptosis and epithelial‐mesenchymal transition (EMT) in NPC. Colony formation analyses and CCK8 assays were used to assess the proliferative capacity of NPC cells. Transmission electron microscopy (TEM) was used to identify autophagosomes. Autophagic flux was monitored using the tandem monomeric RFP‐GFP‐tagged LC3 (tfLC3) assay. The rate of apoptosis in NPC cells was analysed by flow cytometry. Western blot analysis was used to evaluate the activation of autophagy and the signalling status of the AKT/mTOR pathway. Our study reveals that knocking out PLAC8 (koPLAC8) induces autophagy and apoptosis, while suppressing NPC cell proliferation and EMT. However, inhibition of autophagy with 3‐methyladenine or by knocking down Beclin‐1 reverses the cell proliferation, apoptosis and EMT influenced by koPLAC8. We find that koPLAC8 inhibits the phosphorylation of AKT and its downstream target, mTOR. Moreover, immunofluorescence and co‐immunoprecipitation reveal complete PLAC8/AKT colocalization and PLAC8/AKT interaction, respectively. Furthermore, knockout of PLAC8 induced autophagy and inactivated AKT/mTOR signalling pathway of NPC xenografts. Overall, our findings demonstrate that koPLAC8 induces autophagy via the AKT/mTOR pathway, thereby inhibiting cell proliferation and EMT, and promoting apoptosis in NPC cells.     

AKT Inhibitors Promote Cell Death in Cervical Cancer through Disruption of mTOR Signaling and Glucose Uptake

Background

PI3K/AKT pathway alterations are associated with incomplete response to chemoradiation in human cervical cancer. This study was performed to test for mutations in the PI3K pathway and to evaluate the effects of AKT inhibitors on glucose uptake and cell viability.

Experimental Design

Mutational analysis of DNA from 140 pretreatment tumor biopsies and 8 human cervical cancer cell lines was performed. C33A cells (PIK3CAR88Q and PTENR233*) were treated with increasing concentrations of two allosteric AKT inhibitors (SC-66 and MK-2206) with or without the glucose analogue 2-deoxyglucose (2-DG). Cell viability and activation status of the AKT/mTOR pathway were determined in response to the treatment. Glucose uptake was evaluated by incubation with ¹⁸F-fluorodeoxyglucose (FDG). Cell migration was assessed by scratch assay.

Results

Activating PIK3CA (E545K, E542K) and inactivating PTEN (R233*) mutations were identified in human cervical cancer. SC-66 effectively inhibited AKT, mTOR and mTOR substrates in C33A cells. SC-66 inhibited glucose uptake via reduced delivery of Glut1 and Glut4 to the cell membrane. SC-66 (1 µg/ml-56%) and MK-2206 (30 µM-49%) treatment decreased cell viability through a non-apoptotic mechanism. Decreases in cell viability were enhanced when AKT inhibitors were combined with 2-DG. The scratch assay showed a substantial reduction in cell migration upon SC-66 treatment.

Conclusions

The mutational spectrum of the PI3K/AKT pathway in cervical cancer is complex. AKT inhibitors effectively block mTORC1/2, decrease glucose uptake, glycolysis, and decrease cell viability in vitro. These results suggest that AKT inhibitors may improve response to chemoradiation in cervical cancer.