Phosphorylation p-Akt Research Articles

A modular microfluidic platform to study how fluid shear stress alters estrogen receptor phenotype in ER+ breast cancer cells

Metastatic breast cancer leads to poor prognoses and worse outcomes in patients due to its invasive behavior and poor response to therapy. It is still unclear what biophysical and biochemical factors drive this more aggressive phenotype in metastatic cancer; however recent studies have suggested that exposure to fluid shear stress in the vasculature could cause this. In this study a modular microfluidic platform capable of mimicking the magnitude of fluid shear stress (FSS) found in human vasculature was designed and fabricated. This device provides a platform to evaluate the effects of FSS on MCF-7 cell line, an estrogen receptor positive (ER+) breast cancer cell line, during circulation in the vessels. Elucidation of the effects of FSS on MCF-7 cells was carried out utilizing two approaches: single cell analysis and bulk analysis. For single cell analysis, cells were trapped in a microarray after exiting the serpentine channel and followed by immunostaining on the device (on-chip). Bulk analysis was performed after cells were collected in a microtube at the outlet of the microfluidic serpentine channel for western blotting (off-chip). It was found that cells exposed to an FSS magnitude of 10 dyn/cm2 with a residence time of 60 s enhanced expression of the proliferation marker Ki67 in the MCF-7 cell line at a single cell level. To understand possible mechanisms for enhanced Ki67 expression, on-chip and off-chip analyses were performed for pro-growth and survival pathways ERK, AKT, and JAK/STAT. Results demonstrated that after shearing the cells phosphorylation of p-AKT, p-mTOR, and p-STAT3 were observed. However, there was no change in p-ERK1/2. AKT is a mediator of ER rapid signaling, analysis of phosphorylated ERα was carried out and no significant differences between sheared and non-sheared populations were observed. Taken together these results demonstrate that FSS can increase phosphorylation of proteins associated with a more aggressive phenotype in circulating cancer cells. These findings provide additional information that may help inform why cancer cells located at metastatic sites are usually more aggressive than primary breast cancer cells.

Discovery of (S)-N1-(thiazol-2-yl) pyrrolidine-1,2-dicarboxamide derivatives targeting PI3Ka/HDAC6 for the treatment of cancer

Recently, PI3K and HDAC have been considered as promising targets for the cancer therapy. A couple of pan-PI3K/HDAC dual inhibitors have been developed as a new class of anticancer agents. Herein, we discovered a new series of (S)-N1-(thiazol-2-yl) pyrrolidine-1,2-dicarboxamide derivatives targeting PI3Kα/HDAC6. All the derivatives exerted dual-target inhibitory activities. Particularly, in the enzymatic selectivity assay, compound 21j was identified as a subtype-selective PI3Kα/HDAC6 dual inhibitor (IC50 = 2.9 and 26 nM against PI3Kα and HDAC6, respectively), which displayed high potency against L-363 cell line with IC50 value of 0.17 μM. In addition, 21j significantly inhibited phosphorylation of pAkt(Ser473) and induced accumulation of acetylated α-tubulin while having a negligible effect on the levels of acetylated Histone H3 and H4 at nanomolar level. Attributed to its favorable in vitro performance, 21j has the potential to alleviate the adverse effects resulted from pan-PI3K inhibition and pan-HDAC inhibition. It is valuable for further functional investigation as an anti-cancer agent.

Differential impact of PI3K/AKT/mTOR signaling on tumor initiation and progression in animal models of prostate cancer.

The PI3K/AKT/mTOR signaling pathway is essential for initiation and progression of prostate cancer. However, there has been no a comprehensive comparison for the role of these signaling nodes in prostate tumor initiation and progression. With genetically engineered animal models, we compared the impact of prostate-specific deletions of Pten, Tsc1, and Tsc2 and activation of Akt1 on tumor initiation and progression. Also, we assessed the expression and genetic alterations of PTEN, AKT1, TSC1, and TSC2 in human primary prostate cancers. For the genetically engineered mice, prostate conditional knockout (cKO) of Pten, Tsc1, and Tsc2 led to initiation and progression of mouse prostatic neoplasia hyperplasia (mPIN). Akt1 transgenic mice developed more aggressive mPINs than mice with Tsc1 or Tsc2 single-cKO, but the effect was more moderate than that for Pten single-cKO or Tsc1/Tsc2 double-cKO mice. Functional analyses showed that Pten single-cKO, AKT1 activation, and Tsc1/Tsc2 double-cKO induced cell proliferation more than Tsc1 or Tsc2 single-cKO, but only Pten single-cKO and AKT1 activation reduced epithelial adhesion. All cKO or AKT1 activation enhanced the phosphorylation of p-S6 (S235/236) but only Pten single-cKO and Tsc1/Tsc2 double-cKO enhanced the phosphorylation of p-AKT (S473) and p-4EBP1 (T37/46/70). In human prostate cancers, PTEN, but not AKT1, TSC1, or TSC2 had frequent genetic alterations. However, as key signaling nodes, AKT1, TSC1, and TSC2 may be responsible for PTEN loss-mediated tumor initiation and progression. Our results for genetically engineered mouse models suggest a differential role of the PI3K/AKT/mTOR signaling nodes in prostate cancer initiation and progression, but the underlying molecular mechanisms remain unaddressed.

