RACGAP1 promotes lung cancer cell proliferation through the PI3K/AKT signaling pathway
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Abstract We aimed to investigate the expression and clinic significance of Rac GTPase Activating Protein 1 (RACGAP1) in human lung adenocarcinoma (LUAD). Online database analysis revealed a significant increase in RACGAP1 mRNA expression among 26 types of tumor tissues, including LUAD tissues. Online database and tissue microarray analyses indicated that RACGAP1 expression was significantly upregulated in LUAD tissues. Genetic variation analysis identified four different genetic variations of RACGAPs in LUAD. Moreover, online database analysis showed that RACGAP1 upregulation was correlated with shorter survival in patients with LUAD. After silencing RACGAP1 expression in A549 cells using siRNA and assessing its protein levels via Western blotting, we found that RACGAP1 knockdown inhibited cell growth and induced apoptosis determined using the Cell Counting Kit-8 assay, colony formation assay, and flow cytometry. Mechanistically, western blot analysis indicated that Bax expression increased, whereas Bcl-2 expression decreased. Moreover, RACGAP1 knockdown attenuated PI3K/AKT pathway activation in lung cancer cells. Taken together, our findings showed that RACGAP1 was overexpressed in LUAD tissues and played an important role in lung cancer by increasing cell growth through the PI3K/AKT signaling pathway. This study suggests recommends evaluating RACGAP1 in clinical settings as a novel biomarker and potential therapeutic target for lung cancer.The PI3K / AKT / mTOR pathway is an important regulator of a wide range of cellular processes including survival, proliferation, growth, metabolism, angiogenesis and metastasis. PI3K induces translocation of Ser / Thr Akt kinase, Akt is activated by PDK1 and mTOR2-dependent phosphorylations. Aberrant activation of the PI3K / AKT / mTOR pathway is frequently observed in many human malignancies and the combination of compounds simultaneously targeting different related molecules in the PI3K / AKT / mTOR pathway leads to synergistic activity. To explore the common AKT / mTOR and AKT / PI3K competitor ATP inhibitors we performed a 2D AKT-SAR model to predict the bioactivity of PI3K and mTOR inhibitors on AKT, the interaction of the best inhibitors was evaluated by docking analysis and compared to that of GSK690693 and Ipatasertib. An AKT-SAR model with a correlation coefficient (R2) of 0.85212 and an RMSE of 0.09266 was obtained, which was validated and evaluated by a cross-validation method LOO, the most predicted inhibitors of PI3K and mTOR respectively PIC50 activities between [9.30 - 8.54], and [9.79 - 8.91]. After docking and several comparisons, inhibitors with better predictions showed better affinity and interaction with AKT compared to GSK690693 and Ipatasertib. We therefore found that 8 PI3K inhibitors and 11 mTOR inhibitors met the Lipinski and Veber criteria and could be future drugs.
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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.The changes of PI3K, p-PI3K, Akt and p-Akt expression in skin wound were detected by immunohistochemistry, Western blotting and real-time PCR.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.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.
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The purpose of this study was to explore the mechanism of Solanine disrupting energy metabolism in human renal cancer ACHN cells and to clarify its target. The specific method was to culture human renal cancer ACHN cell lines, and to intervene with Solanine of high, medium and low concentrations. The content of ATP in cells was measured by ELISA method. The expression of HIF-1α protein and the expression of PI3K, AKT, p-PI3K, p-AKT in PI3K/AKT pathway were detected by Western blotting. The results showed that compared with the control group, the relative expression of p-PI3K and p-AKT showed a downward trend with the increase of Solanine concentration (P 0.05). In addition, the relative expression of HIF-1α also showed a downward trend (P < 0.05). According to the above results, it is suggested that Solanine can significantly inhibit the energy metabolism of renal cancer cells, the main mechanism of which is the down-regulation of HI-1αf downstream of the PI3K/Akt pathway by inhibiting the phosphorylation process of PI3K/p-PI3K and Akt/p-Akt.