MicroRNA-21-5p agomir inhibits apoptosis of oligodendrocyte precursor cell and attenuates white matter injury in neonatal rats

Background and research question/hypothesisExcessive oligodendrocyte precursor cell (OPC) apoptosis occurs during intrauterine infection-induced white matter injury (WMI) in premature infants, preventing excessive apoptosis of OPCs is one of the mechanisms protecting WMI. Micro-RNA-21-5p (miR-21-5p) mediating anti-apoptotic activity was observed in other diseases. Therefore, the aim of this study was to determine whether miR-21-5p protects against WMI by modulating phosphatase and tensin homologue deleted on chromosome 10/phosphatidylinositol-3-kinase/protein kinase B (PTEN/PI3K/Akt) signalling pathway. MethodsA lipopolysaccharide (LPS)-induced neonatal Sprague–Dawley (SD) rat model of preterm WMI was established. To explore the effect of miR-21-5p on WMI, we intraventricularly injected miR-21-5p agomir and miR-21-5p antagomir to activate or inhibit endogenous miR-21-5p. Immunofluorescent labelling of myelin basic protein, immunohistochemical labelling of 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase), and terminal deoxynucleotidyl transferase dUTP nick end labelling assays were conducted to observe pathological white matter changes. The antibody of anti-oligodendrocyte marker 4 (O4) was used to specifically recognise OPCs. The expressions of miR-21-5p and PTEN mRNA in the brain were detected with quantitative real-time polymerase chain reaction (qRT-PCR). PTEN, Akt, and phosphorylated Akt (p-Akt) protein levels were assayed with western blotting, and apoptotic proteins associated with PI3K/Akt signalling were quantified. ResultsIntense white matter dysplasia and excessive OPC apoptosis were observed in the brains of rats with WMI. When the miR-21-5p agonist miR-21-5p agomir was used in the WMI group, apoptosis of OPCs was significantly reduced, and myelin maturation increased. MiR-21-5p agomir relieved WMI. MiR-21-5p agomir inhibited the mRNA and protein expression of PTEN, increased p-Akt phosphorylation, and decreased the expression and activation of related apoptotic proteins.On the other hand, the administration of miR-21-5p specific blocker, miR-21-5p antagomir, reduced the level of p-AKT, increased OPC apoptosis, and worsened WMI. InterpretationOur findings revealed that miR-21-5p agomir had anti-OPC over-apoptotic effects and enhanced myelin development in WMI by modulating the PTEN/Akt signalling pathway.

Downregulation of FAPP2 gene induces cell autophagy and inhibits PI3K/AKT/mTOR pathway in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy. Most patients with T-ALL are treated with high-dose multi-agent chemotherapy due to limited targeted therapeutic options. To further investigate its pathogenesis and establish new therapeutic targets, we studied the role of FAPP2, a Golgi protein, that is, highly expressed in T-ALL, in the growth and function of T-ALL. We found that T-ALL cells underwent reduced cell proliferation and sub-G1 accumulation after knocking down of FAPP2 gene using shRNA systems. Instead, FAPP2 downregulation promoted cell autophagy. The level of autophagy markers, LC3Ⅱ/Ⅰ, Beclin1, and ATG5, was markedly increased, whereas that of P62 decreased after FAPP2 knocking down in T-ALL cells. FAPP2 knocking down led to the accumulation of LC3 in the cytoplasm of T-ALL cells as shown by fluorescence microscopy. In addition, the level of PI(4)P and PI(3,4,5)P decreased and phosphorylation of P-AKT and P-mTOR were downregulated in FAPP2 knock-down cells. In summary, our results show that decreased expression of FAPP2 inhibited cell proliferation, resulted in the sub-G1 phase accumulation of T-ALL cells, and enhanced autophagy of T-ALL cells, likely mediated by PI(4)P, PI(3,4,5)P, and PI3K/AKT/mTOR pathway. Our results provide a new insight into the pathogenesis and development of potential targeted therapy of T-ALL.

Effect of Silencing Information Regulator on Islet β Cells in High Glucose Environment Through Regulation of Phosphatidylinositol 3 Kinase (PI3K)/Protein Kinase B (AKT)/Nuclear Factor-κ-Gene Binding Pathway

Islet β-cell regeneration is beneficial for treating diabetic patients. Silencing information regulator (SIRT1) has a regulatory role in endocrine diseases. However, SIRT1’s role in islet β cells remains unclear. MIN6 cells were cultured and assigned into control group, high glucose group, and SIRT1 group (treated with SIRT1 agonist, Resveratrol) followed by analysis of SIRT1 expression by Real time PCR and ELISA, cell proliferation by MTT assay, apoptosis activity by Caspase3 activity kit, secretion of TNF-α and IL-2 by ELISA, insulin secretion, ROS and SOD generation and expression of PI3K/Akt/NF-κB signaling by Western blot. SIRT1 mRNA was decreased in high glucose environment and its secretion in cell supernatant was reduced, with inhibited cell proliferation, increased Caspase3 activity and secretion of TNF-α and IL-2, decreased insulin secretion and SOD activity, increased ROS content, pAKT phosphorylation and NF-κB expression. Resveratrol significantly promoted SIRT1 expression and cell proliferation, decreased Caspase3 activity and secretion of TNF-α and IL-2, increased insulin secretion and SOD activity, as well as decreased ROS content, pAKT phosphorylation and NF-κB expression (P <0.05). SIRT1 is decreased in high glucose environment, and SIRT1 expression can inhibit islet cell apoptosis, inhibit oxidative stress and inflammation, and promote islet cell proliferation and insulin secretion by regulating PI3K/Akt/NF-κB signaling.

Cytotoxic activity of cholesterol oxidase produced by Streptomyces sp. AKHSS against cancerous cell lines: mechanism of action in HeLa cells.

Re-occurrence of cancer is the major drawback for the currently available anticancer therapies. Therefore, study of an efficient enzyme, cholesterol oxidase produced by various kinds of microbes especially obtained from unexplored marine actinobacterial species against human cancer cell lines and understanding its mechanism of action helps to identify an irreversible and potent anticancer agent. The cytotoxic potential of cholesterol oxidase produced by a marine Streptomyces sp. AKHSS against four different human cancer cell lines was demonstrated through MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Fluorescent confocal microscopy and flow cytometry based experiments were performed to understand the efficiency of the enzymatic action on HeLa cells. Further, the apoptotic related proteins were detected through western blotting. Interestingly, the enzyme exhibited potent cytotoxicity at very low concentrations (0.093-0.327µM) against all the cells tested. Fluorescent confocal microscopy revealed the morphological variations induced by the enzyme on cancer cell lines such as the formation of lipid droplets and condensation of nuclei. The enzyme treated cell-free extracts of HeLa cells analyzed through gas chromatography mass spectrometry showed the depletion of membrane cholesterol and the presence of substituted enzyme oxidized product, cholest-4-ene-3-one. The enzyme had induced significant inhibitory effects on the cell viability such as cell cycle arrest (G1 phase), apoptosis and rise of reactive oxygen species as evident through flow cytometry. Besides, hyperpolarization of mitochondrial membrane, reduced rates of phosphorylation of pAkt and the expression of apoptotic death markers like Fas, Fas L, caspases (8 and 3) and PARP-1 were recorded in the enzyme treated HeLa cells. Thus, cholesterol oxidase purified from a marine Streptomyces sp. AKHSS exhibits potent cytotoxicity at very low concentrations against human cancer cell lines. All the ex vivo experiments portrayed the substantial inhibitory effect of the enzyme on HeLa cells suggesting that cholesterol oxidase of Streptomyces sp. AKHSS could be a prominent cancer chemotherapeutic agent.