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Small interfering RNA (siRNA) molecules achieve sequence- specific gene silencing through a process known as RNA interference (RNAi). Compared to other nucleic acid-based therapeutics aimed at post-transcriptional gene silencing, such as antisense oligodeoxynucleotides, siRNA molecules achieve greater magnitude and duration of gene silencing at significantly lower doses. While the duration of gene knockdown by siRNA typically lasts around 1 week in rapidly dividing cells, recent reports of knockdown lasting for several weeks in nondividing cells indicate that dilution due to cell division may be a limiting factor in rapidly dividing cells. To determine if cell division directly impacts the duration of gene knockdown by siRNA, we chose to investigate the kinetics of siRNA-mediated gene silencing in luciferase-expressing cell lines with different observed doubling times using noninvasive bioluminescent imaging and a mathematical model of siRNA delivery and function. In vitro and in vivo, the duration of gene knockdown is inversely proportional to the rate of cell division. Consistent with previous reports, luciferase protein levels recover to pre-treatment values within less than 1 week in rapidly dividing cell lines, but take longer than 3 weeks to return to steady-state levels in nondividing fibroblasts. Similar results are observed in vivo, with knockdown lasting around 1 week in subcutaneous tumors in A/J mice and 3-4 weeks in the nondividing hepatocytes of BALB/c mice. These data indicate that dilution due to cell division, and not intracellular siRNA half-life, governs the duration of gene silencing under these conditions. Here, we will present our latest results describing the effects of cell doubling time, siRNA stability, and dosing schedule on siRNA- mediated gene silencing. Specifically, we will investigate whether the duration of knockdown using chemically modified siRNA molecules exhibits a similar dependence on cell doubling time. The implications of these findings will be highlighted using model calculations to determine the dosing schedule required to maintain persistent silencing of target proteins and to predict when maximum mRNA or protein knockdown will occur, an especially important factor when trying to observe a therapeutic effect resulting from protein knockdown. The approach of bioluminescent imaging combined with mathematical modeling provides insights into siRNA function that will hopefully be of practical use for both researchers and clinicians alike.
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Abstract Background Due to the frequent dysregulation of the PI3K/AKT/mTOR signaling pathway, mTOR represents a suitable therapeutic target in hepatocellular carcinoma (HCC). However, emerging data from clinical trials of HCC patients indicate that mTOR inhibition by RAD001 (Everolimus) alone has only moderate antitumor efficacy which may be due to the feedback activation of AKT after mTOR inhibition. In this study, we analyzed the effects of dual inhibition of mTOR and AKT on the proliferation of HCC cell lines. In addition, we measured the feedback activation of each of the AKT isoforms after mTOR inhibition in HCC cell lines and their enzymatic activity in primary samples from HCC patients. Methods The activation status of specific AKT isoforms in human HCC samples and corresponding healthy liver tissue was analyzed using an AKT isoform specific in vitro kinase assay. AKT isoform activation after mTOR inhibition was analyzed in three HCC cell lines (Hep3B, HepG2 and Huh7), and the impact of AKT signaling on proliferation after mTOR inhibition was investigated using the novel AKT inhibitor MK-2206 and AKT isoform specific knockdown cells. Results AKT isoforms become differentially activated during feedback activation following RAD001 treatment. The combination of mTOR inhibition and AKT isoform knockdown showed only a weak synergistic effect on proliferation of HCC cell lines. However, the combinatorial treatment with RAD001 and the pan AKT inhibitor MK-2206 resulted in a strong synergism, both in vitro and in vivo . Moreover, by analyzing primary HCC tissue samples we were able to demonstrate that a hotspot mutation (H1047R) of PI3KCA, the gene encoding the catalytic subunit of PI3K, was associated with increased in vitro kinase activity of all AKT isoforms in comparison to healthy liver tissue of the patient. Conclusion Our results demonstrate that dual targeting of mTOR and AKT by use of RAD001 and the pan AKT inhibitor MK-2206 does effectively inhibit proliferation of HCC cell lines. These data suggest that combined treatment with RAD001 and MK-2206 may be a promising therapy approach in the treatment of hepatocellular carcinoma.