Experimental study on nuclear-targeted plasmid-based short hairpin RNA (ShRNA) to hypoxia inducible factor-1α (PshHIF-1α) PshHIF-1α nano-drug carrier system for breast cancer treatment in rats

This study was aimed at exploring the regulatory mechanism of auclear-targeted pshHIF-1α nano-drug carrier system (NPNCS) in the treatment of breast cancer in rats. MDA-MB-231 cell was cultured and DJ-1 and PTEN mRNA level was detected by qRT-PCR along with analysis of cell viability by CCK-8 and apoptosis by means of flow cytometry. The rats were assigned into control group, nuclear-targeted pshHIF-1α nanopharmaceutical system treatment group, and nuclear-targeted pshHIF-1α nano-pharmaceutical system+PTEN inhibitor SF1670 group, followed by analysis of cell proliferation by EdU staining, cell apoptosis by flow cytometry, and the levels of DJ-1, PTEN, and p-AKT. Compared with MDA-MB-231 cells treated with NPNCS, cells without intervention showed decreased PTEN and increased DJ-1 level. NPNCS induced cell apoptosis. Administration of nuclear-targeted pshHIF-1α nano-drug delivery system down-regulated DJ-1 and up-regulated PTEN. Combined treatment with SF1670 promoted p-AKT level and decreased the inhibitory effect of NPNCS on p-AKT level. In conclusion, the nuclear-targeted pshHIF-1α nano-drug delivery system down-regulated DJ-1 and exerted a tumor suppressor and pro-apoptotic effect, which wasrelated to p-AKT phosphorylation and up-regulation of PTEN.

Abstract 1873: Drivers of secondary resistance to anti-EGFR therapy in metastatic colorectal cancer

Abstract Secondary Resistance (SR) evolves in virtually all metastatic colorectal cancers (mCRC) treated with anti-EGFR targeting drugs such as Cetuximab (Cmab). Yet, SR patient-derived xenograft (pdx) models are largely lacking. Here, we established eight SR pdx models through chronic Cmab treatment of drug-sensitive mCRC pdx. All pdx models were initially wild-type for KRAS, NRAS, PI3K and BRAF, as required for Cmab treatment. Post SR establishment, two models showed KRAS mutations and were excluded from further studies. Whole exome sequencing of the remaining six SR pdx models did not reveal any known driver mutations which could confer SR. However, global gene expression analyses identified widespread transcriptional reprograming with many genes able to activate KRAS and/or PI3K signaling pathways. Among the highly dysregulated genes, fibroblast growth factors FGF13, FGF19, FGF20, and insulin-like growth factor IGF2 stood out as potential drivers of SR through upregulation of RTK signaling. Functional molecular analyses of these factors were performed using lentiviral vector mediating gene transduction. Cell viability, proliferation assays and western blot (WB) analyses were applied to assess the molecular functionality of FGFs and IGF2-ligands in vitro. Finally, transgenic cell lines were injected into nude mice to test their therapeutic response towards Cmab treatment following upregulation or downregulation of the chosen candidates. Stable lentiviral overexpression of FGF13 and IGF2 in drug-sensitive cell lines and pdx models triggered SR both in vitro and in vivo. WB analyses showed that FGF13 and IGF2 overexpression induced phosphorylation of pAKT, pS6, and pERK. Conversely, knockdown of IGF2 in a cell line with high endogenous IGF2 expression improved Cmab sensitivity. Lastly, when subjecting a Cmab SR pdx model with high endogenous overexpression of FGF13 and FGF19 to a Pan-FGF inhibitor (LY2874455) in combination with Cmab, we observed a conversion of the SR into partial response. Taken together, these results support a role of IGF2 and FGFs as candidate proteins conferring SR in an autocrine fashion in the setting of KRAS wild-type mCRC under chronic anti-EGFR treatment. Importantly, these results also suggest that potential combination therapies of Cmab and an IGF2 or FGF inhibitor could prevent establishment of SR through these pathways and offer new treatment opportunities for patients with SR mCRC. Citation Format: Soha Hussein Noseir, Stephan Hahn, Abdelouahid Maghnouj, Swetlana Ladigan. Drivers of secondary resistance to anti-EGFR therapy in metastatic colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1873.

Apigenin as an anticancer agent.

Apigenin is an edible plant-derived flavonoid that has been reported as an anticancer agent in several experimental and biological studies. It exhibits cell growth arrest and apoptosis in different types of tumors such as breast, lung, liver, skin, blood, colon, prostate, pancreatic, cervical, oral, and stomach, by modulating several signaling pathways. Apigenin induces apoptosis by the activation of extrinsic caspase-dependent pathway by upregulating the mRNA expressions of caspase-3, caspase-8, and TNF-α. It induces intrinsic apoptosis pathway as evidenced by the induction of cytochrome c, Bax, and caspase-3, while caspase-8, TNF-α, and B-cell lymphoma 2 levels remained unchanged in human prostate cancer PC-3 cells. Apigenin treatment leads to significant downregulation of matrix metallopeptidases-2, -9, Snail, and Slug, suppressing invasion. The expressions of NF-κB p105/p50, PI3K, Akt, and the phosphorylation of p-Akt decreases after treatment with apigenin. However, apigenin-mediated treatment significantly reduces pluripotency marker Oct3/4 protein expression which might be associated with the downregulation of PI3K/Akt/NF-κB signaling.

Abstract 502: Aldo-keto reductase 1B8 (AKR1B8) deficiency causes intestinal immune deficiency

Abstract Background: Intestinal epithelial cells (IECs) are crucial mediators of intestinal immunity. IECs defects may disrupt development and maturation of intestinal immunity. Aldo-keto reductase 1B10 (AKR1B10) is a regulator of de novo fatty acids synthesis and lipid synthesis by mediating acetyl-CoA carboxylase-α (ACCA) stability. Aldo-keto reductase 1B8 (AKR1B8) is an ortholog in mice of human AKR1B10, which has similar regulatory function in fatty acid/lipid synthesis. AKR1B8 deficiency causes IECs defects in proliferation and self-renewal, which may lead to dysregulation of intestinal immunity. Methods: AKR1B8 -/- mice mouse stain was produced by homozygous recombination. Immune cells were isolated from colonic lamina propria (cLP) and mesenteric lymph nodes (MLN). Flow cytometry and immunofluorescence staining were applied for immune cell sorting and measurements. Microbiota composition were quantified by next generation sequencing. Results: AKR1B8 deficiency causes infiltration of neutrophils, mast cells and basophils in the colon. Also, AKR1B8 deficiency causes impaired γδT cells and T helper cells (Th cells) immunity, such as IFNγ, IL17, IL4 or IL22 production, in colon and mesenteric lymph nodes, while enhances distinct arm of CD8 activity and Treg cells. These alterations may be due to the changes of MHCII+ DCs and CD103+ DCs in AKR1B8 deficient mice. Meanwhile, AKR1B8 inhibits the phosphorylation of p-AKT (T308 and S473) and p-ERK (1/2) in IECs, and suppresses the regulation IKKα/NF-κB signaling cascades. Furthermore, AKR1B8 deficiency induces gut microbiota shifts with higher ratio of Firmicutes/Bacteroidetes. Conclusions: AKR1B8 is a critical mediators for IECs signaling and immune homeostasis. Loss of AKR1B8 in IECs induces Th cells immune deficiency. This study identified a new compensatory mechanism under IECs defects for maintaining intestinal immune homeostasis. Citation Format: Xin Wang, Deliang Cao. Aldo-keto reductase 1B8 (AKR1B8) deficiency causes intestinal immune deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 502.

The PI3K inhibitor copanlisib synergizes with sorafenib to induce cell death in hepatocellular carcinoma

Sorafenib, a multikinase inhibitor targeting the Ras/Raf/MAPK (mitogen-activated protein kinase) and vascular endothelial growth factor signaling pathways is an established treatment option for patients with advanced-stage hepatocellular carcinoma (HCC); however, despite its clinical benefit, chemoresistance and disease progression eventually occur almost invariably during treatment. Activation of the PI3K/AKT (phosphatidylinositol-3-kinase/serine/threonine kinase) pathway plays a role in the pathogenesis of HCC and may contribute to determine resistance to sorafenib. We thus evaluated in vitro the effects of the combination of sorafenib and copanlisib, a PI3K inhibitor recently approved for clinical use. The effects of copanlisib alone and in combination with sorafenib were assessed in several HCC cell lines by proliferation and colony formation assays, fluorescence-activated cell sorting analyses, and western blot. In addition, sorafenib-resistant cell clones were used. Copanlisib strongly reduced cell viability and colony formation in different native and sorafenib-resistant HCC cell lines by affecting cyclin D1/CDK4/6 signaling and causing cell cycle arrest. Elevation of phosphorylated (p)-AKT was observed upon incubation with sorafenib and was consistently found in six different unstimulated sorafenib-resistant cell clones. Copanlisib counteracted sorafenib-induced phosphorylation of p-AKT and synergistically potentiated its antineoplastic effect. In summary, copanlisib shows potent anticancer activity as a single agent and acts synergistically in combination with sorafenib in human HCC. Combination of sorafenib with copanlisib represents a rational potential therapeutic option for advanced HCC.