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Everolimus
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The PI3K-AKT pathway is hyperactivated in many human cancers, and several drugs to inhibit this pathway, including the PI3K/mTOR dual inhibitor NVP-BEZ235, are currently being tested in various preclinical and clinical trials. It has been shown that pharmacologic inhibition of the PI3K-AKT pathway results in feedback activation of other oncogenic signaling pathways, which likely will limit the clinical utilization of these inhibitors in cancer treatment. However, the underlying mechanisms of such feedback regulation remain incompletely understood. The PI3K-AKT pathway is a validated therapeutic target in renal cell carcinoma (RCC). Here, we show that FoxO transcription factors serve to promote AKT phosphorylation at Ser473 in response to NVP-BEZ235 treatment in renal cancer cells. Inactivation of FoxO attenuated NVP-BEZ235-induced AKT Ser473 phosphorylation and rendered renal cancer cells more susceptible to NVP-BEZ235-mediated cell growth suppression in vitro and tumor shrinkage in vivo. Mechanistically, we showed that FoxOs upregulated the expression of Rictor, an essential component of MTOR complex 2, in response to NVP-BEZ235 treatment and revealed that Rictor is a key downstream target of FoxOs in NVP-BEZ235-mediated feedback regulation. Finally, we show that FoxOs similarly modulate the feedback response on AKT Ser473 phosphorylation and renal tumor growth by other phosphoinositide 3-kinase (PI3K) or AKT inhibitor treatment. Together, our study reveals a novel mechanism of PI3K-AKT inhibition-mediated feedback regulation and may identify FoxO as a novel biomarker to stratify patients with RCC for PI3K or AKT inhibitor treatment, or a novel therapeutic target to synergize with PI3K-AKT inhibition in RCC treatment.
mTORC2
FOXO3
FOXO1
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Abstract The purpose of this study was to explore the mechanism of Solanine disrupting energy metabolism in human renal cancer ACHN cells and to clarify its target. The specific method was to culture human renal cancer ACHN cell lines, and to intervene with Solanine of high, medium and low concentrations. The content of ATP in cells was measured by ELISA method. The expression of HIF-1α protein and the expression of PI3K, AKT, p-PI3K, p-AKT in PI3K/ AKT pathway were detected by Western blotting. The results showed that compared with the control group, the relative expression of p-PI3K and p-AKT showed a downward trend with the increase of Solanine concentration (P < 0.05), while the relative expression of PI3K and AKT showed no significant change (P > 0.05). In addition, the relative expression of HIF-1α also showed a downward trend (P < 0.05). According to the above results, it is suggested that Solanine can significantly inhibit the energy metabolism of renal cancer cells, the main mechanism of which is the down-regulation of HI-1αf downstream of the PI3K/Akt pathway by inhibiting the phosphorylation process of PI3K/ p-PI3K and Akt/p-Akt.
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Wortmannin
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The Akt/mTOR signaling cascade is a critical pathway involved in various physiological and pathological conditions, including regulation of cell proliferation, survival, invasion, and angiogenesis. In the present study, we investigated the anti-neoplastic effects of casticin (CTC), identified from the plant Vitex rotundifolia L., alone and/or in combination with BEZ-235, a dual Akt/mTOR inhibitor in human tumor cells. We found that CTC exerted a significant dose-dependent cytotoxicity and reduced cell proliferation in a variety of human tumor cells. Also, CTC effectively blocked the phosphorylation levels of Akt (Ser473) and mTOR (Ser2448) proteins as well as induced substantial apoptosis. Additionally treatment with CTC and BEZ-235 in conjunction resulted in a greater apoptotic effect than caused by either agent alone thus implicating the anti-neoplastic effects of this novel combination. Overall, the findings suggest that CTC can interfere with Akt/mTOR signaling cascade involved in tumorigenesis and can be used together with pharmacological agents targeting Akt/mTOR pathway.
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mTORC2
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Objective To develope gene knockdown cell model with artificial microRNA in setting up gene knockdown cell model.Methods We constructed vectors,and prepared siRNA fragments targeting on DJ-1.Then we transciently transfected the artificial miRNA and siRNA into MN9D cells by lipofectamine2000 reagent,the mRNA and protein expression level of DJ-1 gene were detected by RT-PCR and Western blot.Results Compared with control group,DJ-1 expression level was significantly decreased in both artificial miRNA and siRNA groups.DJ-1 was knockdowned and DJ-1 was decreased 90%(P0.05)at mRNA expression level,and decreased 70%~85%(P0.05) at protein level in MN9D cells transfected with the artificial miRNA.While DJ-1 was decreased by 50%~70%(P0.05)at mRNA level,and decreased by 20%~50%(P0.05)at protein level in MN9D cells transfected with siRNA.Comparing with siRNA,miRNA was more effective in silencing DJ-1.Conclusion The artificial miRNA and siRNA are both effective in silencing gene.miRNA has more significant function in knockingdown DJ-1 than siRNA.
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