Abstract P6-17-21: Matching the critical function of the biosimilar ABP 980 and trastuzumab: Totality of evidence and scientific justification for extrapolation across trastuzumab indications

Abstract Background Approval of biosimilars is based on the totality of evidence (TOE) supporting clinical equivalence between the proposed biosimilar and the reference product (RP). TOE comprises comprehensive studies designed to evaluate structural, functional, pharmacokinetic (PK), and clinical similarity. TOE provides scientific justification for possible extrapolation across indications that share a common mechanism of action. ABP 980 has been shown to be biosimilar to trastuzumab using robust analytical and functional assessments. Results from phase 1 and phase 3 studies have shown PK and clinical similarity to trastuzumab. Here, we extend the similarity assessment using multiple orthogonal assays for the primary mechanism of action. Methods Multiple lots of ABP 980, trastuzumab (US) and trastuzumab (EU) were compared in the biological activity assessment. In addition to the primary assessment, we conducted additional characterization assays assessing human epidermal growth factor receptor 2 (HER2) cell binding, inhibition of HER2 signaling, inhibition of HER2 ligand-independent proliferation in gastric cancer cells, and a synergy study conducted with docetaxel chemotherapy in gastric cancer cells. HER2 cell-based binding assays were performed using SKBR-3 target cells and a competitive inhibition format. Inhibition of AKT phosphorylation was assessed by comparing the effect of test samples on basal pAkt in the BT-474 breast cancer line. Inhibition of HER2 ligand-independent proliferation was assessed using the gastric cell line NCI-N87 and specificity of inhibition was evaluated using MCF-7 cells that do not have amplified HER2 expression. The synergy study was conducted using NCI-N87 gastric cancer cells and docetaxel using the Combinatorix™ method. Results Relative cell binding to HER2 was similar (ABP 980, 93%-112%; trastuzumab [EU], 98%-109%; trastuzumab [US], 96%-102%). Synergy scores (mean ± SD) with docetaxel (in NCI-N87 cells) for ABP 980, trastuzumab (US) and trastuzumab (EU), respectively, were 16.1 ± 1.4, 16.4 ± 1.8, and 15.8 ± 1.3. Functionally, ABP 980 and trastuzumab similarly inhibited HER2 ligand-independent proliferation in NCI-N87 gastric cancer cells, exhibited specificity against MCF7 non-amplified HER2 expressing breast cancer cells, and showed similar inhibition of pAkt phosphorylation in BT-474 breast cancer cells. Conclusions In this study, we show similar activity between ABP 980 and trastuzumab RP, either alone or in combination with docetaxel, in both breast cancer and gastric cancer cell lines. Our results add to the TOE supporting the biosimilarity of ABP 980 and trastuzumab, and the justification for extrapolation across its approved indications. Citation Format: Kolberg H-C, Colleoni MA, Tomasevic Z, Ponomarova O, McBride HJ, Tesch H, Hanes V. Matching the critical function of the biosimilar ABP 980 and trastuzumab: Totality of evidence and scientific justification for extrapolation across trastuzumab indications [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-17-21.

KY-226 Protects Blood–brain Barrier Function Through the Akt/FoxO1 Signaling Pathway in Brain Ischemia

KY-226 is a protein tyrosine phosphatase 1B (PTP1B) inhibitor that protects neurons from cerebral ischemic injury. KY-226 restores Akt (protein kinase B) phosphorylation and extracellular signal-regulated kinase (ERK) reduction in transient middle cerebral artery occlusion (tMCAO) damage. However, the mechanisms underlying the neuroprotective effects of KY-226 are unclear. To address this, the effects of KY-226 on blood–brain barrier (BBB) dysfunction were examined in tMCAO mice. KY-226 (10 mg/kg, i.p.) was administered to ICR mice 30 min after 2 h of tMCAO. To assess Akt or ERK involvement, wortmannin (i.c.v.) or U0126 (i.v.), selective inhibitors of PI3K and ERK, respectively, were administered to mice 30 min before ischemia. BBB integrity was assessed by Evans blue leakage 24 h post-reperfusion. The levels of tight junction (TJ) proteins, ZO-1 and occludin, were measured by western blotting; ZO-1 mRNA level was measured by RT-PCR. Compared to vehicle, KY-226 treatment prevented BBB breakdown and reduction in TJ protein levels. KY-226 treatment restored ZO-1 mRNA levels post-reperfusion. Pre-administration of wortmannin or U0126 blocked the protective effects of KY-226 on ZO-1 protein and mRNA reduction in tMCAO mice. In bEnd.3 cells, lipopolysaccharide treatment reduced mRNA and protein levels of ZO-1, an effect rescued by KY-226 treatment. Further, KY-226 treatment restored phosphorylation of pAkt (T308) and its downstream target forkhead box protein O1 (FoxO1) (S256) in bEnd.3 cells. Collectively, we demonstrate that KY-226 protects BBB integrity by restoration of TJ proteins, an effect partly mediated by Akt/FoxO1 pathway activation. Thus, protection of BBB integrity likely underlies KY-226-induced neuroprotection in tMCAO mice.

Effects of aloe emodin on U87MG glioblastoma cell growth: In vitro and in vivo study.

Glioblastoma, the most aggressive and malignant form of glioma, appears to be resistant to various chemotherapeutic agents. Hence other approaches have been investigated to target more pathways involved in glioblastoma development and progression. Here we investigate the anticancer effect of Aloe-Emodin (AE), an anthraquinone compound presents in the leaves of Aloe arborescens, on human glioblastoma cell line U87MG. U87MG were treated with various concentrations of AE (20 and 40 μM) for different times (24, 48, and 72 hr). Cell growth was monitored by daily cell count after treatments. Growth analysis showed that AE significantly decrease proliferation of U87MG in a time and dose dependent manner. FACS analysis demonstrates a block of cell cycle in S and G2/M phase. AE probably induced also apoptosis by releasing of apoptosis-inducing factor: PARP and Lamin activation leading to nuclear shrinkage. In addition, exposure of U87MG to AE reduced pAKT phosphorylation. AE inhibition of U87MG growth is a result of more mechanism together. Here we report that AE has a specific growth inhibition on U87MG also in in vivo. The growth of U87MG, subcutaneously injected in nude mice with severe combined immunodeficiency, is inhibited without any appreciable toxic effects on the animals after AE treatment. AE might represent a conceptually new lead antitumor adjuvant drug.

Human podocytes responses to alternatively spliced Extra domain A Fibronectin in culture.

The interactions of the extracellular matrix (ECM) proteins with cells strongly regulate cell behaviour. The glomerular basement membrane (GBM) is a dynamic structure made up of protein secreted by endothelial cells and podocyte. These proteins could regulate the behaviour of these cells in health and diseases. Extra Domain A + Fn (EDA+Fn) is an alternatively spliced form of Fibronectin (Fn) recently identified in GBM and a recognised marker of various pathologies. In this study for the first time, we have investigated the responses of human podocytes cultured on different composition of GBM proteins which are cellular Fn (EDA+), plasma Fn (EDA-) and collagen IV. Conditionally immortalised human podocyte were grown on the dishes coated with different matrices; collagen IV (Col IV), cellular fibronectin (CFn) containing the EDA Exon, plasma fibronectin (PFn), which lacks the EDA Exon (EDA-Fn). We have performed western blotting to characterise the expression of the different proteins, real time PCR and RT-PCR to look for gene expression and alternative splicing of EDA+Fn. We have used TGFβ1 as a stimulator. We have used HEK-Blue-hTLR4 cells to determine the biological activity of cellular Fn. Conditionally immortalised human podocyte show marked differences in their morphology grown on the dishes coated with different matrices; Col IV, CFn, and PFn. CFn was biologically active as it activated the TLR4 signalling in HEK-Blue-hTLR4 cells. Different matrices effects basal as well as TGFβ1 mediated alternative splicing of EDA+Fn. TGFβ1 was active on different matrices as it induced phosphorylation of pSmad3 however it did not affect phosphorylation of pAkt and p38. Interestingly, different cellular matrices affected basal phosphorylation of pAkt. CFn downregulated gene expression of synaptopodin and increased gene expression of collagen I and Fn. CFn increased cell death in detached human podocytes. Alteration of the constituents of the GBM is likely to significantly alter podocyte cellular responses to growth factors involved in podocytopathies, such as TGFβ.

Pratol, an O-Methylated Flavone, Induces Melanogenesis in B16F10 Melanoma Cells via p-p38 and p-JNK Upregulation.

Tyrosinase is the rate-limiting enzyme critical for melanin synthesis. It controls pigmentation in the skin. Activation of tyrosinase is currently the most common approach in the development of tanning and haircare products. Pratol is a 7-hydroxy-4-methoxyflavone found in Trifolium pratense. In this study, we investigated the effects of pratol on melanogenesis. We also studied the mechanism of action of pratol in B16F10 mouse melanoma cells. The cells were treated with various concentrations (6.25, 12.5, 25, and 50 μM) of pratol to observe its effects. The results showed that pratol significantly increased melanin content and tyrosinase activity in the cells without being cytotoxic. In addition, pratol strongly increased the expression of tyrosinase and tyrosinase-related protein-1 and 2 by enhancing the expression of microphthalmia-associated transcription factor. Furthermore, pratol stimulated melanogenesis via the phosphorylation of p38, c-Jun N-terminal kinases (JNK), and extracellular signal–regulated kinase (ERK). The findings from an assay searching for the inhibitor revealed that SB203580 (a specific p38 inhibitor) or SP600125 (a p-JNK inhibitor) attenuated pratol-induced cellular tyrosinase activity whereas PD98059 (an ERK inhibitor) did not. Additionally, pratol interfered with the phosphorylation of p-AKT. We also found that pratol-induced melanogenesis was reversed by H89, which is a specific protein kinase A inhibitor. The results suggest that, owing to its multi-functional properties, pratol may be a potential tanning agent or a therapeutic agent for hair depigmentation in the cosmetic industry.

CHD5 is a potential tumor suppressor in non small cell lung cancer (NSCLC)

Lung cancer is one of the deadliest types of cancers and genetic and epigenetic alterations play major roles in its development. Chromodomain (CHD) protein family acts in chromatin organization, regulation of transcription and also genomic stability and cancer prevention. Although CHD5, a member of this family was shown to contribute to major cellular events and functions as a tumor suppressor gene in various types of cancer, it is not clear whether CHD5 plays a role in lung carcinogenesis. The aim of this study was to investigate the possible role of CHD5 in progression of non-small cell lung cancer (NSCLC). Expression levels of CHD5 gene in 59 tumor and corresponding non-cancerous lung tissue samples were analyzed by qRT-PCR and the methylation status of the promoter region was investigated by methylation specific PCR (MS-PCR). The Akt phosphorylation levels were investigated by Western Blot (WB). CHD5 was down-regulated in 17 (39.5%) and up-regulated in 24 (55.8%) of tumor specimens. Even though the promoter of CHD5 was hypermethylated in 8 patients, it was not found associated with CHD5 gene expression (p=0.08). Akt phosphorylation was increased in 14 (53.8%) and decreased in 12 (46.2%) of the samples but no significant association was found between p-Akt phosphorylation and CHD5 expression (p=0.67). We suggest that CHD5 may act as a tumor suppressor gene in NSCLC.

Discovery of a series of N-(5-(quinolin-6-yl)pyridin-3-yl)benzenesulfonamides as PI3K/mTOR dual inhibitors

Recently, the phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) have been considered as promising targets for the treatment of cancer. Herein, we synthesized a series of N-(5-(quinolin-6-yl)pyridin-3-yl)benzenesulfonamides as novel PI3K/mTOR dual inhibitors for cancer therapy. In the biological evaluation, compound 17e was identified as a potent PI3K/mTOR dual inhibitor, which significantly inhibit Class I PI3Ks, mTOR and phosphorylation of pAkt(Ser473) at low nanomolar level. Moreover, 17e display high potency against PC-3 cells (IC50 = 80 nM) in the anti-proliferative assay, and showed acceptable pharmacokinetic properties in vivo.

The flavonoid apigenin reduces prostate cancer CD44+ stem cell survival and migration through PI3K/Akt/NF-κB signaling

AimsCancer stem cells (CSCs) are involved in drug resistance, metastasis and recurrence of cancers. The efficacy of apigenin on cell survival, apoptosis, migration and stemness properties were analyzed in CSCs. Main methodsProstate CSCs (CD44+) were isolated from human prostate cancer (PCa) PC3 cells using a magnetic-activated cell sorting system. PC3 and CSCs were treated with various concentrations of apigenin, docetaxel and their combinations for 48h. Key findingsApigenin dose dependently inhibited CSCs and PC3 cell survival, and this was accompanied with a significant increase of p21 and p27. Apigenin induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mRNA expressions of caspases-8, -3 and TNF-α, but failed to regulate the intrinsic pathway as determined by the Bax, cytochrome c (Cyt-c) and APAF-1 in CSCs. In contrary to CSCs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of Bax, Cyt-c and caspase-3 while caspase-8, TNF-α and Bcl-2 levels remained unchanged in PC3 cells. The flavonoid strongly suppressed the migration rate of CSCs compared to untreated cells. Significant downregulation of matrix metallopeptidases-2, -9, Snail and Slug exhibits the ability of apigenin treatment to suppress invasion. The expressions of NF-κB p105/p50, PI3K, Akt and the phosphorylation of pAkt were decreased after apigenin treatment. Moreover, apigenin treatment significantly reduced pluripotency marker Oct3/4 protein expression which might be associated with the down-regulation of PI3K/Akt/NF-κB signaling. SignificanceOur data indicated that, apigenin could be a useful compound to prevent proliferation and migration of cancer cells as well as CSCs.